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musclehealth
New member
I have been reading alot letwly on people saying receptor sensitiivity or receptor downgrade is a myth. What u guys think?
Receptor downgrade? MYTH????????
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bluetwistedsteel67
New member
Total myth. No science behind it at all. People think receptors are the issue when they aren't growing on cycles that made them grow previosly but they forget that they may be 10-15lbs further past their normal genetic limit then the were for the prior cycle. They just need more mg's (don't we all). If Cutler had absolutely fresh receptors does anyone think he would grow on 500mg of test per week? He needs about 3 grams per week in addition to some other candy to even have a chance to grow. So my advice to all that are not growing? EAT! And if that doesn't work? REST! and if that doesn't work? Up your dosage.
pitbullrocco
New member
where's lanky???
Receptors are continuously being regenerated so downregulation is not a factor. If anything, I believe there would be an initial upregulation of the AR. I've read theories that on prolonged AAS use, some downregulation "may/can" occur. However, with the regeneration of AR's, this is sort of moot.
I'm no expert in this area so that is all I will say on the matter. In fact, if the above is wrong, then somebody correct me.
BMJ
I'm no expert in this area so that is all I will say on the matter. In fact, if the above is wrong, then somebody correct me.
BMJ
The argument is that the receptors are replaced but in a weaker state. Like muscle cells subjected to oxidative stress. They regenerate but they are weaker in terms of mitochondrial activity.
That's the arguement. There is no proof it's true in humans. Animals yes, cross-species maybe. Check needstogetas for that answer.
That's the arguement. There is no proof it's true in humans. Animals yes, cross-species maybe. Check needstogetas for that answer.
I'm no Lanky here, but i do know that when i switch compounds when i've been on a previous one for some time, i begin to respond more favorable. So i'm not totally 100% convinced its a myth. The only exclusion to this for me is equipoise, i start responding favorable at >8 weeks.
MTS
New member
"While there are no studies showing downregulation in human skeletal muscle resulting from high-dose AAS use, there are some studies in cell culture, and sometimes in vivo, which seem to indicate that downregulation can occur, though not as a result of increase in androgen from normal to supraphysiological.
This is seen both by measurement of AR mRNA, which is in an indicator of the rate of AR production, and in measurement of receptor number.
All of these studies, however, are flawed from the perspective of the bodybuilder wishing to know if downregulation of the AR has ever been observed in any cell in response to increase of androgen from normal to supranormal levels.
Range of measurement
First, the question is, downregulation relative to what? What is the control?
Unfortunately, the control for in vivo studies is castration, not the normal state. The bodybuilder really doesn’t care if normal testosterone levels may result in fewer ARs for some cell types than would be seen with castration. We would not want to get castrated just to have more ARs than in the intact condition, if for no other reason than that the decrease in androgen level would be more significant than any possible increase in AR number.
In vitro studies have generally been done with zero androgen as the control, not normal androgen.
It cannot be projected that if AR number decreased as testosterone level was increased from zero to normal, that therefore it would continue to decrease as level was increased yet further. For example, the cause of this might be that there is a promotion mechanism increasing AR mRNA production as testosterone levels fall to zero. That would not mean that there would be any loss as testosterone levels increase past normal. Or if it is a repression mechanism that comes into play as testosterone levels rise past zero, that mechanism might be fully saturated by the time levels reach normal, and no further repression might occur as levels go past normal.
In fact, papers which report downregulation, even in their titles, often show in the actual data that the range of downregulation was entirely between zero and normal, or even zero and a subnormal level. Thus they give no evidence whatsoever of downregulation occurring with supraphysiological levels of androgen relative to normal levels.
Estrogen
Testosterone can aromatize to estrogen, which can itself lead to downregulation of the AR. Thus, if a study used testosterone but did not verify that the same results were seen with nonaromatizing androgen, or did not verify that use of an aromatase inhibitor did not change results, there is no way to know if any observed downregulation is due to androgen or not. It might be due to estrogen.
Assay
Unfortunately, AR concentrations are very low in cells, and mRNA is not so easily measured. It is possible for measurements to be misleading.
In Biochemical and Biophysical Research Communications (1991) 177 488, Takeda, Nakamoto, Chang et al. determined, "Our immunostaining [for amount of ARs] and in situ hybridization data [for amount of AR mRNA] indicated that in rat and mouse prostate, androgen-withdrawal decreased both androgen receptor content and androgen receptor mRNA level, and that injection of androgen restored normal levels, a process termed ‘upregulation’….However, Northern blot data of Quarmby et al. in rat prostate have shown a different result, downregulation: the amount of androgen receptor mRNA increased by androgen withdrawal and decreased below the control level after androgen stimulation. Our preliminary Northern blot data (unpublished data) also showed the same tendency, downregulation." [emphasis added]
The authors go on to explain in detail, somewhat beyond the scope of this article, why Northern blot analysis can lead to false results. The in situ hybridization method is indisputably a superior, more accurate method.
Many of the studies claiming downregulation depend on Northern blot data as the sole "proof." This study, however, shows that such measurement might be entirely wrong. In any case, regulation properly refers to control of the number of receptors. Production of mRNA is one of the contributing factors, but ultimately what must be measured to determine the matter is the number of receptors. This has been done in some experiments.
Specific papers often cited to support downregulation of the AR
Endocrinology (1981) 104 4 1431. This paper compares the normal state of the rat to the castrated state, and the muscle cytosol AR concentrations of the female rat to the intact (sham-operated) male rat.
Objections to this study include the fact that the effect of supraphysiological levels of androgen was not studied; that cytosolic measurements of AR are unreliable since varying percentages of ARs may concentrate in the nuclear region, and these are more indicative of activity; and that castration of rats is notorious for producing false conclusions. The cells, and indeed the entire system of the animal, undergo qualitative change (e.g., cessation of growth) from the castration relative to the sham-operated animals. Testosterone levels are not the only thing which change upon castration. Another objection is that estrogen was not controlled and the effects of estrogen were not determined or accounted for. Estrogen levels certainly were not constant in this experiment.
Molecular Endocrinology (1990) 4 22. AR mRNA level, in vitro, was seen to increase as androgen levels were reduced below normal. Supraphysiological levels were not tested. Northern blot analysis was used. AR levels were not measured.
Molecular and Cellular Endocrinology (1991) 76 79. In human prostate carcinoma cells, in vitro, androgen resulted in downregulation of AR mRNA relative to zero androgen levels. Levels of androgen receptor, however, increased, relative to when androgen level was zero, by a factor of two. The researchers noted, "At 49 hours, androgen receptor protein increased 30% as assayed by immunoblots and 79% as assayed by ligand binding" [the later method is the more reliable and indicative of biological effect.]
Molecular Endocrinology (1993) 7 924. In vitro, it was determined by Northern blot analysis that mRNA levels decreased when supraphysiological levels of androgen were compared to zero androgen in cancer cells. Levels of ARs were measured, and there was no observed decrease despite the observed decrease in mRNA level (as measured by Northern blot.)
Molecular and Cellular Endocrinology (1995) 115 177. COS 1 cells were transfected with human AR DNA with the CMV promoter. The authors state that the DNA sequence responsible for downregulation of the AR is encoded within the AR DNA, not the promoter region. Dexamethasone [a glucocorticoid drug similar to cortisol] was observed to result in downregulation of AR mRNA relative to zero dexamethasone level. Androgen also had this effect, but did not result in lower levels of androgen receptors. This was attributed to increase in androgen receptor half life caused by androgen administration. The observed androgen downregulation effect relative to zero androgen ended at a concentration of 0.1 nanomolar of androgen (methyltrienolone) – higher doses, to 100 nanomolar, resulted in no further downregulation of AR mRNA production.
While this list is not complete, I am not omitting any studies that appear to have any better evidence – indeed, any evidence at all – that supraphysiological levels of androgen result in downregulation, relative to normal androgen levels, of the AR The above is a reasonably complete picture of the research evidence that might be used to support the bodybuilding theory of AR downregulation. When analyzed closely, no scientific study provides support for that theory.
Scientific evidence indicating that a biochemical mechanism for upregulation does exist
Even in the above evidence which apparently (at first sight) might seem in favor of downregulation, it was sometimes seen that actual levels of the AR increased, even going from zero to normal (rather than normal to supraphysiological.) This is upregulation of the receptor, since as we recall, regulation is the control of the number of receptors, and this control may be achieved by change in the half life of the receptors. Increased half life of the receptor, all else being equal, or perhaps with change in half-life overcoming other factors, can yield higher receptor numbers. Kemppainen et al. (J Biol Chem 267 968) demonstrated that androgen increases the half life of the AR, which is an upregulating effect.
Endocrinology (1990) 126 1165. In fibroblasts cultured from human genital skin which contained very low amounts of 5-alpha reductase, 2 nanomolar tritium-labeled testosterone [which is sufficient to saturate ARs] produced a 34% increase in androgen receptors as measured by specific AR binding, the best assay method known, and 20 nanomolar tritium-labeled testosterone produced an increase of 64% in number of ARs.
Note: 20 nanomolar free testosterone is approximately 400 times physiological level (normal level in humans is approximately 0.05 nanomolar).
J Steroid Biochemistry and Molecular Biology (1990) 37 553. In cultured adipocytes, methyltrienolone and testosterone demonstrated marked upregulation of AR content upon administration of androgen. 10 nanomolar methyltrienolone increased AR content (as measured by binding to radiolabeled androgen) by more than five times, relative to zero androgen.
J Steroid Biochemistry and Molecular Biology (1993) 45 333. In cultured smooth muscle cells from the penis of the rat, mRNA production was found to be upregulated by high dose testosterone (100 nanomolar) or DHT. When 5-alpha reducatase was inhibited by finasteride, thus blocking metabolism to DHT, AR mRNA production was downregulated in response to testosterone. Blockage of the aromatization pathway to estrogen by fadrozole eliminated this downregulation effect. Estradiol itself was found to downregulate AR mRNA production in these cells.
Endocrinol Japan (1992) 39 235. One nanomolar DHT was demonstrated to increase AR protein by over 100% within 24 hours, relative to zero androgen level. The half life of the AR was demonstrated to increase from 3.3 h to 7.5 h as a result of the androgen administration.
Endocrinology (1996) 137 1385. 100 nanomolar testosterone was found to increase AR levels in vitro in muscle satellite cells, myotubes, and muscle-derived fibroblasts."
This is seen both by measurement of AR mRNA, which is in an indicator of the rate of AR production, and in measurement of receptor number.
All of these studies, however, are flawed from the perspective of the bodybuilder wishing to know if downregulation of the AR has ever been observed in any cell in response to increase of androgen from normal to supranormal levels.
Range of measurement
First, the question is, downregulation relative to what? What is the control?
Unfortunately, the control for in vivo studies is castration, not the normal state. The bodybuilder really doesn’t care if normal testosterone levels may result in fewer ARs for some cell types than would be seen with castration. We would not want to get castrated just to have more ARs than in the intact condition, if for no other reason than that the decrease in androgen level would be more significant than any possible increase in AR number.
In vitro studies have generally been done with zero androgen as the control, not normal androgen.
It cannot be projected that if AR number decreased as testosterone level was increased from zero to normal, that therefore it would continue to decrease as level was increased yet further. For example, the cause of this might be that there is a promotion mechanism increasing AR mRNA production as testosterone levels fall to zero. That would not mean that there would be any loss as testosterone levels increase past normal. Or if it is a repression mechanism that comes into play as testosterone levels rise past zero, that mechanism might be fully saturated by the time levels reach normal, and no further repression might occur as levels go past normal.
In fact, papers which report downregulation, even in their titles, often show in the actual data that the range of downregulation was entirely between zero and normal, or even zero and a subnormal level. Thus they give no evidence whatsoever of downregulation occurring with supraphysiological levels of androgen relative to normal levels.
Estrogen
Testosterone can aromatize to estrogen, which can itself lead to downregulation of the AR. Thus, if a study used testosterone but did not verify that the same results were seen with nonaromatizing androgen, or did not verify that use of an aromatase inhibitor did not change results, there is no way to know if any observed downregulation is due to androgen or not. It might be due to estrogen.
Assay
Unfortunately, AR concentrations are very low in cells, and mRNA is not so easily measured. It is possible for measurements to be misleading.
In Biochemical and Biophysical Research Communications (1991) 177 488, Takeda, Nakamoto, Chang et al. determined, "Our immunostaining [for amount of ARs] and in situ hybridization data [for amount of AR mRNA] indicated that in rat and mouse prostate, androgen-withdrawal decreased both androgen receptor content and androgen receptor mRNA level, and that injection of androgen restored normal levels, a process termed ‘upregulation’….However, Northern blot data of Quarmby et al. in rat prostate have shown a different result, downregulation: the amount of androgen receptor mRNA increased by androgen withdrawal and decreased below the control level after androgen stimulation. Our preliminary Northern blot data (unpublished data) also showed the same tendency, downregulation." [emphasis added]
The authors go on to explain in detail, somewhat beyond the scope of this article, why Northern blot analysis can lead to false results. The in situ hybridization method is indisputably a superior, more accurate method.
Many of the studies claiming downregulation depend on Northern blot data as the sole "proof." This study, however, shows that such measurement might be entirely wrong. In any case, regulation properly refers to control of the number of receptors. Production of mRNA is one of the contributing factors, but ultimately what must be measured to determine the matter is the number of receptors. This has been done in some experiments.
Specific papers often cited to support downregulation of the AR
Endocrinology (1981) 104 4 1431. This paper compares the normal state of the rat to the castrated state, and the muscle cytosol AR concentrations of the female rat to the intact (sham-operated) male rat.
Objections to this study include the fact that the effect of supraphysiological levels of androgen was not studied; that cytosolic measurements of AR are unreliable since varying percentages of ARs may concentrate in the nuclear region, and these are more indicative of activity; and that castration of rats is notorious for producing false conclusions. The cells, and indeed the entire system of the animal, undergo qualitative change (e.g., cessation of growth) from the castration relative to the sham-operated animals. Testosterone levels are not the only thing which change upon castration. Another objection is that estrogen was not controlled and the effects of estrogen were not determined or accounted for. Estrogen levels certainly were not constant in this experiment.
Molecular Endocrinology (1990) 4 22. AR mRNA level, in vitro, was seen to increase as androgen levels were reduced below normal. Supraphysiological levels were not tested. Northern blot analysis was used. AR levels were not measured.
Molecular and Cellular Endocrinology (1991) 76 79. In human prostate carcinoma cells, in vitro, androgen resulted in downregulation of AR mRNA relative to zero androgen levels. Levels of androgen receptor, however, increased, relative to when androgen level was zero, by a factor of two. The researchers noted, "At 49 hours, androgen receptor protein increased 30% as assayed by immunoblots and 79% as assayed by ligand binding" [the later method is the more reliable and indicative of biological effect.]
Molecular Endocrinology (1993) 7 924. In vitro, it was determined by Northern blot analysis that mRNA levels decreased when supraphysiological levels of androgen were compared to zero androgen in cancer cells. Levels of ARs were measured, and there was no observed decrease despite the observed decrease in mRNA level (as measured by Northern blot.)
Molecular and Cellular Endocrinology (1995) 115 177. COS 1 cells were transfected with human AR DNA with the CMV promoter. The authors state that the DNA sequence responsible for downregulation of the AR is encoded within the AR DNA, not the promoter region. Dexamethasone [a glucocorticoid drug similar to cortisol] was observed to result in downregulation of AR mRNA relative to zero dexamethasone level. Androgen also had this effect, but did not result in lower levels of androgen receptors. This was attributed to increase in androgen receptor half life caused by androgen administration. The observed androgen downregulation effect relative to zero androgen ended at a concentration of 0.1 nanomolar of androgen (methyltrienolone) – higher doses, to 100 nanomolar, resulted in no further downregulation of AR mRNA production.
While this list is not complete, I am not omitting any studies that appear to have any better evidence – indeed, any evidence at all – that supraphysiological levels of androgen result in downregulation, relative to normal androgen levels, of the AR The above is a reasonably complete picture of the research evidence that might be used to support the bodybuilding theory of AR downregulation. When analyzed closely, no scientific study provides support for that theory.
Scientific evidence indicating that a biochemical mechanism for upregulation does exist
Even in the above evidence which apparently (at first sight) might seem in favor of downregulation, it was sometimes seen that actual levels of the AR increased, even going from zero to normal (rather than normal to supraphysiological.) This is upregulation of the receptor, since as we recall, regulation is the control of the number of receptors, and this control may be achieved by change in the half life of the receptors. Increased half life of the receptor, all else being equal, or perhaps with change in half-life overcoming other factors, can yield higher receptor numbers. Kemppainen et al. (J Biol Chem 267 968) demonstrated that androgen increases the half life of the AR, which is an upregulating effect.
Endocrinology (1990) 126 1165. In fibroblasts cultured from human genital skin which contained very low amounts of 5-alpha reductase, 2 nanomolar tritium-labeled testosterone [which is sufficient to saturate ARs] produced a 34% increase in androgen receptors as measured by specific AR binding, the best assay method known, and 20 nanomolar tritium-labeled testosterone produced an increase of 64% in number of ARs.
Note: 20 nanomolar free testosterone is approximately 400 times physiological level (normal level in humans is approximately 0.05 nanomolar).
J Steroid Biochemistry and Molecular Biology (1990) 37 553. In cultured adipocytes, methyltrienolone and testosterone demonstrated marked upregulation of AR content upon administration of androgen. 10 nanomolar methyltrienolone increased AR content (as measured by binding to radiolabeled androgen) by more than five times, relative to zero androgen.
J Steroid Biochemistry and Molecular Biology (1993) 45 333. In cultured smooth muscle cells from the penis of the rat, mRNA production was found to be upregulated by high dose testosterone (100 nanomolar) or DHT. When 5-alpha reducatase was inhibited by finasteride, thus blocking metabolism to DHT, AR mRNA production was downregulated in response to testosterone. Blockage of the aromatization pathway to estrogen by fadrozole eliminated this downregulation effect. Estradiol itself was found to downregulate AR mRNA production in these cells.
Endocrinol Japan (1992) 39 235. One nanomolar DHT was demonstrated to increase AR protein by over 100% within 24 hours, relative to zero androgen level. The half life of the AR was demonstrated to increase from 3.3 h to 7.5 h as a result of the androgen administration.
Endocrinology (1996) 137 1385. 100 nanomolar testosterone was found to increase AR levels in vitro in muscle satellite cells, myotubes, and muscle-derived fibroblasts."
redsamurai
Banned
gautho said:
which happens how??.....receptor downgrade is the only plausible answer. I think both arguments are true......yes there is receptor downgrade, BUT....there are also physical limits to the human body....and as you approach those limits....gains come harder and harder....no matter what you put in your system. In the body, there are probably defense mechanisms against hyper muscularity that we don't even know about yet.
That's not receptor downregulation. That's simple genetics. You're limits are set by genetics.
Using steroids 101, again. Ready class??
When you take steroids you increase the activity in your genes, that results in an increase in protein synthesis. Increased protein systhesis means??? GROWTH! Hurray you've now changed your puny genetics into super genetics. Very Good. But what happens when the amount of AS you use hits a limit too? You pour in more and move the bar again in that gene activity, and that results in??? Even better protein synthesis and even more growth. Metaphorically you're feeding your genes more so you grow more. And that's why pros just keep getting bigger. Luckily there is myostatin to limit this growth or they'd weigh 600 or more pounds. But that's another lesson.
Using steroids 101, again. Ready class??
When you take steroids you increase the activity in your genes, that results in an increase in protein synthesis. Increased protein systhesis means??? GROWTH! Hurray you've now changed your puny genetics into super genetics. Very Good. But what happens when the amount of AS you use hits a limit too? You pour in more and move the bar again in that gene activity, and that results in??? Even better protein synthesis and even more growth. Metaphorically you're feeding your genes more so you grow more. And that's why pros just keep getting bigger. Luckily there is myostatin to limit this growth or they'd weigh 600 or more pounds. But that's another lesson.
lanky
Well-known member
The androgen-androgen receptor (AR) signaling pathway plays a key role in proper development and function of male reproductive organs, such as prostate and epididymis, as well as nonreproductive organs, such as muscle, hair follicles, and brain. Abnormalities in the androgen-AR signaling pathway have been linked to diseases, such as male infertility, Kennedy’s disease, and prostate cancer. Regulation of AR activity can be achieved in several different ways: modulation of AR gene expression, androgen binding to AR, AR nuclear translocation, AR protein stability, and AR trans-activation. This review covers mechanisms implicated in the control of AR protein expression and degradation, and their potential linkage to the androgen-related diseases. A better understanding of such mechanisms may help us to design more effective androgens and antiandrogens to battle androgen-related diseases.
Introduction
Top
Abstract
Introduction
Structure of AR
How to control AR...
Linkage of AR expression...
Natural products that regulate...
Concluding remarks and future...
References
ANDROGENS, TESTOSTERONE (T) and its metabolite 5-dihydrotestosterone (DHT), play a key role in proper development and function of male reproductive organs, such as prostate and epididymis, as well as nonreproductive organs, such as muscle, hair follicles, and brain (1, 2, 3). The action of androgens is mediated by androgen receptor (AR), a member of the steroid hormone receptor superfamily (1, 2, 3). AR is nearly ubiquitously expressed in mammalian tissues. Upon T or DHT binding, AR undergoes a series of conformational changes that allows AR to interact with androgen response elements (ARE) in various androgen target genes (4, 5). T, the major form of circulating androgen, is synthesized mainly in the Leydig cells of the testes (6). Circulating androgen plays an important role in the hypothalamus-pituitary-testis loop to maintain the proper concentration of sex steroid hormones (7). Adrenal glands also secrete precursor forms of androgen, dehydroepiandrosterone, and androstenedione (7). DHT, a more potent androgen than T, is synthesized mostly in the peripheral tissues, such as prostate (8). The majority of circulating androgens (97–99%) in serum are complexed with serum proteins, such as SHBG or albumin (9, 10). Thus, only 1–3% of androgen is a free form that can diffuse into membranes and bind to AR. This indicates that AR may be a rate-limiting factor for the androgen-AR signaling pathway in cells.
The functional significance of AR in male sex differentiation has been demonstrated by various natural germline AR mutations that cause partial or complete androgen insensitivity syndromes (11, 12). Patients with complete androgen insensitivity syndrome are phenotypically females with female external genitalia, whereas patients with partial androgen insensitivity syndrome are males with a spectrum of defects that vary from near-normal male phenotypes to near-normal female phenotypes.
While the functional significance of the androgen-AR signaling pathway in the male reproduction system is well studied, that in the female has been little understood( females are very complex, not that i needed to tell you guys that) due to the lack of a proper model system. The generation of an AR knockout (ARKO) female mouse model has long been hampered, because the AR gene is located on the X chromosome (13, 14). Recently, a cre-lox conditional KO has been successfully used to generate ARKO female mice (15). The study demonstrated that homozygous ARKO female mice produced a smaller number of pups per litter with an average of four, whereas the counterpart wild-type female mice produced an average of eight pups per litter. This result indicates that AR may also play a role in female fertility and/or ovulation (15). Further study indicated that homozygous ARKO female mice suffer from defective folliculogenesis due to poor follicular maturation and defective implantation due to subnormal uterine development (Wang, C., and C. Chang, unpublished results).
Structure of AR
Top
Abstract
Introduction
Structure of AR
How to control AR...
Linkage of AR expression...
Natural products that regulate...
Concluding remarks and future...
References
The AR is a member of the steroid receptor superfamily that is composed of a variable NH2-terminal domain, a highly conserved DNA-binding domain (DBD), a hinge domain, and a ligand-binding domain (LBD) (16, 17). The apparent molecular mass of AR is 110 kDa, with approximately 918 amino acids (Fig. 1) (1, 2, 3). The most characteristic features of the AR NH2-domain are the polyglutamine and polyglycine repeats that result in variations in the length of the AR (18, 19). The polyglutamine repeat normally ranges in length from 8–31, with an average length of 20. The length of the polyglutamine repeat is inversely correlated with AR transcriptional activity in vitro (20), in other words, reduced AR trans-activation with long polyglutamine repeats. The inverse correlation of AR trans-activation activity with the polyglutamine repeat length might result from the influence of the repeat on interaction with coregulators (21). Alternatively, short polyglutamine repeats may interfere with the AR NH2-terminal domain phosphorylation or interdomain interaction of AR (22, 23). The inverse correlation between length of the polyglutamine repeat and AR mRNA and protein expression in vitro has been also reported (24). Expansion of the polyglutamine repeat length to over 40 causes Kennedy’s disease, a neuromuscular disorder associated with decreased virilization (25). In contrast, yet still controversial, short polyglutamine repeats have been linked to a higher risk factor of developing prostate cancer (reviewed in Refs. 11 , 12 , and 18).
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FIG. 1. Structures of the AR gene and protein. A, The exons of the AR gene are depicted in rectangles. The regulatory elements in the promoter, CRE, polypurine/polypyrimidine (Pu/Py) tract, and Sp1, are shown. B, The structural domains of AR are shown by amino acid position.
The polyglycine repeat length of AR ranges from 10–30 in the normal population (19, 26). Complete deletion of the polyglycine repeat resulted in marked reduction of AR trans-activation in vitro (27). Studies of the polyglycine repeat length as a risk factor of prostate cancer are highly conflicting. Although Stanford and colleagues reported increased prostate cancer risk with short length (16) of the polyglycine repeats, others reported no link between the polyglycine repeat length and prostate cancer risk (28).
The AR activation function-1 (AF-1) domain, amino acids 360–494, shows ligand-independent activation when artificially separated from the LBD (29). A recent study demonstrated that the WXXLF motif in the AR AF-1 domain interacts with the AR LBD, resulting in AR stabilization (30, 31, 32, 33). In addition to the AF-1 domain, the other domain located in amino acids 141–338, is required for full ligand-inducible AR activity (29, 34). Various transcription factors, such as amino-terminal enhancer of split, AR N-terminal domain transactivating protein-1, AR coregulator, AR trapped cloned 27, transcription factor IIF, and transcription factor IIH, have been demonstrated to interact with the AR NH2-terminal domain and modulate AR trans-activation (35). The AR NH2-terminal domain can be phosphorylated by various kinases (36, 37). Certain phosphorylation pathways of the AR NH2-terminal domain have been reported to play an important role in AR trans-activation (37). In addition to phosphorylation, the AR NH2-terminal domain has been reported to be covalently modified by SUMO-1 (sumoylation) (38). Mutation of two sumoylation sites, lysine residues K386 and K520, in AR enhances AR trans-activation, indicating that SUMO-1 inhibits AR activity. The K386 is strongly sumoylated as a master switch for sumoylation of AR, whereas K520 is poorly sumoylated. Mutation of K520 alone showed little enhancement of AR trans-activation, but the double mutation of K386/K520 showed higher enhancement of AR trans-activation than mutation of K386 alone. The sumoylation consensus sequence (I/L/V)KXE has been identified in the other steroid hormone receptors, indicating that reversible sumoylation is one mechanism to modulate steroid receptor activity.
Like other steroid receptors, the AR DBD consists of two zinc fingers. In the case of the inverted ARE, the first zinc finger of AR recognizes and binds to DNA with further stabilization of DNA-AR interaction through the second zinc finger interaction with the phosphate backbone (39, 40). In the case of direct repeat ARE, the second zinc finger and a part of the hinge region interact directly with DNA (41). In addition to the DNA-receptor interaction, amino acids from 617–633 in the AR DBD and hinge region contain a nuclear localization signal (42, 43). Recently, the PEST (proline-, glutamate-, serine-, and threonine-rich) sequence, a degradation motif, has been identified in the AR hinge domain (44). The PEST sequence is found in proteins with short half-lives that are degraded by the ubiquitin-mediated proteasome system (45).
The AR LBD domain, like progesterone receptor (PR) and estrogen receptor (ER), consists of 12 -helixes forming the ligand binding pocket (46, 47, 48, 49). X-Ray crystallographic analyses of ligand-bound and -unbound ER and PR have demonstrated that ligand binding induces a conformational change in the LBD in which helix 12 and the AF-2 domain fold back across the ligand binding pocket, forming a ligand-dependent interaction surface for coregulators (50, 51). Although the crystal structure of the ligand-bound AR LBD is similar to that of the ligand-bound ER or PR (47, 48), functional analyses of the full-length receptors suggest distinct differences in the coregulator interaction with AR and ER. Although the structure of the ligand-bound AF-2 domain of ER or PR is important for interaction with a coregulator steroid receptor coactivator 1 (SRC-1) (52, 53), that of AR is not essential for interaction with SRC-1 (31, 54). Instead, the primary interaction sites of SRC-1 are in the AR NH2-terminal and DBD (31). A recent study indicates that the AR AF-2 domain may stabilize the overall structure of AR to allow the AR NH2-terminal domain to interact with appropriate coregulators (55).
An AR isoform (AR-A) with an apparent molecular mass of 87 kDa, which is an NH2-terminally truncated form of a major AR (AR-B), was reported (56, 57). That study reported that the AR-A isoform comprised 4–26% of the total immunoreactive AR protein in tissues, judged by an immunoblot analysis using the antibodies against the AR NH2-terminal domain. Although the study suggested that the use of initiation methionine at amino acid 188 would yield the AR-A isoform, the possibility that the AR-A isoform might be a proteolyzed product of AR could not be ruled out. As no follow-up studies were reported about the N-terminally truncated isoform of AR, the concept of AR isoforms is not widely accepted. This is in contrast to other steroid receptors, such as PR and ER (58, 59). PR isoforms, PR-A and PR-B, result from alternative usage of initiation codons of the same gene, whereas the ER isoforms, ER and ERß, result from two different genes.
How to control AR expression
Top
Abstract
Introduction
Structure of AR
How to control AR...
Linkage of AR expression...
Natural products that regulate...
Concluding remarks and future...
References
Regulation of AR mRNA level by androgen.
Androgens regulate AR at the level of both mRNA and protein. Regulation of AR protein by androgens is discussed below. Prolonged treatment of human prostate cancer LNCaP as well as breast cancer T47D cells with androgens for 48 h or longer decreased AR mRNA about 2-fold (60, 61, 62, 63). Northern blot assays demonstrated that down-regulation of AR mRNA by androgen in LNCaP cells is at the transcriptional level (62). These results indicate that androgens down-regulate AR expression to limit the androgen response in certain cell types. In contrast, up-regulation of AR mRNA by androgens has been reported in other cell types, including human hepatocellular carcinoma and osteoblastic cell lines (64, 65). Possible up-regulation of AR mRNA by androgens has also been reported in rat hippocampus (66).
In contrast to down-regulation of AR mRNA by androgen in prostate cancer LNCaP cells, Takeda et al. (67) reported that AR mRNA levels in rat or mouse prostate ventral lobe epithelial cells were markedly decreased 3 d after castration, as judged by in situ hybridization. Treatment of the castrated animals with DHT restored the AR mRNA level, indicating up-regulation of AR mRNA by androgens (67). However, this report is in contrast with another report showing that castration increased AR mRNA levels in rat prostate ventral lobes, as judged by Northern blot assay (68). The reasons for these discrepancies in the effect of androgen on the AR mRNA level in rat prostate ventral lobe might be explained by the techniques used. Castration causes drastic changes in total RNA and protein contents in prostate, a highly androgen-dependent organ (69). Therefore, normalization of total RNA in Northern blot analysis may result in complicated interpretations.
Modulation of AR expression at the transcriptional level. 5' Upstream regulatory elements in the AR promoter:
The AR 5' upstream promoter region does not contain a typical TATA box, but contains Sp1-binding sites at -46 bp (70, 71, 72). The TATA box is a binding site for general transcription factor IID (TFIID)/TFIID binding protein and is required for directing accurate transcription initiation sites (73). Sp1 has been reported to recruit TFIID/TFIID binding protein and mediate the formation of the transcription preinitiation complex on the TATA-less promoters (74). S1 nuclease protection of AR mRNA demonstrated two transcription initiation sites separated by 12 bp (75, 76). Transcription from multiple initiation sites is typical for TATA-less promoters. Inhibition of Sp1 activity resulted in a marked reduction of AR protein level in human prostate cancer LNCaP cells (71, 77), indicating that Sp1 plays a key role in the regulation of AR mRNA synthesis. The highly conserved homopurine-homopyrimidine region is located upstream of the Sp1 site in the AR promoter (78), and deletion of this region of the human AR promoter resulted in 3- to 4-fold reduction of AR promoter activity (72, 78).
Another key regulatory element for AR mRNA expression is the cAMP response element (CRE) located at -508 bp in the human AR gene (72, 79). Whereas Sp1 and homopurine-homopyrimidine regions are conserved among the human, rat, and mouse AR gene promoters, the CRE has not been identified in the rat or mouse AR gene promoters. Treatment with the cAMP analog dibutyryl cAMP enhanced AR promoter activity about 4- to 6-fold in a reporter gene assay and increased AR mRNA expression in LNCaP cells (72). It has been proposed that the AR mRNA in rat Sertoli cells is up-regulated by FSH, which may function through cAMP-CRE interaction (80, 81).
The 5' upstream regulatory elements for down-regulation of AR mRNA expression have not been well studied in the human AR gene. The NF1 site of the rat AR promoter has been reported to suppress AR mRNA expression (82). Mutation of the NF1 site increased rat AR promoter activity about 8-fold in transfection assays. However, deletion of the NF1 site of the human AR promoter did not significantly change AR promoter activity (72), indicating species specificity in regulation of the AR promoter region. Age-related down-regulation of AR mRNA expression was observed in rat hepatic cells (83, 84), with mRNA level in senescent rats 70-fold lower than those in young rats. An increase in nuclear factor-B (NF-B) and a decrease in age-dependent factor contributed to the down-regulation of AR mRNA in senescent rats (84).
Masked AR expression
The AR gene contains a CpG island that covers the proximal promoter region and the first exon (72, 85). Hypermethylation of the CpG island in the promoters has been associated with transcriptional inactivation (86). The CpG island of the AR gene is heavily methylated in androgen-independent human prostate cancer DU145 cells, resulting in masked AR expression (85, 87, 88). Treatment of the demethylation reagent 5-aza-2'-deoxycytidine restored AR expression in DU145 cells (87). Most primary and androgen-independent prostate tumors continue to express the AR and androgen-responsive genes such as prostate-specific antigen (PSA), suggesting that androgen-independent cancer cells may retain a functional AR signaling pathway at the castration level of androgen. However, masked AR expression in certain androgen-independent metastatic prostate cancers has been identified (88). That study showed masked AR expression in two of 15 prostate cancer specimens from patients who died of androgen-independent metastastic prostate cancer. Detailed methylation analyses using bisulfide sequencing found that two methylation consensus sequences in the AR gene, from -131 to -121 and from +44 to +54, are linked to the loss of AR expression in metastatic hormone-refractory prostate cancer (88). Sp1 binding to the AR gene promoter has been shown to prevent the CpG island from being methylated (89, 90).
Recently, we took advantage of C2C12 myoblast cells stably transfected with the AR cDNA under the control of the 3.6-kb natural human AR promoter, named C2C12/npAR, to analyze AR function in skeletal muscle tissue. We found that the AR protein level in C2C12/npAR myoblast cells was undetected, whereas the AR protein level was markedly up-regulated in terminally differentiated C2C12/npAR myotubes (Lee, D. K., and C. Chang, unpublished results). Nuclear run-on transcription and pulse-chase labeling assays indicated that the marked up-regulation of AR expression does not result from the altered AR protein stability, but from transcriptional induction of AR mRNA (Lee, D. K., and C. Chang, unpublished results). Further study is required for analyses of the mechanisms implicated in AR mRNA induction during skeletal muscle cell differentiation.
Modulation of AR expression at the posttranscriptional level.
In addition to controlling AR mRNA levels at the transcriptional level, androgens also regulate AR expression at the posttranscriptional level. Androgens have been reported to modulate both stability and translation efficiency of the AR mRNA. Androgens decrease AR mRNA levels, but increase AR protein levels in both prostate cancer LNCaP and breast cancer MDA453 cells (91). Androgens down-regulate AR mRNA transcription in LNCaP cells. However, androgens do not change AR mRNA transcription in MDA453 cells, but destabilize AR mRNA, indicating that androgens down-regulate AR mRNA level at the posttranscriptional level in MDA453 cells (91).
Studies to date indicate that the AR 5'- and 3'-untranslated regions (UTRs) play a role in controlling AR mRNA level at the posttranscriptional level. The human AR mRNA has a relatively long 5'-UTR of about 1100 nt (75). Reporter gene assays using chloramphenicol transferase (CAT) reporter plasmids fused with the AR 5'-UTR between the simian virus 40 promoter and the CAT-coding region demonstrated that the AR 5'-UTR is absolutely required for induction of CAT activity (92). Further deletion analysis indicated that 180 bp from +21 to +202, including a stem-loop secondary structure, were sufficient for the induction of CAT activity. S1 nuclease protection experiments demonstrated that the 180 bp in the fused reporter genes did not increase CAT mRNA levels, indicating that it enhanced the efficiency of translation (92). This report suggests that the 5'-UTR of AR mRNA may be required for efficient translation of AR mRNA.
AR mRNA is expressed as two transcripts with (10.5 kb) or without (8 kb) the 3'-UTR (62, 76, 93). A recent study demonstrates that the 3'-UTR of AR mRNA contains a highly conserved UC-rich region that is a target for cytoplasmic and nuclear RNA-binding proteins, such as poly(C)-binding protein-1 (CP-1) and Hu protein R (HuR) (94). Immunoprecipitation assays demonstrated that these proteins indeed associated with the UC-rich region of AR 3'-UTR (94). HuR is ubiquitously expressed and is involved in the stabilization of several mRNAs (95, 96). CP-1 has been reported to control mRNA turnover and translation (97, 98). Association of HuR and CP-1 with the UC-rich region in AR 3'-UTR indicates that the AR 3'-UTR may play a role in posttranscriptional regulation of AR expression. RT-PCR and sequence analysis from a patient with partial androgen insensitivity syndrome demonstrated that the patient expressed a variant of AR mRNA with the complete coding region, but a shortened 3'-UTR, indicating the physiological significance of AR 3'-UTR in the androgen-AR signaling pathway (99).
Modulation of AR expression at the protein level. Degradation of receptors to control hormone responsiveness: AR vs. ER, GR, and PR.
Numerous studies demonstrate that protein degradation is required not only for elimination of misfolded or unfolded proteins, but also for maintenance of cellular function (100). Steroid receptors have relatively short half-lives, indicating that the amount and duration of steroid receptor ligand effect in cells are regulated by systematic protein activation and degradation (Fig. 2). Proteins with a short half-life, such as certain transcription factors and cell cycle regulators, undergo systematic protein degradation by the ubiquitin-proteasome pathway (100, 101).
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FIG. 2. Mechanisms to limit hormone responsiveness by AR and ER. AR limits hormone responsiveness by ligand dissociation and receptor recycling, whereas ER limits by degradation of ER protein. Although AR does not use a proteasome-ubiquitin pathway to limit hormone responsiveness, a systematic proteasome-ubiquitin pathway may regulate overall AR protein level in cells.
Ligand-dependent and proteasome-mediated degradation has been detected in ER (102, 103, 104), PR (105), and GR (106). MG132, a specific proteasome inhibitor, inhibited ER-mediated transcription, but not p53- or Sp1-mediated transcription (103, 104). Integrity of ER function is required for 26S proteasome-mediated receptor degradation, as deletion of the last 61 amino acids, including helix 12, abolished ligand-dependent and proteasome-mediated down-regulation of the receptor (104). As the last 61 amino acids of ER serve as a binding domain for various coregulators, it is likely that ligand-dependent down-regulation may be dependent on the protein-protein interaction of ER with coactivators (104). Interestingly, the ER mutants lacking an ability to bind DNA also showed ligand-dependent down-regulation, indicating that DNA binding of ER or engagement of ER in the transcription complex is not necessary for the ligand-dependent down-regulation (104). Treatment of cells with the general transcription inhibitor, actinomycin D, or the translation inhibitor, cycloheximide, prevented ER from ligand-dependent down-regulation (104), indicating that continuous synthesis of proteins is required for the ligand-dependent down-regulation of ER.
However, the AR protein level is not down-regulated in the presence of androgens. Instead, androgens have been shown to increase AR protein levels in various cell contexts (91). This result indicates that AR may use another mechanism to limit hormone responsiveness. A recent study using green fluorescent protein technology demonstrates that AR migrates to the subnuclear compartment in the presence of androgen within 15–60 min (107). AR migrates rapidly back to the cytoplasm upon androgen withdrawal and maintains its ability to reenter the nucleus for at least four rounds of AR recycling after the initial androgen treatment (107). This indicates that AR inactivation and migration into the cytoplasm due to ligand dissociation, as well as AR recycling rather than receptor degradation, may control the hormone responsiveness of AR (107).
Modulation of AR protein half-life
AR, like other members of the steroid hormone receptor family, requires dimerization to execute its function (16). Detailed analyses using a two-hybrid protein interaction indicated that the ligand-binding domain interacts with the NH2-terminal domain plus the DBD or the full-length AR in a ligand-dependent manner (31, 32, 33). The androgen-dependent intermolecular interaction between the NH2-terminal domain and the COOH-terminal domain of AR decreases a dissociation rate of the bound androgen and increases AR protein half-life. Systematic mutational analyses showed that two motifs, 23FXXLF (27) and 433WXXLF (4, 37), in the NH2-terminal domain may be required for the AR NH2-terminal interaction with the ligand-binding domain (30). Two-hybrid protein interaction and glutathione-S-transferase pull-down assays showed that mutations of any conserved amino acid sequences in the motifs resulted in marked reduction of the AR NH2-terminal/COOH-terminal domain interaction (30).
The half-life of AR protein varies depending on the context of the cell environment. Androgens have been reported to stabilize AR protein in various cell contexts (91). The half-life of AR in LNCaP cells is approximately 3 h in the absence of androgens and is longer than 10 h in the presence of 10 nM DHT. Interestingly, the half-life of AR from the androgen-independent subline of LNCaP that was grown without androgen treatment, LNCaP-C4–2, is 7 h in the absence of androgens (108). A prolonged half-life of AR protein in the absence of androgens was also observed in recurrent CWR22 tumors (108). In the absence of androgens, AR in the recurrent tumor was detected in nuclei, whereas AR in LNCaP cells was predominantly detected in cytoplasm. These results indicate that AR stabilization and nuclear translocalization in the absence of androgens may activate the AR signaling pathway, resulting in prostate cancer cell proliferation at the castrated level of androgens.
Mechanisms of AR degradation
Systematic protein degradation by the ubiquitin-proteasome system plays important roles in the maintenance of cellular functions, such as cell cycle and antigen presentation. Protein ubiquitination provides the recognition signal for the 26S proteasome, leading to protein degradation (101). Protein ubiquitination requires the activities of three different enzymes, ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzymes (E2s), and ubiquitin ligases (E3s) (101). The E3 ubiquitin ligation step often determines the specificity of protein ubiquitination and serves as a rate-limiting step. Phosphorylation of target proteins has been reported to be necessary for recognition by E3 ligases (109). A good example is IB, which associates with NF-B and inhibits NF-B activity (110, 111). Upon induction by cytokine treatment, IB is phosphorylated at the two specific serine residues, Ser32 and Ser36, leading to NF-B activation via degradation of IB. Mutations of the serine residues prevent IB phosphorylation as well as IB degradation.
Although AR does not undergo ligand-dependent down-regulation of the protein level, recent studies indicate that the AR protein level in cells is regulated by systemic protein degradation pathways. Inhibition of the ubiquitin-proteasome degradation pathway by MG132 has been reported to increase endogenous AR protein levels in HepG2 and LNCaP cells (44). The PEST sequence, a signature motif for ubiquitin-proteasome degradation, has been identified in the hinge region of AR (44). To be degraded by the ubiquitin-proteasome system, AR should be recognized by E3 ligases. A recent study demonstrates that Mdm2 is an E3 ligase for the ubiquitin-proteasome degradation of AR (112). AR associates with Mdm2 in cells, as shown by the coimmunoprecipitation assay. Detailed biochemical binding assays using a glutathione-S-transferase pull-down assay demonstrate that Mdm2 interacts with the AR NH2-terminal domain plus DBD (112). Phosphorylation of target proteins often serves as a recognition signal for E3 ligases (109). The study demonstrates that phosphorylation of AR at Ser213 and Ser791 by the phosphatidylinositol-3-hydroxy kinase (PI3K)/Akt pathway is necessary for AR degradation by Mdm2 E3 ligase. Transfection of constitutively active Akt into LNCaP cells promoted AR degradation, resulting in suppression of PSA expression (112). However, treatment with LY29402, an inhibitor of PI3K, increased the AR protein level in LNCaP cells. When the Mdm2-null cell line was used, AR degradation by the ubiquitin-proteasome pathway was markedly impaired, indicating that phosphorylation-dependent AR ubiquitination and degradation by the PI3K/Akt pathway require Mdm2 E3 ligase.
Phosphorylation of AR at amino acid positions Ser213 and Ser791 by the PI3K/Akt signaling pathway has been suggested to be required for androgen-independent survival and growth of prostate cancer LNCaP cells (113). Based on our study (Lin, H., and C. Chang, unpublished results), the Akt pathway suppresses AR trans-activation in LNCaP cells of low passage numbers (<30), whereas it increases AR trans-activation in LNCaP cells of high passage numbers (60). Due to the report indicating that the expression of prostate-specific phosphatase in LNCaP cells is androgen dependent in low passage numbers, but becomes androgen independent in higher passage numbers (114), it is likely that Akt may be able to differentially regulate AR activity in different passage numbers of LNCaP cells.
A recent study indicates that AR activity can be suppressed in LNCaP cells by phosphatase and tensin homolog deleted from chromosome 10 (PTEN), a tumor suppressor (Lin, H., and C. Chang, unpublished results). However, overexpression of constitutively active Akt in LNCaP cells or treatment of cells with a PI3K inhibitor did not block PTEN-mediated AR suppression, indicating that AR is inhibited by a PI3K/Akt independent pathway. Immunocytofluorescence analyses demonstrate that the expression of wild-type PTEN, but not that of the mutant form of PTEN, Cys124Ser, in LNCaP cells interferes with AR translocation into the nucleus, resulting in AR in the cytoplasm in the presence of 10 nM DHT. Moreover, the expression of the wild-type, but not the mutant, form of PTEN promotes AR protein degradation, resulting in the suppression of endogenous PSA expression in LNCaP cells. Interestingly, addition of the caspase-3 inhibitor DEVD-CHO suppressed PTEN-mediated AR degradation, whereas addition of the proteasome inhibitor MG132 did not (Lin, H., and C. Chang, unpublished results). This suggests that PTEN promotes AR degradation through the caspase-3-dependent pathway. Detailed biochemical binding assays demonstrate that the AR DBD interacts with the PTEN phosphatase domain. Taken together, these data indicate that PTEN may interact with the AR DBD, leading to AR retention in the cytoplasm, followed by AR degradation. In conclusion, AR may be degraded by two independent pathways, Akt-proteasome and PTEN-caspase-3 pathways (Fig. 3).
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FIG. 3. Two different pathways for AR degradation. A, Growth factors or cytokines may induce phosphorylation of AR through the PI3K/Akt pathway. AR undergoes ubiquitination by Mdm2 E3 ligase, followed by AR degradation by proteasome. B, The tumor suppressor PTEN interacts with AR and interferes with AR nuclear translocation. AR in the cytoplasm is degraded by the caspase-3-dependent pathway.
Linkage of AR expression and degradation with androgen-related diseases
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Abstract
Introduction
Structure of AR
How to control AR...
Linkage of AR expression...
Natural products that regulate...
Concluding remarks and future...
References
AR degradation in Kennedy’s disease.
The most notorious proteolyzed form of AR can be found in Kennedy’s disease or spinal bulbar muscular atrophy (SBMA) patients (115). SBMA is a neurodegenerative disease caused by AR mutations with longer polyglutamine repeats in the NH2-terminus. Transfection of the mutant AR with longer polyglutamine repeats (44 Gln) generated a distinct degraded form of AR containing polyglutamine repeats with an apparent molecular mass of 75 kDa (116, 117). The degraded form was not detected when AR with the normal polyglutamine repeat length (20 Gln) was used. The 75-kDa degraded AR is nuclear-associated and resistant to proteolysis even in the presence of 2 M urea in vitro, indicating that the proteolyzed product may be resistant to further degradation in cells (116). Attempts to identify the exact amino acid length of the 75-kDa degraded AR were not successful. Although the molecular mass of 75 kDa could extend over the AR DBD and hinge region, the 75-kDa AR was detected by antibody against AR peptide amino acids 1–21, but not by antibodies against AR peptides containing amino acids 301–320 or 535–547 (115, 116). The explanation for this puzzling result may be that the 75-kDa degraded AR may be a covalent isodipeptide, as shown by the transglutaminase-catalyzed reaction (118), or that epitopes in the C-terminal domain of the 75-kDa polypeptide, after the polyglutamine repeats, are masked due to the rigid structure of the polypeptide. The latter possibility can be supported by the fact that the 75-kDa degraded AR was resistant to tryptic proteolysis even in the presence of 2 M urea.
Accumulation of the misfolded polypeptides containing polyglutamine repeats due to the disruption of normal degradation process in cells generates insoluble aggregates of the peptide, resulting in cellular toxicity (119). Cells containing the expanded polyglutamine repeat aggregates undergo apoptosis in 24 h, twice as fast as cells without the aggregates (119). As the insoluble aggregates appear in unaffected tissues of SBMA patients, such as dermis, testis, and kidney, at a much lower frequency, the frequent appearance of the aggregates in motor neurons of the spinal cord and brainstem and in sensory neurons of dorsal root ganglia is tissue selective. The mechanism by which the expanded polyglutamine repeats result in cytotoxicity is not well understood. It was reported that cleavage of AR at Asp146 by the caspase-3 subfamily protease is critical for cytotoxicity, as mutation of the Asp residue blocks the formation of perinuclear aggregates (120). In contrast to that report, we found that mutation of Asp146 in green fluorescent protein-fused AR did not block the formation of perinuclear aggregates (Hu, Y.-C., and C. Chang, unpublished results). Nevertheless, it is clear that neurotoxicity of the aggregates result from the inability of cells to eliminate the aggregates. Overexpression of molecular chaperones heat shock proteins 70 and 40 increased the solubility of the aggregates and enhanced the degradation of polyglutamine repeats (119). A recent study using a transgenic mouse model system indicates that T contributes to nuclear translocation of the mutant AR and that castration markedly reduces SBMA phenotypes, indicating that the androgen-AR signaling pathway plays a key role in the development of Kennedy’s disease (121).
AR gene amplification and mutation in prostate cancer.
Prostate cancer is the most commonly diagnosed cancer and is the leading cause of cancer death among American men (122). One vital factor for prostate cancer growth is the AR. The AR mediates androgen action through androgen-AR interaction that can activate or repress its target genes (5). Based on this fact, androgen ablation therapy with various antiandrogens has been applied to prostate cancer patients (123). However, most prostate cancer from patients treated with androgen ablation therapy still progresses from an androgen-dependent to an androgen-independent state, eventually leading to death (124). It should be noted that most primary tumors and androgen-independent prostate cancers continue to express the AR and androgen-responsive genes such as PSA, suggesting that androgen-independent cell proliferation of prostate cancer might be due to alterations in AR function. Thus, androgen-independent prostate cancer cells retain a functional AR signaling pathway at the castration level of androgen. Mutations in the AR LBD (125), cofactors (126), AR gene amplification, enhanced expression of Bcl-2 and p21 (127, 128, 129, 130), and signal transduction cross-talk (37) have all been suggested to explain androgen-independent prostate cancer progression. AR gene amplification and mutations in androgen-independent prostate cancer will be discussed in this review.
The AR gene is a single copy gene composed of eight exons in about 90 kb of genomic DNA (131, 132). It has been mapped to q11–12 of the human X chromosome (13, 14). Dual labeling fluorescence in situ hybridization has been successfully applied to identify gene amplification (133). A common DNA amplification site among hormone-refractory prostate cancer patients has been identified in Xq11–13 (134, 135). Further studies demonstrated that the AR gene is amplified in 20–30% of recurrent prostate cancer patients, but not in untreated androgen-dependent prostate cancer patients (136). A recent study using real-time RT-PCR demonstrated that AR gene amplification resulted in increased AR mRNA levels (134), indicating increased AR gene expression in the hormone-refractory patients. However, AR gene amplification is not observed before androgen ablation therapy, indicating that AR gene amplification does not initiate prostate cancer, but occurs as a result of selection during androgen ablation therapy (136, 137). Thus, AR gene amplification has been proposed to explain the mechanisms implicated in the development of androgen-independent prostate cancer.
More than 60 different somatic mutations of the AR gene arising after tumor development have been reported in prostate cancer specimens (138). Studies to date have shown AR mutations in prostate cancer with a frequency of 2–8% (139). In general, the frequency of AR mutations increases with stage, being higher in hormonally relapsed and metastastic cancer. Compared with the wild-type AR, mutant forms of AR often show altered ligand binding specificity or altered binding affinity for hormones other than DHT/T (125, 140). Antiandrogens, such as hydroxyflutamide, can bind to the most notorious AR mutant, AR T877A, and activate AR function (125). A recent study has shown that the AR mutant L701H has a lower affinity to DHT, but a markedly higher affinity to glucocorticoids, compared with the wild-type AR (141). This explains the ability of the AR mutants to stimulate prostate cancer growth despite androgen ablation therapy.
AR protein expression vs. prostate cancer cell growth.
Activation of the androgen-AR signaling pathway has been suggested for the growth of prostate epithelium cells (142). Treatment of LNCaP cells, the mostly widely used human AR-positive prostate cancer cell line, with 10-10 M DHT resulted in cell growth enhancement (143). However, if we forced AR overexpression in LNCaP cells by stable transfection of the AR gene under the control of a strong viral promoter, 10-10 M DHT inhibited cell growth (Lee, D. K., and C. Chang, unpublished data). Similarly, the growth of PC-3(AR)2 cells, an AR negative prostate cancer PC-3 cell line stably transfected with AR, was inhibited in the presence of androgens (144). As the expression of AR in PC-3(AR)2 is under the control of a strong viral promoter, the growth-suppressing effect of PC-3(AR)2 by the androgen-AR signaling pathway may not reflect the physiological environment. Thus, we stably transfected PC-3 cells with the AR cDNA under the control of the 3.6-kb natural AR promoter, PC-3(AR)9. Although androgen induced cell growth arrest or inhibition of cell proliferation of PC-3(AR)2 cells, it slightly increased cell growth of PC-3(AR)9 (Altuwajiri, S., and C. Chang, unpublished results). In addition, we found that the protein stability of a cyclin-dependent kinase inhibitor p27Kip1 was markedly increased in PC-3(AR)2 cells, but not in PC-3(AR)9 cells, in response to androgen (Altuwajiri, S., and C. Chang, unpublished results). This suggests that overexpression of AR in cells may cause up-regulation of cell cycle inhibitors, such as p27, leading to cell growth arrest or suppression. Taken together, alteration of the AR expression level in cells may change the rates of cell proliferation and/or apoptosis in response to androgen.
Natural products that regulate AR expression
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Abstract
Introduction
Structure of AR
How to control AR...
Linkage of AR expression...
Natural products that regulate...
Concluding remarks and future...
References
Natural dietary products have historically been used to prevent diseases in many cultures (145). Epidemiological studies show that natural products containing curcumin, vitamin D, vitamin E, selenium, lycopene, phytoestrogens, resveratrol, quercetin, long-chain -3 polyunsaturated fatty acids, and silymarin have been used to prevent or delay prostate cancer development (145). Efforts have been made to develop analogs with better anticancer properties than the natural products. The antiprostate cancer properties of many natural products may be linked to their anti-AR activity. For example, vitamin E succinate (VES), a derivative of vitamin E, has been demonstrated to inhibit not only the synthesis of AR mRNA, but also the translation of AR mRNA, resulting in inhibition of LNCaP cell proliferation (146). In contrast, HF, an antiandrogen widely used to treat prostate cancer patients, showed little effect on AR expression in LNCaP cells. VES in combination with HF resulted in more efficient inhibition of LNCaP cell growth than VES alone (146). Thus, the future of pharmaceutical application of natural product analogs is promising. Quercetin, a flavonoid found in apples, onions, red wine, and tea, has been reported to inhibit AR expression, resulting in inhibition of LNCaP cell growth. Resveratrol, abundant in grape skin, also interferes with androgen action in LNCaP cells by inhibition of AR expression (147). As natural products other than curcumin have been extensively discussed previously (145), only curcumin and its analogs will be discussed in detail here.
Curcumin, used as a yellow coloring and flavoring food agent, has been reported to decrease the cell growth rate of prostate cancer. In an attempt to develop curcumin analogs with better inhibitory effects on prostate cancer cell growth, various curcumin analogs were screened as potential antiandrogenic compounds. One of the derivatives, JC-15, interfered with AR trans-activation, as judged by decreased PSA expression and reporter gene activity in LNCaP cells (Miyamoto, H., and C. Chang, unpublished results). Detailed analyses indicate that JC-15 inhibits AR dimerization by AR NH2-/COOH-terminal interaction, resulting in decreased AR protein levels through destabilization of AR protein. In addition, JC-15 interferes with AR nuclear translocation, as judged by immunocytofluorescence microscopy analysis (Miyamoto, H., and C. Chang, unpublished results). JC-15 decreases both DHT-mediated and HF-mediated cell growth rates of LNCaP, whereas HF increases the cell growth rate of LNCaP. This is particularly important in light of HF withdrawal syndrome.
Concluding remarks and future direction
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Abstract
Introduction
Structure of AR
How to control AR...
Linkage of AR expression...
Natural products that regulate...
Concluding remarks and future...
References
Extensive research in the field of steroid receptors for the last 3 decades provides considerable understanding of the physiological and pathological roles of steroid receptors. Steroid receptors share many functional and structural similarities, yet display apparent differences among steroid receptors. Nevertheless, a breakthrough finding in one steroid receptor often leads to a new era of research for the other steroid receptors. AR is the steroid receptor cloned most recently and has been extensively studied due to its involvement in the male reproductive system, Kennedy’s disease, and prostate cancer. Molecular and animal/clinical studies provide an understanding of how the androgen-AR signaling pathway plays key roles in these diseases. However, continuous and devoted research is required to further understand the physiological and pathological roles of the androgen-AR signaling pathway as well as to develop better therapeutic drugs for patients.
Abnormalities in the androgen-AR signaling pathway have been linked to the development of male infertility, Kennedy’s disease, and prostate cancer. Regulation of AR activity can be achieved in several different ways: modulation of AR gene expression, androgen binding to AR, AR protein stability, AR nuclear translocation, and AR trans-activation. In this review we extensively discussed modulation of AR gene expression and AR protein stability. The analysis of each stage in modulation of AR activity is a prerequisite to understanding the physiological and pathological roles of the androgen-AR signaling pathway. In addition, drug screens targeting one or multiple stages could lead to the development of effective therapeutic drugs to treat patients.
a little wordy,
Introduction
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Abstract
Introduction
Structure of AR
How to control AR...
Linkage of AR expression...
Natural products that regulate...
Concluding remarks and future...
References
ANDROGENS, TESTOSTERONE (T) and its metabolite 5-dihydrotestosterone (DHT), play a key role in proper development and function of male reproductive organs, such as prostate and epididymis, as well as nonreproductive organs, such as muscle, hair follicles, and brain (1, 2, 3). The action of androgens is mediated by androgen receptor (AR), a member of the steroid hormone receptor superfamily (1, 2, 3). AR is nearly ubiquitously expressed in mammalian tissues. Upon T or DHT binding, AR undergoes a series of conformational changes that allows AR to interact with androgen response elements (ARE) in various androgen target genes (4, 5). T, the major form of circulating androgen, is synthesized mainly in the Leydig cells of the testes (6). Circulating androgen plays an important role in the hypothalamus-pituitary-testis loop to maintain the proper concentration of sex steroid hormones (7). Adrenal glands also secrete precursor forms of androgen, dehydroepiandrosterone, and androstenedione (7). DHT, a more potent androgen than T, is synthesized mostly in the peripheral tissues, such as prostate (8). The majority of circulating androgens (97–99%) in serum are complexed with serum proteins, such as SHBG or albumin (9, 10). Thus, only 1–3% of androgen is a free form that can diffuse into membranes and bind to AR. This indicates that AR may be a rate-limiting factor for the androgen-AR signaling pathway in cells.
The functional significance of AR in male sex differentiation has been demonstrated by various natural germline AR mutations that cause partial or complete androgen insensitivity syndromes (11, 12). Patients with complete androgen insensitivity syndrome are phenotypically females with female external genitalia, whereas patients with partial androgen insensitivity syndrome are males with a spectrum of defects that vary from near-normal male phenotypes to near-normal female phenotypes.
While the functional significance of the androgen-AR signaling pathway in the male reproduction system is well studied, that in the female has been little understood( females are very complex, not that i needed to tell you guys that) due to the lack of a proper model system. The generation of an AR knockout (ARKO) female mouse model has long been hampered, because the AR gene is located on the X chromosome (13, 14). Recently, a cre-lox conditional KO has been successfully used to generate ARKO female mice (15). The study demonstrated that homozygous ARKO female mice produced a smaller number of pups per litter with an average of four, whereas the counterpart wild-type female mice produced an average of eight pups per litter. This result indicates that AR may also play a role in female fertility and/or ovulation (15). Further study indicated that homozygous ARKO female mice suffer from defective folliculogenesis due to poor follicular maturation and defective implantation due to subnormal uterine development (Wang, C., and C. Chang, unpublished results).
Structure of AR
Top
Abstract
Introduction
Structure of AR
How to control AR...
Linkage of AR expression...
Natural products that regulate...
Concluding remarks and future...
References
The AR is a member of the steroid receptor superfamily that is composed of a variable NH2-terminal domain, a highly conserved DNA-binding domain (DBD), a hinge domain, and a ligand-binding domain (LBD) (16, 17). The apparent molecular mass of AR is 110 kDa, with approximately 918 amino acids (Fig. 1) (1, 2, 3). The most characteristic features of the AR NH2-domain are the polyglutamine and polyglycine repeats that result in variations in the length of the AR (18, 19). The polyglutamine repeat normally ranges in length from 8–31, with an average length of 20. The length of the polyglutamine repeat is inversely correlated with AR transcriptional activity in vitro (20), in other words, reduced AR trans-activation with long polyglutamine repeats. The inverse correlation of AR trans-activation activity with the polyglutamine repeat length might result from the influence of the repeat on interaction with coregulators (21). Alternatively, short polyglutamine repeats may interfere with the AR NH2-terminal domain phosphorylation or interdomain interaction of AR (22, 23). The inverse correlation between length of the polyglutamine repeat and AR mRNA and protein expression in vitro has been also reported (24). Expansion of the polyglutamine repeat length to over 40 causes Kennedy’s disease, a neuromuscular disorder associated with decreased virilization (25). In contrast, yet still controversial, short polyglutamine repeats have been linked to a higher risk factor of developing prostate cancer (reviewed in Refs. 11 , 12 , and 18).
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FIG. 1. Structures of the AR gene and protein. A, The exons of the AR gene are depicted in rectangles. The regulatory elements in the promoter, CRE, polypurine/polypyrimidine (Pu/Py) tract, and Sp1, are shown. B, The structural domains of AR are shown by amino acid position.
The polyglycine repeat length of AR ranges from 10–30 in the normal population (19, 26). Complete deletion of the polyglycine repeat resulted in marked reduction of AR trans-activation in vitro (27). Studies of the polyglycine repeat length as a risk factor of prostate cancer are highly conflicting. Although Stanford and colleagues reported increased prostate cancer risk with short length (16) of the polyglycine repeats, others reported no link between the polyglycine repeat length and prostate cancer risk (28).
The AR activation function-1 (AF-1) domain, amino acids 360–494, shows ligand-independent activation when artificially separated from the LBD (29). A recent study demonstrated that the WXXLF motif in the AR AF-1 domain interacts with the AR LBD, resulting in AR stabilization (30, 31, 32, 33). In addition to the AF-1 domain, the other domain located in amino acids 141–338, is required for full ligand-inducible AR activity (29, 34). Various transcription factors, such as amino-terminal enhancer of split, AR N-terminal domain transactivating protein-1, AR coregulator, AR trapped cloned 27, transcription factor IIF, and transcription factor IIH, have been demonstrated to interact with the AR NH2-terminal domain and modulate AR trans-activation (35). The AR NH2-terminal domain can be phosphorylated by various kinases (36, 37). Certain phosphorylation pathways of the AR NH2-terminal domain have been reported to play an important role in AR trans-activation (37). In addition to phosphorylation, the AR NH2-terminal domain has been reported to be covalently modified by SUMO-1 (sumoylation) (38). Mutation of two sumoylation sites, lysine residues K386 and K520, in AR enhances AR trans-activation, indicating that SUMO-1 inhibits AR activity. The K386 is strongly sumoylated as a master switch for sumoylation of AR, whereas K520 is poorly sumoylated. Mutation of K520 alone showed little enhancement of AR trans-activation, but the double mutation of K386/K520 showed higher enhancement of AR trans-activation than mutation of K386 alone. The sumoylation consensus sequence (I/L/V)KXE has been identified in the other steroid hormone receptors, indicating that reversible sumoylation is one mechanism to modulate steroid receptor activity.
Like other steroid receptors, the AR DBD consists of two zinc fingers. In the case of the inverted ARE, the first zinc finger of AR recognizes and binds to DNA with further stabilization of DNA-AR interaction through the second zinc finger interaction with the phosphate backbone (39, 40). In the case of direct repeat ARE, the second zinc finger and a part of the hinge region interact directly with DNA (41). In addition to the DNA-receptor interaction, amino acids from 617–633 in the AR DBD and hinge region contain a nuclear localization signal (42, 43). Recently, the PEST (proline-, glutamate-, serine-, and threonine-rich) sequence, a degradation motif, has been identified in the AR hinge domain (44). The PEST sequence is found in proteins with short half-lives that are degraded by the ubiquitin-mediated proteasome system (45).
The AR LBD domain, like progesterone receptor (PR) and estrogen receptor (ER), consists of 12 -helixes forming the ligand binding pocket (46, 47, 48, 49). X-Ray crystallographic analyses of ligand-bound and -unbound ER and PR have demonstrated that ligand binding induces a conformational change in the LBD in which helix 12 and the AF-2 domain fold back across the ligand binding pocket, forming a ligand-dependent interaction surface for coregulators (50, 51). Although the crystal structure of the ligand-bound AR LBD is similar to that of the ligand-bound ER or PR (47, 48), functional analyses of the full-length receptors suggest distinct differences in the coregulator interaction with AR and ER. Although the structure of the ligand-bound AF-2 domain of ER or PR is important for interaction with a coregulator steroid receptor coactivator 1 (SRC-1) (52, 53), that of AR is not essential for interaction with SRC-1 (31, 54). Instead, the primary interaction sites of SRC-1 are in the AR NH2-terminal and DBD (31). A recent study indicates that the AR AF-2 domain may stabilize the overall structure of AR to allow the AR NH2-terminal domain to interact with appropriate coregulators (55).
An AR isoform (AR-A) with an apparent molecular mass of 87 kDa, which is an NH2-terminally truncated form of a major AR (AR-B), was reported (56, 57). That study reported that the AR-A isoform comprised 4–26% of the total immunoreactive AR protein in tissues, judged by an immunoblot analysis using the antibodies against the AR NH2-terminal domain. Although the study suggested that the use of initiation methionine at amino acid 188 would yield the AR-A isoform, the possibility that the AR-A isoform might be a proteolyzed product of AR could not be ruled out. As no follow-up studies were reported about the N-terminally truncated isoform of AR, the concept of AR isoforms is not widely accepted. This is in contrast to other steroid receptors, such as PR and ER (58, 59). PR isoforms, PR-A and PR-B, result from alternative usage of initiation codons of the same gene, whereas the ER isoforms, ER and ERß, result from two different genes.
How to control AR expression
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Abstract
Introduction
Structure of AR
How to control AR...
Linkage of AR expression...
Natural products that regulate...
Concluding remarks and future...
References
Regulation of AR mRNA level by androgen.
Androgens regulate AR at the level of both mRNA and protein. Regulation of AR protein by androgens is discussed below. Prolonged treatment of human prostate cancer LNCaP as well as breast cancer T47D cells with androgens for 48 h or longer decreased AR mRNA about 2-fold (60, 61, 62, 63). Northern blot assays demonstrated that down-regulation of AR mRNA by androgen in LNCaP cells is at the transcriptional level (62). These results indicate that androgens down-regulate AR expression to limit the androgen response in certain cell types. In contrast, up-regulation of AR mRNA by androgens has been reported in other cell types, including human hepatocellular carcinoma and osteoblastic cell lines (64, 65). Possible up-regulation of AR mRNA by androgens has also been reported in rat hippocampus (66).
In contrast to down-regulation of AR mRNA by androgen in prostate cancer LNCaP cells, Takeda et al. (67) reported that AR mRNA levels in rat or mouse prostate ventral lobe epithelial cells were markedly decreased 3 d after castration, as judged by in situ hybridization. Treatment of the castrated animals with DHT restored the AR mRNA level, indicating up-regulation of AR mRNA by androgens (67). However, this report is in contrast with another report showing that castration increased AR mRNA levels in rat prostate ventral lobes, as judged by Northern blot assay (68). The reasons for these discrepancies in the effect of androgen on the AR mRNA level in rat prostate ventral lobe might be explained by the techniques used. Castration causes drastic changes in total RNA and protein contents in prostate, a highly androgen-dependent organ (69). Therefore, normalization of total RNA in Northern blot analysis may result in complicated interpretations.
Modulation of AR expression at the transcriptional level. 5' Upstream regulatory elements in the AR promoter:
The AR 5' upstream promoter region does not contain a typical TATA box, but contains Sp1-binding sites at -46 bp (70, 71, 72). The TATA box is a binding site for general transcription factor IID (TFIID)/TFIID binding protein and is required for directing accurate transcription initiation sites (73). Sp1 has been reported to recruit TFIID/TFIID binding protein and mediate the formation of the transcription preinitiation complex on the TATA-less promoters (74). S1 nuclease protection of AR mRNA demonstrated two transcription initiation sites separated by 12 bp (75, 76). Transcription from multiple initiation sites is typical for TATA-less promoters. Inhibition of Sp1 activity resulted in a marked reduction of AR protein level in human prostate cancer LNCaP cells (71, 77), indicating that Sp1 plays a key role in the regulation of AR mRNA synthesis. The highly conserved homopurine-homopyrimidine region is located upstream of the Sp1 site in the AR promoter (78), and deletion of this region of the human AR promoter resulted in 3- to 4-fold reduction of AR promoter activity (72, 78).
Another key regulatory element for AR mRNA expression is the cAMP response element (CRE) located at -508 bp in the human AR gene (72, 79). Whereas Sp1 and homopurine-homopyrimidine regions are conserved among the human, rat, and mouse AR gene promoters, the CRE has not been identified in the rat or mouse AR gene promoters. Treatment with the cAMP analog dibutyryl cAMP enhanced AR promoter activity about 4- to 6-fold in a reporter gene assay and increased AR mRNA expression in LNCaP cells (72). It has been proposed that the AR mRNA in rat Sertoli cells is up-regulated by FSH, which may function through cAMP-CRE interaction (80, 81).
The 5' upstream regulatory elements for down-regulation of AR mRNA expression have not been well studied in the human AR gene. The NF1 site of the rat AR promoter has been reported to suppress AR mRNA expression (82). Mutation of the NF1 site increased rat AR promoter activity about 8-fold in transfection assays. However, deletion of the NF1 site of the human AR promoter did not significantly change AR promoter activity (72), indicating species specificity in regulation of the AR promoter region. Age-related down-regulation of AR mRNA expression was observed in rat hepatic cells (83, 84), with mRNA level in senescent rats 70-fold lower than those in young rats. An increase in nuclear factor-B (NF-B) and a decrease in age-dependent factor contributed to the down-regulation of AR mRNA in senescent rats (84).
Masked AR expression
The AR gene contains a CpG island that covers the proximal promoter region and the first exon (72, 85). Hypermethylation of the CpG island in the promoters has been associated with transcriptional inactivation (86). The CpG island of the AR gene is heavily methylated in androgen-independent human prostate cancer DU145 cells, resulting in masked AR expression (85, 87, 88). Treatment of the demethylation reagent 5-aza-2'-deoxycytidine restored AR expression in DU145 cells (87). Most primary and androgen-independent prostate tumors continue to express the AR and androgen-responsive genes such as prostate-specific antigen (PSA), suggesting that androgen-independent cancer cells may retain a functional AR signaling pathway at the castration level of androgen. However, masked AR expression in certain androgen-independent metastatic prostate cancers has been identified (88). That study showed masked AR expression in two of 15 prostate cancer specimens from patients who died of androgen-independent metastastic prostate cancer. Detailed methylation analyses using bisulfide sequencing found that two methylation consensus sequences in the AR gene, from -131 to -121 and from +44 to +54, are linked to the loss of AR expression in metastatic hormone-refractory prostate cancer (88). Sp1 binding to the AR gene promoter has been shown to prevent the CpG island from being methylated (89, 90).
Recently, we took advantage of C2C12 myoblast cells stably transfected with the AR cDNA under the control of the 3.6-kb natural human AR promoter, named C2C12/npAR, to analyze AR function in skeletal muscle tissue. We found that the AR protein level in C2C12/npAR myoblast cells was undetected, whereas the AR protein level was markedly up-regulated in terminally differentiated C2C12/npAR myotubes (Lee, D. K., and C. Chang, unpublished results). Nuclear run-on transcription and pulse-chase labeling assays indicated that the marked up-regulation of AR expression does not result from the altered AR protein stability, but from transcriptional induction of AR mRNA (Lee, D. K., and C. Chang, unpublished results). Further study is required for analyses of the mechanisms implicated in AR mRNA induction during skeletal muscle cell differentiation.
Modulation of AR expression at the posttranscriptional level.
In addition to controlling AR mRNA levels at the transcriptional level, androgens also regulate AR expression at the posttranscriptional level. Androgens have been reported to modulate both stability and translation efficiency of the AR mRNA. Androgens decrease AR mRNA levels, but increase AR protein levels in both prostate cancer LNCaP and breast cancer MDA453 cells (91). Androgens down-regulate AR mRNA transcription in LNCaP cells. However, androgens do not change AR mRNA transcription in MDA453 cells, but destabilize AR mRNA, indicating that androgens down-regulate AR mRNA level at the posttranscriptional level in MDA453 cells (91).
Studies to date indicate that the AR 5'- and 3'-untranslated regions (UTRs) play a role in controlling AR mRNA level at the posttranscriptional level. The human AR mRNA has a relatively long 5'-UTR of about 1100 nt (75). Reporter gene assays using chloramphenicol transferase (CAT) reporter plasmids fused with the AR 5'-UTR between the simian virus 40 promoter and the CAT-coding region demonstrated that the AR 5'-UTR is absolutely required for induction of CAT activity (92). Further deletion analysis indicated that 180 bp from +21 to +202, including a stem-loop secondary structure, were sufficient for the induction of CAT activity. S1 nuclease protection experiments demonstrated that the 180 bp in the fused reporter genes did not increase CAT mRNA levels, indicating that it enhanced the efficiency of translation (92). This report suggests that the 5'-UTR of AR mRNA may be required for efficient translation of AR mRNA.
AR mRNA is expressed as two transcripts with (10.5 kb) or without (8 kb) the 3'-UTR (62, 76, 93). A recent study demonstrates that the 3'-UTR of AR mRNA contains a highly conserved UC-rich region that is a target for cytoplasmic and nuclear RNA-binding proteins, such as poly(C)-binding protein-1 (CP-1) and Hu protein R (HuR) (94). Immunoprecipitation assays demonstrated that these proteins indeed associated with the UC-rich region of AR 3'-UTR (94). HuR is ubiquitously expressed and is involved in the stabilization of several mRNAs (95, 96). CP-1 has been reported to control mRNA turnover and translation (97, 98). Association of HuR and CP-1 with the UC-rich region in AR 3'-UTR indicates that the AR 3'-UTR may play a role in posttranscriptional regulation of AR expression. RT-PCR and sequence analysis from a patient with partial androgen insensitivity syndrome demonstrated that the patient expressed a variant of AR mRNA with the complete coding region, but a shortened 3'-UTR, indicating the physiological significance of AR 3'-UTR in the androgen-AR signaling pathway (99).
Modulation of AR expression at the protein level. Degradation of receptors to control hormone responsiveness: AR vs. ER, GR, and PR.
Numerous studies demonstrate that protein degradation is required not only for elimination of misfolded or unfolded proteins, but also for maintenance of cellular function (100). Steroid receptors have relatively short half-lives, indicating that the amount and duration of steroid receptor ligand effect in cells are regulated by systematic protein activation and degradation (Fig. 2). Proteins with a short half-life, such as certain transcription factors and cell cycle regulators, undergo systematic protein degradation by the ubiquitin-proteasome pathway (100, 101).
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FIG. 2. Mechanisms to limit hormone responsiveness by AR and ER. AR limits hormone responsiveness by ligand dissociation and receptor recycling, whereas ER limits by degradation of ER protein. Although AR does not use a proteasome-ubiquitin pathway to limit hormone responsiveness, a systematic proteasome-ubiquitin pathway may regulate overall AR protein level in cells.
Ligand-dependent and proteasome-mediated degradation has been detected in ER (102, 103, 104), PR (105), and GR (106). MG132, a specific proteasome inhibitor, inhibited ER-mediated transcription, but not p53- or Sp1-mediated transcription (103, 104). Integrity of ER function is required for 26S proteasome-mediated receptor degradation, as deletion of the last 61 amino acids, including helix 12, abolished ligand-dependent and proteasome-mediated down-regulation of the receptor (104). As the last 61 amino acids of ER serve as a binding domain for various coregulators, it is likely that ligand-dependent down-regulation may be dependent on the protein-protein interaction of ER with coactivators (104). Interestingly, the ER mutants lacking an ability to bind DNA also showed ligand-dependent down-regulation, indicating that DNA binding of ER or engagement of ER in the transcription complex is not necessary for the ligand-dependent down-regulation (104). Treatment of cells with the general transcription inhibitor, actinomycin D, or the translation inhibitor, cycloheximide, prevented ER from ligand-dependent down-regulation (104), indicating that continuous synthesis of proteins is required for the ligand-dependent down-regulation of ER.
However, the AR protein level is not down-regulated in the presence of androgens. Instead, androgens have been shown to increase AR protein levels in various cell contexts (91). This result indicates that AR may use another mechanism to limit hormone responsiveness. A recent study using green fluorescent protein technology demonstrates that AR migrates to the subnuclear compartment in the presence of androgen within 15–60 min (107). AR migrates rapidly back to the cytoplasm upon androgen withdrawal and maintains its ability to reenter the nucleus for at least four rounds of AR recycling after the initial androgen treatment (107). This indicates that AR inactivation and migration into the cytoplasm due to ligand dissociation, as well as AR recycling rather than receptor degradation, may control the hormone responsiveness of AR (107).
Modulation of AR protein half-life
AR, like other members of the steroid hormone receptor family, requires dimerization to execute its function (16). Detailed analyses using a two-hybrid protein interaction indicated that the ligand-binding domain interacts with the NH2-terminal domain plus the DBD or the full-length AR in a ligand-dependent manner (31, 32, 33). The androgen-dependent intermolecular interaction between the NH2-terminal domain and the COOH-terminal domain of AR decreases a dissociation rate of the bound androgen and increases AR protein half-life. Systematic mutational analyses showed that two motifs, 23FXXLF (27) and 433WXXLF (4, 37), in the NH2-terminal domain may be required for the AR NH2-terminal interaction with the ligand-binding domain (30). Two-hybrid protein interaction and glutathione-S-transferase pull-down assays showed that mutations of any conserved amino acid sequences in the motifs resulted in marked reduction of the AR NH2-terminal/COOH-terminal domain interaction (30).
The half-life of AR protein varies depending on the context of the cell environment. Androgens have been reported to stabilize AR protein in various cell contexts (91). The half-life of AR in LNCaP cells is approximately 3 h in the absence of androgens and is longer than 10 h in the presence of 10 nM DHT. Interestingly, the half-life of AR from the androgen-independent subline of LNCaP that was grown without androgen treatment, LNCaP-C4–2, is 7 h in the absence of androgens (108). A prolonged half-life of AR protein in the absence of androgens was also observed in recurrent CWR22 tumors (108). In the absence of androgens, AR in the recurrent tumor was detected in nuclei, whereas AR in LNCaP cells was predominantly detected in cytoplasm. These results indicate that AR stabilization and nuclear translocalization in the absence of androgens may activate the AR signaling pathway, resulting in prostate cancer cell proliferation at the castrated level of androgens.
Mechanisms of AR degradation
Systematic protein degradation by the ubiquitin-proteasome system plays important roles in the maintenance of cellular functions, such as cell cycle and antigen presentation. Protein ubiquitination provides the recognition signal for the 26S proteasome, leading to protein degradation (101). Protein ubiquitination requires the activities of three different enzymes, ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzymes (E2s), and ubiquitin ligases (E3s) (101). The E3 ubiquitin ligation step often determines the specificity of protein ubiquitination and serves as a rate-limiting step. Phosphorylation of target proteins has been reported to be necessary for recognition by E3 ligases (109). A good example is IB, which associates with NF-B and inhibits NF-B activity (110, 111). Upon induction by cytokine treatment, IB is phosphorylated at the two specific serine residues, Ser32 and Ser36, leading to NF-B activation via degradation of IB. Mutations of the serine residues prevent IB phosphorylation as well as IB degradation.
Although AR does not undergo ligand-dependent down-regulation of the protein level, recent studies indicate that the AR protein level in cells is regulated by systemic protein degradation pathways. Inhibition of the ubiquitin-proteasome degradation pathway by MG132 has been reported to increase endogenous AR protein levels in HepG2 and LNCaP cells (44). The PEST sequence, a signature motif for ubiquitin-proteasome degradation, has been identified in the hinge region of AR (44). To be degraded by the ubiquitin-proteasome system, AR should be recognized by E3 ligases. A recent study demonstrates that Mdm2 is an E3 ligase for the ubiquitin-proteasome degradation of AR (112). AR associates with Mdm2 in cells, as shown by the coimmunoprecipitation assay. Detailed biochemical binding assays using a glutathione-S-transferase pull-down assay demonstrate that Mdm2 interacts with the AR NH2-terminal domain plus DBD (112). Phosphorylation of target proteins often serves as a recognition signal for E3 ligases (109). The study demonstrates that phosphorylation of AR at Ser213 and Ser791 by the phosphatidylinositol-3-hydroxy kinase (PI3K)/Akt pathway is necessary for AR degradation by Mdm2 E3 ligase. Transfection of constitutively active Akt into LNCaP cells promoted AR degradation, resulting in suppression of PSA expression (112). However, treatment with LY29402, an inhibitor of PI3K, increased the AR protein level in LNCaP cells. When the Mdm2-null cell line was used, AR degradation by the ubiquitin-proteasome pathway was markedly impaired, indicating that phosphorylation-dependent AR ubiquitination and degradation by the PI3K/Akt pathway require Mdm2 E3 ligase.
Phosphorylation of AR at amino acid positions Ser213 and Ser791 by the PI3K/Akt signaling pathway has been suggested to be required for androgen-independent survival and growth of prostate cancer LNCaP cells (113). Based on our study (Lin, H., and C. Chang, unpublished results), the Akt pathway suppresses AR trans-activation in LNCaP cells of low passage numbers (<30), whereas it increases AR trans-activation in LNCaP cells of high passage numbers (60). Due to the report indicating that the expression of prostate-specific phosphatase in LNCaP cells is androgen dependent in low passage numbers, but becomes androgen independent in higher passage numbers (114), it is likely that Akt may be able to differentially regulate AR activity in different passage numbers of LNCaP cells.
A recent study indicates that AR activity can be suppressed in LNCaP cells by phosphatase and tensin homolog deleted from chromosome 10 (PTEN), a tumor suppressor (Lin, H., and C. Chang, unpublished results). However, overexpression of constitutively active Akt in LNCaP cells or treatment of cells with a PI3K inhibitor did not block PTEN-mediated AR suppression, indicating that AR is inhibited by a PI3K/Akt independent pathway. Immunocytofluorescence analyses demonstrate that the expression of wild-type PTEN, but not that of the mutant form of PTEN, Cys124Ser, in LNCaP cells interferes with AR translocation into the nucleus, resulting in AR in the cytoplasm in the presence of 10 nM DHT. Moreover, the expression of the wild-type, but not the mutant, form of PTEN promotes AR protein degradation, resulting in the suppression of endogenous PSA expression in LNCaP cells. Interestingly, addition of the caspase-3 inhibitor DEVD-CHO suppressed PTEN-mediated AR degradation, whereas addition of the proteasome inhibitor MG132 did not (Lin, H., and C. Chang, unpublished results). This suggests that PTEN promotes AR degradation through the caspase-3-dependent pathway. Detailed biochemical binding assays demonstrate that the AR DBD interacts with the PTEN phosphatase domain. Taken together, these data indicate that PTEN may interact with the AR DBD, leading to AR retention in the cytoplasm, followed by AR degradation. In conclusion, AR may be degraded by two independent pathways, Akt-proteasome and PTEN-caspase-3 pathways (Fig. 3).
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FIG. 3. Two different pathways for AR degradation. A, Growth factors or cytokines may induce phosphorylation of AR through the PI3K/Akt pathway. AR undergoes ubiquitination by Mdm2 E3 ligase, followed by AR degradation by proteasome. B, The tumor suppressor PTEN interacts with AR and interferes with AR nuclear translocation. AR in the cytoplasm is degraded by the caspase-3-dependent pathway.
Linkage of AR expression and degradation with androgen-related diseases
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Abstract
Introduction
Structure of AR
How to control AR...
Linkage of AR expression...
Natural products that regulate...
Concluding remarks and future...
References
AR degradation in Kennedy’s disease.
The most notorious proteolyzed form of AR can be found in Kennedy’s disease or spinal bulbar muscular atrophy (SBMA) patients (115). SBMA is a neurodegenerative disease caused by AR mutations with longer polyglutamine repeats in the NH2-terminus. Transfection of the mutant AR with longer polyglutamine repeats (44 Gln) generated a distinct degraded form of AR containing polyglutamine repeats with an apparent molecular mass of 75 kDa (116, 117). The degraded form was not detected when AR with the normal polyglutamine repeat length (20 Gln) was used. The 75-kDa degraded AR is nuclear-associated and resistant to proteolysis even in the presence of 2 M urea in vitro, indicating that the proteolyzed product may be resistant to further degradation in cells (116). Attempts to identify the exact amino acid length of the 75-kDa degraded AR were not successful. Although the molecular mass of 75 kDa could extend over the AR DBD and hinge region, the 75-kDa AR was detected by antibody against AR peptide amino acids 1–21, but not by antibodies against AR peptides containing amino acids 301–320 or 535–547 (115, 116). The explanation for this puzzling result may be that the 75-kDa degraded AR may be a covalent isodipeptide, as shown by the transglutaminase-catalyzed reaction (118), or that epitopes in the C-terminal domain of the 75-kDa polypeptide, after the polyglutamine repeats, are masked due to the rigid structure of the polypeptide. The latter possibility can be supported by the fact that the 75-kDa degraded AR was resistant to tryptic proteolysis even in the presence of 2 M urea.
Accumulation of the misfolded polypeptides containing polyglutamine repeats due to the disruption of normal degradation process in cells generates insoluble aggregates of the peptide, resulting in cellular toxicity (119). Cells containing the expanded polyglutamine repeat aggregates undergo apoptosis in 24 h, twice as fast as cells without the aggregates (119). As the insoluble aggregates appear in unaffected tissues of SBMA patients, such as dermis, testis, and kidney, at a much lower frequency, the frequent appearance of the aggregates in motor neurons of the spinal cord and brainstem and in sensory neurons of dorsal root ganglia is tissue selective. The mechanism by which the expanded polyglutamine repeats result in cytotoxicity is not well understood. It was reported that cleavage of AR at Asp146 by the caspase-3 subfamily protease is critical for cytotoxicity, as mutation of the Asp residue blocks the formation of perinuclear aggregates (120). In contrast to that report, we found that mutation of Asp146 in green fluorescent protein-fused AR did not block the formation of perinuclear aggregates (Hu, Y.-C., and C. Chang, unpublished results). Nevertheless, it is clear that neurotoxicity of the aggregates result from the inability of cells to eliminate the aggregates. Overexpression of molecular chaperones heat shock proteins 70 and 40 increased the solubility of the aggregates and enhanced the degradation of polyglutamine repeats (119). A recent study using a transgenic mouse model system indicates that T contributes to nuclear translocation of the mutant AR and that castration markedly reduces SBMA phenotypes, indicating that the androgen-AR signaling pathway plays a key role in the development of Kennedy’s disease (121).
AR gene amplification and mutation in prostate cancer.
Prostate cancer is the most commonly diagnosed cancer and is the leading cause of cancer death among American men (122). One vital factor for prostate cancer growth is the AR. The AR mediates androgen action through androgen-AR interaction that can activate or repress its target genes (5). Based on this fact, androgen ablation therapy with various antiandrogens has been applied to prostate cancer patients (123). However, most prostate cancer from patients treated with androgen ablation therapy still progresses from an androgen-dependent to an androgen-independent state, eventually leading to death (124). It should be noted that most primary tumors and androgen-independent prostate cancers continue to express the AR and androgen-responsive genes such as PSA, suggesting that androgen-independent cell proliferation of prostate cancer might be due to alterations in AR function. Thus, androgen-independent prostate cancer cells retain a functional AR signaling pathway at the castration level of androgen. Mutations in the AR LBD (125), cofactors (126), AR gene amplification, enhanced expression of Bcl-2 and p21 (127, 128, 129, 130), and signal transduction cross-talk (37) have all been suggested to explain androgen-independent prostate cancer progression. AR gene amplification and mutations in androgen-independent prostate cancer will be discussed in this review.
The AR gene is a single copy gene composed of eight exons in about 90 kb of genomic DNA (131, 132). It has been mapped to q11–12 of the human X chromosome (13, 14). Dual labeling fluorescence in situ hybridization has been successfully applied to identify gene amplification (133). A common DNA amplification site among hormone-refractory prostate cancer patients has been identified in Xq11–13 (134, 135). Further studies demonstrated that the AR gene is amplified in 20–30% of recurrent prostate cancer patients, but not in untreated androgen-dependent prostate cancer patients (136). A recent study using real-time RT-PCR demonstrated that AR gene amplification resulted in increased AR mRNA levels (134), indicating increased AR gene expression in the hormone-refractory patients. However, AR gene amplification is not observed before androgen ablation therapy, indicating that AR gene amplification does not initiate prostate cancer, but occurs as a result of selection during androgen ablation therapy (136, 137). Thus, AR gene amplification has been proposed to explain the mechanisms implicated in the development of androgen-independent prostate cancer.
More than 60 different somatic mutations of the AR gene arising after tumor development have been reported in prostate cancer specimens (138). Studies to date have shown AR mutations in prostate cancer with a frequency of 2–8% (139). In general, the frequency of AR mutations increases with stage, being higher in hormonally relapsed and metastastic cancer. Compared with the wild-type AR, mutant forms of AR often show altered ligand binding specificity or altered binding affinity for hormones other than DHT/T (125, 140). Antiandrogens, such as hydroxyflutamide, can bind to the most notorious AR mutant, AR T877A, and activate AR function (125). A recent study has shown that the AR mutant L701H has a lower affinity to DHT, but a markedly higher affinity to glucocorticoids, compared with the wild-type AR (141). This explains the ability of the AR mutants to stimulate prostate cancer growth despite androgen ablation therapy.
AR protein expression vs. prostate cancer cell growth.
Activation of the androgen-AR signaling pathway has been suggested for the growth of prostate epithelium cells (142). Treatment of LNCaP cells, the mostly widely used human AR-positive prostate cancer cell line, with 10-10 M DHT resulted in cell growth enhancement (143). However, if we forced AR overexpression in LNCaP cells by stable transfection of the AR gene under the control of a strong viral promoter, 10-10 M DHT inhibited cell growth (Lee, D. K., and C. Chang, unpublished data). Similarly, the growth of PC-3(AR)2 cells, an AR negative prostate cancer PC-3 cell line stably transfected with AR, was inhibited in the presence of androgens (144). As the expression of AR in PC-3(AR)2 is under the control of a strong viral promoter, the growth-suppressing effect of PC-3(AR)2 by the androgen-AR signaling pathway may not reflect the physiological environment. Thus, we stably transfected PC-3 cells with the AR cDNA under the control of the 3.6-kb natural AR promoter, PC-3(AR)9. Although androgen induced cell growth arrest or inhibition of cell proliferation of PC-3(AR)2 cells, it slightly increased cell growth of PC-3(AR)9 (Altuwajiri, S., and C. Chang, unpublished results). In addition, we found that the protein stability of a cyclin-dependent kinase inhibitor p27Kip1 was markedly increased in PC-3(AR)2 cells, but not in PC-3(AR)9 cells, in response to androgen (Altuwajiri, S., and C. Chang, unpublished results). This suggests that overexpression of AR in cells may cause up-regulation of cell cycle inhibitors, such as p27, leading to cell growth arrest or suppression. Taken together, alteration of the AR expression level in cells may change the rates of cell proliferation and/or apoptosis in response to androgen.
Natural products that regulate AR expression
Top
Abstract
Introduction
Structure of AR
How to control AR...
Linkage of AR expression...
Natural products that regulate...
Concluding remarks and future...
References
Natural dietary products have historically been used to prevent diseases in many cultures (145). Epidemiological studies show that natural products containing curcumin, vitamin D, vitamin E, selenium, lycopene, phytoestrogens, resveratrol, quercetin, long-chain -3 polyunsaturated fatty acids, and silymarin have been used to prevent or delay prostate cancer development (145). Efforts have been made to develop analogs with better anticancer properties than the natural products. The antiprostate cancer properties of many natural products may be linked to their anti-AR activity. For example, vitamin E succinate (VES), a derivative of vitamin E, has been demonstrated to inhibit not only the synthesis of AR mRNA, but also the translation of AR mRNA, resulting in inhibition of LNCaP cell proliferation (146). In contrast, HF, an antiandrogen widely used to treat prostate cancer patients, showed little effect on AR expression in LNCaP cells. VES in combination with HF resulted in more efficient inhibition of LNCaP cell growth than VES alone (146). Thus, the future of pharmaceutical application of natural product analogs is promising. Quercetin, a flavonoid found in apples, onions, red wine, and tea, has been reported to inhibit AR expression, resulting in inhibition of LNCaP cell growth. Resveratrol, abundant in grape skin, also interferes with androgen action in LNCaP cells by inhibition of AR expression (147). As natural products other than curcumin have been extensively discussed previously (145), only curcumin and its analogs will be discussed in detail here.
Curcumin, used as a yellow coloring and flavoring food agent, has been reported to decrease the cell growth rate of prostate cancer. In an attempt to develop curcumin analogs with better inhibitory effects on prostate cancer cell growth, various curcumin analogs were screened as potential antiandrogenic compounds. One of the derivatives, JC-15, interfered with AR trans-activation, as judged by decreased PSA expression and reporter gene activity in LNCaP cells (Miyamoto, H., and C. Chang, unpublished results). Detailed analyses indicate that JC-15 inhibits AR dimerization by AR NH2-/COOH-terminal interaction, resulting in decreased AR protein levels through destabilization of AR protein. In addition, JC-15 interferes with AR nuclear translocation, as judged by immunocytofluorescence microscopy analysis (Miyamoto, H., and C. Chang, unpublished results). JC-15 decreases both DHT-mediated and HF-mediated cell growth rates of LNCaP, whereas HF increases the cell growth rate of LNCaP. This is particularly important in light of HF withdrawal syndrome.
Concluding remarks and future direction
Top
Abstract
Introduction
Structure of AR
How to control AR...
Linkage of AR expression...
Natural products that regulate...
Concluding remarks and future...
References
Extensive research in the field of steroid receptors for the last 3 decades provides considerable understanding of the physiological and pathological roles of steroid receptors. Steroid receptors share many functional and structural similarities, yet display apparent differences among steroid receptors. Nevertheless, a breakthrough finding in one steroid receptor often leads to a new era of research for the other steroid receptors. AR is the steroid receptor cloned most recently and has been extensively studied due to its involvement in the male reproductive system, Kennedy’s disease, and prostate cancer. Molecular and animal/clinical studies provide an understanding of how the androgen-AR signaling pathway plays key roles in these diseases. However, continuous and devoted research is required to further understand the physiological and pathological roles of the androgen-AR signaling pathway as well as to develop better therapeutic drugs for patients.
Abnormalities in the androgen-AR signaling pathway have been linked to the development of male infertility, Kennedy’s disease, and prostate cancer. Regulation of AR activity can be achieved in several different ways: modulation of AR gene expression, androgen binding to AR, AR protein stability, AR nuclear translocation, and AR trans-activation. In this review we extensively discussed modulation of AR gene expression and AR protein stability. The analysis of each stage in modulation of AR activity is a prerequisite to understanding the physiological and pathological roles of the androgen-AR signaling pathway. In addition, drug screens targeting one or multiple stages could lead to the development of effective therapeutic drugs to treat patients.
a little wordy,
pitbullrocco
New member
lanky said:
lolhere goes another for the favorites....
lanky
Well-known member
Ulter said:Lanky, rather than copying and pasting your test book why not give an easy to understand summary. (see: ulter's post)
i would not know where to begin on this one..lol,,,it is more of an informative article,,not really to prove a point ,,just an overview of the AR, this is not a good article teaching choice on my part.
If you think this is complex,,wait until you try and understand womens Estrogen receptor, progestin action, androgen co regulation and other stuff that i have no idea on,,,womens hormonal system is crazy...but i love what it does to them
, other times of the month not so much 
,,,they are still crazy.
redsamurai
Banned
medical said:
i think we know "some".....the obvious ones. But I truly do think there's more going on....
bigtasty25
New member
Oh man...this thread is getting out of control LOL. I think we've established the basis that receptors arent a plausable cause to limiting one's muscle growth...this argument is revealed well in "Building the Perfect Beast"...and right from the beginning of the book as well, and hinted at throughout the entire text. In essence...it all has to do with catabolism vs anabolism...THATS IT FOLKS...say...youve been off 3-4 months...you go back on...after about 4-6 weeks (give or take a couple depending on the individual) anabolism starts wearing down and catabolism creaps up on you..and they begin to equal each other out...creating HOMEOSTASIS...which is what are body wants...and i believe this may have to do with myostatin as well. If you can make sense of the above...the whole steroid argument anabolism vs catabolism and gaining muscle will make ALOT more sense...forget receptors...youll always have them..and even if they are full, your body produces them all the time...so thats not what matters in muscle growth...it all has to do with tricking the body...
now going on 20 week cycles and just changing the steroids...does not make any sense...how is that tricking the body? ...it might trick it a little with a different compound but the dosages are constant...and after 4-8 weeks your body's anabolism will come to a halt (due to catabolism equaling it...=homeostasis)...instead of 20 weeks...for example...id do 6 weeks on...6 weeks off...6 weeks on...then maybe 12 weeks off followed by a bigger cycle and 6 weeks on etc..
shit...im giving out my secrets...gotta stop this =x..lol.
now going on 20 week cycles and just changing the steroids...does not make any sense...how is that tricking the body? ...it might trick it a little with a different compound but the dosages are constant...and after 4-8 weeks your body's anabolism will come to a halt (due to catabolism equaling it...=homeostasis)...instead of 20 weeks...for example...id do 6 weeks on...6 weeks off...6 weeks on...then maybe 12 weeks off followed by a bigger cycle and 6 weeks on etc..
shit...im giving out my secrets...gotta stop this =x..lol.
"now going on 20 week cycles and just changing the steroids...does not make any sense...how is that tricking the body?"
Tricking the body is not a very good explanation for why changing gear extends a cycle's hypergrowth period. I assume from your post that you've never done this. I suggest you try it. In the 20 years I have used steroids switching gears has been my most effect means of continuing growth on a cycle.
Tricking the body is not a very good explanation for why changing gear extends a cycle's hypergrowth period. I assume from your post that you've never done this. I suggest you try it. In the 20 years I have used steroids switching gears has been my most effect means of continuing growth on a cycle.
T
T-Matt
Guest
Makavelli said:
Not to mention the myth about how methandriol dipropionate is supposed to 'clean out' and 'upregulate your receptors'..
The hardest thing in life is to keep things simple.. If you stop growing consume more calories, take more gear, and change your training up a bit.. If Kcal coming in > Kcal going out = weight gain .. Your training, drug and food choice, and genetics will determine whether that weight will be fat or muscle..
T-Matt
pitbullrocco
New member
i think this horse has been beat down..............
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