Stimulation of Collagen Synthesis by the Anabolic Steroid
Stanozolol
Vincent Falanga,*† Adam S. Greenberg,* Linda Zhou,* Sofia M. Ochoa,* Anita B. Roberts,‡ Anna Falabella,* and
Yuji Yamaguchi*
*University of Miami School of Medicine, Department of Dermatology, †Miami Veterans Affairs Medical Center Miami, Florida, U.S.A.; ‡Laboratory of Cell
Regulation and Carcinogenesis, National Cancer Institute, Bethesda, Maryland, U.S.A.
There is evidence that anabolic steroids, which are derived
from testosterone and have markedly less androgenic
activity, promote tissue growth and enhance tissue repair;
however, the mechanisms involved in their anabolic
activities remain unclear. In this report, we measured the
effect of the anabolic steroid stanozolol on cell replication
and collagen synthesis in cultures of adult human dermal
fibroblasts. Stanozolol (0.625–5 mg per ml) had no effect
on fibroblast replication and cell viability (p J 0.764)
but enhanced collagen synthesis (p < 0.01) in a dosedependent
manner (r J 0.907). Stanozolol also increased
(by 2-fold) the mRNA levels of a1(I) and a1(III) procollagen
and, to a similar extent, upregulated transforming
Anabolic steroids are synthetic derivatives of testosterone
produced to dissociate testosterone’s anabolic and androgenic
action (Wilson, 1990). Although the abuse of
anabolic steroids for increasing muscle mass and for
improving physical performance is well documented,
the accepted therapeutic indications of these agents are seemingly
unrelated to their anabolic effects; stanozolol is an accepted treatment
for angiedema and endometriosis (Wilson, 1990; Helfman and Falanga,
1995). In recent years, however, there has been increasing interest in
the anabolic actions of these drugs, with emphasis on such clinical
applications as aging (Helfman and Falanga, 1995), wound repair
(Browse et al, 1977; Falanga et al, 1991), and HIV-related wasting
myopathy (Berger et al, 1993). The potential use of anabolic steroids
in wound healing has received particular attention. When administered
either preoperatively or postoperatively, anabolic steroids reverse the
deleterious effects of corticosteroids on experimental intestinal anastomotic
healing (Kim et al, 1993) and bone repair (Helfman and Falanga,
1995). Recently, we have shown that stanozolol, a synthetic anabolic
steroid with one of the largest anabolic/androgenic ratios (Wilson,
1990), causes dramatic healing of certain types of ischemic ulcerations
(Falanga et al, 1991; Kirsner et al, 1993). There are, however, few
investigations focused on the mechanisms of action of stanozolol and
other anabolic steroids. An in vivo study reported that anabolic steroids
enhance tensile strength of wounds by increasing the amount of
chondroitin sulfate (Watts et al, 1965). Using dermal fibroblast cultures,
Manuscript received January 23, 1998; revised June 15, 1998; accepted for
publication August 29, 1998.
Reprint requests to: Prof. Vincent Falanga, Boston University, Chairman of
Dermatology, Roger Williams Medical Center, Elmhurst Building, 50 Maude
Street, Providence, RI 02908.
0022-202X/98/$10.50 · Copyright © 1998 by The Society for Investigative Dermatology, Inc.
1193
growth factor-b1 (TGF-b1) mRNA and peptide levels
(p < 0.001). There was no stimulation of collagen synthesis
by testosterone. The stimulatory effects of stanozolol on
collagen synthesis were blocked by a TGF-b1 anti-sense
oligonucleotide, by antibodies to TGF-b, and in dermal
fibroblast cultures derived from TGF-b 1 knockout mice.
We conclude that collagen synthesis is increased by the
anabolic steroid stanozolol and that, for the most part,
this effect is due to TGF-b1. These findings point to a
novel mechanism of action of anabolic steroids. Key
words: fibroblasts/transforming growth factor- b/wound healing.
J Invest Dermatol 111:1193–1197, 1998
others have shown that stanozolol stimulates procollagenase production
(Wright et al, 1989). In this report, we determined the effect of
stanozolol on human dermal fibroblast replication and collagen synthesis.
The results shown here indicate that stanozolol stimulates
collagen synthesis and that, for the most part, this effect is due to the
action of transforming growth factor-b1 (TGF-b1).
MATERIALS AND METHODS
Fibroblast cultures and cell counts Adult human dermal fibroblasts were
cultured from the dorsal forearm of a total of seven healthy donors, as previously
described (Falanga et al, 1987). When so specified, we also used neonatal
foreskin fibroblasts in their first two in vitro passages. Cultures were established
and expanded in T-75 flasks (Costar, Cambridge, MA) with Dulbecco’s modified
Eagle’s medium (DMEM) and 10% fetal bovine serum (GIBCO, Grand Island,
NY) at 37°C, 95% air, and 5% CO2. For actual experiments, AIM V
serum-free media (GIBCO) was used. In all experiments involving collagen
measurements, 100 mM ascorbic acid (Sigma, St. Louis, MO) were added. For
measurements of cell replication, fibroblasts were seeded at a density of 5000
cells per well into 24 micro well dishes. After seeding, cultures were stimulated
at days 1 and 4 with either control media or stanozolol (Research Plus, Bayonne,
NJ), dissolved in dimethylsulfoxide (maximal concentration of dimethylsulfoxide
of 0.001%). In these and other experiments the same concentration of
dimethylsulfoxide was added to control media. Stanozolol was added in
concentrations ranging from 0.625 (1.9 3 10–6M) to 5 mg per ml. Cell numbers
were measured with a hemacytometer. Cell viability was determined by color
exclusion after mixing equal volumes of cell suspension with trypan blue
solution (0.4% in 0.81% NaCl) in the absence of serum.
For the purpose of establishing cultures of TGF-b1 knockout dermal
fibroblasts, skin from TGF-b1 knockout mice and control litter mates (Kulkarni
et al, 1993) was provided by one of us (A.B. Roberts). Dermal fibroblasts
cultured from mouse skin were maintained in DMEM plus 10% fetal bovine
serum until actual experiments, at which time cultures were washed extensively
with DMEM, and serum-free medium (AIM-V) was added.
Measurements of collagenous protein Fibroblasts were seeded into 24
micro well culture dishes at a density of 50,000 cells per well and at confluence
1194 FALANGA ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
(2–3 d later) were stimulated with stanozolol (0.625–5 mg per ml). Replicate
cultures were stimulated with human TGF-b1 (R&D Systems, Minneapolis,
MN). At this time, [3H]proline (99 Ci per mmol; Amersham, Arlington
Heights, IL) was added to each well at a concentration of 20 mCi per well.
The total volume in each well was 0.4 ml. After 48 h, [3H]proline incorporation
into pepsin-resistant, salt precipitable extracellular collagen was determined as
previously described (Webster and Harvey, 1979; Takagi et al, 1995). Results
were expressed as cpm of [3H]collagen per cell number, as determined with a
hemacytometer.
TGF-b1 peptide levels These were measured, as recently described (Hasan
et al, 1997), with an enzyme-linked immunosorbent assay kit from R&D
Systems and following the manufacturer’s protocol. Six-well plates were seeded
with 2.5 3 105 per well of fibroblasts in 0.2 ml of DMEM supplemented with
10% fetal bovine serum. The medium was then changed to DMEM without
serum supplemented with 200 mg crystalline bovine serum albumin (Sigma)
per ml, with four changes of medium over 24 h to remove serum and excess
TGF-b1. Cultures were then incubated for an additional 24 h in DMEM
without serum. At the end of this 24 h period, the supernatant from each
culture (conditioned media) was collected, centrifuged at 10,000 r.p.m. for
10 min, and stored at –70°C. Prior to freezing, each sample received 2 mg
aprotinin, leupeptin, and pepstatin A per ml, plus 120 mg phenylmethylsulfonyl
fluoride (all from Sigma) per ml. For measurements, the samples were thawed
and TGF-b in samples was activated by adding 0.2 ml of 1 N HCl to each
1.0 ml of conditioned media to give a final concentration of 0.167 N HCl.
After 10 min at room temperature, each acidified sample was neutralized with
0.2 ml of 1.2 N NaOH/0.5 M HEPES. Thereafter, 0.2 ml of each conditioned
media sample was added per well into a 96 well plate, the bottom of which
was coated with recombinant TGF-b soluble Type II receptor. A 1:1 dilution
series for TGF-b1 standards was prepared starting at 2000 pg per ml (2000,
1000, 500, 250, 125, 62.5, 31.2, and 0 pg per ml). Standards were assayed in
duplicates, and samples were measured in quadruplicate. Each sample and
standard were incubated in each receptor-coated well for 3 h at room
temperature. The supernatant from each well was then aspirated and discarded
and each well was washed three times with a wash buffer (buffered surfactant,
as per the manufacturer). To each well were then added 0.2 ml of polyclonal
antibody against TGF-b1 conjugated to horseradish peroxidase. After 1.5 h,
the supernatant was removed and each well was washed three times with wash
buffer. A hydrogen peroxide-chromogen mixture (0.2 ml) was then added to
each well for 20 min at room temperature. The reaction was stopped by adding
0.05 ml of 2N sulfuric acid, and optical density was measured at 450 nm.
RNA extraction and northern analysis For all experiments, fibroblasts
were grown to near confluence, to a density ranging from 0.75 to 1.0 3 106
cells per T-75 flask. Total cellular RNA from cells was isolated by extraction
in guanidium isothiocyanate using the method of Chomczynski and Sacchi
(1987). It was then separated for northern blot analysis on 1% agarose gels
containing 5% formaldehyde and transferred to a nylon membrane (Schleicher
and Schuell, Keene, NH) in a gradient of 203to 103sodium citrate/chloride
buffer. The following cDNA probes were used: a 1.5 kb EcoR1 fragment of
cDNA from the original clone Hf677 for the a1(I) procollagen chain (Chu
et al, 1982); a 1.4 kb Pst1 fragment of the cDNA clone pH III 33 coding
region for the a1(III) procollagen chain (Miskulin et al, 1986); and a 1.1 kb
EcoR1 fragment of the TGF-b1 cDNA (Derynck et al, 1985). Plasmids for
these cDNA and for GAPDH cDNA (used as a housekeeping gene) were
obtained from the American Tissue Culture Collection (ATCC, Rockville,
MD). Probes were labeled with 32P by random priming and used for northern
blot analysis as previously described (Falanga et al, 1993). For RNA electrophoresis,
10 mg of total RNA was loaded per lane, as measured by absorbance at
260 nm. Confirmation of uniformity of RNA loading was obtained by staining
the nylon blots with methylene blue (Falanga et al, 1993). Northern hybridization
was performed at 42°C in a solution containing 50% formamide, 63sodium
citrate/chloride buffer, 53Denhardt’s reagent, 0.5% sodium dodecyl sulfate,
and the labeled cDNA probe (2 3 10–8 cpm per mg). The blots were washed
at room temperature one time in 13sodium citrate/chloride buffer, 0.1%
sodium dodecyl sulfate for 20 min, followed by three washes at 68°C in
0.23sodium citrate/chloride buffer, 0.1% sodium dodecyl sulfate for 20 min
each. Autoradiography was generally carried out overnight at –70°C.
Anti-sense oligonucleotides We used 0.1–10 mM of a TGF-b1 199mer
anti-sense oligonucleotide (59 gAg ggC ggC ATg ggg gAg g 39), which overlaps
the promoter and transcriptional start site of the TGF-b1 gene. This same
sequence, which is specific for the TGF-b1 isoform, has been used successfully
to block TGF-b1 transcription in vitro (Murata et al, 1997) and in vivo (Brunet
et al, 1995). Confluent fibroblast cultures were either left untreated in AIM-V,
or treated with stanozolol alone or in combination with three different
concentrations of the anti-sense oligonucleotide. Sense oligonucleotide served
Figure 1. Stanozolol does not stimulate fibroblast replication. Stanozolol
was added to cultures on days 1 and 4 after seeding 5000 adult dermal fibroblasts
per well. The results represent the mean 6 SD from quadruplicate wells. The
arrows show the time points at which stanozolol was added to the cultures.
as an additional control. RNA isolation and measurements of procollagen
mRNA levels were made 24 h later.
TGF-b1 antibodies We used a polyclonal neutralizing antibody to TGF-b
(1D11 antibody, , Cambridge, MA), which has been previously used
to block the activity of this cytokine in vitro (Falanga et al, 1993). This antibody
is not specific for the TGF-b1 isoform, as it also blocks the effect of TGF-b2.
Fibroblast cultures were washed extensively with 0.1% bovine serum albumin
in DMEM to remove excess serum and TGF-b peptides. They were then
either left untreated in AIM-V or treated for 24 h with stanozolol alone or in
combination with the antibody.
Statistical analysis Data were entered in a computerized statistical analysis
program (InStat; GraphPAD Software, San Diego, CA). The Student’s t test
and one-way analysis of variance test were used for parametric results, whereas
linear regression analysis was employed to determine correlation coefficients
(r). Statistical significance was defined as a p value of 0.05 or less.
RESULTS
Stanozolol has no effect on fibroblast proliferation We tested
different concentrations of stanozolol, at different seeding densities,
and with varying dosing schedules. We found no effect of stanozolol
on cell replication, and Fig 1 shows a representative experiment. Cell
viability was not affected by stanozolol, as determined by trypan blue
exclusion in more than 95% of the cells.
Stanozolol increases collagen synthesis and a1(I) procollagen
mRNA levels We determined the effect of stanozolol on [3H]proline
incorporation into pepsin-resistant, salt precipitable extracellular collagen,
using a previously reported method (Takagi et al, 1995). As seen
in the representative experiment shown in Fig 2, stanozolol increased
collagen synthesis by 35% (p , 0.01). In the same experiment, higher
concentrations of stanozolol (1.25 and 2.5 mg per ml) were as effective
in stimulating collagen synthesis as TGF-b1 (5 ng per ml), which was
used here as an additional (positive) control. These results indicate that
stanozolol increases overall collagenous protein synthesis. Total protein
released in the culture medium was not increased by stanozolol
(p . 0.05; data not shown).We next determined the effect of stanozolol
on mRNA levels of type I and type III procollagen. For these
experiments, fibroblasts were grown to confluence in DMEM plus
10% fetal bovine serum. Cultures were then washed twice with
phosphate-buffered saline and the media were changed to serum-free
AIM-V plus different concentrations of stanozolol for 24 h. These
experiments were done four times, and with similar results. A representative
experiment is shown in Fig 3, where it can be seen that stanozolol
caused a dose-dependent increase in the mRNA levels of a1(I)
and a1(III) procollagen, and TGF-b1. These stimulatory effects of
stanozolol on procollagen are most likely due to its anabolic rather
than androgenic properties. Thus, as shown in Fig 4, similar molar
concentrations of testosterone (0.625–5 mg per ml) failed to stimulate
VOL. 111, NO. 6 DECEMBER 1998 TGF-b1 IS NEEDED FOR THE ANABOLIC EFFECTS OF STANOZOLOL 1195
Figure 2. Increased collagenous protein in response to stanozolol. Adult
dermal fibroblasts were seeded at 50,000 cells per 2.1 cm2 well and, after 2 d
(at confluence), were pulsed for 24 h with 20 uCi [3H]proline. Results represent
the mean 6 SD from quadruplicate wells.
Figure 3. Stanozolol increases mRNA levels of a1(I) and a1(III)
procollagen and TGF-b1. Total cellular RNA was isolated from duplicate
confluent cultures of adult dermal fibroblasts after 48 h of exposure to stanozolol.
The figure shows the northern analysis and densitometric determination of
each band.
Figure 4. Testosterone fails to increase a1(I) procollagen mRNA levels.
Total cellular RNA was isolated from duplicate confluent cultures of adult
dermal fibroblasts after 48 h of exposure to testosterone. The figure shows the
northern analysis and densitometric determination of each band.
and actually decreased a1(I) procollagen mRNA levels when compared
with baseline measurements.
The stimulatory effects of stanozolol are due to TGF-b1 We
hypothesized that the action of stanozolol may be mediated by TGF-
b1. TGF-b1 is a potent stimulus for collagen synthesis (Roberts et al,
1986; Varga et al, 1987) and, as our data indicated, its mRNA levels
Figure 5. Stanozolol increases TGF-b1 peptide levels. TGF-b1 peptide
levels were measured by enzyme-linked immunosorbent assay in serum-free
media conditioned for 24 h by cultures of adult dermal fibroblasts in the
presence or absence of stanozolol. The results are the mean 6 SD from
quadruplicate wells.
Figure 6. Stanozolol causes an early increase in TGF-b1 mRNA levels.
Total cellular RNA was isolated from duplicate confluent cultures of adult
dermal fibroblasts after 2 or 6 h of exposure to stanozolol (2.5 mg per ml). The
figure shows the northern analysis and densitometric determination of each
band. The data in the graph have been normalized for GAPDH.
are increased by stanozolol (Fig 3). In the next series of experiments,
we measured TGF-b1 peptide synthesis in response to stanozolol.
TGF-b1 levels were measured by enzyme-linked immunosorbent assay
(R&D Systems), using the type II TGF-b receptor as a substrate and
an antibody specific for TGF-b1 for detection. As shown in Fig 5,
stanozolol increased TGF-b1 peptide levels by as much as 200%
(p , 0.001). Therefore, stanozolol increases both collagen and TGF-
b1 synthesis. In fact, mRNA levels of TGF-b1 are increased as early
as 2 h after exposure of fibroblasts to stanozolol, and before procollagen
mRNA levels are increased (Fig 6). We next asked whether TGF-b1
is a critical factor in the stimulation of collagen synthesis by stanozolol.
1196 FALANGA ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
Figure 7. TGF-b1 knockout fibroblasts fail to respond to stanozolol.
Dermal fibroblasts from TGF-b1 knockout mice and control litter mates were
exposed to stanozolol for 24 h before measuring collagenous protein synthesis.
The results are the mean 6 SD from quadruplicate wells.
Figure 8. TGF-b1 anti-sense oligonucleotide blocks the stimulatory
effect of stanozolol on a1(I) procollagen mRNA. Human dermal fibroblasts
were first exposed for 24 h to increasing concentrations of a 199mer TGF-b1
anti-sense oligonucleotide in serum-free medium. Cultures were then either
left untreated or exposed for an additional 24 h to stanozolol or to sense
oligonucleotide, which served as an additional control. The graph shows the
densitometric analysis of the northern blot for a1(I) procollagen, and the
data are normalized for GAPDH. A-S, anti-sense oligonucleotide; S, sense
oligonucleotide; ST, stanozolol.
We approached this question in three ways. First, we tested the effect
of stanozolol in an environment devoid of TGF-b1. For this purpose,
we derived dermal fibroblast cultures from skin samples of TGF-b1
knockout mice and control litter mates (Kulkarni et al, 1993). We then
used these cultures to measure the effect of stanozolol in the absence
of TGF-b1. As shown in Fig 7, stanozolol increased overall collagen
synthesis in fibroblast cultures from control litter mates (p , 0.002)
but not in fibroblasts from TGF-b1 knockout mice, indicating that
the stimulatory action of stanozolol on collagen synthesis is, in large
part, due to TGF-b1. We next determined whether a TGF-b1 antisense
oligonucleotide would block the stimulatory effect of stanozolol.
We used a 199mer oligonucleotide that has been shown previously to
block TGF-b1 activity (Brunet et al, 1995; Murata et al, 1997). As
shown in Fig 8, the TGF-b1 anti-sense oligonucleotide blocked the
Figure 9. TGF-b antibodies block the stimulatory effect of stanozolol
on a1(I) procollagen mRNA. Confluent cultures of human dermal fibroblasts
were extensively washed to remove excess TGF-b peptides and then either
placed in serum-free media (AIM-V) alone or with stanozolol plus neutralizing
antibody to TGF-b for 24 h. The graph shows the densitometric analysis
of the a1(I) procollagen bands normalized for GAPDH. ST, stanozolol;
Ab, antibodies.
stimulation of a1(I) procollagen mRNA levels by stanozolol in a dosedependent
manner; sense oligonucleotide had no effect. Similarly, as
observed in Fig 9, the stimulation of procollagen mRNA levels by
stanozolol was blocked by antibodies to TGF-b. Taken together, these
results strongly point to TGF-b1 as a critical cytokine involved in the
observed stimulation of collagen synthesis by stanozolol.
DISCUSSION
We report that the anabolic steroid stanozolol stimulated overall
collagen synthesis and increased the mRNA levels of a1(I) and a1(III)
procollagen. These stimulatory effects of stanozolol on collagen synthesis
were not observed with testosterone and were accompanied by
increased synthesis of TGF-b1. The mRNA levels of TGF-b1 were
increased as early as 2 h after exposure of fibroblasts to stanozolol, and
before any increase in procollagen mRNA. We found that stanozolol
failed to stimulate collagen synthesis in TGF-b1 knockout fibroblasts
and after the addition of a TGF-b1 anti-sense oligonucleotide and
antibodies to TGF-b. Taken together, these findings strongly suggest
that the stimulation of collagen synthesis by stanozolol is due, in large
part, to the action of TGF-b1.
Although the increase (35%) in collagenous protein observed in this
study may appear modest at first, it should be noted that the experiments
were done in a defined serum-free media (AIM-V) without added
growth factors, so as to avoid binding of stanozolol to steroid binding
proteins. Moreover, the amount of collagen synthesis observed in
response to stanozolol was the same as that measured after the addition
of TGF-b1 and, importantly, the stimulatory actions of stanozolol
appear to be specific, in that the parent compound testosterone actually
decreased collagen synthesis.
Evidence linking stanozolol or other anabolic steroids to increased
extracellular matrix formation has been limited thus far. In one report,
stanozolol was found to enhance procollagenase production by dermal
fibroblasts but not in synovial fibroblast cultures (Wright et al, 1989);
VOL. 111, NO. 6 DECEMBER 1998 TGF-b1 IS NEEDED FOR THE ANABOLIC EFFECTS OF STANOZOLOL 1197
however, collagenous protein was not measured in that study. Stanozolol
has also been shown to stimulate prostaglandin E2 (PGE2) synthesis
and to inhibit fibroblast growth factor-induced DNA synthesis in
human skin fibroblasts (Ellis et al, 1994). In animal studies, stanozolol
has been reported to increase muscle protein synthesis without a direct
effect on protein degradation (Gribbin and Flavell Matts, 1976; Helfman
and Falanga, 1995). In vivo studies in humans have not specifically
addressed the effects of anabolic steroids on extracellular matrix
formation, but do point to overall anabolic activity. For example, a
short-term study of 16 patients, eight of whom received 10 mg of
stanozolol orally each day for 14–21 d, showed an increase in the bulk
of type I (oxidative) fibers in response to stanozolol (Hosegood and
Franks, 1988). Other reports suggest that stanozolol may be effective
in the treatment of osteoporosis, in improving nitrogen balance in
wasting diseases such as muscular dystrophy, and in postoperative
trauma (Glueck et al, 1995). Stanozolol has also been shown to cause
dramatic healing of painful cutaneous ulcerations due to cryofibrinogenemia
(Falanga et al, 1991; Kirsner et al, 1993); however, it is unclear
whether the beneficial effects of stanozolol in these dermal ischemic
wounds is due to direct stimulation of tissue repair or to the fibrinolytic
action of stanozolol (Browse et al, 1977).
TGF-b1 is an established stimulus for the formation of extracellular
matrix both in vitro (Roberts et al, 1986; Varga et al, 1987) and in vivo
(Roberts et al, 1986; Mustoe et al, 1987). The results shown here point
to TGF-b1 being critically involved in the stimulatory action of
anabolic steroids on collagen synthesis. This observation is of interest
when one considers the possibility of using anabolic steroids to offset
the deleterious effects of corticosteroids on wound healing. For
example, in one study of experimental cutaneous wounds in rats, it
was shown that the systemic administration of TGF-b1 reversed the
inhibitory effects of corticosteroids on healing (Beck et al, 1993). In
another report of anastomotic healing of intestinal wounds, stanozolol
reversed the inhibition of healing caused by corticosteroids (Kim et al,
1993). Therefore, it is plausible that TGF-b1 plays an important role
in the way anabolic steroids work, or at least in the way they oppose
some of the effects of corticosteroids.
In conclusion, we provide evidence that stanozolol stimulates
collagen synthesis. If these effects are mediated through TGF-b1, as
our results indicate, we should start thinking of anabolic steroids as
acting pharmacologically to increase the synthesis of potent growth
factors and cytokines.
This work was supported by grants from the National Institutes of Health (AR42936,
AG10998), the national Dermatology Foundation, and the Dermatology Foundation
of Miami.
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