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Reproductive Endocrine Unit and National Center for Infertility Research, Massachusetts General Hospital, Boston, Massachusetts 02114
Address all correspondence and requests for reprints to: Alan Schneyer, Ph.D., Reproductive Endocrine Unit, BHX-5, Massachusetts General Hospital, Boston, Massachusetts 02114. E-mail: schneyer.alan{at}mgh.harvard.edu
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Abstract |
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TT cells, derived from a
p53-/-/
-inhibin-/- mouse testicular
tumor, respond to activin by proliferating, a response that can be
inhibited by follistatin (FS) treatment. Using semiquantitative RT-PCR
methods, we characterized steady state messenger RNA (mRNA) levels for
the inhibin/activin subunits, FS, and activin receptor subtypes under
basal conditions and in the presence of activin or FS. These cells
produced ample immunoreactive activin A and FS, necessitating higher
treatment doses to observe any modulation of cellular proliferation.
Furthermore, in the presence of exogenous activin, mRNA levels for
activin receptor type IIA (ACTRIIA) and ßA were significantly and
profoundly suppressed. In addition, both ACTR1B and ACTRIIB were
detectable and down-regulated by exogenous activin, although not to the
degree observed for ACTRIIA and ßA. Finally, activin treatment at the
higher doses, which decreased activin receptor mRNA levels, resulted in
inhibition of cellular proliferation. Taken together with previous
observations, our results support the model that these tumor cells
respond to an autocrine activin signal by proliferating, whereas
exogenous or excess activin results in down-regulation of activin
receptor and activin biosynthesis, suggesting a potential
autocrine/paracrine mechanism by which activin can modulate its own
signal.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-subunit (1, 2, 3). In addition to endocrine regulation of pituitary FSH release, activins and inhibins have been implicated in the local autocrine/paracrine regulation of gonadal function (4, 5). In the testis, inhibins are mainly secreted by Sertoli cells (6, 7, 8, 9), where they appear to decrease spermatogonial mitotic activity (10) and enhance LH-stimulated testosterone production (11). Activins appear to be produced by Sertoli cells, germ cells, and immature Leydig cells (4, 6, 12, 13) and have been implicated in regulation of germ cell mitotic and differentiation activities (14, 15). Furthermore, biosynthesis of inhibin and activin subunits appears to be tightly regulated during embryogenesis as well as during defined stages of the spermatogenic cycle (13, 16, 17, 18).
Follistatin (FS), the activin-regulating protein, and the activin receptors are also expressed in a cell type- and developmental stage-specific manner (19, 20), providing additional evidence for an important regulatory role for this protein family. FS is a monomeric glycoprotein found in tissues (21, 22), human follicular fluid (23), and serum (24, 25, 26) whose only known actions are mediated through nearly irreversible binding and neutralization of activin (21, 27, 28).
Members of the TGFß superfamily act at the cellular level through a complex of two related serine/threonine kinase receptor subfamilies collectively known as type I and type II receptors (reviewed in Ref.29). Current evidence supports a model in which ligand binds to one or more type II receptors that then phosphorylate type I receptors, which, in turn, transduce an intracellular signal (30). At least two type I and two type II activin receptors (ACTRI and ACTRII, respectively) have been biochemically and functionally characterized (29). Atlhough type I-II receptor complexes may exist in the unliganded state, complex formation is necessary for signal transduction (29), so that the relative numbers of each type of activin receptor on the cell surface at any moment may be a critical factor in determining the cell’s responsiveness to a particular activin dose. Thus, delineating the mechanisms by which biosynthesis of these receptors is regulated could be critical for understanding the functions of activin in vivo.
We, therefore, undertook studies to determine whether activin regulates
steady state levels of its receptor messenger RNA (mRNA) in an
activin-responsive cell line derived from a testicular tumor generated
in an -inhibin/p53 knockout mouse (31, 32). This TT cell
line was previously demonstrated to increase proliferation in response
to activin, an effect that could be partially abrogated by
administration of FS, suggesting that activin was critical to the
maintenance of growth in these tumor cells (32). In addition to further
characterizing activin and FS biosynthesis in these cells, our studies
identified all four activin receptor subtypes. We further observed that
activin administration acutely decreased both ßA-subunit and activin
receptor steady state mRNA levels while inhibiting proliferation of
these cells at higher doses. Taken together, these results suggest a
mechanism through which activin acts to regulate growth, an effect that
can be modulated by changes in activin biosynthesis, receptor
availability, and/or production and action of FS.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-inhibin subunit (31) and p53 (32).
Production and initial characterization demonstrated that these cells
express ACTRI and ACTRII and proliferate in response to exogenous
activin, whereas both basal and activin-stimulated proliferation can be
inhibited by exogenous FS 288 (32). These cells were provided by Dr.
Aaron Hsueh (Stanford University, Stanford, CA). To avoid potentially
confounding effects of growth factors, such as insulin, that were used
in previous studies (32), cells were grown in DMEM-Ham’s F-12 (1:1)
supplemented with 10% heat-inactivated FBS (Life Technologies, Grand
Island, NY), 2 mM L-glutamine, and
antibiotics (100 IU/ml penicillin and 100 µg/ml streptomycin sulfate)
at 37 C in a 5% carbon dioxide atmosphere. Except as detailed in
experimental protocols, medium was replaced every 3–5 days.
Characterization of FS and activin production by TT cells
TT cells were plated into 100-mm dishes and incubated until
confluent (
48 h). Medium was removed and analyzed for activin A and
FS content.
Total activin A was measured using an anti-ßA-subunit monoclonal antibody (E4, Serotec, Oxford, UK) (33) and procedures described by Knight et al. (34). The antibody was oxidized and plated as previously described (35) at 250 ng/well for use as capture antibody and biotinylated using biotin-LC (Pierce Chemical Co., Rockford, IL) according to the manufacturer’s directions for detection of captured activin molecules. Antibody-coated wells were blocked with 1% BSA in 0.1 M Tris (pH 7.5) plus 0.1% sodium azide for 1 h, followed by two washes with wash buffer (0.05 M Tris plus 0.15 M NaCl, 0.1% sodium azide, and 0.05% Tween-20, pH 7.5). Samples were incubated for 10 min at 95 C with an equal volume of 20% SDS, followed by cooling and treatment with hydrogen peroxide as previously described (35). After washing the plates, 25 µl enzyme immunoassay diluent (0.1 M Tris, 10% BSA, 5% Triton X-100, and 0.1% sodium azide), 100 µl of samples or standards, and 25 µl biotinylated E4 antibody (35 ng/ml) were added. After overnight incubation at 4 C, the plates were washed, and strepavidin-AP (Boehringer Mannheim, Indianapolis, IN) was added (1:2000 dilution) for 2 h. After 10 additional washes, 100 µl p-nitrophenylphosphate in 0.05 M ethanolamine buffer (pH 9.8) were added and incubated at room temperature until color developed in control wells, at which time the assay was read in a microtiter plate reader (Molecular Devices, Menlo Park, CA). Standards were prepared from recombinant human activin A provided by the Genentech reagent distribution program (San Francisco, CA) and analyzed by four-parameter logistics software (SoftMax, Molecular Devices). The minimum detectable dose of activin in this assay was 5 ng/ml, determined as the dose of activin giving an absorbance reading 2 SD above the zero dose in five consecutive assays. Recovery of exogenous recombinant human activin A from culture medium was estimated at 75% over a range of activin doses with an intraassay coefficient of variation of 7%. The activin levels reported are a mean of three separate determinations.
FS was analyzed by two methods. First, total FS in conditioned medium
was assessed by RIA as previously described in detail (25), using a
mouse polyclonal antibody that recognizes both free and complexed FS
from all species tested. Second, free FS in TT cell-conditioned medium
was assessed using gel filtration chromatography as previously
described (23, 24). Conditioned or unconditioned TT cell medium (100
µl) was incubated with radiolabeled activin (200,000 cpm; SA, 35
µCi/µg) (24) for 2 h at room temperature, followed by
overnight incubation at 4 C. Samples were chromatographed on a Superose
(Pharmacia, Piscataway, NJ) gel filtration column (two tandem
10 x 30-cm columns). The column was calibrated with low molecular
mass standards (Pharmacia), including ferritin (440,000 kDa), aldolase
(158,000 kDa), BSA (66,000 kDa), and radiolabeled activin, as shown in
Fig. 1
. The resulting activin binding
(FS) peak eluted at a position similar to that previously observed
in both human serum and recombinant huamn FS 315-conditioned medium
(24).
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10 nM activin and 28 nM FS) (32),
keeping in mind that these cells secrete significant amounts of both
activin and FS. To examine the concentration dependence of the effect
of activin on ßA-subunit, ßB-subunit, FS, and activin receptor mRNA
levels, cells were treated on day 2 of culture with 0, 0.125, 1.25, or
12.5 nM activin A for 24 h. For both sets of
experiments, cellular RNA was extracted for RT-PCR analyses.
RNA extraction and RT-PCR procedures
Total RNA was extracted using the guanidine
isothiocyanate/mercaptoethanol method (36). RNA integrity was confirmed
by agarose gel electrophoresis and ethidium bromide staining as well as
monitoring absorbance at 260/280. RNA concentrations were determined
from the A260 absorbance before each reverse transcription experiment.
Complementary DNA (cDNA) was obtained by RT at 42 C for 30 min in a
20-µl reaction mixture containing 1 µg RNA, 0.5 mM of
each deoxy (d)-NTP, 5 µM oligo(deoxythymidine), 25 U
SuperScript (Life Technologies), and 14 U ribonuclease inhibitor
(Promega). The PCR reaction was performed in a volume of 50 µl
containing 1 x PCR buffer A (Fisher Taq buffer, Fisher
Scientific, Fairlawn, NJ), 5 mM MgCl2, 0.2
mM of each dNTP, 3 µM each of 5'- and
3'-primers, 2.5 U Taq polymerase (Fisher), and 2 µCi
nucleotide triphosphate ([32P]dCTP) to which 10 µl of
the RT reaction product were added. Primer sequence and location as
well as expected product size are listed in Table 1. The amplification profile involved
preincubation at 95 C for 3 min, denaturation at 95 C for 0.5 min,
primer annealing at 50 C for 1.5 min, and extension at 72 C for 1.5
min. In initial experiments, cycle number was optimized for each target
so that signals were always in the exponential portion of the
amplification curve (35 cycles for all, except 24 cycles for ß-actin,
and 40 cycles for FS). Ten microliters of the PCR reaction were
electrophoresed in 5% polyacrylamide gels in Tris-borate-EDTA buffer.
Autoradiography was carried out for 12 h at room temperature, and
the relative concentration of each mRNA was determined by densitometric
scanning of the autoradiograms and normalization to the ß-actin
signal for each sample. As we observed no difference in the loading on
replicate gels in preliminary experiments, each RT-PCR was run on a
single gel. PCR products were verified by Southern blot using an
internal oligonucleotide as a probe (see Table 1).
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), 0.25 µl Taq, and 0.1 µCi
[32P]dCTP. Under these conditions, PCR reactions were in
the exponential range for: 1) actin, 1:10 RT dilution and 21 cycles; 2)
ACTRIIB, 1:10 RT dilution and 31 cycles; and 3) ACTR1B, 1:2 RT dilution
and 31 cycles. The identities of the resulting PCR products were
verified for both by Southern blot as described below.
Southern blot analyses
PCR product identity was verified by Southern blot using
synthetic oligos located internal to the two PCR primers (see Table 1).
PCR-amplified products without radionuclide were fractionated on a
1.5% agarose gel and visualized by UV transillumination, and the
product sizes were estimated by comparison to mol wt markers (PGEM,
Promega). The gels were then denatured in 50 mM NaOH-1.5
M NaCl for 30 min and neutralized in 1 M
Tris-1.5 M NaCl. The DNA was transferred by capillary
action to a nylon membrane (Micron Separations, Westborough, MA) in
20 x SSC (3.0 M NaCl and 0.3 M sodium
citrate, pH 7.0) and subjected to Southern blot analyses using
[32P]dATP-labeled oligonucleotide probes. Hybridization
was performed with each labeled oligonucleotide in 5–10 ml of a
solution containing 5 x SSC, 1 x Denhart’s solution, 0.1%
SDS, and 100 µg/ml salmon sperm DNA overnight at 42 C. The membranes
were washed twice in 0.2 x SSC at 67 C for 15 min each time,
followed by autoradiography.
TT cell proliferation studies
TT cells were plated at 30,000–50,000/well in six-well culture
dishes in 1–2 ml medium containing either 5% or 10% FCS (no
difference was observed with respect to activin response). Increasing
volumes of activin containing medium (see below) or an equivalent
amount of control medium (wild type 293 cell-conditioned medium) were
added to triplicate wells. The cells were further incubated for 24, 48,
72, or 96 h, at which time they were removed (3–5 min of
trypsinization) from the plates and counted in a hemocytometer using
trypan blue to distinguish live cells. After correcting for dilution,
live cell number was plotted vs. time in culture for each
dose of activin or control medium. Results were initially calculated as
the number of live, activin-treated cells/number of live, control
medium-treated cells x 100.
Recombinant activin-containing conditioned medium
Preliminary experiments with lower doses (1–100 ng/ml) of
purified activin from Genentech did not have a significant effect on TT
cell proliferation, in contrast to previous observations of TT cells
(32). To examine higher doses of activin with potentially more
bioactivity, we used conditioned medium from 293 cells stably
transfected with a human inhibin/activin ßA-subunit cDNA (provided by
Genentech). Conditioned medium was concentrated 20-fold using a stirred
cell apparatus with a 10-kDa cut-off membrane (Filtron, Northborough,
MA) and aliquoted. Aliquots were calibrated against purified
recombinant activin from Genentech (lot 18395) and NIH (lot 15365–36)
using a previously described, solid phase, ligand binding assay (SILBA)
(28) as well as by RIA. The RIA used purified activin from Genentech as
radioligand and a rabbit antibody raised to recombinant inhibin A
(M863-8) diluted in PBS to 1:500. The iodination protocol and remaining
assay steps were previously reported for inhibin (23, 37). Using both
RIA and SILBA, the 20-fold concentrated medium, designated MGH-ACT-2,
contains 30 µg/ml activin immunoactivity and FS binding activity with
approximately the same bioactivity as the activin distributed by the
NIH (data not shown). As a control, medium conditioned by wild-type 293
cells was concentrated 20-fold and added to the cultures at volumes
equal to those of the activin-containing medium.
Data analysis
Densitometric results (RT-PCR experiments) were normalized to
the ß-actin level for each sample and are reported as the mean
± SE for two or three independent experiments. For the
dose-response studies (Fig. 4), the results for each dose of activin
were compared with those for the zero dose sample. For
time-course studies (Figs. 3, 5, and 6), the effects of activin or FS
at each time point were compared with an untreated control sample
incubated for the same duration. All statistical differences for these
experiments were determined using t test, comparing each
treatment to the no treatment control. Finally, Fig. 7 represents data
collected from eight different experiments, each covering an
overlapping, but not complete, set of doses such that each dose
represents observations ranging from 1–5. Experiments were combined by
expressing results for each experiment as a percentage of the control
value, and the composite for eight experiments is shown in Fig. 7.
Significant differences were determined using Student’s t
test as indicated by letter codes, with P < 0.05
considered significant for all experiments.
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Results |
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), consistent with the activin BP previously observed in human
serum at approximately 60 kDa (24). In contrast, the BP in
unconditioned TT medium (control) was much smaller and was probably
derived from the FCS (see RIA results above). Thus, it would appear
that at least some free FS is secreted by TT cells. Using a specific two-site activin A immunoassay that measures total activin (free activin plus activin bound to FS), TT cells secrete approximately 15 ng/ml (0.5 nM) activin A/48 h or 4.6 ± 3.4 ng/ml/1.3 million cells·24 h (n = 3). No activin immunoactivity was detected in unconditioned medium.
Steady state mRNA levels
TT cell expression of inhibin/activin ßA- and ßB-subunits, FS,
and activin receptor mRNA by TT cells was examined using RT-PCR
analysis. The identity of the amplified PCR products was demonstrated
by Southern blotting. All PCR products were analyzed on a 1.2% agarose
gel and electrophoresed adjacent to a lane with DNA markers (Fig. 2A). After transfer and hybridization,
each of these PCR products was specifically recognized by an internal
33-mer synthetic oligo probe that did not bind to the adjacent marker
DNA (Fig. 2B).
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).
Regulation of inhibin/activin ß-subunit and activin receptor mRNA
levels by activin A
Activin’s effects on ACTRIIA and ßA mRNA were significant only
for the highest dose (12.5 nM), achieving suppression to
75% and 63% of the untreated control values, respectively (Fig. 4A). This dose of activin was even more
effective in reducing ACTRIIB and ACTRIB mRNA levels, achieving
suppression to 30% and 20% of control, respectively (Fig. 4B).
The time course for the effects of 12.5 nM activin A
treatment on the expression of inhibin/activin subunit and activin
receptor mRNA was analyzed next. Treatment with activin resulted in
50% and 90% suppression of ACTRIIA steady state mRNA levels at 24
(P < 0.01) h of incubation (Fig. 5A). Similarly, this treatment caused a
75% reduction (P < 0.02) of ßA-subunit mRNA at
24 h (Fig. 5A). For both targets, mRNA levels remained below
control levels at 48 h of incubation, although this difference was
not significant.
This 12.5-nM dose of activin A resulted in a maximal 40%
suppression of ACTRIIB mRNA (Fig. 5B) at both 24 and 48 h
(P < 0.05 and P < 0.001,
respectively) as well as 50% and 30% suppressions of ACTR1B mRNA at
24 and 48 h (P < 0.01 and P <
0.05, respectively). Thus, ACTR1A was the only activin receptor subtype
whose expression did not appear to be modulated by treatment with
activin A.
Regulation of ß-subunit, activin receptors, and FS mRNA by
recombinant human FS 288 treatment
Treatment of TT cells with 9 nM recombinant human FS
288 for 24 h induced a 1.5-fold (P < 0.05)
increase in the expression of both ACTRIIA and FS mRNA (Fig. 6). Interestingly, a 25% reduction in
ßB-subunit mRNA (12 h after activin treatment) was found, although
this effect did not reach statistical significance (data not shown). FS
treatment produced a slight, but consistent, increase in the expression
of ACTR1B and ACTRIIB mRNA, but this was only significant at 48 h
(data not shown). No significant effects of FS treatment were observed
for activin ßA and ACTR1A mRNA.
TT cell proliferation studies
At activin doses of 30 nM or greater, significant
inhibition of cellular proliferation was observed after 72 and 96
h of treatment (Fig. 7). These doses
exceed those at which we observed significant decreases in activin
subunit and its receptor mRNA levels. In contrast, we were unable to
document a statistically significant increase in proliferation at any
activin dose or incubation time under the culture conditions used in
these studies. This inhibitory action of activin was not due to
toxicity from the conditioned medium, as equivalent amounts of
wild-type 293 cell-conditioned medium had no effect, and only live
cells were counted. Thus, at higher activin doses, especially those at
which ACTRIIA and ßA-subunit were also reduced, we observed a
significant and dramatic inhibition of cellular proliferation.
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Discussion |
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-inhibin and p53 were deleted by homologous recombination (31),
represents one such tool. Our RT-PCR results demonstrate that these cells express mRNA for all four activin receptor subtypes (29) characterized to date, including ACTRIIA, ACTRIIB, ACTRIA, and ACTRIB. In addition, we quantitated the amounts of activin and FS secreted by these cells and determined the approximate amount of free FS in the medium. Using semiquantitative RT-PCR analysis, we also observed that exogenous activin regulates its own and its receptor mRNA levels. These observations extend the earlier studies by Shikone et al. (32), who demonstrated that TT cells contain ßA, ßB, and FS mRNA as well as ßA-subunit and type I and II activin receptor proteins.
When treated with 12.5 nM activin, a dose necessary to
overcome endogenous activin and FS production, TT cell expression of
ACTRIIA mRNA was decreased by 90%, whereas ßA-subunit mRNA was
decreased by 75%. Similarly, this dose of activin had a significant
negative influence on ACTR1B and ACTRIIB mRNA levels, decreasing steady
state levels by 50% at 24 h. Although we did not observe
consistent effects of activin on TT cell proliferation at this 12.5
nM activin dose, larger doses significantly inhibited
cellular proliferation in a dose-dependent fashion, suggesting that
this decrease in mRNA steady state levels in response to activin was
functional at the cellular effector level. Furthermore, our results are
consistent with the significant 65% reduction in testicular ACTRIIA
mRNA observed in vivo in -inhibin-deficient mice with
elevated circulating activin levels and ßA-subunit production (41, 42). If reductions in activin receptor and ßA-subunit mRNAs are
reflected in secreted protein, our results suggest that the activin
signaling system might become down-regulated in response to high levels
of exogenous or endogenous activin and that this down-regulation is
manifested by inhibition or removal of growth factor support for
cellular proliferation. In addition, this decrease in activin receptor
or ßA-subunit mRNA levels may have important inhibitory consequences
on testicular steroidogenesis and spermatogenesis despite elevated
activin levels. Indeed, Leydig cell numbers decrease and spermatogenic
regression occurs in parallel with tumor growth in
-inhibin-deficient mice with increased circulating activin levels
(41).
Treatment with the activin BP FS 288 reversed activin’s effect on both basal (endogenous) and activin-stimulated proliferation (32). Consistent with this, we observed that FS treatment caused slight increases in both activin receptor and FS mRNA levels. We also observed a significant increase in ACTR1A mRNA levels in untreated cells between 3 and 24 h of incubation. This latter finding suggests that endogenous factors can regulate ACTR1A mRNA expression. Although this endogenous factor could be activin itself, our failure to observe changes in ACTR1A mRNA levels with high doses of activin or FS indicate that other growth regulatory proteins might be responsible.
Using a specific, two-site, total (free plus FS bound) activin A immunoassay, we determined that these cells produce significant amounts of activin on the order of 15 ng/ml conditioned medium/48 h. Immunoreactive total FS (free FS plus FS bound to activin) in a separate batch of conditioned medium was 4.4 ng/ml, suggesting that free activin, i.e. that secreted in excess of FS, might be secreted by TT cells. This would be consistent with the observation by Shikone et al. (32) that addition of FS could suppress basal as well as activin-stimulated proliferation. On the other hand, the peak of radiolabeled activin complexed with FS, as determined by chromatographic analysis of TT cell conditioned medium, suggests that at least some free FS is also secreted by TT cells. As this FS-activin peak eluted at a position similar to that observed in human serum and recombinant human FS 315-conditioned medium (24), the FS secreted by TT cells might contain the 315 form. Although recombinant human FS 288 was previously demonstrated to bind activin with nearly irreversible kinetics (28), FS 315 has been described to bind activin at a lower affinity (43). Thus, the detection of a FS-activin complex in TT cell-conditioned medium might result from exchange of bound unlabeled activin for radiolabeled activin during the incubation before chromatography, rather than representing only free FS. Alternatively, both the total FS RIA and the activin A enzyme-linked immunosorbent assay use human standards, so the mass estimation of secreted mouse FS and activin from TT cells may be inaccurate due to species specificity of the antibodies used in either assay. Nevertheless, our results demonstrate significant production of both activin and FS by TT cells.
Previous studies using TT cells demonstrated that due to the production and secretion of both activin and FS, the use of relatively high doses of activin was necessary to obtain statistically significant increases in cellular proliferation rates (e.g. 1 nM) (32). However, under the growth conditions employed in the present studies, stimulation of proliferation was not observed for any dose of activin, nor did we observe inhibition of basal proliferation for any dose of FS up to 30 nM. The effects of FS and activin on TT cell proliferation previously observed (32) may have resulted from their additional supplementation with growth factors such as insulin and epidermal growth factor. Nevertheless, at higher doses of activin, we consistently observed significant inhibition of proliferation as well as a robust and significant decrease in ßA and activin receptor mRNA levels.
Taken together, the two TT cell studies suggest that activin might influence cellular proliferation in a biphasic manner. Similar biphasic effects on cell growth were observed in response to activin for porcine thyroid cells (44) as well as for connective tissue cells in response to TGFß (45). Perhaps more importantly, this biphasic response to activin has also been reported for testicular cultures, particularly for seminiferous epithelium at stages VIIa–VIIc (46). Interestingly, this phase change in activin action occurred at doses similar to those resulting in reduced ßA and activin receptor mRNA levels in TT cells. Thus, if changes in mRNA correlate with protein expression, our results suggest a mechanism by which activin can modulate its activity level by down-regulating its own and its receptor biosynthesis, thereby suppressing the autocrine growth signal. Alternatively, high, chronic activin levels might induce apoptosis, as recently observed in a human prostate cancer cell line, LNCaP (40), resulting in the observed decrease in proliferation at the activin doses used in this study.
Northern blot analysis of normal mouse testis revealed an equal abundance of ACTRIIA and ACTRIIB (47), whereas a greater abundance of ACTRIIA was detected compared with that of ACTRIIB in testis from adult rat (48). In this study, we identified all four activin receptor subtypes and determined that at least three of them, ACTR1B, ACTRIIA, and ACTRIIB, are regulated by ligand. Differences in the activin receptor content of various activin-responsive cell lines have been recently demonstrated (38, 39). Interestingly, those studies identified ACTRIB as the dominant activin signal transducer. If the same relationship holds for TT cells, then expression of at least the major activin-signaling receptor subtypes appears to be regulated by activin. It would be of interest to determine the prevalence of each possible combination of ACTRI and ACTRII subtypes in primary tumors or in other tumor cell lines that respond to activin with proliferation to understand the potential role of activin in normal reproduction as well as in pathogenesis of infertility and carcinoma. Our results also suggest that TT cells will be an excellent model in which to dissect the intricate relationships among receptor subtype expression, production of autocrine growth factors, and inhibitory binding protein modulation of signal transduction.
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Acknowledgments |
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Footnotes |
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Received August 25, 1997.
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References |
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and ß-B subunits during the
cycle of the rat seminiferous epithelium. Endocrinology 124:987–991-Inhibin is a tumour-suppressor gene with gonadal specificity
in mice. Nature 360:313–319[CrossRef][Medline]
and p53-deficient mice: the role of activin as an autocrine
growth factor. Mol Endocrinol 8:983–995-inhibin deficient mice. Biochem Biophys Res Commun 203:105–112[CrossRef][Medline]
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