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PubMed2003

Activin A induces cell proliferation of fibroblast-like synoviocytes in rheumatoid arthritis.

OBJECTIVE: To investigate the expression of activin A and its receptors in rheumatoid arthritis (RA) synovial tissues, and to determine the effect of activin A on cultured fibroblast-like synoviocytes (FLS). METHODS: The localization of activin A and activin type II receptor (ARII) in synovial tissues of RA patients was analyzed by immunohistochemistry. The expression of activin A and activin receptors in human cultured FLS was examined by reverse transcriptase-polymerase chain reaction and Western blotting. Enzyme-linked immunosorbent assay was used to measure activin A in culture supernatants. The cell growth of FLS was determined by (3)H-thymidine incorporation and MTT assay. RESULTS: Immunohistochemical analysis confirmed the up-regulation of activin A in rheumatoid synovium as compared with osteoarthritis or normal joint tissues. CD68+ macrophage-lineage cells and vimentin-positive FLS were identified as activin-producing cells in rheumatoid synovium. Both cell types also expressed ARII. The expression of activin A and ARII on cultured FLS was confirmed at the protein and messenger RNA levels. Interleukin-1 beta (IL-1 beta), tumor necrosis factor alpha, and transforming growth factor beta activated FLS to secrete activin A. Recombinant activin A accelerated the proliferation of FLS, while follistatin, an endogenous activin antagonist, partially inhibited FLS proliferation induced by IL-1 beta. CONCLUSION: These results suggest that activin A acts as a growth factor of FLS in RA.

Activin Receptors↗
PubMed2006

The activin axis in liver biology and disease.

Activins are a closely related subgroup within the TGFbeta superfamily of growth and differentiation factors. They consist of two disulfide-linked beta subunits. Four mammalian activin beta subunits termed beta(A), beta(B), beta(C), and beta(E), respectively, have been identified. Activin A, the homodimer of two beta(A) subunits, has important regulatory functions in reproductive biology, embryonic development, inflammation, and tissue repair. Several intra- and extracellular antagonists, including the activin-binding proteins follistatin and follistatin-related protein, serve to fine-tune activin A activity. In the liver there is compelling evidence that activin A is involved in the regulation of cell number by inhibition of hepatocyte replication and induction of apoptosis. In addition, activin A stimulates extracellular matrix production in hepatic stellate cells and tubulogenesis of sinusoidal endothelial cells, and thus contributes to restoration of tissue architecture during liver regeneration. Accumulating evidence from animal models and from patient data suggests that deregulation of activin A signaling contributes to pathologic conditions such as hepatic inflammation and fibrosis, acute liver failure, and development of liver cancer. Increased production of activin A was suggested to be a contributing factor to impaired hepatocyte regeneration in acute liver failure and to overproduction of extracellular matrix in liver fibrosis. Recent evidence suggests that escape of (pre)neoplastic hepatocytes from growth control by activin A through overexpression of follistatin and reduced activin production contributes to hepatocarcinogenesis. The role of the activin subunits beta(C) and beta(E), which are both highly expressed in hepatocytes, is still quite incompletely understood. Down-regulation in liver tumors and a growth inhibitory function similar to that of beta(A) has been shown for beta(E). Contradictory results with regard to cell proliferation have been reported for beta(C). The profound involvement of the activin axis in liver biology and in the pathogenesis of severe hepatic diseases suggests activin as potential target for therapeutic interventions.

Activin Receptors↗
PubMed1998

Cell survival effect of activin against heat shock stress on OVCAR3.

Activin has been known as the hormone protein which regulates either cell proliferation or cell differentiation. Recently, it has also been reported that activin may have cell survival function. In this study, we have investigated, 1) the expression of inhibin subunits and activin receptors (ActRs) in ovarian carcinoma cell line (OVCAR3), 2) the binding property between activin and its receptors under the exposure to stress, and 3) the effect of activin on cell proliferation. All of inhibin subunits and ActR Ia, IIa and IIb mRNA were amplified by RT-PCR in OVCAR3. By Western blot analysis, ActR IIa and IIb proteins were detected. The binding property between activin and ActRs was analyzed with the fixed complex, using chemical cross linker. The bigger molecular weight signals, which had been shown to form the heterotrimeric complex among activin, ActR type I and ActR type II were detected after cross linking. These upper signals were apparently increased by rh-Activin and decreased by rh-Follistatin. Therefore, it was suggested that they were resultant from activin and Act-R complex. OVCAR3 was exposed to the stress (42C, 1 hour heat shock), the protein level of ActR IIa increased and ActR IIb decreased from about 3 h to 24 h after the exposure to the heat stress (HS). On the other hand, the complex between activin and ActR IIa and IIb increased from 3 h after the exposure to HS. To investigate the effect of activin and follistatin on OVCAR3 proliferation after the exposure to HS, we counted the cell number at 96 h after the treatment with activin or follistatin in the condition either with or without HS. Proliferation of the cell in the presence of HS was stimulated by rh-Activin and inhibited by rh-Follistatin. These data suggest that activin might have the function to survive and to proliferate OVCAR3, due to, at least in part the increase in its binding capacity to ActRs through either autocrine or paracrine manner.

Activin Receptors↗
PubMed1996

Activin inhibits basal and androgen-stimulated proliferation and induces apoptosis in the human prostatic cancer cell line, LNCaP.

LNCaP cells, derived from an androgen-sensitive cell line widely employed as an in vitro model of human prostate cancer, have been shown to express activin receptors. Activin is a local regulator of cellular growth, appears to play a key role in mesoderm induction and differentiation during development, and has been implicated in gonadal tumorigenesis. Follistatin, a monomeric glycoprotein that specifically binds and neutralizes activin, is often coexpressed with activin and, thus, modulates the autocrine/paracrine biological activity of this potent growth factor. We tested the hypothesis that LNCaP growth is modulated by the activin/follistatin system. Recombinant human activin A inhibited [3H]thymidine incorporation in a dose-dependent fashion with an ED50 of approximately 0.43 +/- 0.3 nM. Activin (0.1-3 nM) also inhibited dihydrotestosterone (DHT)-stimulated [3H]thymidine incorporation in LNCaP cells. Similarly, recombinant human inhibin A inhibited LNCaP proliferation, but was only 1/100th as potent as activin. Furthermore, activin (3 nM) induced a 3-fold increase in the extent of labeling of low mol wt DNA fragments typical of apoptosis. Activin-induced apoptosis was also indicated by an increase in the number of cells with reduced DNA content, as measured by flow cytometry of activin-treated cells. Both activin-mediated inhibition of cell proliferation and induction of apoptosis could be completely blocked by recombinant human follistatin. Based upon these results using an in vitro model, we speculate that activin functions locally to oppose androgen-driven cell proliferation and, thus, is a key factor controlling prostate growth. Reduced activin biosynthesis, increased follistatin secretion, or signaling defects in the activin receptor system should be further investigated in future studies as potential mechanisms underlying enhanced androgen-independent growth of human prostate cancer cells.

Activin Receptors↗
PubMed2003

Both SMAD2 and SMAD3 mediate activin-stimulated expression of the follicle-stimulating hormone beta subunit in mouse gonadotrope cells.

The activins are pleiotropic members of the TGFbeta superfamily. Within the anterior pituitary gland, activins stimulate FSH synthesis in an autocrine/paracrine fashion by stimulating transcription of the FSHbeta subunit gene. Here, the mechanisms mediating this effect were investigated in the murine gonadotrope cell line, LbetaT2. Recombinant activin A and activin B dose- and time-dependently stimulated endogenous FSHbeta mRNA expression. FSHbeta primary transcript and mRNA levels were increased within 30-60 min, but these effects were blocked by preincubation with the transcription inhibitor actinomycin-D, suggesting that the FSHbeta gene is a direct target of the activin signal transduction cascade. In other systems, activin signals are transduced through a heteromeric serine/threonine receptor complex, which includes the signaling activin type IB receptor [activin receptor-like kinase 4 (ALK4)]. Transfection of a constitutively active form of the receptor, ALK4T206D, stimulated FSHbeta mRNA levels. Overexpression of the inhibitory SMAD7 blocked this effect, as well as activin-stimulated FSHbeta transcription. Because SMAD7 functions by preventing access of SMAD2 and SMAD3 to ALK4, these data suggested that both activins and ALK4 require SMAD2 and/or SMAD3 to affect FSHbeta transcription. Consistent with this idea, activin A stimulated SMAD2 and SMAD3 phosphorylation and nuclear translocation within 5-10 min in LbetaT2 cells. Transient transfection of SMAD3, but not SMADs 1, 2, 4, 5, or 8, stimulated endogenous FSHbeta mRNA levels. The results of SMAD2 transfection studies were inconclusive, however, because of a persistent failure to overexpress the full-length SMAD2 protein specifically in LbetaT2 cells. To assess more directly roles for both SMAD2 and SMAD3 in activin-stimulated FSHbeta expression, RNA interference was used to decrease endogenous SMAD protein levels in LbetaT2 cells. Activin A- and ALK4T206D-stimulated transcription of the FSHbeta gene were significantly attenuated by the depletion of either SMAD2 or SMAD3. Collectively, these data suggest that activins use both SMAD2- and SMAD3-dependent mechanisms to stimulate FSHbeta transcription in mouse gonadotrope cells.

Activin Receptors, Type I↗
PubMed2000

Activin A stimulates type IV collagenase (matrix metalloproteinase-2) production in mouse peritoneal macrophages.

The role of activin, a dimer of inhibin beta subunit, in mouse peritoneal macrophages was evaluated. Activin activity in the cultured macrophages was augmented in response to activation by LPS. In Western blot analysis, immunoreactive activin A was detected in the culture medium only when the macrophages were stimulated by LPS. Although mRNA expression of betaA subunit was detected, that of alpha and betaB subunit was not found in macrophages by reverse RT-PCR. The activin betaA mRNA level was increased in macrophages by LPS, suggesting that the activin production augmented by LPS is regulated at the mRNA level of the betaA gene. The mRNAs of four activin receptors (ActRI, ActRIB, ActRII, and ActRIIB) were also detected in the peritoneal macrophages, and the mRNA levels, except for ActRIB, were decreased during the LPS treatment. Exogenous activin A stimulated the mRNA expression and gelatinolytic activity of matrix metalloproteinase-2 (MMP-2) in macrophages in both the presence and the absence of LPS. In contrast, activin did not affect the production of MMP-9 in macrophages. These results suggested that 1) mouse peritoneal macrophages produced activin A; 2) expression of activin A was enhanced with activation of the macrophages; 3) the macrophages also expressed activin receptors; and 4) exogenous activin A stimulated MMP-2 expression and activity, implicating activin A as an positive regulator of MMP-2 expression. Considering that MMP-2 constitutes the rate-limiting proteinase governing the degradation of basement membrane collagens, activin A may be involved in migration and infiltration of macrophages through the basement membrane in an inflammatory state.

Activin Receptors↗
PubMed1993

Expression of activin transcripts in follicle cells and oocytes of Xenopus laevis.

Activin is a potent inducer of axial mesoderm in vitro and may have a similar role in vivo. Xenopus laevis eggs contain significant amounts of activin or activin-like factors, but maternal activin transcripts have not been detected in Xenopus eggs. The maternal activin protein might be translated from activin beta A or beta B mRNAs that are transiently expressed during oogenesis, or activin polypeptides might be transferred from follicle cells to oocytes. To assess these possibilities we studied activin mRNAs in follicle cells and oocytes by reverse transcriptase-polymerase chain reaction (RT-PCR) and RNA blotting. Activin beta A, beta B1, and beta B2 transcripts occur in follicle cells; among them, beta A mRNA is by far the most abundant. Activin beta A and beta B1 mRNAs were not detected by RT-PCR in the corresponding stage IV oocytes, but activin beta B2 transcripts were found at low levels. These observations are consistent with synthesis of activin beta A and possibly beta B polypeptides in follicle cells followed by their secretion and uptake by oocytes, although synthesis of activin beta B2 in the oocytes could make a contribution to the maternal activin pool.

Activins↗
PubMed2002

Decidual activin: its role in the apoptotic process and its regulation by prolactin.

Successful pregnancy requires profound differentiation and reorganization of the uterine tissues including, as pregnancy progresses, extensive apoptosis of decidual tissue to accommodate the developing conceptus. We have previously shown a positive correlation between expression of activin A and apoptosis in the decidua and have also shown that expression of activin A occurs at the time when prolactin (PRL) receptors disappear from decidual cells. The goals of this study were to examine whether activin A plays a role in decidual apoptosis and whether expression of activin A in the decidua is regulated by PRL and placental lactogens. Studies were carried out using primary rat decidual cells, a decidual cell line (GG-AD), and PRL null mice. Treatment of decidual cells with activin A significantly increased DNA degradation, caspase 3 activity, and caspase 3 mRNA expression. However, this effect was observed only in the absence of endogenous activin production by these cells. Addition of follistatin to decidual cells that were producing activin A decreased both caspase 3 activity and mRNA expression. Similarly, addition of activin-blocking antibodies to cultures of GG-AD cells, which also produce activin A, caused a reduction in both DNA degradation and caspase 3 activity. PRL and placental lactogens caused an inhibition of activin A mRNA expression in primary decidual cells. Even more convincingly, decidua of PRL null mice expressed abundant activin A at a time when no expression of this hormone is detected in wild-type mice and treatment of PRL null mice with PRL caused a profound inhibition of activin A mRNA expression. In summary, our investigations into the role and regulation of decidual activin have revealed that activin A can induce cell death in the decidua and that its expression is under tight regulation by PRL and placental lactogens.

Activins↗
PubMed2004

Developmental profiles of activin betaA, betaB, and follistatin expression in the zebrafish ovary: evidence for their differential roles during sexual maturation and ovulatory cycle.

Our recent experiments showed that gonadotropin(s) stimulated activin betaA and follistatin expression through the cAMP-PKA pathway but suppressed betaB via a cAMP-dependent but PKA-independent pathway in cultured zebrafish follicle cells. Given that pituitary gonadotropins are the major hormones controlling the development and function of the ovary, the differential expression of activin betaA and betaB as well as follistatin in response to gonadotropin(s) raises an interesting question about the temporal expression patterns of these molecules in vivo during sexual maturation and ovulatory cycle. Three experiments were performed in the present study. In the first experiment using sexually immature zebrafish, we followed the expression of activin betaA, betaB, and follistatin at the whole ovary level during a 10-day period in which the ovary developed from the primary growth stage to the one with nearly full-grown follicles. Activin betaA expression was very low at the primary growth stage but significantly increased with the growth of the ovary, and its rise was accompanied by an increase in follistatin expression. In contrast, the expression of activin betaB could be easily detected in the ovary of all stages; however, it did not exhibit an obvious trend of variation during the development. The second experiment examined the stage-dependent expression of activin betaA, betaB, and follistatin at the follicle level in the adult mature zebrafish. The expression of activin betaA was again low in the follicles during the primary growth stage, but exhibited a phenomenal increase after the follicles entered vitellogenesis with the peak level reached at midvitellogenic stage; in contrast, activin betaB mRNA could be easily detected at all stages with a slight increase during follicle growth. The expression of follistatin, on the other hand, also increased significantly during vitellogenesis; however, its level dropped sharply after reaching the peak at the midvitellogenic stage. In the third experiment, we investigated the dynamic changes of the ovarian activin betaA, betaB, and follistatin expression during the daily ovulatory cycle. The expression of activin betaA and follistatin gradually increased from 1800 h onward and reached the peak level around 0400 h when the germinal vesicles had migrated to the periphery in the full-grown oocytes. In contrast, activin betaB expression steadily declined, although not statistically significant, during the same period, but increased sharply at 0700 h when mature oocytes started to appear in most of the ovaries collected. In conclusion, activin betaA and betaB exhibit distinct expression patterns during the development of the ovary and the daily ovarian cycle of the zebrafish. It seems that activin betaA is involved in promoting ovary and follicle growth, whereas activin betaB may have a tonic role throughout follicle development but becomes critical at the late stage of oocyte maturation and/or ovulation.

Activins↗
PubMed2001

Activin stimulates proliferation of rat ovarian thecal-interstitial cells.

There is growing evidence that the function of ovarian theca-interstitial (T-I) cells may be modulated by paracrine actions of activin, inhibin, and follistatin. Furthermore, either dysregulation, dysfunction, or both, of these peptides may play a role in conditions associated with T-I hyperplasia, such as polycystic ovary syndrome (PCOS) and hyperthecosis. This study was designed to evaluate the role of activin, inhibin, and follistatin in the modulation of T-I cell proliferation. Interaction of these peptides with insulin-like growth factor-I (IGF-I), a known stimulator of T-I cell proliferation, was also assessed. Purified rat T-I cells were cultured for 48 h in chemically defined media and with or without activin (3-30 ng/ml), inhibin (3-30 ng/ml), follistatin (100 ng/ml), and/or IGF-I (10 nM). T-I cell proliferation was assessed using radiolabeled thymidine incorporation assay. Activin alone stimulated proliferation of T-I cells in a dose-dependent fashion (by up to 320% above control; P < 0.001), whereas inhibin alone or follistatin alone had no significant effect. Inhibin had also no effect on activin-induced proliferation. Follistatin significantly reduced the stimulatory effects of activin and decreased proliferation by up to 46% (P < 0.01) below the level attained in the presence of activin alone. IGF-I (10 nM), at a dose producing a near-maximal effect, increased proliferation by 175% above control (P < 0.001); insulin (10 nM) increased proliferation by 52% above control (P < 0.03). A combination of IGF-I (10 nM) and activin (30 ng/ml) resulted in a 1090% increase of proliferation above control (P < 0.001); this stimulatory effect was significantly greater than that achieved in the presence of either activin alone or IGF-I alone (P < 0.001). Similarly, a combination of insulin (10 nM) and activin (30 ng/ml) increased proliferation by 506% above control levels. Flow cytometry evaluation revealed that activin increased the proportion of actively dividing cells (in S or G2/M phase of the cell cycle) by 42% (P < 0.02), whereas IGF-I had no effect on the proportion of actively dividing cells. The present findings indicate that an activin-follistatin system may be involved in the regulation of the size of ovarian thecal-stromal compartment. In view of the synergy between activin and IGF-I, and the difference in the effects on the cell cycle distribution, stimulation of T-I proliferation by these agents is likely to be mediated via separate transduction pathways. Excess activin or insufficient follistatin may contribute to T-I hyperplasia.

Activins↗
PubMed1993

Follistatin modulates activin activity in a cell- and tissue-specific manner.

The high affinity activin-binding protein, follistatin, has recently been shown to block activin-stimulated activities in several in vitro systems. In the present study we sought to extend these observations and investigate the effects of follistatin on the activity of activin in stimulating the re-aggregation of Sertoli cell monolayers and proliferation of testicular germ cells, as measured by incorporation of [3H]-thymidine in vitro. Germ-Sertoli cell cocultures prepared from 21 day old rats were treated with media alone or media containing recombinant human (rh) activin A or rh activin B with or without follistatin, the low affinity activin-binding protein, alpha 2 macroglobulin, or a monoclonal antibody (mAB) known to block activin B activity. Follistatin blocked the ability of activin A to stimulate reaggregation of Sertoli cell monolayers when present at a 2-fold ratio (wt/wt) to activin. However, in these same cultures, follistatin had no effect on the ability of activin A to stimulate [3H]-thymidine incorporation. In activin B-treated cultures, both responses could be blocked by the addition of a neutralizing mAB directed against activin B. These results suggest that follistatin can modulate activin action in a cell-type specific fashion, and that this protein may play an important role in regulating the bioavailability of activin.

Activins↗
PubMed1993

Effect of an activin A on follicle-stimulating hormone (FSH) receptor messenger ribonucleic acid levels and FSH receptor expressions in cultured rat granulosa cells.

Activin, a dimer of beta-subunits of inhibin, has been found to induce FSH receptor on cultured rat granulosa cells. The effect of activin on FSH receptor messenger RNA (mRNA) levels has not been elucidated. To study the effect of activin on FSH receptor mRNA levels, we used a specific complementary RNA probe to evaluate changes in FSH receptor transcripts in cultured rat granulosa cells. Granulosa cells obtained from immature diethylstilbestrol-treated rats contained two predominant FSH receptor mRNA transcripts (5.5 and 2.4 kilobases). Compared to the control, the treatment of granulosa cells with activin (100 ng/ml) increased FSH receptor mRNA in a time-dependent manner with a maximum of about a 4-fold increase at 24 h. FSH receptor mRNA markedly decreased after 48 h and maintained a level comparable to that found in the control. The FSH receptor expression was also increased by activin. Scatchard analysis of the binding of rat FSH to granulosa cells showed that the increase in FSH binding after activin treatment was due to an increase in the receptor number and not the affinity of binding. Treatment of granulosa cells for 24 h with activin (20-300 ng/ml) increased FSH receptor mRNA in a dose-dependent manner to a maximum of about a 4-fold increase at a concentration of 100-300 ng/ml. We analyzed rat type II activin receptor mRNA transcripts in cultured rat granulosa cells with a specific complementary RNA probe to study the action of activin on granulosa cells. Granulosa cells contained two predominant rat type II activin receptor mRNA transcripts (6.0 and 3.0 kilobases). Furthermore, we measured intracellular cAMP production by activin to examine the mechanism by which activin acts on granulosa cells. In result, activin alone did not increase intracellular cAMP accumulation. In conclusion, this study demonstrates that the effect of activin A on the induction of FSH receptor expression is associated with a change in FSH receptor mRNA levels, suggesting that modulation of follicle development occurs.

Activins↗
PubMed1993

In situ ligand binding of recombinant human [125I] activin-A and recombinant human [125I]inhibin-A to the adult rat ovary.

Inhibin and activin are hormones produced by ovarian follicles. Specific ovarian cells that bind iodinated recombinant human (rh)-activin-A and rh-inhibin-A were identified by in situ ligand binding. Iodinated rh-molecules were incubated with tissue sections from ovaries, uterus, and oviduct, collected on the mornings of metestrus, diestrus, proestrus, and estrus and the evening of proestrus. Additionally, inhibin/activin subunit mRNA and follistatin mRNA accumulation was examined by in situ hybridization of radiolabeled antisense riboprobes. The cellular site of activin/inhibin binding could thus be colocalized to the cellular site of ligand mRNA and binding protein mRNA production. The association of both [125I]rh-activin-A and [125I] rh-inhibin-A with specific cell types varied across the rat estrous cycle. Nonspecific binding was evaluated by competition with a 1000-fold excess of homologous ligand, and low affinity association of the heterologous ligand was evaluated by competition with a 1000-fold excess of heterologous ligand. [125I]rh-activin-A binding was more widespread than was [125I]rh-inhibin-A binding under our experimental conditions. [125I]rh-Activin-A bound to the granulosa cells of all stages of follicles: unrecruited, growing, and Graafian follicles. Thecal cell binding was found in developing follicles (350-500 microns). The granulosa cells of stimulated follicles (evening of proesterus) bound less [125I]rh-activin-A than those of unstimulated follicles. [125I]rh-Activin-A binding was also associated with antral fluid of follicles in each size class. Although early atretic follicles retained some [125I]rh-activin-A binding, late atretic follicles did not bind [125I]rh-activin-A. Corpus luteum present on metestrus and diestrus bound [125I]rh-activin-A; however, corpus lutea present on proestrus and estrus bound little or no [125I]rh-activin-A. [125I]rh-Activin-A-binding sites were also present in the uterus and oviduct in a cycle-dependent manner. The highest levels of binding were found in the muscle wall of the uterus and the epithelial lining of the thick-walled portion of the uterus on metestrus and diestrus. In addition, [125I]rh-activin-A bound to the cumulus-oocyte complex present in the oviduct on metestrus, but did not bind to the oocytes present in developing follicles. Binding of [125I]rh-inhibin-A was restricted to the antral granulosa cells of 450- to 500-microns follicles. No other ovarian, uterine, or oviduct cells bound [125I]rh-inhibin-A. [125I]rh-Activin-A and [125I]rh-inhibin-A ligand binding was associated primarily with follicles coexpressing inhibin/activin subunit and follistatin mRNA.(ABSTRACT TRUNCATED AT 400 WORDS)

Activins↗
PubMed1994

Effect of activin on luteinizing hormone-human chorionic gonadotropin receptor messenger ribonucleic acid in granulosa cells.

Activin (the dimer of inhibin beta-subunit) is involved in the modulation of granulosa cell function. Recent reports have indicated that activin had an effect on LH/human CG (hCG) receptor induction and steroidogenesis in granulosa cells. To characterize the regulation inducing LH/hCG receptor by activin, we investigated messenger RNA (mRNA) levels, the expression of the LH/hCG receptor, and intracellular cAMP accumulation in cultured rat granulosa cells. Northern blot analysis showed an increase in the LH/hCG receptor mRNA level with FSH (30 ng/ml) and activin (100 ng/ml) cotreatment, whereas activin alone could not augment LH/hCG receptor mRNA at all. After the addition of actinomycin D to the culture medium, LH/hCG receptor mRNA was more stable in the presence of FSH plus activin than in the presence of FSH alone. Similarly, a receptor binding assay revealed that the cotreatment with FSH and activin induced more LH/hCG receptor than FSH alone 96 h after exposure to hormone, but that activin (100 ng/ml) alone could not induce the LH/hCG receptor. Since the primary, if not the sole, second messenger mediating the action of FSH in granulosa cells has been shown to be cAMP, intracellular cAMP accumulation was measured in granulosa cells in the presence of FSH (30 ng/ml) and/or activin (100 ng/ml). Although FSH-stimulated cAMP accumulation reached a peak 15 min after incubation, activin did not significantly alter cAMP accumulation in either control nor FSH-stimulated granulosa cells, indicating that the effects of activin on the LH/hCG receptor in granulosa cells are not mediated by the increase in cAMP. These results demonstrate that activin enhances the FSH-induced LH/hCG receptor mRNA, LH/hCG receptor mRNA stability, and LH/hCG binding sites not due to the stimulation of the adenylate cyclase system. Although the signal pathway from the activin receptor has not been elucidated upon yet, activin is capable of increasing LH/hCG receptor levels through the accumulation of LH/hCG receptor mRNA levels.

Activins↗
PubMed1999

Uterine milk protein, a novel activin-binding protein, is present in ovine allantoic fluid.

Activins are pluripotent growth factors that have recently been shown to be present in placental and fetal membrane preparations. Our previous studies have identified and purified activin A from ovine amniotic and allantoic fluids. In this study, ligand blots of side fractions from the isolation of activin A from allantoic fluid suggested the presence of activin-binding proteins other than follistatin. Further purification of one of these fractions involved two sequential reverse phase HPLC steps and a Superose 12HR fractionation. SDS-PAGE revealed a single protein band of 55 kDa, which was identified by NH2-terminal sequencing as ovine uterine milk protein (UTMP), a member of the serine protease inhibitor (serpin) superfamily of proteins. Further binding studies, using ligand blot techniques and Superose 12HR fractionation in the presence of [125I]activin, demonstrated UTMP to be an activin-binding protein with a lower affinity for activin than that of follistatin. A study of the specific binding behavior of UTMP to activin, using surface plasmon resonance, revealed an apparent equilibrium dissociation constant (Kd) of 49 +/- 25 nM, compared with the follistatin-activin Kd of 379 +/- 51 pM. Similar to another activin-binding protein, alpha2-macroglobulin, UTMP was unable to neutralize the bioactivity of activin in a bioassay based on the capacity of activin to inhibit the proliferation of an MPC-11 plasmacytoma cell line. The high concentrations of this protein in uterine fluid during pregnancy and its ability to bind activin suggest that UTMP may act as a low affinity, high capacity binding protein to sequester activin in the local uterine environment.

Activins↗
PubMed2006

Activin A functions as a Th2 cytokine in the promotion of the alternative activation of macrophages.

Activin A, a member of the TGF-beta superfamily, is a pluripotent growth and differentiation factor. In this study, we report that murine Th cells produce activin A upon activation. Activin activity in the cultured CD4+ T cells was induced by anti-CD3 cross-linking. Activin betaA mRNA level was increased in response to activation, indicating that activin production in CD4+ T cells is regulated at the mRNA level. Activin production was detected exclusively in CD4+CD25- T cells, but not in CD4+CD25+ regulatory T cells. When CD4+ T cells were differentiated into Th cell subsets, higher activin secretion was detected when cultured under Th2-skewing conditions. The mRNA level of activin betaA was abundant in Th2, but not in Th1 cells. Furthermore, secretion of activin was significantly higher in activated Th2 clones than in Th1 clones. The activin betaA-proximal promoter contains a binding site for c-Maf, a Th2-specific transcriptional factor, at close proximity with an NF-AT binding site. c-Maf was able to synergize with NF-AT to transactivate activin betaA gene, and both factors are implicated in activin betaA transcription in Th2 cells. Activin A induced macrophages to express arginase-1 (M-2 phenotype), whereas it inhibited inducible NO synthase expression (M-1 phenotype) induced by IFN-gamma. Taken together, these observations suggest that activin A is a novel Th2 cytokine that promotes differentiation of macrophages toward the M-2 phenotype.

Activins↗
PubMed2003

Immunohistochemical localization of inhibin and activin-like proteins in the brain, pituitary gland, and the ovary of thin-lipped grey mullet, Liza ramada (Risso).

Inhibin and activin, dimeric protein hormones originally isolated from mammalian gonads, are involved in the regulation of vertebrate reproduction. In the present study, we investigated the localization of inhibin and activin subunits (alpha, beta(A), and beta(B)) immunoreactivity in the brain, pituitary, and ovary of female Liza ramada using immunohistochemistry. We demonstrate that activin beta(A) subunit immunoreactivity is found in most of the brain regions: telencephalon (Tel), midbrain tegmentum (MT), and cerebellum (C). In the pituitary gland, activin beta(A) subunit immunoreactivity is observed in nerve fibers that are in close contact with somatotrophs and gonadotrophs. Strong immunostaining of activin beta(B) subunit is detected in melanotropin (MSH)-producing cells in pars intermedia (PI), which are in close contact with gonadotrophs. In the ovary, the cytoplasm as well as follicle cells of previtellogenic oocytes exhibited strong immunoreactivity of activin beta(A). However, the activin beta(A) and beta(B) immunoreactivity is decreased when the oocytes entered the vitellogenic stage (recrudescence). The progress in the accumulation of yolk within oocytoplasm is accompanied by a decrease in the activin beta(A) and beta(B) immunoreactivity and eventually the immunoreactivity disappears completely when the oocytes become fully mature. Interestingly, the vitellogenic oocytes of female L. ramada obtained from fresh water (captivity) contain only weak immunoreactivity of activin beta(A) compared to that of saline water (natural habitat). Moreover, immunoreactivity of activin beta(B) is observed in the phagocytic granulosa cells during resorption of oocytes (atresia) within the ovary of female reared in captivity. The immunostaining of inhibin alpha in the ovary is similar to that of activin beta(B) but the reaction intensity is weaker. However, the mature oocytes show a positive reaction for inhibin alpha. These results provide evidence for the presence of inhibin and activin-like molecules within L. ramada and reveal the distribution of these molecules in L. ramada brain-hypophysial-gonadal system. Our observations add some new informations to our current understanding of activin system in fish and suggest that inhibin and activin may play an important role for L. ramada reproduction in the event of oocyte growth and maturation.

Animals↗
PubMed2003

Involvement of cyclic adenosine 3',5'-monophosphate in the differential regulation of activin betaA and betaB expression by gonadotropin in the zebrafish ovarian follicle cells.

Activin is a dimeric protein consisting of two similar but distinct beta-subunits, betaA and betaB. In our previous studies, both activin A (betaAbetaA) and activin B (betaBbetaB) have been demonstrated to stimulate oocyte maturation and promote oocyte maturational competence in the zebrafish. Follistatin, a specific activin-binding protein, can block both activin- and gonadotropin-induced final oocyte maturation in vitro, suggesting that activin is likely a downstream mediator of gonadotropin actions in the zebrafish ovary. In the present study, a full-length cDNA encoding zebrafish ovarian activin betaA was cloned and sequenced. The precursor of zebrafish activin betaA consists of 395 amino acids and its mature region exhibits about 78% homology with that of mammals. Using an in vitro primary culture of the ovarian follicle cells and semiquantitative RT-PCR assays, we examined the regulation of activin betaA and betaB expression by human chorionic gonadotropin (hCG) and its intracellular signal transduction mechanisms. hCG (15 IU/ml) increased the mRNA level of activin betaA-subunit; however, it significantly down-regulated the steady-state expression level of activin betaB in a time- and dose-dependent manner. The differential regulation of the two beta-subunits by hCG could be mimicked by 3-isobutyl-1-methylxanthine, forskolin, and dibutyryl-cAMP, suggesting involvement of the intracellular cAMP pathway. Interestingly, H89 (a specific inhibitor of protein kinase A, PKA) could effectively block hCG- and forskolin-stimulated activin betaA expression at 10 micro M, but it was unable to reverse the inhibitory effects of hCG and forskolin on betaB expression. This suggests that the hCG-stimulated activin betaA expression is dependent on the activation of the cAMP-PKA pathway, whereas the inhibitory effect of hCG on activin betaB expression is likely mediated by PKA-independent pathway(s).

1-Methyl-3-isobutylxanthine↗