TGFB1/INHBA Homodimer/Nodal-SMAD2/3 Signaling Network: A Pivotal Molecular Target in PDAC Treatment.

Pancreatic cancer remains a grueling disease that is projected to become the second-deadliest cancer in the next decade. Standard treatment of pancreatic cancer is chemotherapy, which mainly targets the differentiated population of tumor cells; however, it paradoxically sets the roots of tumor relapse by the selective enrichment of intrinsically chemoresistant pancreatic cancer stem cells that are equipped with an indefinite capacity for self-renewal and differentiation, resulting in tumor regeneration and an overall anemic response to chemotherapy. Crosstalk between pancreatic tumor cells and the surrounding stromal microenvironment is also involved in the development of chemoresistance by creating a supportive niche, which enhances the stemness features and tumorigenicity of pancreatic cancer cells. In addition, the desmoplastic nature of the tumor-associated stroma acts as a physical barrier, which limits the intratumoral delivery of chemotherapeutics. In this review, we mainly focus on the transforming growth factor beta 1 (TGFB1)/inhibin subunit beta A (INHBA) homodimer/Nodal-SMAD2/3 signaling network in pancreatic cancer as a pivotal central node that regulates multiple key mechanisms involved in the development of chemoresistance, including enhancement of the stem cell-like properties and tumorigenicity of pancreatic cancer cells, mediating cooperative interactions between pancreatic cancer cells and the surrounding stroma, as well as regulating the deposition of extracellular matrix proteins within the tumor microenvironment.

Inhibin, beta A regulates the transforming growth factor-beta pathway to promote malignant biological behaviour in colorectal cancer.

Inhibin, beta A (INHBA) is a member of the transforming growth factor (TGF-ß) family. The carcinogenic mechanisms of INHBA during the development of colorectal cancer (CRC) remain unclear. In the present study, we further elucidated the role of INHBA in CRC. We analysed the expression of INHBA in CRC and its relationship with patient prognosis using data from public databases. INHBA expression was evaluated in CRC tissues and cell lines using immunohistochemistry and western blotting. After inhibiting the expression of INHBA, the effect of INHBA on the function of CRC cells was evaluated in vitro. We found that INHBA was upregulated in CRC. High INHBA expression is closely related to poor prognosis in patients with CRC. Knockdown of INHBA in vitro can inhibit the proliferation, migration, and invasion of CRC cells. In terms of mechanism, we found that high INHBA expression activates the TGF-ß pathway. SIGNIFICANCE OF THE STUDY: INHBA acts as an oncogene in the progression of CRC and may, therefore, be a potential therapeutic target for CRC.

Potential treatment of keloid pathogenesis with follistatin 288 by blocking the activin molecular pathway.

Keloids are benign tumours caused by abnormal wound healing driven by increased expression of cytokines, including activin A. This study compared effects of activins on normal and keloid-derived human dermal fibroblasts and investigated a novel treatment for keloids using follistatin. Normal skin and keloid tissue samples from 11 patients were used to develop primary fibroblast cultures, which were compared in terms of their histology and relevant gene (qRT-PCR and RNAseq) and protein (ELISA) expression. Activin A (INHBA) and connective tissue growth factor (CTGF) gene expression were significantly upregulated in keloid fibroblasts, as was activin A protein expression in cell lysates and culture medium. Activator protein 1 inhibitor (SR11302) significantly decreased INHBA and CTGF expression in keloid fibroblasts and a single treatment of follistatin over 5 days significantly inhibited activin and various matrix-related genes in keloid fibroblasts when compared to controls. Follistatin, by binding activin A, suppressed CTGF expression suggesting a novel therapeutic role in managing keloids and perhaps other fibrotic diseases.

Targeting INHBA in Ovarian Cancer Cells Suppresses Cancer Xenograft Growth by Attenuating Stromal Fibroblast Activation.

INHBA-encoded inhibin ß A is a member of the transforming growth factor-ß (TGF-ß) superfamily. INHBA has been reported to be implicated in the progression of multiple types of cancer including ovarian cancer (OC). However, the mechanisms by which INHBA affects OC progression are not well-characterized. The aim of our study was to explore the prognostic value of INHBA for different stages and grades of OC and to identify the possible mechanisms by which INHBA promotes OC progression. Our results demonstrated that INHBA was specifically expressed in OC epithelium, and higher expression was associated with higher risk of mortality in patients with advanced and higher-grade serous OC (SOC). In addition, knockdown of INHBA in cancer cells impaired cancer xenograft growth through reducing OC stromal fibroblast activation in vivo. Further results confirmed that Smad2 signaling pathway was involved in INHBA-induced stromal fibroblast activation, and inhibiting this pathway could effectively reverse activation of stromal fibroblasts. In summary, our results showed that blocking INHBA in cancer cells may be a potential therapeutic strategy to inhibit SOC progression.

Mechanisms involved in follistatin-induced hypertrophy and increased insulin action in skeletal muscle.

BACKGROUND: Skeletal muscle wasting is often associated with insulin resistance. A major regulator of muscle mass is the transforming growth factor ß (TGF-ß) superfamily, including activin A, which causes atrophy. TGF-ß superfamily ligands also negatively regulate insulin-sensitive proteins, but whether this pathway contributes to insulin action remains to be determined. METHODS: To elucidate if TGF-ß superfamily ligands regulate insulin action, we used an adeno-associated virus gene editing approach to overexpress an activin A inhibitor, follistatin (Fst288), in mouse muscle of lean and diet-induced obese mice. We determined basal and insulin-stimulated 2-deoxy-glucose uptake using isotopic tracers in vivo. Furthermore, to evaluate whether circulating Fst and activin A concentrations are associated with obesity, insulin resistance, and weight loss in humans, we analysed serum from morbidly obese subjects before, 1 week, and 1 year after Roux-en-Y gastric bypass (RYGB). RESULTS: Fst288 muscle overexpression markedly increased in vivo insulin-stimulated (but not basal) glucose uptake (+75%, P < 0.05) and increased protein expression and intracellular insulin signalling of AKT, TBC1D4, PAK1, pyruvate dehydrogenase-E1α, and p70S6K, while decreasing TBC1D1 signaling (P < 0.05). Fst288 increased both basal and insulin-stimulated protein synthesis, but no correlation was observed between the Fst288-driven hypertrophy and the increase in insulin-stimulated glucose uptake. Importantly, Fst288 completely normalized muscle glucose uptake in insulin-resistant diet-induced obese mice. RYGB surgery doubled circulating Fst and reduced activin A (-24%, P < 0.05) concentration 1 week after surgery before any significant weight loss in morbidly obese normoglycemic patients, while major weight loss after 1 year did not further change the concentrations. CONCLUSIONS: We here present evidence that Fst is a potent regulator of insulin action in muscle, and in addition to AKT and p70S6K, we identify TBC1D1, TBC1D4, pyruvate dehydrogenase-E1α, and PAK1 as Fst targets. Circulating Fst more than doubled post-RYGB surgery, a treatment that markedly improved insulin sensitivity, suggesting a role for Fst in regulating glycaemic control. These findings demonstrate the therapeutic potential of inhibiting TGF-ß superfamily ligands to improve insulin action and Fst's relevance to muscle wasting-associated insulin-resistant conditions in mice and humans.

Regional localization of activin-ßA, activin-ßC, follistatin, proliferation, and apoptosis in adult and developing mouse prostate ducts.

Activins and inhibins, members of the TGF-ß superfamily, are growth and differentiation factors involved in the regulation of several biological processes, including reproduction, development, and fertility. Previous studies have shown that the activin-ßA subunit plays a pivotal role in prostate development. Activin-A inhibits branching morphogenesis in the developing prostate, and its expression is associated with increased apoptosis in the adult prostate. Follistatin, a structurally unrelated protein to activins, is an antagonist of activin-A. A balance between endogenous activin-A and follistatin is required to maintain prostatic branching morphogenesis. Deregulation of this balance leads to branching inhibition or excessive branching and increased maturation of the stroma surrounding the differentiating epithelial ducts. Recent work identified another member of the TGF-ß superfamily, the activin-ßC subunit, as a novel antagonist of activin-A. Over-expression of activin-C (ßC-ßC) alters prostate homeostasis, by interfering with the activin-A signaling. The current study characterized the spatiotemporal localization of activin-A, activin-C and follistatin in the adult and developing mouse prostate using immunohistochemical analysis. Results showed activin-C and follistatin are differentially expressed during prostate development and suggested that the antagonistic property of follistatin is secondary to the action of activin-C. In conclusion, the present study provides evidence to support a role of activin-C in prostate development and provides new insights in the spatiotemporal localization of activins and their antagonists during mouse prostate development.

RNAi-mediated knockdown of INHBB increases apoptosis and inhibits steroidogenesis in mouse granulosa cells.

Inhibins are members of the TGFß superfamily and act as suppressors of follicle stimulating hormone (FSH) secretion from pituitary glands via a negative feedback mechanism to regulate folliculogenesis. In this study, the INHBB gene was knocked down by three RNAi-Ready pSIREN-RetroQ-ZsGreen vector- mediated recombinant plasmids to explore the effects of INHBB silencing on granulosa cell (GC) cell cycle, apoptosis and steroid production in vitro. Quantitative real-time polymerase chain reaction, Western blot, flow cytometry and ELISA were performed to evaluate the role of INHBB in the mouse GC cell cycle, apoptosis and steroid production in vitro. The results showed that the relative mRNA and protein expression of INHBB in mouse GCs can be significantly reduced by RNAi with pshRNA-B1, pshRNA-B2 and pshRNA-B3 plasmids, with pshRNA-B3 having the best knockdown efficiency. Downregulation of the expression of INHBB significantly arrests cells in the G1 phase of the cell cycle and increases the apoptosis rate in GCs. This was further confirmed by downregulation of the protein expressions of Cyclin D1, Cyclin E and Bcl2, while the protein expression of Bax was upregulated. In addition, specific downregulation of INHBB markedly decreased the concentration of estradiol and progesterone, which was further validated by the decrease in the mRNA levels of CYP19A1 and CYP11A1. These findings suggest that inhibin ßB is important in the regulation of apoptosis and cell cycle progression in granulosa cells. Furthermore, the inhibin ßB subunit has a role in the regulation of steroid hormone biosynthesis. Evidence is accumulating to support the concept that inhibin ßB is physiologically essential for early folliculogenesis in the mouse.

Myostatin/activin blocking combined with exercise reconditions skeletal muscle expression profile of mdx mice.

Duchenne muscular dystrophy is characterized by muscle wasting and decreased aerobic metabolism. Exercise and blocking of myostatin/activin signaling may independently or combined counteract muscle wasting and dystrophies. The effects of myostatin/activin blocking using soluble activin receptor-Fc (sActRIIB-Fc) administration and wheel running were tested alone or in combination for 7 weeks in dystrophic mdx mice. Expression microarray analysis revealed decreased aerobic metabolism in the gastrocnemius muscle of mdx mice compared to healthy mice. This was not due to reduced home-cage physical activity, and was further downregulated upon sActRIIB-Fc treatment in enlarged muscles. However, exercise activated pathways of aerobic metabolism and counteracted the negative effects of sActRIIB-Fc. Exercise and sActRIIB-Fc synergistically increased expression of major urinary protein, but exercise blocked sActRIIB-Fc induced phosphorylation of STAT5 in gastrocnemius muscle. In conclusion, exercise alone or in combination with myostatin/activin blocking corrects aerobic gene expression profiles of dystrophic muscle toward healthy wild type mice profiles.

Sex specific retinoic acid signaling is required for the initiation of urogenital sinus bud development.

The mammalian urogenital sinus (UGS) develops in a sex specific manner, giving rise to the prostate in the male and the sinus vagina in the embryonic female. Androgens, produced by the embryonic testis, have been shown to be crucial to this process. In this study we show that retinoic acid signaling is required for the initial stages of bud development from the male UGS. Enzymes involved in retinoic acid synthesis are expressed in the UGS mesenchyme in a sex specific manner and addition of ligand to female tissue is able to induce prostate-like bud formation in the absence of androgens, albeit at reduced potency. Functional studies in mouse organ cultures that faithfully reproduce the initiation of prostate development indicate that one of the roles of retinoic acid signaling in the male is to inhibit the expression of Inhba, which encodes the ßA subunit of Activin, in the UGS mesenchyme. Through in vivo genetic analysis and culture studies we show that inhibition of Activin signaling in the female UGS leads to a similar phenotype to that of retinoic acid treatment, namely bud formation in the absence of androgens. Our data also reveals that both androgens and retinoic acid have extra independent roles to that of repressing Activin signaling in the development of the prostate during fetal stages. This study identifies a novel role for retinoic acid as a mesenchymal factor that acts together with androgens to determine the position and initiation of bud development in the male UGS epithelia.

Follistatin, an activin antagonist, ameliorates renal interstitial fibrosis in a rat model of unilateral ureteral obstruction.

Activin, a member of the TGF-ß superfamily, regulates cell growth and differentiation in various cell types. Activin A acts as a negative regulator of renal development as well as tubular regeneration after renal injury. However, it remains unknown whether activin A is involved in renal fibrosis. To clarify this issue, we utilized a rat model of unilateral ureteral obstruction (UUO). The expression of activin A was significantly increased in the UUO kidneys compared to that in contralateral kidneys. Activin A was detected in glomerular mesangial cells and interstitial fibroblasts in normal kidneys. In UUO kidneys, activin A was abundantly expressed by interstitial α-SMA-positive myofibroblasts. Administration of recombinant follistatin, an activin antagonist, reduced the fibrotic area in the UUO kidneys. The number of proliferating cells in the interstitium, but not in the tubules, was significantly lower in the follistatin-treated kidneys. Expression of α-SMA, deposition of type I collagen and fibronectin, and CD68-positive macrophage infiltration were significantly suppressed in the follistatin-treated kidneys. These data suggest that activin A produced by interstitial fibroblasts acts as a potent profibrotic factor during renal fibrosis. Blockade of activin A action may be a novel approach for the prevention of renal fibrosis progression.

WNT4/beta-catenin pathway maintains female germ cell survival by inhibiting activin betaB in the mouse fetal ovary.

Female germ cells are essential for organogenesis of the ovary; without them, ovarian follicles do not form and functional and structural characteristics of the ovary are lost. We and others showed previously that when either Wnt4 or beta-catenin was inactivated in the fetal ovary, female germ cells underwent degeneration. In this study, we set out to understand whether these two factors belong to the same pathway and how they maintain female germ cell survival. We found that activation of beta-catenin in somatic cells in the Wnt4 knockout ovary restored germ cell numbers, placing beta-catenin downstream of WNT4. In the absence of Wnt4 or beta-catenin, female germ cells entered meiosis properly; however, they underwent apoptosis afterwards. Activin betaB (Inhbb), a subunit of activins, was upregulated in the Wnt4 and beta-catenin knockout ovaries, suggesting that Inhbb could be the cause for the loss of female germ cells, which are positive for activin receptors. Indeed, removal of Inhbb in the Wnt4 knockout ovaries prevented female germ cells from undergoing degeneration. We conclude that WNT4 maintains female germ cell survival by inhibiting Inhbb expression via beta-catenin in the somatic cells. Maintenance of female germ cells hinge upon a delicate balance between positive (WNT4 and beta-catenin) and negative (activin betaB) regulators derived from the somatic cells in the fetal ovary.

INHBA overexpression promotes cell proliferation and may be epigenetically regulated in esophageal adenocarcinoma.

INTRODUCTION: The expression, mechanisms of regulation, and functional impact of activin (INHBA) in esophageal adenocarcinoma (EAC) have not been fully defined. METHODS: INHBA expression was examined in 46 esophageal samples (nine Barrett's metaplasia (BM); seven BM/low-grade dysplasia; eight low-grade dysplasia; seven high-grade dysplasia; 15 EAC) using oligonucleotide microarrays and real-time reverse transcription-polymerase chain reaction (RT-PCR) and in 90 tissue samples (79 EAC; 8 dysplastic; 3 BM) using immunohistochemistry (IHC). The proliferation of EAC cell lines FLO and OE-33 was examined after treatment with exogenous activin. The proliferation of OE-33 was also examined after treatment with the activin inhibitor follistatin and INHBA-targeting siRNA. OE-33 and FLO cells were treated with 5-aza-2'deoxycytidine (5-AZA) and trichostatin A to investigate the role of epigenetic regulation in INHBA expression. RESULTS: Primary EACs expressed 5.7-times more INHBA mRNA than BM samples on oligonucleotide microarray. Transcript overexpression in EAC relative to BM was confirmed on real-time RT-PCR. IHC suggested higher INHBA protein expression in EAC (69.6%) than in the dysplastic (37.5%) and BM samples (33.3%). FLO and OE-33 treated with activin demonstrated increased proliferation, and OE-33 cells treated with follistatin and INHBA-targeting siRNA demonstrated reduced proliferation, relative to untreated controls. Treatment of FLO cells with trichostatin A and 5-AZA up-regulated INHBA mRNA and protein production by real time RT-PCR and IHC. CONCLUSIONS: INHBA is overexpressed in EAC relative to dysplastic and BM tissue. INHBA overexpression may promote cell proliferation and may be affected by promoter demethylation and histone acetylation in EAC cell lines.

Identification of tocopherol-associated protein as an activin/TGF-beta-inducible gene in mast cells.

Previous studies have demonstrated that treatment with activin A and TGF-beta(1), members of the TGF-beta family, stimulated maturation of mouse bone marrow-derived cultured mast cells (BMMC), which was characterized by morphology and gene expression of mouse mast cell proteases (mmcps). In order to gain a better understanding of activin A- and TGF-beta(1)-induced maturation in mast cells, we investigated the genes that were up-regulated in response to treatment with these two members of the TGF-beta family. The cDNA microarray analyses indicated that in BMMC, five genes were induced by treatment with 4 nM activin A for 2 h. Tocopherol-associated protein (Tap) was one of the induced genes, and the Tap induction in response to activin A treatment was confirmed by real-time RT-PCR analyses. Treatment with TGF-beta(1) at 200 pM but not BMP-2 at 4 nM also increased Tap gene transcript in BMMC. Activin A-induced Tap expression was detected in BMMC but not in RAW264 macrophage-like cells, B16 melanoma cells or P19 embryonic carcinoma cells. Treatment with >1 muM SB431542, an inhibitor of activin and TGF-beta type I receptors ALK4/5, reduced responsiveness of Tap expression to TGF-beta(1), whereas <0.5 microM SB431542 effectively reduced TGF-beta(1)-induced expression of mmcp-1 and mmcp-7. These results suggest that inhibitory effects of SB431542 are different between TGF-beta-induced genes. Reporter assays indicated that Tap expression enhances transcription mediated by the activin/TGF-beta pathway. Thus, the present results suggest that Tap induction in response to activin/TGF-beta occurs predominantly in mast cells and serves as a positive regulator in activin/TGF-beta signaling.

Structural basis for the inhibition of activin signalling by follistatin.

The secreted, multidomain protein follistatin binds activins with high affinity, inhibiting their receptor interaction. We have dissected follistatin's domain structure and shown that the minimal activin-inhibiting fragment of follistatin is comprised of the first and second Fs domains (Fs12). This protein can bind to activin dimer and form a stable complex containing two Fs12 molecules and one activin dimer. We have solved crystal structures of activin A alone and its complex with Fs12 fragment to 2 A resolution. The complex structure shows how Fs12 molecules wrap around the back of the 'wings' of activin, blocking the type II receptor-binding site on activin A. Arginine 192 in Fs2 is a key residue in this interaction, inserting itself in between activin's fingers. Complex formation imposes a novel orientation for the EGF- and Kazal-like subdomains in the Fs2 domain and activin A shows further variation from the canonical TGF-beta family fold. The structure provides a detailed description of the inhibitory mechanism and gives insights into interactions of follistatin with other TGF-beta family proteins.

Testing the antagonistic effect of follistatin on BMP family members in ovine granulosa cells.

Follistatin was first demonstrated as an activin-binding protein, neutralizing its actions. However, there is emerging evidence that follistatin inhibits the action of other members of the transforming growth factor beta(TGFbeta) / bone morphogenetic protein (BMP) superfamily. Recently, numerous BMP factors have been shown to play important roles in regulating folliculogenesis and ovulation rate in mammals, and such a potential antagonistic role of follistatin is of particular interest in the context of ovarian function. Using a biological test based on progesterone production by ovine primary granulosa cells in culture, we show that follistatin was a strong antagonist of activin A, but not BMP-2 or BMP-4 actions. In contrast, noggin, a known specific BMP antagonist, had no effect on activin A but strongly neutralized BMP-2 and BMP-4 actions. BMP-6 action was only slightly reduced by both follistatin and noggin. Our data led to the conclusion that follistatin would not represent a determinant physiological modulator of the biological effect of BMP factors on granulosa cells.

Attenuation of bleomycin-induced pulmonary fibrosis by follistatin.

RATIONALE: Activins are members of the transforming growth factor-beta superfamily thought to be involved in repair processes after tissue injury. OBJECTIVES: The aim of this study was to clarify whether activin and its antagonist, follistatin, played a significant role in lung injury and fibrosis. METHODS AND RESULTS: In bleomycin (BLM)-treated rat lung, mRNA for the beta(A) subunit of activin was upregulated on Days 3 and 7 and decreased gradually thereafter. Immunoreactive activin A was abundantly expressed in macrophages infiltrated in the lung, and was detected in fibroblasts accumulated in the fibrotic area on Day 28. We then administered follistatin, an activin antagonist, to BLM-treated rats. Follistatin significantly reduced the number of macrophages and neutrophils in bronchoalveolar lavage and reduced the protein content. Histologically, follistatin markedly reduced the number of infiltrating cells, ameliorated the destruction of lung architecture on Day 7, and attenuated lung fibrosis on Day 28. The hydroxyproline content was significantly lower in follistatin-treated rats. In cultured lung fibroblasts, production of activin A was augmented by transforming growth factor-beta, and activin antagonist follistatin significantly inhibited transforming growth factor-beta-induced fibroblast activation. These results suggest that activin A was produced in the lung after BLM treatment and promoted acute inflammation and subsequent fibrosis. CONCLUSIONS: Follistatin is effective in treating acute lung injury and BLM-induced fibrosis by blocking the actions of activin and transforming growth factor-beta.

Intact signaling by transforming growth factor beta is not required for termination of liver regeneration in mice.

Transforming growth factor beta (TGF-beta) is a potent inhibitor of hepatocyte proliferation in vitro and is suggested to be a key negative regulator of liver growth. To directly address the role of TGF-beta signaling in liver regeneration in vivo, the TGF-beta type II receptor gene (Tgfbr2) was selectively deleted in hepatocytes by crossing "floxed" Tgfbr2 conditional knockout mice with transgenic mice expressing Cre under control of the albumin promoter. Hepatocytes isolated from liver-specific Tgfbr2 knockout (R2LivKO) mice were refractory to the growth inhibitory effects of TGF-beta1. The peak of DNA synthesis after 70% partial hepatectomy occurred earlier (36 vs. 48 hours) and was 1.7-fold higher in R2LivKO mice compared with controls. Accelerated S-phase entry by proliferating R2LivKO hepatocytes coincided with the hyperphosphorylation of Rb protein and the early upregulation of cyclin D1 and cyclin E. However, by 120 hours after partial hepatectomy, hepatocyte proliferation was back to baseline in both control and R2LivKO liver. Regenerating R2LivKO liver showed evidence of increased signaling by activin A and persistent activity of the Smad pathway. Blockage of activin A signaling by the specific inhibitor follistatin resulted in increased hepatocyte proliferation at 120 hours, particularly in R2LivKO livers. In conclusion, TGF-beta regulates G(1) to S phase transition of hepatocytes, but intact signaling by TGF-beta is not required for termination of liver regeneration. Increased signaling by activin A may compensate to regulate liver regeneration when signaling through the TGF-beta pathway is abolished, and may be a principal factor in the termination of liver regeneration.

Pituitary follistatin gene expression in female rats: evidence that inhibin regulates transcription.

Follistatin (FS), along with the members of the transforming growth factor beta family activin and inhibin, are important regulators of FSH secretion and messenger RNA production. While activin and inhibin appear to function as tonic modulators of FSH (stimulatory and inhibitory, respectively), dynamic changes in FS are noted through the estrous cycle and under varying physiological experimental paradigms. This suggests that FS is a major contributor to the precisely coordinated secretion of FSH that maintains reproductive function. The aim of this study was to investigate changes in FS, in particular the early (<12 h) rise observed after ovariectomy (OVX), and to determine whether these changes were as a consequence of variations in gene transcription rates. FS primary transcript (PT) and mRNA were found to increase 3-fold 12 h post-OVX, indicating increased gene transcription during this time period. Replacement with estradiol and/or blockade of GnRH had only modest effects on FS PT concentration. Inhibin immunoneutralization of intact rats resulted in a 3-fold increase in FS PT 12 h after administration of inhibin alpha antisera. Significant increases in FS mRNA at both 2 and 12 h also suggested that inhibin also may have effects on message stability. After administration of recombinant human inhibin A, there was a prompt decline in both FS PT and mRNA. These results indicate that inhibin is a major regulator of FS, both by transcriptional and nontranscriptional mechanisms.

The p38 MAPK inhibitor, PD169316, inhibits transforming growth factor beta-induced Smad signaling in human ovarian cancer cells.

Transforming growth factor beta (TGFbeta) can signal through a variety of Smad-independent pathways, including the p38 MAPK pathway. Recent work has shown that inhibitors of p38 MAPK, such as SB203580 and SB202190, can inhibit signaling induced by TGFbeta. Here we show that another p38 MAPK inhibitor, PD169316, abrogates signaling initiated by both TGFbeta and Activin A, but not bone morphogenetic protein (BMP) 4. Inhibition of TGFbeta signaling is dose dependent and results in reduced Smad2 and Smad3 phosphorylation, nuclear translocation, and up-regulation of the TGFbeta target gene Smad7. Reduced TGFbeta signaling is not due to abrogation of p38 MAPK activity, since blocking p38 MAPK activity with a dominant negative form of p38 MAPK has no effect on TGFbeta/Smad signaling. Our results show that use of PD169316 at 5 MICROM or higher can block TGFbeta signaling activity and thus caution must be used when attributing cellular activities exclusively to p38 MAPK signaling when these inhibitors are used experimentally.

Activin-A up-regulates type I activin receptor mRNA levels in human immortalized extravillous trophoblast cells.

Activin is known to play an important regulatory role in reproduction, including pregnancy. To further examine the role and signaling mechanism of activin in regulating placental function, the steady-state level of activin type I receptor (ActRI) mRNA in immortalized extravillous trophoblasts (IEVT) cells was measured using competitive PCR (cPCR). An internal standard of ActRI cDNA for cPCR was constructed for the quantification of ActRI mRNA levels in IEVT cells. ActRI mRNA levels were increased in a dose-dependent manner by activin-A with the maximal effect observed at the dose of 10 ng/ml. Time course studies revealed that activin-A had maximal effects on ActRI mRNA levels at 6 hours after treatment. The effects of activin-A on ActRI mRNA levels was blocked by follistatin, an activin binding protein, in a dose-dependent manner. In addition, inhibin-A inhibited basal, as well as activin-A-induced ActRI mRNA levels. These findings provide evidence, for the first time, that activin-A modulates ActRI mRNA levels in human trophoblast cells.