INHBA Expression Research Articles

MiR‐376c Inhibits Metastasis by Controlling RUNX2‐INHBA Axis in Oral Squamous Cell Carcinoma

Aberrant microRNA expression is a common phenotype in cancer cell progression. MicroRNA is the negative regulator of hundred to thousand gene expressions. Here we report chr14q32.2 miRNA cluster significantly down‐regulated in clinical OSCC samples. 19 miRNAs were found with coordinated silencing in OSCC patients. However, their specific target and regulation mechanism have not clearly identified. Here we showed miR‐376c is the most significant microRNA down‐regulated by RT‐Q‐PCR validation in the miRNA cluster. Ectopic expression of miR‐376c not only inhibited oral cancer cells proliferation, but also migration and invasion. MiR‐376c also inhibited the in vivo tumorigenesis and metastasis. Furthermore, miR‐376c was found directly binding to the 3′‐UTR of oncogenic transcription factor, RUNX2, which was found significantly up‐regulated in OSCC patients. Silencing of RUNX2 inhibited OSCC proliferation and invasion through INHBA expression. Taken together, our study demonstrates the tumor suppressor role of miR‐376c, which regulates RUNX2 transcriptomes in OSCC. Moreover, our results indicate RUNX2‐INHBA axis may play important roles in OSCC progression.

Bone morphogenetic proteins are mediators of luteolysis in the human corpus luteum.

Bone morphogenetic proteins (BMPs), members of the transforming growth factor β (TGFβ) superfamily, play important roles in folliculogenesis in various species; however, little is known about their role in luteal function. In this study, we investigated the expression, regulation, and effects of BMP2, BMP4, and BMP6 in carefully dated human corpora lutea and cultured human luteinized granulosa cells. The mRNA abundance of BMPs was increased in the regressing corpus luteum in vivo (P<.01-.001). Human chorionic gonadotropin (hCG) down-regulated BMP2, BMP4, and BMP6 transcripts both in vivo (P=.05-.001) and in vitro (P<.001), and decreased the mRNA abundance of BMP receptors (BMPR1A, BMPR1B, BMPR2; P<.05-.01) in vitro. Three BMPs were regulated by differential signaling pathways. H89, a protein kinase A inhibitor, increased the expression of both BMP2 (P<.05) and BMP4 (P<.05) while decreasing BMP6 (P<.01). PMA, a protein kinase C activator, decreased both BMP4 and BMP6 expression (P<.0001) while enhancing the mRNA abundance of BMP2 (P<.01). BMPs significantly down-regulated transcripts for LH/choriogonadotropin receptor (LHCGR; P<.001) and steroidogenic acute regulatory protein (STAR; P<.001), whereas up-regulating those of follicular stimulating hormone receptor (FSHR; P<.01) and aromatase (CYP19A1; P<.05-.01) in vitro, possessing an effect opposite to hCG but similar to Activin A. Like Activin A, BMP4 and BMP6 stimulated the expression of Inhibin/Activin subunits with a marked effect on INHBB expression (P<.05-.01). These data confirm that BMPs are increased during luteal regression and negatively regulated by hCG via differential mechanisms, suggesting that BMPs are one of the mediators of luteolysis in women.

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

Inhibins are members of the TGFβ 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 CYP19A1and CYP11A1. These findings suggest that inhibin βB is important in the regulation of apoptosis and cell cycle progression in granulosa cells. Furthermore, the inhibin βB subunit has a role in the regulation of steroid hormone biosynthesis. Evidence is accumulating to support the concept that inhibin βB is physiologically essential for early folliculogenesis in the mouse.

170 GENE SILENCING OF BMPRII IN BOVINE GRANULOSA CELLS

Granulosa cells (GC) are important constituents of the follicular environment for oocyte competence acquisition. However, their functionality depends on oocyte-derived factors, such as GDF9 and BMP15, which act through BMPRII receptor signalling. Gene silencing using lipofection has been used as an important tool to investigate the role of cell genes and proteins. The aim of this study was to establish the ideal conditions for lipofection in bovine GC and starting from this protocol to establish a methodology for silencing of the BMPRII gene by RNA interference, and to use this strategy to study the functions of BMPRII in GDF9 signalling. GC were obtained from slaughterhouse ovaries by aspiration of follicles (2 to 6 mm) and subsequently cultured in DMEM medium at 38.5°C and 5% CO2 in air. All data analyzes were performed by GraphPad Prism® version 5.0 software (GraphPad Software Inc., La Jolla, CA, USA), using one-way ANOVA followed by Tukey's test. For optimizing the conditions for lipofection, the GC were treated with different amounts of lipofection agents Lipofectamine® RNAiMAX (Invitrogen, Carlsbad, CA, USA; 1, 2, and 3 µL) or LipofectamineTM 2000 (Invitrogen; 1, 2, and 3 µL) and the transfection indicators Siglo® (GE Healthcare, Waukesha, WI, USA; 30 to 100 nM) or FUGW transgenic plasmid (100 to 900 nM) during 24 and 48 h. The highest efficiency of lipofection was observed at 24 h of culture with 2 μL of Lipofectamine™ 2000 + 100 nM of Siglo®. Based on these conditions, different concentrations of siBMPRII (100 to 500 pM) were tested during 24 h of culture and subsequently, different incubation times (0, 6, 12, 18, and 24 h) with the best siRNA concentration in order to establish optimum conditions for gene silencing. GC were evaluated for the relative abundance of mRNA for BMPRII using PPIA and β-actin as endogenous controls by real-time PCR. All concentrations provided similar and highly significant transcript reduction in comparison to control (P &lt; 0.001), so the lowest of them was adopted (100 pM). For different incubation times with 100 pM siBMPRII also a similar decrease was also seen, which was more significant at 24 h (P &lt; 0.01). The best concentration (100 pM) and incubation time (24 h) with the siRNA were analysed by Western blotting, which confirmed the BMPRII reduction also at protein level (P &lt; 0.05). For functional evaluation, GC submitted or no to gene silencing were incubated with or without GDF9 (100 ng) and assessed for expression of genes controlled by GDF9 through its BMPRII receptor (LHR, INHA, and INHBA), besides the CYP17 gene, a marker of potential theca cells contamination. LHR expression was reduced in silenced groups (P &lt; 0.05) and highly suppressed by GDF9 in non-silenced groups (P &lt; 0.001), while INHA and INHBA expression remained constant in the different groups (P &gt; 0.05), suggesting that the control of these genes in bovine does not behave as reported in other species. The lack of amplification CYP17 indicates the nonexistence of the contamination. In conclusion, this study allowed the establishment of an efficient methodology for the silencing of BMPRII by lipofection in bovine GC, which may be used as a tool for the functional study of BMPRII and potentially for other genes of interest. Research was supported by FAPESP and CNPq.

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 βA 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.

Melatonin regulates the development and function of bovine Sertoli cells via its receptors MT1 and MT2

Melatonin and its receptors are found in the testis of many species, where they mediate testicular functions. The present study aimed to investigate the expression of melatonin receptors (MT1 and MT2) in bovine Sertoli cells (SCs), using reverse transcription polymerase chain reaction (RT-PCR) and western blot. In addition, we assessed the mRNA levels of spermatogenesis-related genes (real-time PCR) and secretion of inhibin B after treatment with various concentrations (0, 80, 160, and 320pg/mL) of melatonin at different time points (24, 48, or 72h). We found that bovine SCs express MT1 and MT2 receptors, which were regulated by melatonin in time- and dose-dependent manners after treatment with melatonin. Exogenous melatonin up-regulated the expression of spermatogenesis-related genes, including Cyclin D1, Cyclin E, Pdgfa, Dhh, Occludin, and Claudin, and decreased the mRNA levels of P21 and Kit1 in a time or dose-dependent manner. Meanwhile, melatonin supplementation significantly affected Inhba, Inhbb and Inha mRNA expression. These findings were consistent with inhibin B levels detected in the culture medium. In conclusion, exogenous melatonin acts via its receptors and appears to play regulatory roles in the development and function of bovine SCs.

Activin A regulation under global hypoxia in developing mouse brain

Activin A is a multifunctional growth and differentiation factor with pronounced neuroprotective properties that is strongly up-regulated in various forms of acute brain disorders and injuries including epilepsy, stroke and trauma. In a pediatric context, activin A has been advanced as a potential marker for the severity of perinatal hypoxic–ischemic brain injury. Here we investigated the regulation of activin A under global hypoxia without ischemia in primary cultures of cortical neurons and in neonatal and adult mice of two strains (C57BL/6 and CD-1). From birth to adulthood, activin βA subunit, activin receptors, and functional activin antagonists were all expressed at roughly similar mRNA levels in the brain of C57BL/6 mice. Independent of mouse line and age, we found both moderate (11% O2, 2h) and severe hypoxia (8%, 6h) to be consistently associated with normal or even reduced levels of activin βA (Inhba) mRNA. The surprising unresponsiveness of Inhba expression to hypoxia was confirmed at the protein level. In situ hybridization did not indicate regional, hypoxia-related differences in Inhba expression. Pharmacologic stabilization of hypoxia inducible factors with the prolyl hydroxylase inhibitor FG-4497 did not influence Inhba mRNA levels in neonatal mice. Our data indicate that pure hypoxia differs from other, more complex types of brain damage in that it appears not to recruit activin A as an endogenous neuroprotective agent.

Characterization of the testicular, epididymal and endocrine phenotypes in the Leuven Vdr-deficient mouse model: Targeting estrogen signalling

Vitamin D is a key factor for calcium and bone homeostasis, but signalling through the vitamin D receptor (VDR) seems also to be important for testicular function. To test the functional role of vitamin D signalling we examined the male reproductive system of the Leuven Vdr-ablated (Vdr−/−) mice, previously established as a model for hereditary vitamin D-resistant rickets. We investigated reproductive hormones, changes in gene expression and histological phenotype of eleven Vdr−/−, eight Vdr+/− and nine Vdr+/+ mice. Testicular and epididymal histology were grossly normal in Vdr−/− mice. Accordingly, no differences were found in serum concentrations of testosterone, estradiol, LH, and FSH or testicular expression of Cyp19a1, Ersα, Cyp17a1, Star, Insl3, Inhbb, and Amh. However, a significantly lower ERβ expression was found in testis of Vdr+/− and Vdr−/− mice, conversely epididymal expressions of ERα and the estrogen-target gene Aqp9 were higher. In conclusion, vitamin D seems dispensable for murine spermatogenesis and sex hormone production, but aberrant estrogen-signalling may elicit some of the VDR-mediated effects on male reproduction.

Depot differences in gene expression in response to dexamethasone in human adipose tissue

Hypercortisolemia leads to a preferential fat deposition in visceral adipose tissues. We previously noted that the glucocorticoid receptor agonist dexamethasone (Dex, 25 nM) regulated many metabolic and inflammatory gene pathways in human omental (Om) and abdominal subcutaneous (Sc) adipose tissues. To determine whether differential sensitivity to Dex contributes to depot differences in global gene expression, fragments of Sc and Om adipose tissues from severely obese females (n=3) were cultured in the presence of 7nM insulin plus 0 (control), 1, 10, or 1000 nM Dex for 7 days. 1 and 10 nM Dex regulated, respectively, 88 and 414 genes in Om, and 173 and 594 genes in Sc by 2‐fold or more. Thus, almost 2‐fold more genes were regulated by Dex in Sc than Om at low Dex. We noted and confirmed by qPCR that INHBA, a gene known to inhibit adipogenesis, was ~6‐fold higher in Om and was suppressed in both, but remained higher in Om at Dex 1 and 10 nM. In primary cultured predipocytes (n=3), INHBA was similar in confluent preadipocytes, but was less suppressed when a standard differentiation cocktail containing Dex was added. At day 14 of differentiation, INHBA expression was ~50‐fold higher in Om cultures in parallel to poorer differentiation. Overall, our data suggest that differential sensitivity to physiologically relevant concentrations of Dex can contribute to depot differences in adipocyte differentiation and function.

Non-Canonical Smads Phosphorylation Induced by the Glutamate Release Inhibitor, Riluzole, through GSK3 Activation in Melanoma

Riluzole, an inhibitor of glutamate release, has shown the ability to inhibit melanoma cell xenograft growth. A phase 0 clinical trial of riluzole as a single agent in patients with melanoma resulted in involution of tumors associated with inhibition of both the mitogen-activated protein kinase (MAPK) and phophoinositide-3-kinase/AKT (PI3K/AKT) pathways in 34% of patients. In the present study, we demonstrate that riluzole inhibits AKT-mediated glycogen synthase kinase 3 (GSK3) phosphorylation in melanoma cell lines. Because we have demonstrated that GSK3 is involved in the phosphorylation of two downstream effectors of transforming growth factor beta (TGFβ), Smad2 and Smad3, at their linker domain, our aim was to determine whether riluzole could induce GSK3β-mediated linker phosphorylation of Smad2 and Smad3. We present evidence that riluzole increases Smad2 and Smad3 linker phosphorylation at the cluster of serines 245/250/255 and serine 204 respectively. Using GSK3 inhibitors and siRNA knock-down, we demonstrate that the mechanism of riluzole-induced Smad phosphorylation involved GSK3β. In addition, GSK3β could phosphorylate the same linker sites in vitro. The riluzole-induced Smad linker phosphorylation is mechanistically different from the Smad linker phosphorylation induced by TGFβ. We also demonstrate that riluzole-induced Smad linker phosphorylation is independent of the expression of the metabotropic glutamate receptor 1 (GRM1), which is one of the glutamate receptors whose involvement in human melanoma has been documented. We further show that riluzole upregulates the expression of INHBB and PLAU, two genes associated with the TGFβ signaling pathway. The non-canonical increase in Smad linker phosphorylation induced by riluzole could contribute to the modulation of the pro-oncogenic functions of Smads in late stage melanomas.

P1-016 - Activin-A Directly Inhibits Vascular Endothelial Cell Growth VIA TGF-b-Dependent Signal Pathway

ABSTRACT Activin-A is a homodimeric protein consisting of two INHBA (inhibin bA subunit) and belongs to the large transforming growth factor b (TGF-b) superfamily. In cancer, it is known that activin-A is overexpressed in various cancers such as esophageal, pancreatic and ovarian cancers; however, their involvement in angiogenesis remains largely unclear. Our microarray analysis for the clinical specimen of gastric cancer revealed that the mRNA expression of INHBA was overexpressed in gastric cancer cells. To clarify the biological function of activin-A for angiogenesis, we conducted the following experiments. Real-time RT–PCR revealed that INHBA mRNA was overexpressed in several cancer cell lines. Secreted activin-A protein was also confirmed by ELISA and the concentration varied markedly by individual cell lines. Activin-A and TGF-b directly inhibited the cell growth of HUVEC (human umbilical vascular endothelial cells) in a dose-dependent manner. Both activin-A and TGF-b increased the phosphorylation levels of smad on HUVEC cells, suggesting that the TGF-b signal pathway actually functioned in HUVEC cells. Flowcytometry revealed that activin-A and TGF-b induced cell cycle arrest in HUVEC cells. These results suggest that activin-A has a growth inhibitory effect on the vascular endothelial cell and the overexpression of activin-A in gastric cancer may contribute anti-angiogenic roles for tumor growth.

Activin A Stimulates AKR1C3 Expression and Growth in Human Prostate Cancer

Local androgen synthesis in prostate cancer (PC) may contribute to the development of castration-resistant PC (CRPC), but pathways controlling intratumoral steroidogenic enzyme expression in PC are unknown. We investigated the effects of activin, a factor involved in the regulation of PC growth and steroidogenic enzyme expression in other steroidogenic tissues, on intratumoral steroidogenesis in PC. Activin A effects and regulation of the activin-signaling pathway molecules were studied in the PC cell lines LNCaP, VCaP, and PC-3 and in 13 individual PC xenograft models. Also, expression levels of inhibin βA- and βB-subunits (INHBA and INHBB) and of the activin antagonist follistatin were quantitated in patient PC tissues. Activin A induced the expression and enzyme activity of 17β-hydroxysteroid dehydrogenase enzyme AKR1C3 in LNCaP and VCaP cells. Inhibition of endogenous activin A action in the PC-3 cell line decreased AKR1C3 levels and consequently testosterone synthesis. In return, androgens suppressed INHBA expression in both VCaP cells and the PC xenograft models. The antiproliferative effects of activin A were opposed by physiological concentrations of androstenedione in LNCaP cells. In patient PC tissues, expression levels of INHBA were increased in CRPC samples and correlated with AKR1C3 levels. Moreover, a high ratio of activin subunits to follistatin was associated with a worse metastasis-free survival in patients. In conclusion, activin A is controlled by androgens in PC models and regulates local androgen production. Activin A thus seems to mediate (residual) intratumoral androgen levels and could form a novel therapeutic target in CRPC.

Expression patterns of activin, inhibin and follistatin variants in the adult male mouse reproductive tract suggest important roles in the epididymis and vas deferens

Activin A and its inhibitors follistatin and inhibin play key roles in development and function of the male reproductive tract. Quantitative (q) polymerase chain reaction (PCR) was used to evaluate the expression of Inhba (the gene encoding activin A subunits), Inha and Inhbb (genes encoding the inhibin B subunits), as well as the genes for follistatin (Fst) and follistatin-like 3 (Fstl3) and the activin receptor subunits, in the male mouse reproductive tract. A qPCR assay that discriminated between the two follistatin variants of Fst288 (tissue-bound form) and Fst315 (circulating) was established. Activin A protein was measured by ELISA, whereas the inhibin α-subunit and total follistatin proteins were measured by radioimmunoassay (RIA). A screen of 22 tissues demonstrated tissue-specific regulation of the follistatin variants, with Fst288 highly expressed in the vas deferens and Fst315 most highly expressed in the skin. The expression of Fst288 and Fst315 and follistatin protein levels increased progressively from the testis through to the distal vas deferens. Inhba and the activin receptors were highly expressed in the epididymis, but activin A protein was elevated in both the epididymis and vas deferens. Inhibin α-subunit mRNA and protein and Inhbb expression were highest in the testis. These results indicate a role for activin A within the epididymis, but also that activin A bioactivity may be increasingly inhibited by follistatin distally along the male reproductive tract.

Epidermal growth factor differentially regulates activin subunits in the zebrafish ovarian follicle cells via diverse signaling pathways

Epidermal growth factor (EGF) promotes oocyte maturation in the zebrafish and its effect is mediated via the activin system. However, the mechanisms by which EGF regulates activin subunits in the follicle cells remain unknown. The present study demonstrated that EGF controlled expression of three activin subunits (inhbaa, inhbab and inhbb) in the follicle cells via diverse signaling pathways. The expression of inhbaa and inhbb was often co-regulated via similar pathways. Suppression of MAPK3/1, p38 MAPK, PKC and PKA each blocked or partially reduced the stimulatory effects of EGF on the expression of inhbaa and inhbb while up-regulated that of inhbab. Conversely, inhibition of PI3K did not have any effect on the expression of inhbaa and inhbb but significantly suppressed the stimulatory effect of EGF on inhbab. In summary, EGF action in the zebrafish ovary involves activin system and its regulation of activin subunits is mediated by diverse signaling pathways downstream of EGFR.

Gene markers of progression of Barrett’s metaplasia to carcinoma.

46 Background: The incidence of esophageal adenocarcinoma (EAC) has increased strongly during the past 30 years and 5-year survival remained poor at approximately 20%. Barrett’s esophagus (BE) metaplasia is considered to be the key precursor lesion of EAC. The aim of this study was to generate and validate biomarkers to stratify patients with BE in terms of their risk for developing cancer. Methods: We studied gene expression profiling of normal esophageal squamous epithelium (NE), BE, intraepithelial neoplasia (IEN) and esophageal adenocarcinoma (EAC). Laser capture microdissection (LMD) technique was applied to procure cells from defined regions. Microarray results were validated by quantitative real-time polymerase chain reaction (qRT-PCR) in a second and independent cohort. Furthermore, immunohistochemistry was performed on a third independent cohort. Results: The expression profile of BE was compared to EAC and NE resulting in a short list of 16 genes which were differentially expressed among all 3 groups. Hierarchical clustering of all samples based on the expression of these 16 candidate marker genes allowed a clear discrimination of NE, BE and EAC. Validation of the microarray results in an independent patient cohort by qRT-PCR confirmed that the genes CTHRC1, INHBA, PROCR and ADH7 expression correlate with the histological progression to EAC. Accordingly, immunohistochemistry showed that the presence of CTHRC1 and INHBA was high only in EAC. Conclusions: Here, we focus on neoplastic progression and describe a set of genes representing potential biomarkers to predict oesophageal Barrett’s adenocarcinoma at its early stage. These biomarkers can be determined by quantitative expression analysis, which will complement conventional semi-quantitative immunohistochemical analysis in the future. This work was feasible only through intensive cross-discipline collaboration between clinical researchers, pathologists, and basic molecular researchers and is thought to be the starting point for a long-term multicenter study on the clinical validation of the putative markers described here.

Progesterone stimulates expression of follistatin splice variants Fst288 and Fst315 in the mouse uterus

Follistatin, an inhibitor of activin A, has key regulatory roles in the female reproductive tract. Follistatin has two splice variants: FST288, largely associated with cell surfaces, and FST315, the predominant circulating form. The mechanism regulating uterine expression of these variants is unknown. Quantitative RT-PCR was used to measure expression of follistatin splice variants (Fst288, Fst315), the activin βA subunit (Inhba) and the inhibin α subunit (Inha) in uterine tissues during early pregnancy (days 1–4, preimplantation) and in response to exogenous 17β-oestradiol (single s.c. injection) and progesterone (three daily s.c. injections) in ovariectomized mice. Uterine Fst288, Fst315 and Inhba expression increased during early pregnancy, with greater increases in Fst315 relative to Fst288 suggesting differential regulation of these variants. Fst288, Fst315, Inhba and Inha all increased in response to progesterone treatment. Fst288, but not Fst315, mRNA decreased in response to 17β-oestradiol treatment, whereas Inhba increased. A comparison of the absolute concentrations of uterine follistatin mRNA using crossing thresholds indicated that both variants were more highly expressed in early pregnancy in contrast to the hormone treatment models. It is concluded that progesterone regulates uterine expression of both follistatin variants, as well as activin A, during early pregnancy in the mouse uterus.Follistatin and activin are proteins with important functions in female reproduction. There are two forms of follistatin (FST288 and FST315), which interact with activin in different ways and subsequently may have different functions. We are interested in the function of these two forms of follistatin in the uterus. In this study, we examined how the expression of the two follistatin variants and activin changed in the uterus during early pregnancy in mice, as well as in response to the female hormones oestrogen and progesterone. We saw an increase in the expression of the genes for FST288, FST315 and activin in the uterus during early pregnancy. We also saw an increase in these proteins in the mouse uterus in response to progesterone treatment. We concluded that the hormone progesterone regulates expression of both follistatin variants, as well as activin, during early pregnancy in the mouse uterus.

IL-1β Stimulates Activin βA mRNA Expression in Human Skin Fibroblasts Through the MAPK Pathways, the Nuclear Factor-κB Pathway, and Prostaglandin E2

During mouse skin wound healing, mRNAs encoding IL-1, activins, and TGF-βs significantly increased. To elucidate involvement of IL-1 in the regulation of activins and related factors in the wounded skin, human foreskin fibroblasts were stimulated with IL-1β, and levels of mRNAs encoding activins, TGF-βs, and follistatin family proteins were examined by quantitative real-time PCR. IL-1β increased activin βA (INHBA) and follistatin (FST) mRNA expression within 6 h. A p38 MAPK inhibitor, SB202190, a MAPK/ERK kinase inhibitor, U0126, and an nuclear factor κB pathway inhibitor, SC-514, significantly suppressed the IL-1β-stimulated INHBA and FST mRNA expression. A prostaglandin-endoperoxide synthase inhibitor indomethacin, a potent inhibitor of prostaglandin E(2) (PGE(2)) synthesis, also significantly suppressed the IL-1β-stimulated INHBA but not FST mRNA expression. Furthermore, stimulation of fibroblasts with PGE(2) significantly increased INHBA mRNA. The PGE(2)-induced INHBA mRNA expression was significantly suppressed by U0126 and a protein kinase C inhibitor, Gö 6983. Although IL-1β stimulated FST mRNA in an acute phase, long-term exposure of fibroblasts to IL-1β revealed time-dependent stimulatory and inhibitory effects of IL-1β on FST mRNA expression. On the other hand, coculture with keratinocytes significantly increased INHBA mRNA expression in dermal equivalents. In summary, the present study indicates that the p38 MAPK, the MAPK/ERK kinase, the nuclear factor κB pathway, and PGE(2) mediate the effects of IL-1β on INHBA mRNA expression. Furthermore, it is indicated that keratinocyte-derived factor of factors stimulate INHBA mRNA expression during wound healing.

Upregulated INHBA expression is associated with poor survival in gastric cancer

Expression microarrays are widely used for investigating the candidate molecular targets in human cancer. While genome-wide expression signatures screened by gene set enrichment analysis (GSEA) were not performed in Chinese gastric cancer (GC). To gain new molecular targets for GC, GSEA analysis was performed. In the present study, GSEA were used to pick out differentially expressed gene sets of our database. Total RNA of paired tissue samples (n = 48) and a tissue microarray containing 132 paired tissues were used to further validate expression levels of INHBA and its correction with clinicopathological factors. Upregulated INHBA expression in gastric cancer was screened and further confirmed by qPCR and immunostaining analysis. Increased INHBA expression was significantly correlated with the diameter of cancer and depth of tumor invasion. Patients with higher expression levels of INHBA had a shorter disease-free survival rate. It was effective to gain new molecular targets for GC by GSEA analysis. INHBA may be a poor survival indicator of GC.

The maturation-inducing hormone 17α,20β-dihydroxy-4-pregnen-3-one regulates gene expression of inhibin β A and bambi (bone morphogenetic protein and activin-membrane-bound inhibitor) in the rainbow trout ovary

Transforming growth factor-beta (TGFβ) superfamily members are important paracrine and autocrine regulators of ovarian development and steroidogenesis in mammals and birds, but their reproductive roles in fish are not well understood. The activin system, Tgfb, and bone morphogenetic protein 15 (Bmp15) participate in the regulation of follicle maturation in some fish species. In addition, transcript levels of TGFβ superfamily members and their inhibitor, bambi ( bmp and activin-membrane-bound inhibitor), change in the rainbow trout ( Oncorhynchus mykiss) ovary during reproductive development including the transition from vitellogenesis to follicle maturation. The objective of the present study was to determine if the maturation-inducing hormone (MIH) in trout, 17α,20β-dihydroxy-4-pregnen-3-one, regulates gene expression of TGFβ superfamily members and their inhibitors. Transcript levels of inhibin β A subunit ( inhba) were increased and bambi decreased in isolated follicles incubated overnight without hormones compared to abundance in freshly excised tissues from the same fish, suggesting systemic factors influenced transcript abundance. Incubation with MIH decreased inhba and increased bambi expression in a dose-dependant manner and MIH was the most potent steroid examined. The transcripts’ responses to incubation with and without MIH were observed in maturationally competent follicles, which are follicles competent to resume meiosis in response to MIH, and incompetent follicles, although the responses to MIH were greater in competent follicles. In summary, MIH regulates inhba and bambi expression in a stage specific manner supporting a role for MIH regulation of the TGFβ superfamily system and participation of the TGFβ superfamily system in the regulation of follicle maturation in rainbow trout.

WNT4/β-Catenin Pathway Maintains Female Germ Cell Survival by Inhibiting Activin βB 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 β-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 β-catenin in somatic cells in the Wnt4 knockout ovary restored germ cell numbers, placing β-catenin downstream of WNT4. In the absence of Wnt4 or β-catenin, female germ cells entered meiosis properly; however, they underwent apoptosis afterwards. Activin βB (Inhbb), a subunit of activins, was upregulated in the Wnt4 and β-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 β-catenin in the somatic cells. Maintenance of female germ cells hinge upon a delicate balance between positive (WNT4 and β-catenin) and negative (activin βB) regulators derived from the somatic cells in the fetal ovary.