Expression of inhibin/activin proteins and receptors in the human hypothalamus and basal forebrain.

The inhibin/activin family of proteins is known to have a broad distribution of synthesis and expression in many species, as well as a variety of functions in reproductive and other physiological systems. Yet, our knowledge regarding the production and function of inhibin and activin in the central nervous system is relatively limited, especially in humans. The present study aimed to explore the distribution of inhibin/activin protein subunits and receptors in the adult human brain. The human hypothalamus and surrounding basal forebrain was examined using post-mortem tissues from 29 adults. Immunocytochemical studies were conducted with antibodies directed against the inhibin/activin α, ßA, and ßB subunits, betaglycan and the activin type IIA and IIB receptors. An immunoassay was also utilised to measure dimeric inhibin A and B levels in tissue homogenates of the infundibulum of the hypothalamus. Robust ßA subunit immunoreactivity was present in the paraventricular, supraoptic, lateral hypothalamic, infundibular, dorsomedial and suprachiasmatic nuclei of the hypothalamus, in the basal ganglia, and in the nucleus basalis of Meynert. A similar staining distribution was noted for the ßB subunit, betaglycan and the type II receptor antibodies, whereas α subunit staining was not detected in any of the major anatomical regions of the human brain. Inhibin B immunoreactivity was present in all tissues, whereas inhibin A levels were below detectable limits. These studies show for the first time that the inhibin/activin protein subunits and receptors can be co-localised in the human brain, implicating potential, diverse neural functions.

Expression and cyclic change of betaglycan in the rat oviduct.

Although the fallopian tube provides a sufficient environment for fertilization and early embryo development, the mechanism by which it does this is unclear. It is known that the transforming growth factor beta (TGFbeta) superfamily plays important roles in various reproductive functions. Betaglycan, originally characterized as a TGF beta type III receptor lacking a clearly identifiable signaling motif, has been shown to be important for the high-affinity binding of TGF betas to the type II receptor. To our knowledge, there has been no study showing expression of betaglycan in the rat oviduct. Therefore, in this study, we examined the distribution of betaglycan in various rat tissues and its expression patterns in the oviduct during the estrous cycle. Northern blot analysis of various rat tissues showed that the adrenal gland, ovary and oviduct contained abundant amounts of 6.4-kb betaglycan mRNA. Furthermore, the mRNA level of betaglycan was highest after the LH surge that induced ovulation. The betaglycan protein, detected using immunohistochemistry, was especially abundant in the epithelium of the oviduct. Furthermore, in pregnant mare serum gonadotropin (PMSG) primed-rats, the expression of betaglycan was increased significantly by stimulation with human chorionic gonadotropin (hCG). RT-PCR analysis showed co-localization of other TGF beta family receptors (TGF beta types I and II, activin receptor types Ia and Ib and activin receptor types IIa and IIb) with betaglycan in the oviduct. Since betaglycan along with other TGF beta family receptors is abundantly expressed in the epithelium of the oviduct and its expression changes during estrous, it may also play an important role in the oviduct.

Regulation of baboon fetal ovarian folliculogenesis by estrogen.

Although it is well established that formation of the pool of follicles available for ovarian function and fertility in adulthood in human and non human primates occurs in utero, our understanding of the regulation of fetal ovarian development is incomplete. Our laboratories have been instrumental in establishing the baboon as a model for the study of human reproductive endocrinology and showed that estrogen plays a central integrative role in regulating fetal-placental development. Therefore, we adapted our baboon model to study the role of estrogen on fetal ovarian development. Estrogen receptors alpha and beta were expressed in pregranulosa cells and interfollicular nests of the baboon fetal ovary. In baboons in which estrogen levels had been suppressed by administration of an aromatase inhibitor throughout the second half of gestation, fetal ovarian follicle numbers were reduced by 50%, whereas the number of interfollicular nests comprised of oocytes and pregranulosa cells was increased. The decrease in follicles in estrogen-deprived animals was associated with a marked upregulation of expression of alpha-inhibin, but not activins or activin receptors and signaling molecules. Moreover, the majority of the follicles formed in ovaries of estrogen-depleted fetuses appeared unhealthy and contained oocytes with a marked reduction/depletion in microvilli, structures essential for uptake of substrates from surrounding granulosa cells. We propose that estrogen regulates fetal ovarian folliculogenesis and formation of healthy oocytes by controlling the intraovarian activin:inhibin ratio and the development of oocyte microvilli. These findings demonstrate a need for translational research studies of the impact of impairment of estrogen action/availability on reproductive function in adulthood.

Patients with hereditary hemorrhagic telangiectasia have increased plasma levels of vascular endothelial growth factor and transforming growth factor-beta1 as well as high ALK1 tissue expression.

BACKGROUND AND OBJECTIVES: Hereditary hemorrhagic telangiectasia (HHT), an inherited vascular dysplasia, is caused by mutations in endoglin or activin receptor-like kinase (ALK)-1. Haploinsufficiency for these genes is thought to result in an imbalanced angiogenic activity. The aim of this study was to evaluate the plasma levels and the expression profiles of angiogenic and angiogenesis-related factors in the context of HHT. DESIGN AND METHODS: Vascular endothelial growth factor (VEGF) and transforming growth factor (TGF)-beta1 plasma concentrations were determined in 31 HHT patients and 40 healthy controls by ELISA. VEGF and TGF-beta1 plasma concentrations were correlated with the patients' clinicopathological features. Tissue expression of angiogenic and angiogenesis related proteins was determined by immunostaining on nasal cryostat sections from 13 HHT patients and 5 healthy controls. RESULTS: Of the 31 patients, 29 had statistically significantly raised plasma concentrations of VEGF and TGF-beta1 but there was no correlation with specific clinicopathological features. Increased VEGF, TGF-beta1 and ALK1 immunostaining was seen in all 13 investigated patients. beta-smooth muscle actinin immunostaining was increased in 12 patients. Increased endoglin immunostaining was seen in only 9 patients. INTERPRETATION AND CONCLUSIONS: This study provides evidence of the role of VEGF and TGF-beta1 in the pathogenesis of HHT. Plasma concentrations of these two factors may serve as further diagnostic criteria for HHT. For the first time, we report increased TGF-beta1 plasma concentrations and increased TGF-beta1 and ALK1 tissue expression in HHT, which appear not to be specifically associated with either endoglin or ALK1 mutations. The data suggest that HHT is an angiogenic disorder characterized by an over-expression of VEGF, TGF-beta1 and ALK1.

Developmental analysis of activin-like kinase receptor-4 (ALK4) expression in Xenopus laevis.

The type I transforming growth factor-beta (TGFbeta) receptor, activin-like kinase-4 (ALK4), is an important regulator of vertebrate development, with roles in mesoderm induction, primitive streak formation, gastrulation, dorsoanterior patterning, and left-right axis determination. To complement previous ALK4 functional studies, we have analyzed ALK4 expression in embryos of the frog, Xenopus laevis. Results obtained with reverse transcriptase-polymerase chain reaction indicate that ALK4 is present in both the animal and vegetal poles of blastula stage embryos and that expression levels are relatively constant amongst embryos examined at blastula, gastrula, neurula, and early tail bud stages. However, the tissue distribution of ALK4 mRNA, as assessed by whole-mount in situ hybridization, was found to change over this range of developmental stages. In the blastula stage embryo, ALK4 is detected in cells of the animal pole and the marginal zone. During gastrulation, ALK4 is detected in the outer ectoderm, involuting mesoderm, blastocoele roof, dorsal lip, and to a lesser extent, in the endoderm. At the onset of neurulation, ALK4 expression is prominent in the dorsoanterior region of the developing head, the paraxial mesoderm, and midline structures, including the prechordal plate and neural folds. Expression in older neurula stage embryos resolves to the developing brain, somites, notochord, and neural crest; thereafter, additional sites of ALK4 expression in tail bud stage embryos include the spinal cord, otic placode, developing eye, lateral plate mesoderm, branchial arches, and the bilateral heart fields. Together, these results not only reflect the multiple developmental roles that have been proposed for this TGFbeta receptor but also define spatiotemporal windows in which ALK4 may function to modulate fundamental embryological events.

Activin and follistatin in rat mammary gland.

Mammary gland morphogenesis and differentiation are mediated through the combined activities of systemic hormones and locally synthesized growth factors. Activin, a member of the transforming growth factor (TGF)-beta superfamily, is known to regulate the growth and differentiation of several cell types. In the present study, we investigated the role of activin in rat mammary gland on different stages of development. We found that activin A in vitro inhibits the proliferation of isolated acini, and this effect increases with the development of the gland. This factor also produces in vitro an inhibition of the final differentiation of acini obtained from 19th day pregnant rats. We also report the expression of activin receptors IIA and IIB mRNA in whole rat mammary gland and acini, with decreased levels of expression of type IIA (in both compartments) and IIB (in acini) during pregnancy and lactogenesis. In addition, we show that activin betaB-subunit mRNA decreases throughout pregnancy, and that the mRNA levels of follistatin (Fst) (its ligand protein) are high in cycling rats and at the beginning of pregnancy and diminish thereafter, having the acini higher levels of expression. Our data show that activin betaB-subunit, follistatin and ActRIIA and IIB transcripts are expressed in rat mammary gland at appropriate times and locations during development, allowing an interplay that might regulate activin action on growth and differentiation of the gland.

Busulfan induces activin A expression in vitro and in vivo: a possible link to venous occlusive disease.

PURPOSE: Hepatic venous occlusive disease is a severe side effect after administration of busulfan before hematopoietic stem cell transplantation. The syndrome is characterized by liver enlargement, fluid retention, jaundice, and weight gain. Endothelial injury has been described as the precipitating factor. The link between busulfan administration and endothelial damage has not been established thus far. METHODS: Complementary deoxyribonucleic acid expression arrays were used to screen for busulfan responsive genes in ECV304 cells. Specific messenger ribonucleic acid and protein levels were examined by real-time polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. Serum samples of 15 pediatric patients with leukemia were analyzed for busulfan and cytokine levels. RESULTS: We identified a member of the transforming growth factor beta superfamily, activin A, to be induced in the human cell line ECV304 after exposure to busulfan in a time- and concentration-dependent manner. Maximum effects were observed at 120 and 168 hours for activin A messenger ribonucleic acid and protein, respectively. Preincubation with the protein kinase C inhibitor bisindolylmaleimide I (10 nmol/L) abolished activin A induction by busulfan (P <.05). Activin receptors were detected in ECV304. Both tissue factor and cyclooxygenase 2 were significantly induced by busulfan (P <.05). In a parallel in vivo study a significant increase in serum activin A concentration was found 4.5 hours after the second dose of busulfan. CONCLUSION: The data demonstrate that busulfan induces activin A both in vitro and in vivo. In view of the multiple targets of activin A (inflammation, proliferation, apoptosis, and coagulation), these findings may be of relevance to our understanding of venous occlusive disease.

Expression and function of activin receptors in human endometrial adenocarcinoma cells.

Menstrual cycle-dependent expressions of activin A in normal human endometrial tissues have been reported. Expression of activin receptor mRNAs and increased activin A production were also observed in human endometrial adenocarcinoma tissues, suggesting that activin A might enhance cell proliferation and inhibit apoptotic signaling in endometrial cancer cells. In this study, we have examined the effects of activin A on cell proliferation, anticancer drug-induced apoptosis and Fas-mediated apoptosis in 3 differentiated human endometrial adenocarcinoma cell lines, namely HEC-1, HHUA and Ishikawa. Flow cytometric analyses revealed moderate expressions of all 4 types of activin receptor subunits on the cell surfaces of the 3 cell lines. The proliferations of the 3 endometrial cancer cells were completely unaffected by activin A, whereas it suppressed the cell proliferation of a human ovarian endometrioid adenocarcinoma cell line, OVK-18, in a dose-dependent manner. Moreover, activin A did not affect the apoptotic changes in the 3 endometrial adenocarcinoma cells treated with 4 different anticancer drugs, namely CDDP, paclitaxel, etoposide and SN38. The apoptotic changes in HHUA cells treated with anti-Fas IgM were also unaffected by activin A. These results indicate that the increased activin A production in human endometrial adenocarcinoma tissues in vivo may not stimulate carcinoma cell proliferation or inhibit apoptotic signaling in carcinoma cells. Insensitivity to the usual growth suppression signals induced by activin A might be one of the mechanisms of immortality of human endometrial adenocarcinoma cells.

Activin signaling pathways in ovine pituitary and LbetaT2 gonadotrope cells.

In the pituitary, activin stimulates the synthesis and release of FSH. However, the activin receptor signaling pathways that mediate these effects are poorly known. We investigated these mechanisms in primary ovine pituitary cells (POP) and in the murine LbetaT2 gonadotrope cell line. POP cells and LbetaT2 cells express the different activin receptors (types IA, IB, IIA, and IIB) and the Smad proteins (Smad-2, -3, -4, and -7). In both POP and LbetaT2 cells, activin activated several signaling pathways: Smad-2, extracellular regulated kinase-1/2 (ERK1/2), p38, and phosphatidylinositol 3'-kinase (PI3K)/Akt. Phosphorylation of ERK1/2 and p38 were stimulated (3- to 6-fold) rapidly in 5 min, whereas activation of both Smad-2 and Akt (3- to 5-fold) occurred later, in 60 min. Activin also increased the association of activin receptor IIB with PI3K. Using specific inhibitors, we demonstrated that the activation of Smad-2 was partially blocked by the inhibition of PI3K but not by the inhibition of ERK1/2 or p38, suggesting a cross-talk between the Smad and PI3K/Akt pathways. In both POP and LbetaT2 cells, FSH expression and secretion in response to activin were not altered by the inhibition of PI3K/Akt, ERK1/2, or p38 pathways, whereas they were reduced by about 2-fold by expression of a dominant negative of Smad-2 or the natural inhibitory Smad-7 in LbetaT2 cells. These results indicate that activin activates several signaling pathways with different time courses in both POP and LbetaT2 cells, but only the Smad-2 pathway appears to be directly implicated in FSH expression and release in LbetaT2 cells.

Expression and localization of activin receptors, Smads, and beta glycan to the postnatal rat ovary.

Despite understanding the molecular basis of activin/TGF beta and bone morphogenetic protein (BMP) signaling, this study is the first to characterize multiple, sequential elements of these pathways in the ovary concurrently. The expression of activin/BMP receptor, Smad, and beta glycan mRNAs by postnatal rat ovaries were investigated by real-time PCR. Activin/BMP receptors (ActRIA, ActRIB, ActRIIA, and ActRIIB), beta glycan, and Smad 1-8 mRNAs were expressed by the ovary. Activin receptor and Smad 1, 2, 4, 5, and 7 mRNAs declined up to 4-fold between postnatal d 4-8, coinciding with secondary follicle formation. The emergence of antral follicles (postnatal d 12) saw ActRIA, ActRIIB, and Smad 2 mRNA expression return to d 4 levels, whereas ActRIB, ActRIIA, and Smads 1, 4, 5, and 7 remained at lower levels. beta glycan mRNA levels increased 2-fold between d 8 and 12, suggesting expression by the developing theca. Smad 3, 6, and 8 mRNAs were unchanged. Activin receptor and Smad proteins were present in oocytes at all stages of follicular development; granulosa cells of primary-antral follicles, and theca cells. beta glycan protein was present in oocytes, granulosa cells, and theca cells at all stages of folliculogenesis. The colocalization of receptors and Smads supports the notion that activin/TGF beta and BMP signaling pathways are functional in the cellular compartments of the follicle.

Expression of activin receptors, follistatin and betaglycan by human endometrial stromal cells; consistent with a role for activins during decidualization.

Decidualization of the human endometrium is critical for implantation, but the mechanisms involved are largely unknown. Activin subunits are expressed in endometrium during decidualization. From its known actions in cell differentiation and tissue remodelling, we hypothesized that activin A is involved in the paracrine regulation of decidualization. We examined the expression of activin receptors (ActRs) by semi-quantitative and real-time RT-PCR. mRNA for all ActR subtypes (Ia, Ib, IIa and IIb) was detected in endometrium, with maximal expression in the early secretory phase and in early pregnancy. ActR protein was localized exclusively to stromal and endothelial cells. This expression pattern was confirmed by in-situ hybridization. Activin bioavailability is locally regulated by its binding protein, follistatin, and also by the antagonist, inhibin. Inhibin competition for ActRII binding is enhanced by the binding protein, betaglycan. Follistatin and betaglycan were also detected in the endometrium, localized to stromal and epithelial cells. This co-expression of activin subunits, receptors and binding proteins indicates that stromal cells are capable of responding to activin, and that there is tight local regulation of activin action within the endometrium. As activin production is up-regulated in decidual cells, this provides further evidence for an involvement of activins during stromal cell decidualization.

Seven BMPs and all their receptors are simultaneously expressed in osteosarcoma cells.

Members of the bone morphogenetic protein (BMP) family and their receptors (BMPRs and activin receptors-ActRs) promote the development of bones with a fine regulation of their expression. Mutations in BMPs or BMPRs cause several diseases, as shown in knockout mice, such as skeletal defects, familial primary pulmonary hypertension and neoplasias. Osteosarcoma is the most frequent primary malignant tumor of bone. Due to their importance in bone development, BMPs, BMPRs and ActRs could also play a role in osteosarcoma growth and development. Previous data have shown that the overexpression of the BMPR-II was related to poor prognosis in malignant and metastatic bone tumors. We evaluate by reverse transcription-linked polymerase chain reaction analysis (RT-PCR) the expression pattern of BMPs, BMPRs and ActRs in five different human osteosarcoma cell lines (MG63, G292, HOS, SaOS and U2). Moreover, we performed the mutational screening of the complete BMPR-II mRNA by automated sequencing of the correspondent cDNA to evaluate the presence of point mutations in osteosarcoma cell lines. All the osteosarcoma cell lines studied simultaneously expressed the BMPs, BMPRs and ActRs investigated. No mutations were detected in the BMPR-II cDNA. Our results suggest the presence of a mechanism involving the simultaneous activation of the BMPs and their receptors in osteosarcoma cell lines.