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.     

Serum and liver iron differently regulate the bone morphogenetic protein 6 (BMP6)-SMAD signaling pathway in mice

The bone morphogenetic protein 6 (BMP6)-SMAD signaling pathway is a central regulator of hepcidin expression and systemic iron balance. However, the molecular mechanisms by which iron is sensed to regulate BMP6-SMAD signaling and hepcidin expression are unknown. Here we examined the effects of circulating and tissue iron on Bmp6-Smad pathway activation and hepcidin expression in vivo after acute and chronic enteral iron administration in mice. We demonstrated that both transferrin saturation and liver iron content independently influence hepcidin expression. Although liver iron content is independently positively correlated with hepatic Bmp6 messenger RNA (mRNA) expression and overall activation of the Smad1/5/8 signaling pathway, transferrin saturation activates the downstream Smad1/5/8 signaling cascade, but does not induce Bmp6 mRNA expression in the liver. Hepatic inhibitory Smad7 mRNA expression is increased by both acute and chronic iron administration and mirrors overall activation of the Smad1/5/8 signaling cascade. In contrast to the Smad pathway, the extracellular signal-regulated kinase 1 and 2 (Erk1/2) mitogen-activated protein kinase (Mapk) signaling pathway in the liver is not activated by acute or chronic iron administration in mice. CONCLUSION: Our data demonstrate that the hepatic Bmp6-Smad signaling pathway is differentially activated by circulating and tissue iron to induce hepcidin expression, whereas the hepatic Erk1/2 signaling pathway is not activated by iron in vivo.     

Engagement of activin and bone morphogenetic protein signaling pathway Smad proteins in the induction of inhibin B production in ovarian granulosa cells

In the mammalian ovary cell growth and differentiation is regulated by several members of the transforming growth factor beta (TGF beta) superfamily including activins, inhibins, growth differentiation factors and bone morphogenetic proteins (BMPs). The effects of TGF beta family members are mediated to the target cells via heteromeric complexes of type I and II serine/threonine kinase receptors which activate Smad signaling protein pathways in various cell types. We have previously shown that inhibin B, a hormonally important product from human granulosa cells, is up regulated by activin and BMPs. Here, we report the use of adenoviral gene transfer methodology to manipulate the TGF beta growth factor signaling system in primary cultures of human granulosa cells. These cells are exceedingly difficult to transfect by conventional transfection methods, but were virtually 100% infected with recombinant adenoviruses expressing green fluorescent protein (GFP). Adenoviruses expressing constitutively active forms of the seven known mammalian type I activin receptor-like kinase receptors (Ad-caALK1 through Ad-caALK7) cause activation of endogenous and adenovirally transferred Smad signaling proteins so that Ad-caALK1/2/3/6 and Ad-caALK4/5/7 induced phosphorylation of the Smad1 and Smad2 pathways, respectively. Activin A and BMP-2 activated the Smad1 and Smad2 pathways as well as inhibin B production as did all the Ad-caALKs. Furthermore, overexpression of adenoviral Smad1 and Smad2 proteins without exogenously added ligands induced inhibin B production. The inhibitory Smad7 protein suppressed BMP-2 and activin induced inhibin B production. Collectively, the present data demonstrate that adenoviral gene transfer provides an effective approach for dissecting the TGF beta signaling pathways in primary ovarian cells in vitro and more specifically indicate that the Smad1 and Smad2 pathways are involved in the regulation of inhibin B production by TGF beta family ligands in the ovary.     

Localization of activin and inhibin subunits,receptors and SMADs in the mouse mammary gland

Activin and inhibin, two closely related protein hormones, are members of the transforming growth factor beta (TGF beta) superfamily of growth factors. Activin and TGF beta have been associated with mouse mammary gland development and human breast carcinogenesis. TGF beta expression in the mammary gland has been previously described, and was found to be expressed in nonparous tissue and during pregnancy, down-regulated during lactation, and then up-regulated during involution. The expression pattern of activin subunits, receptors and cytoplasmic signaling molecules has not been thoroughly described in post-natal mammary gland development. We hypothesize that activin signaling components are dynamically regulated during mammary gland development, thereby permitting activin to have distinct temporal growth regulatory actions on this tissue. To examine the activin signal transduction system in the mammary gland, tissue from CD1 female mice was dissected from nonparous, lactating day 1, 10, and 20 and post-weaning day 4 animals. The expression of the activin receptors (ActRIIA, ActRIIB and ActRIB), the inhibin co-receptor (betaglycan), and ligand subunit (alpha, beta A and beta B), mRNA was measured by semi-quantitative RT-PCR in these tissues. In addition, the cellular compartmentalization of the activin signaling proteins, including the cytoplasmic signaling co-activators, Smads 2, 3 and 4, were examined by immunohistochemistry. Generally, mRNA abundance of activin signaling components was greatest in the nonparous tissue, and then decreased, whereas protein immunoreactivity for activin signaling components increased during lactation and decreased during involution. The alpha-subunit protein was detected in nonparous and lactating day 1 tissue only. Importantly, Smad 3, but not Smad 2, was detected in epithelial cell nuclei during all time points examined, indicating that activin signaling is mediated by Smad 3 at these times. These findings suggest that activin's growth regulatory role during lactation may be distinguished from that of TGF beta during post-natal mammary development. Future studies will focus on determining the exact role this ligand plays in mammary tissue differentiation and neoplasia.     

Expression of activin/inhibin signaling components in the human adrenal gland and the effects of activins and inhibins on adrenocortical steroidogenesis and apoptosis

Activins and inhibins are structurally related glycoprotein hormones modulating pituitary FSH secretion and gonadal steroidogenesis. Activins and inhibins are also produced in the adrenal cortex where their physiological role is poorly known. Hormonally active human adrenocortical tumors express and secrete inhibins, while in mice adrenal inhibins may function as tumor suppressors. To clarify the significance of adrenal activins and inhibins we investigated the localization of activin/inhibin signaling components in the adrenal gland, and the effects of activins and inhibins on adrenocortical steroidogenesis and apoptosis.Activin receptor type II/IIB and IB, activin signal transduction proteins Smad2/3, and inhibin receptor betaglycan were expressed throughout the adrenal cortex, whereas Smad4 expression was seen mainly in the zona reticularis and the innermost zona fasciculata as evaluated by immunohistochemistry. Treatment of cultured adrenocortical carcinoma NCI-H295R cells with activin A inhibited steroidogenic acute regulatory protein and 17alpha-hydroxylase/17,20-lyase mRNA accumulation as evaluated by the Northern blot technique, and decreased cortisol, androstenedione, dehydroepiandrosterone and dehydroepiandrosterone sulfate secretion as determined by specific enzyme immunoassays. Activin A increased apoptosis as measured by a terminal deoxynucleotidyl transferase in situ apoptosis detection method. Inhibins had no effect on steroidogenesis or apoptosis.In summary, activin/inhibin signaling components are coexpressed in the zona reticularis and the innermost zona fasciculata indicating full signaling potential for adrenal activins and inhibins in these layers. Activin inhibits steroidogenic enzyme gene expression and steroid secretion, and increases apoptosis in human adrenocortical cells. Thus, the activin-inhibin system may have a significant role in the regulation of glucocorticoid and androgen production and apoptotic cell death in the human adrenal cortex.     

mRNA expression of type I and type II receptors for activin, transforming growth factor-beta, and bone morphogenetic protein in the murine erythroleukemic cell line, F5-5.fl

OBJECTIVE: Intracellular signaling of activin and transforming growth factor-beta (TGF-beta) is thought to be mediated by the same molecules (Smad2/3 and Smad4). Although differentiation of murine erythroleukemia F5-5.fl cells is induced by activin, it is not induced by TGF-beta, suggesting that at some point TGF-beta signaling is defective. The aim of this study was to investigate the unresponsiveness of F5-5.fl cells to TGF-beta. DESIGN: mRNA expression of ligands, receptors, and signal mediators for the TGF-beta family was examined in F5-5.fl cells using RT-PCR. RESULTS: Activin induced erythrodifferentiation of F5-5.fl cells in a dose-dependent manner. Neither TGF-beta1 nor bone morphogenetic protein (BMP)-4 affected the differentiation of F5-5.fl cells in the presence or absence of activin. Although mRNAs of TGF-betas (TGF-beta1, TGF-beta2 and TGF-beta3) were detected, those of inhibin/activin (alpha-, betaA- and betaB-subunits) and BMPs (BMP-2, BMP-4 and BMP-7) could not be detected in the cells, suggesting that neither activins nor BMPs are produced in F5-5.fl cells. The expression of both type I (ALK-4/ActRIB) and type II (ActRII) receptors for activin was detected in F5-5.fl cells. In contrast, while the expression of type I receptor for TGF-beta (ALK-5/TbetaRI) was detected, that of type II receptor (TbetaRII) was not. The mRNA of all Smads examined was detected in F5-5.fl cells. CONCLUSIONS: A defect in the type II receptor might cause unresponsiveness to TGF-beta in F5-5.fl cells. An erythrodifferentiation assay using F5-5.fl cells would be useful for measuring net activin activity because it would not be necessary to consider endogenous activins and BMPs.     

Synergistic activity of activin A and basic fibroblast growth factor on tyrosine hydroxylase expression through Smad3 and ERK1/ERK2 MAPK signaling pathways

Activin has previously been shown to act as a nerve cell survival factor and to have neurotrophic effects on neurons. However, the role of activin in regulating neurotransmitter expression in the central nervous system and the exact mechanisms involved in this process are poorly understood. In the present study, we report that activin A and basic fibroblast growth factor (bFGF) synergistically increased the protein level of tyrosine hydroxylase (TH), and also greatly increased the TH mRNA level, in both mouse E14 striatal primary cell cultures and the hippocampal neuronal cell line HT22. Activin A and bFGF cooperatively stimulated nuclear translocation of Smad3 and specifically activated ERK1/2, but not p38 or JNK. Interestingly, a specific inhibitor for MEK, U0126, efficiently blocked the induction of TH promoter activity by activin A and bFGF, indicating that activin A collaborated with bFGF signaling to induce the TH gene through selective activation of ERK-type MAP kinase in mouse striatal and HT22 cells. These data suggest that activin A may act in concert with bFGF for the development of TH-positive neurons.     

Activin A inhibits organization of sarcomeric proteins in cardiomyocytes induced by leukemia inhibitory factor

Cytokine systems are activated in heart failure, and it is believed that interaction between such systems may be important during progression of this disorder. We have previously shown that failing hearts have increased levels of the interleukin-6 related cytokine leukemia inhibitory factor (LIF) and activin A, a member of the transforming growth factor-beta family. The aim of this study was to examine the effects of activin A on cardiomyocytes and a potential interaction with LIF-mediated changes in cell signaling and growth. Cardiomyocytes were isolated from 1- to 3-day-old Wistar rats, and the cells were treated with LIF, activin A or a combination thereof. Our main findings were: (i) activin A treatment reduced the LIF-mediated increase in cardiomyocyte length, perimeter and sarcomeric organization and was accompanied by a substantially decreased alpha-skeletal actin gene expression. (ii) The activin A-mediated phosphorylation of Smad2 was markedly enhanced by LIF. (iii) Activin A markedly induced SOCS3 gene expression, while LIF potently increased the expression of Smad7 mRNA, representing inhibitors of LIF and activin A signaling pathways, respectively. (iv) Inhibiting activation of the Smad2/3 pathway abolished the effects of activin A on LIF-induced changes in cell length, perimeter and sarcomeric organization. In conclusion, activin A markedly attenuates LIF-induced changes in cardiomyocytes, reflecting a potentially important role for both activin A and the Smad2/3 pathway in regulation of myocardial remodeling.     

Heparin: a potent inhibitor of hepcidin expression in vitro and in vivo

Hepcidin is a major regulator of iron homeostasis, and its expression in liver is regulated by iron, inflammation, and erythropoietic activity with mechanisms that involve bone morphogenetic proteins (BMPs) binding their receptors and coreceptors. Here we show that exogenous heparin strongly inhibited hepcidin expression in hepatic HepG2 cells at pharmacologic concentrations, with a mechanism that probably involves bone morphogenetic protein 6 sequestering and the blocking of SMAD signaling. Treatment of mice with pharmacologic doses of heparin inhibited liver hepcidin mRNA expression and SMAD phosphorylation, reduced spleen iron concentration, and increased serum iron. Moreover, we observed a strong reduction of serum hepcidin in 5 patients treated with heparin to prevent deep vein thrombosis, which was accompanied by an increase of serum iron and a reduction of C-reactive protein levels. The data show an unrecognized role for heparin in regulating iron homeostasis and indicate novel approaches to the treatment of iron-restricted iron deficiency anemia.     

Bone morphogenetic protein 6 (BMP6) and BMP7 inhibit estrogen-induced proliferation of breast cancer cells by suppressing p38 mitogen-activated protein kinase activation

Estrogen is involved in the development and progression of breast cancer. Here, we investigated the effects of bone morphogenetic proteins (BMPs) on breast cancer cell proliferation caused by estrogen using human breast cancer MCF-7 cells. MCF-7 cells express estrogen receptors (ESR1 and ESR2), BMP receptors, and SMAD signaling molecules. Estradiol and membrane-impermeable estradiol stimulated MCF-7 cell proliferation. Estradiol also reduced mRNA levels of ESR1, aromatase, and steroid sulfatase. Treatment with BMPs and activin had no effects on MCF-7 cell proliferation. However, BMP2, BMP4, BMP6, BMP7, and activin suppressed estradiol-induced cell mitosis, with the effects of BMP6, BMP7, and activin being more prominent than those of BMP2 and BMP4. Activin decreased ESR1 mRNA expression, while BMP6 and BMP7 impaired steroid sulfatase expression in MCF-7 cells. Interestingly, SMAD1,5,8 activation elicited by BMP6 and BMP7, but not by BMP2 and BMP4, was preserved even under the exposure of a high concentration of estradiol. The difference of BMP responsiveness was likely due to the differential modulation of BMP receptor expression induced by estradiol. In this regard, estradiol decreased the expression levels of BMPR1A, BMPR1B, ACVR2A, and ACVR2B but did not affect ACVR1 and BMPRII, leading to the sustained effects of BMP6 and BMP7 in estrogen-treated MCF-7 cells. Estradiol rapidly activated MAPK phosphorylation including extracellular signal-regulated kinase 1/2, p38, and stress-activated protein kinase/c-Jun NH2-terminal kinase pathways and BMP6, BMP7, and activin preferentially inhibited estradiol-induced p38 phosphorylation. SB203580, a selective p38 MAPK inhibitor effectively suppressed estradiol-induced cell mitosis, suggesting that p38 MAPK plays a key role in estrogen-sensitive breast cancer cell proliferation. Thus, a novel interrelationship between estrogen and the breast cancer BMP system was uncovered, in which inhibitory effects of BMP6 and BMP7 on p38 signaling and steroid sulfatase expression were functionally involved in the suppression of estrogen-induced mitosis of breast cancer cells.