Activin and TGFbeta limit murine primordial germ cell proliferation.
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Mammalian primordial germ cells (PGCs) proliferate as they migrate from their initial location in the extraembryonic mesoderm to the genital ridge, the gonadal anlage. Once in the genital ridge, PGCs cease dividing and differentiate according to their gender. To identify ligands that might limit PGC proliferation, we analyzed growth factor receptors encoded in RNA obtained from purified germ cells shortly after their arrival in the genital ridge. Receptors for two members of the TGFbeta superfamily were found, TGFbeta1 and activin. As the signal-transducing domains of both receptor systems are highly conserved, the effects of both TGFbeta1 and activin on PGCs would be expected to be similar. We found that both ligands limited the accumulation of germ cells in primary PGC cultures. BrdU incorporation assays demonstrated that either ligand inhibits PGC proliferation. These results suggest that these signal transduction pathways are important elements of the mechanism that determines germ cell endowment. (+info)
Expression of inhibin/activin subunits and their receptors and binding proteins in human preimplantation embryos.
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PURPOSE: Our purpose was to study the role of inhibin/activin during embryogenesis. METHODS: Transcripts of inhibin/activin subunits (alpha, beta A, beta B), activin receptors (types I and II), and follistatin were detected by a reverse transcriptase-polymerase chain reaction in human reproductive cells and preembryos cultured alone or co-cultured with human endometrial cells. RESULTS: Transcripts of alpha, beta A, beta B subunits were all detected in granulosa luteal cells, but only beta A units were detected in endometrial stromal and decidualized cells. In human preimplantation embryos, none of these subunits were detected in embryos from the four-cell to the morula stage and only beta A subunits were detectable in blastocyst embryos. Activin receptors were detectable in all of the studied embryos and cells. Transcripts of beta A, activin receptors, and follistatin were differentially expressed in human preimplantation embryos cultured in vitro and their expressions were significantly enhanced with the presence of endometrial stromal cells. CONCLUSIONS: Our data suggest that there is a possible endometrium-embryo interaction via endometrial activins and preimplantation embryo receptors and that the embryonic expressions of these activins, their receptors, and binding proteins are dependent on embryonic stage. (+info)
Assignment of transforming growth factor beta1 and beta3 and a third new ligand to the type I receptor ALK-1.
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Germ line mutations in one of two distinct genes, endoglin or ALK-1, cause hereditary hemorrhagic telangiectasia (HHT), an autosomal dominant disorder of localized angiodysplasia. Both genes encode endothelial cell receptors for the transforming growth factor beta (TGF-beta) ligand superfamily. Endoglin has homology to the type III receptor, betaglycan, although its exact role in TGF-beta signaling is unclear. Activin receptor-like kinase 1 (ALK-1) has homology to the type I receptor family, but its ligand and corresponding type II receptor are unknown. In order to identify the ligand and type II receptor for ALK-1 and to investigate the role of endoglin in ALK-1 signaling, we devised a chimeric receptor signaling assay by exchanging the kinase domain of ALK-1 with either the TGF-beta type I receptor or the activin type IB receptor, both of which can activate an inducible PAI-1 promoter. We show that TGF-beta1 and TGF-beta3, as well as a third unknown ligand present in serum, can activate chimeric ALK-1. HHT-associated missense mutations in the ALK-1 extracellular domain abrogate signaling. The ALK-1/ligand interaction is mediated by the type II TGF-beta receptor for TGF-beta and most likely through the activin type II or type IIB receptors for the serum ligand. Endoglin is a bifunctional receptor partner since it can bind to ALK-1 as well as to type I TGF-beta receptor. These data suggest that HHT pathogenesis involves disruption of a complex network of positive and negative angiogenic factors, involving TGF-beta, a new unknown ligand, and their corresponding receptors. (+info)
Bone morphogenetic proteins regulate the developmental program of human hematopoietic stem cells.
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The identification of molecules that regulate human hematopoietic stem cells has focused mainly on cytokines, of which very few are known to act directly on stem cells. Recent studies in lower organisms and the mouse have suggested that bone morphogenetic proteins (BMPs) may play a critical role in the specification of hematopoietic tissue from the mesodermal germ layer. Here we report that BMPs regulate the proliferation and differentiation of highly purified primitive human hematopoietic cells from adult and neonatal sources. Populations of rare CD34(+)CD38(-)Lin- stem cells were isolated from human hematopoietic tissue and were found to express the BMP type I receptors activin-like kinase (ALK)-3 and ALK-6, and their downstream transducers SMAD-1, -4, and -5. Treatment of isolated stem cell populations with soluble BMP-2, -4, and -7 induced dose-dependent changes in proliferation, clonogenicity, cell surface phenotype, and multilineage repopulation capacity after transplantation in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. Similar to transforming growth factor beta, treatment of purified cells with BMP-2 or -7 at high concentrations inhibited proliferation yet maintained the primitive CD34(+)CD38(-) phenotype and repopulation capacity. In contrast, low concentrations of BMP-4 induced proliferation and differentiation of CD34(+) CD38(-)Lin- cells, whereas at higher concentrations BMP-4 extended the length of time that repopulation capacity could be maintained in ex vivo culture, indicating a direct effect on stem cell survival. The discovery that BMPs are capable of regulating repopulating cells provides a new pathway for controlling human stem cell development and a powerful model system for studying the biological mechanism of BMP action using primary human cells. (+info)
A quantitative analysis of signal transduction from activin receptor to nucleus and its relevance to morphogen gradient interpretation.
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Previous work has shown that Xenopus blastula cells sense activin concentration by assessing the absolute number of occupied receptors per cell (100 and 300 molecules of bound activin activate Xbra and Xgsc transcription, respectively; a difference of only 3-fold). We now ask how quantitative differences in the absolute number of occupied receptors lead to the qualitatively distinct gene responses in the nucleus through SMAD2, a transducer of concentration-dependent gene responses to activin. We show that the injection of 0.2 or 0.6 ng of Smad2 mRNA activates Xbra or Xgsc transcription, respectively, involving, again, only a 3-fold difference. Furthermore, Xbra transcription is down-regulated by overexpression of SMAD2 as it is after activin signaling. We have developed a method to isolate nuclei from animal cap cells and subsequently have quantified the amount of nuclear SMAD2 protein. We find that the injection of 0.2 or 0.6 ng of Smad2 mRNA into an egg leads to only a 3-fold difference in the amount of SMAD2 protein in the nuclei of the blastula cells that express Xbra or Xgsc. We conclude that a 3-fold difference in the absolute number of occupied activin receptors can be maintained only as a 3-fold difference in the level of nuclear SMAD2 protein. Therefore, in this example of morphogen action, there appears to be no amplification of a key cytoplasmic transduction response, and a small but developmentally important change in extracellular signal concentration is relayed directly to the nucleus. (+info)
Activin family members in the developing chick retina: expression patterns, protein distribution, and in vitro effects.
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We have investigated whether the activin family of growth factors is involved in the regulation of retinal cell differentiation. Immunocytochemistry and in situ hybridization have shown that activin/inhibin subunits alpha, betaA, and betaB; receptors II and IIB; follistatin; and a follistatin-like gene are expressed in different regions of the chick embryo retina in developmentally regulated patterns. When tested in dissociated retinal cultures, activin did not appear to affect cell survival or proliferation, but it exerted marked inhibitory effects on the differentiation of photoreceptors, while stimulating the differentiation of nonphotoreceptor neurons; both effects were concentration-dependent and follistatin-sensitive. The results are consistent with the possibility that activin family members play significant roles in the regulation of retinal development. (+info)
Smad3 inhibits transforming growth factor-beta and activin signaling by competing with Smad4 for FAST-2 binding.
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Transcriptional regulation by transforming growth factor-beta and activin is mediated by interaction of Smad2 and Smad3 with specific transcription factors and/or DNA elements. However, Smad3 behaves differently from Smad2 in regulating transcription by a winged-helix transcription factor, FAST-2, on an activin-responsive element (ARE) in the Xenopus Mix.2 promoter. Smad3 alone was able to stimulate the ARE through FAST-2, but inhibited the ARE transactivation mediated by Smad2/Smad4 following receptor activation. We characterized the functional domains that are involved in these two activities of Smad3. Deletion of the MH1 domain as well as mutations of four lysine residues in the MH1 domain abrogated the inhibitory activity of Smad3, but did not compromise the self-stimulatory function. In contrast, deletion of the MH2 domain or a point mutation of glycine 379 within this domain obliterated the self-stimulatory activity of Smad3, but not the inhibitory activity. In an electrophoretic mobility shift assay, we found that Smad3 was able to associate with the FAST-2.ARE complex and that this association was dependent on FAST-2. In addition, Smad3 was not able to directly bind the ARE in a DNase I protection assay, in which FAST-2 binds the ARE around a motif (TGTGTATT) previously characterized to associate with the human FAST-1 protein. Interestingly, Smad4 was also able to directly associate with the FAST-2.ARE complex through binding with FAST-2. In a gel shift assay, the association of FAST-2 with Smad4 was mutually exclusive from the association with Smad3. Taken together, these data indicate that Smad3 exerts the inhibitory activity by competitive association with FAST-2. (+info)
Human activin-A is expressed in the atherosclerotic lesion and promotes the contractile phenotype of smooth muscle cells.
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Activin is a member of the transforming growth factor-beta superfamily, and it modulates the proliferation and differentiation of various target cells. In this study, we investigated the role of activin in the initiation and progression of human atherosclerosis. The expression of activin, its physiological inhibitor follistatin, and activin receptors were assayed in human vascular tissue specimens that represented various stages of atherogenesis. In situ hybridization experiments revealed activin mRNA in endothelial cells and macrophages and a strong induction of activin expression in neointimal smooth muscle cells from the early onset of atherogenesis. We developed an "in situ free-activin binding assay" by using biotinylated follistatin, which allowed us to detect bioactive activin at specific sites in atherosclerotic lesions. The mRNAs encoding the activin receptors are expressed similarly in normal and atherosclerotic tissue, which indicates that activin-A signaling in atherogenesis is most likely dependent on changes in growth factor concentrations rather than on receptor levels. In vitro, activin induces the contractile, nonproliferative phenotype in cultured smooth muscle cells, as is reflected by increased expression of smooth muscle-specific markers (SMalpha-actin and SM22alpha). Our data provide evidence that activin induces redifferentiation of neointimal smooth muscle cells, and we hypothesize that activin is involved in plaque stabilization. (+info)