Characterization of receptors for osteogenic protein-1/bone morphogenetic protein-7 (OP-1/BMP-7) in rat kidneys.
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BACKGROUND: Osteogenic protein-1/bone morphogenetic protein-7 (OP-1/BMP-7), a member of the transforming growth factor-beta superfamily, has been shown to prevent kidney damage from ischemia/reperfusion injury in rats. The molecular events involved in OP-1 action on kidney are not yet understood. METHODS: In this study, we evaluated the biodistribution of (125)I-labeled OP-1 in rat kidneys. Adult rats received a single intravenous injection of 250 microg (125)I-labeled OP-1 per kg body wt, a dose that was effective in protecting kidneys from ischemic injury. Tissue localization, in situ hybridization, and immunostaining with a specific receptor antibody were performed to identify OP-1 cellular targets. Also, isolated plasma membranes from kidney cortex and medulla regions were analyzed to identify and characterize receptor structural components that recognize OP-1. RESULTS: At 10 and 180 minutes following injection, the relative uptake of (125)I-labeled OP-1 was consistently higher in kidney cortex than in medulla region. Upon autoradiography, kidney tissue sections revealed that OP-1 bound to the convoluted tubule epithelium, glomeruli, and collecting ducts. Moreover, in situ hybridization and immunostaining methods have shown localization of mRNA transcripts and the protein for BMP receptor type II in the cortex and medulla in similar areas as (125)I-labeled OP-1. Bulk membranes (enriched with plasma membranes) isolated from the cortex and medulla regions of kidney each bound specifically to (125)I-OP-1, and the binding of (125)I-labeled OP-1 was inhibited by unlabeled OP-1 in a dose-dependent manner. However, platelet-derived growth factor, transforming growth factor-beta, insulin-like growth factor, fibroblast growth factors, and other members of BMP family such as BMP-2 and cartilage-derived morphogenetic protein-1/growth and differentiation factor-5 (CDMP-1/GDF-5) failed to inhibit the binding of (125)I-labeled OP-1 to receptors, suggesting a high degree of specificity with which OP-1 bound to kidney receptors. Scatchard analysis of quantitative binding data indicated that the OP-1 receptors of kidney contained a single class of high-affinity binding sites for OP-1 with an association constant (Ka) of 2.26 x 109 mol/L-1 and a binding capacity of 1.01 pmol of OP-1 per mg membrane protein. When analyzed by a ligand blot technique, plasma membranes isolated from kidney cortex and medulla each showed the presence of a prominent specific band with a relative molecular mass (Mr) of 100 kD. Further analysis by Western blotting indicated that an antibody raised against BMP type II receptor effectively recognized the 100 kD OP-1 binding component of kidney plasma membranes. CONCLUSIONS: We demonstrated, to our knowledge for the first time, the presence of membrane-bound, specific, high-affinity OP-1 receptors in rat kidney tissues, which are likely to mediate OP-1 actions in the kidney. The major OP-1-binding component of the kidney appears to be a long form of BMP type II receptor with a Mr of 100 kD. In vivo and in vitro evidence suggests that the cellular targets for OP-1 are convoluted tubule epithelium, glomeruli, and collecting ducts. OP-1 does not share receptor binding properties with other growth factors, including BMP-2 and CDMP-1, suggesting that its mode of action in kidney appears to be specific. (+info)
BMP and FGF regulate the development of EGF-responsive neural progenitor cells.
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Temporal changes in progenitor cell responses to extrinsic signals play an important role in development, but little is known about the mechanisms that determine how these changes occur. In the rodent CNS, expression of epidermal growth factor receptors (EGFRs) increases during embryonic development, conferring mitotic responsiveness to EGF among multipotent stem cells. Here we show that cell-cell signaling controls this change. Whereas EGF-responsive stem cells develop on schedule in explant and aggregate cultures of embryonic cortex, co-culture with younger cortical cells delays their development. Exogenous BMP4 mimics the effect of younger cells, reversibly inhibiting changes in EGFR expression and responsiveness. Moreover, blocking endogenous BMP receptors in progenitors with a virus transducing dnBMPR1B accelerates changes in EGFR signaling. This involves a non-cell-autonomous mechanism, suggesting that BMP negatively regulates signal(s) that promote the development of EGF-responsive stem cells. FGF2 is a good candidate for such a signal, as we find that it antagonizes the inhibitory effects of younger cortical cells and exogenous BMP4. These findings suggest that a balance between antagonistic extrinsic signals regulates temporal changes in an intrinsic property of neural progenitor cells. (+info)
Transcriptional mechanisms of bone morphogenetic protein-induced osteoprotegrin gene expression.
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Osteoprotegerin (OPG), an osteoblast-secreted decoy receptor, specifically binds to osteoclast differentiation factor and inhibits osteoclast maturation. Members of the transforming growth factor-beta superfamily including bone morphogenetic proteins (BMPs) stimulate OPG mRNA expression. In this study, we have characterized the transcription mechanism of BMP-induced OPG gene expression. Transfection of Smad1 and a constitutively active BMP type IA receptor ALK3 (Q233) stimulated the OPG promoter. Deletion analysis of the OPG promoter identified two Hoxc-8 binding sites that respond to BMP stimulation. Glutathione S-transferase-Hoxc-8 protein binds to these two Hox sites specifically. Consistent with the transfection results of the native promoter, ALK3 or Smad1 linker region, which interacts with Hoxc-8, stimulated the activation of the reporter construct with the two Hox sites. Overexpression of Hoxc-8 inhibited the induced promoter activity. When the two Hox binding sites were mutated, ALK3 or Smad1 linker region no longer activated the transcription. Importantly, Smad1 linker region induced both OPG promoter activity and endogenous OPG protein expression in 2T3 osteoblastic cells. The medium from cells transfected with Smad1 linker region expression plasmid effectively inhibited osteoclastogenesis. Collectively, our data indicate that Hox sites mediate both OPG promoter construct activity and endogenous OPG gene expression in response to BMP stimulation. (+info)
Noggin antagonizes BMP signaling to create a niche for adult neurogenesis.
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Large numbers of new neurons are born continuously in the adult subventricular zone (SVZ). The molecular niche of SVZ stem cells is poorly understood. Here, we show that the bone morphogenetic protein (BMP) antagonist Noggin is expressed by ependymal cells adjacent to the SVZ. SVZ cells were found to express BMPs as well as their cognate receptors. BMPs potently inhibited neurogenesis both in vitro and in vivo. BMP signaling cell-autonomously blocked the production of neurons by SVZ precursors by directing glial differentiation. Purified mouse Noggin protein promoted neurogenesis in vitro and inhibited glial cell differentiation. Ectopic Noggin promoted neuronal differentiation of SVZ cells grafted to the striatum. We thus propose that ependymal Noggin production creates a neurogenic environment in the adjacent SVZ by blocking endogenous BMP signaling. (+info)
Lost-a-fin encodes a type I BMP receptor, Alk8, acting maternally and zygotically in dorsoventral pattern formation.
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TGFbeta signaling pathways of the bone morphogenetic protein (BMP) subclass are essential for dorsoventral pattern formation of both vertebrate and invertebrate embryos. Here we determine by chromosomal mapping, linkage analysis, cDNA sequencing and mRNA rescue that the dorsalized zebrafish mutant lost-a-fin (laf) is defective in the gene activin receptor-like kinase 8 (alk8), which encodes a novel type I TGFbeta receptor. The alk8 mRNA is expressed both maternally and zygotically. Embyros that lack zygotic, but retain maternal Laf/Alk8 activity, display a weak dorsalization restricted to the tail and die by 3 days postfertilization. We rescued the laf dorsalized mutant phenotype by alk8 mRNA injection and generated homozygous laf/alk8 mothers to investigate the maternal role of Laf/Alk8 activity. Adult fish lacking Laf/Alk8 activity are fertile, exhibit a growth defect and are significantly smaller than their siblings. Embryos derived from homozygous females, which lack both maternal and zygotic Laf/Alk8 activity, display a strongly dorsalized mutant phenotype, no longer limited to the tail. These mutant embryos lack almost all gastrula ventral cell fates, with a concomitant expansion of dorsal cell types. During later stages, most of the somitic mesoderm and neural tissue circumscribe the dorsoventral axis of the embryo. Zygotic laf/alk8 mutants can be rescued by overexpression of the BMP signal transducer Smad5, but not the Bmp2b or Bmp7 ligands, consistent with the Laf/Alk8 receptor acting within a BMP signaling pathway, downstream of a Bmp2b/Bmp7 signal. Antibodies specific for the phosphorylated, activated form of Smad1/5, show that BMP signaling is nearly absent in gastrula lacking both maternal and zygotic Laf/Alk8 activity, providing further evidence that Laf/Alk8 transduces a BMP signal. In total, our work strongly supports the role of Laf/Alk8 as a type I BMP receptor required for the specification of ventral cell fates. (+info)
Restricting Bmp-4 mediated apoptosis in hindbrain neural crest.
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The segregation of the rhombencephalic neural crest into three streams, destined for particular pharyngeal arches, is a prominent feature of the developing vertebrate head and is likely necessary for normal morphogenesis. At least in part, the segregation of the crest into these streams involves the focal depletion of neural crest from rhombomeres 3 and 5 through large-scale apoptosis, mediated by the induction of Bmp-4 expression in the crest primordia of these two rhombomeres. Previously we found that in contrast to rhombomeres 3 and 5, the intervening segment rhombomere 4 was not susceptible to Bmp-4-sponsored neural crest cell death. To analyse the reason for this difference, we have isolated clones for the necessary components of the Bmp-4 signal transduction apparatus. We find that the hindbrain neural crest generally is primed to respond to Bmp-4 and that in vitro, besides rhombomere 3 and 5, rhombomeres 2 and 6 are also sensitive to Bmp-4-sponsored death. As before, however, we find that rhombomere 4 will not respond to this factor. Our analysis of the Bmp-4 antagonists has uncovered a reason for this. Rhombomere 4 expresses elevated levels of the antagonist Noggin at its dorsal midline just as crest production from this segment commences and, as development proceeds, in the crest that migrates away from there. At these later stages, expression also becomes apparent in the migratory post-otic crest as it flows by the otic vesicle. An interesting feature of these domains of Noggin expression is that they lie between territories of Bmp-4 expression, suggesting that Noggin is acting to protect from Bmp-4-mediated cell death. (+info)
Synergistic effects of different bone morphogenetic protein type I receptors on alkaline phosphatase induction.
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Bone morphogenetic proteins (BMPs) are members of the transforming growth factor-beta superfamily, which regulate the differentiation of osteoprogenitor cells. Here we show that among members of the BMP family, BMP-4 and growth/differentiation factor 5 (GDF-5) induce osteoblast differentiation through the activation of three receptor-regulated Smads (i.e. Smad1, Smad5 and Smad8). By contrast, BMP-6 and BMP-7 induce alkaline phosphatase activity through Smad1 and Smad5, but not through Smad8. Consistent with these findings, BMP-4 induced phosphorylation and nuclear translocation of Smad1, Smad5 and Smad8, but BMP-6 activated only Smad1 and Smad5. BMP-4 and GDF-5 are known to bind to activin receptor-like kinase 3 (ALK-3) and/or ALK-6 (also termed BMP type IA and type IB receptors, respectively), whereas BMP-6 and BMP-7 preferentially bind to ALK-2. Compared with the effects induced by only one of the type I receptors, the combination of constitutively active forms of ALK-2 and ALK-3 (or ALK-6) more strongly induced alkaline phosphatase activity in C2C12 cells. Moreover, addition of BMP-4 and BMP-6 to C2C12 cells resulted in higher alkaline phosphatase activity than that of only one of these BMPs. The combination of ALK-2 and ALK-3 also induced higher transcriptional activity than either receptor alone. Thus, ALK-2 and ALK-3 (or ALK-6) might synergistically induce osteoblast differentiation of C2C12 cells, possibly through efficient activation of downstream signaling pathways. (+info)
Mutation in bone morphogenetic protein receptor-IB is associated with increased ovulation rate in Booroola Merino ewes.
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Ewes from the Booroola strain of Australian Merino sheep are characterized by high ovulation rate and litter size. This phenotype is due to the action of the FecB(B) allele of a major gene named FecB, as determined by statistical analysis of phenotypic data. By genetic analysis of 31 informative half-sib families from heterozygous sires, we showed that the FecB locus is situated in the region of ovine chromosome 6 corresponding to the human chromosome 4q22-23 that contains the bone morphogenetic protein receptor IB (BMPR-IB) gene encoding a member of the transforming growth factor-beta (TGF-beta) receptor family. A nonconservative substitution (Q249R) in the BMPR-IB coding sequence was found to be associated fully with the hyperprolificacy phenotype of Booroola ewes. In vitro, ovarian granulosa cells from FecB(B)/FecB(B) ewes were less responsive than granulosa cells from FecB(+)/FecB(+) ewes to the inhibitory effect on steroidogenesis of GDF-5 and BMP-4, natural ligands of BMPR-IB. It is suggested that in FecB(B)/FecB(B) ewes, BMPR-IB would be inactivated partially, leading to an advanced differentiation of granulosa cells and an advanced maturation of ovulatory follicles. (+info)