BMP2 is required for early heart development during a distinct time period.
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BMP2, like its Drosophila homologue dpp, is an important signaling molecule for specification of cardiogenic mesoderm in vertebrates. Here, we analyzed the time-course of BMP2-requirement for early heart formation in whole chick embryos and in explants of antero-lateral plate mesoderm. Addition of Noggin to explants isolated at stage 4 and cultured for 24 h resulted in loss of NKX2.5, GATA4, eHAND, Mef2A and vMHC expression. At stages 5-8 the individual genes showed differential sensitivity to Noggin addition. While expression of eHAND, NKX2.5 and Mef2A was clearly reduced by Noggin vMHC was only marginally affected. In contrast, GATA4 expression was enhanced after Noggin treatment. The developmental period during which cardiac mesoderm required the presence of BMP signaling in vivo was assessed by implantation of Noggin expressing cells into stage 4-8 embryos which were then cultured until stage 10-11. Complete loss of NKX2.5 and eHAND expression was observed in embryos implanted at stages 4-6, and expression was still suppressed in stages 7 and 8 implanted embryos. GATA4 expression was also blocked by Noggin at stage 4, however increased at stages 5, 6 and 7. Explants of central mesendoderm, that normally do not form heart tissue were employed to study the time-course of BMP2-induced cardiac gene expression. The induction of cardiac lineage markers in central mesendoderm of stage 5 embryos was distinct for different genes. While GATA4, -5, -6 and MEF2A were induced to maximal levels within 6 h after BMP2 addition, eHAND and dHAND required 12 h to reach maximum levels of expression. NKX2.5 was induced by 6 h and accumulated over 48 h. vMHC and titin were induced at significant levels only after 48 h of BMP2 addition. These results indicate that cardiac marker genes display distinct expression kinetics after BMP2 addition and differential response to Noggin treatment suggesting complex regulation of myocardial gene expression in the early tubular heart. (+info)
MFH-1 is required for bone morphogenetic protein-2-induced osteoblastic differentiation of C2C12 myoblasts.
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Mesenchyme forkhead-1 (MFH-1), a winged helix/forkhead transcription factor, is expressed in developing cartilaginous tissues, kidney and arch arteries, and is essential for the normal development of the axial skeleton and aortic arch formation of mice. To investigate the possible role of MFH-1 in osteogenesis and osteoblast differentiation, we examined expression of MFH-1 induced by bone morphogenetic protein-2 (BMP-2) in C2C12 myoblasts, and found that MFH-1 protein and also MFH-1 mRNA increased markedly in C2C12 cells after treatment with BMP-2. To confirm the hypothesis that BMP-2 induced osteoblastic differentiation of C2C12 cells by increasing MFH-1 expression, we lowered the endogenous MFH-1 level by stably transfecting C2C12 cells with antisense MFH-1 sequence, and found that in antisense MFH-1 cell lines, both alkaline phosphatase (ALP) activity and production of osteocalcin induced by BMP-2 decreased markedly in comparison with control cell lines. Our results suggest that the BMP-2-induced MFH-1 protein may play a key role in regulating the commitment to osteoblastic differentiation of C2C12 myoblasts and production of osteoblast markers including ALP and osteocalcin. (+info)
Embryonic stem cell-derived chondrogenic differentiation in vitro: activation by BMP-2 and BMP-4.
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Differentiation of mouse embryonic stem (ES) cells via embryoid bodies was established as a suitable model to study development in vitro. Here, we show that differentiation of ES cells in vitro into chondrocytes can be modulated by members of the transforming growth factor-beta family (TGF-beta(1), BMP-2 and -4). ES cell differentiation into chondrocytes was characterized by the appearance of Alcian blue-stained areas and the expression of cartilage-associated genes and proteins. Different stages of cartilage differentiation could be distinguished according to the expression pattern of the transcription factor scleraxis, and the cartilage matrix protein collagen II. The number of Alcian-blue-stained areas decreased slightly after application of TGF-beta(1), whereas BMP-2 or -4 induced chondrogenic differentiation. The inducing effect of BMP-2 was found to be dependent on the time of application, consistent with its role to recruit precursor cells to the chondrogenic fate. (+info)
IHG-2, a mesangial cell gene induced by high glucose, is human gremlin. Regulation by extracellular glucose concentration, cyclic mechanical strain, and transforming growth factor-beta1.
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We used cloning in silico coupled with polymerase chain reaction to demonstrate that IHG-2 is part of the 3'-untranslated region of gremlin, a member of the DAN family of secreted proteins that antagonize the bioactivities of members of the transforming growth factor (TGF)-beta superfamily. Mesangial cell gremlin mRNA levels were induced by high glucose, cyclic mechanical strain, and TGF-beta1 in vitro, and gremlin mRNA levels were elevated in the renal cortex of rats with streptozotocin-induced diabetic nephropathy in vivo. gremlin expression was observed in parallel with induction of bone morphogenetic protein-2 (BMP-2), a target for gremlin in models of cell differentiation. Together these data indicate that (a) IHG-2 is gremlin, (b) gremlin is expressed in diabetic nephropathy in vivo, (c) both glycemic and mechanical strain stimulate mesangial cell gremlin expression in vitro, (d) high glucose induces gremlin, in part, through TGFbeta-mediated pathways, and (e) Gremlin is a potential endogenous antagonist of BMPs within a diabetic glomerular milieu. (+info)
BMP2-induced apoptosis is mediated by activation of the TAK1-p38 kinase pathway that is negatively regulated by Smad6.
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Bone morphogenetic protein 2 (BMP2), a member of the transforming growth factor-beta (TGF-beta) superfamily, regulates a variety of cell fates and functions. At present, the molecular mechanism by which BMP2 induces apoptosis has not been fully elucidated. Here we propose a BMP2 signaling pathway that mediates apoptosis in mouse hybridoma MH60 cells whose growth is interleukin-6 (IL-6)-dependent. BMP2 dose-dependently induces apoptosis in MH60 cells even in the presence of IL-6. BMP2 has no inhibitory effect on the IL-6-induced tyrosine phosphorylation of STAT3, and the bcl-2 gene expression which is known to be regulated by STAT3, suggesting that BMP2-induced apoptosis is not attributed to alteration of the IL-6-mediated bcl-2 pathway. We demonstrate that BMP2 induces activation of TGF-beta-activated kinase (TAK1) and subsequent phosphorylation of p38 stress-activated protein kinase. In addition, forced expression of kinase-negative TAK1 in MH60 cells blocks BMP2-induced apoptosis. These results indicate that BMP2-induced apoptosis is mediated through the TAK1-p38 pathway in MH60 cells. We also show that MH60-derived transfectants expressing Smad6 are resistant to the apoptotic signal of BMP2. Interestingly, this ectopic expression of Smad6 blocks BMP2-induced TAK1 activation and p38 phosphorylation. Moreover, Smad6 can directly bind to TAK1. These findings suggest that Smad6 is likely to function as a negative regulator of the TAK1 pathway in the BMP2 signaling, in addition to the previously reported Smad pathway. (+info)
Integration of FGF and TWIST in calvarial bone and suture development.
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Mutations in the FGFR1-FGFR3 and TWIST genes are known to cause craniosynostosis, the former by constitutive activation and the latter by haploinsufficiency. Although clinically achieving the same end result, the premature fusion of the calvarial bones, it is not known whether these genes lie in the same or independent pathways during calvarial bone development and later in suture closure. We have previously shown that Fgfr2c is expressed at the osteogenic fronts of the developing calvarial bones and that, when FGF is applied via beads to the osteogenic fronts, suture closure is accelerated (Kim, H.-J., Rice, D. P. C., Kettunen, P. J. and Thesleff, I. (1998) Development 125, 1241-1251). In order to investigate further the role of FGF signalling during mouse calvarial bone and suture development, we have performed detailed expression analysis of the splicing variants of Fgfr1-Fgfr3 and Fgfr4, as well as their potential ligand Fgf2. The IIIc splice variants of Fgfr1-Fgfr3 as well as the IIIb variant of Fgfr2 being expressed by differentiating osteoblasts at the osteogenic fronts (E15). In comparison to Fgf9, Fgf2 showed a more restricted expression pattern being primarily expressed in the sutural mesenchyme between the osteogenic fronts. We also carried out a detailed expression analysis of the helix-loop-helix factors (HLH) Twist and Id1 during calvaria and suture development (E10-P6). Twist and Id1 were expressed by early preosteoblasts, in patterns that overlapped those of the FGF ligands, but as these cells differentiated their expression dramatically decreased. Signalling pathways were further studied in vitro, in E15 mouse calvarial explants. Beads soaked in FGF2 induced Twist and inhibited Bsp, a marker of functioning osteoblasts. Meanwhile, BMP2 upregulated Id1. Id1 is a dominant negative HLH thought to inhibit basic HLH such as Twist. In Drosophila, the FGF receptor FR1 is known to be downstream of Twist. We demonstrated that in Twist(+/)(-) mice, FGFR2 protein expression was altered. We propose a model of osteoblast differentiation integrating Twist and FGF in the same pathway, in which FGF acts both at early and late stages. Disruption of this pathway may lead to craniosynostosis. (+info)
Regulation of glial cell line-derived neurotrophic factor responsiveness in developing rat sympathetic neurons by retinoic acid and bone morphogenetic protein-2.
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There are several lines of evidence suggesting that, in addition to neurotrophins, member(s) of glial cell line-derived neurotrophic factor (GDNF) family play important roles in the development of sympathetic neurons. However, the mechanism regulating the responsiveness of the neurons to GDNF family members is not known. Previously, we reported on the cooperative roles of bone morphogenetic protein-2 (BMP2) and retinoic acid (RA) in the enhancement of neurotrophin-3 (NT3) responsiveness in cultured sympathetic neurons dissociated from perinatal rat superior cervical ganglia (SCG). In the present study, we further examined the effects of BMP2 and RA on the regulation of the responsiveness of SCG neurons to GDNF family members. Consequently, we found that RA alone induced the responsiveness of SCG neurons specifically to GDNF by upregulating the ligand-specifying receptor for GDNF (GFRalpha-1) at both the mRNA and protein levels. The expression levels of mRNAs for other ligand-specifying receptors for GDNF family (GFRalpha-2 and GFRalpha-3) were unaffected by RA. Although the upregulation of signal-transducing receptor Ret by the RA treatment was rather small, this treatment significantly increased the efficacy of tyrosine phosphorylation of Ret by GDNF. Experiments using synthetic retinoids suggested that RA acts through alpha-type of nuclear retinoic acid receptor to exert the induction of GDNF responsiveness. On the other hand, BMP2, which had no significant effect by itself on the GDNF responsiveness, promoted the action of RA to upregulate GFRalpha-1 and enhance the GDNF responsiveness. These results indicate that RA and BMP2 play important roles in the induction of GDNF responsiveness, as well as NT3 responsiveness, of developing SCG neurons. (+info)
Cyclic AMP signaling functions as a bimodal switch in sympathoadrenal cell development in cultured primary neural crest cells.
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Cells of the vertebrate neural crest (crest cells) are an invaluable model system to address cell fate specification. Crest cells are amenable to tissue culture, and they differentiate to a variety of neuronal and nonneuronal cell types. Earlier studies have determined that bone morphogenetic proteins (BMP-2, -4, and -7) and agents that elevate intracellular cyclic AMP (cAMP) stimulate the development of the sympathoadrenal (SA, adrenergic) lineage in neural crest cultures. To investigate whether interactive mechanisms between signaling pathways influence crest cell differentiation, we characterized the combinatorial effects of BMP-2 and cAMP-elevating agents on the development of quail trunk neural crest cells in primary culture. We report that the cAMP signaling pathway modulates both positive and negative signals influencing the development of SA cells. Specifically, we show that moderate activation of cAMP signaling promotes, in synergy with BMP-2, SA cell development and the expression of the SA lineage-determining gene Phox2a. By contrast, robust activation of cAMP signaling opposes, even in the presence of BMP-2, SA cell development and the expression of the SA lineage-determining ASH-1 and Phox2 genes. We conclude that cAMP signaling acts as a bimodal regulator of SA cell development in neural crest cultures. (+info)