Bone morphogenetic proteins (BMPs) and activins are members of the TGFbeta superfamily of growth factors, a crucial group of regulators of induction and patterning of embryonic germ layers in metazoa. In early Xenopus embryos, activin, Vgl, and nodal are potent inducers of dorsal mesoderm, whereas BMPs can ventralize mesoderm, repress neural fate, and induce blood differentiation. These characteristic responses rely on ligand-specific signaling pathways, encompassing transmembrane kinase receptors and signal transducers belonging to the Smad family. The overexpression in Xenopus embryos of BMP-activated Smad1 and of activin/Vg1/ nodal-activated Smad2 is sufficient to specifically recapitulate ligand responses. In a search for determinants of a Smad specificity code, we have identified two small regions within the conserved carboxyl-domain that are necessary and sufficient for specific Smad action. Swapping both residue clusters (C1 and C2) between Smadl and Smad2 completely switches Smad effects in vivo. Thus, Smadl with swapped Smad2 clusters responds specifically to BMP but elicits an activin response, while a Smad2 protein containing the Smadl clusters is activated by activin and elicits a BMP response. Furthermore, association between Smads and FAST-1, a mediator of mesoderm induction by activin, is dependent upon the presence of the Smad2 C1 sequence. Finally, the Smadl-specific antagonist Smad6 can inhibit a Smad2 molecule harboring Smadl C1 and C2 sequences. Thus, the C1 and C2 regions of Smads specify the association between Smads and pathway-specific partners, such as FAST-1 and Smad6, and account for activin- and BMP- specific responses in vertebrate embryogenesis. (+info)
Synergistic signaling in fetal brain by STAT3-Smad1 complex bridged by p300.
The cytokines LIF (leukemia inhibitory factor) and BMP2 (bone morphogenetic protein-2) signal through different receptors and transcription factors, namely STATs (signal transducers and activators of transcription) and Smads. LIF and BMP2 were found to act in synergy on primary fetal neural progenitor cells to induce astrocytes. The transcriptional coactivator p300 interacts physically with STAT3 at its amino terminus in a cytokine stimulation-independent manner, and with Smad1 at its carboxyl terminus in a cytokine stimulation-dependent manner. The formation of a complex between STAT3 and Smad1, bridged by p300, is involved in the cooperative signaling of LIF and BMP2 and the subsequent induction of astrocytes from neural progenitors. (+info)
Cross-talk between the Smad1 and Ras/MEK signaling pathways for TGFbeta.
Our previous data demonstrated that Ras activation was necessary and sufficient for transforming growth factor-beta (TGFbeta)-mediated Erk1 activation, and was required for TGFbeta up-regulation of the Cdk inhibitors (CKI's) p27(Kip1) and p21(Cip1) (KM Mulder and SL Morris, J. Biol. Chem., 267, 5029-5031, 1992; MT Hartsough and KM Mulder, J. Biol. Chem., 270, 7117-7124, 1995; MT Hartsough et al., J. Biol. Chem., 271, 22368-22375, 1996 and J Yue et al., Oncogene, 17, 47-55, 1998). Here we examined the role of Ras in TGFbeta-mediated effects on a rat homolog of Smad1 (termed RSmad1). We demonstrate that both TGFbeta and bone morphogenetic protein (BMP) can induce endogenous Smad1 phosphorylation in intestinal epithelial cells (IECs). The combination of transient expression of RSmad1 and TGFbeta treatment had an additive effect on induction of the TGFbeta-responsive reporter 3TP-lux. Either inactivation of Ras by stable, inducible expression of a dominant-negative mutant of Ras (RasN17) or addition of MAP and ERK kinase (MEK) inhibitor PD98059 to cells significantly decreased the ability of both TGFbeta and BMP to induce phosphorylation of endogenous Smad1 in IECs. Moreover, either inactivation of Ras or addition of PD98059 to IEC 4-1 cells inhibited the ability of RSmad1 to regulate 3TP luciferase activity in both the presence and absence of TGFbeta. Collectively, our data indicate that TGFbeta can regulate RSmad1 function in epithelial cells, and that the Ras/MEK pathway is partially required for TGFbeta-mediated regulation of RSmad1. (+info)
Smad1 interacts with homeobox DNA-binding proteins in bone morphogenetic protein signaling.
Bone morphogenetic proteins (BMP) transduce their signals into the cell through a family of mediator proteins known as Smads. Upon phosphorylation by the BMP receptors, Smad1 interacts with Smad4 and translocates into the nucleus where the complex recruits DNA-binding protein(s) to activate specific gene transcription. However, the DNA-binding protein(s) involved in BMP signaling has not been identified. Using a yeast two-hybrid approach, we found that Smad1 interacts with Hoxc-8, a homeodomain transcription factor. The interaction between Smad1 and Hoxc-8 was confirmed by a "pull-down" assay and a co-immunoprecipitation experiment in COS-1 cells. Interestingly, purified Smad1 inhibited Hoxc-8 binding to the osteopontin Hoxc-8 site in a concentration-dependent manner. Transient transfection studies showed that native osteopontin promoter activity was elevated upon BMP stimulation. Consistent with the gel shift assay, overexpression of Hoxc-8 abolished the BMP stimulation. When a wild type or mutant Hoxc-8 binding element was linked to an SV40 promoter-driven reporter gene, the wild type but not the mutant Hoxc-8 binding site responded to BMP stimulation. Again, overexpression of Hoxc-8 suppressed the BMP-induced activity of the wild type reporter construct. Our findings suggest that Smad1 interaction with Hoxc-8 dislodges Hoxc-8 from its DNA binding element, resulting in the induction of gene expression. (+info)
Screening SMAD1, SMAD2, SMAD3, and SMAD5 for germline mutations in juvenile polyposis syndrome.
BACKGROUND AND AIMS: Juvenile polyps occur in several Mendelian disorders, whether in association with gastrointestinal cancer alone (juvenile polyposis syndrome, JPS) or as part of known syndromes (Cowden, Gorlin, and Bannayan-Zonana) in association with developmental abnormalities, dysmorphic features, or extraintestinal tumours. Recently, some JPS families were shown to harbour germline mutations in the SMAD4 (DPC4) gene, providing further evidence for the importance of the TGFbeta signalling pathway in colorectal cancer. There remains, however, considerable, unexplained genetic heterogeneity in JPS. Other members of the SMAD family are excellent candidates for JPS, especially SMAD2 (which, like SMAD4, is mutated somatically in colorectal cancers), SMAD3 (which causes colorectal cancer when "knocked out" in mice), SMAD5, and SMAD1. METHODS: SMAD1, SMAD2, SMAD3, and SMAD5 were screened for germline mutations in 30 patients with JPS and without SMAD4 mutations. RESULTS: No mutations were found in any of these genes. A G-A C89Y polymorphism with possible effects on protein function was found in SMAD3, but the frequencies of the G and A alleles did not differ between patients with JPS and controls. CONCLUSIONS: It remains to be determined whether or not this polymorphism is involved in a minor predisposition to colorectal or other carcinomas. SMAD4 may be the only member of the SMAD family which causes JPS when mutant in the germline. The other genes underlying JPS remain to be identified. (+info)
Roles of bone morphogenetic protein type I receptors and Smad proteins in osteoblast and chondroblast differentiation.
The biological effects of type I serine/threonine kinase receptors and Smad proteins were examined using an adenovirus-based vector system. Constitutively active forms of bone morphogenetic protein (BMP) type I receptors (BMPR-IA and BMPR-IB; BMPR-I group) and those of activin receptor-like kinase (ALK)-1 and ALK-2 (ALK-1 group) induced alkaline phosphatase activity in C2C12 cells. Receptor-regulated Smads (R-Smads) that act in the BMP pathways, such as Smad1 and Smad5, also induced the alkaline phosphatase activity in C2C12 cells. BMP-6 dramatically enhanced alkaline phosphatase activity induced by Smad1 or Smad5, probably because of the nuclear translocation of R-Smads triggered by the ligand. Inhibitory Smads, i.e., Smad6 and Smad7, repressed the alkaline phosphatase activity induced by BMP-6 or the type I receptors. Chondrogenic differentiation of ATDC5 cells was induced by the receptors of the BMPR-I group but not by those of the ALK-1 group. However, kinase-inactive forms of the receptors of the ALK-1 and BMPR-I groups blocked chondrogenic differentiation. Although R-Smads failed to induce cartilage nodule formation, inhibitory Smads blocked it. Osteoblast differentiation induced by BMPs is thus mediated mainly via the Smad-signaling pathway, whereas chondrogenic differentiation may be transmitted by Smad-dependent and independent pathways. (+info)
Smad1 domains interacting with Hoxc-8 induce osteoblast differentiation.
Bone morphogenetic proteins are potent osteotropic agents that induce osteoblast differentiation and bone formation. The signal transduction of bone morphogenetic proteins has recently been discovered to involve Smad proteins. Smad1 is an essential intracellular component that is specifically phosphorylated by bone morphogenetic protein receptors and translocated into the nucleus upon ligand stimulation. Previously, we have reported that Smad1 activates osteopontin gene expression in response to bone morphogenetic protein simulation through an interaction with a homeodomain transcription factor, Hoxc-8. In the present study, the interaction domains between the two proteins were characterized by deletional analysis in both yeast two-hybrid and gel shift assays. Two regions within the amino-terminal 87 amino acid residues of Smad1 were mapped to interact with Hoxc-8, one of which binds to the homeodomain. Overexpression of recombinant cDNAs encoding the Hoxc-8 interaction domains of Smad1 effectively activated osteopontin gene transcription in transient transfection assays. Furthermore, stable expression of these Smad1 fragments in 2T3 osteoblast precursor cells stimulated osteoblast differentiation-related gene expression and led to mineralized bone matrix formation. Our data suggest that the interaction of amino-terminal Smad1 with Hoxc-8 mimics bone morphogenetic protein signaling and is sufficient to induce osteoblast differentiation and bone cell formation. (+info)
Smad and AML proteins synergistically confer transforming growth factor beta1 responsiveness to human germ-line IgA genes.
Transcription of germ-line immunoglobulin heavy chain genes conditions them to participate in isotype switch recombination. Transforming growth factor-beta1 (TGF-beta1) stimulates promoter elements located upstream of the IgA1 and IgA2 switch regions, designated Ialpha1 and Ialpha2, and contributes to the development of IgA responses. We demonstrate that intracellular Smad proteins mediate activation of the Ialpha1 promoter by TGF-beta. TGF-beta type 1 receptor (ALK-5), activin type IB receptor (ALK-4), and the "orphan" ALK-7 trans-activate the Ialpha1 promoter, thus raising the possibility that other members of the TGF-beta superfamily can also modulate IgA synthesis. Smads physically interact with the AML family of transcription factors and cooperate with them to activate the Ialpha1 promoter. The Ialpha1 element provides a canape of interspersed high and low affinity sites for Smad and AML factors, some of which are indispensable for TGF-beta responsiveness. While AML.Smad complexes are formed in the cytoplasm of DG75 and K562 cells constitutively, only after TGF-beta receptor activation, novel Smad3.Smad4.AML complexes are detected in nuclear extracts by EMSA with Ialpha1 promoter-derived probes. Considering the wide range of biological phenomena that AMLs and Smads regulate, the physical/functional interplay between them has implications that extend beyond the regulation of class switching to IgA. (+info)