Characterization of a bone morphogenetic protein-responsive Smad-binding element

Bone morphogenetic proteins (BMPs) are pleiotropic growth and differentiation factors belonging to the transforming growth factor-beta (TGF-beta) superfamily. Signals of the TGF-beta-like ligands are propagated to the nucleus through specific interaction of transmembrane serine/threonine kinase receptors and Smad proteins. GCCGnCGC has been suggested as a consensus binding sequence for Drosophila Mad regulated by a BMP-like ligand, Decapentaplegic. Smad1 is one of the mammalian Smads activated by BMPs. Here we show that Smad1 binds to this motif upon BMP stimulation in the presence of the common Smad, Smad4. The binding affinity is likely to be relatively low, because Smad1 binds to three copies of the motif weakly, but more repeats of the motif significantly enhance the binding. Heterologous reporter genes (GCCG-Lux) with multiple repeats of the motif respond to BMP stimulation but not to TGF-beta or activin. Mutational analyses reveal several bases critical for the responsiveness. A natural BMP-responsive reporter, pTlx-Lux, is activated by BMP receptors in P19 cells but not in mink lung cells. In contrast, GCCG-Lux responds to BMP stimulation in both cells, suggesting that it is a universal reporter that directly detects Smad phosphorylation by BMP receptors.     

An Atypical Canonical Bone Morphogenetic Protein (BMP) Signaling Pathway Regulates Msh Homeobox 1 (Msx1) Expression during Odontogenesis

Bone morphogenetic protein (BMP) signaling plays an essential role in early tooth development, evidenced by disruption of BMP signaling leading to an early arrested tooth development. Despite being a central mediator of BMP canonical signaling pathway, inactivation of Smad4 in dental mesenchyme does not result in early developmental defects. In the current study, we investigated the mechanism of receptor-activated Smads (R-Smads) and Smad4 in the regulation of the odontogenic gene Msx1 expression in the dental mesenchyme. We showed that the canonical BMP signaling is not operating in the early developing tooth, as assessed by failed activation of the BRE-Gal transgenic allele and the absence of phospho-(p)Smad1/5/8-Smad4 complexes. The absence of pSmad1/5/8-Smad4 complex appeared to be the consequence of saturation of Smad4 by pSmad2/3 in the dental mesenchyme as knockdown of Smad2/3 or overexpression of Smad4 led to the formation of pSmad1/5/8-Smad4 complexes and activation of canonical BMP signaling in dental mesenchymal cells. We showed that Smad1/5 but not Smad4 are required for BMP-induced expression of Msx1 in dental mesenchymal cells. We further presented evidence that in the absence of Smad4, BMPs are still able to induce pSmad1/5/8 nuclear translocation and their binding to the Msx1 promoter directly in dental mesenchymal cells. Our results demonstrate the functional operation of an atypical canonical BMP signaling (Smad4-independent and Smad1/5/8-dependent) pathway in the dental mesenchyme during early odontogenesis, which may have general implication in the development of other organs.     

Regulation of Hex gene expression by a Smads-dependent signaling pathway

The homeobox gene Hex is expressed in multiple cell types during embryogenesis and is required for liver and monocyte development. Hex is expressed in the foregut region of late gastrula avian and mammalian embryos in a pattern that overlaps with expression of bone morphogenetic proteins (BMPs). Here we investigate the relationship between BMP signaling and Hex gene expression. We find that Hex expression in avian anterior lateral endoderm is regulated by autocrine BMP signaling. Characterization of the mouse Hex gene promoter identified a 71-nucleotide BMP-responsive element (BRE) that is required for up-regulation of Hex by an activated BMP signaling pathway. The Hex BRE binds Smad4 and Smad1-Smad4 complexes in vitro, and in transfection assays, it is responsive to Smad1 and Smad4 but not to Smad2 and Smad4 or Smad3 and Smad4. The BRE contains two copies of a GCCGnCGC-like motif that in Drosophila is the binding site for Mad and Madea followed by two CAGAG boxes that are similar to sequences required for transforming growth factor-beta/activin responsiveness of several vertebrate genes. Mutation of the GC elements, but not the two CAGAG boxes, abolishes Smads responsiveness in the intact Hex promoter, whereas mutations in both the GC elements and CAGAG boxes show that they act cooperatively to confer Smads responsiveness to the Hex promoter. The Hex BRE can confer Smads responsiveness to a heterologous promoter, and in this context, both the GC-rich elements and the CAGAG boxes are required for Smads-dependent promoter activity. An element almost identical to the Hex BRE is present within the BMP-responsive Nkx2-5 gene promoter, suggesting that the Hex BRE represents a common response element for genes regulated by BMP signaling in the foregut region of the embryo.     

Drosophila Nemo antagonizes BMP signaling by phosphorylation of Mad and inhibition of its nuclear accumulation

Drosophila Nemo is the founding member of the Nemo-like kinase (Nlk) family of serine/threonine protein kinases that are involved in several Wnt signal transduction pathways. Here we report a novel function for Nemo in the inhibition of bone morphogenetic protein (BMP) signaling. Genetic interaction studies demonstrate that nemo can antagonize BMP signaling and can inhibit the expression of BMP target genes during wing development. Nemo can bind to and phosphorylate the BMP effector Mad. In cell culture, phosphorylation by Nemo blocks the nuclear accumulation of Mad by promoting export of Mad from the nucleus in a kinase-dependent manner. This is the first example of the inhibition of Drosophila BMP signaling by a MAPK and represents a novel mechanism of Smad inhibition through the phosphorylation of a conserved serine residue within the MH1 domain of Mad.     

MSX2 stimulates chondrocyte maturation by controlling Ihh expression

Several studies indicated that a homeobox gene, Msx2, is implicated in regulation of skeletal development by controlling enchondral ossification as well as membranous ossification. However, the molecular basis by which Msx2 conducts chondrogenesis is currently unclear. In this study, we examined the role of Msx2 in chondrocyte differentiation using mouse primary chondrocytes and embryonic metatarsal explants. Treatment with BMP2 up-regulated the expression of Msx2 mRNA along with chondrocyte differentiation in murine primary chondrocytes. Overexpression of wild-type Msx2 stimulated calcification of primary chondrocytes in the presence of BMP2. We also found that constitutively active Msx2 (caMsx2) enhanced BMP2-dependent calcification more efficiently than wild-type Msx2. Consistently, caMsx2 overexpression up-regulated the expression of alkaline phosphatase and collagen type X induced by BMP2. Furthermore, organ culture experiments using mouse embryonic metatarsals indicated that caMsx2 clearly stimulated the maturation of chondrocytes into the prehypertrophic and hypertrophic stages in the presence of BMP2. In contrast, knockdown of Msx2 inhibited maturation of primary chondrocytes. The stimulatory effect of Msx2 on chondrocyte maturation was enhanced by overexpression of Smad1 and Smad4 but inhibited by Smad6, an inhibitory Smad for BMP2 signaling. These data suggest that Msx2 requires BMP2/Smad signaling for its chondrogenic action. In addition, caMsx2 overexpression induced Ihh (Indian hedgehog) expression in mouse primary chondrocytes. Importantly, treatment with cyclopamine, a specific inhibitor for hedgehogs, blocked Msx2-induced chondrogenesis. Collectively, our results indicated that Msx2 promotes the maturation of chondrocytes, at least in part, through up-regulating Ihh expression.     

Msk is required for nuclear import of TGF-{beta}/BMP-activated Smads

Nuclear translocation of Smad proteins is a critical step in signal transduction of transforming growth factor beta (TGF-beta) and bone morphogenetic proteins (BMPs). Using nuclear accumulation of the Drosophila Smad Mothers against Decapentaplegic (Mad) as the readout, we carried out a whole-genome RNAi screening in Drosophila cells. The screen identified moleskin (msk) as important for the nuclear import of phosphorylated Mad. Genetic evidence in the developing eye imaginal discs also demonstrates the critical functions of msk in regulating phospho-Mad. Moreover, knockdown of importin 7 and 8 (Imp7 and 8), the mammalian orthologues of Msk, markedly impaired nuclear accumulation of Smad1 in response to BMP2 and of Smad2/3 in response to TGF-beta. Biochemical studies further suggest that Smads are novel nuclear import substrates of Imp7 and 8. We have thus identified new evolutionarily conserved proteins that are important in the signal transduction of TGF-beta and BMP into the nucleus.     

Flexible interaction of Drosophila Smad complexes with bipartite binding sites

A subset of BMP-responsive enhancer elements are characterized by pairing of a GC-rich Smad1 binding site and an SBE-type Smad4 binding site. Such paired, or bipartite, sites are in some cases just 5 bp apart and thus might be contacted by a single Smad1-Smad4 complex. Other potential pairings are separated as much as 60 bp but it is not known whether such longer distances can be spanned by a Smad1-Smad4 complex, indeed binding of native Smad1-Smad4 complexes to any of these bipartite elements has yet to be reported. Here we report that a complex of the homologous Drosophila Smad proteins, Mad and Medea, is capable of concerted binding to GC-rich and SBE sites separated by as much as 20 bp. The wider the separation, the more severely binding affinity was reduced by shortening of the linker region that tethers the DNA binding domain of Medea. In contrast, length of the Mad linker did not affect the allowed distance between paired sites, rather it contributes specifically to Mad contact with the GC-rich site. Finally, we show that Smad1 and Smad4 can participate in binding to bipartite sites.