SANE,a novel LEM domain protein,regulates bone morphogenetic protein signaling through interaction with Smad1

Bone morphogenetic proteins (BMPs) are members of the transforming growth factor-beta (TGF-beta) superfamily that play important roles in bone formation, embryonic patterning, and epidermal-neural cell fate decisions. BMPs signal through pathway specific mediators such as Smads1 and 5, but the upstream regulation of BMP-specific Smads has not been fully characterized. Here we report the identification of SANE (Smad1 Antagonistic Effector), a novel protein with significant sequence similarity to nuclear envelop proteins such as MAN1. SANE binds to Smad1/5 and to BMP type I receptors and regulates BMP signaling. SANE specifically blocks BMP-dependent signaling in Xenopus embryos and in a mammalian model of bone formation but does not inhibit the TGF-beta/Smad2 pathway. Inhibition of BMP signaling by SANE requires interaction between SANE and Smad1, because a SANE mutant that does not bind Smad1 does not inhibit BMP signaling. Furthermore, inhibition appears to be mediated by inhibition of BMP-induced Smad1 phosphorylation, blocking ligand-dependent nuclear translocation of Smad1. These studies define a new mode of regulation for intracellular BMP/Smad1 signaling.     

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.     

Galectin-9 induces osteoblast differentiation through the CD44/Smad signaling pathway

Galectin-9 is a β-galactoside-binding lectin expressed in various tissues. It binds various glycoconjugates and modulates a variety of biological functions in various cell types. Although galectin-9 is expressed in bone, its function in human osteoblasts remains unclear. We demonstrate that galectin-9 induces osteoblast differentiation through the CD44/Smad signaling pathway in the absence of bone morphogenetic proteins (BMPs). Galectin-9 increases alkaline phosphatase activities in human osteoblasts and induces the phosphorylation of Smad1/5/8 and translocation of Smad4 to the nucleus in the absence of BMPs. Galectin-9 also induces binding of Smad4 to the Id1 promoter and increases its activity. Anti-CD44 antibody inhibits Smad1/5/8 phosphorylation by galectin-9. Galectin-9 binds to CD44 and induces the formation of a CD44/BMP receptor complex. Because Smad1 is phosphorylated by BMP receptors, we propose that formation of the CD44/BMP receptor complex induced by galectin-9 may provide a trigger for the activation of Smads.     

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.