Molecules mimicking Smad1 interacting with Hox stimulate bone formation

Bone morphogenetic proteins (BMPs) induce osteoblast differentiation and bone formation. Smads, a group of functionally and structurally related intracellular effectors, mediate signaling initiated by BMPs and regulate cell definite commitment. Previously, we showed that Smad1 activates osteopontin and osteoprotegerin gene expression by dislodging Hoxc-8 from its DNA binding sites. A domain of Smad1, termed Smad1C, was characterized as interacting with Hoxc-8 and then crippling its DNA-binding ability. Ectopic expression of Smad1C is able to bypass BMP signaling in the induction of osteoblast differentiation and bone formation in vitro. To test the function of Smad1C on osteogenesis in vivo, we generated transgenic mice in which Smad1C expression was induced with doxycycline and localized in bone by using a tetracycline-inducible expression system (Tet-on) modified with a bone-specific gene promoter, type I collagen alpha1. The mice expressing Smad1C showed increased skeletal bone mineral density compared with their littermates. Bone histomorphometric analysis of mouse tibiae showed that Smad1C significantly increases trabecular bone area and length of trabecular surface covered with osteoid and up-regulates bone marker gene (OPN, Cbfa1, Col I alpha1, BSP, ALP) expression in vivo. Moreover, stromal cells isolated from mice expressing Smad1C displayed a higher potential for differentiating into osteoblasts than the other mice. These results indicate that Smad1C mimics BMPs in the induction of osteogenesis in vivo. Most important, using a high throughput screening assay based on mimicking Smad1C's displacement of Hoxc-8 binding to DNA, we identified chemical entities that exhibit bone anabolic activity in cell and bone organ cultures, suggesting the possibility that the compounds may be used as bone anabolic agents to treat bone pathologies.     

Mouse smad8 phosphorylation downstream of BMP receptors ALK-2, ALK-3, and ALK-6 induces its association with Smad4 and transcriptional activity

Smads are intracellular signaling mediators for TGF-beta superfamily. Smad1 and Smad5 are activated by BMP receptors. Here, we have cloned mouse Smad8 and functionally characterized its ability to transduce signals from BMP receptors. Constitutively active BMP type I receptors, ALK-3 and ALK-6, as well as ALK-2, were phosphorylated Smad8 and induced Smad8 interaction with Smad4. Nuclear translocation of Smad8 was stimulated by constitutively active BMP type I receptors. In contrast, constitutively active TGF-beta type I receptor, ALK-5, did not exhibit any action on Smad8. Smad8 and Smad4 cooperatively induced the promoter of Xvent2, a homeobox gene that responds specifically to BMP signaling. Dominant-negative Smad8 was shown to inhibit the increase of alkaline phosphatase activity induced by BMP-2 on pluripotent mesenchymal C3H10T1/2 and myoblastic C2C12 cell lines. The presence of Smad8 mRNA in mouse calvaria cells and osteoblasts suggests a role of Smad8 in the osteoblast differentiation and maturation.     

SnoN facilitates ALK1–Smad1/5 signaling during embryonic angiogenesis

In endothelial cells, two type I receptors of the transforming growth factor β (TGF-β) family, ALK1 and ALK5, coordinate to regulate embryonic angiogenesis in response to BMP9/10 and TGF-β. Whereas TGF-β binds to and activates ALK5, leading to Smad2/3 phosphorylation and inhibition of endothelial cell proliferation and migration, BMP9/10 and TGF-β also bind to ALK1, resulting in the activation of Smad1/5. SnoN is a negative regulator of ALK5 signaling through the binding and repression of Smad2/3. Here we uncover a positive role of SnoN in enhancing Smad1/5 activation in endothelial cells to promote angiogenesis. Upon ligand binding, SnoN directly bound to ALK1 on the plasma membrane and facilitated the interaction between ALK1 and Smad1/5, enhancing Smad1/5 phosphorylation. Disruption of this SnoN–Smad interaction impaired Smad1/5 activation and up-regulated Smad2/3 activity. This resulted in defective angiogenesis and arteriovenous malformations, leading to embryonic lethality at E12.5. Thus, SnoN is essential for TGF-β/BMP9-dependent biological processes by its ability to both positively and negatively modulate the activities of Smad-dependent pathways.     

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.     

Serine-204 in the linker region of Smad3 mediates the collagen-I response to TGF-β in a cell phenotype-specific manner

Regulation of TGF-β1/Smad3 signaling in fibrogenesis is complex. Previous work by our lab suggests that ERK MAP kinase phosphorylates the linker region (LR) of Smad3 to enhance TGF-β-induced collagen-I accumulation. However the roles of the individual Smad3LR phosphorylation sites (T179, S204, S208 and S213) in the collagen-I response to TGF-β are not clear. To address this issue, we tested the ability of Smad3 constructs expressing wild-type Smad3 or Smad3 with mutated LR phosphorylation sites to reconstitute TGF-β-stimulated COL1A2 promoter activity in Smad3-null or -knockdown cells. Blocking ERK in fibroblasts and renal mesangial cells inhibited both S204 phosphorylation and Smad3-mediated COL1A2 promoter activity. Mutations replacing serine at S204 or S208 in the linker region decreased Smad3-mediated COL1A2 promoter activity, whereas mutating T179 enhanced basal COL1A2 promoter activity and did not prevent TGF-β stimulation. Interestingly, mutation of all four Smad3LR sites (T179, S204, S208 and S213) was not inhibitory, suggesting primacy of the two inhibitory sites. These results suggest that in these mesenchymal cells, phosphorylation of the T179 and possibly S213 sites may act as a brake on the signal, whereas S204 phosphorylation by ERK in some manner releases that brake.     

Pbx raises the DNA binding specificity but not the selectivity of antennapedia Hox proteins.

We have used a binding site selection strategy to determine the optimal binding sites for Pbx proteins by themselves and as heterodimeric partners with various Hox gene products. Among the Pbx proteins by themselves, only Pbx3 binds with high affinity, as a monomer or as a homodimer, to an optimal binding site, TGATTGATTTGAT. An inhibitory domain located N terminal of the Pbx1 homeodomain prevents intrinsic Pbx1 binding to this sequence. When complexed with Hoxc-6, each of the Pbx gene products binds the same consensus sequence, TGATTTAT, which differs from the site bound by Pbx3 alone. Three members of the Antennapedia family, Hoxc-6, Hoxb-7, and Hoxb-8, select the same binding site in conjunction with Pbx1. The affinities of these proteins as heterodimeric partners with Pbx1 for the selected optimal binding site are similar. However, the binding specificity of Hox proteins for optimal binding sites is increased, compared to nonspecific DNA, in the presence of Pbx proteins. Thus, while cooperative DNA binding involving heterodimers of Pbx and Hox gene products derived from members within the Antennapedia family does not increase binding site selectivity, DNA binding specificity of the Hox gene products is significantly enhanced in the presence of Pbx.     

XMAN1, an inner nuclear membrane protein,antagonizes BMP signaling by interacting with Smad1 in Xenopus embryos

A family of inner nuclear membrane proteins is implicated in gene regulation by interacting with chromatin, nuclear lamina and intranuclear proteins; however, the physiological functions of these proteins are largely unknown. Using a Xenopus expression screening approach with an anterior neuroectoderm cDNA library, we have identified an inner nuclear membrane protein, XMAN1, as a novel neuralizing factor that is encoded by the Xenopus ortholog of human MAN1. XMAN1 mRNA is expressed maternally, and appears to be restricted to the entire ectoderm at the early gastrula stage, then to the anterior neuroectoderm at the neurula stage. XMAN1 induces anterior neural markers without mesoderm induction in ectodermal explants, and a partial secondary axis when expressed ventrally by dorsalizing the ventral mesoderm. Importantly, XMAN1 antagonizes bone morphogenetic protein (BMP) signaling downstream of its receptor Alk3, as judged by animal cap assays, in which XMAN1 blocks expression of downstream targets of BMP signaling (Xhox3 and Msx1), and by luciferase reporter assays, in which XMAN1 suppresses BMP-dependent activation of the Xvent2 promoter. Deletion mutant analyses reveal that the neuralizing and BMP-antagonizing activities of XMAN1 reside in the C-terminal region, and that the C-terminal region binds to Smad1, Smad5 and Smad8, which are intracellular mediators of the BMP pathway. Interference with endogenous XMAN1 functions with antisense morpholino oligos leads to the reduction of anterior neuroectoderm. These results provide the first evidence that the nuclear envelope protein XMAN1 acts as a Smad-interacting protein to antagonize BMP signaling during Xenopus embryogenesis.