Osterix, a zinc-finger transcription factor, is specifically expressed in osteoblasts and osteocytes of all developing bones. Because no bone formation occurs in Osterix null mice, Osterix is thought to be an essential regulator of osteoblast differentiation. We report that bone morphogenetic protein-2 (BMP-2) induces an increase in Osterix expression, which is mediated through a homeodomain sequence located in the proximal region of the Osterix promoter. Our results demonstrate that induction of Dlx5 by BMP-2 mediates Osterix transcriptional activation. First, BMP-2 induction of Dlx5 precedes the induction of Osterix. Second, Dlx5 binds to the BMP-responsive homeodomain sequences both in vitro and in vivo. Third, Dlx5 overexpression and knock-down assays demonstrate its role in activating Osterix expression in response to BMP-2. Furthermore, we show that Dlx5 is a novel substrate for p38 MAPK in vitro and in vivo and that Ser-34 and Ser-217 are the sites phosphorylated by p38. Phosphorylation at Ser-34/217 increases the transactivation potential of Dlx5. Thus, we propose that BMP activates expression of Osterix through the induction of Dlx5 and its further transcriptional activation by p38-mediated phosphorylation.Bone is a dynamic tissue that is constantly remodeled throughout life. Bone remodeling activity is dependent on a strict coupling mechanism of osteoclast resorption and new matrix deposition by osteoblasts, and an imbalance between these two activities leads to pathological states such as osteoporosis and osteosclerosis. Commitment to the osteoblast phenotype is ultimately controlled by a specific set of transcription factors activated by signals and regulatory pathways. Among them, bone morphogenetic protein (BMP) 5 signaling has been shown to be involved in bone regeneration and osteoblast differentiation in vitro and in vivo (1-3). BMP target genes include a growing number of tissue-determining transcription factors that promote differentiation of mesenchymal precursors toward the osseous cell phenotypes. For instance, osteogenic induction of bone marrow mesenchymal stem cells or premyogenic C2C12 cells has identified several types of transcription factors such as Id1, several homeodomain proteins, ATF4, the runt homology domain factor Runx2 (Cbfa1) and Osterix (Osx) (for review, see Refs. 4 -6). Runx2 and Osx have been widely accepted as master osteogenic factors since neither Cbfa1 nor Osx null mice form mature osteoblasts (7,8). Nakashima et al. (8) identified Osx as a zinc-finger SP1 family member induced by BMP-2 in C2C12 cells that is specifically expressed in osteoblasts and osteocytes of all developing bones. In Osx-null mice, no bone formation takes place, although Runx2 is expressed, suggesting that Osx acts downstream of Runx2 during bone development (8). It has been suggested that Runx2 would function from the commitment step to the point where osteochondroprogenitors appear, whereas Osx would have a role in the segregation of osteoblasts from osteochondroprogenitors (5). Supporting ...

Background: TGF␤1-induced pY654-␤-catenin correlates with epithelial mesenchymal transition (EMT) and pulmonary fibrosis, but whether pY654-␤-catenin is functionally important is unknown. Results: ␤-Catenin point mutants reveal that pY654 is critical to EMT, and pY654-␤-catenin accumulation is blocked by axin-dependent ␤-catenin turnover. Conclusion: Raised axin levels in vivo attenuate EMT and fibrosis after bleomycin injury. Significance: Targeting axin levels could attenuate fibrosis without blocking TGF␤1 homeostatic functions.

Osteoblast differentiation depends on the coordinated network of evolutionary conserved transcription factors during bone formation and homeostasis. Evidence indicates that bone morphogenetic protein (BMP) and Wnt proteins regulate several steps of skeletal development. Here, we provide a molecular description of the cooperative effects of BMP and Wnt canonical pathway on the expression of the early osteogenic genes Dlx5, Msx2, and Runx2 in C2C12 cells, primary cultures of bone marrow-mesenchymal stem cells, and organotypic calvarial cultures. Coordinated regulation of these genes leads to the cooperative activation of their downstream osteogenic target gene osterix. Induction of these genes is mediated through enhancer regions with an evolutionary conserved structure encompassing both Smad and TCF/LEF1 DNA-binding sites. Formation of a cooperative complex is mediated through DNA binding of Smads and TCF4/b-catenin to their cognate sequences, as well as protein-protein interactions between them. The formation of these cooperative transcriptional complexes results in a more efficient recruitment of coactivators such as p300. We propose that evolutionary conserved regulatory regions in specific osteogenic master genes are key integrative modules during osteogenesis. ß

Hypoxia is linked to epithelial mesenchymal transition (EMT) and tumor progression in numerous carcinomas. Responses to hypoxia are thought to operate via hypoxia-inducible factors (HIFs), but the importance of co-factors that regulate HIF signaling within tumors is not well understood. Here we elucidate a signaling pathway that physically and functionally couples tyrosine phosphorylation of β-catenin to hypoxia-inducible factor-1α (HIF1α) signaling and HIF1α-mediated tumor EMT. Primary human lung adenocarcinomas accumulate pY654-β-catenin and HIF1α. All pY654-β-catenin, and only the tyrosine phosphorylated form, was found complexed with HIF1α and active Src, both within human tumors and in lung tumor cell lines exposed to hypoxia. Phosphorylation of Y654, generated by hypoxia mediated, reactive oxygen species (ROS)-dependent Src kinase activation, was required for β-catenin to interact with HIF1α and Src, to promote HIF1α transcriptional activity, and for hypoxia-induced EMT. Mice bearing hypoxic pancreatic islet adenomas, generated by treatment with anti-vascular endothelial growth factor antibodies, accumulate HIF1α/pY654-β-catenin complexes and develop an invasive phenotype. Concurrent administration of the ROS inhibitor N-acetylcysteine abrogated β-catenin/HIF pathway activity and restored adenoma architecture. Collectively, the findings implicate accumulation of pY654-β-catenin specifically complexed to HIF1α and Src kinase as critically involved in HIF1α signaling and tumor invasion. The findings also suggest that targeting ROS-dependent aspects of the pY654-β-catenin/ HIF1α pathway may attenuate untoward biological effects of anti-angiogenic agents and tumor hypoxia.

Summary Alternative splicing is a prevalent mechanism of gene regulation that is modulated in response to a wide range of extracellular stimuli. Stress-activated protein kinases (SAPKs) play a key role in controlling several steps of mRNA biogenesis. Here, we show that osmostress has an impact on the regulation of alternative splicing (AS), which is partly mediated through the action of p38 SAPK. Splicing network analysis revealed a functional connection between p38 and the spliceosome component SKIIP, whose depletion abolished a significant fraction of p38-mediated AS changes. Importantly, p38 interacted with and directly phosphorylated SKIIP, thereby altering its activity. SKIIP phosphorylation regulated AS of GADD45α, the upstream activator of the p38 pathway, uncovering a negative feedback loop involving AS regulation. Our data reveal mechanisms and targets of SAPK function in stress adaptation through the regulation of AS.

No abstract