MAP3K7 (Phospho-Thr187) ELISA Kit

Nearly every extracellular ligand that has been found to play a role in regulating bone biology acts, at least in part, through MAPK pathways. Nevertheless, much remains to be learned about the contribution of MAPKs to osteoblast biology in vivo. Here we report that the p38 MAPK pathway is required for normal skeletogenesis in mice, as mice with deletion of any of the MAPK pathway member-encoding genes MAPK kinase 3 (Mkk3), Mkk6, p38a, or p38b displayed profoundly reduced bone mass secondary to defective osteoblast differentiation. Among the MAPK kinase kinase (MAP3K) family, we identified TGF-beta-activated kinase 1 (TAK1; also known as MAP3K7) as the critical activator upstream of p38 in osteoblasts. Osteoblast-specific deletion of Tak1 resulted in clavicular hypoplasia and delayed fontanelle fusion, a phenotype similar to the cleidocranial dysplasia observed in humans haploinsufficient for the transcription factor runt-related transcription factor 2 (Runx2). Mechanistic analysis revealed that the TAK1-MKK3/6-p38 MAPK axis phosphorylated Runx2, promoting its association with the coactivator CREB-binding protein (CBP), which was required to regulate osteoblast genetic programs. These findings reveal an in vivo function for p38 beta and establish that MAPK signaling is essential for bone formation in vivo. These results also suggest that selective p38 beta agonists may represent attractive therapeutic agents to prevent bone loss associated with osteoporosis and aging.

We recently reported that increased NADPH oxidase 4 (NOX4) expression and activity during aging results in enhanced cellular and mitochondrial oxidative stress, vascular inflammation, dysfunction, and atherosclerosis. The goal of the present study was to elucidate the molecular mechanism(s) for these effects and determine the importance of NOX4 modulation of proinflammatory gene expression in mouse vascular smooth muscle cells (VSMCs). A novel peptide-mediated siRNA transfection approach was used to inhibit Nox4 expression with minimal cellular toxicity. Using melittin-derived peptide p5RHH, we achieved significantly higher transfection efficiency (92% vs. 85% with Lipofectamine) and decreased toxicity (p < 0.001 vs. Lipofectamine in MIT and p < 0.0001 vs. Lipofectamine in LDH assays) in VSMCs. TGF01 significantly upregulates Nox4 mRNA (p < 0.01) and protein (p < 0.01) expression in VSMCs. p5RHH-mediated Nox4 siRNA transfection greatly attenuated TGF beta 1-induced upregulation of Nox4 mRNA (p < 0.01) and protein (p < 0.0001) levels and decreased hydrogen peroxide production (p < 0.0001). Expression of pro-inflammatory genes Cc12, Cc15, 116, and Vcaml was significantly upregulated in VSMCs in several settings cells isolated from aged vs. young wild-type mice, in atherosclerotic arteries of Apoe(-/-) mice, and atherosclerotic human carotid arteries and correlated with NOX4 expression. p5RHH-mediated Nox4 siRNA transfection significantly attenuated the expression of these pro-inflammatory genes in TGF beta 1-treated mouse VSMCs, with the highest degree of inhibition in the expression of 116. p5RHH peptide-mediated knockdown of TGFO-activated kinase 1 (TAK1, also known as Map3k7),Jun, and Rela, but not Nfkb2, downregulated TGF beta 1-induced Nox4 expression in VSMCs. Together, these data demonstrate that increased expression and activation of NOX4, which might result from increased TGF beta 1 levels seen during aging, induces a proinflammatory phenotype in VSMCs, enhancing atherosclerosis. (C) 2016 Published by Elsevier Ltd.