u-0126 and beta-glycerophosphoric-acid

U0126 has been reported as a specific inhibitor of the ERK1/2 signaling pathway, which plays a vital role during the osteogenic differentiation of mesenchymal stem cells (MSCs). We report the positive effect of U0126 on the osteogenesis of rat MSCs. We find that U0126 promotes the osteogenic differentiation of rat MSCs as demonstrated by the quantitative real-time polymerase chain reaction for osteogenic markers, alkaline phosphatase activity and calcium nodule formation. Our data indicate that U0126 enhances the BMP/Smad signaling pathway in rat MSCs, while inhibiting the ERK1/2 signaling pathway. Furthermore, Western blot results demonstrate that U0126 increases Smad1/5/8 phosphorylation synergistically with β-glycerophosphate. In addition, U0126 significantly increases the expression of BMP2 during the process of osteogenesis in rat MSCs and the level of phosphorylated Smad1/5/8 is significantly reduced by BMP2 antibody, suggesting that U0126 also promotes the expression of BMP2 to enhance Smad proteins phosphorylation. Thus, we demonstrate a novel function for U0126 in promoting osteogenic differentiation of rat MSCs by the activation of the BMP/Smad signaling pathway.

Topics: Animals; Bone Marrow Cells; Bone Morphogenetic Protein 2; Butadienes; Cell Survival; Glycerophosphates; Mesenchymal Stem Cells; Nitriles; Osteogenesis; Phenotype; Phosphorylation; Rats, Sprague-Dawley; Real-Time Polymerase Chain Reaction; Signal Transduction; Smad Proteins; Up-Regulation

Although basic fibroblast growth factor (FGF-2) had been shown to inhibit type I collagen gene expression in osteoblast, its inhibitory mechanism is unknown. In the present study, we investigated the underlying mechanisms by which growth factors downregulate type I collagen gene expression. Treatment of mouse osteoblastic MC3T3-E1 cells with okadaic acid (40 ng/ml), an inhibitor of phosphoserine/threonine-specific protein phosphatase and activator of ERK1/2, for 24 h and 48 h completely inhibited steady-state mRNA levels of type I collagen. FGF-2 (30 ng/ml), platelet-derived growth factor-BB (PDGF-BB), 30 ng/ml, and serum, which activate ERK mitogen-activated protein kinase (MAPK) pathway also inhibited collagen type I gene expression, suggesting that the activation of ERK pathway mediates inhibition of type I collagen mRNA. This observation was further confirmed by experiments using inhibitors of the ERK pathway (i.e., PD and U0126), which increased type I collagen mRNA in MC3T3-E1 cells, indicating that the inhibition of ERK pathway upregulates type I collagen gene expression. Low serum (0.3%) markedly increased type I collagen mRNA. MEK inhibitor PD inhibited c-fos induction by FGF-2 and PDGF-BB, suggesting that c-fos is the downstream target of ERK pathway. Our data have clearly demonstrated for the first time that the ERK MAPK pathway play an important role in the regulation of type I collagen gene expression in osteoblastic cells. Results also showed that one of the mechanisms by which FGF-2 and PDGF-BB downregulate type I collagen gene expression in the osteoblast is through the activation of ERK signaling pathway.

Topics: 3T3 Cells; Animals; Ascorbic Acid; Becaplermin; Butadienes; Down-Regulation; Enzyme Inhibitors; Fibroblast Growth Factor 2; Flavonoids; Genes, fos; Glycerophosphates; Humans; Mice; Mitogen-Activated Protein Kinases; Nitriles; Okadaic Acid; Osteoblasts; Platelet-Derived Growth Factor; Procollagen; Proto-Oncogene Proteins c-sis; Recombinant Proteins; RNA, Messenger; Signal Transduction