u-0126 and Calcinosis

Tumor necrosis factor-alpha (TNF-alpha) is a skeletal catabolic agent that stimulates osteoclastogenesis and inhibits osteoblast function. Although TNF-alpha inhibits the mineralization of osteoblasts, the effect of TNF-alpha on mesenchymal stem cells (MSC) is not clear. In this study, we determined the effect of TNF-alpha on osteogenic differentiation of stromal cells derived from human adipose tissue (hADSC) and the role of NF-kappaB activation on TNF-alpha activity. TNF-alpha treatment dose-dependently increased osteogenic differentiation over the first 3 days of treatment. TNF-alpha activated ERK and increased NF-kappaB promoter activity. PDTC, an NF-kappaB inhibitor, blocked the osteogenic differentiation induced by TNF-alpha and TLR-ligands, but U102, an ERK inhibitor, did not. Overexpression of miR-146a induced the inhibition of IRAK1 expression and inhibited basal and TNF-alpha- and TLR ligand-induced osteogenic differentiation. TNF-alpha and TLR ligands increased the expression of transcriptional coactivator with PDZ-binding motif (TAZ), which was inhibited by the addition of PDTC. A ChIP assay showed that p65 was bound to the TAZ promoter. TNF-alpha also increased osteogenic differentiation of human gastroepiploic artery smooth muscle cells. Our data indicate that TNF-alpha enhances osteogenic differentiation of hADSC via the activation of NF-kappaB and a subsequent increase of TAZ expression.

Topics: Acyltransferases; Adipose Tissue; Binding Sites; Butadienes; Calcinosis; Cell Differentiation; Cells, Cultured; Enzyme Activation; Extracellular Signal-Regulated MAP Kinases; Female; Humans; Interleukin-1 Receptor-Associated Kinases; Male; Mesenchymal Stem Cells; MicroRNAs; Middle Aged; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; NF-kappa B; Nitriles; Osteogenesis; Promoter Regions, Genetic; Protein Kinase Inhibitors; Pyrrolidines; Recombinant Proteins; RNA Interference; Thiocarbamates; Time Factors; Toll-Like Receptors; Transcription Factor RelA; Transcription Factors; Transfection; Tumor Necrosis Factor-alpha; Up-Regulation

Much remains to be discovered about the etiology of heart valve disease and the molecular level mechanisms that drive it. The MAPK/ERK pathway influences calcification in many cell types and has been linked to the expression of a contractile phenotype in valvular interstitial cells (VICs). However, a direct correlation between MAPK/ERK pathway activity and VIC calcification has not been previously described. Thus the role of the MAPK pathway in the calcification of VIC cultures was investigated by measuring ERK activation in both calcifying and noncalcifying VIC environments and then, conversely, analyzing the effects of ERK pathway inhibition on VIC calcification and phenotype. Prolonged elevation of phosphorylated ERK-1/2 was found in calcifying VIC cultures, whereas directly blocking phosphorylation of ERK-1/2 resulted in a dramatic decrease in nodule number, nodule size, and total calcified area. Application of the ERK pathway inhibitor was also associated with a dramatic decrease in apoptosis, which may have contributed to the decreased nodule formation obtained via ERK inhibition. Real-time PCR analysis revealed that calcified samples exhibited significantly elevated expression of several myofibroblastic and osteoblastic markers, while ERK inhibition substantially reduced the expression of these markers, often to levels comparable to the noncalcifying control. These data suggest that the MAPK pathway plays an important role in regulating the phenotype and calcification of VICs, wherein sustained pathway activation is associated with increased VIC calcification. These findings may be used to further elucidate the mechanisms of valvular disease and identify potential treatment targets.

Topics: Animals; Apoptosis; Biomarkers; Butadienes; Calcinosis; Cell Count; Cell Movement; Enzyme Inhibitors; Extracellular Matrix; Flavonoids; Gene Expression; Heart Valve Diseases; JNK Mitogen-Activated Protein Kinases; MAP Kinase Kinase 1; MAP Kinase Kinase 2; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Nitriles; Phenotype; Swine; Tissue Culture Techniques

Synovial fluid basic calcium phosphate (BCP) crystals are markers of severe joint degeneration in osteoarthritis. These crystals are mitogenic and induce protooncogene expression and matrix metalloproteinase (MMP) synthesis and secretion in human fibroblasts, effects that are specifically blocked by phosphocitrate (PC). We have recently determined that crystals transduce signals to the nucleus via the activation of the p42 and p44 mitogen-activated protein (MAP) kinases (Nair et al., 1997, J Biol Chem 272:18920-18925). Treatment of human fibroblasts (HF) with BCP induces phosphorylation of p42/44 MAPK, which is inhibited by PC in a dose-dependent manner. Blocking of p42/44 MAPK signal transduction with an inhibitor (PD98059) of MEK1, an upstream activator of MAPKs, reduces crystal-induced p42/44 MAPK activation and significantly inhibits crystal-induced cell proliferation. Based on these findings, we sought to determine the role of the p42/44 MAPK signal transduction pathway in crystal-induced expression of matrix MMPs. We demonstrate suppression of crystal-induced MMPs via the utilization of two different MEK inhibitors: PD98059 and the recently described U0126, a novel inhibitor of MEK1 and MEK2. Treatment of HF with PD98059 blocks the induction of crystal-stimulated collagenase 1 (MMP-1) and stromelysin (MMP-3) expression. PD98059 and PC reduced the level of crystal-induced MMP-1 and MMP-3 mRNA expression to that observed in nonstimulated cells. Likewise, PD98059 treatment of HF blocked the epidermal growth factor (EGF)- and crystal-induced increases in MMP-1 and MMP-3 protein expression and secretion as demonstrated by Western blotting and zymography. Treatment of HF with U0126 inhibits EGF-induced phosphorylation of p42/44 MAPK as well as crystal- and EGF-induced upregulation of MMP-1 mRNA. Additionally, we demonstrate that treatment of HF with BCP, EGF, or PD98059 does not significantly alter levels of gelatinase A (MMP-2) mRNA and protein expression.

Topics: Blotting, Northern; Butadienes; Calcinosis; Calcium Phosphates; Calcium-Calmodulin-Dependent Protein Kinases; Cells, Cultured; Collagenases; Crystallization; Dose-Response Relationship, Drug; Enzyme Inhibitors; Flavonoids; Gelatinases; Gene Expression Regulation, Enzymologic; Humans; Matrix Metalloproteinase 1; Matrix Metalloproteinase 2; Matrix Metalloproteinase 3; Metalloendopeptidases; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Nitriles; RNA, Messenger; Signal Transduction; Skin