title: c-Src-dependent transactivation of PDGFR contributes to TNF-α-induced MMP-9 expression and functional impairment in osteoblasts.
abstract: Matrix metalloproteinases (MMPs), MMP-9 especially, have been shown to be induced by cytokines, including tumor necrosis factor-α (TNF-α) and may contribute to bone inflammatory diseases and postnatal bone modeling and remodeling. However, the mechanisms underlying MMP-9 expression induced by TNF-α in osteoblasts remain unclear. Here, we showed that in MC3T3-E1 cells, TNF-α induced MMP-9 gene expression determined by real-time PCR, zymography, and promoter assay. TNF-α-mediated responses were attenuated by pretreatment with the inhibitor of protein tyrosine kinase (PTK; genistein), c-Src (PP1), PDGFR (AG1296), PI3K (LY294002), Akt (SH-5), MEK1/2 (U0126), p38 MAPK (SB202190), JNK1/2 (SP600125), or AP-1 (Tanshinone IIA) and transfection with siRNA of c-Src, PDGFR, p85, Akt, c-Jun, or ATF2. Moreover, TNF-α also time-dependently stimulated phosphorylation of c-Src and PDGFR and c-Src/PDGFR complex formation, which were reduced by pretreatment with PP1 or AG1296. TNF-α-stimulated Akt phosphorylation was inhibited by genistein, PP1, AG1296, LY294002, or SH5. We further demonstrated that TNF-α stimulated ERK1/2, p38 MAPK, and JNK1/2 phosphorylation via a c-Src-dependent PDGFR/PI3K/Akt pathway. TNF-α stimulated AP-1 activation, including c-Jun and ATF2 phosphorylation and AP-1 transcription activity via MAPK-dependent pathways. In addition, TNF-α-induced MMP-9 promoter activity was mediated through an AP-1 binding domain of the MMP-9 promoter region. Finally, we found that up-regulation of MMP-9 contributes to MMP-mediated type I collagen degradation and osteoblasts detachment. These results suggested that TNF-α-induced MMP-9 expression is mediated through a c-Src-dependent PDGFR transactivation and PI3K/Akt cascade linking to MAPK-mediated activation of AP-1 (c-Jun/ATF2) and leading to functional impairment in osteoblasts.
authors.affiliation: Department of Physiology and Pharmacology and Health Ageing Research Center, College of Medicine, Chang Gung University, Tao-Yuan, Taiwan; Department of Nursing, Division of Basic Medical Sciences, Chang Gung University of Science and Technology, Tao-Yuan, Taiwan.
authors.forename: Chia-Lan
authors.initials: CL
authors.lastname: Tsai
authors.affiliation: Department of Physiology and Pharmacology and Health Ageing Research Center, College of Medicine, Chang Gung University, Tao-Yuan, Taiwan.
authors.forename: Wei-Chung
authors.initials: WC
authors.lastname: Chen
authors.affiliation: Department of Physiology and Pharmacology and Health Ageing Research Center, College of Medicine, Chang Gung University, Tao-Yuan, Taiwan; Department of Anesthetics, Chang Gung Memorial Hospital at Lin-Kou and College of Medicine, Chang Gung University, Kwei-San, Tao-Yuan, Taiwan.
authors.forename: I-Ta
authors.initials: IT
authors.lastname: Lee
authors.affiliation: Department of Physiology and Pharmacology and Health Ageing Research Center, College of Medicine, Chang Gung University, Tao-Yuan, Taiwan.
authors.forename: Pei-Ling
authors.initials: PL
authors.lastname: Chi
authors.affiliation: Department of Physiology and Pharmacology and Health Ageing Research Center, College of Medicine, Chang Gung University, Tao-Yuan, Taiwan.
authors.forename: Shin-Ei
authors.initials: SE
authors.lastname: Cheng
authors.affiliation: Department of Physiology and Pharmacology and Health Ageing Research Center, College of Medicine, Chang Gung University, Tao-Yuan, Taiwan. Electronic address: chuenmao@mail.cgu.edu.tw.
authors.forename: Chuen-Mao
authors.initials: CM
authors.lastname: Yang
mesh_terms: D000818:Animals; D000081247:CSK Tyrosine-Protein Kinase; D002448:Cell Adhesion / Q000187:drug effects; D002465:Cell Movement / Q000187:drug effects; D002478:Cells, Cultured; D003094:Collagen / Q000378:metabolism; D006801:Humans; D020780:Matrix Metalloproteinase 9 / Q000235:genetics* / Q000378:metabolism; D051379:Mice; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D010006:Osteoblasts / Q000187:drug effects / Q000201:enzymology*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D059748:Proteolysis / Q000187:drug effects; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D051381:Rats; D020796:Receptor, Platelet-Derived Growth Factor alpha / Q000235:genetics* / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D018808:Transcription Factor AP-1 / Q000378:metabolism; D014158:Transcription, Genetic / Q000187:drug effects; D015533:Transcriptional Activation / Q000187:drug effects* / Q000235:genetics; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*; D019061:src-Family Kinases / Q000378:metabolism*
chemical_list: D000081247:CSK Tyrosine-Protein Kinase; D003094:Collagen; D020780:Matrix Metalloproteinase 9; D020928:Mitogen-Activated Protein Kinases; D019869:Phosphatidylinositol 3-Kinases; D051057:Proto-Oncogene Proteins c-akt; D020796:Receptor, Platelet-Derived Growth Factor alpha; D018808:Transcription Factor AP-1; D014409:Tumor Necrosis Factor-alpha; D019061:src-Family Kinases; C000631346:CSK protein, human
keywords: Cell-mediated type I collagen degradation; Cytokine; MMP-9; Osteoblast-like MC3T3-E1 cells; PDGFR transactivation