☆ 4.6 Article

c-Src-dependent transactivation of PDGFR contributes to TNF-α-induced MMP-9 expression and functional impairment in osteoblasts

BONE (2014)

Journal

BONE

Volume 60, Issue -, Pages 186-197

Publisher

ELSEVIER SCIENCE INC

DOI: 10.1016/j.bone.2013.12.014

Keywords

Cytokine; MMP-9; Osteoblast-like MC3T3-E1 cells; Cell-mediated type I collagen degradation; PDGFR transactivation

Funding

National Science Council, Taiwan [NSC-101-2320-B-182-039-MY3, NSC-102-2321-B-182-011]
Ministry of Education, Taiwan [EMRPD1C0261, EMRPD1C0271]
Chang Gung Medical Research Foundation, Taiwan [CMRPD1C0102, CMRPD1B0382, CMRPD1C0561]

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Abstract

Matrix metalloproteinases (MMPs), MMP-9 especially, have been shown to be induced by cytokines, including tumor necrosis factor-alpha (TNF-alpha) and may contribute to bone inflammatory diseases and postnatal bone modeling and remodeling. However, the mechanisms underlying MMP-9 expression induced by TNF-alpha in osteoblasts remain unclear. Here, we showed that in MC3T3-E1 cells, TNF-alpha induced MMP-9 gene expression determined by real-time PCR, zymography, and promoter assay. TNF-alpha-mediated responses were attenuated by pretreatment with the inhibitor of protein tyrosine kinase (PM; 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 transfecdon with siRNA of c-Src, PDGFR, p85, Akt, c-Jun, or ATF2. Moreover, TNF-alpha 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-alpha-stimulated Akt phosphorylation was inhibited by genistein,PP1,AG1296,LY294002, or SH5. We further demonstrated that TNF-alpha stimulated ERK1/2, p38 MAPK, and JNK1/2 phosphorylation via a c-Src-dependent PDGFR/PI3K/Akt pathway. TNF-alpha stimulated AP-1 activation, including c-Jun and ATF2 phosphorylation and AP-1 transcription activity via MAPK-dependent pathways. In addition, TNF-alpha-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-alpha-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. (C) 2013 Elsevier Inc. All rights reserved.

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Evaluation of MC3T3-E1 Cell Osteogenesis in Different Cell Culture Media

Makoto Izumiya, Miyu Haniu, Katsuya Ueda, Haruka Ishida, Chuang Ma, Hirokazu Ideta, Atsushi Sobajima, Koki Ueshiba, Takeshi Uemura, Naoto Saito, Hisao Haniu

Summary: The study evaluated the osteogenesis of MC3T3-E1 cells under different culture media conditions, measuring cell proliferation, alkaline phosphatase activity, expression of osteoblast markers, and calcification. The research found that the type of base medium, the presence or absence of vitamin C, and the freshness of the medium may impact biomaterial regeneration.

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