Mechanisms underlying tumor necrosis factor-alpha-induced expression of matrix metalloproteinases-9 in murine osteoblast-like cell line MC3T3-E1 cells

• Main Authors: Chia Lan Tsai, 蔡佳蘭
• Other Authors: C. M. Yang
• Format: Others
• Published: 2014
• Online Access: http://ndltd.ncl.edu.tw/handle/69698964972791504784

博士 === 長庚大學 === 生物醫學研究所 === 102 === Mature bone mass is maintained by bone remodeling cycle. However, most skeletal diseases are due to excess osteoclast bone resorption, leading to an imbalance in bone remodeling. In osteoblast, 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. Nevertheless, the mechanisms underlying MMP-9 expression induced by TNF-alpha in osteoblast-like MC3T3-E1 cell remain unclear. TNF-alpha activates a variety of intracellular signaling cascades that lead to the induction of inflammatory responses. Elevated levels of TNF-alpha have been detected in several bone diseases, which up-regulates MMP-9 expression in various cell types. MMP-9 expression induced by TNF-alpha is involved in bone inflammatory diseases and bone destruction. Therefore, our study was focused on investigating the mechanisms underlying the intracellular signals involved in the MMP-9 expression induced by TNF-alpha in MC3T3-E1 cells. We applied gelatin zymography, Westen blot, RT-PCR, real-time PCR, selective pharmacological inhibitors including transcription (actinomycin D, Act.D), translation (cycloheximide, CHI), c-Src (PP1), PDGF receptor (AG1296), PI3K (LY294002), Akt (SH-5), MEK1/2 (U0126), p38 (SB202190), JNK (SP600125), AP-1 (tanshinone IIA), and NF-κB (Bay11-7082), transfection with respective siRNAs, promoter assay, immunofluorescence staining, ELISA, cell-mediated type I collagen dissolution assay and cell spreading assay to investigate the mechanisms by which TNF-alpha induced MMP-9 expression, sICAM-1 release, MMP-mediated type I collagen degradation and osteoblasts detachment in MC3T3-E1 cells. Here we demonstrated that TNF-α induced the expression of MMP-9 protein and mRNA which were attenuated by inhibitor of Act.D, CHI, PP1, AG1296, LY294002, SH-5, U0126, SB202190, SP600125, Bay11-7082, or Tanshinone IIA and transfection with siRNA of TRAF2, c-Src, PDGFR, p85, Akt, ERK2, p38, JNK2, c-Jun, or ATF2. TNF-alpha-stimulated TNFR1, TRAF2, and c-Src complex formation was revealed by immunoprecipitation and Western blot. TNF-alpha-stimulated phosphorylation and translocation of NF-κB into the nucleus was blocked by Bay11-7082, but not by PP1, U0126, SB202190, and SP600125. 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. Furthermore, 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 phosporylation and AP-1 transcription activity via MAPKs-dependent pathways. TNF-alpha time-dependently induced MMP-9 promoter activity which was also inhibited by PP1, AG1296, LY294002, SH5, U0126, SB202190, SP600125, Bay11-7082, or Tanshinone IIA. In addition, TNF-alpha-induced MMP-9 promoter activity was mediated through NF-kappaB and AP-1 binding domain of the MMP-9 promoter region. These results suggested that TNF-alpha-induced MMP-9 expression is mediated through c-Src-dependent PDGFR transactivation and PI3K/Akt cascade linking to MAPKs-mediated activation of AP-1 (c-Jun/ATF2) and NF-kappaB pathways in MC3T3-E1 cells. TNF-alpha has been shown to promote solube ICAM-1 release and a proteolytic enzyme involved in ICAM-1 cleavage. In this study, sICAM-1 ELISA analysis showed TNF-alpha-induced release of sICAM-1 in MC3T3-E1 cells, which were attenuated by the inhibitors of GM6001 (MMPs), MMP2/9i (MMP2/9), JNK (SP600125), p38 MAPK (SB202190), MEK1/2 (U0126) and NF-kappa B (Bay11-7082). Finally, we found that up-regulation of MMP-9 may contribute to MMP-mediated type I collagen degradation and osteoblasts detachment. Increased understanding of signal transduction mechanisms underlying MMP-9 gene regulation creates opportunities for the development of bone remodeling diseases therapeutic strategies. Moreover, the interplay between MMP-9 expression, MMP-mediated ECM degradation and sICAM-1 release may exert an important role in the regulation of bone inflammatory diseases.