title: TNF-alpha induces MMP-9 expression via activation of Src/EGFR, PDGFR/PI3K/Akt cascade and promotion of NF-kappaB/p300 binding in human tracheal smooth muscle cells.
abstract: TNF-alpha has been shown to induce matrix metalloproteinase-9 (MMP-9) expression, which, in turn, degrades extracellular matrix in the inflammatory responses. However, the inductive mechanisms of the MMP-9 by TNF-alpha remain unclear. In human tracheal smooth muscle cells, TNF-alpha induced MMP-9 expression and Akt phosphorylation in a time-dependent manner, which was attenuated by the inhibitors of Src (PP1), epidermal growth factor receptor (AG1478), PDGFR (AG1296), and PI3K (LY294002), respectively, revealed by reporter gene assay, RT-PCR, zymographic, and Western blot analyses. Transfection with the dominant negative mutants of c-Src (KM, K295M [kinase inactive mutant]), p85, and Akt (KA, K179A) also reduced MMP-9 expression. These findings indicated that MMP-9 expression was regulated by PI3K/Akt via the transactivation of growth factor receptors. Furthermore, LY294002 or wortmannin inhibited Akt phosphorylation but had no effect on NF-kappaB translocation, which was blocked by helenalin. Mutated NF-kappaB DNA binding element in the MMP-9 promoter and helenalin also attenuated MMP-9 expression, suggesting that PI3K/Akt and NF-kappaB independently regulated MMP-9 expression. To support this notion, immunofluorescence staining and immunoprecipitation were applied to characterize the transcription factors involved in these responses. The results showed that LY294002 and curcumin blocked Akt translocation into nucleus. In contrast, p300, acetyl-histone (H3), and NF-kappaB p65 were found to be coimmunoprecipitated with the phosphorylated Akt, indicating that these components associated with the MMP-9 promoter are revealed by chromatin immunoprecipitation assay. Thus, our study provides a new insight into the molecular mechanisms that TNF-alpha-stimulated Akt phosphorylation mediated through transactivation of Src and growth factor receptors may stimulate the recruitment of p300, assemble transcription factor (p65), and then lead to MMP-9 expression.
authors.affiliation: Department of Physiology and Pharmacology, Chang Gung Memorial Hospital, Chang Gung University, 259 Wen-Hwa 1st Rd., Kwei-San, Tao-Yuan, Taiwan.
authors.forename: Chiang-Wen
authors.initials: CW
authors.lastname: Lee
authors.forename: Chih-Chung
authors.initials: CC
authors.lastname: Lin
authors.forename: Wei-Ning
authors.initials: WN
authors.lastname: Lin
authors.forename: Kao-Chih
authors.initials: KC
authors.lastname: Liang
authors.forename: Shue-Feng
authors.initials: SF
authors.lastname: Luo
authors.forename: Chow-Bin
authors.initials: CB
authors.lastname: Wu
authors.forename: Shyi-Wu
authors.initials: SW
authors.lastname: Wang
authors.forename: Chuen-Mao
authors.initials: CM
authors.lastname: Yang
mesh_terms: D015153:Blotting, Western; D002478:Cells, Cultured; D047369:Chromatin Immunoprecipitation; D050881:E1A-Associated p300 Protein / Q000235:genetics / Q000378:metabolism*; D066246:ErbB Receptors / Q000235:genetics / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D006801:Humans; D047468:Immunoprecipitation; D020780:Matrix Metalloproteinase 9 / Q000235:genetics / Q000378:metabolism*; D032389:Myocytes, Smooth Muscle / Q000378:metabolism; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D010766:Phosphorylation; D011401:Promoter Regions, Genetic; D051057:Proto-Oncogene Proteins c-akt / Q000235:genetics / Q000378:metabolism*; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D017479:Receptors, Platelet-Derived Growth Factor; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D014132:Trachea / Q000166:cytology / Q000378:metabolism*; D014158:Transcription, Genetic; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
chemical_list: D016328:NF-kappa B; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; D050881:E1A-Associated p300 Protein; C494360:EP300 protein, human; D019869:Phosphatidylinositol 3-Kinases; D066246:ErbB Receptors; D017479:Receptors, Platelet-Derived Growth Factor; D016392:Proto-Oncogene Proteins pp60(c-src); D051057:Proto-Oncogene Proteins c-akt; D020780:Matrix Metalloproteinase 9