title: Epigallocatechin-3-gallate diminishes CCL2 expression in human osteoblastic cells via up-regulation of phosphatidylinositol 3-Kinase/Akt/Raf-1 interaction: a potential therapeutic benefit for arthritis.
abstract: OBJECTIVE
  To assess the effects of epigallocatechin-3-gallate (EGCG) on oncostatin M (OSM)-induced CCL2 synthesis and the associated signaling pathways in human osteoblastic cells. The therapeutic effect of EGCG on collagen-induced arthritis (CIA) in rats was also studied.
  
  
  METHODS
  CCL2 and c-Fos messenger RNA expression was analyzed by Northern blotting. The modulating effects of EGCG on the activation of Raf-1, Akt, and phosphatidylinositol 3-kinase (PI 3-kinase) were examined by coimmunoprecipitation, Western blotting, and PI 3-kinase activity assay. Interactions between c-Fos and CCL2 promoter were evaluated by electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) assay. The effect of EGCG on CIA in rats was examined clinically and immunohistochemically.
  
  
  RESULTS
  EGCG inhibited OSM-stimulated CCL2 expression in primary human osteoblasts and MG-63 cells. In MG-63 cells, EGCG alleviated the OSM-induced phosphorylation of Raf-1 at Ser338 but restored the dephosphorylation of Raf-1 at Ser259. EGCG increased the activity of PI 3-kinase, the level of phosphorylated Akt (Ser473), and binding between Raf-1 and active Akt. EMSA and ChIP assay revealed that EGCG attenuated activator protein 1 (AP-1)-CCL2 promoter interaction, possibly by reducing c-Fos synthesis. Codistribution of CD68+ macrophages and CCL2+ osteoblasts in osteolytic areas was obvious in the CIA model. Administration of EGCG markedly diminished the severity of CIA, macrophage infiltration, and the amount of CCL2-synthesizing osteoblasts.
  
  
  CONCLUSIONS
  By stimulating PI 3-kinase activity, EGCG promoted Akt/Raf-1 crosstalk, resulting in decreased AP-1 binding to CCL2 promoter, and finally reduced CCL2 production in osteoblasts. EGCG alleviated the severity of CIA, probably by suppressing CCL2 synthesis in osteoblasts to diminish macrophage infiltration. Our data support the therapeutic potential of EGCG on arthritis.
authors.affiliation: National Taiwan University Hospital, Department of Veterinary Medicine, College of Bio-Resources and Agriculture, National Taiwan University, Taipei, Taiwan.
authors.forename: Sze-Kwan
authors.initials: SK
authors.lastname: Lin
authors.forename: Hao-Hueng
authors.initials: HH
authors.lastname: Chang
authors.forename: Yunn-Jy
authors.initials: YJ
authors.lastname: Chen
authors.forename: Chih-Chiang
authors.initials: CC
authors.lastname: Wang
authors.forename: Deborah L
authors.initials: DL
authors.lastname: Galson
authors.forename: Chi-Yuan
authors.initials: CY
authors.lastname: Hong
authors.forename: Sang-Heng
authors.initials: SH
authors.lastname: Kok
mesh_terms: D000818:Animals; D000975:Antioxidants / Q000494:pharmacology*; D001169:Arthritis, Experimental / Q000188:drug therapy; D001172:Arthritis, Rheumatoid / Q000188:drug therapy*; D002392:Catechin / Q000031:analogs & derivatives* / Q000494:pharmacology; D002478:Cells, Cultured; D018932:Chemokine CCL2 / Q000187:drug effects*; D015536:Down-Regulation; D006801:Humans; D008297:Male; D051058:Oncogene Protein v-akt / Q000187:drug effects; D010006:Osteoblasts / Q000187:drug effects*; D019869:Phosphatidylinositol 3-Kinases / Q000187:drug effects; D019908:Proto-Oncogene Proteins c-raf / Q000187:drug effects; D051381:Rats; D015398:Signal Transduction / Q000187:drug effects*
chemical_list: D000975:Antioxidants; C111835:CCL2 protein, human; D018932:Chemokine CCL2; D002392:Catechin; C045651:epigallocatechin gallate; D019869:Phosphatidylinositol 3-Kinases; D051058:Oncogene Protein v-akt; D019908:Proto-Oncogene Proteins c-raf