Repression of IGF-I-induced osteoblast migration by (-)-epigallocatechin gallate through p44/p42 MAP kinase signaling

Abstract

Polyphenolic compounds in beverages may have benefits in the prevention of osteoporosis. It has been demonstrated previously that insulin-like growth factor-I (IGF-I) could stimulate the migration of osteoblasts. In the present study, it was investigated whether chlorogenic acid, a major polyphenol in coffee, and (-)-epigallocatechin gallate (EGCG), a major polyphenol in green tea, could affect this IGF-I-stimulated migration of osteoblast-like MC3T3-E1 cells. The IGF-I-stimulated osteoblast migration, evaluated by Transwell cell migration and wound-healing assays, was inhibited by EGCG but not chlorogenic acid. IGF-I induced the phosphorylation of p44/p42 mitogen-activated protein (MAP) kinase, p70 S6 kinase and Akt. The IGF-I-induced migration was suppressed by PD98059, a MAP kinase kinase 1/2 inhibitor, and deguelin, an Akt inhibitor, but not rapamycin, an inhibitor of the upstream kinase of p70 S6 kinase (mammalian target of rapamycin). EGCG attenuated the IGF-I-induced phosphorylation of p44/p42 MAP kinase but not Akt. Taken together, the present results suggest that EGCG inhibits IGF-I-induced osteoblast migration via p44/p42 MAP kinase.

Incretin accelerates platelet-derived growth factor-BB-induced osteoblast migration via protein kinase A: The upregulation of p38 MAP kinase

“…Normal human osteoblasts (NHOsts) isolated from human tissue obtained under "informed consent" were obtained from CAMBREX (Charles, IA, USA) and cultured under conditions similar to those for MC3T3-E1 cells, as previously described 25 . The migration of MC3T3-E1 cells or NHOsts was analyzed by a wound-healing assay as previously described 22 . In brief, the cells were seeded at 10 × 10 4 cells/well into an Ibidi Culture-Insert 2 Well (Ibidi, Martinsried, Germany) with a 500-μm margin from the side of the well and cultured for 24 h. After removing the insert and performing pretreatment with GLP-1, GIP, 8-bromo cAMP or Bt2 cAMP for 60 min, the cells were stimulated by PDGF-BB for the indicated periods.…”

“…After 5 days, the medium was exchanged for α-MEM containing 0.3% FBS. After 48 h, the cells were used for a Western blot analysis as previously described 22 . The stimulation with GLP-1 or PDGF-BB to the cultured cells were performed in 1 ml of 0.3% FBS-containing α-MEM for a variety of times indicated.…”

“…In the indicated experiments, the pretreatment with H89 or GIP to the cells were performed for 60 min prior to the stimulation. The cells were then lysed, homogenized and sonicated in a lysis solution composed of 62.5 mM Tris/HCl, pH 6.8, 2% sodium dodecyl sulfate (SDS), 50 mM dithiothreitol and 10% glycerol 22 . SDS-polyacrylamide gel electrophoresis (PAGE) was performed by the method of Laemmli 26 in 10% polyacrylamide gels 22 .…”

“…The cells were then lysed, homogenized and sonicated in a lysis solution composed of 62.5 mM Tris/HCl, pH 6.8, 2% sodium dodecyl sulfate (SDS), 50 mM dithiothreitol and 10% glycerol 22 . SDS-polyacrylamide gel electrophoresis (PAGE) was performed by the method of Laemmli 26 in 10% polyacrylamide gels 22 . The protein was fractionated and transferred onto an Immun-Blot PVDF membrane (Bio-Rad, Hercules, CA, USA) 22 .…”

“…SDS-polyacrylamide gel electrophoresis (PAGE) was performed by the method of Laemmli 26 in 10% polyacrylamide gels 22 . The protein was fractionated and transferred onto an Immun-Blot PVDF membrane (Bio-Rad, Hercules, CA, USA) 22 . The membranes were blocked with 5% fat-free dry milk in Tris-buffered saline-Tween (TBS-T; 20 mM Tris-HCl, pH 7.6, 137 mM NaCl, 0.1% Tween 20) for 1 h before incubation with primary antibodies 22 .…”