H-Ras/mitogen-activated protein kinase pathway inhibits integrin-mediated adhesion and induces apoptosis in osteoblasts.
We have studied the relevance of H-Ras and its downstream effectors to osteoblast functions. 1) Purified human osteoblasts highly expressed integrins beta1, alpha4, alpha5, alpha6 and the activation epitope of beta1. However, these molecules were markedly down-regulated on osteoblasts transfected with expression vector encoding fully activated H-Ras(V12), H-Ras(V12)T35S, activating Raf-1/mitogen-activated protein kinase (MAPK), or an active Raf-1 but not on cells having H-Ras(V12)Y40C, a phosphoinositide 3-kinase (PI3K)-binding mutant. 2) Although osteoblasts spontaneously adhered to fibronectin and laminin in beta1-dependent manner, the expression of H-Ras(V12) or H-Ras(V12)T35S, but not H-Ras(V12)Y40C, in osteoblasts reduced their adhesion. 3) Osteoblasts bearing H-Ras(V12), H-Ras(V12)T35S, or Raf-1 failed to proliferate, whereas those with H-Ras(V12)Y40C proliferated well. (4) The up-regulation of Fas and down-regulation of Bcl-2 were observed in osteoblasts expressing H-Ras(V12), H-Ras(V12)T35S, or Raf-1. (5) Most of the cells having H-Ras(V12), H-Ras(V12)T35S, or Raf-1 became annexin-V(high)/propidium iodide (PI)(high or low) and terminal deoxynucleotidyl-transferase-mediated dUTP nick-end labeling (TUNEL)(high)/PI(low) after 24 and 72 h incubation, respectively. Thus, we propose that H-Ras signals followed by Raf-1/MAPK pathway but not PI3K not only reduces beta(1)-mediated adhesion of osteoblasts to matrix proteins but induces apoptosis presumably via the Fas up-regulation and Bcl-2 down-regulation.
Tanaka
Yoshiya
Y
First Department of Internal Medicine, University of Occupational and Environmental Health School of Medicine, Kitakyushu 807-8555, Japan. tanaka@med.uoeh-u.ac.jp
Nakayamada
Shingo
S
Fujimoto
Hiroko
H
Okada
Yosuke
Y
Umehara
Hisanori
H
Kataoka
Tohru
T
Minami
Yasuhiro
Y
D017209:Apoptosis; D002448:Cell Adhesion; D002455:Cell Division; D002478:Cells, Cultured; D024042:Collagen Type I / Q000378:metabolism; D015536:Down-Regulation; D000939:Epitopes; D005353:Fibronectins / Q000378:metabolism; D005456:Fluorescent Dyes / Q000494:pharmacology; D011905:Genes, ras / Q000235:genetics*; D006801:Humans; D020287:In Situ Nick-End Labeling; D019012:Integrin beta1 / Q000096:biosynthesis; D016023:Integrins / Q000378:metabolism*; D007797:Laminin / Q000378:metabolism; D020935:MAP Kinase Signaling System; D009154:Mutation; D009841:Oligonucleotides / Q000378:metabolism; D010006:Osteoblasts / Q000378:metabolism / Q000473:pathology*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D011485:Protein Binding; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D015398:Signal Transduction; D014162:Transfection; D015854:Up-Regulation; D019096:Vitronectin / Q000378:metabolism; D019014:fas Receptor / Q000378:metabolism; D018631:ras Proteins / Q000378:metabolism*
D024042:Collagen Type I; D000939:Epitopes; D005353:Fibronectins; D005456:Fluorescent Dyes; D019012:Integrin beta1; D016023:Integrins; D007797:Laminin; D009841:Oligonucleotides; D019253:Proto-Oncogene Proteins c-bcl-2; D019096:Vitronectin; D019014:fas Receptor; D019869:Phosphatidylinositol 3-Kinases; D018631:ras Proteins