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H-Ras/mitogen-activated protein kinase pathway inhibits integrin-mediated adhesion and induces apoptosis in
Yoshiya Tanaka1, Shingo Nakayamada, Hiroko Fujimoto
1First Department of Internal Medicine, University of Occupational and Environmental Health School of Medicine, Kitakyushu 807-8555, Japan. tanaka@med.uoeh-u.ac.jp
Abstract:
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.
Insights
Activated H-Ras signaling, particularly via Raf-1/MAPK, impairs osteoblast adhesion and proliferation by down-regulating integrins and inducing apoptosis. This pathway, distinct from PI3K signaling, affects key cell functions.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Osteoblasts are crucial for bone formation and maintenance.
- H-Ras signaling pathways play a role in cellular functions.
- Integrins mediate cell adhesion to the extracellular matrix, influencing cell behavior.
Purpose of the Study:
- To investigate the role of H-Ras and its downstream effectors in osteoblast functions.
- To determine how activated H-Ras impacts osteoblast adhesion, proliferation, and apoptosis.
- To elucidate the specific signaling pathways involved in H-Ras-mediated effects on osteoblasts.
Main Methods:
- Transfection of human osteoblasts with expression vectors for activated H-Ras mutants and Raf-1.
- Assessment of integrin expression levels via Western blotting.
- Cell adhesion assays using fibronectin and laminin.
- Proliferation assays.
- Apoptosis detection using Annexin-V/propidium iodide staining and TUNEL assay.
- Analysis of Fas and Bcl-2 expression.
Main Results:
- Activated H-Ras (H-Ras(V12), H-Ras(V12)T35S) and Raf-1 down-regulated key integrins (beta1, alpha4, alpha5, alpha6) and reduced osteoblast adhesion to fibronectin and laminin.
- H-Ras(V12)Y40C, a phosphoinositide 3-kinase (PI3K)-binding mutant, did not affect integrin expression or adhesion.
- Osteoblasts expressing H-Ras(V12), H-Ras(V12)T35S, or Raf-1 failed to proliferate, while H-Ras(V12)Y40C-expressing cells proliferated well.
- Fas was up-regulated and Bcl-2 was down-regulated in osteoblasts expressing H-Ras(V12), H-Ras(V12)T35S, or Raf-1, correlating with increased apoptosis.
Conclusions:
- The Raf-1/MAPK pathway, downstream of H-Ras, is critical for H-Ras-mediated effects on osteoblasts.
- H-Ras signaling via Raf-1/MAPK reduces beta(1)-integrin-dependent adhesion and inhibits proliferation.
- This pathway induces osteoblast apoptosis through up-regulation of Fas and down-regulation of Bcl-2.