The Effects Of Mechanical Stimulation On The Function And Signal Transduction In Osteoblasts

Objective: To observe the effects of mechanical stimulation on proliferation and differentiation in MG-63 osteoblast-like cellsMethods: Alkaline phoaphatase (ALP) activity was determined by pnitrophenyl phosphate assay. The mRNA levels of Collagenâ… (COLâ… ), osteocalcin (OC) and osteopontin (OPN) were examined using semi-quantitive RT-PCR. The protein levels of proliferating cell nuclear antigen (PCNA) and COLâ… were evaluated by Western Blot. The mineralization of MG-63 osteoblast-like cells was stained using Alizarin Red-S method.Results: Mechanical stimulation promoted the expression of PCNA, ALP, COL, OC, OPN and the formation of mineralization nodules in MG-63 osteoblast-like cells.Conclusions: Mechanical stimulation promoted the markers expression of proliferation and differentiation such as PCNA, COLâ… , OC and OPN in MG-63 osteoblast-like cells. It indicated mechanical stimulation may play an important role in proliferation and differentiation of osteoblasts. Objective: To examined the role of mechanical stimulation and MAP kinase signaling in stimulating CTGF.Methods: CTGF protein levels were determined by Western Blot and immunocytochemistry. MAPK (including ERK, JNK and p38) and AKT activities were evaluated by Western Blot. The mRNA levels of CTGF were examined using semi-quantitive RT-PCR.Results: Both CTGF mRNA and protein levels were increased by mechanical stimulation in MG-63 osteoblasts-like cells. Mechanical stimulation activated ERK and JNK, but not p38 MAP kinase in MG63 cells. While inhibition of ERK and p38 MAP kinase did not affect cyclic stretch-induced CTGF expression, inhibition of JNK suppressed stretch-induced CTGF expression in MG63 cells. In addition, inhibition of PI3-kinase (PI3K) abolished stretch-induced CTGF expression and JNK activation.Conelusions: The results suggest that mechanical stimulation induces CTGF expression, through PI3K signaling-mediated activation of the JNK signaling pathway. Objective: To examined the effects of mechanical stimulation on OPG/RANKL/RANK systemMethods: OPG, RANKL, RANK expressions were examined by Western Blot analysis and semi-quantitive RT-PCR. MAPK activities (including ERK, JNK and p38) were determined by Western Blot analysis. RAW264.7 cells were treated with or without RANKL for 6d, and then the multinucleated cells (MNCs) were observed with TRAP staining.Results: Mechanical stimulation did not affect RANKL expression, but with an increase in both OPG mRNA and protein levels in MG-63 osteoblasts-like cells. Mechanical stimulation activated ERK and JNK, but not p38 MAP kinase in MG63 cells. While inhibition of JNK did not affect cyclic stretch-induced OPG expression, inhibition of ERK suppressed stretch-induced OPG expression in MG-63 cells. In addition, mechanical stimulation did not affacte RANK expression in RAW264.7 cells, but it dramatically inhibited the RANKL-induced differentiation of RAW264.7 cells.Conclusions: The results suggest that mechanical stimulation promote bone formation and suppresse bone absorption through induction of OPG expression in osteoblasts and inhibition of RANKL-induced osteoclast differentiation of precursor bone marrow cells. Objective: The purposes of this study were to observe the expression of integrin-β₁ during MG-63 osteoblast-like cells differentiation, and if so to examine the effect of mechanical stimulation on integrin-β₁ expression and the role of integrin-β₁ in stretch-induced FAK and ERK activation.Methods: Integrin-β₁ protein and mRNA expressions were examined by Western Blot analysis and Real-Time PCR. FAK and ERK activities were determined by Western Blot analysis. The interation between integrin-β₁ and FAK was determined by Co-immunoprecipitation.Results: Integrin-β₁ expression demonstrated a bimodal distribution, being high during proliferation and mineralization in MG-63 cells. Mechanical stimulation promoted integrin-β₁ expression, induced FAK and ERK activation and increased the interation between integrin-β₁ and FAK. The inhibition of interation between integrin-β₁ and ECM (external cellular matrix) suppressed stretch-induced FAK and ERK activation.Conclusions: Integrin-β₁ mediates the stretch-induced FAK and ERK activation and integrin-β₁/FAK/ERK may play an important role in mechanotransduction.