| ObjectivesWe assessed insulin promoted osteoblastic differentiation of calcifying vascular smooth muscle cells(CVSMCs)and calcification of CVSMCs and investegated the mechanism involved.Methods6 weeks old male rats were sacrificed, the aorta removed. Clonally lines were identified as CVSMCs by their positive staining with monoclonal antibody a-actin, and by their ability to express high levels of alkaline phosphatase(ALP)and form calcified nodules. ALP activity was assayed by spectrophotometric measurement of p-nitrophenol release at 37℃.Osteocalcin released into the culture media was measured using a specific radioimmunoassay kit. By comparing the experimental group and the control, investeged the dose-dependent effect of insulin on receptor activator of NF-κB Ligand(RANKL)mRNA levels in CVSMCs. Real-time quantitative PCR assay for RANKL mRNA expression. The extent of mineralized matrix was determined by Alizarin Red S staining. In order to illuminate whether and how RANKL,extracellular signal-regulated kinase 1/2(ERK 1/2),and phosphatidylinositol 3-kinase (PI3K)/Akt signaling involved in the regulation of osteoblastic differentiation of CVSMCs, we used siRNA for blocking RANKL,and signal inhibitor for blocking ERK 1/2,and PI3K/Akt signal pathway, respectively, and then observe the expression of RANKL mRNA and ALP activity. Western blot was performed using anti-p-ERK1/2,ERK 1/2,p-Akt, and Akt.Resultsour experiments demonstrated that insulin could promote alkaline phosphatase activity, osteocalcin expression and the formation of mineralized nodules in CVSMCs.The dose response of effects of insulin on the RANKL activity in cultured CVSMCs. Suppression of with small interfering RNA (siRNA) abolished the insulin-induced alkaline phosphatase activity. Insulin induced activation of ERK 1/2 mitogen-activated protein kinase (MAPK) and Akt. Furthermore, pretreatment of human osteoblasts with the ERK 1/2 inhibitor PD98059, but not PI3K inhibitor LY294002 or Akt inhibitor HIMO, abolished the insulin-induced ALP secretion and blocked the promoting effect of insulin on osteoblastic differentiation of CVSMCs.ConclusionsThese data demonstrated that insulin could promote osteoblastic differentiation of CVSMCs by increased RANKL expression through ERK 1/2 activation, but not PI3K/Akt activation. |
