The Biological Effects Of CGRP On MC3T3-E1 Cell Line And The Relationship With Wnt/β-catenin Signaling Pathway

Objective:To investigate the effect of calcitonin gene-related peptide(CGRP) on the differentiation of MC3T3E1 cells, and to explore the possible mechanism by investigating the Wnt/β-catenin signaling pathway.Methods:MC3T3E1 cells were supplied by PUMC College Cell Bank, cultured to the 6 generation. The 6th generation cells were divided into four groups,CGRP group,CGRP+CGRP receptor antagonist(CGRP8-37) group,CGRP+DKK1 group and the control group(the same amount of PBS),each group were cultured in osteogenic environment. The expression level of bone markers and Wnt/β-catenin signaling gene,such as ALP,OC,COLⅠ,RUN X2,C-myc, Lef, Tcf and β-catenin, were detected at culture 4,7,14 day using real time PCR. The expression level ofβ-catenin protein was also detected by Western blot at the same point.After culturing 7 day, location of β-catenin in cells were detected by immunofluorescence.Results:1. Using alizarin red, ALP staining staining were observed in MC3T3E1 cell extracellular matrix calcium deposition and MC3T3E1 cell alkaline phosphatase(ALP) were positive. ALP activity and presence of Ca deposits in extracellular matrix.2.Q-PCR results show that 10-8 mol/L CGRP can significantly improve the expression of MC3T3-E1 related genes(ALP, OC, COL I, X2 RUN)and Wnt/ beta-catenin signaling pathway related genes(C-myc,-catenin,TCF, LEF) in cells.3.Western blot results showed that CGRP can significantly promote the expression of beta-catenin protein and its aggregation in the nucleus,while the CGRP receptor antagonist and DKK1 obviously inhibit this effecConclusions:MC3T3E1 cells possess osteogenic properties of osteoblast. 10-8 mol/L CGRP can significantly promote differentiation of MC3T3E1 in osteogenic environment, Wnt/β-catenin signaling pathway may be the underlying mechanism.