The influence of RAMP1 overexpression on CGRP‐induced osteogenic differentiation in MG‐63 cells in vitro: An experimental study

Abstract

The aim of this study was to elucidate the influence of receptor activity modifying protein 1 (RAMP1) overexpression on the expression and distribution of calcitonin receptor-like receptor (CRLR) in MG-63 cells. Our research also focused on whether RAMP1 overexpression enhanced the promoting effect of exogenous CGRP on osteogenic differentiation in MG-63 cells. We first constructed a eukaryotic expression vector containing human RAMP1 and stably transfected it into MG-63 cells. Real-time PCR and Western blotting were used to determine the expression levels of RAMP1 and CRLR mRNA and protein, respectively. Immunofluorescence analysis was employed to compare the distribution of CRLR in transfected cells. After treatment with CGRP, the extent of osteogenic differentiation was evaluated by simultaneous monitoring of alkaline phosphatase activity, the expression patterns of osteoblastic markers and mineralisation staining. We found that RAMP1 was more highly expressed in the transfected group compared with the control groups (P < 0.01). The CRLR expression was significantly higher than that in the control groups (P < 0.05). In addition, after 7 days of CGRP treatment to induce osteogenic differentiation, the expression of collagen I mRNA was markedly increased in the transfected group (P < 0.05). The transfected group exhibited more granular precipitation in the cytoplasm with alkaline phosphatase staining after 7 and 14 days of differentiation. When stained with Alizarin Red, cells overexpressing RAMP1 were darker and formed many mineralised nodules with clear boundaries and calcium deposition typical of mineralised bone matrix structures at 28 days post-induction of differentiation. The CGRP-induced ALP activity in the RAMP1 overexpression group was significantly higher 3, 6 and 9 days after induction than that in the two control groups (P < 0.05). RAMP1 overexpression promotes CRLR expression, localisation on the cell membrane and enhanced CGRP-mediated differentiation of MG-63 cells. This study contributes to a better understanding of the molecular mechanisms governing CGRP-induced MG-63 differentiation.

Meteorin-Like Shows Unique Expression Pattern in Bone and Its Overexpression Inhibits Osteoblast Differentiation

“…These cells display several osteoblastic traits that are typical of a relatively immature osteoblast and can be induced to mineralize by adding ascorbate-2-phosphate, β-glycerophosphate, and dexamethasone[ 20 ]. In our study, control cells overexpressing EGFP formed mineralizing nodules as detected by von Kossa staining after 21 days induction, consistent with previous reports[ 20 , 21 ]. MG63 cells overexpressing METRNL-EGFP did not exhibit matrix mineralization.…”

Effects of αCGRP on the Adhesion, Proliferation and Differentiation of Osteoblasts Cultured on Titanium Surfaces

“…Previous studies have suggested that αCGRP can directly activate bone-forming osteoblasts because these cells express the calcitonin receptor-like receptor and RAMP1 dimer complex, which serve as the binding site for αCGRP 12) . Moreover, αCGRP was also observed to express on osteoblasts in previous studies 8,13,14) . Other studies have also reported that osteoblast αCGRP receptor activation stimulates adenylyl-cyclase 15) , which induces the activation of protein kinase A (PKA) and phospholipase C 16) , thereby increasing intracellular free calcium 17) with subsequent protein kinase C activation.…”

Receptor activity‐modifying protein 1 regulates the phenotypic expression of BMSCs via the Hippo/Yap pathway

“…Zhang et al, 2017). Moreover, Zhao et al, (2013) confirmed that RAMP1 overexpression promoted exogenous CGRPenhanced osteogenic differentiation in MG-63 cells. These data indicated that RAMP1 may be a potential therapeutic target in regulating CGRP-mediated effects during bone fracture healing (Y.…”