Calcitonin gene-related peptide stimulates BMP-2 expression and the differentiation of human osteoblast-like cells in vitro

Abstract

Aim: To investigate whether bone morphogenic protein-2 (BMP-2) expression was involved in calcitonin gene-related peptide (CGRP)-induced osteogenesis in human osteoblast-like cells in vitro. Methods: MG-63 osteogenic human osteosarcoma cells were treated with CGRP (10 -8 mol/L) for 48 h. Cell cycle phases were determined using flow cytometry assay. The protein levels of BMP-2, ALP, Osteocalcin, ColIa1, CREB, and pCREB were measured with Western blotting, while the mRNA level of BMP-2 was measured with qR-T PCR. The expression of ALP in MG-63 cells was also studied using immunofluorescence staining. The level of cAMP was measured with ELISA assay. Results: CGRP treatment significantly stimulated proliferation of MG-63 cells, and increased the expression of BMP-2 and the osteogenic proteins ALP, Osteocalcin and ColIa1. Pretreatment with the BMP signaling inhibitor Noggin (100 ng/mL) did not affect CGRPstimulated proliferation and BMP-2 expression, but abolished the CGRP-induced increases of the osteogenic proteins ALP, Osteocalcin and ColIa1. Furthermore, CGRP treatment markedly increased cAMP level in MG-63 cells, whereas pretreatment with the cAMP pathway inhibitor H89 (5 μmol/L) abolished the CGRP-induced increases of cAMP level and BMP-2 expression. Conclusion: In MG-63 cells, the BMP pathway is involved in CGRP-induced osteogenic differentiation but not in proliferation, whereas the cAMP/pCREB pathway is involved in the expression of BMP-2.Keywords: c calcitonin gene-related peptide; Noggin; H89; MG-63 human osteosarcoma cell; osteogenesis; bone morphogenic protein; cAMP/pCREB pathway Acta Pharmacologica Sinica (2013Sinica ( ) 34: 1467Sinica ( -1474 doi: 10.1038/aps.2013 published online 27 May 2013 Original Article IntroductionSensory nerve activation has a centrally mediated but poorly understood action on bone. The relative importance and interactions between autonomic, sensory, and peripheral nervous system actions on bone mass are not clear in healthy individuals and even less so in pathologic states. Understanding how the central nervous system integrates homeostatic signals with the regulation of bone homeostasis is an exciting research area.Experimental evidence suggests that the nervous system is involved in bone remodeling. In response to fracture or other trauma, peripheral peptidergic neurons can influence osteoclast formation through the production of several neuropeptides. Calcitonin gene-related peptide (CGRP), a 37-residue peptide generated in specific neurons by alternative splicing of the calcitonin gene, is an important neuropeptide expressed * To whom correspondence should be addressed.E-mail tanyhoms@yahoo.cn Received 2012-10-30 Accepted 2013-03-25 in nerve fibers during bone development and repair [1,2] .Numerous in vivo studies have suggested that CGRP is associated with bone development, metabolism and repair. In vitro studies have demonstrated that CGRP stimulates osteoblast proliferation, differentiation and maturation in both osteoblast cell lines and bone marrow mesenchyme strom...

Isoflavones rich cowpea and vitamin D induces the proliferation and differentiation of human osteoblasts via BMP‐2/Smad pathway activation: Mechanistic approach

“…BMP antagonist diminished the gene expression of the markers namely ALP, Collagen OPN, and total and phosphorylated Smad‐1/5/8 levels. The findings of our study with CP and VD were coherent with earlier reports where noggin influenced the expression levels of the genes ALP, Collagen, OPN and t‐Smad‐1/5/8 and p‐Smad‐1/5/8 . However, the expression levels of the OSX transcription factor were quite unpredicted after noggin treatment because the expression was completely abolished in all combination and individual groups of Dz and VD and combination groups of CP and Ge alone.…”

“…Serine/threonine kinases receptors and other intracellular signaling proteins like Smads play a major role in triggering the effects at cellular level by BMP's . Amid the different BMP's, BMP‐2 both by in vivo and in vitro studies have paraded to be a crucial regulator during the bone formation and also stimulation of the differentiation of osteoblasts in an osteogenic way …”

Implant-derived magnesium induces local neuronal production of CGRP to improve bone-fracture healing in rats

“…6d). Further study is required to understand why RUNX2 is not responsive to magnesium or CGRP and whether other reported CREB1-regulated pathways (for example, BMP2, Wnt/β-catenin) are involved 48,49 .…”

NPY and CGRP Inhibitor Influence on ERK Pathway and Macrophage Aggregation during Fracture Healing

“…Zhang et al claimed that CGRP participated in bone metabolism and promoted swifter fracture healing [14]. Moreover, in a recent study, Tian et al confirmed that CGRP promoted osteogenic differentiation as well as proliferation [15]. Despite various proposed mechanisms, it is certain that CGRP is a pro-osteogenic neurotransmitter.…”