Calcitonin-gene-related peptide stimulates stromal cell osteogenic differentiation and inhibits RANKL induced NF-κB activation, osteoclastogenesis and bone resorption

Abstract

Previously we observed that capsaicin treatment in rats inhibited sensory neuropeptide signaling, with a concurrent reduction in trabecular bone formation and bone volume, and an increase in osteoclast numbers and bone resorption. Calcitonin gene-related peptide (CGRP) is a neuropeptide richly distributed in sensory neurons innervating the skeleton and we postulated that CGRP signaling regulates bone integrity. In this study we examined CGRP effects on stromal and bone cell differentiation and activity in vitro. CGRP receptors were detected by immunocytochemical staining and real time PCR assays in mouse bone marrow stromal cells (BMSCs) and bone marrow macrophages (BMMs). CGRP effects on BMSC proliferation and osteoblastic differentiation were studied using BrdU incorporation, PCR products, alkaline phosphatase (ALP) activity, and mineralization assays. CGRP effects on BMM osteoclastic differentiation and activity were determined by quantifying tartrate-resistant acid phosphatase positive (TRAP + ) multinucleated cells, Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. NIH Public Access Author ManuscriptBone. Author manuscript; available in PMC 2011 May 1. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript pit erosion area, mRNA levels of TRAP and cathepsin K, and nuclear factor-κB (NF-κB) nuclear localization. BMSCs, osteoblasts, BMMs, and osteoclasts all expressed CGRP receptors. CGRP (10 -10 -10 -8 M) stimulated BMSC proliferation, up-regulated the expression of osteoblastic genes, and increased ALP activity and mineralization in the BMSCs. In BMM cultures CGRP (10 -8 M) inhibited receptor activator of NF-κB ligand (RANKL) activation of NF-κB. CGRP also down-regulated osteoclastic genes like TRAP and cathepsin K, decreased the numbers of TRAP+ cells, and inhibited bone resorption activity in RANKL stimulated BMMs. These results suggest that CGRP signaling maintains bone mass both by directly stimulating stromal cell osteoblastic differentiation and by inhibiting RANKL induced NF-κB activation, osteoclastogenesis, and bone resorption.

Increase of CGRP-Containing Nerve Fibers in the Rat Periodontal Ligament After Luxation

“…Osteoclast-like cells from co-cultures of mouse bone marrow cells and spleen cells are devoid of RAMP1 (Granholm et al 2008). However, CGRP is known to have effects on rat osteoclasts (Naot and Cornish 2008;Wang et al 2010). Thus, there appears to be species difference about osteoclastic RAMP1 expression.…”

“…These findings suggest that CGRP has functions to RAMP1-IR cells after experimental luxation injury. Previous studies have indicated that CGRP inhibits osteoclast activity and bone resorption (Naot and Cornish 2008;Wang et al 2010). This peptide is also known to stimulate osteoblast proliferation in vitro and increase bone formation in vivo (Wang et al 2010).…”

“…Previous studies have indicated that CGRP inhibits osteoclast activity and bone resorption (Naot and Cornish 2008;Wang et al 2010). This peptide is also known to stimulate osteoblast proliferation in vitro and increase bone formation in vivo (Wang et al 2010). By affecting osteoclasts and osteoblasts, CGRP may suppress bone resorption and promote bone formation after the experimental luxation injury.…”

“…At 5-7 days after the treatment, the number of CGRP-containing nerve fibers increases in the PDL. In vitro experiments, CGRP has been shown to regulate the activity of osteoblasts and osteoclasts (Naot and Cornish 2008;Wang et al 2010). Therefore, this peptide is also considered to be involved in bone remodeling during experimental tooth movement.…”

Corneal confocal microscopy detects severe small fiber neuropathy in diabetic patients with Charcot neuroarthropathy

“…Since then, the role of small fiber neuropathy has received little attention in relation to CN, despite the observation that there are significant numbers of substance P and calcitonin gene‐related peptide (CGRP)‐expressing nerves in the periosteum and bone marrow14. Indeed, there is a substantial literature on the role of non‐synaptically released substance P and CGRP, regulating osteoblast and osteoclast proliferation15, and inhibiting pro‐inflammatory cytokines16, key players in the pathophysiology of CN. Whereas in a study of 23 patients with CN, small fiber integrity, assessed by the hyperemic response to heating skin, was found to be preserved17, a subsequent study from the same group showed an impaired nerve–axon reflex in patients with CN18.…”