Calcitonin-gene-related peptide stimulates stromal cell osteogenic differentiation and inhibits RANKL induced NF-kappaB activation, osteoclastogenesis and bone resorption.
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, pit erosion area, mRNA levels of TRAP and cathepsin K, and nuclear factor-kappaB (NF-kappaB) 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-kappaB ligand (RANKL) activation of NF-kappaB. 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-kappaB activation, osteoclastogenesis, and bone resorption.
Wang
Liping
L
Physical Medicine and Rehabilitation Service, Veterans Affairs Palo Alto Health Care System, Palo Alto, CA, USA. lipingw@stanford.edu
Shi
Xiaoyou
X
Zhao
Rong
R
Halloran
Bernard P
BP
Clark
David J
DJ
Jacobs
Christopher R
CR
Kingery
Wade S
WS
D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D015153:Blotting, Western; D001862:Bone Resorption / Q000139:chemically induced / Q000188:drug therapy*; D015740:Calcitonin Gene-Related Peptide / Q000494:pharmacology* / Q000627:therapeutic use; D002454:Cell Differentiation / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D016328:NF-kappa B / Q000378:metabolism*; D010010:Osteoclasts / Q000166:cytology* / Q000187:drug effects; D010012:Osteogenesis / Q000187:drug effects*; D016133:Polymerase Chain Reaction; D053245:RANK Ligand / Q000494:pharmacology*; D017154:Stromal Cells / Q000166:cytology* / Q000187:drug effects
D016328:NF-kappa B; D053245:RANK Ligand; D000469:Alkaline Phosphatase; D015740:Calcitonin Gene-Related Peptide