Calcitonin gene-related peptide induces proliferation and monocyte chemoattractant protein-1 expression via extracellular signal-regulated kinase activation in rat osteoblasts.
BACKGROUND
Calcitonin gene-related peptide (CGRP), a sensory neuropeptide, affects osteoblast proliferation and bone formation. However, the mechanisms are not fully understood. Monocyte chemoattractant protein-1 (MCP-1) is a chemokine that stimulates the migration of monocytes and plays important roles in regulating bone remolding during fracture repair. In this study, we investigated the effects of CGRP on proliferation and MCP-1 expression in cultured rat osteoblasts.


METHODS
Primary rat osteoblasts were isolated from fetal rats calvariae. Cells were exposed to gradient concentrations (10(-9) to 10(-7) mol/L) of CGRP. Protein and mRNA levels of MCP-1 were quantified by Western blotting and semiquantitative reverse transcription-polymerase chain reaction, respectively. The protein level of MCP-1 was investigated and compared in cell culture media by enzyme linked immunosorbent assay (ELISA). Phospho-extracellular signal-regulated kinase (ERK) expression was detected by Western blotting. Cell proliferative activity was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and BrdU assay. The effects of MAPK/ERK kinase (MEK)-inhibitor U0126 on CGRP-induced MCP-1 expression in primary rat osteoblasts were examined.


RESULTS
CGRP effectively enhanced primary rat osteoblast proliferation and led to significant increases in the expression of MCP-1 mRNA and protein in time- and dose-dependent manners. CGRP activated the ERK pathway. Pretreatment of cultured rat osteoblasts with MEK inhibitor U0126 resulted in dose-dependent inhibitions of CGRP-induced MCP-1 mRNA and protein levels. Thus, CGRP promoted cell proliferation and stimulated MCP-1 expression in cultured rat osteoblasts.


CONCLUSIONS
These studies document novel links between CGRP and MCP-1 and illuminate the effects of CGRP in regulating bone remodeling.
Han
Na
N
Department of Orthopaedics and Trauma, Peking University People's Hospital, Beijing 100044, China.
Zhang
Dian-Ying
DY
Wang
Tian-Bing
TB
Zhang
Pei-Xun
PX
Jiang
Bao-Guo
BG
D000818:Animals; D015153:Blotting, Western; D002070:Butadienes / Q000494:pharmacology; D015740:Calcitonin Gene-Related Peptide / Q000494:pharmacology*; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D018932:Chemokine CCL2 / Q000235:genetics / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004797:Enzyme-Linked Immunosorbent Assay; D048049:Extracellular Signal-Regulated MAP Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D009570:Nitriles / Q000494:pharmacology; D010006:Osteoblasts / Q000187:drug effects* / Q000378:metabolism*; D051381:Rats
D002070:Butadienes; D018932:Chemokine CCL2; D004791:Enzyme Inhibitors; D009570:Nitriles; C113580:U 0126; D048049:Extracellular Signal-Regulated MAP Kinases; D015740:Calcitonin Gene-Related Peptide