title: Calcitonin gene-related peptide stimulates BMP-2 expression and the differentiation of human osteoblast-like cells in vitro.
abstract: OBJECTIVE
  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 CGRP-stimulated 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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Stomatology, Xinqiao Hospital Affiliated to the Third Military Medicine College, Chongqing 400037, China.
authors.forename: Gang
authors.initials: G
authors.lastname: Tian
authors.forename: Gang
authors.initials: G
authors.lastname: Zhang
authors.forename: Ying-hui
authors.initials: YH
authors.lastname: Tan
mesh_terms: D015153:Blotting, Western; D055396:Bone Morphogenetic Protein 2 / Q000235:genetics*; D015740:Calcitonin Gene-Related Peptide / Q000008:administration & dosage / Q000378:metabolism*; D002352:Carrier Proteins / Q000494:pharmacology; D002453:Cell Cycle; D002454:Cell Differentiation; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D000242:Cyclic AMP / Q000378:metabolism; D017362:Cyclic AMP Response Element-Binding Protein / Q000235:genetics*; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D006801:Humans; D010006:Osteoblasts / Q000378:metabolism*; D012516:Osteosarcoma / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D000099176:Noggin Protein
chemical_list: D055396:Bone Morphogenetic Protein 2; D015740:Calcitonin Gene-Related Peptide; D002352:Carrier Proteins; D000242:Cyclic AMP; D017362:Cyclic AMP Response Element-Binding Protein; D012333:RNA, Messenger; D000099176:Noggin Protein; C525718:BMP2 protein, human