title: Bezafibrate enhances proliferation and differentiation of osteoblastic MC3T3-E1 cells via AMPK and eNOS activation.
abstract: OBJECTIVE
  To investigate the effects of bezafibrate on the proliferation and differentiation of osteoblastic MC3T3-E1 cells, and to determine the signaling pathway underlying the effects.
  
  
  METHODS
  MC3T3-E1 cells, a mouse osteoblastic cell line, were used. Cell viability and proliferation were examined using MTT assay and colorimetric BrdU incorporation assay, respectively. NO production was evaluated using the Griess reagent. The mRNA expression of ALP, collagen I, osteocalcin, BMP-2, and Runx-2 was measured using real-time PCR. Western blot analysis was used to detect the expression of AMPK and eNOS proteins.
  
  
  RESULTS
  Bezafibrate increased the viability and proliferation of MC3T3-E1 cells in a dose- and time-dependent manner. Bezafibrate (100 μmol/L) significantly enhanced osteoblastic mineralization and expression of the differentiation markers ALP, collagen I and osteocalcin. Bezafibrate (100 μmol/L) increased phosphorylation of AMPK and eNOS, which led to an increase of NO production by 4.08-fold, and upregulating BMP-2 and Runx-2 mRNA expression. These effects could be blocked by AMPK inhibitor compound C (5 μmol/L), or the PPARβ inhibitor GSK0660 (0.5 μmol/L), but not by the PPARα inhibitor MK886 (10 μmol/L). Furthermore, GSK0660, compound C, or N(G)-nitro-L-arginine methyl ester hydrochloride (L-NAME, 1 mmol/L) could reverse the stimulatory effects of bezafibrate (100 μmol/L) on osteoblast proliferation and differentiation, whereas MK886 only inhibited bezafibrate-induced osteoblast proliferation.
  
  
  CONCLUSIONS
  Bezafibrate stimulates proliferation and differentiation of MC3T3-E1 cells, mainly via a PPARβ-dependent mechanism. The drug might be beneficial for osteoporosis by promoting bone formation.
authors.affiliation: Department of Endocrinology, First Affiliated Hospital, Sun Yat-sen University, Guangzhou 510080, China.
authors.forename: Xing
authors.initials: X
authors.lastname: Zhong
authors.forename: Ling-ling
authors.initials: LL
authors.lastname: Xiu
authors.forename: Guo-hong
authors.initials: GH
authors.lastname: Wei
authors.forename: Yuan-yuan
authors.initials: YY
authors.lastname: Liu
authors.forename: Lei
authors.initials: L
authors.lastname: Su
authors.forename: Xiao-pei
authors.initials: XP
authors.lastname: Cao
authors.forename: Yan-bing
authors.initials: YB
authors.lastname: Li
authors.forename: Hai-peng
authors.initials: HP
authors.lastname: Xiao
mesh_terms: D016475:3T3 Cells; D055372:AMP-Activated Protein Kinases / Q000378:metabolism; D000818:Animals; D001629:Bezafibrate / Q000008:administration & dosage / Q000494:pharmacology*; D002454:Cell Differentiation / Q000187:drug effects*; D049109:Cell Proliferation / Q000187:drug effects*; D002470:Cell Survival / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D005786:Gene Expression Regulation / Q000187:drug effects; D051379:Mice; D009569:Nitric Oxide / Q000378:metabolism; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism; D010006:Osteoblasts / Q000187:drug effects* / Q000378:metabolism; D048068:PPAR-beta / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D013997:Time Factors
chemical_list: D048068:PPAR-beta; D009569:Nitric Oxide; D052250:Nitric Oxide Synthase Type III; D055372:AMP-Activated Protein Kinases; D001629:Bezafibrate