Diosmetin induces human osteoblastic differentiation through the protein kinase C/p38 and extracellular signal-regulated kinase 1/2 pathway.
BACKGROUND
The survival of osteoblasts is one of the determinants of the development of osteoporosis. This study is the first to investigate the osteoblastic differentiation induced by diosmetin, a flavonoid derivative, in osteoblastic cell lines MG-63, hFOB, and MC3T3-E1 and bone marrow stroma cell line M2-10B4.


METHODS
Osteoblastic differentiation was determined by assaying alkaline phosphatase (ALP) activity and mineralization degree and measuring various osteoblast-related markers using ELISA. Expression and phosphorylation of Runt-related transcription factor 2 (Runx2), protein kinase Cdelta (PKCdelta), extracellular signal-regulated kinase (ERK), p38, and c-jun-N-terminal kinase (JNK) was assessed by immunoblot. Rac1 activity was determined by immunoprecipitation, and Runx2 activity was assessed by EMSA. Genetic inhibition was performed by small hairpin RNA plasmids or small interfering RNA (siRNA) transfection.


RESULTS
Diosmetin exhibited an effect on osteoblastic maturation and differentiation by means of ALP activity, osteocalcin, osteopontin, and type I collagen production, as well as Runx2 upregulation. Induction of differentiation by diosmetin was associated with increased PKCdelta phosphorylation and the activations of Rac1 and p38 and ERK1/2 kinases. Blocking PKCdelta by siRNA inhibition significantly decreased osteoblastic differentiation by inhibiting Rac1 activation and subsequently attenuating the phosphorylation of p38 and ERK1/2. In addition, blocking p38 and ERK1/2 by siRNA transfection also suppressed diosmetin-induced cell differentiation.


CONCLUSIONS
In this study, we show that diosmetin induced osteoblastic differentiation through the PKCdelta-Rac1-MEK3/6-p38 and PKCdelta-Rac1-MEK1/2- ERK1/2-Runx2 pathways and that it is a promising agent for treating osteoporosis.
Hsu
Ya-Ling
YL
Cell Biology Laboratory, Department of Biotechnology, Chia-Nan University of Pharmacy and Science, Tainan, Taiwan.
Kuo
Po-Lin
PL
D000818:Animals; D001853:Bone Marrow / Q000187:drug effects; D002454:Cell Differentiation / Q000187:drug effects*; D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects; D050659:Core Binding Factor Alpha 1 Subunit / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D005419:Flavonoids / Q000494:pharmacology*; D006801:Humans; D020935:MAP Kinase Signaling System / Q000187:drug effects; D051379:Mice; D019950:Mitogen-Activated Protein Kinase 1 / Q000235:genetics / Q000378:metabolism*; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism*; D020929:Mitogen-Activated Protein Kinase Kinases / Q000378:metabolism; D010006:Osteoblasts / Q000166:cytology* / Q000187:drug effects / Q000201:enzymology*; D010766:Phosphorylation / Q000187:drug effects; D051745:Protein Kinase C-delta / Q000235:genetics / Q000378:metabolism*; D017154:Stromal Cells / Q000166:cytology / Q000187:drug effects; D020830:rac1 GTP-Binding Protein / Q000378:metabolism
D050659:Core Binding Factor Alpha 1 Subunit; D005419:Flavonoids; D051745:Protein Kinase C-delta; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020929:Mitogen-Activated Protein Kinase Kinases; D020830:rac1 GTP-Binding Protein; C039602:diosmetin