Genistein Induced Murine Bone Marrow Mesenchymal Stem Cell Proliferation And Differentiation To Osteoblasts

Aim: The natural isoflavone phytoestrogen genistein (Gen) has been shown to stimulate osteoblastic bone mineralization in vitro and in vivo. The pharmacological mechanisms of Gen that acts on bone metabolism, however, have not been fully clarified. In the present study, we examined the effect of Gen on the proliferation and osteoblastic differentiation of primary mouse bone marrow derived mesenchymal stem cells (BMSCs).Materials and Methods: Mouse primary BMSCs were cultured in phenol red-free a-MEM supplemented with 1 0% charcoal-stripped fetal bovine serum and treated with 0.01 to lμM Gen with or without different antagonists. The cell proliferation was measured by [3H]-thymidine incorporation. The temporal sequence of osteoblastic differentiation in BMSCs was assessed by measuring alkaline phosphatase (ALP) activity and calcium deposition. Nitric oxide (NO) production, nitric oxide synthesase (NOS) activity and cyclic GMP (cGMP) level were measured using commercial NO, NOS and cGMP radioimmunoassay kit, respectively. Moreover, we determined core binding factor al (Cbfa1) gene expression by using semi-quantitative RT-PCR and mouse Cbfa1 promoter/luciferase reporter construct ,which was stably transfected into C2C12 cell line..Results: Gen (0.01～1μM) resulted in a dose-dependent increase in nitrite formation and cGMP content in the conditioned media of BMSCs cultures, consistent with increase of NOS activity. Gen treatment was associated with increased [3H] thymidine incorporation, ALP activity, calcium deposition and Cbfa\ gene expression in BMSCs cultures. Concurrent treatment with ICI 182,780 (0.1 μM), an estrogen receptor antagonist, or NG-monomethyl-L-arginine (L-NMMA, ImM), a NOS inhibitor or lH-[l,2,4]oxadiazolo[4,3,-a] quinoxalin-1-one (ODQ, 1 μM), a soluble guanylyl cyclase inhibitor abolished the Gen (lμ,M)-mediated increase in NO production and cGMP content, eliminated the stimulation of BMSCs on the proliferation and osteoblastic differentiation and significantly reduced the up-regulation of Cbfa1 gene expression by Gen. Furthermore, these results were similar to that observed with 17β-estradiol (E2, 0.0 lμM) and could be reproduced by treating BMSCs with 8-bromoguanosine 3',5'-cyclicmonophosphate (8-bromo-c GMP, 10μM), which were not blocked by the antagonists mentioned above, andConclusion: Our results indicated that genistein has estrogen-like activity and stimulates the proliferation and osteoblastic differentiation of mouse BMSCs through NO/cGMP pathway.