High concentration of tacrolimus inhibits proliferation and osteoblastic differentiation of human mesenchymal stem cells / 中国药理学与毒理学杂志

OBJECTIVE To investigate the effect of tacrolimus on cell proliferation and osteoblastic differentiation of primary human bone marrow-derived mesenchymal stem cells (hBMSCs). METHODS hBMSCs were cultured with tacrolimus 0.001-5 μmol·L-1. BrdU incorporation was used to assess the cell proliferation while cellular alkaline phosphatase (ALP) activity and calcium deposition were measured to evaluate the osteoblastic differentiation of hBMSCs cultures. The calcineurin (CaN) activity was also examined using commercial CaN assay kit, and core binding factor 1 alpha subunit (Cbfα1) protein level was determined by Western blotting. RESULTSTacrolimus 0.001-0.1 μmol·L-1 promoted BrdU incorporation but had no effect on ALP activity and calcium deposition, whereas tacrolimus 0.5-5 μmol·L-1 resulted in significant decrease in both cell proliferation and osteoblastic maturation, by reducing BrdU incorporation, ALP activity, and calcium deposition of hBMSCs cultures in a concentration-dependent manner. In addition, tacrolimus 0.5-5 μmol·L-1 led to concentration-dependent decrement in CaN activity, which was consistent with down-regulated Cbfα1 protein in the tacrolimus treated cells. CONCLUSION High concentration of tacrolimus might inhibit the cell proliferation and osteoblastic differentiation of hBMSCs cultures through a CaN/Cbfα1 pathway.

Runx2 regulation and its isoforms / 中国药理学通报

Runx2 is a transcription factor essential for osteoblast differentiation and bone formation.Expression of Runx2 is initiated from two promoters,the distal P1 promoter and the proximal P2 promoter.These promoters give rise to two major protein isoforms with distinct amino termini,named as Runx2-TypeI and Runx2-TypeII.Here we review some integrated complex pathways(Wnt/LRP5/?-catenin,BMP/Smads/DPC4,Vitamin D/VDR/VDRE pathway,etc) and several key proteins(Msx2,Dlx5,Twists,etc) necessary for regulating Runx2 activity and bone formation,which give us new hints on controlling osteoblast differentiation by drug therapy and treating osteoporosis by gene therapy.

Phytoestrogen Genistein induces osteoblastic differentiation of mouse bone marrow-derived mesenchymal stem cells through p38 MAPK / 中国药理学通报

Aim To investigate the role of p38 mitogen-activated protein kinases(MAPKs) in genistein-induced osteoblastic differentiation of mouse bone marrow-derived mesenchymal stem cells(BMSCs).Methods Mouse BMSCs cultured in phenol red-free ?-MEM containing 10% V/V FBS,were added ?-glycerophosphate and ascorbic acid for inducing osteoblastic differentiation,and treated with 0.01~1 ?mol?L~(-1) genistein and/or SB203580 and PD98059.The temporal sequence of osteoblastic differentiation in BMSCs cultures was assayed by measuring alkaline phosphatase activity(ALP) and calcium deposition.The MAPK phosphorylation level was detected by Western-blot.Results Genistein(0.01~1 ?mol?L~(-1)) showed a dose-dependent effect on osteoblastic differentiation as evidenced by increased alkaline phosphatase(ALP) activity and calcium deposition in mouse BMSCs cultures.Genistein(1 ?mol?L~(-1))-induced osteoblastic differentiation of BMSCs was diminished by p38 MAPK inhibitor but not the p44/42 MAPK inhibitor.The effects of Genistein were associated with rapid and sustained activation of p38 MAPK in the BMSCs cultures,which was blocked by SB203580(1 ?mol?L~(-1)).In contrast,Genistein treatment resulted in inactivation of p42/44MAPK,which was further attenuated by PD98059(25 ?mol?L~(-1)).Conclusion p38 MAPK plays an important role in genistein-induced osteoblastic differentiation of mouse BMSCs cultures.

Progress in Cbf?1 gene and its regulation / 中国药理学通报

Cbf?1 gene encodes an osteoblast specific transcription factor that regulates osteoblast differentiation and bone formation. The Cbf?1 gene contains 9 exons, however, the alternative splicing of its exons leads to genesis of multiple Cbf?1 isoforms with differences in transactivation potentials. Several signal transduction pathways, such as Ras MAPK pathway, cAMP pathway, or Smads DPC4 pathway, are involved in regulating Cbf?1 activity or its expression. These studies open new doors in pharmacological control of bone formation and gene or cell therapy of slowing the progression of osteoporosis.