Rapamycin inhibits osteoblast proliferation and differentiation in MC3T3-E1 cells and primary mouse bone marrow stromal cells.
While the roles of the mammalian target of rapamycin (mTOR) signaling in regulation of cell growth, proliferation, and survival have been well documented in various cell types, its actions in osteoblasts are poorly understood. In this study, we determined the effects of rapamycin, a specific inhibitor of mTOR, on osteoblast proliferation and differentiation using MC3T3-E1 preosteoblastic cells (MC-4) and primary mouse bone marrow stromal cells (BMSCs). Rapamycin significantly inhibited proliferation in both MC-4 cells and BMSCs at a concentration as low as 0.1 nM. Western blot analysis shows that rapamycin treatment markedly reduced levels of cyclin A and D1 protein in both cell types. In differentiating osteoblasts, rapamycin dramatically reduced osteoblast-specific osteocalcin (Ocn), bone sialoprotein (Bsp), and osterix (Osx) mRNA expression, ALP activity, and mineralization capacity. However, the drug treatment had no effect on osteoblast differentiation parameters when the cells were completely differentiated. Importantly, rapamycin markedly reduced levels of Runx2 protein in both proliferating and differentiating but not differentiated osteoblasts. Finally, overexpression of S6K in COS-7 cells significantly increased levels of Runx2 protein and Runx2 activity. Taken together, our studies demonstrate that mTOR signaling affects osteoblast functions by targeting osteoblast proliferation and the early stage of osteoblast differentiation.
Singha
Ujjal K
UK
Division of Hematology/Oncology, Department of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania 15240, USA.
Jiang
Yu
Y
Yu
Shibing
S
Luo
Min
M
Lu
Yi
Y
Zhang
Jian
J
Xiao
Guozhi
G
D000818:Animals; D001854:Bone Marrow Cells / Q000166:cytology / Q000187:drug effects; D019556:COS Cells / Q000378:metabolism; D002454:Cell Differentiation / Q000187:drug effects; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line / Q000166:cytology / Q000187:drug effects; D002522:Chlorocebus aethiops; D050659:Core Binding Factor Alpha 1 Subunit / Q000096:biosynthesis / Q000235:genetics; D019925:Cyclin A / Q000096:biosynthesis / Q000235:genetics; D056741:Cyclin D; D016213:Cyclins / Q000096:biosynthesis / Q000235:genetics; D005786:Gene Expression Regulation / Q000187:drug effects; D058574:Integrin-Binding Sialoprotein; D051379:Mice; D010006:Osteoblasts / Q000166:cytology / Q000187:drug effects*; D015675:Osteocalcin / Q000096:biosynthesis / Q000235:genetics; D038762:Ribosomal Protein S6 Kinases, 70-kDa / Q000096:biosynthesis / Q000235:genetics; D012795:Sialoglycoproteins / Q000096:biosynthesis / Q000235:genetics; D020123:Sirolimus / Q000494:pharmacology*; D000076182:Sp7 Transcription Factor; D017154:Stromal Cells / Q000166:cytology / Q000187:drug effects; D014157:Transcription Factors / Q000096:biosynthesis / Q000235:genetics
D050659:Core Binding Factor Alpha 1 Subunit; D019925:Cyclin A; D056741:Cyclin D; D016213:Cyclins; C546872:Ibsp protein, mouse; D058574:Integrin-Binding Sialoprotein; C493716:Runx2 protein, mouse; D012795:Sialoglycoproteins; D000076182:Sp7 Transcription Factor; C479466:Sp7 protein, mouse; D014157:Transcription Factors; D015675:Osteocalcin; D038762:Ribosomal Protein S6 Kinases, 70-kDa; C448959:ribosomal protein S6 kinase, 70kD, polypeptide 2; D020123:Sirolimus