Effects of 25‐hydroxyvitamin D 3 on proliferation and osteoblast differentiation of human marrow stromal cells require CYP27B1/1α‐hydroxylase

1,25‐Dihydroxyvitamin D 3 [1,25(OH) 2 D 3 ] has many noncalcemic actions that rest on inhibition of proliferation and promotion of differentiation in malignant and normal cell types. 1,25(OH) 2 D 3 stimulates osteoblast differentiation of human marrow stromal cells (hMSCs), but little is known about the effects of 25‐hydroxyvitamin D 3 [25(OH)D 3 ] on these cells. Recent evidence shows that hMSCs participate in vitamin D metabolism and can activate 25(OH)D 3 by CYP27B1/1α‐hydroxylase. These studies test the hypothesis that antiproliferative and prodifferentiation effects of 25(OH)D 3 in hMSCs depend on CYP27B1. We studied hMSCs that constitutively express high (hMSCs hi‐1α ) or low (hMSCs lo‐1α ) levels of CYP27B1 with equivalent expression of CYP24A1 and vitamin D receptor. In hMSCs hi‐1α , 25(OH)D 3 reduced proliferation, downregulated proliferating cell nuclear antigen (PCNA), upregulated p21 Waf1/Cip1 , and decreased cyclin D1. Unlike 1,25(OH) 2 D 3 , the antiapoptotic effects of 25(OH)D 3 on Bax and Bcl‐2 were blocked by the P450 inhibitor ketoconazole. The antiproliferative effects of 25(OH)D 3 in hMSCs hi‐1α and of 1,25(OH) 2 D 3 in both samples of hMSCs were explained by cell cycle arrest, not by increased apoptosis. Stimulation of osteoblast differentiation in hMSCs hi‐1α by 25(OH)D 3 was prevented by ketoconazole and upon transfection with CYP27B1 siRNA. These data indicate that CYP27B1 is required for 25(OH)D 3 's action in hMSCs. Three lines of evidence indicate that CYP27B1 is required for the antiproliferative and prodifferentiation effects of 25(OH)D 3 on hMSCs: Those effects were not seen (1) in hMSCs with low constitutive expression of CYP27B1 , (2) in hMSCs treated with ketoconazole, and (3) in hMSCs in which CYP27B1 expression was silenced. Osteoblast differentiation and skeletal homeostasis may be regulated by autocrine/paracrine actions of 25(OH)D 3 in hMSCs. © 2011 American Society for Bone and Mineral Research.