Department of Orthopedic Surgery, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115, USA.
J Bone Miner Res. 2011 May;26(5):1145-53. doi: 10.1002/jbmr.298.
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.
1,25-二羟维生素 D(3)[1,25(OH)(2)D(3)]具有许多非钙调作用，这些作用依赖于抑制恶性和正常细胞类型的增殖和促进分化。1,25(OH)(2)D(3)刺激人骨髓基质细胞(hMSCs)的成骨细胞分化，但对 25-羟维生素 D(3)[25(OH)D(3)]对这些细胞的影响知之甚少。最近的证据表明，hMSCs 参与维生素 D 代谢，并可通过 CYP27B1/1α-羟化酶激活 25(OH)D(3)。这些研究检验了这样一个假设，即 25(OH)D(3)在 hMSCs 中的抗增殖和促分化作用依赖于 CYP27B1。我们研究了高表达（hMSCs(hi-1α)）或低表达（hMSCs(lo-1α)）CYP27B1 的 hMSCs，其 CYP24A1 和维生素 D 受体的表达水平相同。在 hMSCs(hi-1α)中，25(OH)D(3)降低增殖，下调增殖细胞核抗原（PCNA），上调 p21(Waf1/Cip1)，并降低 cyclin D1。与 1,25(OH)(2)D(3)不同，酮康唑抑制 25(OH)D(3)对 Bax 和 Bcl-2 的抗凋亡作用。25(OH)D(3)在 hMSCs(hi-1α)中的抗增殖作用和 1,25(OH)(2)D(3)在 hMSCs 两种样本中的抗增殖作用都可以通过细胞周期阻滞来解释，而不是通过增加细胞凋亡来解释。25(OH)D(3)刺激 hMSCs(hi-1α)中的成骨细胞分化被酮康唑和 CYP27B1 siRNA 转染所阻止。这些数据表明 CYP27B1 是 25(OH)D(3)在 hMSCs 中作用所必需的。有三条证据表明 CYP27B1 是 25(OH)D(3)对 hMSCs 的抗增殖和促分化作用所必需的：（1）在 CYP27B1 基础表达水平较低的 hMSCs 中未见这些作用，（2）在酮康唑处理的 hMSCs 中未见这些作用，（3）在 CYP27B1 表达被沉默的 hMSCs 中未见这些作用。成骨细胞分化和骨骼内稳态可能受 hMSCs 中 25(OH)D(3)的自分泌/旁分泌作用调节。
