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

Abstract

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.

Fig. 1

Expression of CYP27B1 and CYP24A1 genes and 1α-hydroxylase activity in hMSCs. (A) Gel electrophoretogram shows RT-PCR products CYP27B1, VDR, and GAPDH in 7 representative specimens of hMSCs. Labels for lanes indicate age and gender. (B) Gel electrophoretogram shows RT-PCR products for CYP27B1, CYP24A1, VDR, and GAPDH in selected hMSCs^hi-1α (from a 42-year-old man) and hMSCs^lo-1α (from a 46-year-old man). (C) 1,25(OH)₂D₃ synthesis was measured in hMSCs^hi-1α and hMSCs^lo-1α. Cultures were treated with or without 1000 nM 25(OH)D₃ in serum-free α-MEM supplemented with 1% ITS⁺¹, 10 µM 1,2-dianilinoethane (N,N'-diphenylethylene diamine) for 24 hours. Cellular 1,25(OH)₂D₃ production (three replicate wells) was determined by EIA. There was no detectable (ND) 1,25(OH)₂D₃ in cultures without 25(OH)D₃ exogenous substrate. ***p < .001. (D) Gel electrophoretogram shows RT-PCR products for CYP24A1 and GAPDH in hMSC^hi-1α and hMSC^lo-1α cultures after 3 days in standard growth medium with 10% FBS-HI in the absence or presence of 1,25(OH)₂D₃ or 25(OH)D₃.

Fig. 2

Relative effects of 25(OH)D₃ and 1,25(OH)₂D₃ on proliferation of hMSCs. (A) Photomicrographs show hMSC^hi-1α and hMSC^lo-1α cultures after 3 days in the absence or presence of 100 nM 1,25(OH)₂D₃ or 25(OH)D₃ (×200 magnification). (B) Cell number was determined in hMSC^hi-1α and hMSC^lo-1α cultures after 3 days in the absence or presence of 1, 10, 100 nM 1,25(OH)₂D₃ or 25(OH)D₃. Results are expressed as mean ± SEM (12 replicate wells). *p < .05; **p < .01; ***p < .001. (C) Western immunoblots show proliferating cell nuclear antigen and β-actin levels in hMSC^hi-1α and hMSC^lo-1α cultures after 3 days in the absence or presence of 1,25(OH)₂D₃ or 25(OH)D₃. (D) Western immunoblots show p21, cyclin D1, and β-actin in hMSC^hi-1α and hMSC^lo-1α cultures after 3 days in the absence or presence of 1,25(OH)₂D₃ or 25(OH)D₃.

Fig. 3

Relative effects of 25(OH)D₃ and 1,25(OH)₂D₃ on Bax/Bcl-2 ratios in hMSCs. (A) Western immunoblots show Bax, Bcl-2, and β-actin in hMSC^hi-1α and hMSC^lo-1α cultures after 3 days in the absence or presence of 1,25(OH)₂D₃ or 25(OH)D₃. The bar graphs represent the Bax/Bcl-2 ratios after each densitometric value was normalized to β-actin. (B) Gel electrophoretograms show RT-PCR products for Bax, Bcl-2, and GAPDH in hMSC^hi-1α and hMSC^lo-1α cultures after 3 days in the absence or presence of 1, 10, or 100 nM 1,25(OH)₂D₃ or 1000 nM 25(OH)D₃. (C) Gel electrophoretogram shows RT-PCR products for Bax, Bcl-2, and GAPDH in hMSCs^hi-1α after 3 days in the absence or presence of 10 nM 1,25(OH)₂D₃ or 1000 nM 25(OH)D₃ ± 10 µM ketoconazole.

Fig. 4

Comparison of effects of 25(OH)D₃ and 1,25(OH)₂D₃ on osteoblast differentiation in hMSCs. (A) Alkaline phosphatase enzymatic activity (6 replicate wells) was measured in hMSCs^hi-1α and hMSCs^lo-1α in the absence or presence of 10 nM 1,25(OH)₂D₃ (open bars) or 25(OH)D₃ (closed bars) in osteogenic medium with 1% FBS-HI for 7 days. Results are reported relative to control (Rx/control) with horizonal dashed line as 1.0; mean ± SEM. (B) Gel electrophoretogram shows RT-PCR products of osteoblast signature genes (Runx2, AlkP, and BSP) and GAPDH in hMSCs^hi-1α after 0, 3, and 7 days in standard osteogenic medium with 10% FBS-HI. (C) Gel electrophoretogram shows RT-PCR products of osteoblast signature genes (Runx2, AlkP, and BSP) and GAPDH in hMSCs^hi-1α after 3 days in the absence or presence of 1, 10, or 100 nM 1,25(OH)₂D₃ in standard growth medium with 10% FBS-HI. (D) Gel electrophoretogram shows RT-PCR products of osteoblast signature genes (Runx2, AlkP, and BSP) and GAPDH in hMSCs^hi-1α after 3 days in the absence or presence of 1000 nM 25(OH)D₃ ± 10 µM ketoconazole in standard growth medium with 10% FBS-HI.

Fig. 5

Effect of CYP27B1 siRNA on the stimulation of osteoblast differentiation by 25(OH)D₃. Four groups were treated by electroporation with PBS (C = control), with nonsilencing control siRNA (NC), or with 10 or 100 pmol of CYP27B1 siRNA. (A) Photomicrographs show cultures of control and transfected hMSCs^hi-1α (×200 magnification). (B) Gel electrophoretogram shows CYP27B1 and GAPDH in controls and in transfected cells. (C) Western immunoblot shows CYP27B1 and β-actin protein levels in controls and in transfected cells. (D) Cells transfected with nonsilencing siRNA and 100 pmol of CYP27B1 siRNA were treated with or without 1000 nM 25(OH)D₃ in serum-free α-MEM supplemented with 1% ITS⁺¹, 10 µM 1,2-dianilinoethane (N,N'-diphenylethylene diamine) for 24 hours. Cellular 1,25(OH)₂D₃ production was determined by EIA as described under “In vitro biosynthesis of 1,25(OH)₂D₃ by hMSCs.” Results are shown as the mean ± SEM (3 replicate wells). There was no detectable (ND) 1,25(OH)₂D₃ in cultures without 1000 nM 25(OH)D₃ exogenous substrate. ***p< .001. (E) Gel electrophoretogram shows Runx2, AlkP, BSP, and GAPDH in controls and in transfected hMSCs^hi-1α after 3 days ± 1000 nM 25(OH)D₃. (F) Alkaline phosphatase enzymatic activity was measured in control and transfected hMSCs^hi-1α (100 pmol of CYP27B1 siRNA) after 7 days ± 10 nM 25(OH)D₃ in osteogenic medium. Values represent the mean ± SEM (6 replicate wells). ***p< .001.