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      Effects of 25-Hydroxyvitamin D 3 on Proliferation and Osteoblast Differentiation of Human Marrow Stromal Cells Require CYP27B1/1α-Hydroxylase

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          Abstract

          1,25-Dihydroxyvitamin D 3 [1,25(OH) 2D 3] has many noncalcemic actions that rest on inhibition of proliferation and promotion of differentiation in malignant and normal cell types. 1,25(OH) 2D 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) 2D 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) 2D 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.

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          Age-related intrinsic changes in human bone-marrow-derived mesenchymal stem cells and their differentiation to osteoblasts.

          Shuanhu Zhou, Joel Greenberger, Michael Epperly … (2008)
          In vivo and in vitro studies indicate that a subpopulation of human marrow-derived stromal cells (MSCs, also known as mesenchymal stem cells) has potential to differentiate into multiple cell types, including osteoblasts. In this study, we tested the hypothesis that there are intrinsic effects of age in human MSCs (17-90 years). We tested the effect of age on senescence-associated beta-galactosidase, proliferation, apoptosis, p53 pathway genes, and osteoblast differentiation in confluent monolayers by alkaline phosphatase activity and osteoblast gene expression analysis. There were fourfold more human bone MSCs (hMSCs) positive for senescence-associated beta-galactosidase in samples from older than younger subjects (P < 0.001; n = 17). Doubling time of hMSCs was 1.7-fold longer in cells from the older than the younger subjects, and was positively correlated with age (P = 0.002; n = 19). Novel age-related changes were identified. With age, more cells were apoptotic (P = 0.016; n = 10). Further, there were age-related increases in expression of p53 and its pathway genes, p21 and BAX. Consistent with other experiments, there was a significant age-related decrease in generation of osteoblasts both in the STRO-1+ cells (P = 0.047; n = 8) and in adherent MSCs (P < 0.001; n = 10). In sum, there is an age-dependent decrease in proliferation and osteoblast differentiation, and an increase in senescence-associated beta-galactosidase-positive cells and apoptosis in hMSCs. Up-regulation of the p53 pathway with age may have a critical role in mediating the reduction in both proliferation and osteoblastogenesis of hMSCs. These findings support the view that there are intrinsic alterations in human MSCs with aging that may contribute to the process of skeletal aging in humans.
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            Transcriptional activation of the Cdk inhibitor p21 by vitamin D3 leads to the induced differentiation of the myelomonocytic cell line U937.

            Hye Sun Lee, M Bommakanti, Gen Liu … (1996)
            The hormonal form of vitamin D, 1,25-dihydroxyvitamin D3, acting through its cognate nuclear receptor (vitamin D3 receptor, VDR) will induce myeloid leukemic cell lines to terminally differentiate into monocytes/macrophages. Because VDR acts by transcriptionally regulating responsive genes in a ligand-dependent manner, we sought target genes of the receptor that initiate, the differentiation process in response to ligand. We screened a cDNA library prepared from the myelomonocytic U937 cell line with probes generated from either 1,25-dihydroxyvitamin D3-treated or untreated cells. We report here that a candidate clone that hybridized differentially is the Cdk inhibitor p21WAF1, CIP1. Furthermore, we show that p21 is transcriptionally induced by 1,25-dihydroxyvitamin D3 in a VDR-dependent, but not p53-dependent, manner, and we identify a functional vitamin D response element in the p21 promoter. Transient overexpression of p21 and/or the related Cdk inhibitor p27 in U937 cells in the absence of 1,25-dihydroxyvitamin D3 results in the cell-surface expression of monocyte/macrophage-specific markers, suggesting that ligand-modulated transcriptional induction of the p21 gene facilitates the induced differentiation of this monoblastic cell line. We believe that this is the first report demonstrating that the ectopic overexpression of a Cdk inhibitor such as p21 or p27 directly leads to a terminal differentiation program.
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              Regulation of the human p21(waf1/cip1) gene promoter via multiple binding sites for p53 and the vitamin D3 receptor

              Anna Saramaki, Claire M. Banwell, Moray J. Campbell … (2006)
              The main regulator of the human tumor suppresser gene p21(waf1/cip1) is the transcription factor p53, but more recently it has been suggested to be a primary anti-proliferative target for the nuclear receptor VDR in the presence of its ligand 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3). To identify VDR responding regions, we analyzed 20 overlapping regions covering the first 7.1 kb of the p21(waf1/cip1) promoter in MCF-7 human breast cancer cells using chromatin immuno-precipitation assays (ChIP) with antibodies against p53 and VDR. We confirmed two known p53 binding regions at approximate positions −1400 and −2300 and identified a novel site at position −4500. In addition, we found three VDR-associated promoter regions at positions −2300, −4500 and −6900, i.e. two regions showed binding for both p53 and VDR. In silico screening and in vitro binding assays using recombinant and in vitro translated proteins identified five p53 binding sites within the three p53-positive promoter regions and also five 1α,25(OH)2D3 response elements within the three VDR-positive regions. Reporter gene assays confirmed the expected responsiveness of the respective promoter regions to the p53 inducer 5-fluorouracil and 1α,25(OH)2D3. Moreover, re-ChIP assays confirmed the functionality of the three 1α,25(OH)2D3-reponsive promoter regions by monitoring simultaneous occupancy of VDR with the co-activator proteins CBP, SRC-1 and TRAP220. Taken together, we demonstrated that the human p21(waf1/cip1) gene is a primary 1α,25(OH)2D3-responding gene with at least three VDR binding promoter regions, in two of which also p53 co-localizes.
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                Author and article information

                Journal
                Journal ID (nlm-ta): J Bone Miner Res
                Journal ID (publisher-id): jbmr
                Title: Journal of Bone and Mineral Research
                Publisher: Wiley Subscription Services, Inc., A Wiley Company
                ISSN (Print): 0884-0431
                ISSN (Electronic): 1523-4681
                Publication date (Print): May 2011
                Publication date (Electronic): 23 November 2010
                Volume: 26
                Issue: 5
                Pages: 1145-1153
                Affiliations
                [1 ]simpleDepartment of Orthopedic Surgery, Brigham and Women's Hospital, Harvard Medical School Boston, MA, USA
                [2 ]simpleDepartment of Orthopedic Surgery, First Affiliated Hospital of Harbin Medical University, Harbin Medical University Harbin, Heilongjiang, People's Republic of China
                Author notes
                Address correspondence to: Julie Glowacki, PhD, Department of Orthopedic Surgery, Brigham and Women's Hospital, 75 Francis Street, Boston, MA 02115, USA. E-mail: jglowacki@ 123456rics.bwh.harvard.edu

                This study was presented in part at the Annual Meeting of the American Society for Bone and Mineral Research, in Toronto, Ontario, Canada, October 15–19, 2010.

                Article
                DOI: 10.1002/jbmr.298
                PMC ID: 3179303
                PubMed ID: 21542014
                SO-VID: 488de0a1-18cf-44f8-b091-7158b256b231
                Copyright © Copyright © 2011 American Society for Bone and Mineral Research
                License:

                Re-use of this article is permitted in accordance with the Creative Commons Deed, Attribution 2.5, which does not permit commercial exploitation.

                History
                Date received : 09 July 2010
                Date revision received : 21 September 2010
                Date accepted : 11 November 2010
                Categories
                Subject: Original Article

                ScienceOpen disciplines: Human biology
                Keywords: osteoblast differentiation,vitamin d,proliferation,bone marrow stromal cells,apoptosis
                Data availability:
                ScienceOpen disciplines: Human biology
                Keywords: osteoblast differentiation, vitamin d, proliferation, bone marrow stromal cells, apoptosis

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