Species difference exists in the effects of 1α,25(OH)2D3 and its analogue 2-methylene-19-nor-(20S)-1,25-dihydroxyvitamin D3 (2MD) on osteoblastic cells

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Abstract

The direct effect of 1α,25(OH)2D3 on osteoblasts remains unclear. In this study, we evaluated the in vitro effects of 1α,25(OH)2D3 and its analogue, 2-methylene-19-nor-(20S)-1,25-dihydroxyvitamin D3 (2MD), on osteoblasts from three different species, i.e. bone marrow stromal cells from the Sprague–Dawley (SD) rat, from the C57BL/6 mouse, as well as human osteoblast NHOst cells and human osteosarcoma derived MG-63 cells. We found that in rat cells, both compounds increased cell proliferation, inhibited cell apoptosis and increased alkaline phosphatase (ALP) activity. In mouse cells, however, both compounds initiated cell apoptosis and inhibited ALP activity. In human cells, although cell proliferation was inhibited by both compounds, cell apoptosis was inhibited and ALP activity was enhanced. In each species, 2MD was much more potent than 1α,25(OH)2D3. To summarize, species differences should be taken into account in studies of vitamin D effects. However, in all tested species – rat, mouse and human – 2MD is considerably more potent in its effects on osteoblastic cells in vitro than 1α,25(OH)2D3.

Introduction

The physiologically active form of vitamin D, 1α,25(OH)2D3, is a seco-steroid hormone. Its effects are mediated primarily via the vitamin D receptor (VDR), which is a member of the nuclear receptor superfamily. When bound by its ligands, the VDR dimerizes with the retinoic X receptor (RXR) and binds to promoter regions of responsive genes to either activate or repress transcription [1], [2]. It is generally agreed that 1α,25(OH)2D3 affects bone formation mainly by indirect mechanisms on calcium homeostasis [2]. Direct effects on osteoblasts have been reported but the findings have sometimes been contradictory, for example, 1α,25(OH)2D3 has been shown to increase the production of osteocalcin and alkaline phosphatase (ALP) in rat and human osteoblasts [3], [4], while in mouse osteoblasts 1α,25(OH)2D3 down-regulated expression of Phex, a mature osteoblast marker, and blocked in vitro mineralization [5], [6]. We felt that the role of species differences in this controversy needed to be clarified further. As a synthetic 1α,25(OH)2D3 analogue, 2-methylene-19-nor-(20S)-1α,25(OH)2D3 (2MD) differs structurally from 1α,25(OH)2D3 by the absence of the 19-methylene carbon and the addition of a methylene group in the 2-position between the 3β-hydroxyl and 1α-hydroxyl. In addition, the C-20 configuration is changed to 20S compared with a 20R configuration in naturally occurring vitamin D compounds [7]. In rat studies, 2MD stimulated bone formation without triggering noticeable hypercalcemia and hypercalciuria; additionally, although 2MD was about 30–100-fold more potent than 1α,25(OH)2D3 in bone calcium mobilization it was no more effective in promoting intestinal calcium absorption [8]. Further studies in ovariectomized rats showed that low doses of 2MD increased total bone mass and promoted the synthesis of both trabecular and cortical bone with high quality, whereas much higher doses of 1α,25(OH)2D3 only prevented bone loss over the same period [9]. Such bone anabolic effects imply that 2MD increases bone formation through a direct action on bone cells.

The bone forming osteoblasts originate from pluripotent mesenchymal stem cells [10]. After maturing and participating in bone remodeling most of the cells undergo apoptosis [11], [12]. In the present study, we used osteoblastic cells from different species, i.e. bone marrow stromal cells from Sprague–Dawley (SD) rat and C57BL/6 mouse, as well as human osteoblast NHOst and osteosarcoma MG-63 cells. We compared herein the effects of 1α,25(OH)2D3 and 2MD on cell proliferation, apoptosis and alkaline phosphatase activity.

Section snippets

Compounds

2MD was synthesized by Tetrionics (Madison, WI) by methods previously described [7] and was kindly supplied by Dr. HF DeLuca. 1α,25(OH)2D3 was purchased from Calbiochem (Darmstadt, Germany). Both reagents were diluted in 99% ethanol in stock concentrations for experimental use.

Cell culture

Rat bone marrow stromal cells were obtained from 6-week-old male Sprague–Dawley rats. Briefly, rats were euthanized using 4% isofluorane in CO2, and the bones were aseptically excised from the hindlimbs. External soft

Effects of 1,25(OH)2D3 and 2MD on growth of osteoblastic cells

In order to investigate the effects of 1α,25(OH)2D3 and 2MD on proliferation of osteoblastic cells, we cultured the cells in bone inducing medium for 8 days in the presence of different concentrations of the two VDR-ligands. In rat cells (Fig. 1A), both 1α,25(OH)2D3 and 2MD stimulated cell growth most apparently on day 8. However, in mouse cells (Fig. 1B), the results were apparently opposite to that in rat cells with all tested doses of 2MD, as well as the highest dose of 1α,25(OH)2D3 which

Discussion

In the present study osteoblastic cells from different species, rat, mouse and human were evaluated regarding the responses to two VDR ligands, 1α,25(OH)2D3 and 2MD. Our study focused on two important issues about the direct effects of VDR ligands on osteoblasts: the species difference and the comparison between 2MD and 1α,25(OH)2D3.

Cells from different species respond to VDR ligands differently. The rat is the most commonly used animal for vitamin D studies. Similar to humans, rats respond to

Acknowledgements

Åsa-Lena Dackland is gratefully acknowledged for technical support with the FACScan equipment. We thank Hector De Luca for supplying us with 2MD. This study was supported by the Karolinska Institute and the Swedish Foundation for Strategic Research.

References (27)

  • G.A. Rodan et al.

    The missing bone

    Cell

    (1997)
  • H. Tanaka et al.

    Direct action of 1,25-dihydroxyvitamin D on bone: VDRKO bone shows excessive bone formation in normal mineral condition

    J. Steroid. Biochem. Mol. Biol.

    (2004)
  • M.R. Haussler et al.

    The nuclear vitamin D receptor: biological and molecular regulatory properties revealed

    J. Bone Miner Res.

    (1998)
  • G. Jones et al.

    Current understanding of the molecular actions of vitamin D

    Physiol. Rev.

    (1998)
  • A. Gurlek et al.

    Regulation of osteoblast growth by interactions between transforming growth factor-beta and 1alpha,25-dihydroxyvitamin D3

    Crit. Rev. Eukaryot. Gene Expr.

    (2001)
  • M. van Driel et al.

    Evidence that both 1alpha,25-dihydroxyvitamin D3 and 24-hydroxylated D3 enhance human osteoblast differentiation and mineralization

    J. Cell. Biochem.

    (2006)
  • B. Ecarot et al.

    1,25-(OH)2D3 down-regulates expression of Phex, a marker of the mature osteoblast

    Endocrinology

    (1999)
  • E. Berger et al.

    Differentiation of cultured mice bone marrow into osteoblast-like cells results in acquisition of sex-specific responsiveness to gonadal steroids

    J. Endocrinol. Invest.

    (2004)
  • R.R. Sicinski et al.

    New 1alpha,25-dihydroxy-19-norvitamin D3 compounds of high biological activity: synthesis and biological evaluation of 2-hydroxymethyl, 2-methyl, and 2-methylene analogues

    J. Med. Chem.

    (1998)
  • N.K. Shevde et al.

    A potent analog of 1alpha,25-dihydroxyvitamin D3 selectively induces bone formation

    Proc. Natl. Acad. Sci. U.S.A.

    (2002)
  • L.A. Plum et al.

    2MD, a new anabolic agent for osteoporosis treatment

    Osteoporos Int.

    (2006)
  • R.L. Jilka et al.

    Osteoblast programmed cell death (apoptosis): modulation by growth factors and cytokines

    J. Bone Miner Res.

    (1998)
  • R.L. Jilka et al.

    Increased bone formation by prevention of osteoblast apoptosis with parathyroid hormone

    J. Clin. Invest.

    (1999)
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