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Evidence for auto/paracrine actions of vitamin D in bone: 1-hydroxylase expression and activity in human bone cells

M. van Driel^*, M. Koedam^*, C. J. Buurman^*, M. Hewison, H. Chiba, A. G. Uitterlinden^*, H. A. P. Pols^* and J. P. T. M. van Leeuwen^*,1

^* Department of Internal Medicine, Erasmus MC, Rotterdam, the Netherlands;

Division of Medical Sciences, University of Birmingham, Birmingham, UK; and

Department of Pathology, Sapporo Medical University School of Medicine, University of Sapporo, Sapporo, Japan

¹Correspondence: Department of Internal Medicine, Rm. Ee585, Erasmus MC, Dr. Molewaterplein 50, 3015 GE Rotterdam, The Netherlands. E-mail: j.vanleeuwen{at}erasmusmc.nl

SPECIFIC AIMS

Vitamin D is an important regulator of mineral homeostasis and bone metabolism. 1-Hydroxylation of 25-(OH)D₃ to form the bioactive vitamin D hormone, 1,25-(OH)₂D₃, is classically considered to take place in the kidney. However, 1-hydroxylase has also been reported at extrarenal sites. Whether bone is a 1,25-(OH)₂D₃ synthesizing tissue is not univocal.

The aim of this study was to investigate expression and activity of 1-hydroxylase in osteoblasts to define a possible autocrine/paracrine function for localized synthesis of 1,25-(OH)₂D₃ in bone.

PRINCIPAL FINDINGS

1. 1-Hydroxylase is expressed in both human and mouse osteoblasts and osteoclasts
CYP27B1 mRNA was found in human bone biopsies as well as in cultured human and murine osteoblasts and osteoclasts. Expression of 1-hydroxylase protein was demonstrated by Western blot analysis of extracts from human osteoblasts. Besides 1-hydroxylase, human osteoblasts expressed other components of the 1,25-(OH)₂D₃ endocrine system. These include the two membrane proteins, megalin and cubilin, and two P450 enzymes, CYP2R1 and CYP3A4, identified to have 25-hydroxylase activity.

2. 1-Hydroxylase is active in human osteoblasts
1-Hydroxylase activity was shown by the production of 400 pM 1,25-(OH)₂D₃ after incubation of human osteoblasts (SV-HFO, MG-63) with 1000 nM 25-(OH)D₃ for 48 h (Fig. 1 A). Time- and dose-dependent incubations with 25-(OH)D₃ on SV-HFO cells reveal production of 1,25-(OH)₂D₃ after 1 h and at lower concentrations (Km: 370 nM, V_max: 33 pmol/h/mg protein) (Fig. 1B ). The 1-hydroxylase activity and production of 1,25-(OH)₂D₃ by osteoblasts was completely blocked by the P450 inhibitor ketoconazole (Fig. 1B ).

3. 1-Hydroxylase activity is dependent on osteoblast differentiation
At the different stages of ostoblast differentiation, no changes in the expression of CYP27B1 of either human and murine osteoblasts were observed. There was a significant increase in 1-hydroxylase activity measured during culture in the supernatant (maximum 1,25-(OH)₂D₃ production of 800 fmol/ml). However, when 1,25-(OH)₂D₃ production was corrected for cell number (DNA), this translated into a small progressive decrease in production per cell over time. mRNA levels of megalin and cubilin did not change throughout the 3 wk culture period. In contrast to osteoblasts, induction of murine and human osteoclast differentiation by the NF-B activator RANKL resulted in an increase in 1-hydroxylase mRNA levels. 1-Hydroxylase activity and CYP27B1 expression were unaffected by 1,25-(OH)₂D₃ pretreatment.

4. 1-Hydroxylase expression in human osteoblasts is not regulated by PTH or calcium, but is increased by interleukin (IL) 1ßbeta;
Incubation with increasing doses of 25-(OH)D₃ had no effect on CYP27B1 mRNA expression. Neither PTH nor calcium changed 1-hydroxylase mRNA expression or activity in human osteoblasts. Albeit the production of 1,25-(OH)₂D₃ tended to increase at higher calcium concentrations. Incubation with IL-1ßbeta; significantly increased CYP27B1 expression.

5. 1-Hydroxylase activity results in biologically active 1,25-(OH)₂D₃ in osteoblasts
Short-term (48 h) incubations with 25-OHD₃ induced osteocalcin (OC) (Fig. 2 A,C) and CYP24 mRNA expression and alkaline phosphatase (ALP) activity (Fig. 2B ). This effect was stronger compared with directly added 1,25-(OH)₂D₃ (10 nM) for 48 h. Induction of OC and CYP24 mRNA expression by 25-(OH)D₃ was dose dependent (Fig. 2C ). OC protein measurements also demonstrated a stimulation following incubation with 25-(OH)D₃. Blocking 1,25-(OH)₂D₃ production by ketoconazole almost completely blocked the effect of 25-(OH)D₃ on OC (Fig. 2C ) and CYP24 mRNA expression. Mineralization was not affected after short-term incubation of 25-(OH)D₃ or 1,25-(OH)₂D₃.

Long-term (continuous) incubations showed no effect on total DNA, but treatment with 100 nM 25-(OH)D₃ significantly increased ALP activity at days 7 and 14 and significantly increased mineralization in the early phase of mineralization at day 14.

CONCLUSIONS AND SIGNIFICANCE

We show that in human osteoblasts 1-hydroxlase is expressed, as well as the vitamin D binding protein receptors megalin and cubilin, which in the kidney are known to be important for cellular uptake of 25-(OH)D₃ associated with vitamin D binding protein (DBP). Two P450 enzymes, CYP2R1 and CYP3A4, identified to have 25-hydroxylase activity and to convert vitamin D₃ into 25-(OH)D₃, were also expressed in human osteoblasts. These data implicate that human osteoblasts can function as an almost autonomous 1,25-(OH)₂D₃ producing cell (Fig. 3 ).

To date the only tissue that has been shown to be entirely autonomous in synthesizing 1,25-(OH)₂D₃ is human skin, as it can produce vitamin D₃ from 7-dehydrocholestrol.

1-Hydroxylase activity was shown by a time- and dose-dependent production of 1,25-(OH)₂D₃ after incubation of human osteoblasts with 25-(OH)D₃. Kinetics were on the same order of magnitude as that reported for cultured human renal proximal tubule cells. The 1-hydroxylase activity and production of 1,25-(OH)₂D₃ by osteoblasts were completely blocked by the P450 inhibitor ketoconazole, which is in line with data in kidney cells.

In contrast to osteoblasts, induction of murine and human osteoclast differentiation, which is dependent on the critical osteoclast inducer RANKL that acts via NF-B, resulted in an increase in 1-hydroxylase mRNA levels.

Regulation by 1,25-(OH)₂D₃ itself provides an elegant feedback mechanism to control 1-hydroxylase activity. However, autocrine/paracrine 1-hydroxylase activity in extrarenal tissues is modulated differently and is less sensitive to autoregulation by 1,25-(OH)₂D₃. Our data support this notion, as in human osteoblasts 1-hydroxylase activity and CYP27B1 expression were unaffected by 1,25-(OH)₂D₃, which resembles data obtained in the skin.

CYP27B1 mRNA expression and activity in osteoblasts were not affected by incubation with increasing doses of 25-(OH)D₃ or by two major regulators of renal 1-hydroxylase activity: PTH and calcium. These data suggest alternative regulatory mechanisms in target tissues. We found that a potent regulator of CYP27B1 mRNA in human osteoblasts appeared to be the NF-B activator IL-1ßbeta;. This further substantiates the significance of immune modulatory cytokines and the 1,25-(OH)₂D₃ endocrine system. Studies have reported both positive and negative effects of NF-B on 1-hydroxylase expression; however, the current IL-1ßbeta; data together with the effects of RANKL incubations implicate a role for NF-B in regulation of 1-hydroxylase in bone cells. All in all, this indicates the existence of a local (autocrine/paracrine) regulation of 1-hydroxylase by factors (growth factors, cytokines) derived from bone cells and/or cells in the bone marrow.

The functional biological significance of CYP27B1 expression and 1-hydroxylase activity in osteoblasts is shown by the positive short-term effects of 25-(OH)D₃ on OC and CYP24 mRNA expression and ALP activity, and by positive long-term effects on ALP activity and mineralization. What is remarkable is that the biological effects are much stronger when derived from the locally produced 1,25-(OH)₂D₃ after 25-(OH)D₃ treatment compared with directly added exogenous 1,25-(OH)₂D₃. This is all the more interesting because the self-produced amounts of 1,25-(OH)₂D₃ (400–800 pM as shown in the different 1,25-(OH)₂D₃ production experiments) are much lower than the 10 nM of 1,25-(OH)₂D₃, which is added directly. An explanation for this may include aspects related to the balance between synthesis and catabolism of 1,25-(OH)₂D₃ when continuous production of 1,25-(OH)₂D₃ takes place in the presence of 25-(OH)D₃ and direct availability at the site of action (binding to the vitamin D receptor in the osteoblasts).

The significance of 1-hydroxylase activity for the eventual biological response of 25-(OH)D₃ is shown by the studies using ketoconazole. Blocking 1,25-(OH)₂D₃ production by the P450 inhibitor ketoconazole almost completely blocked the effect of 25-(OH)D_3. This indicates that the enzyme that is blocked is 1-hydroxylase, because the only substrate added is 25-(OH)D_3. However, direct effects of 25-(OH)D₃ itself may contribute to the overall effect because 25-(OH)D₃ can bind to the vitamin D receptor, though with a 50- to 600-fold lower affinity, depending on cell origin.

In conclusion, this study provides the unequivocal answer that human osteoblasts contain all the components necessary to fulfill an autocrine/paracrine action of vitamin D in bone (Fig. 3) . In a general perspective, this study contributes to the currently emerging concept of steroid hormone production within its target tissues. This implicates a transition from a hormone acting at a distant site of synthesis to a local factor acting in an auto/paracrine manner.

View larger version (13K): [in this window] [in a new window]   Figure 1. Activity of 1-hydroxylase in human osteoblasts. A) The human osteoblast cell lines SV-HFO and MG-63 were incubated for 48 h with 1000 nM 25-(OH)D3 and next production of 1,25-(OH)2D3 was measured. B) SV-HFO cells were incubated with various concentrations of 25-(OH)D3 for 48 h in the absence (•) or presence () of 100 µM ketoconazole after which 1,25-(OH)2D3 production was measured. Data shown are means ± SD.

View larger version (12K): [in this window] [in a new window]   Figure 2. Effect of 25-(OH)D3 incubation on human osteoblast gene expression and osteoblast function. Human pre-osteoblasts were induced to differentiate and at various time points during differentiation treated for 48 h with 1000 nM 25-(OH)D3 (), 10 nM 1,25-(OH)2D3 (), or vehicle (), then analyzed for effects on A) OC mRNA expression by real-time PCR and B) ALP activity. C) OC mRNA expression was measured after incubation with various concentrations of 25-(OH)D3 and 100 µM of ketoconazole for 24 h on day 14 of SV-HFO culture. Data are shown as means ± SD. *P < 0.05 and **P < 0.01 vs. control on the same day.

View larger version (16K): [in this window] [in a new window]   Figure 3. Schematic diagram of auto/paracrine actions of vitamin D in bone.The 25-(OH)D3-vitamin D binding protein (DBP) complex binds to its receptors cubilin and megalin on the membrane of osteoblasts and is internalized. The complex is dissociated and DBP degraded while 25-(OH)D3 is converted into 1,25-(OH)2D3 in the mitochondria. Next, 1,25-(OH)2D3 can bind to its receptor (VDR) and modify cellular activity. The eventual result shown in this manuscript is induction of CYP24 and osteocalcin mRNA expression, an increase in osteocalcin protein, alkaline phosphatase activity, and enhancement of mineralization.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.06-6374fje

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This Article Abstract Full Text Full Text (PDF) All Versions of this Article: fj.06-6374fjev1 20/13/2417    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by van Driel, M. Articles by van Leeuwen, J. P. T. M. Search for Related Content PubMed PubMed Citation Articles by van Driel, M. Articles by van Leeuwen, J. P. T. M.