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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. Ee526, Erasmus MC, Dr. Molewaterplein 50, 3015 GE Rotterdam, the Netherlands. E-mail: j.vanleeuwen{at}erasmusmc.nl

ABSTRACT

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)₂D_3, is classically considered to take place in the kidney. However, 1-hydroxylase has 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 an autocrine/paracrine function for 1,25-(OH)₂D₃ in bone. We show that 1-hydroxlase is expressed in human osteoblasts, as well as the vitamin D binding protein receptors megalin and cubilin. Functional analyses demonstrate that after incubation with the 1-hydoxylase substrate 25-(OH)D_3, the osteoblasts can produce sufficient 1,25-(OH)₂D₃ to modulate osteoblast activity, resulting in induced alkaline phosphatase (ALP) activity, osteocalcin (OC) and CYP24 mRNA expression, and mineralization. The classical renal regulators of 1-hydroxylase, parathyroid hormone, and ambient calcium do not regulate 1-hydroxylase in osteoblasts. In contrast, interleukin (IL)-1ß strongly induces 1-hydroxylase. Besides the bone-forming cells, we demonstrate 1-hydroxylase activity in the bone resorbing cells, the osteoclasts. This is strongly dependent on osteoclast inducer RANKL. This study showing expression, activity, and functionality of 1-hydoxylase unequivocally demonstrates that vitamin D can act in an auto/paracrine manner in bone.—van Driel, M., Koedam, M., Buurman, C. J., Hewison, M., Chiba, H., Uitterlinden, A. G., Pols, H. A. P., van Leeuwen, J. P. T. M. Evidence for auto/paracrine actions of vitamin D in bone: 1-hydroxylase expression and activity in human bone cells.

Key Words: calcium homeostasis • PBMCs • osteoblasts • osteoclasts

VITAMIN D IS ONE OF THE MAJORfactors involved in calcium homeostasis via actions on intestine, kidney, parathyroid gland, and bone (1) . The biologically most active vitamin D molecule is 1,25-dihydroxyvitamin D₃ (1,25-(OH)₂D₃). In bone, 1,25-(OH)₂D₃ is important for mineralization, either indirectly via control of calcium absorption in intestine and reabsorption in the kidney, but also via direct action on osteoblasts (2 and unpublished results). 1,25-(OH)₂D₃ is formed from the parental vitamin D molecule by sequential hydroxylations in the liver (25-hydroxylation) and kidney (1-hydroxylation): 25-hydroxyvitamin D₃ (25-(OH)D₃) formed in the liver is transported to the kidney bound to the vitamin D binding protein (DBP) and is then metabolized to 1,25-(OH)₂D₃ by the renal cytochrome P450 enzyme 25-hydroxyvitamin D₃-1-hydroxylase (3) _.

However, 1-hydroxylase expression and activity are not restricted to the kidney. The first evidence for extrarenal 1-hydroxylase was based on studies of patients with sarcoidosis in whom conversion from 25-(OH)D₃ to 1,25-(OH)₂D₃ was demonstrated in affected lymph nodes and pulmonary alveolar macrophages (4 , 5) . More recently it was reported that 1-hydroxylase expression was increased in pathological parathyroid glands (6 , 7) and up-regulated in various other malignant conditions (8 9 10) . Prostate cells also express 1-hydroxylase, but here the activity of the enzyme is decreased in cancer cells (11 12 13 14) .

1-Hydroxylase expression has also been demonstrated in a variety of normal extrarenal tissues like skin, lymph nodes, colon, pancreas, adrenal medulla, dendritic cell, endothelial cells, brain, hypothalamus, and placenta (15 16 17 18 19 20) . Remarkably, 25 years ago 1-hydroxylase activity was reported in human bone cells in culture (21 22 23) but bone has never been considered a vitamin D-synthesizing tissue. The aim of the present study was to settle this issue and comprehensively investigate the expression and activity of 1-hydroxylase and other components of the vitamin D metabolic pathway in osteoblasts and to define the potential biological activity in these cells. An in vitro model of osteoblast differentiation and bone formation was used to examine the impact of osteoblast maturation on the synthesis of 1,25-(OH)₂D₃ and to assess 1-hydroxylase functionality in bone cell activity.

MATERIALS AND METHODS

Cell culture
Human SV-HFO cells were seeded at a density of 10 x 10³ vital cells per cm² in phenol-red free -minimal essential medium (-MEM Gibco BRL, Paisley, U.K.), pH 7.5, supplemented with 20 mM HEPES (Sigma, St. Louis, MO, USA), 100 IU/ml penicillin, 100 µg/ml streptomycin (Life Technologies, Breda, the Netherlands), 1.8 mM CaCl₂^.2H₂O (Sigma), and 2% heat-inactivated charcoal-treated FCS at 37°C and 5% CO₂ in a humidified atmosphere. Medium was replaced every 2–3 days supplemented with freshly diluted 10 mM ß-glycerophosphate (Sigma) and 1 µM dexamethasone (9-fluoro-16-methylprednisolone, Sigma). Cells and culture supernatant from duplicate wells were collected at multiple days (indicated in figures) during culture. Medium was stored at –20°C and cells were scraped in PBS containing 0.1% triton X-100 and stored in –80°C. Before use, cell lysates were sonicated on ice in a sonifier cell disrupter for 2 x 15 s. Human osteoblast-like MG-63 cells were maintained in MEM medium supplemented with 2 mM L-glutamine, 0.1% glucose (Glc), 100 IU/ml penicillin, 100 IU/ml streptomycin, and 10% FCS at 37°C in 5% CO₂ in a humidified atmosphere. Cells were seeded at 100,000 cells/cm² in MEM containing 10% FCS and cultured for 6 days. The cDNAs from KS483 murine osteoblasts at different time points during culture were generously provided by Dr. M. Karperien (Leiden University Medical Center, the Netherlands).

Human osteoclast cultures
Peripheral blood mononuclear cells (PBMCs) were obtained from healthy donors and induced to osteoclasts as described earlier (24) .

RAW 264.7 monocytic cells were cultured in -MEM containing 10% FCS, with or without 20 ng/ml mRANKL (R&D Systems, Abingdon, UK).

Human femoral head biopsies
Human bone material was obtained from two femoral head biopsies (of ostearthritic bone). These have been collected within a clinical trial, which was approved by the medical ethical commission (MEC No. 204.287). Human biopsy samples were homogenized using a Mikro Dismembrator S (Sartorius, Goettingen, Germany).

Cell culture treatments
25-(OH)D₃ and 1,25-(OH)₂D₃ were generously provided by Dr. L. Binderup, Leo Pharmaceuticals, Ballerup, Denmark. 25-(OH)D₃ and 1,25-(OH)₂D₃ were incubated for 24 h, 48 h, or continuously in concentrations indicated in the legends of the figures. Cells were incubated with interleukin-1 (IL-1ß) (Peprotech, London, UK), PTH (Bachem, Weil am Rhein, Germany) and different concentrations of CaCl₂ (Sigma-Aldrich) for 24 h. 1,25-(OH)₂D₃ production were measured using a gamma-B 1,25-dihydroxy vitamin D RIA according to the manufacturer’s protocol (IDS, Boldon, UK); cross-reactivity with 25-(OH)D₃ is 0.001%.

RNA isolation
Total RNA was isolated using RNA-Bee solution (Tel-Test, Friendswood, TX, USA) according to the manufacturer’s protocol and processed for real-time polymerase chain reaction (PCR) or Affymetrix array-based gene expression. RNA was washed and precipitated overnight at –20°C with equal volumes of 100 mM EDTA and 8 M LiCl. Total RNA was quantified using the Ribogreen assay (Molecular Probes, Eugene, OR, USA). One µg of RNA was reverse transcribed into cDNA, using 0.5 µg oligo(dT)₁₈, 0.2 µg random hexamer primers, and Moloney murine leukemia virus according to the protocol of the manufacturer (MBI fermentas, St. Leon-Rot, Germany).

Gene expression (real-time PCR)
Quantitative real-time PCR was carried out using an ABI Prism 7700 sequence detection system (PE Biosystems, Rotkreuz, Switzerland). Reactions were performed in 25 µl volumes using a qPCR^TM core kit (Eurogentec, Seraing, Belgium). Reaction mixes contained 2–40 ng cDNA, 5 mM MgCl₂, 200 µM dNTPs, and 0.025 U/µl Hot GoldStar enzyme. Primer and probe sets were designed using the Primer Express software (Version 1.5; Applied Biosystems, Foster City, CA, USA) and Biolegio (Malden, the Netherlands). Probes were labeled at the 3'-end with the quencher dye 6-carboxytetramethylrhodamine and at the 5'-end with the reporter dye 6-carboxyfluorescein (Eurogentec, Seraing, Belgium). Primer and probe sequences and concentrations are listed in Table 1 . Cycling conditions were 50°C for 2 min, 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. Data are presented as relative mRNA levels calculated by the equation 2^{– Ct} ( Ct=Ct of target gene minus Ct of GAPDH).

Gene expression (microarrays)
To get a complete overview on the expression of other components of the vitamin D metabolism/endocrine system, Affymetrix-based microarray studies were performed, according to the manufacturer’s protocol.

Western blotting
Cells were scraped in PBS phenylmethylsulfonylfluoride (PMSF) 0.5 mM. Lysates were obtained via three freeze thaw cycles and stored in –80°C. Western blot analysis was performed as described previously (25) .

DNA measurement and ALP activity
SV-HFO cell lysates were treated with heparin and RNase A (50 µg/ml in PBS) for 30 min at 37°C. DNA content was measured according to the ethidium bromide method of Karsten and Wollenberger (26) . ALP activity was assayed by determining the release of paranitrophenol from paranitrophenylphosphate (20 mM in 1 M diethanolamin buffer supplemented with 1 mM MgCl₂ at pH 9.8) in the cell lysates for 10 min at 37°C (27) . Adding 0.1 M NaOH stopped the reaction and absorption was measured at 405 nm using a Packard Spectra Count. Results were adjusted for DNA content of the corresponding cell lysates.

Mineralization
SV-HFO cell lysates were incubated overnight with 0.25 M HCl at 4°C. Calcium content was colorimetrically determined after addition of 1M ethanolamine buffer (pH 10.6) 0.35 mM o-cresolphtalein complexone, 19.8 mM 8-hydroxyquinoline, and 0.6 mM hydrochloric acid at 595 nm (Packard Spectra Count). Results were adjusted for DNA content of the corresponding cell lysates.

Statistical analyses
Differences between groups were tested for significance using the Student’s t test. Values were considered significantly different at P < 0.05.

RESULTS

Figure 1 A shows the presence of CYP27B1 mRNA 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 (Fig. 1B ). 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, as found by Affymetrix gene expression analyses (Fig. 1C ).

View larger version (25K): [in this window] [in a new window]   Figure 1. Expression of CYP27B1, 1-hydroxylase protein, DPB receptors, and vitamin D3-25-hydroxylases in bone cells. A) Real-time PCR analyses of CYP27B1 expression in human (SV-HFO, day 14) and murine (KS463, day 14) osteoblasts, the human osteosarcoma cell line MG-63, a human bone biopsy and human osteoclasts. Values are 2^-delta ct (delta ct=ct of target gene minus ct of GAPDH). B) Western blot analyses of 1-hydroxylase in SV-HFO human osteoblasts at day 14 of culture. C) Affymetrix-based gene expression analyses of the DBP receptors megalin and cubilin and the vitamin D3-25-hydroxylases CYP2R1 and CYP3A4 in human osteoblasts (SV-HFO) at day 14 of culture. Data are presented as means ± SD.

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

View larger version (11K): [in this window] [in a new window]   Figure 2. 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 1000 nM 25-(OH)D3 for various periods and C) 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.

We studied the relationship with differentiation in the differentiating human preosteoblasts (SV-HFO). These cells express maximal ALP activity at day 14 of culture coinciding with the onset of mineralization (Fig. 3 A, B). Treatment with 1,25-(OH)₂D₃ during this differentiation sequence caused an increase in ALP activity in the first phase of culture and an earlier maximum calcification. At the different stages of differentiation no changes in the expression of CYP27B1 of both human and murine osteoblasts were observed (data not shown). Despite this, there was a significant increase in 1-hydroxylase activity during culture and osteoblast differentiation, with a maximum 1,25-(OH)₂D₃ production of 800 fmol/ml at day 16 (Fig. 4 A). 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 (Fig. 4B ). However, the CYP27B1 mRNA levels and megalin and cubilin mRNA expression did not change throughout the 3 wk culture period (data not shown). In contrast to osteoblasts, induction of both murine and human osteoclast differentiation resulted in an increase in 1-hydroxylase mRNA levels (Fig. 4C, D ). 1-Hydroxylase activity (Fig. 4B ) and CYP27B1 expression (data not shown) were unaffected by 1,25-(OH)₂D₃ pretreatment.

View larger version (16K): [in this window] [in a new window]   Figure 3. Effect of 1,25-(OH)2D3 on human osteoblast activity. SV-HFO human preosteoblasts were induced to differentiate, and at various time points during differentiation ALP activity (A) and mineralization (B) were measured after incubation with vehicle or 10 nM 1,25-(OH)2D3. Data shown are means ± SD.

View larger version (13K): [in this window] [in a new window]   Figure 4. Regulation of 1-hydroxylase activity. A) Total production of 1,25-(OH)2D3 at various time points during human osteoblast differentiation, incubated for 48 h with 1000 nM 25-(OH)D3 (*P<0.01 vs. day 7). B)1,25-(OH)2D3 production corrected for total number of cells and effect of 1,25-(OH)2D3 preincubation on 1-hydroxylase activity during human osteoblast differentiation. C) Real-time PCR analysis of CYP27B1 expression in murine RAW 264.7 monocytic cells () and during osteoclast development (+ RANKL) () at days 3, 5, and 7. D) Real-time PCR analysis of CYP27B1 expression in human monocytes (M) and osteoclasts (M-CSF/RANKL induced) (O).

Incubation with increasing doses of 25-(OH)D₃ at day 14 of culture for 24 h had no effect on CYP27B1 mRNA expression (Fig. 5 A). In addition, PTH (data not shown) and calcium (Fig. 5B ) did not change 1-hydroxylase mRNA expression or activity in human osteoblasts. However, production of 1,25-(OH)₂D₃ showed a tendency to increase at higher calcium concentrations. Incubation with IL-1ß significantly increased CYP27B1 expression (Fig. 5C ).

View larger version (12K): [in this window] [in a new window]   Figure 5. Effects on CYP27B1 mRNA expression. Relative CYP27B1 mRNA expression was measured at day 14 of SV-HFO cell culture after 24 h incubation with A) various concentrations of 25-(OH)D3, B) various concentrations of CaCl2 (bars), 1,25-(OH)2D3 production under the same conditions was also measured (line), and C) 10 ng/ml IL-1ß. Data are presented as means ± SE. *P < 0.005 vs. control.

Short-term (48 h) incubations with 25-(OH)D₃ induced OC (Fig. 6 A, B) and CYP24 (Fig. 6C, D ) mRNA expression and ALP activity (Fig. 6E ) to a greater extent than when 1,25-(OH)₂D₃ (10 nM) was directly added for 48 h. Induction of OC and CYP24 mRNA expression increased with osteoblast differentiation and was dose dependent. OC protein measurements also demonstrated a stimulation following incubation with 25-(OH)D₃ (data not shown). Blocking 1,25-(OH)₂D₃ production by ketoconazole (Fig. 2C ) almost completely blocked the effect of 25-(OH)D₃ on both OC (Fig. 6B ) and CYP24 (Fig. 6D ) mRNA expression. ALP activity was induced by incubation with 25-(OH)D₃ to a similar extent as when 1,25-(OH)₂D₃ was directly added (Fig. 6E ). Mineralization was not affected after short-term incubation of 25-(OH)D₃ or 1,25-(OH)₂D₃ (data not shown).

View larger version (11K): [in this window] [in a new window]   Figure 6. Effect of 25-(OH)D3 incubation on human osteoblast gene expression and osteoblast function. Human preosteoblasts were induced to differentiate and at various time points during differentiation treated for 48 h with 1000 nM 25-(OH)D3 () or 10 nM 1,25-(OH)2D3 () (vehicle ()) and analyzed for effects on A) OC mRNA expression and C) CYP24 mRNA expression by real-time PCR and calculated as described in Materials and Methods. B) OC and D) CYP24 mRNA expression was measured after incubation with various concentrations of 25-(OH)D3 and 100 µM of ketoconazole for 24 h at day 14 of SV-HFO culture, E) ALP activity. Data are shown as means ± SD. *P < 0.05; **P < 0.01, and #P < 0.005 vs. control at the same day.

Long-term (continuous) incubations showed no effect on total DNA (Fig. 7 A), but treatment with 100 nM 25-(OH)D₃ significantly increased ALP activity at days 7 and 14 (Fig. 7B ) and significantly increased mineralization in the early phase of mineralization at day 14, but not at day 21 (Fig. 7C ).

View larger version (22K): [in this window] [in a new window]   Figure 7. Effect of long-term incubation of 25-(OH)D3 on human osteoblast differentiation and mineralization. Human preosteoblasts were induced to differentiate and from day 2 on continuously incubated with various concentrations of 25-(OH)D3: 1 nM, 10 nM, 100 nM vs. vehicle (dotted line). Effects were measured on days 7, 14 and 21 on A) total DNA, B) ALP activity; *P < 0.05 vs. control of the same day, and C) mineralization; P < 0.025 vs. control of the same day.

DISCUSSION

The current study clearly demonstrates the production of 1,25-(OH)₂D₃ by bone cells. Studies of the autocrine/paracrine actions of 1-hydroxylase in bone have been relatively limited. To date there are only a few reports from 25 years ago and a recent paper indicating expression of CYP27B1 in rat femur (21 22 23 , 28) . Our current study has taken this further by demonstrating the presence of 1-hydroxylase mRNA in human bone biopsies as well as in cultured human and murine osteoblasts and osteoclasts. 1-Hydroxylase protein was detected in human osteoblasts by using the same antibody (Ab) as that shown to detect 1-hydroxylase expression in human kidney cells (29) . A relationship between malignant status and 1-hydroxylase expression has been described for several extrarenal tissues like colon tumors (higher expression) (30 , 31) and prostate cancers (lower expression) (14 , 32) . In bone cells, the expression of CYP27B1 in the osteoblast-like osteosarcoma cell line MG-63 is highly increased compared with normal 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, which in the kidney are known to be important for cellular uptake of 25-(OH)D₃ associated with DBP (33 34 35) . Two P450 enzymes, CYP2R1 and CYP3A4, identified to have 25-hydroxylase activity and to convert vitamin D₃ into 25-(OH)D₃ (36) , were also expressed in human osteoblasts. These data imply that human osteoblasts can function as an almost autonomous 1,25-(OH)₂D₃ producing cell. To date the only tissue that has been shown to be entirely autonomous in synthesizing 1,25-(OH)₂D₃ is human skin, as this can also produce vitamin D₃ from 7-dehydrocholestrol (15 , 37) . In mouse skin this may be different, as no 1-hydroxylase promoter activity was found recently in the skin of CYP27B1 null mice (38) .

1-Hydroxylase activity was shown by a time- and dose-dependent production of 1,25-(OH)₂D₃ after incubations of human osteoblasts with 25-(OH)D_3. Kinetics were on the same order of magnitude as that reported earlier for cultured human renal proximal tubule cells (25) . The 1-hydroxylase activity and production of 1,25-(OH)₂D₃ by osteoblasts was completely blocked by the P450 inhibitor ketoconazole, which is in line with data in kidney cells (25) .

In keratinocytes there has been found an inverse relation between 1,25-(OH)₂D₃ formation and differentiation (15 , 16) , but no differences in CYP27B1 expression at different stages of differentiation of either human or murine osteoblasts were observed. However, a small progressive decrease was observed in 1,25-(OH)₂D₃ production per cell over time, suggesting an inverse relationship between 1-hydroxylase activity and osteoblast differentiation. However, the CYP27B1 mRNA levels did not change throughout the 3 wk culture period, suggesting an additional differentiation-dependent regulatory step. The current data do not suggest a decrease in 1-hydroxylase activity and 1,25-(OH)₂D₃ formation due to decreased DPB binding as both megalin and cubilin mRNA expression did not change during osteoblast differentiation. In contrast to osteoblasts, induction of both 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. This follows a similar pattern to that observed for other monocyte-derived cells such as macrophages and dendritic cells (19 , 39) .

Regulation by 1,25-(OH)₂D₃ itself provides an elegant feedback mechanism to control 1-hydroxylase activity (40 41 42 43) . However, autocrine/paracrine 1-hydroxylase activity in extrarenal tissues is modulated differently and is less sensitive to autoregulation by 1,25-(OH)₂D₃ (44) . Our data support this notion, as in human osteoblasts 1-hydroxylase activity and CYP27B1 expression were unaffected by 1,25-(OH)₂D₃. This is in line with the observation that CYP27B1 expression in rat femur is independent of circulating 1,25-(OH)₂D₃ levels (28) and resembles data obtained in the skin (37) .

CYP27B1 mRNA expression and activity 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-1ß. This further substantiates the significance of immune modulatory cytokines and the 1,25-(OH)₂D₃ endocrine system (17 , 19) . Studies have reported both a positive (19) and negative (45) effect of NF-B on 1-hydroxylase expression. However, the current IL-1ß 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, it 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 the positive long-term effects on ALP activity and mineralization. It is remarkable that the biological effects are much stronger when derived from the locally produced 1,25-(OH)₂D₃ after 25-(OH)D₃ treatment than 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 that is directly added. 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). These data strongly support a two-step model similar to that which has already been demonstrated in skin cells (37) and in prostate cancer cells, where incubation with 25-(OH)D₃ also leads to regulation of cellular activity (46) .

The absence of long-term effects on mineralization at day 21 is in line with that observed after direct 1,25-(OH)₂D₃ incubations and can be attributed to a limitation of the culture system, i.e., full mineralization has been reached in the culture well at day 21.

The significance of 1-hydroxylase activity for the eventual biological response of 25-(OH)D₃ is shown by 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 (47) , 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. Specifically, we have demonstrated the expression of 1-hydroxylase mRNA and protein, enzyme activity, and the induction of 1,25-(OH)₂D₃-mediated responses in osteoblasts incubated with the inactive precursor molecule 25-(OH)D₃. It has been postulated that vitamin D plays a role in tissues that have a barrier function that is either immunological or truly physical (39) . It is tempting to speculate that vitamin D in osteoblasts serves a similar role and regulates the mineralization process mediated by osteoblasts that cover the bone surface as a barrier. The current data provide a solid basis for further in vivo analyses to address these issues and clearly warrant development of osteoblast- as well as osteoclast-specific CYP27B1 knockout mice. These findings also hold a possible clue to explain the association studies between serum 25-(OH)D₃ levels and bone parameters like bone mineral density and fractures (48) . 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.

ACKNOWLEDGMENTS

We thank T. Schoenmakers and Dr. T. de Vries (Periodontology, ACTA, Amsterdam, the Netherlands) for providing cDNA samples from human osteoclast cultures and Dr. M. Karperien (Internal Medicine, LUMC, Leiden, the Netherlands) for providing us with human trabecular bone sections.

Received for publication April 28, 2006. Accepted for publication June 12, 2006.

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