Interrelationship between signal transduction pathways and 1,25(OH)2D3 in UMR106 osteoblastic cells

Wen Yang, Sven Johan Hyllner, and Sylvia Christakos

Department of Biochemistry and Molecular Biology, University of Medicine and Dentistry of New Jersey, New Jersey Medical School, Newark, New Jersey 07103


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In this study, the interrelationship between signal transduction pathways and 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] action was examined in UMR106 osteoblastic cells. Treatment of these cells with 8-bromo-cAMP (1 mM) resulted in an upregulation of the vitamin D receptor (VDR) and an augmentation in the induction by 1,25(OH)2D3 of 25(OH)D3 24-hydroxylase [24(OH)ase] and osteopontin (OPN) mRNAs as well as gene transcription. Transfection with constructs containing the vitamin D response element devoid of other promoter regulatory elements did not alter the cAMP-mediated potentiation, suggesting that cAMP-enhanced transcription is due, at least in part, to upregulation of VDR. Treatment with phorbol ester [12-O-tetradecanoyl-phorbol-13-acetate (TPA) 100 nM], an activator of protein kinase C, significantly enhanced 1,25(OH)2D3-induced OPN mRNA and transcription but had no effect on VDR or on 24(OH)ase mRNA or transcription. Studies using OPN promoter constructs indicate that TPA-enhanced OPN transcription is mediated by an effect on the OPN promoter separate from an effect on VDR. Thus interactions with signal transduction pathways can enhance 1,25(OH)2D3 induction of 24(OH)ase and OPN gene expression, and, through different mechanisms, changes in cellular phosphorylation may play a significant role in determining the effectiveness of 1,25(OH)2D3 on transcriptional control in cells expressing skeletal phenotypic properties.

osteopontin; 25-hydroxyvitamin D3 24-hydroxylase; vitamin D receptor; protein kinase A; protein kinase C


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THERE IS INCREASING EVIDENCE that the different pathways involved in steroid receptor action and second messenger signaling do not function independently. The action of steroid receptors can be influenced, for example, by an increase in the activity of cAMP-dependent protein kinase A (PKA) or phospholipase/protein kinase C (PKC). PKA has been reported to enhance steroid hormone-dependent transcriptional activation, to phosphorylate a number of steroid receptors, and to switch steroid receptor antagonists into agonists (5, 13, 21, 25, 26, 35). However, activation of PKC and activation protein (AP)1 transcription factors has been reported to result in inhibitory or stimulatory effects on steroid receptor-mediated gene expression and transcriptional activation (1, 8, 21, 24, 40, 41). Although these findings suggest an interrelationship between signal transduction pathways and steroid hormone action, the mechanisms underlying these interactions are not well understood. They may involve an effect on the regulation of the steroid receptor, on the promoter of the target gene through AP1 sites or cAMP response elements (CREs), or an effect on other transcription factors.

With regard to the regulation of calcium homeostasis, cooperativity between second messenger systems and steroid hormone action is indicated by the interrelationship between parathyroid hormone (PTH) and 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] action. PTH, a peptide hormone that can stimulate both PKA and PKC activity (19, 33, 45), and 1,25(OH)2D3, which acts analogously to the steroid hormones, are the major, physiologically important calciotropic hormones that act in an interrelated manner. Under conditions of hypocalcemia, PTH enhances the synthesis of 1,25(OH)2D3 in the kidney (18). In addition, 1,25(OH)2D3 potentiates PTH-dependent calcium transport in the distal tubule, and PTH has been reported to sensitize long bones to stimulation of resorption by 1,25(OH)2D3 (15, 46). Because the receptors for both hormones are found in osteoblasts rather than in osteoclasts, there is general agreement that 1,25(OH)2D3 and PTH regulate bone resorption via a primary action on the osteoblast that includes, in part, the stimulation of osteoclast-differentiating factor (or RANK ligand; Ref. 50). Cross talk between 1,25(OH)2D3 and signal transduction pathways is also noted by the modulation of the expression of 1,25(OH)2D3 target proteins by phorbol ester [12-O-tetradecanoyl-phorbol-13-acetate (TPA)], PTH, growth factors, and activation of PKA. For example, PKA has been reported to upregulate VDR, and fibroblast growth factor and activation of PKC have been reported to downregulate vitamin D receptor (VDR) in NIH 3T3 mouse fibroblasts (24, 25). In addition, PTH, mainly due to the activation of PKA, has been reported to enhance VDR abundance in osteoblastic cells (2, 23). Because up- or downregulation of VDR in these studies resulted in a corresponding alteration of a functional response, it was suggested that regulation of VDR by signal transduction pathways plays an important role in modulating cell responsiveness to 1,25(OH)2D3. However, opposite findings concerning the effect of PKA and PKC on VDR have also been reported (37, 38). Thus effects of signaling pathways not only on the VDR but also on endogenous VDR function and on the promoter of the 1,25(OH)2D3 target genes need to be considered.

To investigate the interaction between signaling pathways and 1,25(OH)2D3 action, we examined, in UMR106 osteoblastic cells, the effect of activation of PKA or PKC on the modulation of the expression of 24(OH)ase and osteopontin (OPN), target genes regulated by 1,25(OH)2D3 in bone. We addressed the mechanisms of these effects by use of transfected reporter constructs. Previous studies have suggested functions for 24(OH)ase and OPN in bone (44, 12). In bone, a major function of 24(OH)ase is to inactivate 1,25(OH)2D3, preventing elevated intracellular 1,25(OH)2D3 levels that may adversely affect mineralization (44). Thus 1,25(OH)2D3 self-induces its metabolism by inducing the 24(OH)ase enzyme. Recent studies by St-Arnaud et al. (44) indicated that mice deficient in 24(OH)ase have markedly elevated 1,25(OH)2D3 levels and impaired bone formation at specific sites. It was suggested that elevated 1,25(OH)2D3 levels, acting through VDR at specific sites, were responsible for the abnormalities in bone. OPN, also induced in response to 1,25(OH)2D3, is a highly phosphorylated, secreted protein, abundant in the bone matrix, which has been reported to modulate both resorption and mineralization (12). Our findings demonstrate for the first time that activation of PKA and PKC can enhance 1,25(OH)2D3 induction of OPN in cells expressing skeletal phenotypic properties and that the enhancement by PKC is due to an effect on the OPN promoter separate from an effect on VDR. Thus the effect of second messenger signaling pathways on 1,25(OH)2D3-regulated genes is not necessarily correlated with a similar change in VDR. However, the enhancement by cAMP of 1,25(OH)2D3-induced OPN and 24(OH)ase transcription was not due to an effect on a CRE in the promoter of these genes but rather may be due, at least in part, to upregulation of VDR. Thus our data indicate that interactions with signal transduction pathways can enhance 1,25(OH)2D3 induction of 24(OH)ase and OPN gene expression, and, through different mechanisms, cellular phosphorylation may play a significant physiological role in determining the effectiveness of 1,25(OH)2D3 action in mineral homeostasis.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Materials. [14C]chloramphenicol (50 mCi/mmol) and [32P]dCTP (3,000 Ci/mmol, 370 MBq/ml) were obtained from Du Pont-New England Nuclear Products (Boston, MA). Oligonucleotides, RadPrime DNA labeling system, and all restriction enzymes were purchased from GIBCO-BRL-Life Technologies (Gaithersburg, MD). Biotrans nylon membranes were obtained from ICN Biochemicals (Costa Mesa, CA). Oligo(dT) cellulose was purchased from Boehringer Mannheim Biochemicals (Indianapolis, IN). 8-Bromo-cAMP, acetyl-coenzyme A, and phorbol-12-myristate-13-acetate (PMA) were obtained from Sigma (St. Louis, MO). Phenol, formamide, and guanidinium isothiocyanate were purchased from International Biotechnologies (IBI, New Haven, CT). Rat anti-vitamin D receptor antibody was from Affinity BioReagents (Neshanic Station, NJ). Chemically synthesized 1,25(OH)2D3 was provided by Dr. M. Uskokovic of Hoffmann- LaRoche (Nutley, NJ).

Northern blot analysis. Total RNA was prepared from UMR106-01 cells by the guanidium thiocyanate-phenol-chloroform method described by Chomczynski and Sacchi (9). Polyadenylated [poly(A+)] RNA was isolated by oligo(deoxythymidine)-cellulose chromatography. Northern blot analysis was performed as described previously (48). The blots were hybridized to specific 32P-labeled cDNA probes for 16 h at 42°C, washed, and subjected to autoradiography as previously described (48). Labeled probes were prepared using the RadPrime DNA labeling system according to the random prime method (4). To be normalized for sample variation, blots were probed with 32P-labeled beta -actin cDNA. The blots were air dried and exposed to Kodak XAR-5 film at -80°C in the presence of intensifying screens. All autoradiograms were analyzed by densitometric scanning using the Dual-Wavelength Flying Spot Scanner (Shimadzu Scientific Instruments, Princeton, NJ). The relative optical densities obtained using the test probes were divided by the relative optical density obtained after probing with the control probe (beta -actin) to normalize for sample variation.

Cell culture, cell transfection and assay of chloramphenicol acetyltransferase activity. UMR106-01 cells were obtained from the American Type Culture Collection. These cells were maintained in DMEM-F-12 (GIBCO-BRL-Life Technologies) supplemented with 5% heat-inactivated fetal bovine serum (FBS) (Gemini, Calabasas, CA) at 37°C in a 5% CO2 atmosphere. Cells were grown to 60-70% confluence in 100-mm tissue culture dishes, and 24 h before the start of experiments, medium was changed to 2% charcoal-stripped serum-containing medium. Cells were treated with vehicle or the compounds noted at the concentrations indicated and for various times (1-24 h). Studies using different concentrations of TPA or 8-bromo-cAMP indicated that the enhancement of 1,25(OH)2D3 induction of OPN mRNA by TPA was maximal at 25 nM TPA (a plateau in responsiveness was observed between 25 and 100 nM) and that enhancement of 1,25(OH)2D3 induction of both OPN and 24(OH)ase mRNA was maximal at 1 mM 8-bromo-cAMP. Thus maximally effective concentrations of TPA (100 nM) and 8-bromo-cAMP (1 mM) were used. For transfections, cells were plated at a density of 1 × 106 cells/100-mm plate 24 h before transfection. UMR106-01 cells were cotransfected with reporter plasmid (8 µg) and the beta -galactosidase expression vector pCH110 (4 µg; from Pharmacia), an internal control for transfection efficiency, by use of the calcium phosphate DNA precipitation method (4). Cells were transfected for 16 h, shocked for 1 min with 10% dimethyl sulfoxide in phosphate-buffered saline (PBS), washed with PBS, and treated for 24 h with vehicle (0.1% ethanol) or test compound at the concentrations indicated. After 24-h incubation, cells were harvested, and cell extracts were prepared by freeze-thaw (-80°C freeze, 37°C thaw; three times, 5 min each). The chloramphenicol acetyltransferase (CAT) assay was performed at constant beta -galactosidase activity with standard protocols (4, 16) and quantitated by densitometric scanning of TLC autoradiograms. For some experiments, several autoradiographic exposure times were needed for densitometric analysis. CAT activity was also quantitated by scanning TLC plates by means of the Packard Constant Imager System (Packard Instrument, Meriden, CT).

Complementary DNA probes, 24(OH)ase, and OPN-CAT constructs. The 1-kb mouse OPN cDNA was generated by digestion with HindIII and was a gift from D. Denhardt (Rutgers University, Piscataway, NJ) (42). A 1.7-kb rat VDR cDNA was obtained by digestion of pIBI76 with EcoRI (34). The 3.2-kb rat 24(OH)ase cDNA was obtained by EcoRI digestion and was a gift from K. Okuda (Hiroshima University School of Dentistry, Hiroshima, Japan) (31). beta -Actin cDNA was from Clontech (Palo Alto, CA).

For transfection studies, constructs of a chimeric gene in which the rat 24(OH)ase promoter (-671/+74; containing both vitamin D response elements (VDREs) at -258/-244 and -151/-137) was linked to the CAT gene, as previously described (22), were used. A proximal rat 24(OH)ase VDRE thymidine kinase (tk) CAT reporter construct, prepared by introducing multiple copies of the proximal VDRE (-151/-137) of the rat 24(OH)ase promoter with the CAT plasmid (49), was also used. The mouse OPN promoter CAT construct (-777/+74; OPN VDRE at -757/-743) was obtained from D. Denhardt (10). The OPN VDRE tk-CAT construct, containing multiple copies of the OPN VDRE, was a gift of Dr. L. Freedman (Sloan Kettering Cancer Center, New York, NY).

Western blot analysis. For nuclear extracts for Western blot analysis of VDR, cells were harvested by trypsinization and washed with PBS. Each dish of cells was resuspended in 1 ml of sonication buffer [10 mM Tris, pH 7.4, 1.5 mM EDTA, and 1.0 mM dithiothreitol (TED), 10 mM sodium molybdate, 500 KIU/ml Trasylol, 300 µM phenylmethylsulfonylfluoride (PMSF), and 200 µg/ml soybean trypsin inhibitor] and sonicated. Cell extracts were centrifuged at 12,000 g for 10 min at 4°C. Crude nuclear pellets were resuspended in 5 ml of washing buffer (TED, 300 µM PMSF, and 0.5% Triton X-100), vortexed, and centrifuged at 13,000 g. After the third wash, the pellets were resuspended in 1 ml of resuspension buffer (0.3 M KCl, TED, 10 mM sodium molybdate, 500 KIU/ml Trasylol, 300 µM PMSF) and vortexed well. The protein concentration was determined by the method of Bradford (6). Protein (30 µg) from each sample was used for electrophoresis on a 12% SDS polyacrylamide gel. After electrophoresis, proteins were transferred electrophoretically to a nitrocellulose membrane (Hybond-ECL; Amersham) that was incubated with anti-VDR monoclonal antibody 9A7 (1:2,000 dilution) in Tris-buffered saline (TBS; pH 7.5) for 12 h at 4°C. After washing with TBS, the membrane was incubated with secondary antibody [goat anti-rat IgG conjugated to horseradish peroxidase (Sigma), 1:2,000 dilution] for 1 h at room temperature. After being washed with TBS, the antigen-antibody complex was detected using the enhanced chemiluminescent Western blotting detection system (Amersham) according to the manufacturer's protocol.

Statistical analysis. Results are expressed as means ± SE, and significance was determined by Student's t-test for two-group comparison or analysis of variance (ANOVA) for multiple group comparison. In conjunction with ANOVA, a posttest analysis by Dunnett's multiple t statistic was used with a significance level of 0.05.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

To explore the mechanisms by which 1,25(OH)2D3 and signal transduction pathways coordinately regulate the expression of vitamin D3-responsive genes, we examined the effects of 1,25(OH)2D3, 8-bromo-cAMP (an activator of PKA), and TPA (an activator of PKC) on the mRNA expression of VDR, 24(OH)ase, and OPN in UMR106-01 cells (Fig. 1). UMR106-01 cells were treated with 1,25(OH)2D3 (10-8 M) in the presence or absence of 8-bromo-cAMP (1 mM) or TPA (100 nM). The treated cells were harvested at the indicated time points and mRNA was isolated. Results of Northern blot analyses are shown in Fig. 1. The first significant induction of VDR mRNA by 1,25(OH)2D3 was observed at 9 h. In the presence of both cAMP and 1,25(OH)2D3, VDR mRNA was induced as early as 3 h. Thus cAMP enhanced the rapidity of VDR mRNA expression induced by 1,25(OH)2D3. 8-Bromo-cAMP also enhanced the response of 24(OH)ase and OPN mRNA to 1,25(OH)2D3. The time course of response of VDR and 24(OH)ase mRNAs to 1,25(OH)2D3 was similar in the presence or absence of TPA, indicating that the PKC signaling pathway does not affect VDR and 24(OH)ase expression in UMR106 cells. However, TPA shifted the time course of response of OPN to 1,25(OH)2D3 to the left and resulted in an increase in the response of OPN to 1,25(OH)2D3 (Fig. 1).


View larger version (69K):
[in this window]
[in a new window]
 
Fig. 1.   Time-dependent effects of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] or 1,25(OH)2D3 + cAMP or 12-O-tetradecanoyl-phorbol-13-acetate (TPA) on levels of vitamin D receptor (VDR), 24-hydroxylase [24(OH)ase], and osteopontin (OPN) mRNAs in UMR cells. A: Northern analysis was performed using 8 µg poly(A+) RNA per lane from UMR cells that had been treated with vehicle control or 10-8 M 1,25(OH)2D3 alone or in combination with 8-bromo-cAMP (1 mM) or TPA (100 nM) and harvested at various times after treatment. The filter was hybridized with 32P-labeled rat VDR cDNA; then blots were stripped and rehybridized with 32P-labeled rat 24(OH)ase, mouse OPN, and beta -actin cDNAs sequentially. B: graphic representation of Northern blot analyses of time-dependent effects of 1,25(OH)2D3 in the presence or absence of cAMP or TPA on VDR, 24(OH)ase, and OPN mRNAs in UMR106 cells. Data were normalized on the basis of results obtained on rehybridization with beta -actin cDNA. Data represent means ± SE from 3 independent experiments.

The modulation of 1,25(OH)2D3 induction of VDR, 24(OH)ase, and OPN by PKA and PKC signaling pathways was further characterized in experiments performed using different concentrations of 1,25(OH)2D3 (Fig. 2, A, B, and D). Cells were treated for 9 h in these studies, because enhancement by cAMP of the 1,25(OH)2D3 response [VDR, OPN, and 24(OH)ase mRNAs] was observed at this time, and enhancement by TPA of 1,25(OH)2D3 induction of OPN mRNA was also observed after treatment for 9 h (Fig. 1). 1,25(OH)2D3 treatment (10-10-10-7 M) of UMR106 cells resulted in an upregulation of VDR mRNA, and cAMP enhanced the 1,25(OH)2D3 induction at all concentrations of 1,25(OH)2D3. 8-Bromo-cAMP also potentiated the induction of 24(OH)ase mRNA by 1,25(OH)2D3. In addition, the induction of OPN mRNA by 1,25(OH)2D3 was increased by cAMP but only in the presence of higher concentrations of 1,25(OH)2D3 [10-8-10-7 M]. In contrast to cAMP, TPA had no effect at any concentration of 1,25(OH)2D3 on VDR or 24(OH)ase gene expression. However, TPA enhanced OPN expression at all concentrations of 1,25(OH)2D3 (Fig. 2, A, B, and D). The effect of TPA in enhancing 1,25(OH)2D3-induced OPN expression appeared to be independent of the 1,25(OH)2D3 concentration. We also noted that 8-bromo-cAMP alone induced VDR mRNA levels but not 24(OH)ase or OPN mRNA levels (Fig. 2, C and D). TPA alone had no effect on VDR or 24(OH)ase gene expression. However, OPN mRNA is induced by TPA alone (Fig. 2, C and D).


View larger version (70K):
[in this window]
[in a new window]
 
Fig. 2.   Concentration-dependent effects of 1,25(OH)2D3 in the presence or absence of cAMP or TPA and effects of cAMP or TPA alone on levels of VDR, 24(OH)ase, and OPN mRNAs in UMR cells. Northern analysis was performed using 8 µg poly(A+) RNA/lane from UMR cells that had been treated for 9 h with the indicated concentration of 1,25(OH)2D3 in the presence or absence of 8-bromo-cAMP (1 mM; A) or TPA (100 nM; B) or 8-bromo-cAMP alone (1 mM) or TPA alone (100 nM) compared with treatment with vehicle control or 1,25(OH)2D3 (10-8 M) for 9 h (C). The filters were hybridized with 32P-labeled rat VDR cDNA; then blots were stripped and rehybridized with 32P-labeled rat 24(OH)ase, mouse OPN, and beta -actin cDNAs sequentially. D: graphic representation of Northern blot analyses of dose-dependent effects of 1,25(OH)2D3 and/or 8-bromo-cAMP or TPA on VDR, 24(OH)ase, and OPN mRNAs in UMR cells. Data were normalized on the basis of results obtained on rehybridization with beta -actin cDNA. Data are from 3 independent experiments (means ± SE). In the presence of 8-bromo-cAMP, VDR and 24(OH)ase mRNAs were significantly induced above the levels obtained with 1,25(OH)2D3 at all concentrations of 1,25(OH)2D3. OPN mRNA was significantly induced by 8-bromo-cAMP above the levels obtained with 1,25(OH)2D3 alone at 10-8 and 10-7 M 1,25(OH)2D3 (P < 0.05; ANOVA and posttest analysis by Dunnett's). No significant induction in VDR mRNA or 24(OH)ase mRNA above the levels obtained in the presence of 1,25(OH)2D3 was observed in the presence of TPA. In the presence of TPA, OPN mRNA was significantly induced above the levels obtained with 1,25(OH)2D3 alone at all concentrations of 1,25(OH)2D3 (P < 0.05; ANOVA and posttest analysis by Dunnett's).

Northern analysis of the time course of response indicated that, in the presence of cAMP, VDR mRNA is induced by 1,25(OH)2D3 before 24(OH)ase and OPN mRNAs, suggesting that cAMP may mediate the enhanced induction of these target genes by upregulating VDR levels. Therefore, we examined the effects of cAMP, TPA, and 1,25(OH)2D3 on VDR protein expression (Fig. 3). We observed that 1,25(OH)2D3 and cAMP increased VDR protein levels by 3.3- and 3.8-fold, respectively, and that TPA did not change VDR protein levels. These findings suggest that cAMP may enhance 1,25(OH)2D3-induced gene expression, at least in part, by upregulation of VDR levels.


View larger version (58K):
[in this window]
[in a new window]
 
Fig. 3.   Effect of 1,25(OH)2D3, cAMP, or TPA on VDR protein content. UMR106 cells were treated with vehicle 0.1% ethanol (-D), 10-8 M 1,25(OH)2D3 (+D), 8-bromo-cAMP (1 mM), or TPA (100 nM) for 12 h. Top: nuclear proteins were prepared as described in MATERIALS AND METHODS. Western blot analysis was performed using 30 µg of protein and probed with polyclonal antibody against rat VDR. Molecular size markers are indicated at right. VDR is visualized as a 50-kDa immunoreactive band. Bottom: graphic representation of results obtained from 3 separate experiments. Levels of VDR were significantly induced above control levels by 1,25(OH)2D3 (1,25D) or cAMP treatment (P < 0.05; ANOVA and posttest analysis by Dunnett's).

To determine whether effects on the promoter, separate from effects on VDR, could account for at least part of the effects of the second messenger systems, UMR106 cells were transiently transfected with plasmids incorporating various regions of the rat 24(OH)ase or mouse OPN promoter linked to CAT. By use of the 24(OH)ase promoter construct -671/+74 phCAT that contains both VDREs and three consensus CREs (determined by computational analysis) (22, 49), cAMP (1 mM 8-bromo-cAMP) was found to significantly potentiate the dose-dependent activation of transcription by 1,25(OH)2D3, indicating that the 24(OH)ase modulation by cAMP of 24(OH)ase mRNA is at the transcriptional level (Fig. 4, top). Transfection of UMR106 cells with a construct containing multiple copies of the proximal 24(OH)ase VDRE devoid of other 24(OH)ase promoter regulatory elements did not alter the cAMP-mediated potentiation of 1,25(OH)2D3-dependent 24(OH)ase transcription (Fig. 4, bottom). In contrast, TPA had no significant effect on activation by 1,25(OH)2D3 of 24(OH)ase transcription (Fig. 4, top and bottom). By use of the -777/+79 phCAT OPN promoter construct (VDRE at -757/-743 and AP1 sites at -9/-3 and -75/-69) (29, 10), both cAMP and TPA potentiated the activation of transcription of the OPN gene by 1,25(OH)2D3 (Fig. 5, top). However, experiments done using an OPN VDRE tk-CAT construct indicated that this construct responds to 1,25(OH)2D3 and cAMP but not to TPA (Fig. 5, bottom). Thus the VDRE of the mouse OPN gene, in the absence of other sequences in the OPN promoter, is sufficient to confer cAMP, but not TPA, enhancement.


View larger version (32K):
[in this window]
[in a new window]
 
Fig. 4.   Effect of cAMP or TPA on 1,25(OH)2D3-induced transcription of rat 24(OH)ase promoter (CAT) construct -671/+74 (top) or 24(OH)ase VDRE (-151/-137) thymidine kinase (tk) chloramphenicol acetyltransferase (CAT) construct (bottom). Transfected UMR106 cells were treated with the indicated concentrations of 1,25(OH)2D3 alone (open circle ) or in the presence of 1 mM 8-bromo-cAMP (), or 100 nM TPA (black-triangle) for 24 h. CAT assays were performed, and results are expressed as a percentage of maximal response (means ± SE of 3 separate experiments). Cells treated with 1,25(OH)2D3 + cAMP had significantly increased CAT activity (P < 0.05; ANOVA and posttest analysis by Dunnett's) at all doses of 1,25(OH)2D3 [except 10-10 M with the -671/+74 24(OH)ase promoter CAT construct].



View larger version (42K):
[in this window]
[in a new window]
 
Fig. 5.   Effect of cAMP or TPA on 1,25(OH)2D3-induced transcription of mouse OPN promoter CAT construct -777/+79 or OPN VDRE (-757/-743) tk-CAT construct. Transfected UMR106 cells were treated with 10-8 M 1,25(OH)2D3 alone or in the presence of 1 mM 8-bromo-cAMP or 100 nM TPA for 24 h. CAT assays were performed, and results are expressed as relative CAT activity (means ± SE of 3 separate experiments). Cells transfected with the -777/+79 OPN promoter CAT construct (top) had significantly increased CAT activity in the presence of cAMP or TPA compared with treatment with 1,25(OH)2D3 alone (P < 0.05). Cells transfected with the OPN VDRE tk-CAT (bottom) had significantly increased CAT activity in the presence of cAMP compared with treatment with 1,25(OH)2D3 (P < 0.05). By use of the OPN tk-CAT construct, cotreatment with TPA did not alter CAT activity compared with treatment with 1,25(OH)2D3 alone. Comparisons with treatment with 1,25(OH)2D3 were by ANOVA and posttest analysis by Dunnett's.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Our results suggest that, through different mechanisms, changes in cellular phosphorylation mediated by activation of PKA or PKC play an important role in determining the effectiveness of 1,25(OH)2D3 action in osteoblastic cells. In our study, treatment with either cAMP or TPA resulted in an enhancement of 1,25(OH)2D3-induced OPN expression, although cAMP resulted in an upregulation of VDR but treatment with TPA did not. Thus cAMP and TPA do not necessarily have opposite effects on 1,25(OH)2D3 action, and the enhancement of VDR function by second messenger signaling pathways is not necessarily correlated with a similar increase in VDR levels. In previous studies, modulation by second messenger signaling pathways of 1,25(OH)2D3 induction of osteocalcin transcription was examined in NIH 3T3 mouse fibroblasts transfected with the human osteocalcin VDRE (-558/-338) fused to a tk-CAT construct. Up- or downregulation of VDR in NIH 3T3 cells by forskolin or PMA treatment respectively resulted in a corresponding enhancement or attenuation of 1,25(OH)2D3-induced transcription (24, 25). Although TPA treatment resulted in an inhibition of 1,25(OH)2D3-induced transcription with the human osteocalcin VDRE (24), in our study we found that 1,25(OH)2D3-induced OPN expression and transcription were enhanced by TPA in UMR106 cells. Previous studies have also noted an enhancement of OPN expression after TPA treatment, and functional AP1 sites in the OPN promoter have been identified (10, 11). Thus, in our study, promoter-specific effects contributed more significantly than receptor regulation to modulation of vitamin D-induced OPN transcription by PKC. The effect of TPA was gene specific, since 1,25(OH)2D3-induced 24(OH)ase expression and transcription were unaffected by TPA treatment.

Concerning the cAMP enhancement of 1,25(OH)2D3-induced 24(OH)ase transcription, the 24(OH)ase VDRE tk-CAT construct was able to confer cAMP enhancement, suggesting that regions in the 24(OH)ase promoter other than the VDRE are not primarily involved in the cAMP responsiveness. An OPN VDRE tk-CAT construct was also able to confer cAMP responsiveness (Fig. 5). Because 8-bromo-cAMP treatment resulted in an increase in VDR protein levels as well as an enhancement of 1,25(OH)2D3 induction of VDR mRNA levels in UMR cells, regulation of VDR levels by cAMP may be one mechanism by which cAMP modulates 1,25(OH)2D3-induced transcription of 24(OH)ase and OPN. Similar to our studies, Krishnan et al. (23) and Van Leeuwen et al. (47) also found that changes in VDR by agents that raise intracellular cAMP in UMR106 cells correspond to an enhanced functional response. In addition, preliminary studies in our laboratory using osteoblastic cells isolated from calvaria of 3- to 4-day-old mice indicate that upregulation of VDR mRNA by cAMP as well as the cAMP-mediated enhancement of 24(OH)ase and OPN mRNAs can be observed in primary osteoblasts as well as in UMR cells (K. Gengaro, M. Huening, and S. Christakos, unpublished observation). Because CREs have been identified by sequence homology in the promoter of the VDR gene (28), it will be of interest in future studies to determine the role of transcriptional regulation of VDR by cAMP in the interaction between the PKA signaling pathway and 1,25(OH)2D3. Because the time course and magnitude of the enhancement by cAMP differed for 24(OH)ase and OPN mRNAs (Figs. 1 and 2), more than one mechanism may be involved in the effect of cAMP. Phosphorylation of VDR or phosphorylation of other coactivators involved in VDR-mediated transcription may also play a role in the enhancement by cAMP of 1,25(OH)2D3-induced target gene expression. Because the enhancement by cAMP is observed even after 24 h of treatment for OPN gene expression but only at earlier times for 24(OH)ase gene expression, it is also possible that the PKA signaling pathway may have effects on the stability of OPN mRNA. Effects of PTH (mediated by cAMP) on the stability of osteocalcin mRNA in osteoblast-like cells have previously been reported (30). Thus the effect of PKA may involve not only an upregulation of VDR but also an effect on mRNA stability, an effect on the phosphorylation of the VDR or on the phosphorylation of another protein that is involved in VDR-mediated gene transcription.

In our studies in UMR106 cells, TPA treatment did not affect 1,25(OH)2D3-induced 24(OH)ase mRNA or transcription. However, in primary cultures of rat renal cells as well as in intestinal epithelial cells, treatment with phorbol ester enhances 1,25(OH)2D3-induced 24(OH)ase expression (1, 8). Thus cell type or tissue-specific factors can alter the effect of signal transduction pathways on VDR-mediated transcription. The lack of response to TPA of the 24(OH)ase promoter may be due to the absence of specific PKC-activated proteins in UMR cells, needed for enhanced 24(OH)ase transcription, that are present in intestinal and renal cells. Because the OPN promoter was sensitive to TPA, the response to TPA is not only cell type specific but is also gene specific. In future studies, it will be of interest to identify cell type-specific factors that are involved in the modulation by signal transduction pathways of the transcription of specific 1,25(OH)2D3-regulated genes.

The effect of second messenger signaling pathways on 24(OH)ase expression may have physiological importance. 24(OH)ase, an important enzyme involved in vitamin D metabolism, hydroxylates 25(OH)D3 and 1,25(OH)2D3 (32). The 24-hydroxylation of 1,25(OH)2D3 has been reported to be involved in the catabolism of 1,25(OH)2D3 (36). 1,25(OH)2D3 at high concentrations self-induces its deactivation by inducing the 24(OH)ase enzyme. Recent studies using 24(OH)ase-deficient mice have provided the first direct in vivo evidence for a role for 24(OH)ase in the catabolism of 1,25(OH)2D3 (44). Mice deficient in 24(OH)ase have not only impaired 1,25(OH)2D3 catabolism but also impaired bone formation at specific sites (calvaria, mandible, clavicle, and periosteum of long bones) (44). 24,25(OH)2D3 supplementation failed to correct most of the bone abnormalities, arguing against a direct role of 24,25(OH)2D3 on bone formation. Because crossing the 24(OH)ase-deficient mice with VDR-ablated mice totally rescued the bone phenotype, the authors suggested that elevated 1,25(OH)2D3 levels, acting through VDR at specific sites, were responsible for the abnormalities observed in bone development (44). Thus 24(OH)ase is an important modulator of 1,25(OH)2D3 action in bone cells, and its activation at lower, physiological concentrations of 1,25(OH)2D3 via cross talk with the PKA signaling pathway may be important for preventing elevated, intracellular 1,25(OH)2D3 levels that may adversely affect mineralization.

In contrast to the modulation of 1,25(OH)2D3 induction of 24(OH)ase expression only by PKA activation, both PKA and PKC are involved in the modulation of OPN expression in UMR cells. Although the specific functions of OPN have not been fully understood, OPN has been reported to modulate bone resorption and mineralization (7, 12, 39). OPN is a ligand for a subset of integrins including alpha vbeta 3, and the OPN-integrin interaction has been reported to be important in adherence of the osteoclast to bone and bone resorption (39). Studies in OPN-deficient mice indicate significantly fewer osteoclasts in the deficient mice, confirming the in vitro findings of a role for OPN in osteoclast recruitment (3). OPN is also believed to play a role in the process of matrix mineralization by influencing the rate of mineralization (43). In addition, mechanical stress has been reported to result in elevated OPN mRNA levels (17, 27), consistent with a function for OPN in bone remodeling. Because 1,25(OH)2D3 affects bone resorption, it is possible that PKA and PKC are involved in enhancing the effect of 1,25(OH)2D3 by enhancing OPN expression and its reported effects more specifically on bone resorption. When considering the effects of kinases, it is of interest to note that OPN is a highly phosphorylated protein (12), and different forms of OPN (phosphorylated vs. dephosphorylated) have been reported to have different functional roles. For example, when OPN is dephosphorylated by tartrate-resistant acid phosphatase, which is secreted by osteoclasts, the dephosphorylated OPN is unable to bind to osteoclasts (14). On the other hand, phosphorylation increases the capacity of OPN to stimulate osteoclast attachment (20). Whether the different signal transduction pathways act by regulating not only the increase in the synthesis of OPN but also by regulating different forms of OPN remains to be determined.

In summary, it is becoming evident that signal transduction pathways and steroid receptors do not function independently in the cell and that understanding their interaction is critical to understanding the biological response. Our findings show that activation of either PKA or PKC can enhance 1,25(OH)2D3 induction of OPN in cells expressing skeletal phenotypic properties and that the effect of PKC is due to an effect on the OPN promoter separate from an effect on VDR. However, cAMP-mediated enhancement of 1,25(OH)2D3-induced OPN and 24(OH)ase transcription is not due to an effect on a CRE in the promoter of these genes but rather may be due, at least in part, to upregulation of VDR. Thus our findings suggest that, through different mechanisms, these transmembrane signaling pathways (that can be activated by PTH and by growth factors) can play an important role in modulating the responsiveness of osteoblastic cells to 1,25(OH)2D3.


    ACKNOWLEDGEMENTS

The assistance of Kristen Gengaro, Michael Huening, and Xiaorong Peng in certain aspects of this investigation is gratefully acknowledged. This work was supported by National Institutes of Health Grant DK-38961 (to S. Christakos).


    FOOTNOTES

Address for reprint requests and other correspondence: S. Christakos, Dept. of Biochemistry and Molecular Biology, UMDNJ-New Jersey Medical School, 185 S. Orange Ave., Newark, NJ 07103-2714 (E-mail: christak{at}umdnj.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 13 July 2000; accepted in final form 28 February 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Armbrecht, HJ, Hodam TL, Boltz MA, and Chen ML. Phorbol ester markedly increases the sensitivity of intestinal epithelial cells to 1,25-dihydroxyvitamin D3. FEBS Lett 327: 13-16, 1993[ISI][Medline].

2.   Armbrecht, HJ, Hodam TL, Boltz MA, Partridge NC, Brown AJ, and Kumar VB. Induction of the vitamin D 24-hydroxylase (CYP24) by 1,25 dihydroxyvitamin D3 is regulated by parathyroid hormone in UMR106 osteoblastic cells. Endocrinology 139: 3375-3381, 1998[Abstract/Free Full Text].

3.   Asou, Y, Rittling S, Yoshitake H, Tsuji K, Shinomiya K, Nifuji A, Denhardt DT, and Noda M. Osteopontin facilitates angiogenesis, accumulation of osteoclasts, and resorption in ectopic bone. Endocrinology 142: 1325-1332, 2001[Abstract/Free Full Text].

4.   Ausubel, FM, Brent R, Kingston RE, Moore DD, Steidman JG, Smith JA, and Struhl K. Current Protocols in Molecular Biology. New York: Wiley, 1992.

5.   Beck, CA, Weigel NL, Moyer ML, Nordeen SK, and Edwards DP. The progesterone antagonist RU486 acquires agonist activity upon stimulation of cAMP signaling pathways. Proc Natl Acad Sci USA 90: 4441-4445, 1993[Abstract].

6.   Bradford, MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding. Anal Biochem 72: 248-254, 1976[ISI][Medline].

7.   Chen, J, Singh K, Mukherjee BB, and Sodek J. Developmental expression of osteopontin (OPN) mRNA in rat tissues: evidence for a role for OPN in bone formation and resorption. Matrix 13: 113-123, 1993[ISI][Medline].

8.   Chen, ML, Boltz MA, and Armbrecht HJ. Effects of 1,25-dihydroxyvitamin D3 and phorbol ester on 25-hydroxyvitamin D3 24-hydroxylase cytochrome P450 messenger ribonucleic acid levels in primary cultures of renal cells. Endocrinology 132: 1782-1788, 1993[Abstract].

9.   Chomczynski, P, and Sacchi N. Single step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162: 156-159, 1987[ISI][Medline].

10.   Craig, AM, and Denhardt DT. The murine gene encoding secreted phosphoprotein 1 (osteopontin): promoter structure, activity and induction in vivo by estrogen and progesterone. Gene 100: 163-171, 1991[ISI][Medline].

11.   Craig, AM, Smith JH, and Denhardt DT. Osteopontin, a transformation-associated cell adhesion phosphoprotein, is induced by 12-O-tetradecanoylphorbol 13-acetate in mouse epidermis. J Biol Chem 264: 9682-9689, 1989[Abstract/Free Full Text].

12.   Denhardt, DT, and Noda M. Osteopontin expression and function: role in bone remodeling. J Cell Biochem Suppl 30/31: 92-102, 1998.

13.   Denner, LA, Schrader WT, O'Malley BW, and Weigel NL. Hormonal regulation and identification of chicken progesterone receptor phosphorylation sites. J Biol Chem 265: 16548-16555, 1990[Abstract/Free Full Text].

14.   Dodds, RA, Connor JR, James IE, Rykaczewski EL, Appelbaum E, Dul E, and Gowen M. Human osteoclasts, not osteoblasts, deposit osteopontin onto resorption surfaces: an in vitro and ex vivo study of remodeling bone. J Bone Miner Res 10: 1666-1680, 1995[ISI][Medline].

15.   Friedman, PA, and Gesek FA. Vitamin D3 accelerates PTH-dependent calcium transport in distal convoluted tubule cells. Am J Physiol Renal Fluid Electrolyte Physiol 265: F300-F308, 1993[Abstract/Free Full Text].

16.   Gorman, C, Moffat LF, and Howard BH. Recombinant genomes which express chloramphenicol acetyl transferase in mammalian cells. Mol Cell Biol 2: 1044-1051, 1982[ISI][Medline].

17.   Harter, LV, Hruska KA, and Duncan RL. Human osteoblast-like cells respond to mechanical strain with increased bone matrix protein production independent of hormonal regulation. Endocrinology 136: 528-535, 1995[Abstract].

18.   Henry, HL, Dutta C, Cunningham N, Blanchard R, Penny R, Tang C, Marchetto G, and Chou SY. The cellular and molecular regulation of 1,25(OH)2D3 production. J Steroid Biochem Mol Biol 41: 401-407, 1992[ISI][Medline].

19.   Hruska, KA, Moskowitz D, Esbrit P, Civitelli R, Westbrook S, and Huskey M. Stimulation of inositol trisphosphate and diacylglycerol production in renal tubular cells by parathyroid hormone. J Clin Invest 79: 230-239, 1987[ISI][Medline].

20.   Katayama, Y, House CM, Udagawa N, Kazama JJ, McFarland RJ, Martin TJ, and Findlay DM. Casein kinase 2 phosphorylation of recombinant osteopontin enhances adhesion of osteoclasts but not osteoblasts. J Cell Physiol 176: 179-187, 1998[ISI][Medline].

21.   Katzenellenbogen, BS. Estrogen receptors: bioactivities and interactions with cell signaling pathways. Biol Reprod 54: 287-293, 1996[Abstract].

22.   Kerry, DM, Dwivedi PP, Hahn CN, Morris HA, Omdahl JL, and May BK. Transcriptional synergism between vitamin D responsive elements in the rat 25-hydroxyvitamin D3 24-hydroxylase (CYP24) promoter. J Biol Chem 271: 29715-29721, 1996[Abstract/Free Full Text].

23.   Krishnan, AV, Cramer SD, Binghurst FR, and Feldman D. Regulation of 1,25dihydroxyvitamin D3 receptors by parathyroid hormone in osteoblastic cells: role of second messenger pathways. Endocrinology 136: 705-712, 1995[Abstract].

24.   Krishnan, AV, and Feldman D. Activation of protein kinase-C inhibits vitamin D receptor gene expression. Mol Endocrinol 5: 605-612, 1991[Abstract].

25.   Krishnan, AV, and Feldman D. Cyclic adenosine 3'5' monophosphate upregulates 1,25dihydroxyvitamin D3 receptor gene expression and enhances hormone action. Mol Endocrinol 6: 198-206, 1992[Abstract].

26.   LeGoff, P, Montano MM, Schodin DJ, and Katzenellenbogen BS. Phosphorylation of the human estrogen receptor: identification of hormone-regulated sites and examination of their influence on transcriptional activity. J Biol Chem 269: 4458-4466, 1994[Abstract/Free Full Text].

27.   Miles, RR, Turner CH, Santerre R, Tu Y, McClelland P, Argot J, DeHoff BS, Mundy CW, Rosteck PR, Bidwell J, Sluka JP, Hock J, and Onyia JE. Analysis of differential gene expression in rat tibia after an osteogenic stimulus in vivo: mechanical loading regulates osteopontin and myeloperoxidase. J Cell Biochem 68: 355-365, 1998[ISI][Medline].

28.   Miyamoto, K, Kesterson RA, Yamamoto H, Taketani Y, Nishiwaki E, Tatsumi S, Inoue Y, Morita K, Takeda E, and Pike JW. Structural organization of the human vitamin D receptor chromosomal gene and its promoter. Mol Endocrinol 11: 1165-1179, 1997[Abstract/Free Full Text].

29.   Noda, M, Vogel RL, Craig AM, Prahl J, DeLuca HF, and Denhardt DT. Identification of a DNA sequence responsible for binding of the 1,25-dihydroxyvitamin D3 receptor and 1,25-dihydroxyvitamin enhancement of mouse secreted phosphoprotein 1 (Spp-1 or osteopontin) gene expression. Proc Natl Acad Sci USA 87: 9995-9999, 1990[Abstract].

30.   Noda, M, Yoon K, and Rodan GA. Cyclic AMP-mediated stabilization of osteocalcin mRNA in rat osteoblast-like cells treated with parathyroid hormone. J Biol Chem 263: 18574-18577, 1988[Abstract/Free Full Text].

31.   Ohyama, Y, Noshiro M, and Okuda K. Cloning and expression of cDNA encoding 25-hydroxyvitamin D3 24-hydroxylase. FEBS Lett 278: 195-198, 1991[ISI][Medline].

32.   Omdahl, J, and May B. The 25-hydroxyvitamin 24-hydroxylase D. In: Vitamin D, edited by Feldman D, Glorieux D, and Pike JW. San Diego: Academic, 1997, p. 69-86.

33.   Partridge, NC, Bloch SR, and Pearman AT. Signal transduction pathways mediating parathyroid hormone regulation of osteoblastic gene expression. J Cell Biochem 55: 321-327, 1994[ISI][Medline].

34.   Pike, JW, Kesterson RA, Scott RA, Kerner SA, McDonnell DP, and O'Malley BW. Vitamin D receptors: molecular structures of the protein and its chromosomal gene. In: Vitamin D, Molecular, Cellular and Clinical Endocrinology: Proceedings of the Seventh Workshop on Vitamin D, edited by Norman AW, Schaefer K, Grigoleit HG, and Herrath D. New York: de Gruyter, 1988, p. 215-224.

35.   Rangarajan, PN, Umesono K, and Evans RM. Modulation of glucocorticoid receptor function by protein kinase A. Mol Endocrinol 6: 1451-1457, 1992[Abstract].

36.   Reddy, GS, and Tserng KY. Calcitroic acid, endproduct of renal metabolism of 1,25-dihydroxyvitamin D3 through C-24 oxidation pathway. Biochemistry 28: 1763-1769, 1989[ISI][Medline].

37.   Reinhardt, TA, and Horst RL. Parathyroid hormone down-regulates 1,25-dihydroxyvitamin D3 receptors (VDR) and VDR messenger ribonucleic acid in vitro and blocks homologous up-regulation of VDR in vivo. Endocrinology 127: 942-948, 1990[Abstract].

38.   Reinhardt, TA, and Horst RL. Phorbol 12-myristate 13-acetate and 1,25-dihyroxyvitamin D3 regulate 1,25-dihydroxyvitamin D3 receptors synergistically in rat osteosarcoma cells. Mol Cell Endocrinol 101: 159-165, 1994[ISI][Medline].

39.   Ross, FP, Chappel J, Alvarez JI, Sander D, Butler WT, Farach-Carson MC, Mintz KA, Robey PG, Teitelbaum SL, and Cheresh DA. Interactions between the bone matrix proteins osteopontin and bone sialoprotein and the osteoclast integrin alpha vbeta 3 potentiate bone resorption. J Biol Chem 268: 9901-9907, 1993[Abstract/Free Full Text].

40.   Schule, R, Rangarajan P, Kliewer S, Ransone LJ, Bolado J, Yang N, Verma IM, and Evans RM. Functional antagonism between oncoprotein c-jun and the glucocorticoid receptor. Cell 62: 1217-1226, 1990[ISI][Medline].

41.   Shemshedini, L, Knauthe R, Sassone-Corsi P, Pornon A, and Gronemeyer H. Cell-specific inhibitory and stimulatory effects of fos and jun on transcription activation by nuclear receptors. EMBO J 10: 3839-3849, 1991[Abstract].

42.   Smith, JH, and Denhardt DT. Molecular cloning of a tumor promoter-inducible mRNA found in JB6 mouse epidermal cells: induction is stable at high, but not at low, cell densities. J Cell Biochem 34: 13-22, 1987[ISI][Medline].

43.   Sodek, J, Chen J, Kasugai S, Nagata T, Zhang Q, McKee MD, and Nanci A. Elucidating the functions of bone sialoprotein and osteopontin in bone formation. In: Chemistry and Biology and Mineralized Tissues, edited by Slavkin H, and Price P. New York: Elsevier, 1992, p. 297-306.

44.   St-Arnaud, R, Arabian A, Travers R, Barletta F, Raval-Pandya M, Chapin K, Depovere J, Mathieu C, Christakos S, Demay MB, and Glorieux FH. Deficient mineralization of intramembranous bone in vitamin D-24-hydroxylase-ablated mice is due to elevated 1,25-dihydroxyvitamin D and not to the absence of 24,25-dihydroxyvitamin D. Endocrinology 141: 2658-2666, 2000[Abstract/Free Full Text].

45.   Tyson, DR, Swarthout JT, and Partridge NC. Increased osteoblastic c-fos expression by parathyroid hormone requires protein kinase A phosphorylation of the cyclic adenosine 3',5' monophosphate response element binding protein at serine 133. Endocrinology 140: 1255-1261, 1999[Abstract/Free Full Text].

46.   Van Leeuwen, JPTM, Birkenhager JC, Bos MP, van der Bemd GJCM, Herrmann-Erlee MPM, and Pols HAP Parathyroid hormone sensitizes long bones to the stimulation of bone resorption by 1,25-dihydroxyvitamin D3. J Bone Miner Res 7: 303-309, 1992[ISI][Medline].

47.   Van Leeuwen, JPTM, Birkenhager JC, van den Bemd GCM, and Pols HAP Evidence for coordinated regulation of osteoblast function by 1,25-dihydroxyvitamin D3 and parathyroid hormone. Biochim Biophys Acta 1312: 54-62, 1996[Medline].

48.   Varghese, S, Lee S, Huang YC, and Christakos S. Analysis of rat vitamin D-dependent calbindin-D28k gene expression. J Biol Chem 263: 9776-9784, 1988[Abstract/Free Full Text].

49.   Yang, W, Friedman PA, Kumar R, Omdahl JL, May BK, Siu-Caldera ML, Reddy GS, and Christakos S. Expression of 25(OH)D3 24-hydroxylase in distal nephron: coordinate regulation by 1,25(OH)2D3 and cAMP or PTH. Am J Physiol Endocrinol Metab 276: E793-E805, 1999[Abstract/Free Full Text].

50.   Yasuda, H, Shima N, Nakagawa N, Yamaguchi K, Kinosaki M, Mochizuki S, Tomoyasu A, Yano K, Goto M, Murakami A, Tsuda E, Morinaga T, Higashio K, Udagawa N, Takahashi N, and Suda T. Osteoclast differentiation factor is a ligand for osteoprotegerin/osteoclastogenesis-inhibitory factor and is identical to TRANCE/RANKL. Proc Natl Acad Sci USA 95: 3597-3602, 1998[Abstract/Free Full Text].


Am J Physiol Endocrinol Metab 281(1):E162-E170
0193-1849/01 $5.00 Copyright © 2001 the American Physiological Society