Retinoic acid modulation of 1,25(OH)2 vitamin D3 receptors and bioresponse in bone cells: species differences between rat and mouse

Retinoic acid (RA) caused a reduction in the level of 1,25(OH)2D3 receptors to 1/3 of control in rat osteoblast-like cells (ROB) while increasing the receptor level to 3-fold the control in mouse osteoblast-like cells (MOB). Scatchard analysis of receptor binding indicated that there was no change in affinity for 1,25(OH)2D3. The changes in receptor levels required time to develop and were dose-dependent. RA also modulated the ability of cells to respond to 1,25(OH)2D3 as measured by the induction of the enzyme 25(OH)D3-24 hydroxylase. Induction of enzyme activity by 1,25(OH)2D3 closely paralleled receptor level established by RA pretreatment. In MOB, the up-regulation of the receptor occurred despite the action of RA to inhibit DNA, RNA and protein synthesis. However, RA stimulation of 1,25(OH)2D3 receptor levels was blocked by the addition of cycloheximide or actinomycin D, indicating that the up-regulation required protein and RNA synthesis. The opposite effect of RA on mouse and rat cells suggests that important species-dependent factors modulate the action of retinoids on mammalian cells.     

1,25-dihydroxyvitamin D(3) stimulated protein kinase C phosphorylation of type VI adenylyl cyclase inhibits parathyroid hormone signal transduction in rat osteoblastic UMR 106-01 cells

1,25-Dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) treatment of osteoblastic cells was shown previously to attenuate Parathyroid hormone (PTH) response by inhibiting adenylyl cyclase (AC) activity. In this study, we have investigated the mechanism by which 1,25(OH)(2)D(3) inhibits AC in rat osteoblastic UMR 106-01 cells. 1,25(OH)(2)D(3) treatment inhibited both PTH and forskolin-stimulated AC activity by 25%-50% within 12 min in a concentration-dependent manner suggesting a direct inhibition of the AC enzyme. Treatment with 25(OH)D(3) had no effect on basal or stimulated AC activity. We determined the profile of AC subtypes expressed in UMR cells and found AC VI to be the dominant subtype accounting for 50% of AC mRNA. Since AC VI can be inhibited by protein kinase C (PKC) phosphorylation, we examined 1,25(OH)(2)D(3) activation of various PKC isoforms. 1,25(OH)(2)D(3) increased the membrane translocation of PKC-betaI, -delta, and -zeta with a concomitant increase in PKC activity. The translocation of PKC-betaI and -delta was blocked by the PLC inhibitor U73122 whereas that of PKC-zeta was abolished by the PI-3 kinase inhibitor wortmannin. The attenuation of cAMP production by 1,25(OH)(2)D(3) was antagonized by the PKC inhibitors Go6850, calphostin C, and wortmannin, but not by a calmodulin kinase II (CaMKII) inhibitor. Treatment with 1,25(OH)(2)D(3) for 20 min increased AC VI phosphorylation by 10.8-fold and this was blocked partially by Go6850 and partially by wortmannin but was unaffected by CaMKII inhibitor. These results demonstrate that 1,25(OH)(2)D(3) activation of PKC isoforms leads to phosphorylation of AC VI and inhibition of PTH-activation of this pathway in osteoblasts.     

Involvement of endogenously produced 1,25-dihydroxyvitamin D-3 in the growth and differentiation of human keratinocytes.

In this study, we investigated the possibility that cultured keratinocytes from normal human adult skin produce 1,25-dihydroxyvitamin D-3 (1,25(OH)2D3, a biologically active form of vitamin D-3) from 25-hydroxyvitamin D-3 [25(OH)D3], and that 1,25(OH)2D3 endogenously produced by keratinocytes is involved in the self regulation of their growth and differentiation. To determine whether 1,25(OH)2D3 is produced from 25(OH)D3 by skin keratinocytes, 25(OH)[3H]D3 was added to keratinocyte cultures and incubated for 1 h and 5 h. The intracellular and extracellular metabolites were analyzed by three chromatographic systems. The three chromatograms revealed that the major metabolite produced from 25(OH)D3 was 1,25(OH)2D3. Most of the 1,25(OH)2D3 endogenously produced from 25(OH)D3 remained within the cells. To examine the time course of 1,25(OH)2D3 production, the amount of 1,25(OH)[3H]D3 was measured at 15 min, 1 h, 5 h and 10 h, being at a maximum 1 h after the addition of 25(OH)D3. These data indicate that keratinocytes rapidly convert 25(OH)D3 to 1,25(OH)2D3 and that 1,25(OH)2D3 is not released into the medium. To determine whether endogenously produced 1,25(OH)2D3 is involved in the regulation of growth and differentiation of normal human keratinocytes, we examined the effects of 1,25(OH)2D3 and 25(OH)D3 on their growth and differentiation. Keratinocyte growth was inhibited to 52.6% and 23.4% by 10(-8) M and 10(-7) M 1,25(OH)2D3 and to 80.5% and 23.9% by 10(-8) M and 10(-7) M 25(OH)D3, respectively. Differentiation of these cells was evaluated by quantifying the number which express involucrin, a precursor protein of cornified envelope. The population of involucrin expressing cells (differentiated cells) increased from 6.2% to 14.5% by 2.5.10(-7) M 1,25(OH)2D3, and to 11.8% by 2.5.10(-7) M 25(OH)D3. These results clearly indicate that 25(OH)D3 is as effective on human keratinocytes as 1,25(OH)2D3 in inhibiting growth and inducing differentiation, although to a slightly lesser extent than 1,25(OH)2D3. The possibility that the effect of 25(OH)D3 is mediated through binding to the 1,25(OH)2D3 receptor can be excluded, since a competitive binding assay revealed that the affinity of 25(OH)D3 for the 1,25(OH)2D3 receptor in a cytosolic extract of keratinocytes was 100-times lower than that of 1,25(OH)2D3. Thus, these results suggest that 1,25(OH)2D3 endogenously produced in keratinocytes from 25(OH)D3 is involved in the regulation of their growth and differentiation in vitro.     

All-trans retinoic acid blocks the antiproliferative prodifferentiating actions of 1,25-Dihydroxyvitamin D3 in normal human keratinocytes

1,25-Dihydroxyvitamin D₃ [1,25(OH)₂D₃] and all-trans retinoic acid (RA), the active metabolites of vitamins D and A respectively, regulate the proliferation and differentiation of keratinocytes. Both the vitamin D receptor (VDR) and the retinoic acid receptor family (RAR) bind to DNA response elements as heterodimers with the retinoic X receptor (RXR), suggesting that there are pathways of action that are shared by both compounds. Therefore, we examined the interactions of 1,25(OH)₂D₃ and RA upon the proliferation and differentiation of normal human keratinocytes (NHK) and of a squamous cell carcinoma cell line, SCC4. Although both 1,25(OH)₂D₃ and RA were each able to inhibit NHK proliferation in a dose-dependent manner, when they were administered in combination, proliferation was stimulated, suggesting mutual antagonism. In contrast, SCC4 cells proved insensitive in terms of proliferation to 1,25(OH)₂D₃ and to all but the highest concentration (10⁻⁶ M) of RA. 1,25(OH)₂D₃ exerted a biphasic effect on transglutaminase (TGase) and involucrin (INV) mRNA levels, with maximal stimulation at 10⁻⁹ M. RA inhibited TGase and INV mRNA levels and antagonized the stimulation by 1,25(OH)₂D₃. A similar pattern was observed for TGase protein, but, RA, which, by itself, reduced INV, markedly enhanced the ability of 1,25(OH)₂D₃ to raise INV levels, possibly by inhibiting 1,25(OH)₂D₃-stimulated TGase activity and cross-linking of soluble INV into the insoluble cornified envelope (CE). Thus, in NHK cells, RA antagonizes the antiproliferative prodifferentiating actions of 1,25(OH)₂D₃, but assessment of a single marker, such as INV protein, may be misleading. J. Cell. Physiol. 174:1–8, 1998. © 1998 Wiley-Liss, Inc.     

Bone Gla protein messenger ribonucleic acid is regulated by both 1,25-dihydroxyvitamin D3 and 3',5'-cyclic adenosine monophosphate in rat osteosarcoma cells

1,25-Dihydroxyvitamin D3 [1,25-(OH)2D3] regulates the synthesis of bone gamma-carboxyglutamic acid (Gla) protein (BGP) by osteoblastic cells. In this study we examined the effect of cAMP, alone and in combination with 1,25-(OH)2D3, on the regulation of BGP mRNA levels in ROS 17/2 rat osteosarcoma cells. Elevation of intracellular cAMP levels by cAMP analogs or by isobutylmethylxanthine (IBMX), forskolin, or PTH, resulted in increased BGP mRNA levels and BGP secretion after 1 day of treatment. The effects of these agents were additive with 1,25-(OH)2D3 in stimulating BGP gene expression. After 4 days of treatment, pertussis toxin (PT) and 1,25-(OH)2D3 were synergistic in stimulating BGP mRNA, and the effect of PT could be mimicked by (Bu)2cAMP, IBMX, forskolin, cholera toxin, and to a lesser extent by PTH. The effect of 1-day treatment with cAMP alone and the synergistic effect with 1,25-(OH)2D3 on the stimulation of BGP mRNA were dependent on cell density, while basal and 1,25-(OH)2D3-stimulated synthesis were not. Cyclic AMP inhibited ROS 17/2 cell growth after 1 day of treatment, an effect that was also dependent on initial cell density. After 4 days of treatment, 1,25-(OH)2D3, cAMP, and PT all demonstrated inhibition of cell growth. When cells were treated with actinomycin D, both 1,25-(OH)2D3 and cAMP stimulation of BGP mRNA were blocked. In addition, neither agent was effective in enhancing BGP mRNA stability when prestimulated cells were exposed to actinomycin D.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of human pulmonary surfactant protein gene expression by 1alpha,25-dihydroxyvitamin D3

1alpha,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] has been reported to stimulate lung maturity, alveolar type II cell differentiation, and pulmonary surfactant synthesis in rat lung. We hypothesized that 1,25(OH)(2)D(3) stimulates expression of surfactant protein-A (SP-A), SP-B, and SP-C in human fetal lung and type II cells. We found that immunoreactive vitamin D receptor was detectable in fetal lung tissue and type II cells only when incubated with 1,25(OH)(2)D(3). 1,25(OH)(2)D(3) significantly decreased SP-A mRNA in human fetal lung tissue but did not significantly decrease SP-A protein in the tissue. In type II cells, 1,25(OH)(2)D(3) alone had no significant effect on SP-A mRNA or protein levels but reduced SP-A mRNA and protein in a dose-dependent manner when the cells were incubated with cAMP. SP-A mRNA levels in NCI-H441 cells, a nonciliated bronchiolar epithelial (Clara) cell line, were decreased in a dose-dependent manner in the absence or presence of cAMP. 1,25(OH)(2)D(3) had no significant effect on SP-B mRNA levels in lung tissue but increased SP-B mRNA and protein levels in type II cells incubated in the absence or presence of cAMP. Expression of SP-C mRNA was unaffected by 1,25(OH)(2)D(3) in lung tissue incubated +/- cAMP. These results suggest that regulation of surfactant protein gene expression in human lung and type II cells by 1,25(OH)(2)D(3) is not coordinated; 1,25(OH)(2)D(3) decreases SP-A mRNA and protein levels in both fetal lung tissue and type II cells, increases SP-B mRNA and protein levels only in type II cells, and has no effect on SP-C mRNA levels.     

Membrane actions of vitamin D metabolites 1alpha,25(OH)2D3 and 24R,25(OH)2D3 are retained in growth plate cartilage cells from vitamin D receptor knockout mice

1alpha,25(OH)(2)D(3) regulates rat growth plate chondrocytes via nuclear vitamin D receptor (1,25-nVDR) and membrane VDR (1,25-mVDR) mechanisms. To assess the relationship between the receptors, we examined the membrane response to 1alpha,25(OH)(2)D(3) in costochondral cartilage cells from wild type VDR(+/+) and VDR(-/-) mice, the latter lacking the 1,25-nVDR and exhibiting type II rickets and alopecia. Methods were developed for isolation and culture of cells from the resting zone (RC) and growth zone (GC, prehypertrophic and upper hypertrophic zones) of the costochondral cartilages from wild type and homozygous knockout mice. 1alpha,25(OH)(2)D(3) had no effect on [(3)H]-thymidine incorporation in VDR(-/-) GC cells, but it increased [(3)H]-thymidine incorporation in VDR(+/+) cells. Proteoglycan production was increased in cultures of both VDR(-/-) and VDR(+/+) cells, based on [(35)S]-sulfate incorporation. These effects were partially blocked by chelerythrine, which is a specific inhibitor of protein kinase C (PKC), indicating that PKC-signaling was involved. 1alpha,25(OH)(2)D(3) caused a 10-fold increase in PKC specific activity in VDR(-/-), and VDR(+/+) GC cells as early as 1 min, supporting this hypothesis. In contrast, 1alpha,25(OH)(2)D(3) had no effect on PKC activity in RC cells isolated from VDR(-/-) or VDR(+/+) mice and neither 1beta,25(OH)(2)D(3) nor 24R,25(OH)(2)D(3) affected PKC in GC cells from these mice. Phospholipase C (PLC) activity was also increased within 1 min in GC chondrocyte cultures treated with 1alpha,25(OH)(2)D(3). As noted previously for rat growth plate chondrocytes, 1alpha,25(OH)(2)D(3) mediated its increases in PKC and PLC activities in the VDR(-/-) GC cells through activation of phospholipase A(2) (PLA(2)). These responses to 1alpha,25(OH)(2)D(3) were blocked by antibodies to 1,25-MARRS, which is a [(3)H]-1,25(OH)(2)D(3) binding protein identified in chick enterocytes. 24R,25(OH)(2)D(3) regulated PKC in VDR(-/-) and VDR(+/+) RC cells. Wild type RC cells responded to 24R,25(OH)(2)D(3) with an increase in PKC, whereas treatment of RC cells from mice lacking a functional 1,25-nVDR caused a time-dependent decrease in PKC between 6 and 9 min. 24R,25(OH)(2)D(3) dependent PKC was mediated by phospholipase D, but not by PLC, as noted previously for rat RC cells treated with 24R,25(OH)(2)D(3). These results provide definitive evidence that there are two distinct receptors to 1alpha,25(OH)(2)D(3). 1alpha,25(OH)(2)D(3)-dependent regulation of DNA synthesis in GC cells requires the 1,25-nVDR, although other physiological responses to the vitamin D metabolite, such as proteoglycan sulfation, involve regulation via the 1,25-mVDR.     

The human myelomonocytic cell line U-937 as a model for studying alterations in steroid-induced monokine gene expression: marked enhancement of lipopolysaccharide-stimulated interleukin-1 beta messenger RNA levels by 1,25-dihydroxyvitamin D3.

The active metabolite of vitamin D, 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3], is a potent regulator of human monocyte/macrophage function in vitro. To establish a model for 1,25-(OH)2D3 regulation of human monocyte monokine synthesis, three human cell lines (U-937, THP-1, and HL-60) were examined for: 1) the presence of functional 1,25-(OH)2D3 receptors; 2) the accumulation of interleukin-1 beta (IL-1 beta) mRNA and IL-1 beta protein in response to lipopolysaccharide (LPS); and 3) the regulation of this response by 1,25-(OH)2D3. All three cell lines expressed vitamin D receptor and had increased levels of IL-1 beta mRNA in response to LPS. Preincubation of cells with 1,25-(OH)2D3 augmented IL-1 beta mRNA levels only in U-937 and HL-60 cells. From these data, and taking into consideration their state of differentiation and relative ease of culture, U-937 was chosen over HL-60 and THP-1 as the cell line we further characterized. In U-937 cells, optimum time and dose of pretreatment with 1,25-(OH)2D3 were determined to be 12-24 h at a receptor saturating concentration of 1,25-(OH)2D3 (10 nM). Preincubation of cells with 1,25-(OH)2D3 had no effect on the time course of IL-1 beta mRNA appearance in response to LPS. However, exposure of U-937 cells to 1,25-(OH)2D3 increased by 200% the level of IL-1 beta mRNA detected and decreased by three orders of magnitude the concentration of LPS required to achieve steady state mRNA levels equivalent to those observed in U-937 cells not preincubated with the hormone.2+o     

The mechanism of 1,25-dihydroxyvitamin D(3) autoregulation in keratinocytes

The synthesis of 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) from its precursor, 25-dihydroxyvitamin D(3) (25(OH)D(3)), is catalyzed by the mitochondrial cytochrome P450 enzyme 25-hydroxyvitamin D(3)-1alpha-hydroxylase (1alpha-hydroxylase). It has been generally assumed that 1,25(OH)(2)D(3) inhibits the activity of this enzyme by regulating its expression at the genomic level. We confirmed that 1,25(OH)(2)D(3) reduced the apparent conversion of 25(OH)D(3) to 1,25(OH)(2)D(3) while stimulating the conversion of 1,25(OH)(2)D(3) and 25(OH)D(3) to 1,24,25(OH)(3)D(3) and 24,25(OH)(2)D(3), respectively. However, 1,25(OH)(2)D(3) failed to reduce the abundance of its mRNA or its encoded protein in human keratinocytes. Instead, when catabolism of 1,25(OH)(2)D(3) was blocked with a specific inhibitor of the 25-hydroxyvitamin D(3)-24-hydroxylase (24-hydroxylase) all apparent inhibition of 1alpha-hydroxylase activity by 1,25(OH)(2)D(3) was reversed. Thus, the apparent reduction in 1alpha-hydroxylase activity induced by 1,25(OH)(2)D(3) is due to increased catabolism of both substrate and product by the 24-hydroxylase. We believe this to be a unique mechanism for autoregulation of steroid hormone synthesis.     

Targeted disruption of the 25-hydroxyvitamin D3 1alpha-hydroxylase gene in ras-transformed keratinocytes demonstrates that locally produced 1alpha,25-dihydroxyvitamin D3 suppresses growth and induces differentiation in an autocrine fashion

It has been previously shown that keratinocytes express a high level of 25-hydroxyvitamin D(3) (25-OHD(3)) 1alpha-hydroxylase (1alpha-hydroxylase). 1alpha-Hydroxylase catalyzes the conversion of 25-OHD(3) to 1alpha,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)]. 1,25(OH)(2)D(3) is both antiproliferative (i.e., suppresses cell growth) and prodifferentiative (i.e., induces cell differentiation) in many cell types. We hypothesized that local production of 1,25(OH)(2)D(3) by keratinocytes may suppress their growth and induce their differentiation in an autocrine fashion. To test this hypothesis, we inactivated both 1alpha-hydroxylase alleles in a ras-transformed keratinocyte cell line, HPK1Aras, which typically produces squamous carcinoma in nude mice. To inactivate 1alpha-hydroxylase expression by HPK1Aras cells, we disrupted both alleles of the 1alpha-hydroxylase gene by homologous recombination. Lack of expression and activity of 1alpha-hydroxylase was confirmed by Northern blot analysis and detected conversion of 25-OHD(3) to 1,25(OH)(2)D(3). We then examined the effect of substrate 25-OHD(3) on parameters of growth and differentiation in the double knockout cell line as compared to wild-type HPK1Aras cells in vitro. It was found that 1alpha-hydroxylase inactivation blocked the antiproliferative and prodifferentiative effect of 25-OHD(3). These in vitro effects were further analyzed in vivo by injecting knockout or control cells subcutaneously in severely compromised immunodeficient mice. Tumor growth was accelerated and differentiation was inhibited in mice given injections of knockout cells as compared to control cells in the presence of substrate 25-OHD(3). Our results demonstrate, for the first time, that 1alpha-hydroxylase expression by keratinocytes plays an important role in autocrine growth and differentiation of these cells, and suggest that expression of this enzyme may modulate tumor growth in squamous carcinomas.     

Analysis by high-resolution two-dimensional electrophoresis of differentiation-dependent alterations in cytosolic protein pattern of HL-60 leukemic cells.

HL-60 leukemic cells were differentiated along the neutrophilic pathway with retinoic acid (RA) or along the monocytic pathway with 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). Using a high-resolution two-dimensional electrophoresis technique and subsequent silver staining, differentiation-dependent changes in cytosolic protein pattern of HL-60 cells were analysed and were compared with the cytosolic protein pattern of human neutrophils. The amount of 64 and 50 out of a total of 632 proteins studied was increased or decreased in RA- and 1,25(OH)2D3-differentiated HL-60 cells, respectively, in comparison to undifferentiated HL-60 cells. Thirty-three of these proteins were similarly altered in RA- and 1,25(OH)2D3-differentiated HL-60 cells. Twenty-two and 25 of the proteins altered in amount in RA- or 1,25(OH)2D3-differentiated HL-60 cells versus undifferentiated HL-60 cells were similarly altered in human neutrophils in comparison to undifferentiated HL-60 cells. Seven and 10 of the proteins altered in amount in RA- or 1,25(OH)2D3-differentiated HL-60 cells had specific equivalents in neutrophil cytosol. Our results show (i) that neutrophilic and monocytic differentiation is associated with decreases and increases in amount of cytosolic proteins; (ii) that both differentiation processes share a common set of alterations; and (iii) are associated with specific alterations in protein amount.     

Vitamin D metabolites and analogs induce lipoxygenase mRNA expression and activity as well as reactive oxygen species (ROS) production in human bone cell line

Vitamin D metabolites and its less-calcemic analogs (vitamin D compounds) are beneficial for bone and modulate cell growth and energy metabolism. We now analyze whether 25(OH)D(3) (25D), 1,25(OH)(2)D(3) (1,25D), 24,25(OH)(2)D(3) (24,25D), JKF1624F(2)-2 (JKF) or QW1624F(2)-2 (QW) regulate lipooxygenase (LO) mRNA expression and its products; hydroxyl-eicosatetraenoic acid (12 and 15HETE) formation, as well as reactive oxygen species (ROS) production in human bone cell line (SaOS2) and their interplay with modulation of cell proliferation and energy metabolism. All compounds except 25D increased 12LO mRNA expression and modulated 12 and 15HETE production whereas ROS production was increased by all compounds, and inhibited by NADPH oxidase inhibitors diphenyleneiodonium (DPI) and N-acetylcysteine (NAc). Baicaleine (baic) the inhibitor of 12 and 15LO activity blocked only slightly the stimulation of DNA synthesis by all compounds, whereas DPI inhibited almost completely the stimulation of DNA and CK by all compounds. Treatments of cells with 12 or 15HETE increased DNA synthesis and CK that were only slightly inhibited by DPI. These results indicate that vitamin D compounds increased oxidative stress in osteoblasts in part via induction of LO expression and activity. The increased ROS production mediates partially elevated cell proliferation and energy metabolism, whereas the LO mediation is not essential. This new feature of vitamin D compounds is mediated by intracellular and/or membranal binding sites and its potential hazard could lead to damage due to increased lipid oxidation, although the transient mediation of ROS in cell proliferation is beneficial to bone growth in a yet unknown mechanism.     

Role of vitamin D3 receptor in the synergistic differentiation of WEHI-3B leukemia cells by vitamin D3 and retinoic acid.

WEHI-3B D- cells differentiate in response to 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) but not to all-trans-retinoic acid (RA) or other inducing agents. Combinations of RA with 1,25-(OH)2D3 interact to produce synergistic differentiation of WEHI-3B D- cells. To determine factors involved in the synergistic interaction, expression of the 1,25-(OH)2D3 receptor (VDR) and retinoid receptors, RARalpha and RXRalpha, was measured. No VDR was detected in untreated WEHI-3B D- cells; however, RA and 1,25-(OH)2D3 when used as single agents caused a slight induction of the VDR and in combination produced a marked increase in the VDR. In contrast, no changes in RARalpha and RXRalpha were initiated by these compounds. An RAR-selective agonist combined with 1,25-(OH)2D3 produced synergistic differentiation of WEHI-3B D- cells, whereas an RXR-selective agonist did not. To gain information on the role of the VDR in the synergistic interaction, the VDR gene was transferred into WEHI-3B D+ cells, in which no VDR was detected and no synergism was produced. Expression of the VDR conferred differentiation responsiveness to 1,25-(OH)2D3 in WEHI-3B D+ cells. These findings suggest that (a) induction of VDR expression is a key component in the synergistic differentiation induced by 1,25-(OH)2D3 and RA and (b) RAR and not RXR must be activated for enhanced induction of the VDR and for the synergistic differentiation produced by RA and 1, 25-(OH)2D3.