Characterization of the vitamin D receptor from the Caco-2 human colon carcinoma cell line: effect of cellular differentiation.

The human colon carcinoma cell line, Caco-2, is the only intestinal cell line to spontaneously differentiate in culture to a population exhibiting structural and biochemical characteristics of mature enterocytes. We conducted studies to establish the presence of the vitamin D receptor (VDR), determine changes in VDR concentration and affinity with differentiation and determine whether 1 alpha,25-dihydroxyvitamin D3 (1,25(OH)2D3) mediates a functional response in this cell line. We found that Caco-2 cells possess a specific 1,25(OH)2D3 binding protein similar to the mammalian VDR. It has an equilibrium dissociation constant (Kd) of 0.72 nM, binds vitamin D analogues in order of their biological activities in vivo (1,25(OH)2D3 greater than 25(OH)D3 greater than 24,25(OH)2D3), sediments as a single peak on sucrose density gradients at 3.7 S, and is eluted from a DNA-cellulose column by 0.16 M KCl. The maximum number of binding sites was 2.6-fold greater in the differentiated cell (Day 15) compared to the preconfluent, undifferentiated (Day 4) cell (23 fmol/mg protein vs 56 fmol/mg protein). Cell growth was reduced 59% when exposed to 10(-7) M 1,25(OH)2D3 for 8 days. Alkaline phosphatase activity significantly increased in cultures incubated with 10(-8) M 1,25(OH)2D3 for up to 4 days when treatment was started in both undifferentiated cells (Day 5) and differentiated cells (Day 11). These findings suggest that the VDR present in undifferentiated and differentiated Caco-2 cells is functional. Caco-2 cells provide a unique in vitro model to study vitamin D-regulated functions in differentiated mammalian enterocytes.     

Specific binding of 24R,25-dihydroxyvitamin D3 to chick intestinal mucosa: 24R,25-dihydroxyvitamin D3 is an allosteric effector of 1,25-dihydroxyvitamin D3 binding

We have previously described a significant decrease in the positive cooperativity level and affinity of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] binding to its chick intestinal chromatin receptor induced in vitro by a physiological 10-fold molar excess of (24R)-25-dihydroxyvitamin D3 [24R,25(OH)2D3] [F. Wilhelm and A. W. Norman (1985) Biochem. Biophys. Res. Commun. 126, 496-501]. In this report, we have initiated a comparative study of the binding of 24R,25(OH)2[3H]D3 and 1,25(OH)2[3H]D3 to the the intestinal chromatin fraction obtained from vitamin D-replete birds. 24R,25(OH)2[3H]D3 specific binding to this chromatin fraction was characterized by a dissociation constant (Kd) of 34.0 +/- 6.4 nM, a positive cooperativity level (nH) of 1.40 +/- 0.13, and a capacity (Bmax) of 47 +/- 8 fmol/mg protein. The very low relative competitive index (RCI) of 24R,25(OH)2D3 (0.11 +/- 0.03%) for the 1,25(OH)2D3 binding site/receptor, as well as the inability of 1,25(OH)2D3 to displace 24R,25(OH)2D3 from its binding site at a physiological molar ratio of 1:10, strongly suggest the independence of 24R,25(OH)2D3 and 1,25(OH)2D3 binding sites. Stereospecificity of the 24R,25(OH)2D3 binding sites was attested by the displacement of only 45 +/- 6% of 24R,25(OH)2D3 specific binding by equimolar concentrations of 24S,25(OH)2D3. Collectively these results suggest the existence of a binding domain/receptor for 24,25(OH)2D3 in the chick intestine which is independent of the 1,25(OH)2D3 receptor.     

1,25-Dihydroxyvitamin D3 binds specifically to rat vascular smooth muscle cells and stimulates their proliferation in vitro

Cultured vascular smooth muscle cells (VSMC) derived from rat aorta were found to contain a specific receptor for 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]. Its Kd (5.0 x 10(-11) M) and capacity (22.9 fmol/mg of cytosol protein) for 1,25-(OH)2D3, its sedimentation coefficient on a sucrose density gradient (3.2 S), its relative affinities for various vitamin D3 metabolites [1,25-(OH)2D3 greater than 25-hydroxyvitamin D3 greater than 24,25-dihydroxyvitamin D3 greater than vitamin D3] and its affinity for DNA-cellulose were similar to those reported for the 1,25-(OH)2D3 receptor in other tissues. 1,25-(OH)2D3 at concentrations of more than 10(-10) M caused dose-dependent enhancement of the proliferation of VSMC in DMEM with 10% FCS. 25-Hydroxyvitamin D3 stimulated the proliferation of VSMC only at its highest concentration tested (10(-6) M). These data show that 1,25-(OH)2D3 stimulates the proliferation of VSMC after its binding to a cytoplasmic receptor of the cells in vitro, and support the possibility that VSMC are target cells of the hormone.     

Self-induction of 1,25-dihydroxyvitamin D3 metabolism limits receptor occupancy and target tissue responsiveness

Whole cell 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) receptor (VDR) binding assays, which measure VDR in the presence of the metabolic machinery of the cell, were used in conjunction with a cytosol binding assay for VDR to determine if self-induced metabolism of 1,25-(OH)2D3 limits VDR occupancy, total VDR levels, and target cell responsiveness. Treatment of cells with 0.5 nM 1,25-(OH)2[3H]D3 for 16 h results in up-regulation of total cell VDR from 82 to 170 fmol/mg protein as measured in a cytosol binding assay. Conversely, whole cell binding assays of VDR showed a 1,25-(OH)2D3-mediated apparent down-regulation of VDR from 90 to 40 fmol/mg protein. Scatchard analysis using the cytosol binding assay demonstrated that 1,25-(OH)2D3 treatment increased total cell VDR from 93 to 154 fmol/mg protein. In contrast, Scatchard analysis with the whole cell binding assay demonstrated that 1,25-(OH)2D3 treatment resulted in reduction in total cell VDR from 100 to 64 fmol/mg protein. Initial Kd estimates with the whole cell binding assay suggested that 1,25-(OH)2D3 treatment resulted in a reduction in VDR Kd from 0.6 to 6.2 nM. This apparent reduction in the affinity of VDR for 1,25-(OH)2D3 was due to degradation of free 1,25-(OH)2[3H]D3 which occurred during whole cell saturation assay. Competitive inhibitors of 1,25-(OH)2D3 metabolism were found to reverse the apparent receptor down-regulation observed in whole cell binding assays of treated cells. In addition, the presence of competitive inhibitors amplified responses of cells to 1,25-(OH)2[3H]D3 treatment as measured by an increased occupancy of VDR by 1,25-(OH)2[3H]D3 and increased up-regulation of VDR over that observed without metabolism inhibitors. These data demonstrate that self-induced target tissue deactivation of 1,25-(OH)2D3 regulates 1,25-(OH)2D3 occupancy of VDR and ultimately the biopotency of 1,25-(OH)2D3 in target cells.     

Biological activity of 1 alpha, 25-dihydroxyvitamin D derivatives--24-epi-1 alpha, 25-dihydroxyvitamin D-2 and 1 alpha,25-dihydroxyvitamin D-7

Biological activity of 24-epi-1 alpha,25-dihydroxyvitamin D-2 (24-epi-1,25(OH)2D2) and 1 alpha,25-dihydroxyvitamin D-7 (1,25(OH)2D7), the 22,23-dihydro derivative of the former compound, was investigated. Both of the vitamin D derivatives stimulated intestinal calcium transport and calcium mobilization from bones in rats; however, the effect was about 50% of that of 1 alpha,25-dihydroxyvitamin D-3 (1,25(OH)2D3). On the other hand, 24-epi-1,25(OH)2D2 and 1,25(OH)2D7 inducement of HL-60 human leukemia cell differentiation was comparable to that of 1,25(OH)2D3. Accordingly, the differentiation-inducing activity of 24-epi-1,25(OH)2D2 and 1,25(OH)2D7 was much greater than their ability to stimulate calcium metabolism. In contrast to 1,25(OH)2D3, 24-epi-1,25(OH)2D2 and 1,25(OH)2D7 exerted little hypercalcemic activity in mice. These results suggest that both vitamin D derivatives will be useful as anti-tumor agents.     

Characterization of a specific, high affinity binding macromolecule for 1 alpha, 25-dihydroxyvitamin D3 in cultured chick kidney cells

Cytosol prepared from vitamin D3-deficient kidney cells in culture contains a 3.7 S protein that specifically binds 1,25-dihydroxyvitamin D3 with high affinity and low capacity. Whole kidney homogenate cytosol preparations are shown to possess two 1,25-dihydroxyvitamin D3 binding macromolecules. One of the binding proteins sediments at 3.5 to 3.7 S while the second sediments at 6.0 S. The 6.0 S component has a greater affinity for 25-dihydroxyvitamin D3 than for 1,25-dihydroxyvitamin D3. Cultured cell cytosol was found to have little 6.0 S 25-hydroxyvitamin D3 binding protein. Scatchard analysis of the cultured cell cytosol reveals an equilibrium binding constant (KD) of 5.6 x 10 (-11) with 57 fmol of sites/mg of protein. The receptor-like protein has a Mr = 72,000 and as with other steroid receptors it aggregates in the presence of low potassium concentrations. Analog competition for receptor binding reveals the following potency order: 1,25-dihydroxyvitamin D3 > 25-hydroxyvitamin D3 > 1 alpha-hydroxyvitamin D3 > 24(R),25-dihydroxyvitamin D3; the receptor had no detectable affinity for vitamin D3. The kidney cells respond to 1,25-dihydroxyvitamin D3 by diminishing 25-hydroxyvitamin D3 1 alpha-hydroxylation and increasing 24R-hydroxylation. Cultured cells provide a preparation of cytosol which has allowed extensive characterization of the renal 1,25-dihydroxyvitamin D3 receptor and should facilitate investigations into the role this receptor plays in renal control of vitamin D3 metabolism.     

Up-regulation of the intestinal 1,25-dihydroxyvitamin D receptor during hypervitaminosis D: a comparison between vitamin D2 and vitamin D3

Concentrations of intestinal 1,25-dihydroxyvitamin D receptor were measured in rats receiving pharmacological amounts (25,000 IU/rat daily for 6 days) of either vitamin D2 or vitamin D3. The data showed that both hypervitaminosis D2 and hypervitaminosis D3 resulted in significant up-regulation of intestinal 1,25-dihydroxyvitamin D receptor (fmol/mg protein) relative to controls (409 +/- 24, vitamin D2-treated; 525 +/- 41, vitamin D3-treated; and 249 +/- 19, control). The 1,25-dihydroxyvitamin D receptor enhancement also was accompanied by elevated plasma 25-hydroxyvitamin D and hypercalcemia. These data suggest that increased target-tissue 1,25-dihydroxyvitamin D receptor may play a role in enhancing target-tissue responsiveness and, thus, have a significant role in mediating the toxic effects of hypervitaminosis D.     

Molecular mechanism of 1,25-dihydroxyvitamin D3 inhibition of adipogenesis in 3T3-L1 cells

We have investigated the molecular mechanism whereby 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] inhibits adipogenesis in vitro. 1,25(OH)2D3 blocks 3T3-L1 cell differentiation into adipocytes in a dose-dependent manner; however, the inhibition is ineffective 24-48 h after the differentiation is initiated, suggesting that 1,25(OH)2D3 inhibits only the early events of the adipogenic program. Treatment of 3T3-L1 cells with 1,25(OH)2D3 does not block the mitotic clonal expansion or C/EBPbeta induction; rather, 1,25(OH)2D3 blocks the expression of C/EBPalpha, peroxisome proliferator-activated receptor-gamma (PPARgamma), sterol regulatory element-binding protein-1, and other downstream adipocyte markers. The inhibition by 1,25(OH)2D3 is reversible, since removal of 1,25(OH)2D3 from the medium restores the adipogenic process with only a temporal delay. Interestingly, although the vitamin D receptor (VDR) protein is barely detectable in 3T3-L1 preadipocytes, its levels are dramatically increased during the early phase of adipogenesis, peaking at 4-8 h and subsiding afterward throughout the rest of the differentiation program; 1,25(OH)2D3 treatment appears to stabilize the VDR protein levels. Consistently, adenovirus-mediated overexpression of human (h) VDR in 3T3-L1 cells completely blocks the adipogenic program, confirming that VDR is inhibitory. Inhibition of adipocyte differentiation by 1,25(OH)2D3 is ameliorated by troglitazone, a specific PPARgamma antagonist; conversely, hVDR partially suppresses the transacting activity of PPARgamma but not of C/EBPbeta or C/EBPalpha. Moreover, 1,25(OH)2D3 markedly suppresses C/EBPalpha and PPARgamma mRNA levels in mouse epididymal fat tissue culture. Taken together, these data indicate that the blockade of 3T3-L1 cell differentiation by 1,25(OH)2D3 occurs at the postclonal expansion stages and involves direct suppression of C/EBPalpha and PPARgamma upregulation, antagonization of PPARgamma activity, and stabilization of the inhibitory VDR protein.     

A specific binding moiety for 1,25-dihydroxyvitamin D(3) in basal lateral membranes of carp enterocytes

Carp (Cyprinus carpio), a freshwater fish that lives in a low-calcium environment, and Atlantic cod (Gadus morhua), a seawater fish that lives in a high-calcium environment, were studied for the presence of a novel membrane binding protein ("receptor") for the vitamin D metabolite, 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)]. Basal lateral membranes from enterocytes of either species were prepared and analyzed for specific saturable binding. Membranes from carp revealed a dissociation constant of 1.23 nM with a maximal binding capacity of 212 fmol/mg protein. In comparison, membranes from Atlantic cod enterocytes revealed very low and nonsignificant levels of specific binding. The [(3)H]1,25(OH)(2)D(3) binding activity in carp was further characterized for protein dependence, detergent extractability, and competition for binding with the metabolites 25(OH)D(3) and 24R,25(OH)(2)D(3). Finally, introduction of 1,25(OH)(2)D(3) to isolated carp enterocytes enhanced protein kinase C activity within 5 min, whereas intracellular Ca(2+) concentrations were unaffected by a range of 1,25(OH)(2)D(3) concentrations, as judged by fura 2 fluorescence. Thus the binding moiety may be a putative plasma membrane receptor for vitamin D, because it is functionally coupled to at least one signal transduction pathway.     

1,25-Dihydroxyvitamin D3 induces 25-hydroxyvitamin D3-24-hydroxylase in a cultured monkey kidney cell line (LLC-MK2) apparently deficient in the high affinity receptor for the hormone

A consequence of 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) action in kidney is the enhanced production of 24,25-dihydroxyvitamin D3 (24,25-(OH)2D3). We have studied this apparent induction phenomenon in two established mammalian cell lines of renal origin. A porcine kidney cell line, LLC-PK1, was found to possess typical receptors for 1,25-(OH)2D3 which sediment at 3.3 S and bind to immobilized DNA. Saturation analysis of LLC-PK1 cell cytosol revealed an equilibrium binding constant (Kd) for 1,25-(OH)2D3 of 7.8 X 10(-11) M and a concentration of 5400 binding sites/cell. In the presence of serum, intact LLC-PK1 cells also internalize and bind 1,25-(OH)2D3. In contrast, a monkey kidney cell line, LLC-MK2, was found to contain a negligible concentration of the 1,25-(OH)2D3 receptor by all criteria examined. However, both renal cell lines respond to 1,25-(OH)2D3 with a 2- to 20-fold increase in basal levels of 25-hydroxyvitamin D3-24-hydroxylase (24-hydroxylase) activity. Incubation of viable cell suspensions with 25-hydroxy[26,27-3H]vitamin D3 (0.5 microM) at 37 degrees C for 30 min followed by subsequent analysis of lipid extracts via high performance liquid chromatography was carried out to assess 24,25-(OH)2[3H]D3 formation. Enzyme induction was found to be specific for 1,25-(OH)2D3 in both cell lines with half-maximal stimulation of 24-hydroxylase activity observed at 0.2 and greater than or equal to 1.0 nM 1,25-(OH)2D3 in LLC-PK1 and LLC-MK2, respectively. The response in LLC-PK1 was more rapid (1-4 h) than in LLC-MK2 (4-8 h) following 1,25-(OH)2D3 treatment of cultures in situ. In both cell lines, actinomycin D abolished the 1,25-(OH)2D3-dependent increase in 24-hydroxylase activity. Our results suggest that the high affinity 1,25-(OH)2D3 receptor may not be required for 1,25-(OH)2D3-dependent induction of renal 24-hydroxylase activity. Alternatively, LLC-MK2 cells could contain an atypical form of the 1,25-(OH)2D3 receptor protein which retains functionality but escapes detection by standard binding techniques.     

Effects of 1,25-dihydroxyvitamin D3 on proliferation and differentiation of porcine preadipocyte in vitro

1,25-Dihydroxyvitamin D3, the physiologically active form of vitamin D3, exerts its functions through a receptor-mediated mechanism and plays an important role in the cell differentiation. This study investigated the effects of 1,25-dihydroxyvitamin D3 on the proliferation and differentiation of porcine preadipocyte. Stromal-vascular cells containing preadipocytes were prepared from dorsal subcutaneous adipose tissue of approximately 3-day-old Chinese male crossbred pigs. After confluence, the differentiation was induced by transferrin, dexamethasone and insulin for 2 days, and then subsequently cultured for 6 days. The cells were treated with 1,25-dihydroxyvitamin D3 during the induction of differentiation (the early phase of differentiation) or throughout the differentiation period. The terminal differentiation markers, such as glycerol-3-phosphate dehydrogenase activity and lipid accumulation were measured during the process of cultures. The treatment with 1,25-dihydroxyvitamin D3 severely affected the induction of all differentiation markers throughout the differentiation period. 1,25-Dihydroxyvitamin D3 suppressed the expression of peroxisome proliferator-activated receptor gamma mRNA and interfered with the induction of retinoid X receptor alpha mRNA. The mRNAs of the adipogenesis-related genes, lipoprotein lipase, stearoyl-CoA desaturase, phosphoenolpyruvate carboxykinase, glycerol-3-phosphate dehydrogenase and glucose transporter 4 were reduced when 1,25-dihydroxyvitamin D3 was added into differentiation medium. Also, 1,25-dihydroxyvitamin D3 inhibited preadipocyte differentiation in dose-dependent manner. These results suggested that 1,25-dihydroxyvitamin D3 inhibited porcine preadipocyte differentiation through suppressing PPAR gamma and RXR alpha mRNA expressions and then down regulating the expression of adipogenesis-related genes.     

C-6 functionalized analogs of 25-hydroxyvitamin D3 and 1alpha,25-dihydroxyvitamin D3: synthesis and binding analysis with vitamin D-binding protein and vitamin D receptor.

In this article, we describe the development of a general synthetic strategy to functionalize the C-6 position of vitamin D3 and its biologically important metabolites, i.e. 25-hydroxyvitamin D3 (25-OH-D3) and 1alpha,25-dihydroxyvitamin D3 [1,25(OH)2D3]. We employed Mazur's cyclovitamin D method to synthesize vitamin D3 analogs with several functionalities at the C-6 position. In addition, we synthesized 6-(3-hydroxypropyl) and 6-[(2-bromoacetoxy)propyl] derivatives of 25-OH-D3 15 and 16, respectively, and 6-(3-hydroxypropyl) derivative of 1,25(OH)2D3 17. Competitive binding assays of 15-17 with human serum vitamin D-binding protein showed that all these analogs specifically bound to this protein, although with significantly lower affinity than the 25-OH-D3, the strongest natural binder, but with comparable affinity with 1,25(OH)2D3, the hormone. On the other hand, 6-[3-hydroxypropyl], 1alpha,25-dihydroxyvitamin D3 17 did not show any specific binding for recombinant nuclear vitamin D receptor. These results indicated that the region containing the C-6 position of the parent seco-steroid [1,25(OH)2D3] may be an important recognition marker towards vitamin D receptor binding. Information, delineated in this article, will be important for evaluating structure-activity relationship in synthetic analogs of vitamin D and its metabolites.     

Receptor for 1,25-dihydroxyvitamin D in a vascular smooth muscle cell line derived from rat aorta

The presence of a specific receptor for 1,25-dihydroxyvitamin D (1,25(OH)2D) was investigated in a cell line A7r5 derived from fetal aorta. 1,25(OH)2[3H]D3 binding to cytosol was saturated at 0.6-1 nM, and Scatchard analysis yielded dissociation constant and binding sites, (3.02 +/- 0.4) X 10(-11) M and 33.9 +/- 5.8 fmol/mg protein, respectively. Sucrose density gradient analysis revealed the sedimentation constant 3.2 S. Furthermore, the receptor protein had affinity for DNA-cellulose column and eluted with 0.2 M KCl. These data suggest that vascular smooth muscle cell may be a target tissue of vitamin D.     

Identification of 1,25-dihydroxyvitamin D3 receptors and activities in muscle

Cytosols from cultured myoblast cells (G-8 and H9c2) prepared in high salt (0.3 M KCl) possesses receptor like proteins for 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) that sediment in the 3.2 S region of sucrose gradients. These receptors were characterized as having high affinity (Kd less than 0.1 nM) for 1,25-(OH)2D3 and are in low capacity (less than 80 fmol/mg of cytosol protein). Analog competition for receptor binding revealed that 1,25-(OH)2D3 was more potent than 24,25-(OH)2D3, or 25-(OH)2D3 for displacement of 1,25-(OH)2[3H]D3 from these 3.2 S region sedimenting receptors. Furthermore, the receptor proteins had affinity for DNA and eluted from Sephacryl S-200 as a macromolecule with Stokes radius (Rs) of 32 A. High salt cytosol from collagenase-dispersed skeletal muscle cells was also found to possess a 3.2 S 1,25-(OH)2D3 receptor-like protein. The 1,25-(OH)2D3 receptor concentration in both G-8 and H9c2 myoblast lines was found to down-regulate by 50-70% when cells were stimulated to differentiate to myotubes by lowering fetal calf serum to 5% of the medium. Moreover, we demonstrated that 1,25-(OH)2D3 can inhibit DNA synthesis and cell proliferation of the G-8 myoblast cells in a dose-dependent manner. 1,25-(OH)2D3 was more potent at inhibiting cell proliferation in cells grown in 5% serum than in 20% serum. The data suggest that 1,25-(OH)2D3 can act directly on muscle myoblast via a 1,25-(OH)2D3 receptor that is similar to those found in intestine and bone. The data support the possibility that muscle is a target tissue for 1,25-(OH)2D3 and the hormone may act to initiate terminal differentiation of myoblast cells.     

Rat cytochrome P450C24 (CYP24A1) and the role of F249 in substrate binding and catalytic activity

A high level of functional recombinant rat cytochrome P450C24 enzyme (CYP24A1) was obtained (40-50mg/L) using an Escherichia coli expression system. Purified enzyme was stable with retention of spectral and catalytic activity. The rate of 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] side-chain oxidation and cleavage to the end-product calcitroic acid was directly related to the rate of electron transfer from the ferredoxin redox partner. It was determined from substrate-induced spectral shifts that the 1 alpha- and 25-hydroxyl groups on vitamin D(3) metabolites and analogs were the major determinants for high-affinity binding to CYP24A1. Lowest K(d) values were obtained for 1 alpha-vitamin D(3) (0.06 microM) and 1,25-dihydroxyvitamin D(3) (0.05 microM) whereas unmodified parental vitamin D(3) and the non-secosteroid 25-hydroxycholesterol had lower affinities with K(d) values of 1.3 and 1.9 microM, respectively. The lowest binding affinity for natural vitamin D metabolites was observed for 24,25-dihydroxyvitamin D(3) [24,25(OH)(2)D(3)] (0.43 microM). Kinetic analyses of the two natural substrates 25-hydroxyvitamin D(3) [25(OH)D(3)] and 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] revealed similar K(m) values (0.35 and 0.38 microM, respectively), however, the turnover number was higher for 25(OH)D(3) compared to 1,25(OH)(2)D(3) (4.2 and 1 min(-1), respectively). Mutagenesis of F249 within the F-helix of CYP24A1 altered substrate binding and metabolism. Most notable, the hydrophobic to polar mutant F249T had a strong impact on lowering substrate-binding affinity and catalysis of the final C(23) oxidation sequence from 24,25,26,27-tetranor-1,23-dihydroxyvitamin D(3) to calcitroic acid. Two other hydrophobic 249 mutants (F249A and F249Y) also lowered substrate binding and expressed metabolic abnormalities that included the C(23)-oxidation defect observed with mutant F249T plus a similar defect involving an earlier pathway action for the C(24) oxidation of 1,24,25-trihydroxyvitamin D(3). Therefore, Phe-249 within the F-helix was demonstrated to have an important role in properly binding and aligning substrate in the CYP24A1 active site for C(23) and C(24) oxidation reactions.     

24R,25-dihydroxyvitamin D3 regulates 1,25-dihydroxyvitamin D3 binding to its chick intestinal receptor

We have studied the binding of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] to its crude chromatin chick intestinal receptor in the absence or presence of a ten-fold excess of 24R,25-dihydroxyvitamin D3 [24R,25(OH)2D3] for each concentration of [3H]-1,25(OH)2D3 studied. We have found a significant shift to the right in the binding of 1,25(OH)2D3 to its receptor in the presence of this excess of 24R,25(OH)2D3. As a result, the affinity was found to be significantly reduced, the apparent dissociation constants varied from 0.97 +/- 0.09 (n = 5) to 1.36 +/- 0.04 nM (p less than 0.01). This reduction was related to a significant decrease in the positive cooperativity for the apparent Hill coefficient from nH = 1.49 +/- 0.06 to nH = 1.26 +/- 0.06 (p less than 0.03) in the binding of 1,25(OH)2D3 to its receptor. There was no significant change in the capacity of the receptor (189 +/- 11 compared to 200 +/- 9 fmoles/mg protein). These results suggest that the intestinal 1,25(OH)2D3 receptor must also have a binding recognition site for 24R,25(OH)2D3 which is postulated to play a regulatory role in the 1,25(OH)2D3 receptor's ligand binding properties.     

A receptor-like binding macromolecule for 1 alpha, 25-dihydroxycholecalciferol in cultured mouse bone cells.

1alpha, 25-Dihydroxycholecalciferol (1,25-(OH)2D3), the active form of vitamin D, like other steroid hormones, initiates its action by binding to cytoplasmic receptors in target cells. Although the 1,25-(OH)2D3 receptor has been well studied in intestine, little information beyond sucrose gradient analyses is presently available from mammalian bone. We, therefore, employed primary cultures of mouse calvarial cells to characterize the mammalian receptor in bone. A hypertonic molybdate-containing buffer was found to protect receptor binding. On hypertonic sucrose gradients, the 1,25-(OH)2-[3H]D3 binder sedimented at 3.2 S. Scatchard analysis of specific 1,25-(OH)2[3H]D3 binding sites at 0 degrees C yielded an apparent Kd of 0.26 nM and an Nmax of 75 fmol/mg of cytosol protein. Competitive binding experiments revealed the receptor to prefer 1,25-(OH)2D3 greater than 25-(OH)-D3 = 1 alpha-(OH)-D3 greater than 24R,25-(OH)2D3; vitamin D3, dihydrotachysterol, sex steroids, and glucocorticoids exhibited negligible binding. As shown in other systems, the receptor could be distinguished from a 25-(OH)-[3H]D3 binder which sedimented at approximately 6 S. In summary, cultured mouse calvarial cells possess a macromolecule with receptor-like properties. This system appears to be an ideal model for the investigation of 1,25-(OH)2D3 receptor binding and action in mammalian bone.     

Clonal differences in expression of 25-hydroxyvitamin D(3)-1alpha-hydroxylase,of 25-hydroxyvitamin D(3)-24-hydroxylase,and of the vitamin D receptor in human colon carcinoma cells: effects of epidermal growth factor and 1alpha,25-dihydroxyvitamin D(3)

Human colon carcinoma cells express 25-hydroxyvitamin D(3)-1alpha-hydroxylase (CYP27B1) and thus produce the vitamin D receptor (VDR) ligand 1alpha,25-dihydroxyvitamin D(3) (1,25-D3), which can be metabolized by 25-hydroxyvitamin D(3)-24-hydroxylase (CYP24). Expression of VDR, CYP27B1, and CYP24 determines the efficacy of the antimitotic action of 1,25-D3 and is distinctly related to the degree of differentiation of cancerous lesions. In the present study we addressed the question of whether the effects of epidermal growth factor (EGF) and of 1,25-D3 on VDR, CYP27B1, and CYP24 gene expression in human colon carcinoma cell lines also depend on the degree of cellular differentiation. We were able to show that slowly dividing, highly differentiated Caco-2/15 cells responded in a dose-dependent manner to both EGF and 1,25-D3 by up-regulation of VDR and CYP27B1 expression, whereas in highly proliferative, less differentiated cell lines, such as Caco-2/AQ and COGA-1A and -1E, negative regulation was observed. CYP24 mRNA was inducible in all clones by 1,25-D3 but not by EGF. From the observed clonal differences in the regulatory effects of EGF and 1,25-D3 on VDR and CYP27B1 gene expression we suggest that VDR-mediated growth inhibition by 1,25-D3 would be efficient only in highly differentiated carcinomas even when under mitogenic stimulation by EGF.