Association of 1,25-dihydroxyvitamin D3 with cultured 3T6 mouse fibroblasts. Cellular uptake and receptor-mediated migration to the nucleus

The uptake of 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) by intact cells was investigated using the cultured embryonic 3T6 mouse fibroblast as a model. Suspended cells, incubated for 60-90 min in serum-containing culture medium supplemented with 1,25-(OH)2D3 (2 nM), maximally accumulate hormone which becomes bound to a typical vitamin D 3.3 S receptor protein. Incubation of cells with varying concentrations of 1,25-(OH)2D3 reveals the presence of 21,000 receptor molecules/3T6 cell, with an apparent uptake constant of 6-8 X 10(-10) M at 37 degrees C. This value contrasts with the equilibrium dissociation constant (Kd) for 1,25-(OH)2D3 binding of 6 X 10(-11) M as determined at 2 degrees C in disrupted cell cytosol. The distribution of unoccupied (R0) receptors is predominantly (greater than 85%) cytosolic in the hormone-deprived state (1,25-(OH)2D3 less than 0.05 nM), whereas exposure to 1,25-(OH)2D3 (2 nM) leads to almost complete nuclear localization of the occupied receptor at both 2 and 37 degrees C. This phenomenon was similarly supported through reconstitution of receptor and purified 3T6 nuclei in vitro in which binding also occurs at 2 degrees C. The majority (65%) of intact cell-formed receptor-nuclear complexes can be solubilized by micrococcal nuclease treatment, suggesting the participation of DNA in the acceptor binding site for the 1,25-(OH)2D3 receptor. Consistent with these data, DNA-binding of receptor also occurred in vitro at 2 degrees C and was a characteristic of both occupied (Rs) and unoccupied receptors. However, elution of the latter occurred at reduced ionic strength, implying that the hormone does physically alter the receptor protein. This binding was also sensitive to prior ethidium bromide saturation of DNA-cellulose, but not phosphocellulose. Although the biologic effects of the 1,25-(OH)2D3 hormone in 3T6 fibroblasts are as yet unknown, the present findings support previous work with 1,25-(OH)2D3 receptors and suggest that this cell represents a good model for the study of nuclear events associated with the molecular action of 1,25-(OH)2D3.     

Properties and compartmentalization of the testicular receptor for 1,25-dihydroxyvitamin D3

Adult rat testis contains a specific, high-affinity, low-capacity binding protein for 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) with properties similar to 1,25-(OH)2D3 receptors in other tissues. The receptor sediments at 3.5 +/- 0.2 S20,w in high-salt sucrose density gradients, but aggregates in low-salt gradients. Binding of 1,25-(OH)2D3 was abolished by trypsin, but not by DNase or RNase. Binding was also heavily reduced by the sulfhydryl alkylating agent, N-ethylmaleimide, and by the mercurial reagent, mersalyl, showing that free, reduced SH-groups are necessary for hormone-binding activity. The receptor shows high affinity for 1,25-(OH)2D3 (Kd = 3 X 10(-11) M), but low capacity (Nmax = 8 fmol/mg protein) and is specific for 1,25-(OH)2D3 (Affinity: 1,25-(OH)2D3 greater than 1,24(R),25-(OH)3D3 greater than 25-OH-D3 greater than 1 alpha-OH-D3 greater than 24(R),25-(OH)2D3 much greater than 17 beta-estradiol, testosterone, dexamethasone, R5020, progesterone). With 0.6 nM [3H]1,25-(OH)2D3 and at 0 degrees C, maximum specific binding was achieved after 4 h, and the occupied receptors were stable for more than 24 h. The dissociation of hormone-receptor complexes was temperature-dependent and very slow at low temperature (t1/2 (0 degrees C) much greater than 48 h). At 0 degrees C, the second order association rate constant and the pseudo-first order dissociation rate constant were 2.7 X 10(7) M-1 min-1 and 2 X 10(-5) min-1, respectively. Receptors for 1,25-(OH)2D3 are present in similar amounts in isolated seminiferous tubules and interstitial tissue of adult rats. No specific binding of [3H]1,25-(OH)2D3 could be detected in cultured immature Sertoli cells, cultured immature peritubular (myoid) cells or crude germ cells.     

Regulation of 1,25(OH)2 vitamin D3 receptor content in cultured LLC-PK1 kidney cells limits hormonal responsiveness

Receptor content in cultured kidney (LLC-PK1) cells was found to be modulated following the introduction of a culture medium change, declining to 40% of control values at 18 h. Scatchard analysis indicated that the reduced 1,25(OH)2-[3H]D3 nuclear binding we detected was due to decreased abundance of receptors (3811 vs 1619 sites/cell) with no change in the Kd (0.4-0.5 nM). Cells with reduced receptors exhibited diminished ability to respond to 1,25(OH)2D3 as measured by induction of 25(OH)vitamin D-24-hydroxylase activity. There was a close coupling between decreased receptor levels and diminished hormone responsiveness. The data suggest the absence of "spare" receptors and that receptor abundance is a limiting factor in cell responsiveness to 1,25(OH)2D3.     

Biochemical significance of enhanced activity of fluorinated 1,25-dihydroxyvitamin D3 in human cultured cell lines

Several human cancer cells possess receptors for 1,25-dihydroxyvitamin D3[1,25-(OH)2D3]. In these cells 1,25-(OH)2D3 has a biphasic concentration-dependent regulatory effect on cell replication and specifically induces its own metabolism. We have studied the effects on these parameters of the native hormone together with those of two analogues fluorinated at the 24-carbon and of 1,24R,25-trihydroxyvitamin D3[1,24R,25-(OH)3D3]. The difluorinated analogue 24,24-difluoro-1,25-(OH)2D3[24,24-F2-1,25-(OH)2D3] is an approximately fivefold more potent inhibitor of cellular replication than the native hormone, while 1,24R,25-(OH)3D3 is about fivefold less potent. This enhanced potency of the fluorinated analogue parallels its enhanced potency in in vivo studies of its effects on calcium and mineral metabolism. However, although the analogue retains replication stimulatory activity, it is clearly no more potent than the native hormone in this activity: 1,24R,25-(OH)3D3 has no significant stimulatory activity. Exposure of the cells to 1,25-(OH)2D3 at 0.05 nM for 6 h increases the subsequent conversion of labelled hormone to aqueous phase soluble compounds by 6.7-fold. None of the other compounds had a similar effect at this concentration. At 10 nM all 1-hydroxylated compounds increased aqueous phase radioactivity about equally (13 to 17-fold); this effect is still specific since 25-OH D3 had no such effect even at 10 nM. Studies on the effects of the fluorinated analogues upon receptor binding of hormone in cell cytosols and uptake of hormone by intact cells clearly demonstrate that the enhanced activity of these analogues is not due to higher receptor affinity or more rapid access to intracellular receptor.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Demonstration of 1 alpha,25-dihydroxyvitamin D3 receptor-like molecule in ST 13 and 3T3 L1 preadipocytes and its inhibitory effects on preadipocyte differentiation

The active metabolite of vitamin D3, 1 alpha,25-dihydroxyvitamin D3 (1,25(OH)2D3), inhibited morphologic and enzymatic expression during differentiation of preadipocyte to adipocyte. In the presence of approximately 6.4-20 X 10(-10) M 1,25(OH)2D3, the triacylglycerol accumulation was only 50% of that of fully differentiated control cells. High-affinity binding sites for 1,25-dihydroxyvitamin D3 were detected in two preadipose cell lines. The 1,25(OH)2D3 binding component sediments at 3.3 S in 4-24% (w/v) sucrose gradients prepared in hypertonic buffer. Binding assay revealed that Nmax was 70 fmol/mg protein and 90 fmol/mg protein, and Kd value was 170 pM and 37 pM in cell lines ST 13 and 3T3 L1, respectively. We also found that differentiated adipocytes did not contain specific receptors for 1,25(OH)2D3. 1,25(OH)2D3, 1(OH)D3, 24,25(OH)2D3, and 24(OH)D3 all suppressed differentiation of preadipocytes to adipocytes, and the dose required closely reflected the affinities of the various metabolites and the synthetic derivative for 1,25(OH)2D3 receptor. It is suggested that the action of vitamin D3 on preadipocyte differentiation may result from a receptor-mediated event.     

Human colon cell line HT-29: characterisation of 1,25-dihydroxyvitamin D3 receptor and induction of differentiation by the hormone

The human colon carcinoma cell line HT-29 differentiates into functional enterocytes upon replacement of glucose by galactose in the culture medium. Since the differentiation of other types of cells is associated with the modulation of 1,25-dihydroxycholecalciferol (1,25(OH)2D3) receptor concentrations and since enterocytes are classical target cells for 1,25(OH)2D3 we have examined the HT-29 cells to determine whether the differentiated and undifferentiated stages could be directly linked to the presence of 1,25(OH)2D3 receptors. HT-29 cells were grown in Dulbecco's modified medium containing 10% fetal calf serum (FCS) and glucose or galactose. Cell differentiation was assessed by measuring the brush border hydrolase, maltase. 1,25(OH)2D3 receptors were studied in the cells after 48 h without FCS. Nuclear uptake was measured in intact dispersed cells and the receptor protein was further characterized by vitamin D metabolite binding specificity, sucrose density gradient analysis and binding to DNA-cellulose. Maltase activity was 5-fold greater in differentiated HT-29 cells than in undifferentiated cells. Scatchard analysis showed a highly specific saturable (9500 sites per cell) high affinity (2 x 10(-10) M), binding of 1,25(OH)2D3 in undifferentiated cells. This receptor-like protein sedimented at 3.3S, bound to and eluted from DNA-cellulose and had all the characteristics of a 1,25(OH)2D3 receptor. No specific binding was detected in differentiated HT-29 cells. The presence of 1,25(OH)2D3 receptors in undifferentiated HT-29 cells implies that these cells are targets for vitamin D. The maltase activity increased significantly when undifferentiated cells were exposed to 1,25(OH)2D3 for 5-6 days, indicating that the hormone can promote differentiation of HT-29 cells. These results demonstrate that HT-29 cells can provide a new model for studying steroid receptor regulation and cell differentiation.     

High performance liquid chromatography analysis of 1,25-dihydroxyvitamin D3 receptor in malignant cells. Correlation of effects on cell proliferation and receptor concentration

Malignant cells were assayed for 1,25(OH)2D3 receptors and for the effects of 1,25(OH)2D3 on cell proliferation. The established lines studied were human promyelocytic leukemia (HL-60), T-cell lymphocytic leukemias (Molt-4, RPMI-8402, CEM), mouse leukemia (L1210), breast cancers (HT-39 and MCF-7) and a glioma (C-6) cultures. A TSK 3000 SW (0.75 X 60 cm) HPLC size exclusion column was used to characterize specific 1,25(OH)2D3 binding. We show for the first time that this column is capable of resolving the 3.2-3.5S 1,25(OH)2D3 mammalian receptor (Rs = 32 A) from the 5.5-6.0S form of the mammalian serum 25(OH)D3 transport receptor (Rs = 40 A). The molecular size of the 1,25(OH)2D3 receptors from these cancer cell lines was identical to that from rabbit intestine. HT-39, HL-60, MCF-7, Molt-4, C-6, RPMI-8402 and L1210 cells demonstrated specific 1,25(OH)2[3H]D3 binding (120, 90, 80, 45, 30 and 18 fmoles of sites/mg protein, respectively). Receptors were not detected in the CEM line. 1,25(OH)2D3 inhibited cell proliferation of HT-39, HL-60, MCF-7 and Molt-4 cells by 20% to 70%. In contrast, mouse leukemia (L1210) cells were stimulated to proliferate by this hormone. Proliferation of RPMI and CEM cells was not affected by 1,25(OH)2D3. We demonstrate that size-exclusion HPLC of 1,25(OH)2D3 binding proteins from mammalian intestine and cancer cells provided a rapid method for identification of specific 1,25(OH)2D3 receptors. Furthermore, in the cells studied, the presence and concentration of 1,25(OH)2D3 receptors qualitatively predicted the potency of this hormone to alter cell proliferation. We believe this assay will be useful for rapid analysis of human tumor receptor concentrations.     

Lipopolysaccharide negatively modulates vitamin D action by down-regulating expression of vitamin D-induced VDR in human monocytic THP-1 cells

Vitamin D3, an important seco-steroid hormone for the regulation of body calcium homeostasis, promotes immature myeloid precursor cells to differentiate into monocytes/macrophages. Vitamin D receptor (VDR) belongs to a nuclear receptor super-family that mediates the genomic actions of vitamin D3 and regulates gene expression by binding with vitamin D response elements in the promoter region of the cognate gene. Thus by regulating gene expression, VDR plays an important role in modulating cellular events such as differentiation, apoptosis, and growth. Here we report lipopolysaccharide (LPS), a bacterial toxin; decreases VDR protein levels and thus inhibits VDR functions in the human blood monocytic cell line, THP-1. The biologically active form of vitamin D3, 1alpha,25-dihydroxy vitamin D3 [1,25(OH)2D3], induced VDR in THP-1 cells after 24 h treatment, and LPS inhibited 1,25(OH)2D3-mediated VDR induction. However, LPS and 1,25(OH)2D3 both increased VDR mRNA levels in THP-1 cells 20 h after treatment, as observed by real time RT-PCR. Moreover, LPS plus 1,25(OH)2D3 action on VDR mRNA level was additive and synergistic. A time course experiment up to 60 h showed an increase in VDR mRNA that was not preceded with an increase in VDR protein levels. Although the proteasome pathway plays an important role in VDR degradation, the proteasome inhibitor lactacystin had no effect on the LPS-mediated down-regulation of 1,25(OH)2D3 induced VDR levels. Reduced VDR levels by LPS were accompanied by decreased 1,25(OH)2D3/VDR function determined by VDR responsive 24-hydroxylase (CYP24) gene expression. The above results suggest that LPS impairs 1,25(OH)2D3/VDR functions, which may negatively affect the ability of 1,25(OH)2D3 to induce myeloid differentiation into monocytes/macrophages.     

Evidence that the self-induced metabolism of 1,25-dihydroxyvitamin D-3 limits the homologous up-regulation of its receptor in rat osteosarcoma cells

The osteoblast-like osteosarcoma cell line UMR-106 has been shown to possess high-affinity receptors for 1,25-dihydroxyvitamin D (1,25-(OH)2D3). Also, these cells metabolize 1,25-(OH)2D3 to more polar metabolites. As previously demonstrated (Pols, H.A.P., et al. (1987) Biochim. Biophys. Acta 931, 115-119) the time course of specific binding of 1,25-(OH)2D3 in intact UMR-106 cells was found to be characterised by (a) an ascending phase, representing association with receptor, (b) a maximum at 90-120 min and (c) a rapid descending phase, closely associated with a decrease of medium 1,25-(OH)2D3 due to the metabolism of the hormone. The purpose of the present study was to investigate further the self-induced metabolism of 1,25-(OH)2D3 in relation to the homologous up-regulation of its receptor in these cells. Inhibition of metabolism of 1,25-(OH)2D3 with ketoconazole resulted, after a lag-time of about 90 min, in a sharp increase of receptor accumulation. This increase in receptor level in the presence of ketoconazole was blocked by coincubation with cycloheximide and actinomycin D. Preincubation experiments with unlabeled 1,25-(OH)2D3 showed that the elevation of hormone binding was 1,25-(OH)2D3-concentration dependent (ED50 200-300 pM). Addition of ketoconazole during these preincubations resulted in an even more pronounced accumulation of receptors, whereby the ED50 (50-60 pM) was comparable with the dissociation constant of the 1,25-(OH)2D3 receptor (41.3 +/- 4.3 pM). In summary, these data support the concept that the self-induced metabolism of 1,25-(OH)2D3 has a dual effect: (1) directly, by the regulation of the cellular concentration of and, consequently, receptor occupancy by the active form of vitamin D and (2) indirectly by its ability to modulate the ligand-dependent regulation of the 1,25-(OH)2D3.     

Differential effects of protease inhibitors on 1,25-dihydroxyvitamin D3 receptors

We describe herein two different effects of protease inhibitors and substrates on receptors for 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) obtained from the intestinal mucosa of vitamin D-deficient chicks: inhibition of binding of 1,25(OH)2D3 to its receptor and stabilization of the receptor. Both L-1-tosylamido-2-phenylethyl chloromethyl ketone (TPCK), a chymotrypsin inhibitor, and N alpha-p-tosyl-L-lysine chloromethyl ketone (TLCK), a trypsin inhibitor, block [3H]1,25(OH)2D3 binding to the receptor. Fifty per cent inhibition of binding occurs at 20 microM TPCK, and 100% inhibition at 100-200 microM; TLCK is about 25-fold less effective. At higher concentrations (10-100 mM), the chymotrypsin substrates N alpha-p-tosyl-L-arginine methyl ester and tryptophan methyl ester and the cathepsin B inhibitor leupeptin also inhibit [3H] 1,25(OH)2D3 binding to its receptor. Different inhibitors and substrates interact with the receptor differently: TPCK (20 microM) and N alpha-p-tosyl-L-arginine methyl ester (10 mM) are reversible, noncompetitive inhibitors, L-tryptophan methyl ester (20 mM) is a reversible competitive inhibitor, and phenylmethylsulfonyl fluoride (300 microM) shows no effect on [3H]1,25(OH)2D3 binding to its receptor. The most stable form of unoccupied 1,25(OH)2D3 receptors from chick intestinal mucosa was that obtained from a low salt chromatin preparation (t 1/2 = 6.0 h). The presence of KCl drastically decreased receptor stability (t 1/2 = 1.8 h); and the addition of 2.5 mM CaCl2 further reduced their stability. Phenylmethylsulfonyl fluoride and Trasylol inhibited the KCl-induced receptor instability, but did not prevent the additional instability in the presence of CaCl2. In summary, TPCK and TLCK exert direct effects on the 1,25(OH)2D3 receptor molecule, independent of their protease inhibitor function. These compounds may prove useful as covalent affinity labels for the receptor. On the other hand, phenylmethylsulfonyl fluoride and Trasylol stabilize 1,25(OH)2D3 receptors, probably via inhibition of KCl-activated nuclear protease(s). This receptor stabilization will be advantageous in receptor assays and/or purification procedures.     

Immunological identification of 1,25-dihydroxyvitamin D3 receptors in human promyelocytic leukemic cells (HL-60) during homologous regulation

Immunological techniques were utilized to detect 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) receptor levels and to characterize physical/chemical changes in receptors in human promyelocytic leukemic cells (HL-60) during continuous exposure to hormone. The monoclonal antibody (IVG8C11) raised against the porcine intestinal 1,25-(OH)2D3 receptor immunoprecipitated quantitatively 1,25-(OH)2D3 receptors in nuclear extracts from HL-60 cells. The highly enriched immunoprecipitated receptors were electrophoresed on sodium dodecyl sulfate-polyacrylamide gels and transferred to polyvinylidene difluoride membranes, which were probed with 125I-labeled IVG8C11. The basal receptor from the cells treated with 1,25-(OH)2D3 for 2 h was detected as a single form at 53 kDa. Moreover, receptors were shown to be up-regulated at 12 h and down-regulated at 48 and 72 h in the continuous presence of hormone as evidenced by the ratio of density of the bands, 1.0 (2 h):4.2 (12 h):1.2 (48 h):0.9 (72 h), as measured by laser scanning densitometry. The up- and down-regulated receptors were also detected as single forms and had the same molecular mass as the basal receptor. Therefore, the data presented here strongly support the hypothesis of homologous regulation of 1,25-(OH)2D3 receptors in intact human target cells.     

1,25-Dihydroxyvitamin D3 modulates bone marrow macrophage precursor proliferation and differentiation. Up-regulation of the mannose receptor

1,25-Dihydroxyvitamin D3 (1,25-(OH)2D3), the biologically active form of vitamin D3, has been shown to inhibit proliferation and promote monocytic differentiation of leukemic cell lines. In the present communication, we extend these observations to normal bone marrow macrophage precursors, and 1) identify the stage of monocytic maturation wherein the steroid exerts its antiproliferative effect, and 2) demonstrate that 1,25-(OH)2D3 promotes bone marrow macrophage differentiation as manifest by specific up-regulation of the lineage-specific membrane protein, the mannose-fucose receptor. In these experiments, the 1,25-(OH)2D3-mediated inhibitory effect on colony formation was shown to be independent of attendant levels of colony stimulating factor-1 and targeted through the adherent bone marrow macrophage precursor. Examination of this steroid-sensitive adherent precursor population demonstrates that its specific binding of 125I-mannose bovine serum albumin spontaneously and progressively increases with time in culture. Whereas adherent bone marrow macrophages cultured for 2 days express 3 X 10(4) mannose receptors/cell, the number of binding sites increases to 7 X 10(4)/cell by day 4. When bone marrow macrophage precursors are exposed to 1,25-(OH)2D3, an additional stepwise enhancement of 125I-mannose bovine serum albumin obtains with time. Four days of culture with the steroid results in 1.6 X 10(5) mannose receptors/cell, a 100% increase as compared to control cells. Neither duration of culture nor exposure to 1,25-(OH)2D3 alters the KD of 125I-mannose bovine serum albumin which approximates 3-5 X 10(-9) ml-1. Finally, the "specificity" of vitamin D-mediated up-regulation of the mannose receptor was established by demonstrating that the steroid does not alter binding of 125I-alpha-thrombin by bone marrow-derived macrophage precursors.     

1 alpha,25-Dihydroxyvitamin D3-binding macromolecules in human B lymphocytes: effects on immunoglobulin production

Previous studies have indicated that upon in vitro activation with mitogenic lectins, human peripheral blood T lymphocytes express receptors for the steroid hormone 1 alpha, 25-dihydroxyvitamin D3(1,25(OH)2D3). Furthermore, the hormone can inhibit interleukin 2 production by the activated cells. In this investigation, we report that human peripheral B lymphocytes activated in vitro with the B lymphotropic Epstein-Barr virus (EBV) also express 1,25(OH)2D3 receptor-like macromolecules. These receptors are localized in the cell nucleus and exhibit properties similar to those found in classical target tissues for 1,25(OH)2D3. They sediment on sucrose gradients at 3.3 S, display a dissociation constant (Kd) of 4 X 10(-10) M, and can bind to DNA. In addition to the 1,25(OH)2D3 receptors, however, EBV-activated lymphocytes express a second class of 1,25(OH)2D3-binding proteins that appear to occur mainly in the cell cytosol and exhibit distinct biochemical properties from the receptor, including higher sedimentation coefficients (3.7 S to 4 S) and the lack of ability to bind to DNA. The addition of 1,25(OH)2D3 to cultures of EBV-infected cells inhibited the production of IgM and IgG by the B cells. The vitamin D3 analog 24,25(OH)2D3 did not inhibit Ig production, thus suggesting that the effect is probably mediated through the high affinity receptor macromolecule localized in the nucleus. Because the EBV-induced Ig production is independent of T cell participation, the data also suggest that the effects of 1,25(OH)2D3 are exerted directly on the B cell. The present results add to the evidence of the importance of 1,25(OH)2D3 as an immunoregulatory hormone.     

Monoclonal antibodies to chick intestinal receptors for 1,25-dihydroxyvitamin D3. Interaction and effects of binding on receptor function

Monoclonal antibodies, developed against the chick intestinal receptor for 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), were characterized with respect to their interaction with this protein and for their effects on the polypeptide's hormone-binding and nuclear-binding functions. Antibodies, internally labeled with [35S]methionine, react directly with hormone-labeled receptor, as identified by comigration of both isotopes during sedimentation on hypertonic 10-30% sucrose gradients. Antibodies bound both the unoccupied and occupied forms of the receptor, the latter with equilibrium dissociation constants of 10(-10)-10(-11) M at 4 degrees C. Excess antibody, added to unoccupied receptors prior to incubation with 1,25(OH)2D3, did not affect the receptor's apparent affinity for the hormone (Kd approximately equal to 6 X 10(-11) M). In contrast, all three antibodies, complexed with occupied receptors, significantly reduced the extent of the receptor's association with isolated nuclei (48-64% inhibition). This inhibition most likely represents a general reduction in the affinity of the protein for nuclei under the conditions tested, since the affinity of the occupied 1,25(OH)2D3 receptor for DNA, as well as the ionic strength necessary to elute receptor from both cation and anion exchange resins was significantly reduced by prior incubation with excess antibody. These findings suggest that the epitopes for each of the three monoclonal antibodies may be located in or near the DNA or nuclear binding domain of the 1,25(OH)2D3 receptor. Taken cumulatively, these results indicate that the monoclonal immunoreagents utilized here should prove useful in delineating important biochemical features of this unique sterol hormone receptor.     

Interaction between 1,25-dihydroxyvitamin D3 receptors and intestinal nuclei. Binding to nuclear constituents in vitro

The molecular action of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) is thought to involve its localization within the nucleus of target cells, a process mediated by intracellular receptors. This report probes both the association between chick intestinal 1,25(OH)2D3 receptors and purified homologous nuclei and the interaction between this receptor and nucleic acids. 1,25(OH)2D3 receptors bound to purified nuclei in a apparently saturable manner (Kd = 2.2-4.8 X 10(-10) M) under conditions of intermediate ionic strength and constant protein concentration. Nuclear binding was hormone-dependent; whereas receptor-hormone complex (Rs) binds to nuclei under the ionic conditions employed here (greater than 70%), hormone-free (R0) receptors do not bind (less than 10%). Binding was localized to the nuclear chromatin fraction and was extremely sensitive to KCl concentration both in the incubation medium and during postincubation treatment of nuclei. The interaction appeared to be temperature-independent, suggesting the lack of a classic activation event characteristic of most steroid receptors. Partial digestion of intestinal nuclei with DNase I eliminated subsequent receptor binding by greater than 95%, pointing to the involvement of DNA in the binding interaction. In turn, receptors were found to bind to both DNA and RNA, a characteristic independent of receptor aggregation, but sensitive to disruption with increasing ionic strength buffers. Elution of both Rs and R0 from DNA appeared identical (0.28 M KCl), whereas the strength of interaction with RNA was much less (0.12 M KCl). Thus, while there appeared to be a fundamental difference between R0 and Rs, such that only the binding of receptor-hormone complex to nuclei was allowed under the conditions employed here, this characteristic was not observed during DNA binding. Nevertheless, the possibility exists that the in vivo interaction between 1,25(OH)2D3 receptor and nuclei involves DNA and that this nuclear constituent may be the ultimate site of action of this unique sterol hormone.     

DNA bending is induced by binding of vitamin D receptor-retinoid X receptor heterodimers to vitamin D response elements.

The ability of vitamin D receptor-retinoid X receptor (VDR-RXR) heterodimers to induce a DNA bend upon binding to various vitamin D response elements (VDRE) has been investigated by circular permutation and phasing analysis. Recombinant rat VDR expressed in the baculovirus system and purified recombinant human RXR beta have been used. The VDREs were from 1,25-dihydroxyvitamin D3 (1,25-[OH]2D3) enhanced genes (rat osteocalcin, rOC; mouse osteopontin, mOP, and rat 1,25-dihydroxyvitamin D3-24-hydroxylase, r24-OHase), and a 1,25-(OH)2D3 repressed gene (human parathyroid hormone, hPTH). As shown by circular permutation analysis, VDR-RXR induced a distortion in DNA fragments containing various VDREs. Calculated distortion angles were similar in magnitude (57 degrees, 56 degrees, 61 degrees, and 59 degrees, respectively for rOC, mOP, r24-Ohase, and hPTH). The distortions took place with or without a 1,25-(OH)2D3 ligand. The centers of the apparent bend were found in the vicinity of the midpoint of all VDREs, except for rOC VDRE which was found 4 bp upstream. Phasing analysis was performed with DNA fragments containing mOP VDRE and revealed that VDR-RXR heterodimers induced a directed bend of 26 degrees, not influenced by the presence of hormone. In this study we report that similar to other members of the steroid and thyroid nuclear receptor superfamily, VDR-RXR heterodimers induce DNA bending.     

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.