Factors Affecting the Distribution and Stability of Unoccupied 1,25-Dihydroxyvitamin D3 Receptors

Abstract

Recently our laboratory reported that unoccupied 1,25(OH)₂D₃ receptors are found in the nuclei/chromatin fraction under low salt conditions in vitro (J. Biol. Chem. 255, 6799–6805, 1980). Additionally, various conditions exert differential effects on receptor solubilization and stability in vitro, potentially leading to confusion when these effects are not fully appreciated. In order to facilitate future studies, we have compiled a review of these factors and their effects. The conditions discussed include dilution, ionic strength (KCl and molybdate), protease inhibitors (Trasylol, PMSF and TPCK), temperature, ligand, glycerol, dithiothreitol, and EDTA.     

1,25-Dihydroxyvitamin D3 and the immune system

There is substantial evidence that lymphocytes and monocytes are targets for the actions of the hormonal form of vitamin D, 1,25-(OH)2D3 and that 1,25-(OH)2D3 acts to modulate the proliferation, differentiation, and immune functions of these cells. The effects of the hormone on lymphocytes are mediated directly as well as indirectly via the accessory monocytes. Depending upon the presence or absence of monocytes and the mode of lymphocyte activation, 1,25-(OH)2D3 can either stimulate or suppress lymphocytes. This evidence as well as clinical information and in vivo studies support a role of 1,25-(OH)2D3 in immunobiology. The physiologic, pathophysiologic, and pharmacologic implications of the immunomodulating properties of 1,25-(OH)2D3 however have not been well established.     

1,25-Dihydroxyvitamin D3 receptor in bovine thymus gland

1,25-Dihydroxyvitamin D₃ [1,25-(OH)₂D₃] receptor was characterized after partial purification of thymus cytosol by ammonium sulfate fractionation. The 1,25-(OH)₂D₃ receptor sediments at 3.7S in 5–20% sucrose gradients. The binding of 1,25-(OH)₂D₃ in thymic cytosol was a saturable process with high affinity (Kd = 0.12?0.48 nM) at 4°C. Competition for 1,25-(OH)₂[³H]D₃ receptor by nonradioactive analogs demonstrated the affinities of these analogs to be in order; 1,25-(OH)₂D₃ = 1,24R,25-(OH)₃D₃ = 1,25S,26-(OH)₃D₃ = 1,25R,26-(OH)₃D₃ > 1,25-(OH)₂D₃-26,23 lactone > 25-OHD₃ > 23R,25-(OH)₂D₃ > 24R,25-(OH)₂D₃ > 23S,25-(OH)₂D₃ ? 25-OHD₃-26,23 lactone. The receptor bound to DNA cellulose columns in low salt buffer and eluted as a single peak at 0.21 M KCl. These findings provide evidence that the thymus possesses a 1,25-(OH)₂D₃ receptor with properties indistinguishable from 1,25-(OH)₂D₃ receptors in other tissues.     

1,25—二羟基维生素D3的免疫调节机能

1,25-二羟基维生素D_3是人们熟悉的调节钙磷代谢的重要激素,它可抑制淋巴细胞增殖和抗体生成,诱导巨噬细胞的分化及细胞表面主要组织相容性抗原的表达,是一种新的免疫调节激素。     

1,25-Dihydroxyvitamin D3 inhibits antigen-induced T cell activation

The proliferative response of murine spleen and thymus cells to antigen but not to lectin was inhibited by the active metabolite of vitamin D3, 1,25-(OH)2D3. To directly examine the effect of 1,25-(OH)2D3 on T cell activation in the absence of other complicating interactions, we utilized a panel of cloned Ia-restricted T cell hybridomas that secrete IL 2 on activation by cloned Ia-bearing stimulator cells (TA3) or when stimulated by mitogen. Physiologic concentrations of 1,25-(OH)2D3 (0.01 to 0.1 nm) inhibited the antigen-induced secretion of IL 2 by several of these T cell hybridomas. This inhibition was dependent on the concentration of the free hormone and could be overcome by increasing the number of Ia-bearing stimulator cells used. Pretreatment of the T hybridoma but not the TA3 stimulator cell with 1,25-(OH)2D3 resulted in inhibition of activation. These results are consistent with the finding that specific 1,25-(OH)2D3 receptors are present on the T cell hybridomas but are lacking in TA3 cells. 1,25-(OH)2D3 failed, however, to inhibit the activation of the T cell hybridomas by lectin or by an anti-Thy-1 antibody. These findings suggest that 1,25-(OH)2D3 may be interfering with early events of antigen-induced T cell activation, perhaps by hindering T cell recognition of the relevant antigen on stimulator cell surfaces. This system should prove useful in studying the molecular mechanisms by which 1,25-(OH)2D3 acts to inhibit T cell activation and subsequent IL 2 production.     

1,25-Dihydroxyvitamin D3 receptors in rat kidney cytosol

Rat kidney cytosol contains a 3.3 S high affinity binding component for 1,25-dihydroxyvitamin D₃ as detected by DNA-cellulose chromatography and subsequent sucrose gradient analysis. The semipurified aporeceptor demonstrates specificity for 1,25-dihydroxyvitamin D₃ and an apparent dissociation constant for this sterol-hormone of 3.4 × 10^?10M at 25°C. The physicochemical properties of this binding component are in agreement with those observed for the chick intestinal 1,25-dihydroxyvitamin D₃ receptor, suggesting that this component may function as a specific receptor for the hormone in the kidney.     

1,25-Dihydroxyvitamin D3-stimulated mRNAs in rat small intestine

The technique of differential hybridization has been employed to study gene expression associated with vitamin D action on the mammalian intestine. A cDNA library consisting of 10(6) independent recombinants was constructed from poly(A)+ RNA extracted from vitamin D-deficient rats given 1,25-dihydroxyvitamin D3. A survey of 20,000 clones resulted in identification of four distinct cDNAs whose corresponding mRNAs are significantly increased 12 h after an intrajugular dose of 1,25-dihydroxyvitamin D3 given to vitamin D-deficient rats. DNA sequence analysis identified these mRNAs as mitochondrial ATP synthetase, vitamin D-dependent calcium binding protein, cytochrome oxidase subunit I, and cytochrome oxidase subunit III. The time course of response of three of these mRNAs was similar, with maximum values at 12 h after dosing, while that of cytochrome oxidase subunit I showed two peaks at 6 and 18 h following a single dose of 1,25-dihydroxyvitamin D3. The levels of all four mRNAs were elevated in rats supplied with vitamin D when hypocalcemia was produced by dietary calcium restriction.     

1,25-Dihydroxyvitamin D3 regulates proliferation of activated T-lymphocyte subsets

The biologically active vitamin D metabolite, 1,25-(OH) 2D3 suppressed phytohaemagglutinin (PHA)-induced lymphocyte proliferation dose-dependently (0.1 nM-100 nM), and decreased the OKT4+/OKT8+ ratio and transferrin-receptor-positive (OKT9+) cells. A possible parallelism between expression of 1,25-(OH) 2D3 receptors and interleukin 2 (IL2)-receptors recognized by anti-Tac antibody was not confirmed in this study. However, the addition of exogenous IL2 abolished the inhibitory effects of 1,25-(OH) 2D3 on PHA-stimulated T-cell proliferation, and the decrease of OKT4+ and OKT9+ T-cell in this population. Among various vitamin D3 analogues examined, 1,25-(OH) 2D3 was the most potent anti-proliferative effect, followed in order by 1,24S-(OH) 2D3, 1 alpha OH D3, 25 OH D3 and 24,25-(OH) 2D3.     

1,25-Dihydroxyvitamin D3 regulates tubulin expression in chick intestine

As in many other cell types, autoregulation of tubulin synthesis is evident in the intestinal epithelium of normal (vitamin D-replete) chicks: Suppression of protein (tubulin) synthesis by cycloheximide administration in vivo resulted within 30 min in a two-fold increase in RNA hybridizing with an alpha-tubulin probe. Vitamin D status revealed an additional regulatory component. alpha-Tubulin mRNA was elevated in vitamin D-deficient (-D) chicks and those treated with 1,25(OH)2D3 for 1-10 h prior to sacrifice, but declined precipitously 15-20 h after hormone, and in normal birds. These results suggested hormonally increased tubulin levels which in turn suppressed cellular alpha-tubulin mRNA. Analyses of total tubulin levels by [3H]-colchicine binding revealed low levels of the protein(s) in -D chicks, increased levels at 1-15 h after 1,25(OH)2D3, and maximum binding at 20 h after hormone and in normal birds.     

1,25-Dihydroxyvitamin D3 increases citrate secretion from osteosarcoma cells

Rat osteosarcoma cells respond to 1,25-dihydroxyvitamin D3 with a 6- to 10-fold increase in the secretion of citric acid. The time required to attain a half-maximal response is 12 h, a time course which is consistent with the postulated steroidal hormone action of this vitamin D metabolite. The citrate response is achieved by physiological concentrations of 1,25-dihydroxyvitamin D3, with half of the maximal response at a vitamin concentration of 0.03 ng/ml. Both the time course and the dose dependence of the citrate response closely parallel the previously reported stimulation of bone Gla protein synthesis by 1,25-dihydroxyvitamin D3 in these cells. Citrate and bone Gla protein bind avidly to bone mineral and are numerically the most abundant organic acid and protein in bone. The parallel secretion of both in 1,25-dihydroxyvitamin D3-treated osteoblastic cells suggests that they may act in tandem to mediate an action of this vitamin D metabolite on the mineral phase of bone.     

1,25-Dihydroxyvitamin D3 receptor replenishment in the chicken intestine

1,25-Dihydroxyvitamin D3 intestinal receptor replenishment was examined in rachitic chickens after hormone administration. A single injection of 1,25-dihydroxyvitamin D3 caused an increase in the level of occupied receptors with a concomitant decrease in the amount of unoccupied receptors. Maximum occupancy occurred 1 h after hormone injection. The metabolic inhibitor of protein synthesis, cycloheximide, was employed to obtain additional information concerning the fate of 1,25-dihydroxyvitamin D3 receptor complexes. Cycloheximide, at a dose that effectively blocked protein synthesis, had no effect on the time-course or the magnitude of replenishment of nuclear receptors. Additionally, repletion with vitamin D3 or administration of several injections of 1,25-dihydroxyvitamin D3 did not lead to a lag in replenishment time or a significant decrease in total receptor levels. These findings demonstrate that recycling of receptors plays an important functional role for the replenishment of unoccupied 1,25-dihydroxyvitamin D3 intestinal receptors.     

1,25-Dihydroxyvitamin D3: A novel agent for enhancing wound healing

1,25-Dihydroxyvitamin D₃ (1,25(OH)₂D₃), has diverse effects in a variety of tissues and cell types, including skin. Since 1,25(OH)₂D₃ affects both fibroblast and keratinocytes, we evalauated the effect of 1,25(OH)₂D₃ or wound healing. We investigated the effect of the topically applied 1,25(OH)₂D₃ or vehicle on the healing of cutaneous wounds in rats in a blinded manner. Wound areas were measured by planimetry technique. Healing was expressed as the percentage of the original wound area that was healed. 1,25(OH)₂D₃ at concentrations between 5 and 50 ng/day caused a dose-dependent acceleration of healing. Time course and specificity studies indicated that 1,25(OH)₂D₃ specifically promoted healing between 1–5 days after wounding as compared with vitamin D (0.5 μg/day), which showed no significant improvement over control. Our results suggest that 1,25(OH)₂D₃ and its analogues may be a new class of compounds that could be developed to enhance wound healing. © 1995 Wiley-Liss, Inc.     

1,25-Dihydroxyvitamin D3 target cells in rat mammary gland

In autoradiograms of mammary glands of rats on days 18 and 20 of pregnancy and day 6 of lactation after injection of 3H 1,25-(OH)2 vitamin D3, a nuclear concentration of radioactivity is observed in alveolar and ductal cells, as well as in cells of the epidermis of the nipple. Myoepithelial cells and connective tissue cells do not concentrate the hormone in their nuclei. The nuclear radioactivity appears specific, since 1,25-(OH)2 vitamin D3 but not 25-(OH)2 vitamin D3 prevents the uptake. The results suggest the existence of nuclear receptors for 1,25-(OH)2 vitamin D3 in mammary tissues and thus complements previous biochemical evidence showing the presence of cytoplasmic receptors for the hormone in mouse mammary glands; in addition, our results allow the identification of the cell types possessing the receptors.     

1,25-Dihydroxyvitamin D3 and the regulation of human cancer cell replication

Several human and animal cancer cell lines have been shown to possess specific high affinity receptors for 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3). The replication of several of these cell types has also been shown to be regulated by this hormone, both in vitro and in vivo. To further understand the mechanisms of these actions, we have examined cancer cells in vitro and in vivo. The in vitro studies extend our previous reports on the treatment of human breast cancer cells (T 47D) with 10(-9) to 10(-6) M 1,25-(OH)2D3, which resulted in a dose- and time-dependent decrease in cell numbers over 6 days. Treatment with 10(-8) M 1,25-(OH)2D3, which reduced cell numbers to approximately one half of those found in control cultures at 6 days, was associated with a doubling of the proportion of cells in the G2 + M phase of the cell cycle and was accompanied by a significant decline in the proportion of G0/G1 cells. At higher concentrations there was a significant decline in S phase cells with accumulation of cells in both G0/G1 and G2 + M phases. The antiestrogen, tamoxifen, at a concentration which caused similar effects on cell number, resulted in proportional decreases in both S and G2 + M phase cells and accumulation of G0/G1 cells. The effects of 1,25-(OH)2D3 on T 47D cell proliferation were associated with time- and concentration-dependent reductions in epidermal growth factor receptor levels to a minimum level of about half that seen in control cultures. The in vivo experiments extend our previous studies, which demonstrated marked inhibition of the growth of human cancer xenografts in immunosuppressed mice by 1,25-(OH)2D3. Xenograft growth was inhibited with 1,25-(OH)2D3 (0.1 microgram ip three times per week) but growth was rapidly restored when the 1,25-(OH)2D3 was withdrawn. Thus, there are clear-cut time- and dose-dependent, yet reversible, effects of 1,25-(OH)2D3 on the replication of human cancer cells in vitro and in vivo, which are possibly mediated through changes in growth factor receptor levels. Further study of these effects may advance understanding of the hormonal control of cellular replication in human cancers.     

1,25-Dihydroxyvitamin D3 activates secretion of hydrogen peroxide by human monocytes

Human blood monocytes cultured in the presence of 1,25(OH)2D3 developed enhanced competence for secretion of H2O2 relative to cells suspended in media. This effect was maximal at a concentration of 10(-8) M 1,25(OH)2D3. After 3 days of incubation, monocyte-derived macrophages (MDM) exposed to 1,25(OH)2D3 demonstrated competence for secretion of H2O2 equivalent to cells exposed to recombinant IFN-gamma. Both IFN-gamma and 1,25(OH)2D3 offset decay of this function among cells in culture after 7 days. Simultaneous exposure of cells to 1,25(OH)2D3 and IFN-gamma did not activate competence for H2O2 secretion more than either agent alone. 24,25(OH)2D3 and 25(OH)2D3 activated MDM but at higher concentration than required for 1,25(OH)2D3. Progesterone did not affect H2O2 production. Incubation of MDM with a monoclonal antibody directed against IFN-gamma inhibited activation induced by lymphokine, and to a lesser extent by cells activated with IFN-gamma; this antibody had an insignificant effect on cells treated with 1,25(OH)2D3. These results suggest that 1,25(OH)2D3 exerts a receptor-mediated effect on monocyte function that results in cellular activation as manifested by enhanced competence for secretion of H2O2. It is possible that smaller concentrations of 1,25(OH)2D3 present in serum are permissive for macrophage activation, or that monocytic phagocytes are exposed to high concentrations of vitamin D metabolites under some clinical circumstances.     

25-Hydroxyvitamin D3 and 1,25-Dihydroxyvitamin D3 Promote the Differentiation of Human Subcutaneous Preadipocytes

1,25(OH)₂D₃ inhibits adipogenesis in mouse 3T3-L1 adipocytes, but little is known about its effects or local metabolism in human adipose tissue. We showed that vitamin D receptor (VDR) and 1α-hydroxylase (CYP27B1), the enzyme that activates 25(OH)D₃ to 1,25(OH)₂D₃, were expressed in human adipose tissues, primary preadipocytes and newly-differentiated adipocytes. Preadipocytes and newly-differentiated adipocytes were responsive to 1,25(OH)₂D₃, as indicated by a markedly increased expression of CYP24A1, a primary VDR target. 1,25(OH)₂D₃ enhanced adipogenesis as determined by increased expression of adipogenic markers and triglyceride accumulation (50% to 150%). The magnitude of the effect was greater in the absence of thiazolidinediones. 1,25(OH)₂D₃ was equally effective when added after the removal of differentiation cocktail on day 3, but it had no effect when added only during the induction period (day 0–3), suggesting that 1,25(OH)₂D₃ promoted maturation. 25(OH)D₃ also stimulated CYP24A1 expression and adipogenesis, most likely through its conversion to 1,25(OH)₂D₃. Consistent with this possibility, incubation of preadipocytes with 25(OH)D₃ led to 1,25(OH)₂D₃ accumulation in the media. 1,25(OH)₂D₃ also enhanced adipogenesis in primary mouse preadipocytes. We conclude that vitamin D status may regulate human adipose tissue growth and remodeling.