Ketoconazole inhibits self-induced metabolism of 1,25-dihydroxyvitamin D3 and amplifies 1,25-dihydroxyvitamin D3 receptor up-regulation in rat osteosarcoma cells

Ketoconazole (an inhibitor of vitamin D-24 hydroxylase) was used to study the role of self-induced 1,25-dihydroxyvitamin D3 (1,25-D3) metabolism on cellular responsiveness to 1,25-D3. Eighteen hours of treatment with 1,25-dihydroxy-[26,27-methyl-3H]vitamin D3 (1,25-[3H]D3) increased total 1,25-D3 receptors (VDR) from 60 to 170 fmol mg/protein. In cells treated with both 1,25-[3H]D3 and ketoconazole, up-regulation of VDR was increased by 40% over that observed with cells receiving 1,25-[3H]D3 alone. Ketoconazole alone had no agonistic activity. Treatment of cells with 1 nM 1,25-[3H]D3 plus increasing doses of ketoconazole (0-30 microM) resulted in a dose-dependent increase in occupied VDR and total VDR. This up-regulation was associated with reduced 1,25-[3H]D3 catabolism. 1,25-[3H]D3-induced up-regulation of VDR typically peaked at 14 h and declined thereafter. Ketoconazole lengthened the time to reach peak VDR up-regulation to 20 h. The ability of ketoconazole to increase cell responsiveness (VDR up-regulation) was the result of both increased and prolonged occupancy of VDR by 1,25-[3H]D3. The t1/2 of occupied VDR was 2 h in the absence of ketoconazole and greater than 7 h when ketoconazole was present. Collectively, these results suggested that self-induced catabolism of 1,25-D3 is an important regulator of VDR occupancy and therefore cellular responsiveness to hormone. These data also demonstrate the usefulness of ketoconazole as an inhibitor of vitamin D hydroxylases in intact cells.     

Functional involvement of calcium in the homologous up-regulation of the 1,25-dihydroxyvitamin D3 receptor in osteoblast-like cells

In several cell types 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) causes up-regulation of its receptor. The present study demonstrates that in the osteoblast-like cell line UMR 106 this up-regulation is inhibited by two different calcium channel blockers (nitrendipine, verapamil). Also with chelating extracellular calcium (EGTA) and by inhibition of calcium release from intracellular stores (TMB-8) comparable results were obtained. These findings indicate that calcium is functionally involved in this cellular response to the steroid hormone 1,25(OH)₂D₃. Moreover, data obtained with EGTA show that the 1,25(OH)₂D₃ receptor level is closely regulated by the extracellular calcium concentration.     

Vitamin K-dependent gamma-carboxylation of the 1,25-dihydroxyvitamin D3 receptor.

It has been reported that vitamin K deficiency in the rat markedly increases the 1,25-dihydroxyvitamin D3 receptor (VDR) binding to DNA and that vitamin K-dependent gamma-carboxylation of endogenous substrates of the intestinal and renal cytosol, also containing VDR, sharply reduced that binding (Sergeev, I.N., and Spirichev, V.B. (1989) Nutr. Res. 9, 725-733). In the present study we have evaluated vitamin K-dependent 14CO2 incorporation to VDR quantitated by immunoprecipitation with anti-VDR monoclonal antibodies. The results obtained strongly suggest that VDR in vitro can undergo gamma-carboxylation in the presence of vitamin K1 and that 15-25% of Glu residues in the VDR are carboxylated in vivo. Taking into account our earlier findings, it is likely that the VDR gamma-carboxylation modulates its binding to DNA.     

Stimulation of 1,25-dihydroxyvitamin D3 production by 1,25-dihydroxyvitamin D3 in the hypocalcaemic rat.

Serum 1,25-dihydroxyvitamin D3 concentration and renal 25-hydroxyvitamin D 1 alpha-hydroxylase activity were measured in rats fed various levels of calcium, phosphorus and vitamin D3. Both calcium deprivation and phosphorus deprivation greatly increased circulating levels of 1,25-dihydroxyvitamin D3. The circulating level of 1,25-dihydroxyvitamin D3 in rats on a low-calcium diet increased with increasing doses of vitamin D3, whereas it did not change in rats on a low-phosphorus diet given increasing doses of vitamin D3. In concert with these results, the 25-hydroxyvitamin D 1 alpha-hydroxylase activity was markedly increased by vitamin D3 administration to rats on a low-calcium diet, whereas the same treatment of rats on a low-phosphorus diet had no effect and actually suppressed the 1 alpha-hydroxylase in rats fed an adequate-calcium/adequate-phosphorus diet. The administration of 1,25-dihydroxyvitamin D3 to vitamin D-deficient rats on a low-calcium diet also increased the renal 25-hydroxy-vitamin D 1 alpha-hydroxylase activity. These results demonstrate that the regulatory action of 1,25-dihydroxyvitamin D3 on the renal 25-hydroxyvitamin D3 1 alpha-hydroxylase is complex and not simply a suppressant of this system.     

1,25 二羟基维生素D3 对兔角膜碱烧伤朗格罕氏细胞影响

目的:研究1,25二羟基维生素D3(骨化三醇)对兔角膜碱烧伤后角膜朗格罕氏细胞分布的影响,并初步探讨其作用机制。方法:在兔角膜制作碱烧伤模型,然后实验组局部和全身给予1,25二羟基维生素D3,分别在第3,7,21天时对正常组,实验组和对照组家兔行角膜共聚焦显微镜,HE染色观察角膜病理改变。结果:正常组角膜中央在三个时间点均未检测出朗格罕氏细胞。实验组和对照组碱烧伤后3、7天角膜中央出现朗格罕氏细胞,对照组密度高于实验组(p<0.05);碱烧伤后21天两组朗格罕氏细胞密度相近(p>0.05)。实验组炎性反应程度在第7,21天时轻于对照组。结论:1,25二羟基维生素D3能够在兔角膜碱烧伤早期抑制朗格罕氏细胞的向心性迁移,并且能在一定程度上抑制炎性反应。     

C-terminal proteolysis of the avian 1,25-dihydroxyvitamin D3 receptor

Exposure of the 60 kDa chick intestinal 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) receptor to carboxypeptidase A resulted in a time dependent decrease in receptor hormone-binding; after 2 h, there was no detectable macro-molecular-bound 1,25(OH)2[3H]D3. Upon DNA-cellulose chromatography of this preparation, a 56 kDa protein adsorbed to the column and eluted as a function of para-chloromercuribenzene sulfonate (a sulfhydryl blocking reagent). The 56 kDa fragment was detected by anti-receptor monoclonal antibodies via immunoblot technology. The 1,25(OH)2[3H]D3 eluted in the fall through fractions of the column. Thus, cleavage of up to 40 amino acids from the carboxy-terminus of the 1,25(OH)2D3 receptor results in a protein which no longer binds to hormone, but retains its capacity to interact with DNA-cellulose and monoclonal antibody. These results represent novel biochemical evidence that allows us to orient the 1,25(OH)2D3 binding domain near the C-terminus of the receptor.     

Involvement of cAMP and calcium in the induction of ornithine decarboxylase activity in an osteoblast cell line

The role of cAMP and calcium in the induction of ornithine decarboxylase (ODC, E.C.4.1.1.17) activity in the osteogenic sarcoma cell line, UMR 106-01, was studied, with particular interest for parathyroid hormone (PTH). PTH and forskolin dose-dependently induced the ODC activity and the cAMP production. Protein synthesis is involved in the effect of PTH and forskolin on ODC activity but not on cAMP production. Using quin2 we showed that 20 nM PTH and 10 microM forskolin increased the intracellular ionized calcium concentration ([Ca2+]i), thereby offering the possibility for calcium to play a role as cellular mediator in the action of PTH and forskolin in bone. Data obtained with A23187 showed that solely an increase of the [Ca2+]i is not sufficient to stimulate basal or potentiate PTH- and forskolin-induced ODC activity. However, the effects of calcium channel blockers and EGTA on basal and PTH- and forskolin-induced ODC activity point to a specific role for calcium. Moreover, the effects of calcium channel blockers and EGTA on basal and PTH- and forskolin-induced cAMP production indicate that the involvement of calcium in the induction of ODC activity is primarily located at another site than the adenylate cyclase. These data indicate that calcium is involved in the control of basal ODC activity. Furthermore, these data suggest that both cAMP and calcium are involved in the induction of ODC activity by PTH and forskolin. More precisely, ODC activity in UMR 106-01 cells can be induced by PTH and forskolin via a calcium-dependent cAMP messenger system.     

An immunoradiometric assay for 1,25-dihydroxyvitamin D3 receptor

A ligand-independent, quantitative assay has been developed for the measurement of 1,25-dihydroxyvitamin D receptor utilizing purified receptor from pig intestine as a standard and two high affinity monoclonal antibodies directed to two different epitopes on the receptor. In this assay a fixed amount of 125I-labeled antibody is incubated with a fixed amount of a second antireceptor antibody linked to biotin and increasing amounts of purified receptor protein or sample. Antibody-receptor complexes can then be immunoprecipitated with avidin-Sepharose beads and counted. This method is highly reproducible and can detect 150 pg of 1,25-dihydroxyvitamin D3 receptor in crude extracts with intra- and interassay coefficients of variation of 8.6 and 18.2%. The monoclonal antibodies used recognize both native and denatured receptors from several different species, including human. This immunoradiometric assay should prove useful for studies of receptor regulation, occupancy, distribution, and turnover.     

Molybdate and the 1,25-dihydroxyvitamin D3 receptor from chick intestine

The nature of the 1,25-dihydroxyvitamin D3 receptor from chick intestine was examined in regard to its response to sodium molybdate. Sodium molybdate (10 mM) stabilized the receptor from crude nuclear extract but not that from the supernatant or cytoplasmic fraction, suggesting the molybdate may act by binding to the DNA binding region of the receptor. At a concentration of 50 mM, sodium molybdate prevented aggregation of the nuclear receptor. This concentration of sodium molybdate also inhibited the receptor from binding to DNA cellulose while the same ionic strength KCl (90 mM) did not. These properties also suggest that molybdate interacts with the DNA binding region. Purification of the receptor using DNA cellulose chromatography has also been improved by using a sodium molybdate gradient (0-0.2 M) instead of the KCl gradient used previously.     

An estrogen-stimulated 1,25-dihydroxyvitamin D3 receptor in rat uterus

Sucrose density gradient analysis was utilized to determine whether 1,25-dihydroxyvitamin D₃ receptors are present in the rat uterus. A distinct 3.6S [³H]1,25-dihydroxyvitamin D₃ binding component was observed in chromatin extracts of estrogen-primed, ovariectomized rat uteri. Binding to this putative 1,25-dihydroxyvitamin D₃ receptor was inhibited by excess 1,25-dihydroxyvitamin D₃, but not by 25-hydroxyvitamin D₃, estradiol-17β, promegestone, or cortisol. Low levels of the receptor seemed to be present in the unprimed uterus. Estrogen injection significantly increased the number of 1,25-dihydroxyvitamin D₃ receptors and progesterone co-administration reduced, but did not abolish, this effect.     

Tissue distribution of the 1,25-dihydroxyvitamin D3 receptor in the male rat.

Tissue distribution of 1,25-dihydroxyvitamin D3 receptors was studied in male rats using a quantitative immunoradiometric assay. Extracts were prepared from 16 different rat tissues and assayed for 1,25-dihydroxyvitamin D3 receptor. Measurable levels of receptor were detected in intestine, stomach, kidney, bone thyroid/parathyroid, skin, liver, spleen, heart and lung. The highest levels were found in the proximal small intestine and colon, containing over 1000 fmol/mg total protein, while ileum and kidney contained one-half and one-fourth of this amount, respectively. Other parts of the vitamin D endocrine system, including bone, thyroid/parathyroid and skin, contained moderate levels of receptor of 40 to 80 fmol/mg, while lung, heart, stomach, spleen and liver had levels at or below 20 fmol/mg. No 1,25-dihydroxyvitamin D3 receptor was detected in cerebrum, cerebellum or skeletal muscle. The data support a wide-spread role for 1,25-dihydroxyvitamin D3 on cellular processes and suggest a more important role for vitamin D in colon.     

24-homologated 1,25-dihydroxyvitamin D3 compounds: separation of calcium and cell differentiation activities

A series of 24-homologated 1,25-dihydroxyvitamin D3 compounds have been chemically synthesized and studied with regard to their activity in inducing differentiation of human promyelocyte HL-60 cells to monocytes and in calcium mobilizing activity in vitamin D deficient rats. Homologation of 1,25-dihydroxyvitamin D3 or its delta 22 analogue by one or two carbons increases by 10-fold and three-carbon homologation reduces by half the activity in causing differentiation of HL-60. On the other hand, homologation causes a substantial decrease in in vivo calcium mobilization activity. The addition of each carbon at the 24-position decreases binding to the HL-60 receptor or rat intestinal receptor by 5-10-fold so that binding affinity of the trihomo compound for the receptors is 130 times less that of 1,25-dihydroxyvitamin D3. Thus, binding affinity for the receptor cannot account for the preferential activity of the 24-homologated compounds in inducing cell differentiation.     

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.     

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.     

Hormone-dependent phosphorylation of the 1,25-dihydroxyvitamin D3 receptor in mouse fibroblasts

Experimental results, employing several immunologic techniques, suggest that the mouse receptor for 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) undergoes hormone-dependent phosphorylation in intact cells. Treatment of monolayer cultures of mouse 3T6 fibroblasts with 1,25(OH)2D3 reveals that the occupied 1,25(OH)2D3 receptor displays a minor reduction in electrophoretic mobility as compared to its unoccupied 54,500 dalton counterpart, a change consistent with covalent modification. Similar results were obtained by immunoprecipitation of metabolically-labeled receptors after incubation of 3T6 cells with [35S]methionine. This technique also provided greater insight into the precursor-product relationship between the two receptor forms. [32P]Orthophosphate-labeling of 3T6 cells, followed by immunoprecipitation indicated that only the form exhibiting covalent modification was phosphorylated. The temporal correspondence between the binding of 1,25(OH)2D3 to its cellular receptor and its phosphorylation suggests that the biochemical role of 1,25(OH)2D3 may be to induce a conformational change susceptible to phosphorylation and possibly functional activation.     

Heterologous up-regulation of the 1,25-dihydroxyvitamin D3 receptor by parathyroid hormone (PTH) and PTH-like peptide in osteoblast-like cells

1,25-Dihydroxyvitamin D3 (1,25-(OH)2D3) receptor content in cultured osteogenic sarcoma cells (UMR-106) was found to be increased after treatment with both bovine and human PTH and human PTH-like peptide (hPLP). The dose dependent increase of receptors was preceded by a dose dependent stimulation of cAMP production. This suggests a role for cAMP as mediator of the PTH- and hPLP-induced 1,25-(OH)2D3 receptor up-regulation. Furthermore, evidence was obtained that new mRNA and de novo receptor synthesis is involved in this heterologous 1,25-(OH)2D3 receptor up-regulation.     

Effect of 1,25-dihydroxyvitamin D3 on intestinal calcium absorption in strontium-fed rats.

These studies investigated the initial stimulation of intestinal calcium absorption in the rat by 1,25-dihydroxyvitamin D₃. To produce a functional vitamin D₃-deficiency, rats were fed a diet containing 2.4% strontium. After 10 days on the diet, intestinal calcium uptake, as measured by everted gut sacs, was significantly depressed. Strontium-fed rats were dosed orally with 1,25-dihydroxyvitamin D₃, and changes in intestinal calcium uptake, intestinal alkaline phosphatase activity, and intestinal calcium-binding protein were measured as a function of time after dose. Calcium uptake was significantly increased in the proximal 2.5 cm of the duodenum at 4 h and along the whole duodenum by 7 h. Intestinal alkaline phosphatase activity, measured in a Triton extract of the mucosal homogenate and in isolated brush border complexes, was also increased by 7 h. Using both gel electrophoresis and immunodiffusion against a specific antiserum, an increase in intestinal calcium-binding protein was detected in intestinal supernate at 4 h after dosing. Almost no calcium-binding protein was detectable in strontium-fed rats dosed with propylene glycol only. These time studies are consistent with a role for both alkaline phosphatase and calcium-binding protein in the 1,25-dihydroxyvitamin D₃-stimulated uptake of calcium by the intestine. In addition, the usefulness of strontium feeding for producing a functional vitamin D₃ deficiency in rats is demonstrated.     

Immunomodulatory role of 1,25-dihydroxyvitamin D3.

The active vitamin D metabolite 1,25-dihydroxyvitamin D3 [1,25-D3] is thought to promote many of its actions through interaction with a specific intracellular receptor. The discovery of such receptors in monocytes and activated lymphocytes has led investigators to evaluate the role of the hormone on the immune system. The sterol inhibits lymphocyte proliferation and immunoglobulin production in a dose-dependent fashion. At a molecular level, 1,25-D3 inhibits the accumulation of mRNA for IL-2, IFN-gamma, and GM-CSF. At a cellular level, the hormone interferes with T helper cell (Th) function, reducing Th-induction of immunoglobulin production by B cells and inhibiting the passive transfer of cellular immunity by Th-clones in vivo. The sterol promotes suppressor cell activity and inhibits the generation of cytotoxic and NK cells. Class II antigen expression on lymphocytes and monocytes is also affected by the hormone. When given in vivo, 1,25-D3 has been particularly effective in the prevention of autoimmune diseases such as experimental autoimmune encephalomyelitis and murine lupus but its efficacy has been limited by its hypercalcemic effect. Synthetic vitamin D3 analogues showing excellent 1,25-D3-receptor binding but less pronounced hypercalcemic effects in vivo have recently enhanced the immunosuppressive properties of the hormone in autoimmunity and transplantation.