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H F DeLuca

Publications and source records attributed to H F DeLuca.

At least 109 records · Page 6Linked to original sources
PubMed1994

cDNA cloning and characterization of a vitamin D3 hydroxylase-associated protein.

We previously reported the generation of monoclonal antibodies which immunoprecipitate a fraction of the total chick kidney 1,25-dihydroxyvitamin D3-24R-hydroxylase activity. These antibodies were used to screen a chick kidney lambda gt11 cDNA library resulting in the isolation of a full-length cDNA encoding a protein that is not the 1,25-dihydroxyvitamin D3-24R-hydroxylase but another protein we term the vitamin D3 hydroxylase-associated protein (VDHAP). The deduced amino acid sequence agreed with an NH2-terminal amino acid sequence from the isolated VDHAP. Gene and protein bank search did not identify homology to known sequences or functional domains in the VDHAP cDNA. VDHAP mRNA levels are not altered by conditions which either induce 1,25-dihydroxyvitamin D3-24R-hydroxylase activity (78-fold) or 25-hydroxyvitamin D3-1 alpha-hydroxylase activity (30-fold). Northern analysis of poly(A)+ RNA from chick tissues revealed VDHAP only in kidney. Cellular fractionation experiments demonstrated that VDHAP and the 25-hydroxyvitamin D3-1 alpha-hydroxylase are colocalized in the inner membrane of mitochondria. The VDHAP antibody immunoprecipitates 14% of the total 1,25-dihydroxyvitamin D3-24R-hydroxylase activity (7-fold over background) and immunoprecipitates 21% of the total 25-hydroxyvitamin D3-1 alpha-hydroxylase activity (2-fold over background). VDHAP is a novel chick kidney-specific inner membrane protein of mitochondria, which associates with a fraction of the 1,25-dihydroxyvitamin D3-24R-hydroxylase and 25-hydroxyvitamin D3-1 alpha-hydroxylase.

Amidohydrolases↗
PubMed1994

Effects of increasing doses of 1 alpha-hydroxyvitamin D2 on calcium homeostasis in postmenopausal osteopenic women.

This study is the first reported administration of 1 alpha-hydroxyvitamin D2 (1 alpha-OHD2) to human subjects. A total of 15 postmenopausal osteopenic women were given increasing oral doses of 1 alpha-OHD2, beginning with a low dose of 0.5 microgram/day. In 15 subjects, the doses were raised at weekly intervals to 1.0, 2.0, 4.0, and 5.0 micrograms/day, and in 5 of these subjects, the dose was further increased to 8.0 or 10.0 micrograms/day. Mean urine calcium +/- SEM showed a dose-related increase from 134 +/- 17 mg/24 h on 0.5 microgram/day to 198 +/- 21 mg/24 h on 4.0 micrograms/day (p < 0.05) and to 241 +/- 35 mg/24 h on 5.0 micrograms/day (p < 0.05). No subjects had hypercalciuria (> 350 mg/24 h, the upper limit of the laboratory normal range) at doses less than 5.0 micrograms/day; 5 subjects had hypercalciuria at or above 5.0 micrograms/day (3 at 5.0 micrograms/day, 1 at 8.0 micrograms/day, and 1 at 10.0 micrograms/day). Mean serum calcium increased slightly on the 4.0 micrograms dose only (p < 0.05) but remained well within the normal range. Mean creatinine clearance and BUN, used as measures of renal function, showed no significant changes. Routine blood and urine assays also showed no significant changes.(ABSTRACT TRUNCATED AT 250 WORDS)

Administration, Oral↗
PubMed1994

Calcium uptake by chorioallantoic membrane: effects of vitamins D and K.

The chorioallantoic membrane (CAM) of birds is an epithelial tissue that actively transports large amounts of Ca during embryonic development. In this study the effect of vitamins D and K on Ca uptake by the CAM was studied. Four dietary treatments were used to produce eggs that are the following: deficient in vitamins D and K (-D/-K), sufficient in both (+D/+K), or deficient in one and sufficient in the other (-D/+K or +D/-K). Vitamin D-deficient (-D) Japanese quail embryos (from hens fed 1,25-dihydroxyvitamin D3) do not hatch because of severe Ca deficiency resulting from their inability to obtain Ca from shell, whereas vitamin K deficiency results in only 14% reduction in hatchability. The results demonstrate that Ca uptake by CAM is vitamin D dependent and only slightly vitamin K dependent. Ca-binding activity of CAM extracts was unchanged by vitamin K deficiency, and only a small increase was provided by vitamin D treatment. Vitamin D stimulated both Ca entry and exist from the chorion cells as indicated by the increased accumulated 45Ca in +D embryos. We conclude that vitamin D is essential for the utilization of eggshell Ca by the developing embryo and hence its survival, suggesting that Ca transport across the CAM is largely a vitamin D-dependent process.

Allantois↗
PubMed1994

Impaired homologous upregulation of vitamin D receptor in rats with chronic renal failure.

We studied the homologous regulation of the vitamin D receptor (VDR) in the duodenum of rats with chronic renal failure. Mean basal nuclear 3H-labeled 1 alpha,25-dihydroxyvitamin D3 ([3H]1,25(OH)2D3) binding capacity was 48 and 43 fmol/mg protein for sham-operated and uremic rats with similar dissociation constants (Kd), respectively. These results coincided with those of immunoblot analysis, which found that VDR protein level of uremic rats was 87.6% that of sham-operated rats. In uremic rats, 1,25(OH)2D3, 2.0 micrograms/kg, failed to upregulate VDR protein levels until 24 h, in contrast to the significant increases produced in sham-operated rats at both 12 (1.55-fold) and 24 h (1.75-fold). Baseline level of VDR mRNA in uremic rats, determined by Northern blot analysis, was comparable to that in sham-operated rats. Treatment with 1,25(OH)2D3 slightly decreased VDR mRNA at 6-24 h in the sham-operated rats, in contrast to the increase seen at 6 h in uremic rats. These results suggest that the homologous upregulation of VDR was attenuated in rats with chronic renal failure because of an impairment at a translational and/or posttranslational step.

Analysis of Variance↗
PubMed1994

A single up-stream element confers responsiveness to 1,25-dihydroxyvitamin D3 and tumor necrosis factor-alpha in the rat osteocalcin gene.

Tumor necrosis factor-alpha (TNF alpha) is one of several autocrine/paracrine factors known to exert potent inhibitory effects on bone. We have shown that TNF alpha inhibition of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]-stimulated synthesis of the bone-specific protein osteocalcin (OC) occurs by decreasing steady state levels of OC mRNA, suggesting a pretranslational mechanism. In many genes, TNF alpha action is mediated by the transcription factor NF kappa B. Analysis of OC 5'-flanking DNA revealed a sequence structurally homologous to the previously described NF kappa B-binding site and, thus, a potential TNF alpha response element. Deletion analysis was performed to identify the sequences mediating the response to TNF alpha in osteoblastic ROS 17/2.8 cells by transient transfection with reporter constructs containing rat OC 5'-flanking DNA [chloramphenicol acetyltransferase (CAT)] that retained or deleted homologous NF kappa B sites or a previously defined 1,25-(OH)2D3 response element (VDRE). Transfection with all reporter constructs resulted in low basal CAT activity, measured 72 h after transfection. 1,25-(OH)2D3 stimulated CAT activity 2.8- to 4.5-fold in cells transfected with constructs that included the VDRE. TNF alpha inhibited 1,25-(OH)2D3-stimulated, but not basal, CAT activity. Deletion analysis localized the effect of TNF alpha to a sequence between -522 and -306 relative to the OC transcription start site, an area that included the VDRE but deleted a homologous NF kappa B element. Transfection of cells with a heterologous reporter containing one copy of the OC VDRE inserted in correct orientation or two copies in inverse orientation was sufficient to confer a response to TNF alpha. Gel mobility shift analysis of DNA-nuclear protein interaction revealed that 1,25-(OH)2D3 stimulated an increase in binding of nuclear proteins to an OC 32P-VDRE probe. Preincubation of nuclear extract with specific monoclonal antibodies confirmed that the proteins binding the VDRE included the vitamin D receptor and retinoid-X receptor. TNF alpha treatment of cells inhibited the 1,25-(OH)2D3-stimulated increase in nuclear protein binding to the VDRE. These results suggest 1) the VDRE is sufficient to confer a response to the inhibitory effect of TNF alpha on 1,25-(OH)2D3-stimulated rat OC gene transcription; 2) the action of TNF alpha does not require homologous NF kappa B response elements; and 3) the mechanism of TNF alpha inhibition of 1,25-(OH)2D3-stimulated OC gene expression includes modulation of binding of the vitamin D receptor/retinoid-X receptor heterodimer to the VDRE.

Animals↗
PubMed1994

Separation of two forms of chick 1,25-dihydroxyvitamin D3 and 25-hydroxyvitamin D3 24-hydroxylase.

We previously reported the development of monoclonal antibodies against chick kidney mitochondrial 1,25-dihydroxyvitamin D3 24-hydroxylase. However, these monoclonal antibodies were able to immunoprecipitate only approximately 25% of total 24-hydroxylase activity present in solubilized preparations, suggesting the existence of different forms of the enzyme. Chromatography of the solubilized preparations on a mono-Q column resolved the 24-hydroxylase activity into two peaks. The protein found in the first peak of enzyme activity (peak 1) did not immunoblot with the monoclonal antibody (IVC2F10) that was raised against the partially purified 24-hydroxylase. However, the protein in the second peak (peak 2) reacted on immunoblot with the same antibody. The enzyme for both peaks had similar kinetics, required adrenodoxin and adrenodoxin reductase, and acted on both 1,25-dihydroxyvitamin D3 and 25-hydroxyvitamin D3. It is not yet clear whether these are two distinct enzymes or whether it is the same enzyme complexed with different proteins.

Animals↗
PubMed1994

Differentiation of neoplastic cells toward normal induced by vitamin D3 derivatives.

Two new analogs of 1,25 dihydroxy vitamin D3 (calcitriol, DHCC), the active metabolite of vitamin D3 (cholecalciferol (D3) are less active on calcium metabolism and less toxic than (DHCC). They had increased differentiation-inducing activity towards normal on human myeloblastic leukemia cell line HL-60 and on histiocytic lymphoma cell line U-937 consisting of monoblastoid cells. 1,2 tetradeconyl phorbol 1, 3 acetate (TPA) was used as a positive control in these experiments.

Calcitriol↗
PubMed1993

1 alpha, 25-Dihydroxyvitamin D3 and 19-nor-1 alpha, 25-dihydroxyvitamin D2 suppress immunoglobulin production and thymic lymphocyte proliferation in vivo.

BALB/CANNHSD female mice were intraperitoneally (i.p.) injected with 1,25-dihydroxyvitamin D-3 (1,25-(OH)2D3) (20 ng/day) or 19-nor-1,25-(OH)2D2 (100 ng/day) for 7 days and then given the same dose every other day thereafter until day 17 for measuring cell-mediated immunity and on day 33 for observing immunoglobulin production. To observe Ig production, the mice were immunized with an injection of hemocyanin (150 microgram/0.1 ml, i.p.) on day 8. On the day 7, 15 and 20 after immunization, the IgG1, and on day 17 and 20, the IgG3 levels in 1,25-(OH)2D3 and 19-nor-1,25-(OH)2D2 groups were significantly lower than in the control group. To measure cell-mediated immunity, mice were sensitized with dinitrofluorobenzene (DNFB) on the shaved abdominal skin. The number of thymic lymphocytes and their stimulation index were significantly reduced by 1,25-(OH)2D3 and 19-nor-1,25-(OH)2D2. These results demonstrate that 1,25-(OH)2D3 and 19-nor-1,25-(OH)2D2 can suppress immunoglobulin production and thymic lymphocyte proliferation in vivo. 19-Nor-1,25-(OH)2D2 at doses of 100 ng/day was less effective than 1,25-(OH)2D3 at doses of 20 ng/day.

25-Hydroxyvitamin D 2↗
PubMed1993

Vitamin D-influenced gene expression via a ligand-independent, receptor-DNA complex intermediate.

A lingering question regarding the regulation of target gene expression by 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] has been the delineation of vitamin D receptor (VDR)-DNA binding and transactivation. This report confirms that initial VDR-DNA interaction occurs in a ligand-independent fashion. An electrophoretic mobility-shift analysis demonstrated that VDR, derived from extracts of the small intestines of vitamin D-deficient rats, is capable of binding a vitamin D response element (DRE). Additional mobility-shift studies using either porcine-derived VDR or recombinant rat VDR from insect cells revealed DRE-binding capability in the absence of 1,25-(OH)2D3. The reactions were performed in various salt environments, with the maximum of porcine VDR-DRE and rat VDR-DRE binding detected at 100 mM and 150 mM KCl, respectively. The addition of 1,25-(OH)2D3 to an identical set of reaction mixtures resulted in increased DRE binding with greater affinities exhibited by both VDR types. These two phenomena were confirmed upon examination of an elution profile of VDR bound to DRE-linked Sepharose. When a linear KCl gradient was used for elution without the addition of 1,25-(OH)2D3, the peak of VDR was 205 mM KCl; the presence of exogenous hormone shifted the maximum VDR elution to a position corresponding to 265 mM KCl. Based on these data and previous reports on VDR-mediated transactivation, we propose a model for 1,25-(OH)2D3-influenced target gene expression.

Animals↗
PubMed1993

Vitamin D deficiency suppresses cell-mediated immunity in vivo.

Severe vitamin D deficiency has been produced in mice as evidenced by severe hypocalcemia and an absence of 25-hydroxyvitamin D in blood. Vitamin D deficiency was accompanied by a slight decrease in body weight and food consumption. Vitamin D-deficient and vitamin D-sufficient mice were sensitized with dinitrofluorobenzene (DNFB). Sensitivity to DNFB was determined by treatment of one ear with DNFB. The ratio of thickness of the treated ear to that of nontreated ear was used as an index of cell-mediated immune reaction. The incorporation of [3H]thymidine into the DNA of the ear was also used as an index of cell-mediated immunity as was the response of thymus lymphocytes to concanavalin A. Vitamin D deficiency markedly decreased the ear thickness ratio and the [3H]thymidine incorporation ratio in DNFB-sensitized mice. Similarly, the incorporation of [3H]thymidine into the DNA of concanavalin A-treated thymus lymphocytes from DNFB-sensitive mice was significantly reduced in vitamin D deficiency. These results show that in vivo vitamin D deficiency impairs cell-mediated immunity. The provision of a vitamin D-sufficient diet for 8 weeks corrected the impaired response of the immune system, while vitamin D administration for 3 weeks did not.

Animals↗
PubMed1993

Isolation and expression of human 1,25-dihydroxyvitamin D3 24-hydroxylase cDNA.

Human 1,25-dihydroxyvitamin D3 24-hydroxylase cDNA clones were isolated from an HL-60 cell cDNA library by using a reverse transcription/polymerase chain reaction-generated human cDNA probe. The 24-hydroxylase cDNA consists of a 1539-bp open reading frame encoding a 513-amino acid polypeptide. Protein sequence analysis shows that the human 24-hydroxylase is 90% homologous (82% identical) to that of the rat, with 100% homology in the 21-amino acid heme-binding region. Northern blot analysis showed that the 24-hydroxylase cDNA probe hybridized to a 3.4-kb mRNA species. Treatment of HL-60 cells with 0.1 microM 1,25-dihydroxyvitamin D3 for 24 hr produced a 30-fold increase in the 24-hydroxylase mRNA level. This result is consistent with previous studies in the same cell line, in which 24-hydroxylase activity was elevated to a maximum in 24 hr by a similar treatment with 1,25-dihydroxyvitamin D3. To verify the identity of these isolated cDNA clones, two polymerase chain reaction-amplified human 24-hydroxylase cDNA fragments containing the entire coding region were used to produce 24-hydroxylase enzyme activity in two genetic expression systems. Transient levels of 24-hydroxylase activity were measured in transfected mammalian COS-1 cells and in recombinant baculovirus-infected Spodoptera frugiperda (Sf21) insect cells.

Amino Acid Sequence↗
PubMed1993

The necessity for calcium for increased renal vitamin D receptor in response to 1,25-dihydroxyvitamin D.

To further investigate the regulation of the vitamin D receptor in the kidney of the rat, we analyzed the response of the receptor to 1,25-dihydroxyvitamin D-3 under conditions of calcium supplementation and calcium restriction. Vitamin D-deficient, male weanling rats, fed a calcium-restricted or calcium-supplemented, vitamin D-deficient diet, were treated for 4 weeks with vitamin D (orally) or 1,25-dihydroxyvitamin D-3 (60 pmol/d by mini-osmotic pump). We also extended the treatment to 8 weeks for one group of animals fed the calcium-supplemented diet. Vitamin D compounds decreased the level of renal receptor in rats fed the low calcium diet. However, in animals given a 1.2% calcium diet, both vitamin D and 1,25-dihydroxyvitamin D-3 increased receptor levels by 130%. After 8 weeks, 1,25-dihydroxyvitamin D-3 had increased the receptor level by 260% while vitamin D had no further effect. Thus, dietary calcium is required for vitamin D to up-regulate the renal vitamin D receptor level. Further, 1,25-dihydroxyvitamin D-3 itself appears to have a marked ability to increase vitamin D receptor levels. These results suggest a complex mechanism of homologous regulation of the vitamin D receptor in the kidney.

Animals↗
PubMed1993

Phosphorylation is involved in transcriptional activation by the 1,25-dihydroxyvitamin D3 receptor.

The 1,25-dihydroxyvitamin D3 receptor becomes phosphorylated upon treatment with 1,25-dihydroxyvitamin D3. We have investigated the role of phosphorylation in the transcriptional activity induced by 1,25-dihydroxyvitamin D3 through its receptor. An active 1,25-dihydroxyvitamin D3-dependent transcription system was reconstituted in CV-1 cells by co-transfection of plasmids containing the rat 1,25-(OH)2D3 receptor DNA and a functional vitamin D response element (DRE) in a reporter gene construct. Treatment of these transiently transfected CV-1 cells with modulators of protein kinase A (8-Br-cAMP, PKIA and H-9) and phosphatases (Okadaic acid) resulted in mimicking or abolishing the transcriptional activity of 1,25-dihydroxyvitamin D3 in a receptor-dependent fashion. These modulators directly altered 1,25-dihydroxyvitamin D3 receptor phosphorylation. Therefore, the present results strongly suggest that phosphorylation plays a central role in the transcriptional activity of the 1,25-dihydroxyvitamin D3 receptor.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1993

Vitamin D-dependency rickets type II: truncated vitamin D receptor in three kindreds.

Fibroblasts from three patients with vitamin D-dependency rickets type II were used to study mutations in the 1,25-dihydroxyvitamin D3 receptor responsible for this hereditary disease. Normal human fibroblasts contain 43 +/- 13 fmol receptor/mg protein as determined by immunoradiometric assay and 22 +/- 3 fmol/mg by ligand binding assay. The fibroblasts from the rachitic patients contained no receptor detectable by either method. The 1,25-(OH)2D receptor cDNA for cells from each kindred was produced from total RNA using reverse transcription and polymerase chain reaction amplification. When these cDNAs were sequenced, it was found that each cell line contained a nucleotide substitution resulting in a stop codon in the coding sequence. The predicted resultant receptor protein is 69 amino acids long in one family, and 148 and 291 amino acids long in two other families. These truncated proteins have little or no 1,25-dihydroxyvitamin D3-binding domain accounting for 1,25-dihydroxyvitamin D resistance.

Base Sequence↗
PubMed1993

1,25-dihydroxycholecalciferol analogs cannot replace vitamin D in normocalcemic male rats.

A possible role of vitamin D in the growth and development of rats was investigated. Impaired development was observed in normocalcemic, vitamin D-deficient male and female rats, as revealed by low intestinal calcium transport, low renal vitamin D receptor levels and poor bone mineralization. Analogs of 1,25-dihydroxycholecalciferol, possessing reduced calcium-mobilizing activity in intestine and bone but retaining differentiation activity in cultured cells, were unable to support normal development of normocalcemic, vitamin D-deficient male rats. These results suggest that either the calcium-mobilizing activity alone or both the calcium-mobilizing activity and differentiating activity of vitamin D are required for normal development or that the analogs are inactive in vivo. We also demonstrated sex-related differences in intestinal calcium transport, renal vitamin D receptor regulation and bone mineralization that were independent of vitamin D status.

Animals↗
PubMed1993

Stabilization of the vitamin D receptor in rat osteosarcoma cells through the action of 1,25-dihydroxyvitamin D3.

A regulatory mechanism for the vitamin D receptor (VDR) in rat osteosarcoma cells (ROS 17/2.8) is stabilization of the receptor through binding of its ligand, 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]. Increased transcription of the gene encoding VDR does not occur upon treatment of these osteoblast-like cells with 1,25-(OH)2D3. When 10 nM 1,25-(OH)2D3 was administered to confluent cultures of ROS 17/2.8 cells, no change in receptor mRNA was detected, as measured by a ribonuclease protection assay. VDR abundance was measured using an immunoradiometric assay at varying time points within a 24-h period after 1,25-(OH)2D3 treatment. Receptor protein levels increased rapidly and continued to rise over 24 h. By 2 h, the level of receptor increased 2.5-fold, achieving a maximum level of 8-fold above the baseline at 18 h. The half-life of the receptor protein is 2 h in the absence of hormone, as determined by blockage of translation in cycloheximide-treated cells. In the presence of hormone, however, receptor levels were unchanged for at least 6 h. The administration of 1,25-(OH)2D3 stabilizes the receptor, thereby resulting in its accumulation in ROS 17/2.8 cells.

Animals↗