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H F DeLuca

Publications and source records attributed to H F DeLuca.

At least 145 records · Page 8Linked to original sources
PubMed1992

Evidence of an age-related decrease in intestinal responsiveness to vitamin D: relationship between serum 1,25-dihydroxyvitamin D3 and intestinal vitamin D receptor concentrations in normal women.

Although aged rats reportedly have reduced intestinal vitamin D receptor (VDR) concentrations, it is unclear whether an analogous age-related defect occurs in man. Thus, we assessed the interrelationship among serum 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3], calcium absorption and intestinal VDR in 44 healthy, ambulatory women, ages 20-87 yr. Fractional calcium absorption was measured after oral administration of 45Ca (20 mg CaCl2 as carrier); serum 1,25-(OH)2D3, by the calf thymus binding assay; and serum intact PTH, by a two-site immunochemiluminometric assay. Vitamin D receptor concentration was measured, by a new immunoradiometric assay, in biopsy specimens taken from the second part of the duodenum during gastroduodenoscopy in 35 of the women. Despite an age-related increase in serum PTH (r = 0.48; P less than 0.001) and in serum 1,25-(OH)2D3 concentration (r = 0.32; P less than 0.05), intestinal VDR concentration decreased with age (r = -0.38; P = 0.03) and fractional calcium absorption did not change with age. Although a contribution of decreased 25-hydroxyvitamin D 1 alpha-hydroxylase activity to the blunting of the increase in serum 1,25-(OH)2D3 concentration late in life is not excluded, the data are far more consistent with impaired intestinal responsiveness to 1,25-(OH)2D3 action. This defect could lead to compensatory increases in PTH secretion and 1,25-(OH)2D3 production which maintain calcium absorption and serum ionic calcium, but at the expense of increased bone loss.

Adult↗
PubMed1992

Characterization of 1,25-dihydroxyvitamin D3 receptor interactions with target sequences in the rat osteocalcin gene.

The sequences in the rat osteocalcin gene that bind the 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] receptor and mediate its effects on gene transcription contain three copies of a motif homologous to those found in other steroid response elements. To evaluate the relative importance of these motifs and their flanking sequences, functional properties and receptor binding of a series of mutant response elements were analyzed. Mutations in the third motif, including its deletion, have no effect on receptor binding and only modestly detrimental effects on the ability of the upstream motifs to confer 1,25-(OH)2D3 responsiveness in transfected cells. Mutations in the first two motifs have negative effects on both receptor binding and gene activation. Mutagenesis of bases upstream from each of the first two motifs alters receptor binding and gene activation; these bases are, therefore, an integral part of the rat osteocalcin 1,25-(OH)2D3 response element. Two direct hexameric repeats (GGGTGA ATG AGGACA) and not the palindromic motif thus characterize this 1,25-(OH)2D3 response element.

Animals↗
PubMed1992

Intestine, bone, and mammary gland contributions to maternal plasma calcium increase after abrupt weaning.

The response of maternal plasma calcium concentration to the abrupt and permanent removal of the suckling pups on Day 13 of lactation was investigated. Maternal plasma calcium did not change at 6 hr or 12 hr following pup removal. At 18 hr and 24 hr after weaning, the maternal plasma calcium concentration increased in mothers consuming either 0.47% calcium or 0.02% calcium diets. At 24 hr after weaning, the plasma calcium increase in mothers consuming low dietary calcium was 55% that of mothers consuming adequate dietary calcium. The contribution of the mammary gland to the plasma calcium increase in rats consuming the low dietary calcium was investigated by removing the mammary glands. Following mammary gland removal, plasma calcium increased 50% compared with mothers that had intact mammary glands. The data suggest that intestinal absorption of calcium and bone calcium mobilization remain stimulated by the lactation process for at least 24 hr after removal of the nursing pups.

Animals↗
PubMed1992

1,25-Dihydroxyvitamin D and not calcium is the major regulator of calbindin-D 9-kDa mRNA levels in vivo.

A possible role of calcium in vivo on intestinal calbindin-D 9-kDa mRNA levels has been studied in rats. In vitamin D-deficient rats, a marked increase in dietary calcium has a small but significant effect on calbindin-D 9-kDa mRNA levels, despite a dramatic increase in serum calcium concentration that clearly resulted from increased intestinal absorption of calcium. On the other hand, vitamin D under all circumstances increased calbindin-D 9-kDa mRNA levels, with the greatest levels found in animals on a low calcium diet where little or no calcium is available for absorption. These results strongly support the idea that 1,25-dihydroxyvitamin D is directly responsible for the induction of calbindin-D 9-kDa.

Animals↗
PubMed1991

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.

Animals↗
PubMed1991

Isolation of a mRNA that encodes a putative intestinal alkaline phosphatase regulated by 1,25-dihydroxyvitamin D-3.

Using differential hybridization techniques to screen a rat intestinal cDNA library we isolated a cDNA whose predicted amino acid sequence exhibits a high degree of homology to the alkaline phosphatases. The predicted cDNA sequence has 79% identity at the amino acid level to the rat intestinal alkaline phosphatase, and shows approx. 70% homology to other human and rat alkaline phosphatases. The corresponding mRNA is markedly increased by 6 h after a single dose of 1,25-dihydroxyvitamin D-3. The mRNA is also increased by 24-homologated analogs of 1,25-dihydroxyvitamin D-3 that do not increase calcium transport.

Alkaline Phosphatase↗
PubMed1991

Overproduction of rat 1,25-dihydroxyvitamin D3 receptor in insect cells using the baculovirus expression system.

The rat 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] receptor has been expressed at elevated levels in Spodoptera frugiperda cells using the baculovirus expression vector system. The recombinant 1,25-(OH)2D3 receptor is full-length, binds 1,25-(OH)2D3, and is recognized by a monoclonal antibody specific for 1,25-(OH)2D3 receptor. Densitometric scanning of Coomassie brilliant blue-stained SDS/polyacrylamide gels indicated a recombinant receptor protein level comprising 5% of the total soluble protein from the insect cells. The hydroxylapatite binding assay revealed average levels of 2 nmol of unoccupied 1,25-(OH)2D3 receptor per mg of protein in insect cells at 72 hr after infection with recombinant baculovirus. A measure of total 1,25-(OH)2D3 receptor using a ligand-independent, immunoradiometric assay disclosed average levels of 2.3 nmol of receptor per mg of protein produced by these same cells. A monoclonal antibody directed against the 1,25-(OH)2D3 receptor, and reported to cross-react with this receptor derived from several species, recognized the recombinant rat 1,25-(OH)2D3 receptor upon Western analysis. A monoclonal antibody directed specifically against the porcine receptor failed to recognize the recombinant rat 1,25-(OH)2D3 receptor protein. The cytosolic preparation of insect cells infected with recombinant baculovirus exhibited an equilibrium dissociation constant of 1 x 10(-11) M as determined by a 1,25-(OH)2D3 saturation analysis plotted by the method of Scatchard. This expression system provides an adequate source from which abundant quantities of 1,25-(OH)2D3 receptor can be purified for subsequent x-ray crystallographic analyses.

Animals↗
PubMed1991

Kinetic properties of 25-hydroxyvitamin D- and 1,25-dihydroxyvitamin D-24-hydroxylase from chick kidney.

1,25-Dihydroxyvitamin D3 induces both 25-hydroxyvitamin D3- and 1,25-dihydroxyvitamin D3- 24-hydroxylase activities. However, whether 24-hydroxylation of these substrates is catalyzed by a single enzyme is unknown. We have examined the substrate specificity of the enzyme using the solubilized and reconstituted chick renal mitochondrial 24-hydroxylase enzyme system. The soluble enzyme catalyzes 24-hydroxylation of both substrates. The apparent Km of the 24-hydroxylase for 25-hydroxyvitamin D3 and 1,25-dihydroxyvitamin D3 were 1.47 and 0.14 microM, respectively. Kinetic studies demonstrated that 25-hydroxyvitamin D3 and 1,25-dihydroxyvitamin D3 act as competitive inhibitors with respect to each other. 1,25-Dihydroxyvitamin D3 inhibited the production of 24,25-dihydroxyvitamin D3 with an apparent Ki of 0.09 microM and 25-hydroxyvitamin D3 inhibited the production of 1,24,25-trihydroxyvitamin D3 with an apparent Ki of 3.9 microM. These results indicate that chick 24-hydroxylase preferentially hydroxylates 1,25-dihydroxyvitamin D3 and support the idea that the 24-hydroxylation of these substrates is catalyzed by a single enzyme.

24,25-Dihydroxyvitamin D 3↗
PubMed1991

Sites of phosphorylation and photoaffinity labeling of the 1,25-dihydroxyvitamin D3 receptor.

The 1,25-dihydroxyvitamin D3 receptor is a member of the steroid/thyroid hormone receptor gene family and is thought to act by regulating transcription of specific genes. In this report, we have used peptide mapping of porcine 1,25-dihydroxyvitamin D3 receptor to localize the sites of phosphorylation, photoaffinity labeling, and monoclonal antibody binding. Receptor was immunoprecipitated from [32P]orthophosphate-labeled pig kidney LLC-PK1 cells grown in the absence and presence of 1,25-dihydroxyvitamin D3. Phosphorylation of receptor was induced by 1,25-dihydroxyvitamin D3. The phosphorylated receptor was digested with Staph A V8 protease within Cleveland gels and the 32P label was found entirely in a 23-kDa fragment. Similarly, receptor that was photoaffinity labeled with 1,25-dihydroxy-[26,27-3H]vitamin D3 was subjected to peptide mapping by Cleveland gels. The primary site of photoaffinity label incorporation was in the same 23-kDa peptide. This peptide was localized to a region in the center of the receptor protein, spanning part of the previously designated hinge region and roughly one-half the proposed steroid binding domain. Because phosphorylation did not occur in the DNA binding domain, it may not be involved in the binding of receptor to DNA. The localization of phosphorylation sites to this 23-kDa peptide may suggest that phosphorylation is involved in steroid binding and/or activation of the receptor.

Affinity Labels↗
PubMed1991

1,25-Dihydroxyvitamin D3 receptors in human carcinomas: a pilot study.

1,25-Dihydroxyvitamin D3 [1,25-(OH)2D3] receptor concentration was measured by an accurate immunoradiometric assay in primary tumors from 10 patients with colorectal carcinoma and 11 patients with non-small cell carcinoma of the lung. Measurements were also performed on a noncancerous sample of the same origin as the tumor from each patient. All of the tumors contained receptor with a mean concentration of 123.4 fmol/mg protein for colorectal and 75.1 fmol/mg protein for lung carcinoma. Compared to normal tissue from the same patient, 100% of the lung tumors and 70% of the colorectal tumors had significantly higher levels of the 1,25-dihydroxyvitamin D3 receptor. A correlation was found between well-differentiated colorectal tumors with no or few metastases and high levels of 1,25-dihydroxyvitamin D3 receptor. Receptor concentration was also assayed in metastatic lesions of malignant melanoma from 7 patients. 1,25-Dihydroxyvitamin D3 receptor was present in 85% of the metastases at a mean level of 26 fmol/mg protein. For these patients an inverse correlation was found between receptor level and age. The results obtained in this pilot study suggest that an alteration in 1,25-dihydroxyvitamin D3 receptor regulation may occur in vivo when a cell undergoes malignant transformation.

Aged↗
PubMed1991

Photoaffinity labeling of the 1,25-dihydroxyvitamin D-3 receptor.

Underivatized 1,25-dihydroxy[26,27-3H]vitamin D-3 was successfully used to photoaffinity label the 1,25-dihydroxyvitamin D-3 receptor. The covalent incorporation of tritium into the receptor protein was induced by ultraviolet irradiation of the receptor-1,25-dihydroxy[26,27-3H]vitamin D-3 complex in crude pig intestinal nuclear extract. The amount of incorporated label increased with increasing time of irradiation and was dependent on light of wavelengths 220-280 nm. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and fluorography were used to demonstrate that label was incorporated primarily into the 1,25-dihydroxyvitamin D-3 receptor. In addition, the label incorporation was eliminated by competition with a 100-fold excess nonradioactive 1,25-dihydroxyvitamin D-3, indicating that the label was specific for the steroid binding site. Since 1,25-(OH)2[26,27-3H]vitamin D-3 is readily available and requires no special precautions for its preparation and handling, it should be a useful photoaffinity label for future studies of the receptor.

Affinity Labels↗
PubMed1991

Characteristics of the 25-hydroxyvitamin D3- and 1,25-dihydroxyvitamin D3-24-hydroxylase(s) from HL-60 cells.

1,25-Dihydroxyvitamin D3 induces the human promyelocyte leukemia cell line, HL-60, to differentiate into macrophages/monocytes via a steroid-receptor mechanism. This system is a relevant one for an investigation of the molecular mechanism of 1,25-dihydroxyvitamin D3. We have now examined the effect of 1,25-dihydroxyvitamin D3 on the induction of 1,25-dihydroxyvitamin D3- and 25-hydroxyvitamin D3-24-hydroxylase activities in HL-60 cells. The hydroxylase activities were measured by a periodate-based assay, which was validated by comparison with well-established HPLC analysis. HPLC analysis also suggested that 1,25-dihydroxyvitamin D3 induces a 23-hydroxylase in addition to the 24-hydroxylase. 1,25-Dihydroxyvitamin D3- and 25-hydroxyvitamin D3-24-hydroxylase activities were stimulated as early as 4 h after the addition of 10(-7) M 1,25-dihydroxyvitamin D3 and became maximal by 24 h. 1,25-Dihydroxyvitamin D3 stimulated both activities in a dose-dependent manner up to 10(-6) M. The Km of 24-hydroxylase for 1,25-dihydroxyvitamin D3 and 25-hydroxyvitamin D3 were 2 x 10(-8) M and 5.2 x 10(-7) M, respectively. Cycloheximide (5 microM) inhibited 1,25-dihydroxyvitamin D3-mediated stimulation of 24-hydroxylase activity. Other differentiation inducers, such as retinoic acid and phorbol ester, did not induce either activity. 1,25-Dihydroxyvitamin D3-24-hydroxylase in HL-60 mitochondria was solubilized with 0.6% cholate and reconstituted with NADPH, beef adrenal ferredoxin, and beef adrenal ferredoxin reductase, each component being essential for 24-hydroxylase activity. These results strongly suggest that the 24-hydroxylase in HL-60 cells is a three-component cytochrome P450-dependent mixed-function oxidase.

25-Hydroxyvitamin D3 1-alpha-Hydroxylase↗
PubMed1991

Induction of calbindin-D 9k mRNA but not calcium transport in rat intestine by 1,25-dihydroxyvitamin D3 24-homologs.

The function and precise mechanism of regulation of calbindin-D 9k in intestine is largely unknown. It is suggested that this calcium binding protein is involved in active intestinal calcium transport and that its expression is mainly mediated by 1,25-dihydroxyvitamin D3. We examined the effect of two side chain modified analogs of 1,25-dihydroxyvitamin D3 as compared to 1,25-dihydroxyvitamin D3 itself on the regulation of the calbindin-D 9k at the mRNA level and on intestinal calcium transport in the rat. delta 22-24,24-dihomo-1,25-dihydroxyvitamin D3 at a single dose of 500, 1,000, and 2,000 pmol caused greater than 7.0-fold increase in calbindin-D 9k mRNA without stimulating intestinal calcium transport. A 10,000-pmol dose of delta 22-24,24,24-trihomo-1,25-dihydroxyvitamin D3 caused a 7.6-fold increase in calbindin-D 9k mRNA without significantly increasing intestinal absorption of calcium. In contrast, 1,25-dihydroxyvitamin D3 caused a parallel increase in calbindin-D 9k mRNA and intestinal absorption of calcium. Thus, calbindin 9k is not by itself responsible for 1,25-dihydroxyvitamin D3-mediated increase in intestinal absorption of calcium.

Animals↗
PubMed1991

An estrogen-responsive element mediates the transcriptional regulation of calbindin D-9K gene in rat uterus.

The rat calbindin (molecular mass 9 kDa) gene sequence revealed the presence of an imperfect palindromic estrogen-responsive-like element (ERE) located at position +51 from the transcriptional initiation site. This element has the sequence AGGTCAGGGTGATCT which differs by one nucleotide from the vitellogenin ERE palindromic sequence AGGTCACTGTGACCT. The activity of this element to induce transcription in response to estrogen and the ability to bind estrogen receptor was investigated. This sequence confers estrogen-dependent transcriptional activity when cloned upstream from the tkp-CAT construct in pBLCAT2 plasmid and transfected into T47D cells, similar to the vitellogenin gene ERE activity but to a lesser extent. This element binds to the estrogen receptor in vitro as assessed by gel retardation assay similar to the vitellogenin gene ERE. No such binding was detected when a mutant sequence AGATCACTGTGATCT was used. The specificity of the complex was confirmed using polyclonal antibodies, ER712 raised against the estrogen receptor. Furthermore, competition assays showed that both sequences were able to compete for binding to the estrogen receptor. The in vivo transcriptional regulation of the calbindin D-9K gene by estrogen was also investigated in the rat. Female animals maintained on a vitamin D-sufficient or a vitamin D-deficient diet were ovariectomized, housed for 3 weeks, and then injected with 17 beta-estradiol (0.5 micrograms/kg body weight/day). Slot blot analysis of total RNA showed a marked increased in calbindin D-9K mRNA levels in the uterus but not in the intestine by 26 and 50 h post-injection. These results demonstrate that the calbindin D-9K gene is transcriptionally regulated by estrogen in the uterus mediated by an estrogen-responsive element identified in the gene.

Animals↗
PubMed1991

The Sp1 transcription factor gene (SP1) and the 1,25-dihydroxyvitamin D3 receptor gene (VDR) are colocalized on human chromosome arm 12q and rat chromosome 7.

By means of somatic cell hybrids segregating either human or rat chromosomes, the genes encoding the transcription factor Sp1 (SP1) and the 1,25-dihydroxyvitamin D3 receptor (VDR) were both assigned to human chromosome arm 12q and to rat chromosome 7. This result implies that the locus for the clinical disorder vitamin D dependency rickets type II maps on 12q. The phenylalanine hydroxylase (PAH) and the retinoic acid receptor-gamma (RARG) genes also map on human chromosome arm 12q and rat chromosome 7, indicating that a synteny group is conserved on these chromosomes.

Animals↗
PubMed1991

Serum 25-hydroxyvitamin D concentrations of free-living subjects consuming olestra.

The effect of olestra on vitamin D status was assessed in a 6-wk, double-blind, placebo-controlled study involving 202 free-living adults. Subjects consumed a total of 20 g/d of olestra or triglycerides in cookies eaten at each meal. A 20-micrograms ergocalciferol capsule was taken with each morning meal. Serum 25-hydroxyergocalciferol (25-OHD2) concentrations rose from approximately 5.7 to 39.0 and 31.7 nmol/L in the placebo and olestra groups, respectively, at week 6. At week 6, 25-OHD2 contributed 46-54% to total serum 25-OHD concentration compared with 11% at baseline. The 19% decrease in serum 25-OHD2 concentrations produced by olestra in this study is equivalent to a decrease of approximately 1.2 nmol/L under nonsupplemented dietary conditions. Ingesting 20 g olestra/d in the diet is thus not expected to affect vitamin D nutritional status.

25-Hydroxyvitamin D 2↗