Acute actions of 1,25-dihydroxyvitamin D3 upon chick pancreatic calbindin-D28K

We have compared the relative responsiveness of pancreatic, intestinal and renal tissue calbindin-D28K protein content to the stimulatory actions of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] in vitamin D-deficient (-D) chicks. Tissue concentrations of calbindin-D28K were undetectable in the -D chick intestine but present, albeit at low concentrations (less than 1 microgram CaBP/mg protein) in the -D kidney and pancreas. Intestinal, pancreatic and renal calbindin-D28K content was stimulated 318, 9.8 and 2.9 fold respectively, 48 hours after -D chicks received a single dose of 1,25(OH)2D3 [6.5 nmol/animal]. The pancreatic calbindin-D28K content could be significantly stimulated as early as 5 hours after 1,25(OH)2D3 administrations in vivo. These findings support the contention that the pancreas is a target for vitamin D, and is consistent with the view that calbindin-D28K plays a role in normal pancreatic functions.     

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.     

Modulation of 1,25-dihydroxyvitamin D3 receptor by phospholipids and fatty acids

The structural relationship between several lipids and their respective capacities to inhibit the specific binding of [3H]-1,25 (OH)2 vitamin D3 to chick intestinal cytosol preparations was investigated. The lipids investigated were: synthetic 3-sn-phosphatidylcholine and 3-sn-phosphatidic acid, egg yolk 3-sn-phosphatidylcholine and its corresponding phosphatidic acid, and free unsaturated fatty acids and their esters. The results indicate that at least three structural elements in the phospholipid molecule appear to be important; these are: 1) the structure of the fatty acid, 2) the anionic properties of the phospholipid phosphate group, and 3) the glycerol phosphate portion of the molecule. Our data also demonstrate that the position (1 or 2) and the amount (single vs. double) of unsaturated fatty acids in the phospholipid molecule do not play a major role in the receptor-1,25 (OH)2 vitamin D3 interaction. Furthermore, under equilibrium conditions, kinetic and Scatchard analysis suggest that phospholipids or free fatty acids may bind at a site different from the 1,25 (OH)2 vitamin D3 binding site, and therefore inhibit the hormone binding via a noncompetitive conformational change in the receptor molecule. A model for this phospholipid/free fatty acid binding site is proposed.     

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.     

Stimulation of rat intestinal protein synthesis by 1,25-dihydroxyvitamin D3

To study general stimulatory effects of 1,25-dihydroxyvitamin D3 on intestinal protein synthesis, slices of duodenal villi from 1,25-dihydroxyvitamin D3-treated and vitamin D-deficient rats were incubated in vitro for 90 min at the surface of medium containing [3H]leucine. Incorporation of the [3H]leucine into TCA-precipitated protein, which was shown to be linear for 12 h and 90% inhibited by cycloheximide, was increased by 50-60% at 26 h after a single injection of 125 ng of 1,25-dihydroxyvitamin D3 (three experiments, P less than 0.001). The increase, which was not due to circadian rhythm fluctuations of the intestine, was in synchrony with the second Ca2+ transport response observed by Halloran and DeLuca (Arch. Biochem. Biophys. 208, 477-486, 1981). However, no significant difference in [3H]leucine incorporation was observed before or during the initial Ca2+ transport response observed by Halloran and DeLuca, i.e., at 1.0, 3.0, and 6.5 h following an injection of 1,25-dihydroxyvitamin D3. The late onset of the 1,25-dihydroxyvitamin D3-induced increase in total protein synthesis implies that it is an indirect rather than a direct effect of the hormone.     

Calcium and 1,25-dihydroxyvitamin D3 regulation of adipokine expression

Objective: Obesity is associated with elevated oxidative stress and low‐grade systemic inflammation. We have demonstrated recently that 1α,25‐(OH)₂‐D₃ promotes reactive oxygen species production in cultured adipocytes, whereas suppression of 1α,25‐(OH)₂‐D₃ by increasing dietary calcium down‐regulates diet‐induced oxidative stress in aP2‐agouti transgenic mice. However, whether the anti‐obesity effect of dietary calcium plays a role in regulation of obesity‐associated inflammation is not clear. Research Methods and Procedures: We investigated the role of dietary calcium in the regulation of inflammatory cytokine production in aP2‐agouti transgenic mice fed low‐ and high‐calcium obesigenic diets and in the modulation of cytokine production by 1α,25‐(OH)₂‐D₃ in cultured murine and human adipocytes. Results: The high‐calcium diet inhibited the expression of pro‐inflammatory factors tumor necrosis factor α and interleukin (IL)‐6 by 64% and 51%, respectively (p < 0.001), in visceral fat, stimulated the expression of the anti‐inflammatory factors IL‐15 and adiponectin by 52% (p = 0.001) and 54% (p = 0.025), respectively, in visceral fat, and induced a 2‐fold increase in IL‐15 expression in soleus muscle (p = 0.01) compared with litter mate controls on a low‐calcium diet. 1α,25‐(OH)₂‐D₃ also markedly stimulated the expression of tumor necrosis factor α (p < 0.001) and IL‐6 (p = 0.016) in differentiated 3T3‐L1 adipocytes and increased IL‐6 (p = 0.004) and IL‐8 (p < 0.001) production in differentiated human adipocytes. These effects were blocked by calcium channel antagonism with nifedipine. Discussion: These data demonstrate that 1α,25‐(OH)₂‐D₃ favors inflammatory cytokine expression and inhibits anti‐inflammatory cytokine expression; accordingly, suppression of 1α,25‐(OH)₂‐D₃ by dietary calcium inhibits adipocyte‐derived inflammation associated with obesity.     

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.     

Analysis of rat vitamin D-dependent calbindin-D28k gene expression

We report the use of a cloned cDNA for mammalian calbindin-D28k (28-kDa vitamin D-dependent calcium-binding protein) to study the expression of the rat calbindin gene. Tissue distribution studies, using Northern analysis, indicated that calbindin-D28k-mRNA is detected in rat kidney and brain but is not detected in rat intestine, testes, bone, pancreas, liver, lung, or skeletal muscle. Both rat kidney and brain contain three RNA species (1.9, 2.8, and 3.2 kilobase pairs). The regulation of the gene was characterized by both Northern and slot blot analysis. Hormonal regulation, developmental expression of calbindin-D28k-mRNA, and the effect of dietary alteration were examined. In the kidney all three species of mRNA were dependent on the presence of 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) for their induction. The time course of induction of renal calbindin-D28k-mRNA indicated that a significant increase in calbindin-D-mRNA was detectable as early as 2 h following a single injection of 1,25-(OH)2D3 (200 ng/100 g of body weight), reaching a maximum at 12 h. Unlike the kidney high levels of calbindin-D28k-mRNA were observed in the brain of vitamin D-deficient rats. The concentration of calbindin-D28k-mRNA in brain was unchanged after 1,25-(OH)2D3 administration. Developmental studies indicated that calbindin-D-mRNA in rat kidney and brain is present prior to birth but is developmentally regulated in a tissue-specific manner. The most pronounced changes in the abundance of renal calbindin-D28k-mRNA occur between birth and 1 week of age. Unlike the kidney a large increase in brain calbindin-D28k-mRNA occurs at a later time, between 1 and 2 weeks of age (the period of major synapse formation). In dietary alteration studies results of Northern blot analysis indicate that low dietary phosphorus results in increased calbindin-D-mRNA in kidney but not in brain. These studies represent the first analysis of the rat calbindin-D28k gene and its regulation in vivo. Our findings suggest that in rat kidney and brain there are significant differences both in the expression of the gene for calbindin-D28k and its regulation by 1,25-(OH)2D3.     

Regulation of calbindin-D9k expression by 1,25-dihydroxyvitamin D(3) and parathyroid hormone in mouse primary renal tubular cells

Membranes of the obligate methylotroph Methylobacillus flagellatus KT contained hemes B, O, and C and cytochromes b, o, and c both in batch and in continuous cultures. Neither heme A nor heme D was detected in the membranes. The cytochromes o and bb were the main components reversibly binding carbon monoxide (CO) in the terminal part of the respiratory chain. The alpha-region and especially the alpha-peaks at 568 and 573 nm and the alpha-troughs at 586 and 592 on the CO-difference spectra were diagnostic for the cytochromes o and bb, respectively. The cytochrome o content increased up to 1.8 times upon increasing the dilution rate of the culture from 0.15 to 0.55 h(-1) under methanol limitation. By contrast, the level of the CO-binding cytochrome bb was not affected by methanol concentration but its content increased up to 1.9 times when the level of oxygen decreased from 95 to 21 microM under the constant dilution rate (mu = 0.55 h(-1)). The maximum ratio between the cytochromes o and bb reached 2 during continuous cultivation under methanol-limited conditions (mu = 0.55 h(-1)), whereas the minimum ratio between them was about 0.7 during batch cultivation at stationary phase of growth. The synthesis of the CO-binding cytochrome bb but not of the cytochrome o in M. flagellatus KT was assumed to depend on the ambient redox potential of the medium. The cytochrome o synthesis was supposed to depend on the transmembrane gradient of protons (Delta(mu)H+).     

Evidence for regulation of amelogenin gene expression by 1,25-dihydroxyvitamin D(3) in vivo

The unique hereditary enamel defect clearly related to the disturbance of one enamel matrix protein is X-linked amelogenesis imperfecta (AI), in which several mutations of amelogenin gene have been identified. The clinical phenotype of many of these subjects shows similarities with enamel defects related to rickets. Therefore, we hypothesized that rachitic dental dysplasia is related to disturbances in the amelogenin pathway. In order to test this hypothesis, combined qualitative and quantitative studies in experimental vitamin D-deficient (-D) rat model systems were performed. First, Western blot analysis of microdissected enamel matrix (secretion and maturation stages) showed no clear evidence of dysregulation of amelogenin protein processing in -D rats as compared with the controls. Second, the ultrastructural investigation permitted identification of the internal tissular defect of rachitic enamel, the irregular absence of intraprismatic enamel observed in -D animals, suggesting a possible link between prism morphogenesis and vitamin D. In addition, the steady-state levels of amelogenin mRNAs measured in microdissected dental cells was decreased in -D rats and up-regulated by an unique injection of 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)). The present study shows evidences that amelogenin expression is regulated by vitamin D. This is the first study of an hormonal regulation of tooth-specific genes.     

Effect of hormones and development on the expression of the rat 1,25-dihydroxyvitamin D3 receptor gene. Comparison with calbindin gene expression

We have used specific cDNAs to the rat vitamin D receptor (VDR) and to the mammalian vitamin D-dependent calcium-binding proteins (calbindin-D9k in intestine and calbindin-D28k in kidney) in order to obtain a better understanding of the regulation of the VDR gene and its relationship to calbindin gene expression. Hormonal regulation and development expression of the rat VDR gene were characterized by both Northern and slot blot analyses. Administration of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3; 25 ng/day for 7 days) to vitamin D-deficient rats resulted in an increase in calbindin mRNA in intestine and kidney but no change in VDR mRNA in these tissues. Vitamin D-deficient rats responded to dexamethasone treatment (100 micrograms/100 g of body weight/day for 4 days) with a 2.5-fold increase in intestinal VDR mRNA which was accompanied by a 4-fold decrease in intestinal calbindin-D9k mRNA. Developmental studies indicated a pronounced increase in renal VDR mRNA and calbindin-D28k mRNA between birth and 1 week of age. In the intestine, an induction of VDR and calbindin-D9k gene expression was observed at a later time, during the 3rd postnatal week (the period of increased duodenal active transport of calcium). Taken collectively, our data indicate that in the adult rat, target tissue response to hormone is not modified by a corresponding alteration in new receptor synthesis. However, developmental studies indicate that the induction of 1,25(OH)2D3 receptor mRNA is correlated with the induction of calbindin gene expression. Our results also demonstrate that glucocorticoid administration can result in an alteration in intestinal calbindin and VDR gene expression.     

Molecular mechanism underlying 1,25-dihydroxyvitamin D regulation of nephrin gene expression

Nephrin plays a key role in maintaining the structure of the slit diaphragm in the glomerular filtration barrier. Our previous studies have demonstrated potent renoprotective activity for 1,25-dihydroxyvitamin D (1,25(OH)(2)D(3)). Here we showed that in podocytes 1,25(OH)(2)D(3) markedly stimulated nephrin mRNA and protein expression. ChIP scan of the 6-kb 5' upstream region of the mouse nephrin gene identified several putative vitamin D response elements (VDREs), and EMSA confirmed that the VDRE at -312 (a DR4-type VDRE) could be bound by vitamin D receptor (VDR)/retinoid X receptor. Luciferase reporter assays of the proximal nephrin promoter fragment (-427 to +173) showed strong induction of luciferase activity upon 1,25(OH)(2)D(3) treatment, and the induction was abolished by mutations within -312VDRE. ChIP assays showed that, upon 1,25(OH)(2)D(3) activation, VDR bound to this VDRE leading to recruitment of DRIP205 and RNA polymerase II and histone 4 acetylation. Treatment of mice with a vitamin D analog induced nephrin mRNA and protein in the kidney, accompanied by increased VDR binding to the -312VDRE and histone 4 acetylation. 1,25(OH)(2)D(3) reversed high glucose-induced nephrin reduction in podocytes, and vitamin D analogs prevented nephrin decline in both type 1 and 2 diabetic mice. Together these data demonstrate that 1,25(OH)(2)D(3) stimulates nephrin expression in podocytes by acting on a VDRE in the proximal nephrin promoter. Nephrin up-regulation likely accounts for part of the renoprotective activity of vitamin D.     

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.