Intestinal resistance to 1,25 dihydroxyvitamin D in mice heterozygous for the vitamin D receptor knockout allele

We tested the hypothesis that low vitamin D receptor (VDR) level causes intestinal vitamin D resistance and intestinal calcium (Ca) malabsorption. To do so, we examined vitamin D regulated duodenal Ca absorption and gene expression [transient receptor potential channel, vallinoid subfamily member 6 (TRPV6), 24-hydroxylase, calbindin D(9k) (CaBP) mRNA, and CaBP protein] in wild-type mice and mice with reduced tissue VDR levels [i.e. heterozygotes for the VDR gene knockout (HT)]. Induction of 24-hydroxylase mRNA levels by 1,25 dihydroxyvitamin D(3) [1,25(OH)(2) D(3)] injection was significantly reduced in the duodenum and kidney of HT mice in both time-course and dose-response experiments. TRPV6 and CaBP mRNA levels in duodenum were significantly induced after 1,25(OH)(2) D(3) injection, but there was no difference in response between wild-type and HT mice. Feeding a low-calcium diet for 1 wk increased plasma PTH, renal 1alpha-hydroxylase (CYP27B1) mRNA level, and plasma 1,25(OH)(2) D(3), and this response was greater in HT mice (by 88, 55, and 37% higher, respectively). In contrast, duodenal TRPV6 and CaBP mRNA were not higher in HT mice fed the low-calcium diet. However, the response of duodenal Ca absorption and CaBP protein to increasing 1,25(OH)(2) D(3) levels was blunted by 40% in HT mice. Our data show that low VDR levels lead to resistance of intestinal Ca absorption to 1,25(OH)(2) D(3), and this resistance may be due to a role for the VDR (and VDR level) in the translation of CaBP.     

Calbindin-D28k (CaBP28k) identification and regulation by 1,25-dihydroxyvitamin D3 in human choriocarcinoma cell line JEG-3

Calbindin-D28k (CaBP28k) is a cytosolic calcium (Ca2+)-binding protein expressed in tissues such as intestine, kidneys and placenta. This protein is thought to be involved in Ca2+ homeostasis. While it is well known that CaBP28k is influenced by 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] in the intestine and kidneys, nothing is known regarding the regulation of this protein in trophoblasts of human placenta. We used JEG-3 syncytiotrophoblast-like carcinoma cell line to study the regulation of CaBP28k in correlation with 1,25(OH)2D3 receptor (VDR) following 1,25(OH)2D3 treatments. Our data demonstrated for the first time that both CaBP28k mRNA and protein were highly induced by the addition of 1,25(OH)2D3 in dose-dependent manner. Moreover, the increase and subsequent decrease in the expression of CaBP28k and VDR mRNAs indicates the transient nature of the changes in gene expression in response to 1,25(OH)2D3. This is in contrast with the temporal pattern of increasing protein for CaBP28k and VDR. We also showed that new RNA and protein syntheses are required for 1,25(OH)2D3-induced upregulation of CaBP28k. Furthermore, a 25-carboxylic ester analogue of 1,25(OH)2D3, ZK159222, used as an antagonist of 1,25(OH)2D3 signaling confirmed that indeed 1,25(OH)2D3 was implicated in the induction of CaBP28k. These novel findings are a contribution to the processes that drive CaBP28k expression regulation in human placenta.     

Vitamin D-dependent calcium binding proteins in the kidney and intestine of the X-linked hypophosphatemic mouse: changes with age and responses to 1,25-dihydroxycholecalciferol

We have previously observed decreased intestinal 9 kilodalton (kd) vitamin D-dependent calcium binding protein (CaBP) and decreased calcium absorption in juvenile X-linked hypophosphatemic (Hyp) mice. The present studies were undertaken to examine whether the kidney CaBPs (9 kd and 28 kd) are also affected in young Hyp mice and to investigate the ability of 1,25-dihydroxycholecalciferol [1,25-(OH)2D3] to increase CaBP in the intestine and kidney. The 28 kd CaBP and the 9 kd CaBP were measured in the kidneys and the 9 kd CaBP in the intestines of normal and Hyp mice from 1 week to 40 weeks of age. At all times between 3 and 6 weeks, intestinal CaBP in Hyp mice was decreased by more than 50% (P less than 0.005-0.001) and no significant decrease was present in the adult Hyp mice (12 and 40 weeks of age). By contrast, both kidney CaBPs were decreased only slightly in young Hyp mice. Between 1 and 6 weeks of age, the 9 kd CaBP in Hyp mice was 82% +/- 4% of control (P less than 0.001) and the 28 kd protein was 89% +/- 3% of control (P less than 0.001). Minipumps containing 1,25-(OH)2D3 or vehicle were implanted in 4-week and 13-week-old Hyp mice for 3 days to provide a dose of 0.12 micrograms/kg mouse X day. The 9 kd CaBP was increased approximately 3-fold (P less than 0.001) by 1,25-(OH)2D3 in the intestines of Hyp mice at both ages. The 9 kd kidney CaBP in Hyp mice also was increased by 1,25-(OH)2D3 treatment at both ages, but only by 33-52%. The 28 kd CaBP in the kidney was not affected by 1,25-(OH)2D3 treatment of Hyp mice at either age. We conclude that (9 kd and 28 kd) CaBPs levels in both intestine and kidney are decreased in juvenile Hyp mice although to much different degrees. The administration of 1,25-(OH)2D3 to Hyp mice increases the 9 kd CaBP in both intestine and kidneys, whereas the renal 28 kd CaBP is unaffected.     

Effect of 1,25-dihydroxyvitamin D3 on plasma concentrations of calcium-binding protein in normal and rachitic (vitamin D-dependent rickets type I) pigs

An i.v. injection of calcitriol (1,25-(OH)2D3) had no effect within 2.5 h on plasma concentrations of calbindin-D9K (vitamin D-induced calcium-binding protein; CaBP) in hypocalcaemic pigs with inherited vitamin D-dependent rickets type I or in their normocalcaemic siblings or half-siblings. Three days later the plasma concentration of CaBP had doubled in the hypocalcaemic pigs, but was unaltered in the normocalcaemic siblings and half-siblings. Following daily i.v. injections of 1,25-(OH)2D3 for a further 5 days (days 4-8) plasma concentrations of CaBP increased in both the hypocalcaemic (days 4-8) and normocalcaemic (day 8) pigs, the effect being more rapid and greater in the hypocalcaemic 1,25-(OH)2D3-deficient animals. An i.v. injection of 1,25-(OH)2D3 to pure Yucatan pigs also had no effect on plasma concentrations of CaBP within 1.5 h, but in the following 1 h there was some indication of an increase in plasma CaBP levels. In contrast to the normal pigs, insulin-induced hypoglycaemia did not lead to a peak in plasma CaBP concentrations in the hypocalcaemic pigs. There was also no change in the plasma concentrations of 1,25-(OH)2D3 associated with the peak in plasma CaBP following insulin-induced hypoglycaemia in normocalcaemic pigs. These results suggest that changes in plasma concentrations of 1,25-(OH)2D3 are not directly involved in mediating the increase in plasma CaBP which follows hypoglycaemia induced by insulin in normal pigs, although 1,25-(OH)2D3 probably plays a permissive role.     

Modulation by vitamin D status of the responsiveness of rat bone to gonadal steroids

We have previously demonstrated that gonadal steroids stimulate [3H]thymidine incorporation and creatine kinase specific activity in skeletal tissues. In the present study we report that in 20-day-old vitamin D-deficient Wistar-derived rats, 17 beta-estradiol (E2; 5 micrograms/rat) or testosterone (50 micrograms/rat) failed to stimulate [3H]thymidine incorporation into diaphyses of long bones and that the response to these hormones in terms of increased creatine kinase specific activity was less than half the value in normally fed rats. Two daily ip injections of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3; 0.5 ng/g BW], but not 24,25-(OH)2D3 (5 ng/g BW), partially restored the biological responses to E2 in bone of 21-day-old vitamin D-deficient female rats. Vitamin D deficiency did not impair the responsiveness to gonadal steroids in the epiphysis of long bones, uterus, or prostate, in contrast to its effect on diaphysis. In 21-day-old normally fed female rats, neither vitamin D metabolite enhanced the response to E2. When cultures of rat epiphyseal cells were treated daily for 5 days with either 1,25-(OH)2D3 (1 nM) or 24,25-(OH)2D3 (10 nM), followed by E2 (30 nM) for 24 h, creatine kinase activity was significantly higher than in cultures treated daily for 5 days with vehicle alone, and then with E2. The same treatment of rat embryo calvaria bone cells showed that 1,25-(OH)2D3, but not 24,25-(OH)2D3, significantly increased the creatine kinase activity response to E2. These findings suggest that vitamin D metabolites selectively affect the biological responses of skeletal tissues to gonadal steroids.     

Differential regulation by 1,25-dihydroxyvitamin D3 of calbindin-D9k and calbindin-D28k gene expression in mouse kidney

The mouse kidney is a unique tissue since both vitamin D-dependent calcium binding proteins (calbindin-D9k and calbindin-D28k) are present in the same cells of the distal convoluted tubule. We have used specific complementary DNAs to mouse calbindin-D9k and mouse calbindin-D28k and Northern and slot blot analyses in order to obtain a better understanding of the regulation of two different molecular expressions of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] action in the same cells. Both calbindins were found to be regulated developmentally in a similar manner (an increase in gene expression between birth and 1 week of age, coinciding with nephron differentiation, and a peak at 3 weeks of age). However, the time course of response of the messenger RNA of each calbindin to 1,25(OH)2D3 was markedly different. The peak of induction of renal calbindin-D28k mRNA was at 12 h after a single injection of 1,25(OH)2D3 (200 ng/100 g body wt) to vitamin D-deficient mice, and a decrease was observed at 24 h (similar to the time course of response of other steroid-regulated genes). Interestingly, unlike calbindin-D28k, a delayed response of renal calbindin-D9k mRNA to 1,25(OH)2D3 was observed (the peak of induction was at 24 h after 1,25(OH)2D3 administration). Both genes in mouse kidney did not respond to glucocorticoids, although a dose-dependent decrease (12-86%) of mouse intestinal calbindin-D9k mRNA was observed after dexamethasone treatment, suggesting tissue-specific multiple steroid interactions in the regulation of calbindin gene expression. The finding of a different time course of regulation of each calbindin by 1,25(OH)2D3 suggests that different factors may be regulating the expression of the two different calbindins in mouse kidney and that elucidation of these control mechanisms should provide new insight concerning 1,25(OH)2D3-regulated gene expression.     

Induction of androgen receptor by 1alpha,25-dihydroxyvitamin D3 and 9-cis retinoic acid in LNCaP human prostate cancer cells

We have recently shown that 1alpha,25-dihydroxyvitamin D3 [1,25-(OH)2D3] inhibits proliferation of LNCaP cells, an androgen-responsive human prostate cancer cell line. Also, 1,25-(OH)2D3 increases androgen receptor (AR) abundance and enhances cellular responses to androgen in these cells. In the current study, we have investigated the mechanism by which 1,25-(OH)2D3 regulates AR gene expression and the involvement of AR in the 1,25-(OH)2D3- and 9-cis retinoic acid (RA)-mediated growth inhibition of LNCaP cells. Northern blot analyses demonstrated that the steady-state messenger RNA (mRNA) level of AR was significantly increased by 1,25-(OH)2D3 in a dose-dependent manner. Time-course experiments revealed that the increase of AR mRNA by 1,25-(OH)2D3 exhibited delayed kinetics. In response to 1,25-(OH)2D3, AR mRNA levels were first detected to rise at 8 h and reached a maximal induction of 10-fold over the untreated control at 48 h; the effect was sustained at 72 h. Furthermore, the induction of AR mRNA by 1,25-(OH)2D3 was completely abolished by incubation of cells with cycloheximide, a protein synthesis inhibitor. 1,25-(OH)2D3 was unable to induce expression of an AR promoter-luciferase reporter. Together, these findings indicate that the stimulatory effect of 1,25-(OH)2D3 on AR gene expression is indirect. Western blot analyses showed an increase of AR protein in 1,25-(OH)2D3-treated cells. This increased expression of AR was followed by 1,25-(OH)2D3-induced inhibition of growth in LNCaP cells. Similar to 1,25-(OH)2D3, 9-cis RA also induced AR mRNA expression, and the effect of both hormones was additive. Moreover, 1,25-(OH)2D3 and 9-cis RA acted synergistically to inhibit LNCaP cell growth. These antiproliferative effects of 1,25-(OH)2D3 and 9-cis RA, alone or in combination, were blocked by the pure AR antagonist, Casodex. In conclusion, our results demonstrate that growth inhibition of LNCaP cells by 1,25-(OH)2D3 and 9-cis RA is mediated by an AR-dependent mechanism and preceded by the induction of AR gene expression. This finding, that differentiating agents such as vitamin D and A derivatives are potent inducers of AR, may have clinical implications in the treatment of prostate cancer.     

Evidence for multiple effects of vitamin D3 on calcium absorption: response of rachitic chicks, with or without partial vitamin D3 repletion, to 1,25-dihydroxyvitamin D3.

The effects of vitamin D3 or 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], or both, on the relationship among calcium absorption, vitamin D-induced calcium-binding protein (CaBP), and phospholipid metabolism were examined. When 1,25(OH)2D3 was injected intracardially into D3-deficient chicks, both the stimulation of calcium absorption and the induction of the synthesis of CaBP occurred 2-4 hr later. When 1,25(OH)2D3 was injected into chicks partially repleted with D3, an earlier increase in calcium absorption was observed without a significant change in the concentration of CaBP already present in the duodenal mucosa. Other early events were an increased uptake of calcium by the intestinal tissue and an alteration in phospholipid metabolism. These and other observations support the proposal that at least two phases of calcium absorption are influenced by 1,25(OH)2D3--permeation of calcium across the brush border, and transfer of calcium through and out of the cell. The first phase responds more rapidly to 1,25(OH)2D3 than does the second phase, correlates with changes in phospholipid metabolism, and might not be dependent on de novo protein synthesis. The second phase correlates with CaBP synthesis and therefore is dependent on protein synthesis. Either the first phase or the second phase can constitute the limiting step in calcium absorption.     

Induction of flavokinase (EC 2.7.1.26) by aldosterone in the rat kidney

The effect of aldosterone on rat renal flavokinase (EC 2.7.1.26) enzymic activity and concentration was investigated in bilaterally adrenalectomized male Sprague-Dawley rats. Flavokinase enzymic activity was measured in the 100,000 X g supernatant of renal cortex and red medulla and was increased after 3 h by 19% and 42%, respectively, as a result of aldosterone (1.5 micrograms/100 g BW) administration. Dual isotope labeling studies revealed increases of 20-30% and 20-35% in the incorporation of [3H]- and [35S]methionine into rat renal cortical and red medullary flavokinase, respectively, as a result of aldosterone administration. The aldosterone-dependent increases in methionine incorporation were blocked when the mineralocorticoid receptor antagonist spirolactone (SC 26304; 150 micrograms/100 BW) was administered 30 min before aldosterone. The relative concentrations of renal flavokinase also increased by 40% in both the cortex and red medulla 3 h after aldosterone treatment, as determined by an enzyme-linked immunosorbent assay (ELISA). These increases were abolished by actinomycin D (100 micrograms/100 g BW) and cycloheximide (200 micrograms/100 g BW), given 1 h before aldosterone administration. The mineralocorticoid receptor antagonists SC 26304 (225 micrograms/100 g BW) and progesterone (500 micrograms/100 g BW) were also able to inhibit the aldosterone-dependent increase in renal flavokinase concentration. The effect of aldosterone on renal flavokinase concentration was studied from 30 min to 8 h after aldosterone administration. There appeared to be maximum increases of 23-30% and 25-32% after 2.5-3.5 h in the renal cortex and red medulla, respectively. 5 alpha-Dihydroaldosterone, a metabolite of aldosterone with mineralocorticoid activity, was also able to increase renal flavokinase concentrations by approximately 40%. However, dexamethasone, a potent glucocorticoid with little or no mineralocorticoid activity, appeared to have no effect on renal flavokinase, as observed by ELISA. These data suggest that the increase in the relative concentration of renal flavokinase may be due to increased biosynthesis of flavokinase, and ultimately, that renal flavokinase may be an aldosterone-induced protein whose synthesis is mediated through the mineralocorticoid receptor and RNA synthesis.     

Parathyroid hormone down-regulates 1,25-dihydroxyvitamin D receptors (VDR) and VDR messenger ribonucleic acid in vitro and blocks homologous up-regulation of VDR in vivo

1,25-Dihydroxyvitamin D3 [1,25(OH)2D3) is a known up-regulator of 1,25(OH)2D3 receptor (VDR) both in vitro and in vivo. However, a 5- to 10-fold increase in plasma 1,25(OH)2D3 induced by dietary calcium deficiency does not result in up-regulation of intestinal VDR, and kidney VDR is down-regulated. Under certain physiological stresses, an increase in plasma PTH precedes increased plasma 1,25(OH)2D3. Therefore, the present study examined the effect of PTH on VDR regulation in vitro in ROS 17/2.8 cells and in vivo in male Holtzman rats. Treatment of ROS cells with PTH (0-5 nM) resulted in a dose and time-dependent decline in VDR from 95 +/- 9 to 35 +/- 5 fmol/mg protein at 18 h of exposure. The ED50 for PTH was 1 nM. This decline in VDR protein was attended by a 50% decline in VDR messenger RNA (mRNA). The PTH-mediated down-regulation of VDR occurred without affecting the affinity of VDR for 1,25(OH)2D3 as determined by Scatchard analysis. Also, the effect of PTH on VDR regulation was specific since cell glucocorticoid receptor concentration was not affected by PTH treatment. In accompanying experiments, 1,25(OH)2[3H]D3 treatment of ROS cells was shown to result in a 3- to 4-fold increased expression of VDR and VDR mRNA. The simultaneous addition of PTH and 1,25(OH)2[3H]D3 resulted in inhibition of the 1,25(OH)2[3H]D3-mediated up-regulation of VDR and VDR mRNA. Similarly, PTH also inhibited heterologous up-regulation of VDR and VDR mRNA induced by retinoic acid. In in vivo experiments, rats infused for 5 days with 1,25(OH)2D3 (1.5 ng/h) increased their expression of intestinal VDR, kidney VDR, and kidney 24-hydroxylase by 31, 336, and 4000%, respectively. Coinfusion of PTH (1.8 IU/h) along with 1,25(OH)2D3 completely inhibited the 1,25(OH)2D3-mediated increases in intestinal VDR and kidney 24-hydroxylase and reduced the 1,25(OH)2D3-mediated up-regulation of kidney VDR by more than half. These data suggest that PTH is a potent down-regulator of VDR and that PTH and 1,25(OH)2D3 have opposing effects on the expression of certain genes.