Vitamin D Receptor Gene Expression Research Articles

Clinical significance of the overexpression of the candidate oncogene CYP24 in esophageal cancer

By using array comparative genomic hybridization (CGH), the increased copy number of CYP24 (which encodes vitamin D 24-hydroxylase) at 20q13.2 was previously reported, leading to the identification of CYP24 as a candidate oncogene in breast cancer. CYP24 leads to abrogate growth control mediated by vitamin D. We examined CYP24 expression as well as VDR (vitamin D receptor) gene expression in 42 esophageal cancer cases using semi-quantitative RT-PCR assay. We induced CYP24 in seven esophageal cancer cell lines using 25-hydroxyvitamin D3 [25(OH)D3] and compared cell growth rate, measured using the 3-(4, 5-dimethylthiazol-2-y)-2, 5-diphenyltetrazolium bromide (MTT) assay system. The overall survival rate was significantly higher in 25 cases of lower CYP24 expression than 17 cases of higher CYP24 expression (P <0.05); on the other hand, 23 cases of low VDR expression had a poorer prognosis than 19 cases of high VDR expression. Moreover, we disclosed that the inverse correlation between CYP24 and VDR expression is significant in esophageal cancer cases (P <0.05). Furthermore, the cell growth evaluated by MTT assay was greatly increased in CYP24-induced and VDR-diminished cells than non-responding cells by 25(OH)D3 activity (P <0.01). Overexpression of the candidate oncogene CYP24 is inversely correlated to vitamin D receptor expression, and may play an important role in determination of the malignant potential of esophageal cancer.

Exclusive underexpression of vitamin D receptor exon 1f transcripts in tumors of primary hyperparathyroidism.

Primary hyperparathyroidism (pHPT) is characterized by excessive production of parathyroid hormone (PTH) due to parathyroid adenomas while uremic secondary HPT (sHPT) is caused by parathyroid hyperplasia in response to renal failure. Active vitamin D, 1,25-dihydroxyvitamin D(3) (1,25-(OH)(2)D(3)), with the vitamin D receptor (VDR) is involved in regulation of the calcium homeostasis together with PTH. In a feedback loop, 1,25-(OH)(2)D(3) has a direct action on the parathyroid gland to regulate PTH transcription, PTH secretion and cell proliferation. We have previously demonstrated reduced VDR mRNA expression in parathyroid adenomas and hyperplasia of sHPT using a probe detecting all 14 variant VDR transcripts expressed in parathyroid cells. Here we have assessed which of the 5'-terminal exon 1a, 1d and 1f variant VDR transcripts are reduced in pathological parathyroid glands. The relative VDR/glyceraldehyde-3-phosphate dehydrogenase mRNA levels for each VDR exon were determined by real-time quantitative RT-PCR in five normal parathyroid glands, seventeen parathyroid adenomas and ten hyperplastic glands of sHPT. The results demonstrated exclusive underexpression of VDR exon 1f transcripts in parathyroid adenoma, while all measured VDR transcripts were reduced in secondary hyperplasia. We suggest that exclusive underexpression of VDR exon 1f transcripts in adenomas of pHPT, which derive from a distal promoter active in tIssues involved in calcium regulation by 1,25-(OH)(2)D(3), may either reflect a defective cell type-specific transcription factor or other physiologically important pathway(s) for tIssue-specific VDR gene expression.

Persistent downregulation of calcium-sensing receptor mRNA in rat parathyroids when severe secondary hyperparathyroidism is reversed by an isogenic kidney transplantation.

Experimental severe secondary hyperparathyroidism (HPT) is reversed within 1 wk after reversal of uremia by an isogenic kidney transplantation (KT) in the uremic rats. Abnormal parathyroid hormone (PTH) secretion in uremia is related to downregulation of CaR and vitamin D receptor (VDR) in the parathyroid glands (PG). The aim of this investigation was to examine the expression of CaR and VDR genes after reversal of uremia and HPT in KT rats. 5/6 nephrectomized rats were kept on a normal or high-phosphorus (hP) diet for 8 wk to induce severe HPT (n = 8 in each group). In another group of seven uremic hP rats, uremia was reversed by an isogenic KT and PG were harvested within 1 wk posttransplant. Plasma urea, creatinine, total calcium, phosphorus, and PTH levels were measured. Parathyroid CaR and VDR mRNA were measured by quantitative PCR. Uremic hP rats had significantly elevated levels of creatinine, urea, and phosphorus (P < 0.001) and developed significant hypocalcemia (plasma calcium 1.83 +/- 0.2 mmol/L; P < 0.001) compared with normal control rats. After KT, the levels were normalized from day 3 to 7: creatinine from 0.117 +/- 0.016 to 0.050 +/- 0.002 mmol/L; urea from 23 +/- 4 to 7 +/- 0.3 mmol/L; phosphorus from 3.9 +/- 0.6 to 1.5 +/- 0.06 mmol/L; calcium from 1.8 +/- 0.2 to 2.5 +/- 0.02 mmol/L. Plasma PTH levels fell from 849 +/- 224 to a normal level of 38 +/- 9 pg/ml (P < 0.01). In uremic rats on a standard diet, CaR mRNA was similar to that of normal control rats, whereas VDR mRNA was significantly decreased. In uremic rats kept on hP diet, CaR mRNA was significantly decreased to 26 +/- 7% of control rats (P = 0.01) and VDR mRNA reduced to 36 +/- 11% (P < 0.01). In KT, previously hP uremic rats, both CaR mRNA and VDR mRNA remained severely reduced (CaR, 39 +/- 7%; VDR, 9 +/- 3%; P < 0.01) compared with normal rats. In conclusion, circulating plasma PTH levels normalized rapidly after KT, despite persisting downregulation of CaR and VDR gene expression. This indicates that upregulation of CaR mRNA and VDR mRNA is not necessary to induce the rapid normalization of PTH secretion from hyperplastic parathyroid glands.

Clonal Differences in Expression of 25-Hydroxyvitamin D3-1α-hydroxylase, of 25-Hydroxyvitamin D3-24-hydroxylase, and of the Vitamin D Receptor in Human Colon Carcinoma Cells: Effects of Epidermal Growth Factor and 1α,25-Dihydroxyvitamin D3

Human colon carcinoma cells express 25-hydroxyvitamin D3-1α-hydroxylase (CYP27B1) and thus produce the vitamin D receptor (VDR) ligand 1α,25-dihydroxyvitamin D3 (1,25-D3), which can be metabolized by 25-hydroxyvitamin D3-24-hydroxylase (CYP24). Expression of VDR, CYP27B1, and CYP24 determines the efficacy of the antimitotic action of 1,25-D3 and is distinctly related to the degree of differentiation of cancerous lesions. In the present study we addressed the question of whether the effects of epidermal growth factor (EGF) and of 1,25-D3 on VDR, CYP27B1, and CYP24 gene expression in human colon carcinoma cell lines also depend on the degree of cellular differentiation. We were able to show that slowly dividing, highly differentiated Caco-2/15 cells responded in a dose-dependent manner to both EGF and 1,25-D3 by up-regulation of VDR and CYP27B1 expression, whereas in highly proliferative, less differentiated cell lines, such as Caco-2/AQ and COGA-1A and -1E, negative regulation was observed. CYP24 mRNA was inducible in all clones by 1,25-D3 but not by EGF. From the observed clonal differences in the regulatory effects of EGF and 1,25-D3 on VDR and CYP27B1 gene expression we suggest that VDR-mediated growth inhibition by 1,25-D3 would be efficient only in highly differentiated carcinomas even when under mitogenic stimulation by EGF.

The caudal-related homeodomain protein Cdx-2 regulates vitamin D receptor gene expression in the small intestine.

The actions of 1,25-dihydroxyvitamin D3 (1,25(OH)2 D3) are mediated through the nuclear vitamin D receptor (VDR). The regulation of VDR abundance plays an important role in determining the magnitude of the target cell response to 1,25(OH)2D3. The major physiological activity of 1,25(OH)2D3 is the regulation of calcium absorption in the small intestine, and the level of VDR is an important factor in this regulation. However, the characterization of VDR gene expression in the small intestine remains unknown. In the present study, we investigated the regulation of the human VDR (hVDR) gene expression in the small intestine. The 4.0 kb of the 5'-flanking region of the hVDR gene promoter was cloned and characterized by the measurement of luciferase activity and an electrophoretic mobility-shift assay (EMSA). With the EMSA, we found that Cdx-2 (a homeodomain protein-related caudal) binds to the sequence 5'-ATAAAAACTTAT-3' at -3731 to -3720 bp (hVD-SIF1) relative to the transcription start site of the hVDR promoter. This sequence is very similar to the human sucrase-isomaltase footprint 1 (SIF1) element. With a competition analysis and specific antibodies for Cdx-2, we demonstrated that Cdx-2 is able to activate VDR gene transcription by binding to this element. The mutation of the hVD-SIF1 sequence in the hVDR gene promoter markedly suppressed the transactivation of the reporter gene in Caco-2 cells. In addition, the DNA fragment (-3996 to -3286) containing the hVD-SIF1 binding site increased transcription when placed upstream of the herpes simplex virus thymidine kinase promoter. These findings suggest that Cdx-2 plays an important role in the intestine-specific transcription of the hVDR gene.

1,25‐dihydroxyvitamin D3 regulates the expression of VDR and NGF gene in Schwann cells in vitro

The vitamin D receptor (VDR) is a nuclear receptor that mediates the effect of the active metabolite of vitamin D3, the 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3). To investigate the potential role of this hormone in the peripheral nervous system, we have studied the VDR expression in Schwann cells. The VDR mRNA was detected by Northern blot analysis in rat primary cultures of Schwann cells, and its levels were strongly increased in the presence of 1,25-(OH)2D3. Using the mouse Schwann cell line, MSC80, we showed that concentrations as low as 10-10 M of hormone stimulated the expression of the VDR gene and strongly increased the amounts of activated VDR, capable of binding to the specific vitamin D responsive element (VDRE). We also found that 1,25-(OH)2D3 stimulated the expression of the nerve growth factor gene in MSC80. These data suggest a role for the hormone in the peripheral nervous system, possibly as a mediator actived in trauma. J. Neurosci. Res. 53:742–746, 1998. © 1998 Wiley-Liss, Inc.

1,25-Dihydroxyvitamin D3 regulates the expression of VDR and NGF gene in Schwann cells in vitro.

The vitamin D receptor (VDR) is a nuclear receptor that mediates the effect of the active metabolite of vitamin D3, the 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3). To investigate the potential role of this hormone in the peripheral nervous system, we have studied the VDR expression in Schwann cells. The VDR mRNA was detected by Northern blot analysis in rat primary cultures of Schwann cells, and its levels were strongly increased in the presence of 1,25-(OH)2D3. Using the mouse Schwann cell line, MSC80, we showed that concentrations as low as 10(-10) M of hormone stimulated the expression of the VDR gene and strongly increased the amounts of activated VDR, capable of binding to the specific vitamin D responsive element (VDRE). We also found that 1,25-(OH)2D3 stimulated the expression of the nerve growth factor gene in MSC80. These data suggest a role for the hormone in the peripheral nervous system, possibly as a mediator active in trauma.

1,25-Dihydroxyvitamin D3 regulates the expression of VDR and NGF gene in Schwann cells in vitro.

The vitamin D receptor (VDR) is a nuclear receptor that mediates the effect of the active metabolite of vitamin D3, the 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3). To investigate the potential role of this hormone in the peripheral nervous system, we have studied the VDR expression in Schwann cells. The VDR mRNA was detected by Northern blot analysis in rat primary cultures of Schwann cells, and its levels were strongly increased in the presence of 1,25-(OH)2D3. Using the mouse Schwann cell line, MSC80, we showed that concentrations as low as 10(-10) M of hormone stimulated the expression of the VDR gene and strongly increased the amounts of activated VDR, capable of binding to the specific vitamin D responsive element (VDRE). We also found that 1,25-(OH)2D3 stimulated the expression of the nerve growth factor gene in MSC80. These data suggest a role for the hormone in the peripheral nervous system, possibly as a mediator active in trauma.

Hyperresponsiveness of vitamin D receptor gene expression to 1,25-dihydroxyvitamin D3. A new characteristic of genetic hypercalciuric stone-forming rats.

Hypercalciuria in genetic hypercalciuric stone-forming (GHS) rats is accompanied by intestinal Ca hyperabsorption with normal serum 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] levels, elevation of intestinal, kidney, and bone vitamin D receptor (VDR) content, and greater 1,25(OH)2D3-induced bone resorption in vitro. To test the hypothesis that hyperresponsiveness of VDR gene expression to 1,25(OH)2D3 may mediate these observations, male GHS and wild-type Sprague- Dawley normocalciuric control rats were fed a normal Ca diet (0.6% Ca) and received a single intraperitoneal injection of either 1,25(OH)2D3 (10-200 ng/100 g body wt) or vehicle. Total RNAs were isolated from both duodenum and kidney cortex, and the VDR and calbindin mRNA levels were determined by Northern blot hybridization using specific cDNA probes. Under basal conditions, VDR mRNA levels in GHS rats were lower in duodenum and higher in kidney compared with wild-type controls. Administration of 1,25(OH)2D3 increased VDR gene expression significantly in GHS but not normocalciuric animals, in a time- and dose-dependent manner. In vivo half-life of VDR mRNA was similar in GHS and control rats in both duodenum and kidney, and was prolonged significantly (from 4-5 to > 8 h) by 1,25(OH)2D3 administration. Neither inhibition of gene transcription by actinomycin D nor inhibition of de novo protein synthesis with cycloheximide blocked the upregulation of VDR gene expression stimulated by 1,25(OH)2D3 administration. No alteration or mutation was detected in the sequence of duodenal VDR mRNA from GHS rats compared with wild-type animals. Furthermore, 1,25(OH)2D3 administration also led to an increase in duodenal and renal calbindin mRNA levels in GHS rats, whereas they were either suppressed or unchanged in wild-type animals. The results suggest that GHS rats hyperrespond to minimal doses of 1,25(OH)2D3 by an upregulation of VDR gene expression. This hyperresponsiveness of GHS rats to 1,25(OH)2D3 (a) occurs through an increase in VDR mRNA stability without involving alteration in gene transcription, de novo protein synthesis, or mRNA sequence; and (b) is likely of functional significance, and affects VDR-responsive genes in 1, 25(OH)2D3 target tissues. This unique characteristic suggests that GHS rats may be susceptible to minimal fluctuations in serum 1, 25(OH)2D3, resulting in increased VDR and VDR-responsive events, which in turn may pathologically amplify the actions of 1,25(OH)2D3 on Ca metabolism that thus contribute to the hypercalciuria and stone formation.

Vitamin D receptor expression is required for growth modulation by 1 alpha,25-dihydroxyvitamin D3 in the human prostatic carcinoma cell line ALVA-31.

Epidemiological data suggest that vitamin D3, obtained from dietary sources and sunlight exposure, protects against mortality from prostate cancer (PC). In agreement with this, the most active vitamin D metabolite 1 alpha,25-dihydroxyvitamin D3 [1,25(OH)2 D3] regulates the growth and differentiation of several human PC cell lines. Both genomic and non-genomic signalling pathways for 1,25(OH)2 D3 have been reported, although the mechanism of action in PC cells has not been defined. We now provide data supporting an active role for the nuclear vitamin D receptor (VDR) in mediating the growth-inhibitory effects of 1,25(OH)2 D3 on these cells. In the VDR-rich cell line ALVA-31, the observed changes in growth by 1,25(OH)2 D3 are preceded by significant changes in VDR mRNA expression. In contrast, the cell line JCA-1, containing few VDRs, fails to show both early changes in VDR gene expression and later changes in growth with 1,25(OH)2 D3. To assess the role of the VDR more directly, transfection studies were pursued. ALVA-31 cells were stably transfected with an antisense VDR cDNA construct in an attempt to reduce VDR expression. Antisense mRNA expression among clones was associated with: (a) reduced or abolished sensitivity to the effects of 1,25(OH)2 D3 on growth; (b) decreased numbers of VDRs per cell, as measured by radiolabelled-ligand binding; and (c) a lack of induction of the VDR-regulated enzyme 24-hydroxylase in response to 1,25(OH)2 D3. From these studies we conclude that the antiproliferative effects of 1,25(OH)2 D3 require expression of the nuclear VDR in this system.

Structure-function relationship of human parathyroid hormone in the regulation of vitamin D receptor expression in osteoblast-like cells (ROS 17/2.8).

Studies of the relationship between PTH structure and function in the activation of protein kinases have revealed that different regions within the biologically active PTH-(1-34) peptide are responsible for different functions. The first two N-terminal amino acids are required for plasma membrane adenylyl cyclase stimulation, and the C-terminal region 29-32 is necessary for the translocating activity of protein kinase C. In the present study, we explored the structure-function relationship of human (h) PTH in the regulation of the vitamin D receptor (VDR) in osteoblast-like cells (ROS 17/2.8). VDR-rich cytosol extract was prepared after the confluent cells were incubated with different hPTH fragments for 16 h. hPTH-(1-34) at concentrations of 10(-9)-10(-7) M caused a dose-dependent decrease in VDR content from a control level of 70.2 +/- 2.2 fmol/mg protein to 62.1 +/- 3.3 (-16%) at 10(-9) M, 52.3 +/- 5.3 (-25.5%; P < 0.02) at 10(-8) M, and 45.5 +/- 3.5 fmol/mg protein (-35.3%; P = 0.001) at 10(-7) M (n = 6). hPTH-(1-31) also decreased VDR content from 65.5 +/- 3.6 to 55.2 +/- 7.9 (-19.5%) at 10(-9) M, 44.3 +/- 5.8 (-32.4%; P < 0.05) at 10(-8) M, and 40.6 +/- 3.2 fmol/mg protein (-38.9%; P < 0.05) at 10(-7) M (n = 6). Incubation of ROS 17/2.8 cells with 0.5 nM 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] led to up-regulation of VDR content by 340-370% of the control value. hPTH-(1-34) decreased the VDR up-regulatory effect of 1,25-(OH)2D3 from 340% to 230% of the control value at 10(-8) M (P < 0.0001) and 170% of the control value (P < 0.0001) at 10(-7) M, respectively (n = 6). hPTH-(1-31) also decreased the receptor up-regulatory effect of 1,25-(OH)2D3 from 370% to 286% (P < 0.02) at 10(-8) M and 220% (P < 0.002) at 10(-7) M, respectively (n = 6). hPTH-(3-34) and -(13-34) at concentrations of 10(-9)-10(-7) M did not decrease VDR content in either the absence or presence of 1,25-(OH)2D3. Quantitation of VDR messenger RNA by reverse transcription-polymerase chain reaction showed that PTH-(1-34) and -(1-31) at 10(-7) M, but not PTH-(3-34) and -(13-34), inhibited ROS 17/2.8 cell VDR gene expression in both the absence and presence of 1,25-(OH)2D3.(ABSTRACT TRUNCATED AT 400 WORDS)

Structure-function relationship of human parathyroid hormone in the regulation of vitamin D receptor expression in osteoblast-like cells (ROS 17/2.8)

Studies of the relationship between PTH structure and function in the activation of protein kinases have revealed that different regions within the biologically active PTH-(1-34) peptide are responsible for different functions. The first two N-terminal amino acids are required for plasma membrane adenylyl cyclase stimulation, and the C-terminal region 29-32 is necessary for the translocating activity of protein kinase C. In the present study, we explored the structure-function relationship of human (h) PTH in the regulation of the vitamin D receptor (VDR) in osteoblast-like cells (ROS 17/2.8). VDR-rich cytosol extract was prepared after the confluent cells were incubated with different hPTH fragments for 16 h. hPTH-(1-34) at concentrations of 10(-9)-10(-7) M caused a dose-dependent decrease in VDR content from a control level of 70.2 +/- 2.2 fmol/mg protein to 62.1 +/- 3.3 (-16%) at 10(-9) M, 52.3 +/- 5.3 (-25.5%; P < 0.02) at 10(-8) M, and 45.5 +/- 3.5 fmol/mg protein (-35.3%; P = 0.001) at 10(-7) M (n = 6). hPTH-(1-31) also decreased VDR content from 65.5 +/- 3.6 to 55.2 +/- 7.9 (-19.5%) at 10(-9) M, 44.3 +/- 5.8 (-32.4%; P < 0.05) at 10(-8) M, and 40.6 +/- 3.2 fmol/mg protein (-38.9%; P < 0.05) at 10(-7) M (n = 6). Incubation of ROS 17/2.8 cells with 0.5 nM 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] led to up-regulation of VDR content by 340-370% of the control value. hPTH-(1-34) decreased the VDR up-regulatory effect of 1,25-(OH)2D3 from 340% to 230% of the control value at 10(-8) M (P < 0.0001) and 170% of the control value (P < 0.0001) at 10(-7) M, respectively (n = 6). hPTH-(1-31) also decreased the receptor up-regulatory effect of 1,25-(OH)2D3 from 370% to 286% (P < 0.02) at 10(-8) M and 220% (P < 0.002) at 10(-7) M, respectively (n = 6). hPTH-(3-34) and -(13-34) at concentrations of 10(-9)-10(-7) M did not decrease VDR content in either the absence or presence of 1,25-(OH)2D3. Quantitation of VDR messenger RNA by reverse transcription-polymerase chain reaction showed that PTH-(1-34) and -(1-31) at 10(-7) M, but not PTH-(3-34) and -(13-34), inhibited ROS 17/2.8 cell VDR gene expression in both the absence and presence of 1,25-(OH)2D3.

Vitamin D receptor gene expression in mammalian kidney.

The vitamin D-receptor protein and its mRNA were localized in microscope sections of paraffin-embedded mammalian kidneys by means of immunocytochemistry and in situ hybridization, respectively. A monoclonal antibody against chicken intestinal vitamin D receptor immunostained the nucleus and cytoplasm of cells within the distal convoluted tubule, connecting segment, and initial cortical collecting duct of both rats and pigs. Although fainter, immunostaining also was present over proximal tubular cells. (35S)UTP-labeled cRNA probes were detected over both the proximal and distal portions of the mouse nephron, but silver grain densities were 5.8-fold greater over the latter. In conclusion, localization of both the vitamin D-receptor protein and its mRNA in both the proximal and distal nephron of adult mammals suggests that the gene for this protein is expressed in cells at both of these sites. The intensity of immunostaining and the density of cRNA-associated silver grains suggest that vitamin D-receptor gene expression is greatest in the distal nephron.

Hypercalciuria: lessons from studies of genetic hypercalciuric rats.

Human idiopathic hypercalciuria (IH) is a common cause of hypercalciuria that contributes to calcium oxalate nephrolithiasis. The disorder is characterized by normocalcemia, increased intestinal Ca absorption, and normal or elevated circulating 1,25(OH)2D3. Intestinal Ca hyperabsorption, which is a source of excess urine Ca excretion, may result from either a primary increase in renal 1,25(OH)2D3 production; a primary, vitamin D-independent defect in enterocyte regulation of Ca transport; or a secondary increase in 1,25(OH)2D3 production in response to a defect in renal tubular Ca reabsorption. Breeding male and female Sprague Dawley rats with spontaneous hypercalciuria has resulted in offspring with hypercalciuria, increased intestinal Ca absorption, and normal serum 1,25(OH)2D3. In male IH rats, vitamin D receptor (VDR) content measured by saturation binding and western blotting revealed a twofold increase in VDR number in the duodenum, kidney cortex, and splenic monocytes. The molecular basis for the increase in VDR appears not to be due to increased VDR gene expression, but may result from increased efficiency of translation of the VDR message or prolongation of the half-life of VDR. Comparable migration of normal and IH intestinal VDR on western blots and of intestinal VDR mRNA on northern blots suggests that the abundant VDR in IH rat intestine is not a mutation of the wild-type VDR. These observations strongly suggest that, in IH rats, normal serum 1,25(OH)2D3 and increased VDR results in increased VDR-1,25(OH)2D3 complexes and enhanced biologic actions of 1,25(OH)2D3, including increased intestinal Ca transport. IH in rats may be the first genetic disorder due to a pathologic increase in the VDR.

Regulation of 1,25-dihydroxyvitamin D 3 receptor gene expression by parathyroid hormone and cAMP-agonists

We studied the effect of parathyroid hormone (PTH) and activation of the cAMP signal pathway on vitamin D receptor (VDR) mRNA levels in the phenotypically osteoblast cell line UMR 106. PTH caused a time- and dose-dependent increase of the VDR mRNA content with a maximum after 2 h. After 24 h the VDR mRNA level in PTH-treated cells returned to control level. In contrast, the 1,25-dihydroxyvitamin D 3 (1,25(OH) 2D 3)-induced increase in VDR mRNA did not decline after 24 h. Inhibition of transcription with actinomycin D (10 μg/ml) completely abolished the PTH-induced increase of VDR mRNA and inhibition of translation with cycloheximide (1 μg/ml) resulted in superinduction of VDR mRNA. The role of cAMP in the induction of VDR mRNA was studied with several agents acting via the cAMP pathway. Incubation for 2 and 4 h with forskolin, Bt 2cAMP, PTHrP or prostaglandin E 2 caused an increase in the level of VDR mRNA comparable to that caused by PTH. The calcium ionophore A23187 did not affect VDR mRNA level. The present study demonstrates that PTH and activation of the cAMP signal pathway cause up-regulation of VDR via induction of VDR gene expression. The effect of cAMP on the VDR gene is suggestive for a cAMP responsive element in the VDR gene.

Effect of glucocorticoids and 1,25-dihydroxyvitamin D3 on the developmental expression of the rat intestinal vitamin D receptor gene.

In this study the ontogenesis of rat intestinal vitamin D receptor (VDR) gene expression was examined. When Northern and slot blot analyses were used to examine the expression of intestinal VDR mRNA in 15-, 18-, 22-, and 28-day-old rats, induction of VDR mRNA was not observed until 22 days postpartum. Since little is known, particularly in the neonate, concerning the in vivo regulation of VDR gene expression, we examined the possibility that glucocorticoids and/or 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] could affect the developmental expression of the intestinal VDR gene. To examine the effect of glucocorticoids, rat pups received three sequential injections (one per day) of hydrocortisone (5, 2.5, and 2.5 mg/100 g BW). Hydrocortisone administration before day 14 or on days 19-21 was not effective in inducing VDR mRNA. However, a significant 3.8-fold increase in intestinal VDR mRNA was observed in rats injected with hydrocortisone from days 15-17. The hydrocortisone effective period coincides with the glucocorticoid-sensitive period of rat intestinal development. It should be noted, however, that the up-regulation of VDR was accompanied by an increase in actin mRNA, suggesting that the effect is not specific for VDR. Similarly, when rats were bilaterally adrenalectomized on day 17 (killed on day 22), a 4-fold decrease in VDR mRNA was observed, accompanied by a decrease in actin mRNA. However, when rats were injected with 1,25-(OH)2D3 (25 ng/day.100 g BW) from days 15-17, levels of intestinal VDR mRNA were significantly increased by 1.5-fold, and this change was specific for VDR mRNA. In summary, our results indicate that hydrocortisone and 1,25-(OH)2D3 can precociously induce intestinal VDR mRNA, suggesting the involvement of glucocorticoids and 1,25-(OH)2D3 in the regulation of VDR gene expression in the developing rat intestine. However, our results also indicate that the effect of glucocorticoids (unlike the effect of 1,25-(OH)2D3) is not specific for VDR mRNA, but may reflect general effects of glucocorticoids on intestinal maturation.

Differentiation‐related regulation of 1,25 dihydroxy vitamin D3 receptor mRNA in human leukaemia cells HL‐60

Vitamin D receptor (VDR) is a nuclear protein which mediates the physiological actions of its hormone ligand, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). While it appears that the receptor-hormone complex regulates the expression of hormone-dependent genes involved in mineral homeostasis, its role in induction of differentiation of leukaemic cells is less clear. We have studied the expression of the VDR gene in several sublines of HL-60 leukaemic cells with varying responsiveness to 1,25(OH)2D3. Sublines which rapidly differentiated to monocytic forms were shown to contain elevated steady-state levels of VDR mRNA within 1 h of exposure to high concentration of 1,25(OH)2D3. This up-regulation of the expression of VDR was not apparent in sublines in which monocytic differentiation occurred after a delay of several days. Beginning at approximately 3 h after exposure to 1,25(OH)2D3 in most cases, there was a gradual decline in VDR mRNA levels. Measurement of steady-state levels of mRNA for c-myc and c-fos showed that in sublines of HL-60 cells which respond rapidly to 1,25(OH)2D3, elevation of VDR mRNA is evident prior to the changes in proto-oncogene expression. These data are consistent with the hypothesis that a change in VDR gene expression is one of the steps that promote monocytic differentiation.

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.