VDR mRNA Research Articles

Influence of the vitamin D receptor alleles on human osteoblast-like cells.

We investigated the effect of vitamin D receptor gene (VDRG) polymorphism on the responsiveness to 1,25(OH)2D3 in human osteoblast-like cells. The cells were obtained from the femoral heads of 18 women with osteoarthritis of the hip. Three different restriction enzymes, BsmI, ApaI, and TaqI, were used to analyse the polymorphism. The genotypes of the 18 patients were bbAaTT (8), bbaaTT (6), BbAaTt (3), and BbAATt (1). Our findings showed that there were no differences according to the VDR genotype, but there was a statistically significant difference in the production of osteocalcin between BbAaTt and bbAaTT, and between BbAaTt and bbaaTT. Northern blot analysis of osteocalcin and VDR mRNA showed no significant differences among the three VDR genotypes. These findings suggest that VDR gene polymorphism affects the individual responsiveness of 1,25(OH)2D3.

Influence of the vitamin D receptor alleles on human osteoblast-like cells

We investigated the effect of vitamin D receptor gene (VDRG) polymorphism on the responsiveness to 1,25(OH)2D3 in human osteoblast-like cells. The cells were obtained from the femoral heads of 18 women with osteoarthritis of the hip. Three different restriction enzymes, BsmI, ApaI, and TaqI, were used to analyse the polymorphism. The genotypes of the 18 patients were bbAaTT (8), bbaaTT (6), BbAaTt (3), and BbAATt (1). Our findings showed that there were no differences according to the VDR genotype, but there was a statistically significant difference in the production of osteocalcin between BbAaTt and bbAaTT, and between BbAaTt and bbaaTT. Northern blot analysis of osteocalcin and VDR mRNA showed no significant differences among the three VDR genotypes. These findings suggest that VDR gene polymorphism affects the individual responsiveness of 1,25(OH)2D3.

Reduced parathyroid vitamin D receptor messenger ribonucleic acid levels in primary and secondary hyperparathyroidism.

Vitamin D, via its receptor (VDR), inhibits the hormone secretion and proliferation of parathyroid cells. Vitamin D deficiency and reduced parathyroid VDR expression has been associated with development of hyperparathyroidism (HPT) secondary to uremia. VDR polymorphisms may influence VDR messenger RNA (mRNA) levels and have been coupled to an increased risk of parathyroid adenoma of primary HPT. VDR mRNA relative to glyceraldehyde-3-phosphate dehydrogenase mRNA levels were determined by RNase protection assay in 42 single parathyroid adenomas of patients with primary HPT, 23 hyperplastic glands of eight patients with uremic HPT, and 15 normal human parathyroid glands. The adenomas and hyperplasias demonstrated similar VDR mRNA levels, which were reduced (42 +/- 2.8% and 44 +/- 4.0%) compared with the normal glands (P < 0.0001). Comparison of parathyroid adenoma with a normal-sized parathyroid gland of the same individual (n = 3 pairs) showed a 20-58% reduction in the tumor. Nodularly enlarged glands represent a more advanced form of secondary HPT and showed greater reduction in the VDR mRNA levels than the diffusely enlarged glands (P < 0.005). The reduced VDR expression is likely to impair the 1,25(OH)2D3-mediated control of parathyroid functions, and to be of importance for the pathogenesis of not only uremic but also primary HPT. Circulating factors like calcium, PTH, and 1,25(OH)2D3 seem to be less likely candidates mediating the decreased VDR gene expression in HPT.

Improved Cholecalciferol Nutrition in Rats Is Noncalcemic, Suppresses Parathyroid Hormone and Increases Responsiveness to 1,25-Dihydroxycholecalciferol

We examined how cholecalciferol (vitamin D) nutrition affected serum 25-hydroxycholecalciferol (25(OH)D) and 1, 25-dihydroxycholecalciferol (1,25(OH)(2)D). Rats were fed conventional diet (vitamin D, 4.5 IU/g, or 7 nmol/d) or the same diet plus 18 nmol/d of extra vitamin D for 3 wk. The extra vitamin D resulted in greater serum 25(OH)D (51 +/- 3, vs. control of 21 +/- 2 nmol/L), and kidney mRNA for vitamin D receptor [VDR mRNA] (P = 0. 026) and lower serum 1,25(OH)(2)D (72 +/- 16 vs. control of 161 +/- 10 pmol/L, P = 0.001), and parathyroid hormone (PTH) (89 +/- 4 vs. control of 160 +/- 15 ng/L, P = 0.001). Kidney VDR mRNA relative to GAPDH mRNA correlated inversely with serum 1,25(OH)(2)D (r = -0.714, P = 0.006). There were no differences in serum calcium, phosphate, alkaline phosphatase, or weight gain. Experiment 2 compared groups supplemented with 0.2, 2 or 20 nmol/d of vitamin D orally, or 20 nmol/d dermally to see how vitamin D nutrition influenced the response of 1,25(OH)(2)D to changes in diet calcium. Vitamin D did not affect urinary calcium or pyridinoline excretion, serum calcium, phosphate, vitamin D binding protein or alkaline phosphatase. In groups given 20 nmol/d of vitamin D, renal mitochondrial 25(OH)D-1alpha-hydroxylase was lower (P < 0.01) and 25(OH)D-24-hydroxylase was higher (P < 0.05). Higher 25(OH)D concentration was related to proportionally lower 1,25(OH)(2)D at every calcium intake, indicating greater tissue sensitivity to 1, 25(OH)(2)D. We conclude suppression of 1,25(OH)(2)D and PTH, and higher renal VDR mRNA and 24-hydroxylase did not involve higher free 1,25(OH)(2)D concentration or a first pass effect at the gut. Thus, 25(OH)D or a metabolite other than 1,25(OH)(2)D is a physiological, transcriptionally and biochemically active, noncalcemic vitamin D metabolite.

1,25-Dihydroxyvitamin D3 Hypersensitivity of Osteoclast Precursors from Patients with Paget's Disease

Our previous studies suggested that increased osteoclast formation and activity in Paget's disease may be related in part to increased responsiveness of highly purified osteoclast precursors to 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]. However, the basis for this enhanced sensitivity to 1,25-(OH)2D3 is unclear. To address this question, we examined 24-hydroxylase and 1,25-(OH)2D3 receptor (VDR) messenger RNA (mRNA) expression during human osteoclast differentiation from normal subjects and patients with Paget's disease in response to 1,25-(OH)2D3 as well as VDR content and affinity. Reverse-transcription polymerase chain reaction (RT-PCR) analysis of granulocyte-macrophage colony-forming unit (GM-CFU), the earliest identifiable osteoclast precursor, derived from patients with Paget's disease demonstrated 24-hydroxylase mRNA expression in response to 1,25-(OH)2D3 was induced at concentrations of 1,25-(OH)2D3 that were at least one log less than that required for normal GM-CFU. VDR mRNA and VDR protein were detected in both immature and more differentiated osteoclast precursors, as well as in osteoclast-like multinucleated cells (MNCs). However, VDR expression was lower in MNCs than the mononuclear precursor cells. Osteoclast precursors and MNCs from patients with Paget's disease had levels of VDR expression similar to those of normal subjects but showed increased VDR affinity for 1,25-(OH)2D3. Because the effects of 1,25-(OH)2D3 are in part mediated by induction of expression of RANK ligand on marrow stromal cells, which in turn stimulates osteoclast formation, we examined expression of RANK ligand mRNA by marrow stromal cell lines derived from patients with Paget's disease and normal subjects in response to 1,25-(OH)2D3. RT-PCR analysis showed no difference in sensitivity of marrow stromal cells to 1,25-(OH)2D3 from normal subjects or patients with Paget's disease although the Paget's stromal cells expressed increased basal levels of RANK ligand mRNA. These results show that VDR protein is expressed in early and more differentiated osteoclast precursors, that expression levels of VDR decline with osteoclast differentiation, and that 1,25-(OH)2D3 has direct effects on osteoclast precursors. The enhanced sensitivity to 1,25-(OH)2D3 is an intrinsic property of osteoclast precursors from patients with Paget's disease that distinguishes them from normal osteoclast precursors. Furthermore, our results suggest that an increased affinity of VDR for 1,25-(OH)2D3 may be responsible for the enhanced 1,25-(OH)2D3 sensitivity of osteoclast precursors in patients with Paget's disease compared with normal subjects.

Analysis of 1,25-Dihydroxyvitamin D 3 Receptors (VDR. in Basal Cell Carcinomas

We have analyzed expression of 1,25-dihydroxyvitamin D(3) receptor (VDR) protein and mRNA in basal cell carcinomas (BCC) of human skin. VDR immunoreactivity in BCCs was compared with the staining pattern of the proliferation marker Ki-67 in the same tumors. Additionally, VDR staining was compared to staining pattern of apoptotic cells by terminal UTP nucleotide end labeling assay. Frozen sections of superficial type, nodular type, and fibrosing type BCCs were consistently immunoreactive for VDR (mAb 9A7gamma) with almost every tumor cell labeled (n = 15). In general, VDR staining was pronounced in peripheral tumor cells. VDR immunoreactivity was consistently stronger in tumor cells than in adjacent or unaffected epidermis. No visual correlation was found in BCCs comparing labeling patterns of Ki-67-positive or apoptotic cells and mAb 9A7gamma. VDR mRNA was increased in BCCs (n = 6) compared to normal human skin (n = 5), as revealed by reverse transcription-polymerase chain reaction analysis. Our findings indicate that VDR is strongly expressed in BCCs and may be involved in the growth regulation of this tumour, and VDR mRNA and protein are increased in BCCs as compared to normal human epidermis.

Vitamin D receptor in IMR-90 human fibroblasts and antiproliferative effect of 1,25-dihydroxyvitamin D3.

This study examines the effect of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], serum or forskolin on the proliferation of IMR-90 fetal lung fibroblasts and demonstrates, for the first time, the presence of 1,25(OH)2D3 receptor (VDR) in this cell line. In quiescent, subconfluent cultures neither the treatment with 100 nM 1,25(OH)2D3 nor that with 50 microM forskolin influenced proliferation, while a significant increase was observed after incubation of the cells with 10% fetal bovine serum. Either cell number, determined on growing IMR-90 human fibroblasts after 48 or 72 h incubation with 100 nM 1,25(OH)2D3 or [3H]thymidine incorporation (24, 48 or 72 h incubation) significantly decreased, while protein content per cell increased. Northern blot analysis revealed the expression of the VDR gene, the VDR mRNA bands being prominent in 1,25(OH)2D3, serum or forskolin treated fibroblasts. VDR mRNA levels slightly decreased, when growing fibroblasts were exposed to 100 nM 1,25(OH)2D3 for 48 or 72 h.

Quantification of vitamin D receptor mRNA in tissue sections demonstrates the relative limitations of in situ-reverse transcriptase-polymerase chain reaction.

In situ-reverse transcriptase-polymerase chain reaction (IS-RT-PCR) is a recently described technique that is used to localize low levels of mRNA within cells and tissue sections. One of the major criticisms levelled at this technique is that positive results may be meaningless, as amplification is required to demonstrate the transcripts of interest. The use of IS-RT-PCR to demonstrate mRNA for receptors for 1,25-dihydroxyvitamin D3 (VDR) in sections of human kidney and bone has previously been described. To ascertain whether the levels of VDR mRNA detected following IS-RT-PCR were transcriptionally significant, computerized image analysis was used to determine the mean silver grain density in human kidney and bone cells following conventional in situ hybridization and after various cycles of IS-RT-PCR. Only a few cycles of PCR were needed to produce an optimum signal, but amplification of signal following IS-RT-PCR was found to be relatively inefficient. Following the optimum number of cycles of IS-RT-PCR in kidney sections, there was a less than four-fold increase in signal. Similarly, in bone, the optimum signal detected was only approximately five times greater than that found with conventional in situ hybridization. These results clearly demonstrate that the increase in signal following IS-RT-PCR follows a more linear pattern and is relatively inefficient, compared with the usual exponential increase with conventional solution phase RT-PCR.

Demonstration of vitamin D receptor expression in a human megakaryoblastic leukemia cell line: Regulation of vitamin D receptor mRNA expression and responsiveness by forskolin

We have shown earlier that 1,25-dihydroxyvitamin D 3[1,25(OH) 2D 3] induces cell growth suppression and cell differentiation of a human megakaryoblastic leukemia cell line, HIMeg. However, the molecular mechanism of 1,25(OH) 2 D 3 action is still unknown. Prompted by this, we have searched here for the presence of 1,25(OH) 2 D 3 receptor (VDR) expression in HIMeg cells by reverse transcription-polymerase chain reaction (RT-PCR). The amplified product showed an identical size to the product amplified from the control human VDR cDNA and hybridized specifically with the digoxigenin-labeled human VDR cDNA fragment. As expected, VDR mRNA is also expressed in HOS-8603, a human osteosarcoma cell line. These results represent the first reported evidence that VDR mRNA is expressed in megakaryoblastic cells. In addition, the regulation of VDR mRNA expression in HIMeg cells was studied by quantitative RT-PCR. It was found the VDR mRNA expression in HIMeg cells could be down-regulated rapidly by 1,25(OH) 2 D 3 (10 nM) in a time-dependent manner, reaching a maximal reduction to about 15% of control. However, VDR mRNA expression in HOS-8603 cells was not regulated by 1,25(OH) 2 D 3 at any time-point tested. Treatment of HIMeg cells with forskolin (1 μM), an activator of adenylate cyclase, caused an increase in VDR mRNA levels. Similarly, VDR mRNA expression in HOS-8603 cells was also up-regulated by forskolin. Consistent with the functionality of the VDR in other target cells, we found that the up-regulation of VDR expression in HIMeg cells by forskolin was accompanied by an increased responsiveness of HIMeg cells to 1,25(OH) 2 D 3, even though forskolin alone had no effects. Exposure to 1,25(OH) 2 D 3 in combination with forskolin resulted in a much more significant inhibition of cell proliferation than to 1,25(OH) 2 D 3 alone. Similarly, forskolin could also augment the differentiation induced by 1,25(OH) 2 D 3, reflected by a more evident morphological change and a higher percentage of development of cells with multilobular nuclei. These alterations were accompanied by a loss of clonogenic capacity and a decrease in the number of cells in the S phase. These data establish that HIMeg cells express functional VDR, which served to mediate actions of its ligand on the proliferation and differentiation of these cells.

Simian virus 40-, but not human papillomavirus-, transformation of prostatic epithelial cells results in loss of growth-inhibition by 1,25-dihydroxyvitamin D-3

In addition to its well known calcemic actions, 1,25-dihydroxyvitamin D-3 [1,25(OH)(2)D] exhibits differentiating and antiproliferative effects in several types of cancer cells. 1,25(OH)(2)D receptors (VDR) as well as 1,25(OH)(2)D-mediated growth-inhibition have been demonstrated in human prostate cancer cell lines. In order to further develop model systems for the study of 1,25(OH)(2)D action and to elucidate the mechanism of growth-inhibition, we studied several human prostate cell lines immortalized with either simian virus 40 (SV40) or human papillomavirus type 18 (HPV). The SV40-transformed cell lines P69SV40-T and P153SV40-T were not growth-inhibited by 1,25(OH)(2)D at concentrations as high as 100 nM, whereas the HPV-transformed cells PZ-HPV-7 and CA-HPV-10 were growth-inhibited. All cell lines expressed VDR, and VDR mRNA was demonstrated by Northern blot analysis. All cells exhibited induction of 24-hydroxylase mRNA, a 1,25(OH)(2)D responsive gene, after 1,25(OH)(2)D treatment. In an attempt to understand the apparent dissociation of 1,25(OH)(2)D actions in the SV40-transformed cells, we turned to the human prostate cancer cell line DU 145. These cells, like the SV40-transformed cells, are not growth-inhibited but demonstrate induction of 24-hydroxylase mRNA after 1,25(OH)(2)D treatment. DU 145 cells contain a mutated retinoblastoma gene (Rb) which contributes to their uncontrolled growth, analogous to the disruption of Rb by SV40 and HPV. We compared DU,145 cells to DU 145 cells transfected with normal Rb (DU 145/Rb). Similar to DU 145, DU 145/Rb cells were not growth-inhibited by 1,25(OH)(2)D, while 24-hydroxylase mRNA was induced. These results suggest that divergent pathways mediate the growth-inhibitory effect of 1,25(OH)(2)D and its induction of 24-hydroxylase. It also appears that the antiproliferative effect of 1,25(OH)(2)D is mediated by an Rb-independent mechanism.

17 beta-estradiol increases the receptor number and modulates the action of 1,25-dihydroxyvitamin D3 in human osteosarcoma-derived osteoblast-like cells.

It is well known that 17 beta-estradiol (E2) and 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) have important roles in bone metabolism. This study was undertaken to examine E2 regulation of 1,25(OH)2D3 receptor (VDR) expression and the biological action of 1,25(OH)2D3 in human osteoblast-like cells. When human osteosarcoma-derived osteoblast-like cells were treated with varying concentrations of E2, the VDR levels increased by up to 100% in a dose-dependent manner. VDR levels significantly increased at 10 nM E2 and this increase plateaued at 100 nM E2. E2-dependent increase of VDR was time dependent, plateauing at 24 hours and was maintained for at least 48 hours in human osteoblast-like cells. Scatchard analysis showed that E2 increased the number of VDR (12.3 +/- 0.4 versus 26.5 +/- 0.3 fmol/mg protein; mean +/- SE of three independent experiments) rather than the Kd (0.15 +/- 0.02 versus 0.16 +/- 0.01 nM; mean +/- SE of three independent experiments). Tamoxifen (50 nM), a specific competitor with E2, completely abolished the E2-induced increase of VDR. The levels of VDR mRNA (4.5 kb) from the cells increased in a dose-dependent manner after E2 treatment. With regard to the biological effects, E2 increased by 10-25% the inhibitory effect of 1,25(OH)2D3 on cell growth. However, E2 did not increase the stimulation of alkaline phosphatase activity by 1,25(OH)2D3. The present study suggests that E2 modulates the biological action of 1,25(OH)2D3 through VDR levels in bone cells.

Effects of Presence of an Egg and Calcium Deposition in the Shell Gland on Levels of Messenger Ribonucleic Acid of CaBP-D 28K and of Vitamin D 3 Receptor in the Shell Gland of the Laying Hen

Levels of calbindin-D 28K (CaBP D 28K) mRNA and vitamin D 3, 1α, 25(OH) 2D 3, receptor (VDR) mRNA in the intestine and shell gland were measured in the laying hen during the ovulatory cycle by Northern and slot blot analyses. In addition, effects of presence of an egg and calcium deposition in the shell gland on the levels of both mRNAs were studied either by delay in oviposition (retaining the egg in the shell gland beyond the expected time of oviposition) or premature oviposition (emptying the shell gland and arrest of calcium deposition). During the ovulatory cycle mRNA levels of CaBP-D 28K and of VDR remained relatively constant in the intestine. In contrast, both mRNA levels of the shell gland were low when there was an egg in the infundibulum, magnum, and isthmus, but significantly increased when there was an egg in the shell gland during shell formation. The im injection of indomethacin 3 hr before expected ovulation delayed oviposition but increased neither shell thickness nor mRNA levels of CaBP-D 28K and of VDR. On the other hand, premature oviposition reduced mRNA levels of both CaBP-D 28K and VDR in the shell gland. These results suggest that the presence of an egg and calcium deposition in the shell gland may be a stimulatory factor for synthesis and accumulation of CaBP-D 28K mRNA and VDR mRNA in association with calcification.

1,25(OH)2D3‐dependent regulation of calbindin‐D28k mRNA requires ongoing protein synthesis in chick duodenal organ culture

Organ culture of 19-day-old chick embryo duodena was utilized to evaluate the mechanism of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3)-dependent calbindin-D28k (CaBP) expression. Duodenal CaBP and 1,25(OH)2D3 receptor (VDR) expression were assessed by Western blot analysis, while CaBP and VDR mRNA levels were determined by Northern blot analysis. In untreated duodena, both VDR protein and mRNA were present, while CaBP protein and mRNA were undetectable. Treatment of cultured duodena with 25 nM 1,25(OH)2D3 resulted in detectable CaBP mRNA after 4 h which continued to increase during a 24 h time period. Under these conditions, localization of [3H-1 beta]1 alpha,25(OH)2D3 in duodenal chromatin is rapid (< or = 30 min). Thus, the delayed accumulation of detectable CaBP mRNA cannot be explained by slow nuclear binding of 1,25(OH)2D3. The inclusion of 1.6 microM actinomycin D in the organ culture partially inhibited the 1,25(OH)2D3-regulated increase in CaBP mRNA, which implies that there is a transcriptional component involved in the increased CaBP mRNA levels. Similarly, quantitative polymerase chain reaction studies allowed the detection of CaBP pre-mRNA and mRNA sequences 1 h after hormone treatment, suggesting that CaBP gene transcription is initiated rapidly. Treatment of cultures with 36 microM cycloheximide 1 h prior to 1,25(OH)2D3 addition resulted in superinduction of VDR mRNA levels but sharply reduced CaBP steady-state mRNA levels. This dramatic reduction in CaBP mRNA reveals that 1,25(OH)2D3-mediated CaBP expression is dependent on ongoing protein synthesis. Thus, we propose that a labile auxiliary protein or other cofactor, which may or may not be 1,25(OH)2D3-dependent, is necessary for 1,25(OH)2D3-mediated CaBP gene transcription in chick duodena.

Vitamin D3 receptor expression in N-ethylnitrosourea-induced mouse pulmonary adenomas.

An expanded role for vitamin D (1 alpha,25-(OH)2D3) in mammalian systems has been suggested by recent evidence that its receptor (vitamin D receptor [VDR]) is present not only in classical target organs, but in a variety of normal tissues and organs, tumor tissues, and cancer cell lines. Vitamin D is involved not only in the regulation of calcium homeostasis and bone metabolism, but in the regulation of cell proliferation, differentiation, and immune responses. The role vitamin D may play in normal lung growth, development, and maintenance is unknown. Likewise, its part in lung tumorigenesis is unclear. The present study examined VDR binding activity and VDR expression in normal mouse lung and ethylnitrosourea-induced lung adenomas. Binding of 1 alpha,25-(OH)2D3 was specific and saturable over the concentration range of 0.01 to 0.50 nM, with an affinity (Kd) of 0.93 x 10(-10) M and a total binding capacity (Bmax) of 22 fmol/mg of protein. Scatchard analysis yielded a convex curve, which suggests positive receptor cooperativity. The calculated Hill coefficient equals 1.69, at a receptor concentration of 0.4 nM, consistent with dimerization of the receptor. Western blot analysis showed the presence of 60 kD VDR protein in tumor homogenates, while Northern blot analysis detected the 4.4 kb VDR mRNA in tumor tissue preparations. Immunohistochemistry and in situ hybridization revealed that both adenomatous Clara cells and normal bronchiolar epithelial Clara cells expressed VDR, with the receptor protein present in their nuclei.(ABSTRACT TRUNCATED AT 250 WORDS)

Phorbol 12-myristate 13-acetate and 1,25-dihydroxyvitamin D 3 regulate 1,25-dihydroxyvitamin D 3 receptors synergistically in rat osteosarcoma cells

In this study, we examined the effect of activation of protein kinase C (PKC) pathways on the regulation of 1,25-dihydroxyvitamin D receptors (VDR) in rat osteosarcoma (ROS) 17/2.8 cells. Activation of PKC with phorbol 12-myristate 13-acetate (PMA) resulted in a time- and dose-dependent increase in VDR expression in ROS cells. Treatment of ROS cells with 4α-phorbol 12,13-dedeconate, a PKC-inactive phorbol ester, had no effect on VDR expression. Oleoyl acetyl glycerol (OAG), a synthetic diacylglycerol, stimulated VDR up-regulation in ROS cells. The PKC inhibitors (H-7, staurosporin, and sphingosine) all blocked PMA-mediated up-regulation of VDR in a dose-dependent manner. We next examined the interaction of 1,25(OH) 2D 3 and PKC activation by PMA on the regulation of VDR in ROS cells. We found that PMA or 1,25(OH) 2D 3 treatment alone resulted in a 50 and 200% increase in VDR, respectively. PMA treatment alone resulted in a 50% increase in VDR protein and a marginal 20% increase in VDR mRNA. 1,25(OH) 2D 3 up-regulation of VDR was associated with a 2-fold increase in VDR mRNA. In contrast, co-treatment of ROS cells with PMA and 1,25(OH) 2D 3 resulted in a synergistic 10-fold induction of VDR mRNA and the appearance of a 7.2 kb VDR transcript. VDR protein was also synergistically up-regulated by combined PMA and 1,25(OH) 2D 3 treatment of ROS cells. Scatchard analysis demonstrated that the synergistic effect of PMA and 1,25(OH) 2D 3 on VDR protein expression was not associated with any change in the affinity of VDR for 1,25(OH) 2D 3. The synergistic effect of 1,25(OH) 2D 3 and PMA on VDR expression supports a link between PKC signal pathways and the function of VDR.

Regulation of calcitriol receptor and its mRNA in normal and renal failure rats

Homologous up-regulation of calcitriol receptor (VDR) by calcitriol is believed to be a transcriptional event. In this experiment, we studied the effect of calcitriol on VDR in normal and renal failure rats. The time course of the effect of calcitriol on VDR mRNA showed a biphasic change in VDR mRNA in response to calcitriol. The concentration of intestinal VDR mRNA increased at six hours and reached peak levels approximately 15 hours after calcitriol injection. Thereafter, the mRNA began to decrease and by 48 hours the level had declined to below the control values. The VDR levels also increased, though they lagged behind the VDR mRNA, and nearly plateaued at 24 hours after calcitriol treatment. In renal failure, the concentrations of VDR were lower and the levels of VDR mRNA were higher than the respective values of normal rats, suggesting that VDR synthesis was inhibited at post-transcriptional sites. Chronic administration of calcitriol increased the VDR but lowered the VDR mRNA levels in both normal and renal failure rats. Infusion of uremic ultrafiltrate to normal rats resulted in lower VDR and higher VDR mRNA levels similar to those found in rats with renal failure. The results indicate that uremic toxins are responsible for the low VDR and high VDR mRNA in renal failure.

Synthesis of and response to 1,25 dihydroxycholecalciferol by subpopulations of murine epidermal keratinocytes: existence of a paracrine system for 1,25 dihydroxycholecalciferol.

The epidermis is both a target tissue for and a source of 1,25 dihydroxycholecalciferol. The present study determines which of the epidermal cell populations synthesizes 1,25 dihydroxycholecalciferol and which responds to this hormone. Epidermal keratinocytes from new born rat epidermis were separated by unit gravity sedimentation into poorly differentiated cells, slow-cycling more differentiated cells, actively proliferating cells, and terminally differentiating subpopulations. The keratinocyte populations were characterized by cell size analysis, cell morphology, and DNA and RNA contents (acridine orange flow cytometry). 1,25(OH)2D3 synthesis was studied by measuring the conversion of [3H] 25(OH)D3 to [3H] 1,25(OH)2D3. The purified product was tested for its ability to compete with synthetic [3H] 1,25(OH)2D3 for binding to chick intestinal cytosol. The responses of the keratinocyte subpopulations to exogenous 1,25(OH)2D3 were evaluated by the increase in 25(OH)D3-24 hydroxylase activity. Furthermore the expression of 1,25(OH)2D3 receptors (VDR) was examined in these cell populations. The results show that only the least differentiated cells produced 1,25(OH)2D3. In contrast, immunocytochemical detection of VDR, the VDR mRNA, and a 25(OH)D3-24 hydroxylase response to 1,25(OH)2D3 were mainly found in the more differentiated cells. Thus, the ability of epidermis to synthesize 1,25(OH)2D3 and be simultaneously sensitive to it depends on the state of cell differentiation. This suggests that the mammalian epidermis contains a paracrine system in which the more differentiated keratinocytes are sensitive to the 1,25(OH)2D3 produced locally by neighboring immature ones.

Time-course study of 1,25-(OH)2-vitamin D 3 induction of homologous receptor and c- myc in nontransformed and transformed C3H/10T1/2 cell clones

1. 1. We have used 1,25-dihydroxyvitamin D 3 (1,25-(OH) 2D 3) to investigate autoregulation of homologous receptor and the control of c- myc mRNA and protein expression in C3H/10T1/2 cells. 2. 2. 10 nM 1,25-(OH) 2D 3 stimulated 1,25-(OH) 2D 3 receptor (VDR) synthesis in both non-transformed C3H/10T1/2 C1 8 and in chemically transformed C3H/10T1/2 Cl 16 cells within 4 hr of treatment. Maximal induction was observed between 8 and 24 hr. 3. 3. Two VDR mRNA transcripts, 2.7 and 4.8 kb, were present in both cell types. There were parallel changes in VDR specific mRNA levels and cellular VDR concentration in the C3H/10T1/2 Cl 8 cells indicating that the increase in receptor concentrations was dependent on de novo mRNA synthesis. 4. 4. The increase in VDR mRNA concentration in the chemically transformed C3H/10T1/2 Cl 16 cells was maximal already at 4hr, preceding the maximal increase in receptor concentration by 4–6 hr. 5. 5. Analysis of c- myc mRNA levels also showed cell line specificity. 6. 6. The c- myc mRNA level increased 2.1-fold with 10 nM 1,25-(OH) 2D 3 treatment in C3H/10T1/2 Cl 8 cells after 12 hr while the C3H/10T1/2 C1 16 cells had maximal c- myc mRNA level after 1 hr. 7. 7. The relative amount of c- myc mRNA remained higher than that of unstimulated controls the next 10–12 hr in C3H/10T1/2 Cl 16 cells. 8. 8. The c- myc protein levels were not affected by 1,25-(OH) 2D 3 treatment in either cell line as detected by Western blot analysis. 9. 9. Our data suggest that 1,25-(OH) 2D 3 mediated induction of VDR does not require prior c- myc protein synthesis in the C3H/10T1/2 cells. 10. 10. We found, however, that 1,25-(OH) 2D 3 treatment caused increased levels of c- myc mRNA but with different kinetics in the two cell lines. 11. 11. Accumulation of VDR mRNA in the presence of cycloheximide suggested that de novo synthesis of protein factors was not required for transcriptional activation.

Presence of gene expression of vitamin D receptor and 24-hydroxylase in OK cells

We investigated the gene expression of 1,25-dihydroxy-vitamin D 3 (1,25(OH) 2D 3) receptors (VDR) and 25-hydroxy-vitamin D 3-24-hydroxylase (24-hydroxylase) in opossum kidney (OK) cells. Reverse transcription-polymerase chain reaction demonstrated the presence of VDR gene expression in OK cells. Northern hybridization, using rat VDR cDNA, also demonstrated a 4.4 kbp VDR mRNA expression which was affected by serum but not by 1,25(OH) 2D 3 and/or parathyroid hormone stimulation. Stimulation with 1,25(OH) 2D 3 induced 24-hydroxylase mRNA expression which peaked at 6 h. This study clearly demonstrates for the first time the presence of VDR and 24-hydroxylase gene expression in OK cells, suggesting that OK cells are a useful model for investigating the genetic action mechanisms of 1,25(OH) 2D 3.

1,25(OH) 2-vitamin D 3 mediated changes in mRNA for c- myc and 1,25(OH) 2D 3 receptor in HL-60 cells and related subclones

HL-60 cells have been shown to differentiate into monocyte-like cells as a consequence of the interaction of 1,25-dihydroxyvitamin D 3 (1,25(OH) 2D 3) with a specific intracellular receptor (VDR). Of c-myc it has been reported that it plays a role in cell proliferation and differentiation. We have investigated the expression of VDR and c-myc mRNA of three subclones of HL-60 cells (N, R and MR) after exposure to 1,25(OH) 2D 3. The N cell is a normal clone in which 1,25(OH) 2D 3 inhibited cell proliferation and also induced cell differentiation. The R cell is a resistant clone whose proliferation and differentiation are not affected by 1,25(OH) 2D 3. The MR cell is a mixed response type clone whose proliferation was not inhibited by 1,25(OH) 2D 3, while its differentiation was actually induced by 1,25(OH) 2D 3. The VDR mRNA expression of the N and MR cells reached a peak at 2 h (2.3- and 2.6-fold induction, respectively) and returned to the control level after 24 h treatment of 1,25(OH) 2D 3. The c-myc mRNA expression was significantly inhibited in the l,25(OH) 2D 3-induced N cells, but not in the MR cells. In contrast, 1,25(OH) 2D 3 did not induce any changes of the VDR and c-myc mRNA levels in the R cells. In separate experiments, the level of the VDR in the three clones was determined via Scatchard analysis; the VDR of the N, MR and R cells were found to be 4500/cell, 3570/cell and less than 600/cell, respectively. These results are consistent with an involvement of 1,25(OH) 2D 3 and VDR in differentiation events, and support the notion that c-myc is related to cell proliferation rather than cell differentiation.