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The hormonal metabolite of vitamin D, 1,25-dihydroxyvitamin D3 (1,25D), binds to the vitamin D receptor (VDR) and promotes heterodimerization of VDR with a retinoid-X-receptor (RXR) to genomically regulate diverse cellular processes. Herein, it is revealed for the first time that VDR is post-translationally acetylated, and that VDR immunoprecipitated from human embryonic kidney (HEK293) cells displays a dramatic decrease in acetylated receptor in the presence of 1,25D-ligand, sirtuin-1 (SIRT1) deacetylase, or the resveratrol activator of SIRT1. To elucidate the functional significance of VDR deacetylation, vitamin-d-responsive-element (VDRE)-based transcriptional assays were performed to determine if deacetylase overexpression affects VDR/VDRE-driven transcription. In HEK293 kidney and TE85 bone cells, co-transfection of low amounts (1-5ng) of a SIRT1-expression vector elicits a reproducible and statistically significant enhancement (1.3- to 2.6-fold) in transcription mediated by VDREs from the CYP3A4 and cyp24a1 genes, where the magnitude of response to 1,25D-ligand is 6- to 30-fold. Inhibition of SIRT1 via EX-527, or utilization of a SIRT1 loss-of-function mutant (H363Y), resulted in abrogation of SIRT1-mediated VDR potentiation. Studies with a novel, non-acetylatable VDR mutant (K413R) showed that the mutant VDR possesses enhanced responsiveness to 1,25D, in conjunction with reduced, but still significant, sensitivity to exogenous SIRT1, indicating that acetylation of lysine 413 is relevant, but that other acetylated residues in VDR contribute to modulation of its activity. We conclude that the acetylation of VDR comprises a negative feedback loop that attenuates 1,25D-VDR signaling. This regulatory loop is reversed by SIRT1-catalyzed deacetylation of VDR to amplify VDR signaling and 1,25D actions.

Although hydrophobic bile acids have been demonstrated to exhibit cytotoxic and carcinogenic effects in the colorectum, ursodeoxycholic acid (UDCA) has been investigated as a potential chemopreventive agent. Vitamin D has been shown to play a role in both bile acid metabolism and in the development of colorectal neoplasia. Using a cross-sectional design, we sought to determine whether baseline circulating concentrations of the vitamin D metabolites 25(OH)D and 1,25(OH)2D were associated with baseline fecal bile acid concentrations in a trial of UDCA for the prevention of colorectal adenoma recurrence. We also prospectively evaluated whether vitamin D metabolite concentrations modified the effect of UDCA on adenoma recurrence. After adjustment for age, sex, BMI, physical activity, and calcium intake, adequate concentrations of 25(OH)D (≥30 ng/mL) were statistically significantly associated with reduced odds for high levels of total [OR, 0.61; 95% confidence interval (CI), 0.38–0.97], and primary (OR, 0.61; 95% CI, 0.38–0.96) bile acids, as well as individually with chenodeoxycholic acid (OR, 0.39; 95% CI, 0.24–0.63) and cholic acid (OR, 0.56; 95% CI, 0.36–0.90). No significant associations were observed for 1,25(OH)2D and high versus low fecal bile acid concentrations. In addition, neither 25(OH)D nor 1,25(OH)2D modified the effect of UDCA on colorectal adenoma recurrence. In conclusion, this is the first study to demonstrate an inverse relationship between circulating levels of 25(OH)D and primary fecal bile acid concentrations. These results support prior data demonstrating that vitamin D plays a key role in bile acid metabolism, and suggest a potential mechanism of action for 25(OH)D in colorectal cancer prevention. Cancer Prev Res; 9(7); 589–97. ©2016 AACR.

The active form of vitamin D3, 1,25-dihydroxyvitamin D (1,25D), and serotonin (5-HT) are postulated to play significant roles in abnormal social behavior associated with psychological conditions in...

Vitamin D is a well-studied agent for cancer chemoprevention and treatment. Its chief circulating metabolite, 25-hydroxyvitamin D, is converted into the active hormone 1,25-dihydroxyvitamin D (1,25D) by the cytochrome P450 enzyme CYP27B1 in kidney and other tissues. 1,25D is then deactivated by CYP24A1 and ultimately catabolized. Colorectal carcinoma cells express CYP27B1 and CYP24A1 that locally regulate 1,25D with potential implications for its impact on carcinogenesis. While 1,25D inhibits cancer growth, the effects of polymorphic variations in genes encoding proteins involved in 1,25D homeostasis are poorly understood. Using an RXR-VDR mammalian two-hybrid (M2H) biologic assay system, we measured vitamin D metabolite uptake and activation of the vitamin D receptor (VDR) pathway in colon cancer cells that expressed one of five CYP27B1 single-nucleotide polymorphisms (SNP) or four CYP24A1 SNPs. Compared with the wild-type control, four of five CYP27B1 SNPs reduced enzymatic activity, whereas one (V166L) increased activity. For CYP24A1, all tested SNPs reduced enzyme activity. Quantitative real-time PCR analyses supported the results of M2H experiments. The observed SNP-directed variation in CYP functionality indicated that vitamin D homeostasis is complex and may be influenced by genetic factors. A comprehensive understanding of 1,25D metabolism may allow for a more personalized approach toward treating vitamin D–related disorders and evaluating risk for carcinogenesis. Cancer Res; 73(8); 2563–73. ©2013 AACR.

The hormonal metabolite of vitamin D, 1α,25-dihydroxyvitamin D(3) (1,25D), initiates biological responses via binding to the vitamin D receptor (VDR). When occupied by 1,25D, VDR interacts with the retinoid X receptor (RXR) to form a heterodimer that binds to vitamin D responsive elements in the region of genes directly controlled by 1,25D. By recruiting complexes of either coactivators or corepressors, ligand-activated VDR-RXR modulates the transcription of genes encoding proteins that promulgate the traditional functions of vitamin D, including signaling intestinal calcium and phosphate absorption to effect skeletal and calcium homeostasis. Thus, vitamin D action in a particular cell depends upon the metabolic production or delivery of sufficient concentrations of the 1,25D ligand, expression of adequate VDR and RXR coreceptor proteins, and cell-specific programming of transcriptional responses to regulate select genes that encode proteins that function in mediating the effects of vitamin D. For example, 1,25D induces RANKL, SPP1 (osteopontin), and BGP (osteocalcin) to govern bone mineral remodeling; TRPV6, CaBP(9k), and claudin 2 to promote intestinal calcium absorption; and TRPV5, klotho, and Npt2c to regulate renal calcium and phosphate reabsorption. VDR appears to function unliganded by 1,25D in keratinocytes to drive mammalian hair cycling via regulation of genes such as CASP14, S100A8, SOSTDC1, and others affecting Wnt signaling. Finally, alternative, low-affinity, non-vitamin D VDR ligands, e.g., lithocholic acid, docosahexaenoic acid, and curcumin, have been reported. Combined alternative VDR ligand(s) and 1,25D/VDR control of gene expression may delay chronic disorders of aging such as osteoporosis, type 2 diabetes, cardiovascular disease, and cancer.

1,25-dihydroxyvitamin D (1,25D), through association with the nuclear vitamin D receptor (VDR), exerts control over a novel endocrine axis consisting of the bone-derived hormone FGF23, and the kidney-expressed klotho, CYP27B1, and CYP24A1 genes, which together prevent hyperphosphatemia/ectopic calcification and govern the levels of 1,25D to maintain bone mineral integrity while promoting optimal function of other vital tissues. When occupied by 1,25D, VDR interacts with RXR to form a heterodimer that binds to VDREs in the region of genes directly controlled by 1,25D (e.g., FGF23, klotho, Npt2c, CYP27B1 and CYP24A1). By recruiting complexes of comodulators, activated VDR initiates a series of events that induces or represses the transcription of genes encoding proteins such as: the osteocyte-derived hormone, FGF23; the renal anti-senescence factor and protein co-receptor for FGF23, klotho; other mediators of phosphate transport including Npt2a/c; and vitamin D hormone metabolic enzymes, CYP27B1 and CYP24A1. The mechanism whereby osteocytes are triggered to release FGF23 is yet to be fully defined, but 1,25D, phosphate, and leptin appear to play major roles. The kidney responds to FGF23 to elicit CYP24A1-catalyzed detoxification of the 1,25D hormone while also repressing both Npt2a/c to mediate phosphate elimination and CYP27B1 to limit de novo 1,25D synthesis. Comprehension of these skeletal and renal actions of 1,25D should facilitate the development of novel mimetics to prevent ectopic calcification, chronic renal and vascular disease, and promote healthful aging.

Background Diminished brain levels of two neurohormones, 5-hydroxytryptamine (5-HT; serotonin) and 1,25-dihydroxyvitamin D3 (1,25D; active vitamin D metabolite), are proposed to play a role in the atypical social behaviors associated with psychological conditions including autism spectrum disorders and depression. We reported previously that 1,25D induces expression of tryptophan hydroxylase-2 (TPH2), the initial and rate-limiting enzyme in the biosynthetic pathway to 5-HT, in cultured rat serotonergic neuronal cells. However, other enzymes and transporters in the pathway of tryptophan metabolism had yet to be examined with respect to the actions of vitamin D. Herein, we probed the response of neuronal cells to 1,25D by quantifying mRNA expression of serotonin synthesis isozymes, TPH1 and TPH2, as well as expression of the serotonin reuptake transporter (SERT), and the enzyme responsible for serotonin catabolism, monoamine oxidase-A (MAO-A). We also assessed the direct production of serotonin in cell culture in response to 1,25D. Results Employing quantitative real-time PCR, we demonstrate that TPH-1/-2 mRNAs are 28- to 33-fold induced by 10 nM 1,25D treatment of cultured rat serotonergic neuronal cells (RN46A-B14), and the enhancement of TPH2 mRNA by 1,25D is dependent on the degree of neuron-like character of the cells. In contrast, examination of SERT, the gene product of which is a target for the SSRI-class of antidepressants, and MAO-A, which encodes the predominant catabolic enzyme in the serotonin pathway, reveals that their mRNAs are 51–59% repressed by 10 nM 1,25D treatment of RN46A-B14 cells. Finally, serotonin concentrations are significantly enhanced (2.9-fold) by 10 nM 1,25D in this system. Conclusions These results are consistent with the concept that vitamin D maintains extracellular fluid serotonin concentrations in the brain, thereby offering an explanation for how vitamin D could influence the trajectory and development of neuropsychiatric disorders. Given the profile of gene regulation in cultured RN46A-B14 serotonergic neurons, we conclude that 1,25D acts not only to induce serotonin synthesis, but also functions at an indirect, molecular-genomic stage to mimic SSRIs and MAO inhibitors, likely elevating serotonin in the CNS. These data suggest that optimal vitamin D status may contribute to improving behavioral pathophysiologies resulting from dysregulation of serotonergic neurotransmission.

The activity of beta-catenin, commonly dysregulated in human colon cancers, is inhibited by the vitamin D receptor (VDR), and this mechanism is postulated to explain the putative anti-cancer activity of vitamin D metabolites in the colon. We investigated the effect of a common FokI restriction site polymorphism (F/f) in the human VDR gene as well as the effect of anti-tumorigenic 1,25-dihydroxyvitamin D(3) (1,25D) and pro-tumorigenic lithocholic acid (LCA) VDR ligands on beta-catenin transcriptional activity. Furthermore, the influence of a major regulatory protein of beta-catenin, the APC tumor suppressor gene, on VDR-dependent inhibition of beta-catenin activity was examined. We report herein that beta-catenin-mediated transcription is most effectively suppressed by the VDR FokI variant F/M4 when 1,25D is limiting. Using Caco-2 colorectal cancer (CRC) cells, it was observed that VDR ligands, 1,25D and LCA, both suppress beta-catenin transcriptional activity, though 1,25D exhibited significantly greater inhibition. Moreover, 1,25D, but not LCA, suppressed endogenous expression of the beta-catenin target gene DKK-4 independent of VDR DNA-binding activity. These results support beta-catenin sequestration away from endogenous gene targets by 1,25D-VDR. This activity is most efficiently mediated by the FokI gene variant F/M4, a VDR allele previously associated with protection against CRC. Interestingly, we found the inhibition of beta-catenin activity by 1,25D-VDR was significantly enhanced by wild-type APC. These results reveal a previously unrecognized role for 1,25D-VDR in APC/beta-catenin cross talk. Collectively, these findings strengthen evidence favoring a direct effect on the Wnt-signaling molecule beta-catenin as one anti-cancer target of 1,25D-VDR action in the colorectum.

We investigated the regulation of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]-induced calbindin-D28k (CaBP) and of the vitamin D receptor (VDR) by evaluating CaBP protein, CaBP mRNA, and VDR mRNA under conditions of altered intake of vitamin D, calcium, or phosphorus. Chickens were maintained for 10 days on one of four diets: vitamin D-deficient, normal (1.0% Ca and 1.1% P), low calcium (0.1% Ca and 1.2% P), and low phosphorus (1.1% Ca and 0.3% P). CaBP was undetectable in D-deficient duodena and was elevated above normal values by low-calcium (3.1-fold) and low-phosphorus (2.3-fold) intake. Contradictory to published data, we observed a correlation between CaBP protein and mRNA levels in that the CaBP mRNA was absent in D-deficient intestine and augmented threefold and twofold in low-calcium and low-phosphate duodena, respectively. In contrast, VDR mRNA concentrations were identical in vitamin D-deficient and normal duodena, implying that intestinal VDR is not dependent upon 1,25-(OH)2D3 for basal expression. Chickens fed a low-phosphorus diet displayed a twofold increase in VDR mRNA, but those fed a low-calcium diet exhibited a dramatic decrease in VDR mRNA. These data show that CaBP mRNA and protein levels are modulated in a tightly coupled fashion, and they are consistent with previous conclusions that augmented circulating 1,25-(OH)2D3 stimulates CaBP expression when dietary calcium or phosphorus is limiting. However, a more complex regulation of VDR expression occurs in that low-phosphorus restriction enhances VDR mRNA levels, possibly via increased circulating 1,25-(OH)2D3. Conversely, reduced dietary calcium diminishes VDR mRNA despite increased circulating 1,25-(OH)2D3, indicating that another factor, such as parathyroid hormone, is a predominant downregulator of VDR.

Fifteen hours after the intracardiac administration of a physiological dose (0.65 nmole) of radioactive cholecalciferol (vitamin D3) to rachitic chicks, the bulk of the radioactivity in the plasma, liver, and intestine exists as three polar metabolites. These are designated Metabolites 4A, 4B, and 4C on the basis of their chromatographic mobility on silicic acid columns. After doses of 0.65 to 6.5 nmoles of cholecalciferol, the predominant metabolite found in the plasma is 4A with smaller amounts of 4B and 4C. In the intestine, a primary target tissue of cholecalciferol, the predominant metabolite is 4B, with much smaller amounts of 4A and 4C. Metabolite 4A from both the plasma and intestine is tentatively identified as 25-hydroxycholecalciferol on the basis of its migration with 25-hydroxycholecalciferol during silicic acid column chromatography. The biological activity of Metabolites 4A and 4B was examined with an assay which measures intestinal calcium transport, in vivo. Both 4A and 4B have biological activity significantly greater than equivalent amounts of cholecalciferol. The kinetics of the appearance of Metabolite 4B in the intestine and its binding to intestinal chromatin are consistent with the lag in the physiological response to cholecalciferol. When radioactive Metabolite 4A is administered to rachitic chicks, the bulk of the plasma radioactivity remains as 4A, but the predominant metabolite found in the intestine is 4B. These data support the concept that Metabolite 4B may be the biologically active form of cholecalciferol in the intestine and that 25-hydroxycholecalciferol is an intermediate in its formation.

The human vitamin D receptor (VDR) is a key nuclear receptor that binds nutritionally derived ligands and exerts bioeffects that contribute to bone mineral homeostasis, detoxification of exogenous and endogenous compounds, cancer prevention, and mammalian hair cycling. Liganded VDR modulates gene expression via heterodimerization with the retinoid X receptor and recruitment of coactivators or corepressors. VDR interacts with the corepressor hairless (Hr) to control hair cycling, an action independent of the endocrine VDR ligand, 1,25-dihydroxyvitamin D3. We report novel dietary ligands for VDR including curcumin, γ-tocotrienol, and essential fatty acid derivatives that likely play a role in the bioactions of VDR.

1,25-Dihydroxyvitamin D3 (1,25D) is the renal metabolite of vitamin D that signals through binding to the nuclear vitamin D receptor (VDR). The ligand-receptor complex transcriptionally regulates genes encoding factors stimulating calcium and phosphate absorption plus bone remodeling, maintaining a skeleton with reduced risk of age-related osteoporotic fractures. 1,25D/VDR signaling exerts feedback control of Ca/PO4 via regulation of FGF23, klotho, and CYP24A1 to prevent age-related, ectopic calcification, fibrosis, and associated pathologies. Vitamin D also elicits xenobiotic detoxification, oxidative stress reduction, neuroprotective functions, antimicrobial defense, immunoregulation, anti-inflammatory/anticancer actions, and cardiovascular benefits. Many of the healthspan advantages conferred by 1,25D are promulgated by its induction of klotho, a renal hormone that is an anti-aging enzyme/coreceptor that protects against skin atrophy, osteopenia, hyperphosphatemia, endothelial dysfunction, cognitive defects, neurodegenerative disorders, and impaired hearing. In addition to the high-affinity 1,25D hormone, low-affinity nutritional VDR ligands including curcumin, polyunsaturated fatty acids, and anthocyanidins initiate VDR signaling, whereas the longevity principles resveratrol and SIRT1 potentiate VDR signaling. 1,25D exerts actions against neural excitotoxicity and induces serotonin mood elevation to support cognitive function and prosocial behavior. Together, 1,25D and klotho maintain the molecular signaling systems that promote growth (p21), development (Wnt), antioxidation (Nrf2/FOXO), and homeostasis (FGF23) in tissues crucial for normal physiology, while simultaneously guarding against malignancy and degeneration. Therefore, liganded-VDR modulates the expression of a "fountain of youth" array of genes, with the klotho target emerging as a major player in the facilitation of health span by delaying the chronic diseases of aging.

1,25-Dihydroxyvitamin D 3 (1,25D) is the endocrine metabolite of vitamin D that signals through binding to the vitamin D receptor (VDR). The ligand–receptor complex transcriptionally regulates genes that encode factors promoting intestinal calcium and phosphate absorption plus bone remodeling, maintaining a skeleton with reduced risk of age-related osteoporotic fractures. 1,25D/VDR signaling further exerts feedback control of mineral ions via regulation of FGF23, klotho, and CYP24A1 to prevent age-related, ectopic calcification, and associated pathologies. Vitamin D also elicits xenobiotic detoxification, oxidative stress reduction, antimicrobial defense, immunoregulation, anti-inflammatory/anticancer actions, and cardiovascular benefits. 1,25D exerts neuroprotective actions against excitotoxicity, and induces serotonin mood elevator to support cognitive function and prosocial behavior. Nutrient, low-affinity VDR ligands including curcumin, polyunsaturated fatty acids, and delphinidin/anthocyanidins initiate VDR signaling, whereas longevity agents such as resveratrol and SIRT1 potentiate VDR signaling. Therefore, liganded VDR modulates the expression of a network of genes that facilitates health span by delaying the chronic diseases of aging.

In a closed endocrine loop, 1,25-dihydroxyvitamin D3(1,25D) induces the expression of fibroblast growth factor 23 (FGF23) in bone, with the phosphaturic peptide in turn acting at kidney to feedback repressCYP27B1and induceCYP24A1to limit the levels of 1,25D. In 3T3-L1 differentiated adipocytes, 1,25D repressesFGF23andleptinexpression and inducesC/EBPβ, but does not affectleptin receptortranscription. Conversely, in UMR-106 osteoblast-like cells,FGF23mRNA concentrations are upregulated by 1,25D, an effect that is blunted by lysophosphatidic acid, a cell-surface acting ligand. Progressive truncation of the mouse FGF23 proximal promoter linked in luciferase reporter constructs reveals a 1,25D-responsive region between −400 and −200 bp. A 0.6 kb fragment of the mouse FGF23 promoter, linked in a reporter construct, responds to 1,25D with a fourfold enhancement of transcription in transfected K562 cells. Mutation of either an ETS1 site at −346 bp, or an adjacent candidate vitamin D receptor (VDR)/Nurr1-element, in the 0.6 kb reporter construct reduces the transcriptional activity elicited by 1,25D to a level that is not significantly different from a minimal promoter. This composite ETS1–VDR/Nurr1cis-element may function as a switch between induction (osteocytes) and repression (adipocytes) of FGF23, depending on the cellular setting of transcription factors. Moreover, experiments demonstrate that a 1 kb mouse FGF23 promoter–reporter construct, transfected into MC3T3-E1 osteoblast-like cells, responds to a high calcium challenge with a statistically significant 1.7- to 2.0-fold enhancement of transcription. Thus, the FGF23 proximal promoter harborsciselements that drive responsiveness to 1,25D and calcium, agents that induce FGF23 to curtail the pathologic consequences of their excess.

Nuclear hormone receptor-responsive element binding specificity has been reported to reside predominantly in the proximal box (P-box), three amino acids located in a DNA-recognition alpha-helix situated on the C-terminal side of the first zinc finger. To further define the residues in the vitamin D receptor (VDR) DNA binding domain (DBD) that mediate its interaction as a retinoid X receptor (RXR) heterodimer with the rat osteocalcin vitamin D-responsive element (VDRE), chimeric receptors were created in which the core DBD of VDR was replaced with that of the homodimerizing glucocorticoid receptor (GR). Systematic alteration of GR DBD amino acids in these chimeras to VDR DBD residues identified arg-49 and lys-53, just C-terminal of the P-box within the base recognition alpha-helix of human VDR (hVDR), as the only two amino acids among 36 differences required to convert the GR core zinc finger domain to that of the VDR. Gel mobility shift and 1,25-dihydroxyvitamin D3-stimulated transcription assays verified that an hVDR-GR DBD chimera is functional on the rat osteocalcin VDRE with only the conservative change of lys-49 to arg, and of the negatively charged glu-53 to a basic amino acid (lys or arg). Thus, for RXR heterodimerizing receptors like VDR, the P-box requires redefinition and expansion to include a DNA specificity element corresponding to arg-49 and lys-53 of hVDR. Examination of DNA specificity element amino acids in other nuclear receptors in terms of conservation and base contact in cocrystal structures supports the conclusion that these residues are crucial for selective DNA recognition.

The 1,25-dihydroxyvitamin D3 (1,25D) hormone is derived from vitamin D generated in skin or obtained from the diet, and binds to and activates the vitamin D receptor (VDR) in target tissues including kidney, colon/small intestine, and bone/muscle. We tested resveratrol for its ability to modulate VDR signaling, using vitamin D responsive element (VDRE) and mammalian 2-hybrid (M2H) transcriptional system technology. Via VDRE-based assays in kidney, colon and myoblast cells, VDR-mediated transcription was activated by resveratrol, and a cooperative effect on transactivation was observed with resveratrol plus 1,25D. The M2H assay revealed a modest, resveratrol-induced dimerization of VDR with its retinoid X receptor (RXR) heteropartner. Cells treated with both resveratrol and 1,25D displayed synergistic stimulation of VDR-RXR heterodimerization, while resveratrol antagonized rexinoid-mediated RXR-RXR homodimerization. Increased transactivation in response to resveratrol was also observed with a subset of other nuclear receptors and their respective cognate responsive elements. Evaluation of wild-type versus a ligand-binding domain mutant VDR revealed that hormone-responsiveness to 1,25D was severely depressed, while the response to resveratrol was only moderately attenuated. Moreover, radiolabeled 1,25D-displacement assays demonstrated an increase in VDR-bound 1,25D in the presence of resveratrol. Thus, resveratrol may affect VDR and other nuclear receptors indirectly, likely via the ability of resveratrol to: (1) potentiate 1,25D binding to VDR; (2) activate RXR; and/or (3) stimulate SIRT1, an enzyme known to deacetylate nuclear receptors. The results of this study elucidate a possible pathway for crosstalk between two nutritionally derived lipids, vitamin D and resveratrol, both of which converge on VDR signaling.

The vitamin D receptor (VDR) is a transcription factor believed to function as a heterodimer with the retinoid X receptor (RXR). However, it was reported [Schrader et al., 1994] that, on putative vitamin D response elements (VDREs) within the rat 9k and mouse 28k calcium binding protein genes (rCaBP 9k and mCaBP 28k), VDR and thyroid hormone receptor (TR) form heterodimers that transactivate in response to both 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) and triiodothyronine (T(3)). We, therefore, examined associations of these receptors on the putative rCaBP 9k and mCaBP 28k VDREs, as well as on established VDREs from the rat osteocalcin (rOC) and mouse osteopontin (mOP) genes, plus the thyroid hormone response element (TRE) from the rat myosin heavy chain (rMHC) gene. In gel mobility shift assays, we found no evidence for VDR-TR heterodimer interaction with any tested element. Further, employing these hormone response elements linked to reporter genes in transfected cells, VDR and TR mediated responses to their cognate ligands only from the rOC/mOP and rMHC elements, respectively, while the CaBP elements were unresponsive to any combination of ligand(s). Utilizing the rOC and mOP VDREs, two distinct repressive actions of TR on VDR-mediated signaling were demonstrated: a T(3)-independent action, presumably via direct TR-RXR competition for DNA binding, and a T(3)-dependent repression, likely by diversion of limiting RXR from VDR-RXR toward the formation of TR-RXR heterodimers. The relative importance of these two mechanisms differed in a response element-specific manner. These results may provide a partial explanation for the observed association between hyperthyroidism and bone demineralization/osteoporosis.

To evaluate whether there is an increase in vitamin D receptor (VDR) concentration which could raise intestinal calcium absorption in absorptive hypercalciuric (AH) patients and promote hypercalciuria, we measured VDR concentration and VDR mRNA levels in skin fibroblasts from 16 patients with AH and 17 age-matched normal subjects before and following a 16-hour incubation in the presence of 10–8 M 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. There were no significant differences in VDR concentration between normal subjects and AH patients in the basal state (30 ± 11 vs. 30 ± 15 ng/mg protein, respectively) or following 1,25(OH)2D3-mediated upregulation (43 ± 18 vs. 42 ± 16 ng/mg protein) as measured by immunoblot methodology. Analysis of VDR mRNA/β-actin mRNA ratios demonstrated no significant differences between normal subjects and AH patients prior to (2.1 ± 1.7 vs. 1.8 ± 2.4) or following (2.7 ± 2.8 vs. 1.9 ± 1.8) 1,25(OH)2D3 exposure. As a measure of VDR bioactivity, we quantitated 1,25(OH)2D3-mediated induction of 25-hydroxyvitamin D3-24-hydroxylase. Again, no significant differences were observed between normal subjects and all patients (2.1 ± 1.6 vs. 1.9 ± 1.6 pmol/mg/30 min, respectively). These findings indicate that there is neither an increase in VDR concentration in skin fibroblasts, a recognized vitamin D responsive cell, nor increased sensitivity to upregulation of VDR numbers by 1,25(OH)2D3 in patients with AH. This suggests an alternative cause of intestinal hyperabsorption of calcium in AH other than alteration of the VDR number.

The vitamin D receptor (VDR), but not its hormonal ligand, 1,25-dihydroxyvitamin D3 (1,25D), is required for the progression of the mammalian hair cycle. We studied three genes relevant to hair cycle signaling, DKKL1 (Soggy), SOSTDC1 (Wise), and HR (Hairless), to determine whether their expression is regulated by VDR and/or its 1,25D ligand. DKKL1 mRNA was repressed 49–72% by 1,25D in primary human and CCD-1106 KERTr keratinocytes; a functional vitamin D responsive element (VDRE) was identified at −9590 bp in murine Soggy. Similarly, SOSTDC1 mRNA was repressed 41–59% by 1,25D in KERTr and primary human keratinocytes; a functional VDRE was located at −6215 bp in human Wise. In contrast, HR mRNA was upregulated 1.56- to 2.77-fold by 1,25D in primary human and KERTr keratinocytes; a VDRE (TGGTGAgtgAGGACA) consisting of an imperfect direct repeat separated by three nucleotides (DR3) was identified at −7269 bp in the human Hairless gene that mediated dramatic induction, even in the absence of 1,25D ligand. In parallel, a DR4 thyroid hormone responsive element, TGGTGAggccAGGACA, was identified at +1304 bp in the human HR gene that conferred tri-iodothyronine (T3)-independent transcriptional activation. Because the thyroid hormone receptor controls HR expression in the CNS, whereas VDR functions in concert with the HR corepressor specifically in skin, a model is proposed wherein unliganded VDR upregulates the expression of HR, the gene product of which acts as a downstream comodulator to feedback-repress DKKL1 and SOSTDC1, resulting in integration of bone morphogenic protein and Wnt signaling to drive the mammalian hair cycle and/or influencing epidermal function.

Hereditary hypocalcemic vitamin D-resistant rickets is attributable to defects in the nuclear receptor for 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]. Two novel point mutations (I314S and R391C) identified in the hormone-binding domain of the human vitamin D receptor (VDR) from patients with hereditary hypocalcemic vitamin D-resistant rickets confer the receptor with sharply reduced 1,25-(OH)2D3-dependent transactivation. These natural mutations, especially R391C, also lead to a second specific consequence, namely impaired heterodimeric interaction with retinoid X receptor (RXR). While the transactivation ability of the I314S mutant can be largely restored by providing excess 1,25-(OH)2D3, R391C activity is more effectively restored with exogenous RXR. These observations are reflected also in the clinical course of each patient: the patient bearing the I314S mutation showed a nearly complete cure with pharmacological doses of a vitamin D derivative, whereas the patient bearing R391C responded only partially to such therapy. Further tests with patient fibroblasts and transfected cells show that the activity of the I314S VDR mutant is augmented somewhat by added RXR, while transactivation by the R391C mutant is best corrected by RXR in the presence of excess hormone. Thus, the effects of hormone vs. RXR in bolstering these mutant VDRs, such that they mediate efficient transactivation, are not entirely separable. The unique properties of these genetically altered receptors establish a new subclass of natural human VDR mutants that illustrate, in vivo, the importance of both 1,25-(OH)2D3 binding and heterodimerization with RXR in VDR action.

Isoforms of the mammalian klotho protein serve as membrane co-receptors that regulate renal phosphate and calcium reabsorption. Phosphaturic effects of klotho are mediated in cooperation with fibroblast growth factor receptor-1 and its FGF23 ligand. The vitamin D receptor and its 1,25-dihydroxyvitamin D(3) ligand are also crucial for calcium and phosphate regulation at the kidney and participate in a feedback loop with FGF23 signaling. Herein we characterize vitamin D receptor-mediated regulation of klotho mRNA expression, including the identification of vitamin D responsive elements (VDREs) in the vicinity of both the mouse and human klotho genes. In keeping with other recent studies of vitamin D-regulated genes, multiple VDREs control klotho expression, with the most active elements located at some distance (-31 to -46 kb) from the klotho transcriptional start site. We therefore postulate that the mammalian klotho gene is up-regulated by liganded VDR via multiple remote VDREs. The phosphatemic actions of 1,25-dihydroxyvitamin D(3) are thus opposed via the combined phosphaturic effects of FGF23 and klotho, both of which are upregulated by the liganded vitamin D receptor.

Glucocorticoid treatment of certain lymphoma cell lines and thymocytes activates a self-destructive pathway of programmed cell death referred to as apoptosis. Calcium and calmodulin (CaM) may be important signals in the apoptotic cascade because an early event is a sustained elevation in cytosolic Ca2+ and CaM inhibitors interfere with the death pathway. In the present study, expression of the CaM gene was examined during glucocorticoid-induced apoptosis in WEHI7.2 lymphocytes. Steady state levels of CaM mRNA were increased up to 10-fold following a 4-6-h exposure of WEHI7.2 cells to 10(-6) M dexamethasone. This increase was mediated through the glucocorticoid receptor since the response was not observed in WEHI7.418, a variant line which does not express active glucocorticoid receptor. Induction of CaM mRNA was dose-dependent and highly specific for glucocorticoids, as other steroids were unable to elicit the response. A stringent cell specificity was also observed. Pretreatment of WEHI7.2 lymphocytes with cycloheximide did not interfere with dexamethasone-dependent increases in CaM mRNA levels, and studies with actinomycin D demonstrated that the stability of the transcript was not altered by hormone, Finally, a calmodulin inhibitor elicited a protective effect on WEHI7.2 cells following glucocorticoid exposure. These results indicate that CaM mRNA levels were hormonally controlled in WEHI7.2 lymphocytes and support the putative involvement of CaM in glucocorticoid-induced apoptosis.

Increasing evidence suggests an immunoregulatory function of the potent steroid hormone 1,25-dihydroxyvitamin D 3 (1,25(OH) 2 D 3 ) which has been successfully applied for treatment of psoriasis. The skin is both a site of production and a target of 1,25(OH) 2 D 3 . In vitro, 1,25(OH) 2 D 3 inhibits proliferation and stimulates differentiation of keratinocytes. We investigated the in situ expression of vitamin D-receptors (VDR) in normal and psoriatic skin by immunochemical methods. The VDR were visualized using the monoclonal antibody (MoAb) 9A7g to the VDR and the labeled avidinbiotin technique. Immunoreactivity was consistently confined to nuclei in all skin biopsies. In normal skin specimens (n = 10) VDR antigens were expressed in keratinocytes of all epidermal layers (except those of the stratum corneum) and in cells of the epidermal appendages. Double labeling experiments with MoAb to cluster-defined antigens indicated that melanocytes and approximately 75% of Langerhans cells exhibit 1,25(OH) 2 D 3 receptors in normal skin biopsies (n = 5). Depending on their localization in skin compartments 42–62% of CD11b + positive macrophages and 45–75% of CD3 + T lymphocytes expressed VDR. Non-lesional psoriatic skin specimens (n = 8) revealed nearly identical staining patterns. Lesional psoriatic skin specimens (n = 8) exhibited a significant increase of VDR expression both in basal and suprabasal epidermal layers as measured by computer-assisted morphometry and showed a remarkable change of the immune cell pattern: the densitity and proportion of VDR positive T lymphocytes and macrophages were higher in the epidermal and the perivascular papillary loop compartment. These in vivo findings strongly support the hypothesis that 1,25(OH) 2 D 3 modulates immune response and cell proliferation/differentiation in human skin.

1,25-Dihydroxyvitamin D(3) (1,25D) is known primarily as a regulator of calcium, but 1,25D also promotes phosphate absorption from intestine, reabsorption from kidney, and bone mineral resorption. FGF23 is a newly discovered phosphaturic hormone that, like PTH, lowers serum phosphate by inhibiting renal reabsorption via Npt2a. We show that 1,25D strongly upregulates FGF23 in bone. FGF23 then represses 1alpha-OHase activity in kidney, thus preventing spiraling induction of FGF23 by 1,25D. We also report that LRP5, Runx2, TRPV6, and Npt2c, all anabolic toward bone, and RANKL, which is catabolic, are transcriptionally regulated by 1,25D. This coordinated regulation together with that of FGF23 and PTH allows 1,25D to play a central role in maintaining calcium and phosphate homeostasis and bone metabolism. In the cases of LRP5, Runx2, TRPV6, and Npt2c we show that transcriptional regulation results at least in part from direct binding of VDR near the relevant gene promoter. Finally, because 1,25D induces FGF23, and FGF23 in turn represses 1,25D synthesis, a reciprocal relationship is established with FGF23 indirectly curtailing 1,25D-mediated intestinal absorption and counterbalancing renal reabsorption of phosphate. This newly revealed FGF23/1,25D/Pi axis is comparable in significance to phosphate and bone metabolism as the PTH/1,25D/Ca axis is to calcium homeostasis.

The nuclear vitamin D receptor (VDR) mediates the actions of its 1,25-dihydroxyvitamin D(3) ligand to control gene expression in terrestrial vertebrates. Prominent functions of VDR-regulated genes are to promote intestinal absorption of calcium and phosphate for bone mineralization and to potentiate the hair cycle in mammals. We report the cloning of VDR from Petromyzon marinus, an unexpected finding because lampreys lack mineralized tissues and hair. Lamprey VDR (lampVDR) clones were obtained via RT-PCR from larval protospleen tissue and skin and mouth of juveniles. LampVDR expressed in transfected mammalian COS-7 cells bound 1,25-dihydroxyvitamin D(3) with high affinity, and transactivated a reporter gene linked to a vitamin D-responsive element from the human CYP3A4 gene, which encodes a P450 enzyme involved in xenobiotic detoxification. In tests with other vitamin D responsive elements, such as that from the rat osteocalcin gene, lampVDR showed little or no activity. Phylogenetic comparisons with nuclear receptors from other vertebrates revealed that lampVDR is a basal member of the VDR grouping, also closely related to the pregnane X receptors and constitutive androstane receptors. We propose that, in this evolutionarily ancient vertebrate, VDR may function in part, like pregnane X receptors and constitutive androstane receptors, to induce P450 enzymes for xenobiotic detoxification.

The vitamin D receptor (VDR) mediates the effects of the calcemic hormone 1α,25-dihydroxyvitamin D 3 [1,25(OH) 2 D 3 ]. We show that VDR also functions as a receptor for the secondary bile acid lithocholic acid (LCA), which is hepatotoxic and a potential enteric carcinogen. VDR is an order of magnitude more sensitive to LCA and its metabolites than are other nuclear receptors. Activation of VDR by LCA or vitamin D induced expression in vivo of CYP3A, a cytochrome P450 enzyme that detoxifies LCA in the liver and intestine. These studies offer a mechanism that may explain the proposed protective effects of vitamin D and its receptor against colon cancer.

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.

Summary The effect of 1,25-dihydroxyvitamin D 3 (1,25(OH) 2 D 3 ) administration on duodenal vitamin D receptor (VDR) mRNA levels in hypophosphatemic ( Hyp ) mice, a murine homologue of human X-linked hypophosphatemic rickets, was examined. Basal levels of VDR mRNA in Hyp mice were similar to those of normal littermates and, in normal mice, VDR mRNA levels were up-regulated 1.8–2.7-fold after injection of 1 μg/kg 1,25(OH) 2 D 3 . In contrast, no significant change in VDR mRNA was observed in Hyp mice treated with 1,25(OH) 2 D 3 . To determine the effect of phosphate repletion on VDR mRNA levels, high-phosphate diet was fed to Hyp mice. Although plasma phosphorus concentration was restored to normal, up-regulation of VDR mRNA was not recovered with phosphate supplementation. These results indicate that the vitamin D-resistance in Hyp mice is not caused by hypophosphatemia, per se, and may result from a fundamental molecular defect in vitamin D action at the intestine which could be related to ineffective up-regulation of VDR mRNA by 1,25(OH) 2 D 3 .

The WEHI7.2 thymoma cell line undergoes apoptotic cell death when exposed to glucocorticoids and agents that increase intracellular cAMP. Several lines of evidence indicate that calcium may play an important role in events culminating in lymphocyte apoptosis. In these studies, calbindin-D28K was stably overexpressed in WEHI7.2 cells to determine if increasing the Ca(2+)-binding capacity of the cell interferes with the apoptotic pathway. Indeed, stable expression of calbindin-D28K decreased the apoptotic effects of dexamethasone and forskolin, and the level of resistance to these agents correlated with the relative amount of calbindin expressed in each line. Overexpression of calbindin also increased cell survival in the presence of the calcium ionophore A23187. The stably expressed calcium-binding protein appeared to exert its protective effect subsequent to transcriptional activation, since glucocorticoid- and cAMP-induced gene expression were not affected. These data support the proposal that calcium fluxes are involved in apoptosis and suggest that high level expression of proteins that buffer calcium fluxes can effectively suppress death in apoptosis-susceptible cells.

Clinical observations have demonstrated a positive effect of estrogens and androgens on the maintenance of structural bone integrity. This study examines the direct effects of androgenic hormones on the osteoblast-like human osteosarcoma cell line, HOS TE85. Employing radiolabeled dihydrotestosterone (DHT), 2800 saturable, high-affinity (dissociation constant = 0.66 nM) androgen binding sites were detected per HOS TE85 cell. Androgen binding was specific in that DHT and testosterone (T) displayed significantly greater competition than the progestins, progesterone and medroxyprogesterone. The expression of androgen receptors in HOS TE85 cells was further substantiated by Northern analysis. A human androgen receptor complementary DNA probe revealed a 9.5 kilobase transcript which corresponds to the predominant human androgen receptor transcript detected in human male reproductive tissues. Androgens were also found to elicit biological responses in HOS TE85 cells. Physiological concentrations of DHT and T decreased HOS TE85 cell proliferation as assessed by cell count. This finding suggests that DHT may also play a role in osteoblast differentiation. In support of this hypothesis, treatment with T (24 h, 10 nM) enhanced the abundance of both alpha 1(I)-procollagen messenger RNA (mRNA) (5-fold) and transforming growth factor-beta mRNA (2.2 fold). The nonaromatizable androgen DHT (24 h, 10 nM) elicited an increase in the steady state concentration of alpha 1(I)-procollagen mRNA similar to the increase observed with T treatment. Thus, in addition to the recent discovery of estradiol receptors and estrogenic regulation of HOS TE85 cells, it is now evident that these osteoblast-like osteosarcoma cells also express high affinity androgen binding sites and can respond biologically to androgens.

A finite human cell line was established from trabecular bone explants obtained from a 48-year-old woman. These cells, designated BG688, were characterized as osteoblast-like in phenotype using the following independent criteria: (1) the presence of histochemically detectable alkaline phosphatase (AP) activity; (2) response to the calciotropic hormone 1,25-(OH)2D3 as assessed by increased AP activity; (3) synthesis and secretion of the osteoblast-specific marker bone gla protein; and (4) expression of alpha 1(I)-procollagen and alpha 1(III)-procollagen mRNAs in a pattern similar to that of other osteoblast-like cell lines. In addition to these classic osteoblast markers, BG688 cells also possess approximately 2400 high-affinity (Kd = 0.45 nM) 17 beta-estradiol (E2) binding sites per cell. The binding of E2 to these sites is specific, and of the steroid hormone agonists tested, E2 and diethylstilbestrol elicited the greatest amount of competition with radiolabeled E2. BG688 cells were also shown to respond to a physiologic concentration (10 nM) of E2. In vitro translation products of poly(A)+ RNA obtained from control and hormone-treated cells revealed a pleiotropic influence of E2 on the relative abundance of several mRNAs as assessed by two-dimensional gel electrophoretic analysis of their corresponding peptides. E2 also elicits a twofold increase in the steady-state concentration of alpha 1(I)-procollagen mRNA as demonstrated by northern blot hybridization. Thus, we here extend our previous data obtained in osteoblast-like osteosarcoma cells to indicate that a normal osteoblastic cell line is a target for the action of estrogen.(ABSTRACT TRUNCATED AT 250 WORDS)

Fibroblast Growth Factor-23 (FGF23) is a systemic phosphaturic hormone that inhibits osteoblastic mineralization, blocks renal 25-OH-vitamin D3 bioactivation to 1,25(OH)2D3 (D3) and elicits hypophosphatemia by repressing the renal type IIa sodium-phosphate cotransporter. It has been shown that FGF23 causes a reduction in serum phosphate and D3 concentrations that appear to be caused by a preceding decrease and increase in the expression of 1αOHase and 24OHase genes, respectively. Also, serum FGF23 increases after treatment with D3, suggesting that D3 negatively feedback controls its circulating concentrations by inducing FGF23. We thus undertook this investigation to determine the molecular mechanism by which D3 increases the circulating level of FGF23. To this end we examined by Real-Time PCR the effect of D3 on FGF23 mRNA expression in the rat osteoblastic cell line, UMR-106, as well as in mouse calvaria. The results reveal a dose- and time-dependent stimulation of endogenous FGF23 mRNA level in UMR osteoblasts in response to D3. The maximal increase was 117-fold at 10-6M D3, although for further experiments we chose a 10-7 M concentration of D3, to represent a more physiological concentration of the hormone. The increase of FGF23 mRNA was 55.8-fold after 24 hrs treatment, but statistically significant differences were observed as early as after 4-8 hrs. In addition, using cotreatment with actinomycin D or cyclohexamide, we observed that D3 regulates FGF23 expression at the transcriptional level, likely via the synthesis of an intermediary transfactor. To evaluate the biological significance of vitamin D regulation of FGF23 mRNA expression in vivo, we administered D3 (6μg/kg B.W.) to 4-5 week old C57BL/6 mice by IP injection. Calvaria were collected at 48 hrs after single injection and FGF23 mRNA levels were measured. The expression of FGF23 mRNA in this bone tissue was significantly up-regulated by D3. These results suggest that FGF23 and D3 are reciprocal phosphate regulatory hormones, with D3 initially acting to enhance blood phosphate levels via intestinal absorption and renal reabsorption, followed by a negative feedback effect of D3 to reduce phosphate by inducing FGF23 both diminishing D3 through 1;gaOHase repression and eliciting phosphaturia.

Pseudohypoparathyroidism is a pleiotropic genetic disease of variable expression. Recent observations suggest that the hypocalcemia and bone refractoriness to parathyroid hormone, typical of this disorder, are secondary to impaired production of 1,25-dihydroxycholecalciferol. An incomplete variant of this disease entity, marked by normocalcemia, has been recognized. In this study we investigated whether variable expression of the apparent defect in vitamin D 3 metabolism underlies the normocalcemic variant of this disease. An 18 year old woman with normocalcemic pseudohypoparathyroidism was studied. The diagnosis was confirmed by an absent phosphaturic and urinary cyclic adenosine monophosphate (AMP) response to parathyroid hormone (PTH). Despite this diagnosis the normal serum calcium concentration suggested the presence of normal bone responsiveness. Moreover, the presence of a calcemie response to PTH and a normal serum PTH concentration was consistent with normal PTH bone dynamics. Measurement of 1,25-dihydroxycholecalciferol yielded a normal value in contrast to the decreased concentration of this active vitamin D 3 metabolite present in patients with "hypocalcemic" pseudohypoparathyroidism. The association of a normal serum calcium level, normal bone responsiveness to PTH and normal vitamin D 3 metabolism, in our patient, is consistent with the hypothesis that a decreased serum concentration of 1,25-dihydroxycholecalciferol may be causal of the hypocalcemia in pseudohypoparathyroidism. In addition, these data suggest that variable expression of the apparent defect in vitamin D 3 metabolism may underlie the normocalcemic variant of this disorder.

We developed an immunocytochemical technique to visualize the receptors for 1,25-dihydroxyvitamin D [1,25-(OH)2D receptor] in cryostat sections of normal human tissues, using a rat monoclonal antibody (9A7 gamma) to the chick intestinal receptor, which has been found to react with mammalian 1,25-(OH)2D receptors. Localization of the antigen was predominantly nuclear, with little cytoplasmic immunoreactivity. Specific staining was seen in the nuclei of many normal epithelial tissues, including liver, kidney, thyroid, adrenal, gastrointestinal tract, breast, and skin. No nuclear staining was seen when tissue sections were incubated with normal rat immunoglobulin G or when the monoclonal antibody was preincubated with a receptor-enriched chick intestinal cytosol preparation. Our results demonstrate that the receptor for 1,25-(OH)2D is localized in the nucleus and widely distributed in normal human tissues.

Specific binding of 1α,25-dihydroxycholecalciferol in the cytosol and purified chromatin fractions of chick small intestinal mucosa was studied to investigate the mechanism by which this hormone arrives at its chromatin binding site. The cytosol fraction of the mucosa contains a macromolecule with a high affinity (Kd = 2.2 x 10-9 m) for 1α,25-dihydroxycholecalciferol and has a limited number of binding sites which are saturated at low hormone concentrations (≃ 10-8 m). The association of the sterol with the cytoplasmic component is thermolabile, and is destroyed by treatment with pronase, but not by nucleases, and the receptor component is therefore presumed to be a protein. The protein-sterol complex was isolated and characterized by sucrose gradient centrifugation and sediments at 3 to 3.5 S in the presence of 0.15 m KCl. The interaction of 1α,25-dihydroxy[3H] cholecalciferol with the cytosol protein is reversed by unlabeled 1α,25-dihydroxycholecalciferol g 25-hydroxycholecalciferol g 1α,25-hydroxycholecalciferol g cholecalciferol ≃ estradiol-17β. The subcellular localization of 1α,25-dihydroxycholecalciferol is temperature-dependent. The hormone remains bound to the cytosol fraction at 0° in whole intestines in vitro, and a dramatic movement of 1α,25-dihydroxycholecalciferol to the nuclear chromatin occurs at 37°. The temperature dependence of the hormone localization is also evident in intestinal mucosa homogenates and reconstituted cytosol-chromatin systems. This observation suggests that the cell membrane and nuclear membrane are not involved in the temperature-dependent process. It is postulated that the specific cytosol receptor for 1α,25-dihydroxycholecalciferol functions to transport the hormone to its ultimate site of action in the nuclear chromatin.

The antirachitic effectiveness of la-hydroxyvitamin D3 (lα-OH-D3) was investigated by raising chicks on a vitamin D-deficient diet and chronically administering various levels of this synthetic sterol. Vitamin D3 (D3) and la, 25-dihydroxyvitamin D3 (lα,25-(OH)2-D3) were concurrently tested as dietary supplements in order to compare the potency of these three sterols. Growth, plasma calcium and phosphorus concentration, percentage bone ash, calcium absorption, and radiographic appearance of bone were determined after treatment of the chicks with the sterols for 3 weeks. lα-OH-D3 was 5.1, 3.4 and 3.7 times more potent than D3 in stimulating weight gain, maintenance of plasma calcium and promotion of increased percent bone ash, respectively. Radiographic examination of tibiae and femurs from the various groups revealed that lα-OH-D3 effectively mediates normal bone calcification; lα-OH-D3 was 2–6 times more potent than D3 in this regard. The synthetic lα-OH-D3 was slightly more active than la,25-(OH)2-D3 in ...

A sensitive radioreceptor assay for 1alpha,25-dihydroxyvitamin D3 (1alpha,25-(OH)2D3) is utilized to quantitate the circulating concentration of this sterol in experimental animals and humans. When weanling rats are grown for 2 weeks on low calcium or low phosphate diets, limited availability of either ion elicits a five-fold increase in the plasma level of 1alpha,25-(OH)2D3. The enhancement of 1alpha,25-(OH)2D3 in calcium deficiency is dependent upon the presence of the parathyroid and/or thyroid glands, which is consistent with parathyroid hormone (PTH) mediation of this effect. In contrast, the response to phosphate deficiency is independent of these glands and may result from a direct action of low phosphate on the renal synthesis of 1alpha,25-(OH)2D3. Studies in humans indicate that the normal level of 1alpha,25-(OH)2D is 2.1--4.5 ng/100 ml plasma. Patients with chronic renal failure have markedly lower circulating 1alpha,25-(OH)2D and this kidney hormone is undetectable in anephric subjects, but returns to normal within 1 day after successful renal transplantation. Hypoparathyroidism and pseudohypoparathyroidism are associated with reduced plasma 1alpha,25-(OH)2D while patients with primary hyperparathyroidism have significantly elevated sterol hormone levels. Thus, from measurements in rats and humans, it appears that circulating 1alpha,25-(OH)2D3 is regulated by PTH and/or phosphate and that abnormal plasma 1alpha,25-(OH)2D3 is a part of the pathophysiology of renal osteodystrophy and parathyroid disorders.

The hormonal metabolite of vitamin D3, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), exerts its biological effects by initially binding to a cytosolic receptor protein. Such a protein has been demonstrated in the target organs of vitamin D3 including bone. Although the role of 1,25(OH)2D3 on the skeleton has been extensively studied in normal bone, nothing is known about its effects, if any, in abnormal bone growth. Rat osteogenic sarcoma is a useful model for bone malignancy. Tumor cells retain differentiated functions including the ability to form bone and to respond to parathyroid hormone, prostaglandins, and to a smaller extent to calcitonin with increases in cyclic AMP levels. We have here evaluated osteogenic sarcoma cell lines for the presence of a receptor for 1,25(OH)2D3. We have utilized sucrose gradient sedimentation, saturation analysis, and DNA-cellulose chromatography. Cytosol preparations from these cell lines contain a 3.3 S saturable macromolecule which binds 1,25(OH)2D3 with specificity and high affinity (Kd = 2 x 10(-10) M). The sterol-macromolecule complex binds to DNA-cellulose and its elution profile from this affinity resin is similar to that of the 1,25(OH)2D3 receptor from normal rat bone. These tumor cells should serve as a useful model for studying the action of 1,25(OH)2D3 in bone and the role of this metabolite in the biology of bone malignancy.

We have used a monoclonal antibody (9A7) against the purified avian 1,25-dihydroxyvitamin D3 receptor to develop an immunocytochemical technique for visualization of the protein in fixed tissues and cultured cells. In Bouin's-fixed, chick intestine, 1,25-dihydroxyvitamin D3 receptor-like immunoreactivity was localized mainly in nuclei of epithelial cells and was more abundant in the crypt than in the villar cells. Receptor staining was low or undetectable in liver hepatocytes but was present in nuclei of cells lining the hepatic sinusoids. In rat brain, receptor-like immunoreactivity was abundant and widely distributed, but did not always coincide with the presence of vitamin D-dependent calcium-binding protein; 1,25-dihydroxyvitamin D3 receptor was absent from cerebellar Purkinje cells that contained abundant calcium-binding protein. In disaggregated rat bone cells, receptor immunoreactivity was present in mononuclear cells including osteoblasts and fibroblasts but was absent from osteoclasts. Two separate clones of osteoblast-like, rat osteosarcoma cells, shown in previous studies to be either receptor positive (17/2.8) or negative (24.1), demonstrated nuclear immunoreactivity in exact concordance with receptor levels as determined by ligand binding. The phenomenon of hormone-induced up-regulation of receptor was visualized in receptor-positive 3T6 fibroblasts by demonstration of markedly enhanced nuclear reactivity in cells treated with 10(-7) M 1,25-dihydroxyvitamin D3 for 48 h. Our studies demonstrate the feasibility of the immunocytochemical approach to visualize the 1,25-dihydroxyvitamin D3 receptor in target tissues and show that it is predominantly a nuclear protein in the relatively unoccupied and fully activated states. Moreover, the vitamin D-dependent calcium binding is not a universal marker for 1,25-dihydroxyvitamin D3 action. Rather, our observations suggest that the expression of the 1,25-dihydroxyvitamin D3 receptor may be connected with the state of cellular differentiation.

Chandler et al. (Chandler, J.S., Chandler, S.K., Pike, J.W., and Haussler, M.R. (1984) J. Biol. Chem. 259, 2214-2222) previously demonstrated that 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) caused the induction of 25-hydroxyvitamin D3-24-hydroxylase (24-hydroxylase) in a rhesus monkey kidney cell line (LLC-MK2) apparently deficient in the high affinity 1,25-(OH)2D3 receptor. We have re-examined this phenomenon and report here that 24-hydroxylase induction is mediated by a receptor variant in LLC-MK2 cells with low hormone affinity. Dose response analysis showed that in contrast to LLC-PK1 (a typical receptor-positive cell line), the LLC-MK2 line was less sensitive to 1,25-(OH)2D3 by 2 orders of magnitude. Employing optimal concentrations of 1,25-(OH)2D3 for 24-hydroxylase induction in each cell type, the early time courses of this bioresponse were identical in LLC-MK2 and LLC-PK1 and were consistent with a nuclear action of hormone-receptor complexes. Moreover, the rank order of potency of vitamin D3 congeners as inducers of 24-hydroxylase activity in LLC-MK2 cells agreed well with their relative affinity for the 1,25-(OH)2D3 receptor. An examination of 1,25-(OH)2D3 receptor content via DNA-cellulose chromatography in LLC-MK2 cells incubated at ligand concentrations 10-25-fold higher than the normal 2 nM revealed a minimum of 1600 receptor-like molecules/LLC-MK2 cell. These results show that LLC-MK2 cells possess a variant receptor form with apparent low affinity for 1,25-(OH)2D3. This system should serve as a model for clinical syndromes characterized by the requirement for massive doses of vitamin D to prevent rickets.

A competitive protein binding assay for measurement of the plasma concentration of 1 alpha, 25-dihydroxyvitamin D3 [1alpha, 25-(OH)2D3] has been extended to include the immediate precursor of this hormone, 25-hydroxyvitamin D3 (25-OHD3). In addition, the assay system is capable of measuring the two metabolic products of ergocalciferol, namely. 25-hydroxyvitamin D2 (25-OHD2) and 1alpha, 25-dihydroxyvitamin D2 [1alpha, 25-(OH)2D2]. The target tissue assay system consists of a high affinity cytosol receptor protein that binds the vitamin D metabolites and a limited number of acceptor sites on the nuclear chromatin. By utilizing a series of chromatographic purification steps, a single plasma sample can be assayed for any of the four vitamin D metabolites either individually or combined. Therefore, the assay procedure allows for both the quantitative and qualitative assessment of the total active vitamin D level in a given plasma sample. To show that the binding assay was capable of measuring 1alpha, 25-(OH)2D2 as well as 1alpha, 25 (OH)2D3, two groups of rats were raised. One group, supplemented with vitamin D3, produced assayable material that represented 1alpha, 25-(OH)2D3. The other group, fed only vitamin D2 in the diet, yielded plasma containing only 1alpha, 25-(OH)2D2 as the hormonal form of the vitamin. The circulating concentrations of the two active sterols were nearly identical (15 ng/100 ml) in both groups, indicating that the competitive binding assay can be used to measure both hormonal forms in plasma. In a separate experiment, 1alpha, 25-(OH)2D2 was generated in an in vitro kidney homogenate system using 25-OHD2 as substrate. Comparison of this sterol with 1alpha, 25-(OH)2D3 in the assay system showed very similar binding curves; the D2 form was slightly less efficient (77%). Comparison of the respective 25-hydroxy forms (25-OHD2 vs. 25-OHD3) at concentrations 500-fold that of 1alpha, 25-(OH)2D3, again suggested that the binding of the D2 metabolite was slightly less efficient (71%). Finally, the assay was employed to measure the total active vitamin D metabolite pools in the plasma of normal subjects and patients with varying degrees of hypervitaminosis D. The normal plasma levels of 25-OHD and 1alpha, 25-(OH)2D measured in Tucson adults were 25-40 ng/ml and 2.1-4.5 ng/100 ml, respectively. Both sterols were predominately (greater than 90%) in the form of vitamin D3 metabolites in this environment. Typical cases of hypervitaminosis D exhibited approximately a 15-fold increase in the plasma 25-OHD concentration, and a dramatic changeover to virtually all metabolites existing in the form of D2 vitamins. In contrast, the circulating concentration of 1alpha, 25-(OH)2D was not substantially enhanced in vitamin D-intoxicated patients. We therefore conclude that hypervitaminosis D is not a result of abnormal plasma levels of 1alpha, 25-(OH)2D but may be cuased by an excessive circulating concentration of 25-OHD.

Using both clonal Chinese hamster ovary (CHO) cells in culture and the hen ovary, we have searched for the presence of specific 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] receptors. Receptor analyses were carried out on high salt cytosols of CHO cells and high salt extracts of hen ovarian nuclei that were originally isolated in low salt buffer. Both CHO and hen ovary contain a specific high affinity 1,25-(OH)2D3 receptor (Kd = 10(-10) - 10(-11) M), which sediments (3.3S) in high salt sucrose gradients identically to the chick intestinal receptor for 1,25-(OH)2D3. This 3.3S macromolecule from both sources absorbed to DNA-cellulose at 0.1 M KCl and eluted during a linear salt gradient at 0.2-0.22 M KCl, a property characteristic of the 1,25-(OH)2D3 receptor. Saturation analysis indicated that there are approximately 2000 copies of the receptor molecule per CHO cell. We also investigated the effect of 1,25-(OH)2D3 on ovarian cell growth in monolayer culture of CHO cells. Significant inhibition of CHO cell growth (up to 60%) was observed in the presence of physiological (100 pM) levels of 1,25-(OH)2D3 in the culture medium. This inhibition of growth was dose dependent and was accompanied by a parallel decrease in total cell protein. Concentrations of 25-hydroxyvitamin D3 and 24,25-dihydroxyvitamin D3 as high as 1 nM did not affect CHO cell growth, indicating that, like receptor binding, cell proliferation is selectively influenced by 1,25-(OH)2D3 over other vitamin D metabolites. These data demonstrate that ovarian cells in mammals and birds possess the 1,25-(OH)2D3 receptor which may play a role in the effect of 1,25-(OH)2D3 on the growth of these cells in culture.

Intestinal calcium absorption and plasma levels of 1,25-dihydroxycholecalciferol (1,25(OH)2D3) were measured in lactating and non-lactating rats and the effects of bromocriptine and exogenous prolactin treatment were evaluated. In lactating rats calcium absorption and plasma levels of parathyroid hormone, 1,25(OH)2D3 and alkaline phosphatase activity were significantly increased. Bromocriptine treatment significantly reduced the enhanced calcium absorption and levels of plasma 1,25(OH)2D3 and alkaline phosphatase but had no significant effect on plasma levels of parathyroid hormone. Prolactin administered with bromocriptine to lactating animals prevented all the changes observed with bromocriptine treatment alone. It was concluded that the increased plasma levels of prolactin during lactation lead to high plasma levels of 1,25(OH)2D3 which are responsible for the enhanced intestinal calcium absorption.

The effects of chronic steroid contraceptive therapy on drug clearance from plasma were studied by using plasma antipyrine, phenylbutazone, and cholecalciferol half-lives in women. After 3 mo of oral steroid therapy (Norinyl, 2 mg; norethindrone + mestranol), the antipyrine half-life was increased in 3 of 6 subjects, phenylbutazone half-life was not consistently altered, and vitamin D3 half-life was increased in 3 of 4 patients. After 1 to 7 yr of oral steroid theraphy, the antipyrine half-life was longer while taking the contraceptive than when the contraceptive treatment was discontinued in 4 of 6 subjects, whereas that of phenylbutazone was not consistently altered.

Lactating rats fed a vitamin D-containing diet maintain elevated serum levels of 1,25-dihydroxycholecalciferol [1,25-(OH)2D3] and increased intestinal net calcium absorption (as a percentage of the intake). Rts deprived of vitamin D for only 2 weeks during lactation lack elevated serum levels of 1,25-(OH)2D3 and develop marked hypocalcemia. We have now determined net absorption of Ca for a 3-day period at the end of lactation in rats suckling a second litter of 10-day-old pups. The rats were fed diets containing either no vitamin D (--D) or 5 IU vitamin D3/g diet (+D) from the sixth day of pregnancy. Net Ca absorption was at least 2-fold higher in +D-fed lactating rats than in nonlactating controls. Vitamin D-deprived lactating rats had values approximately the same as the +D-fed lactating rats, even though the former were severely hypocalcemia and lacked elevated levels of 1,25-(OH)2D3 in plasma and intestinal mucosa. The high efficiency of intestinal Ca absorption in --D lactating rats was confirmed in vitro with everted duodenal sacs which developed essentially the same high Ca concentration ratios across the intestinal wall as did sacs from +D rats. Parathyroidectomy did not significantly affect the Ca concentration ratio of sacs obtained from --D lactating rats 2 days after the operation. We conclude that during lactation, 1,25-(OH)2D3 is primarily required for calcium homeostasis, and enhanced calcium absorption does not require elevated serum and intestinal levels of 1,25-(OH)2D3.

To investigate the role of vitamin D metabolites in the pathogenesis of pseudohypoparathyroidism, we studied an elderly man with a unique variant of the disease, which was characterized by hypocalcemia, elevated serum parathyroid hormone (513 +/- 13 pg per milliliter, mean +/- S.E.M., normal, less than 450) but normal renal responses (phosphate and cyclic AMP) to exogenous parathyroid extract. Treatment with parathyroid extract did not produce a calcemic effect, suggesting an isolated skeletal hyporesponsiveness to parathyroid hormone. Although 25-hydroxyvitamin D levels were not reduced, levels of 1,25-dihydroxycholecalciferol were extremely low (0.52 ng per deciliter; normal 3.3 +/- 0.06, S.D.). Treatment with 1,25-dihydroxycholecalciferol (1 microgram by mouth per day for four days) increased circulating levels to normal (4.60 ng per deciliter) and restored to normal the calcemic response to parathyroid (change in calcium 3.0 mg per deciliter). These data suggest that 1,25-dihydroxycholecalciferol deficiency may explain the skeletal resistance, but not the renal resistance, often present in classic pseudohypoparathyroidism.

Major advances in the field of vitamin D metabolism have been achieved in the past decade that have considerably enhanced our understanding of a variety of metabolic bone diseases which include the rachitic and osteomalacic syndromes and parathyroid gland disorders. Delineation of the biochemical structure, metabolic pathways, mechanisms regulating synthesis, and physiologic functions has established that the compound should more appropriately be considered a hormone. Obligatory sequential, two-step hydroxylation of the prohormone, vitamin D, is necessary before physiologic action occurs. Initial hydroxylation occurs mainly in the liver while the second hydroxylation occurs exclusively in the kidney. The physiologically active form of the hormone, 1,25-(OH)2-D, coordinates with parathyroid hormone (and probably calcitonin) in promoting homeostasis of both extra-cellular calcium and phosphorus and the skeletal system. This article reviews the basic metabolism of vitamin D, the modes of physiologic action, the endocrine features of vitamin D and its relationship to parathyroid hormone, and discusses the important medical applications of this information.