Loss of APC function in mesenchymal cells surrounding the Müllerian duct leads to myometrial defects in adult mice

The 1,25-(OH)(2)D metabolite mediates the endocrine actions of vitamin D by regulating in the small intestine the expression of target genes that play a critical role in intestinal calcium absorption. The major role of the vitamin D hormone on bone is indirect and mediated through its endocrine function on mineral homeostasis. However, genetic manipulation of the expression of Cyp27b1 or the VDR in chondrocytes strongly support a direct role for locally synthesized 1,25(OH)(2)D, acting through the VDR, in vascular invasion and osteoclastogenesis during endochondral bone development. Cells from the growth plate respond to the 24,25-(OH)(2)D and 1,25-(OH)(2)D metabolites in a cell maturation-dependent manner and the effects of 1,25-(OH)(2)D are thought to be mediated through binding to the membrane-associated receptor PDIA3 (protein disulfide isomerase associated 3). The physiological relevance of membrane-mediated 1,25-(OH)(2)D signaling is emerging and is discussed. Finally, preliminary results suggest that mice deficient for Cyp24a1 exhibit a delay in bone fracture healing and support a role for 24,25-(OH)(2)D in mammalian fracture repair.     

Vitamin D-mediated growth inhibition of an androgen-ablated LNCaP cell line model of human prostate cancer

1,25-(OH)(2) vitamin D(3) (1,25-(OH)(2) D), the active metabolite of vitamin D, exerts antiproliferative effects on a variety of tumor cells including prostate. This inhibition requires vitamin D receptors (VDRs) as well as downstream effects on the G1 to S phase checkpoint of the cell cycle. Recent data raise the possibility that androgen plays a role in the antiproliferative effects of 1,25-(OH)(2) D in prostate cancer cells; however, this hypothesis has been difficult to test rigorously as the majority of prostate cancer cell lines (unlike human prostate tumors) lack androgen receptors (ARs). We utilized two different models of androgen-independent prostate cancer that express functional ARs and VDRs to evaluate a possible role of androgen in 1,25-(OH)(2) D mediated growth inhibition. We stably introduced the AR cDNA into the human prostate cancer cell line ALVA 31, which expresses functional VDR but is relatively resistant to growth inhibition by 1,25-(OH)(2) D. Neither ALVA-AR nor the control cells, ALVA-NEO, exhibited substantial growth inhibition by 1,25-(OH)(2) D in the presence or absence of androgen. This observation suggests that the basis for the resistance of ALVA 31 to 1,25-(OH)(2) D-mediated growth inhibition is not the lack of AR. The second model was LNCaP-104R1, an AR-expressing androgen independent prostate cancer cell line derived from androgen dependent LNCaP. 1,25-(OH)(2) D inhibited the growth of LNCaP-104R1 cells in the absence of androgen and this effect was not blocked by the antiandrogen Casodex. As was observed in the parental LNCaP cells, this effect was correlated with G1 phase cell cycle accumulation and upregulation of the cyclin dependent kinase inhibitor (CKI) p27, as well as increased association of p27 with cyclin dependent kinase 2. These findings suggest that the antiproliferative effects of 1,25-(OH)(2) D do not require androgen-activated AR but do involve 1,25-(OH)(2) D induction of CKIs required for G1 cell cycle checkpoint control.     

Hereditary 1,25-dihydroxyvitamin D resistant rickets due to a mutation causing multiple defects in vitamin D receptor function

Hereditary vitamin D-resistant rickets (HVDRR) is an autosomal recessive disease caused by mutations in the vitamin D receptor (VDR). We studied a young Saudi Arabian girl who exhibited the typical clinical features of HVDRR, but without alopecia. Analysis of her VDR gene revealed a homozygous T to C mutation in exon 7 that changed isoleucine to threonine at amino acid 268 (I268T). From crystallographic studies of the VDR ligand-binding domain, I268 directly interacts with 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] and is involved in the hydrophobic stabilization of helix H12. We recreated the I268T mutation and analyzed its effects on VDR function. In ligand binding assays, the I268T mutant VDR exhibited an approximately 5- to 10-fold lower affinity for [(3)H]1,25(OH)(2)D(3) compared with the wild-type (WT) VDR. The I268T mutant required approximately a 65-fold higher concentration of 1,25(OH)(2)D(3) to be equipotent in gene transactivation. Both retinoid X receptor heterodimerization and coactivator binding were reduced in the I268T mutant. Analogs of 1,25(OH)(2)D(3) have been proposed as potential therapeutics for patients with HVDRR. Interestingly, in protease sensitivity assays, treatment with the potent vitamin D analog, 20-epi-1,25(OH)(2)D(3), stabilized I268T mutant proteolytic fragments better than 1,25(OH)(2)D(3). Moreover, 20-epi-1,25(OH)(2)D(3) restored transactivation of the I268T mutant to levels exhibited by WT VDR treated with 1,25(OH)(2)D(3). In conclusion, we describe a novel mutation, I268T, in the VDR ligand-binding domain that alters ligand binding, retinoid X receptor heterodimerization, and coactivator binding. These combined defects in VDR function cause resistance to 1,25(OH)(2)D(3) action and result in the syndrome of HVDRR.     

Peripheral blood monocyte vitamin D receptor levels are elevated in patients with idiopathic hypercalciuria

Idiopathic hypercalciuria (IH) is the most common cause of calcium oxalate nephrolithiasis. Increased intestinal calcium absorption and bone resorption and decreased tubule calcium reabsorption may be caused by elevated serum 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] in some patients but not in those with normal serum 1,25(OH)(2)D(3) levels. Because 1,25(OH)(2)D(3) exerts its biological actions through binding to the cellular vitamin D receptor (VDR), the present study was undertaken to test the hypothesis that VDR levels are elevated in IH patients.Ten male IH calcium oxalate stone-formers were paired with controls matched in age within 5 yr and lacking a history of stones or family history of stones. Blood was obtained for serum, peripheral blood monocytes (PBMs) were separated from lymphocytes and other mononuclear cells, and PBM VDR content was measured by Western blotting.The PBM VDR level was 2-fold greater in IH men at 49 +/- 21 vs. 20 +/- 15 fmol/mg protein, mean +/- sd; P < 0.008. Serum 1,25(OH)(2)D(3) levels were not higher than controls (48 +/- 14 vs. 39 +/- 11 pg/ml; P < 0.068). In conclusion, PBM VDR levels are elevated in IH calcium oxalate stone-formers. The elevation could not be ascribed to increased serum 1,25(OH)(2)D(3) levels. These results suggest that the molecular basis for IH involves a pathological elevation of tissue VDR level, which may elevate intestinal calcium absorption and bone resorption and decrease renal tubule calcium reabsorption. The mechanism for increased VDR in IH patients with normal serum 1,25(OH)(2)D(3) levels is unknown.     

Control of autoimmune diseases by the vitamin D endocrine system

1,25-Dihydroxyvitamin D(3) [1,25(OH)(2)D(3)], the biologically active form of vitamin D(3), is a secosteroid hormone essential for bone and mineral homeostasis. It regulates the growth and differentiation of multiple cell types, and displays immunoregulatory and anti-inflammatory properties. Cells involved in innate and adaptive immune responses--including macrophages, dendritic cells, T cells and B cells--express the vitamin D receptor (VDR), and can both produce and respond to 1,25(OH)(2)D(3). The net effect of the vitamin D system on the immune response is an enhancement of innate immunity coupled with multifaceted regulation of adaptive immunity. Epidemiological evidence indicates a significant association between vitamin D deficiency and an increased incidence of several autoimmune diseases, and clarification of the physiological role of endogenous VDR agonists in the regulation of autoimmune responses will guide the development of pharmacological VDR agonists for use in the clinic. The antiproliferative, prodifferentiative, antibacterial, immunomodulatory and anti-inflammatory properties of synthetic VDR agonists could be exploited to treat a variety of autoimmune diseases, from rheumatoid arthritis to systemic lupus erythematosus, and possibly also multiple sclerosis, type 1 diabetes, inflammatory bowel diseases, and autoimmune prostatitis.     

1alpha,25-dihydroxyvitamin D3 inhibits prostate cancer cell growth by androgen-dependent and androgen-independent mechanisms

We recently reported that 1alpha,25-dihydroxyvitamin D3 [1,25-(OH)2D3] inhibits the growth of the LNCaP human prostate cancer cell line by an androgen-dependent mechanism. In the present study we examined the actions and interactions of 1,25-(OH)2D3 and the androgen 5alpha-dihydrotestosterone (DHT) on two new human prostate cancer cell lines (MDA), MDA PCa 2a and MDA PCa 2b. Scatchard analyses revealed that both cell lines express high affinity vitamin D receptors (VDRs) with a binding affinity (Kd) for [3H]1,25-(OH)2D3 of 0.1 nM. However, the MDA cell lines contain low affinity androgen receptors (ARs) with a Kd of 25 nM for [3H]DHT binding. This is 50-fold lower than the AR in LNCaP cells (Kd = 0.5 nM). Their response to DHT is greatly reduced; 2a cells do not respond to 100 nM DHT, and 2b cells show a modest response at that high concentration. 1,25-(OH)2D3 causes significant growth inhibition in both MDA cell lines, greater (for 2b cells) or lesser (for 2a cells) than that in the LNCaP cell line. Moreover, 1,25-(OH)2D3 significantly up-regulates AR messenger RNA in all three cell lines, as shown by Northern blot analysis. The growth inhibitory effect of 1,25-(OH)2D3 on LNCaP cells is blocked by the pure antiandrogen, Casodex, as we previously reported. However, Casodex (at 1 microM) did not block the antiproliferative activity of 1,25-(OH)2D3 in MDA cells. In conclusion, the growth inhibitory action of 1,25-(OH)2D3 in the MDA cell lines appears to be androgen independent, whereas the actions of 1,25-(OH)2D3 in LNCaP cells are androgen dependent. Most importantly, the MDA cell lines, derived from a bone metastasis of human prostate carcinoma, remain sensitive to 1,25-(OH)2D3, a finding relevant to the therapeutic application of vitamin D and its low calcemic analogs in the treatment of advanced prostate cancer.     

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

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

Antiproliferative effects of 1alpha,25-dihydroxyvitamin D(3) and vitamin D analogs on tumor-derived endothelial cells

Although there is abundant evidence that 1alpha,25-dihydroxyvitamin D(3) [1,25-(OH)(2)D(3)] inhibits the growth of several cancer cell types, inhibition of angiogenesis may also play a role in mediating the antitumor effects of 1,25-(OH)(2)D(3.) We examined the ability of 1,25-(OH)(2)D(3) to inhibit the growth of tumor-derived endothelial cells (TDECs) and normal endothelial cells and to modulate angiogenic signaling. 1,25-(OH)(2)D(3) inhibited the growth of TDECs from two tumor models at nanomolar concentrations, but was less potent against normal aortic or yolk sac endothelial cells. The vitamin D analogs Ro-25-6760, EB1089, and ILX23-7553 were also potent inhibitors of TDEC proliferation. Furthermore, the combination of 1,25-(OH)(2)D(3) and dexamethasone had greater activity than either agent alone. 1,25-(OH)(2)D(3) increased vitamin D receptor and p27(Kip1) protein levels in TDECs, whereas phospho-ERK1/2 and phospho-Akt levels were reduced. These changes were not observed in normal aortic endothelial cells. In squamous cell carcinoma and radiation-induced fibrosarcoma-1 cells, 1,25-(OH)(2)D(3) treatment caused a reduction in the angiogenic signaling molecule, angiopoietin-2. In conclusion, 1,25-(OH)(2)D(3) and its analogs directly inhibit TDEC proliferation at concentrations comparable to those required to inhibit tumor cells. Further, 1,25-(OH)(2)D(3) modulates cell cycle and survival signaling in TDECs and affects angiogenic signaling in cancer cells. Thus, our work supports the hypothesis that angiogenesis inhibition plays a role in the antitumor effects of 1,25-(OH)(2)D(3).     

The role of the vitamin D receptor in regulating vitamin D metabolism: a study of vitamin D-dependent rickets, type II

In vitro studies and animal experiments suggest that the production of 1,25-dihydroxyvitamin D [1,25-(OH)(2)D] and 24,25-(OH)(2)D is reciprocally controlled by 1,25-(OH)(2)D. To investigate the role of the vitamin D receptor (VDR) in controlling vitamin D metabolism in humans, we studied 10 patients with vitamin D-dependent rickets type II due to a defective VDR. After a period of high dose calcium therapy, 7 of the patients had normal serum calcium, phosphorus, alkaline phosphatase, and plasma PTH levels (PTH-N), and 3 showed increased serum alkaline phosphatase and plasma PTH (PTH-H). Serum calcium, phosphorus, alkaline phosphatase, PTH, vitamin D metabolites, urinary calcium/creatinine, and renal phosphate threshold concentration were compared with unaffected family members that comprised the control group. Vitamin D metabolites were measured before and after an oral load of 50,000 U/m(2) cholecalciferol. Compared with the control group, 1,25-(OH)(2)D levels were significantly higher and 24,25-(OH)(2)D levels were lower in the PTH-N group and even more so in the PTH-H group. 1alpha-Hydroxylase (1-OHase) and 24-OHase activities were estimated by the product/substrate ratio. In the PTH-N group, 1-OHase activity was higher and 24-OHase activity was lower than in controls. In the PTH-H group, 1-OHase activity was even higher, probably due to an additive effect of PTH. Thus, 1,25-(OH)(2)D-liganded VDR is a major control mechanism for vitamin D metabolism, and PTH exerts an additive effect. Assessment of the influence of 1,25-(OH)(2)D shows reciprocal control of enzyme activity in man, suppressing 1-OHase and stimulating 24-OHase activity.     

Vitamin D Analogs

Patients with chronic renal failure frequently develop secondary hyperparathyroidism, primarily as a result of phosphate retention and low serum 1,25(OH)2D3. Replacement therapy with calcitriol or its synthetic precursor alfacalcidol [1alpha(OH)D3] often produces hypercalcemia, especially when combined with calcium-based phosphate binders. In addition, the natural vitamin D compounds can exacerbate the hyperphosphatemia in patients with chronic renal failure. This combined increase in calcium and phosphate has been correlated with vascular calcification leading to coronary artery disease, the most common cause of mortality in renal patients. Several vitamin D analogs have now been developed that retain the direct suppressive action of calcitriol on the parathyroid glands but have less calcemic activity, thereby offering a safer and more effective means of controlling secondary hyperparathyroidism. Maxacalcitol [22-oxa-1,25(OH)2D3] and falecalcitriol [1,25(OH)2-26,27-F6-D3] are currently available in Japan, and paricalcitol [19-nor-1,25(OH)2D2] and doxercalciferol [1alpha(OH)D2] are available in the US. The mechanisms by which these analogs exert their selective actions on the parathyroid glands are under investigation. The low calcemic activity of maxacalcitol has been attributed to its rapid clearance from the circulation. This prevents sustained effects on intestinal calcium absorption and bone resorption, but still allows a prolonged suppression of parathyroid hormone gene expression. The selectivity of the other analogs is achieved by distinct mechanisms. Understanding how these compounds exert their selective actions on the parathyroid glands will aid in the design of safer, more effective analogs.     

Hydrocortisone and deflazacort induce different effects on vitamin D receptor level increase produced by 1,25-dihydroxyvitamin D3 in rat osteoblast-like UMR-106 cells

OBJECTIVE: The homologous upregulation produced by 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] on vitamin D receptor (VDR) levels, and the effects produced by the heterologous agents hydrocortisone or deflazacort, alone or in conjunction with this vitamin D metabolite, were studied in rat osteoblastic UMR-106 osteosarcoma cells. METHODS: VDR were determined by binding analysis (Bmax and dissociation constant). VDR mRNA expression levels were measured by Northern blot analysis. RESULTS: Incubation with 10 nM 1,25(OH)2D3 produced a significant increase in Bmax with respect to ethanol-treated cells (100.2 +/- 13.2 vs 11.4 +/- 4.8 fmol 3H-1,25(OH)2D3 bound/mg protein) together with a significant increase in VDR mRNA expression (483 +/- 170% vs 100%). The addition of 10 nM hydrocortisone to 1,25(OH)2D3 produced a significant decrease in Bmax (from 100.2 +/- 13.2 to 44 +/- 5.6), with mRNA levels similar to those of basal conditions (116 +/- 25% vs 100%). However, the addition of 10 nM deflazacort did not reduce the activation in Bmax produced by 1,25(OH)2D3 (92.4 +/- 16 vs 100.2 +/- 13.2), maintaining the increase in mRNA levels (430 +/- 10% vs 483 +/- 170%). If 10 nM hydrocortisone or 10 nM deflazacort was added to UMR-106 cells without 1,25(OH)2D3, a similar increase was observed in Bmax with respect to basal conditions (20.4 +/- 1.3 or 20.9 +/- 1.6 vs 11.4 +/- 4.8 in control cells), but hydrocortisone did not produce any significant variation in mRNA VDR levels, while deflazacort itself produced an increase in VDR mRNA expression. CONCLUSION: Our findings of different actions produced by hydrocortisone and deflazacort on the increase of VDR levels produced by 1,25(OH)2D3 could explain some of the different actions produced by both antiinflammatory medications on bone metabolism.     

Functional vitamin D receptor (VDR) in the t-tubules of cardiac myocytes: VDR knockout cardiomyocyte contractility

We have previously shown that the active form of vitamin D, 1,25 dihydroxyvitamin D3 [1,25(OH)(2)D(3)], has both genomic and rapid nongenomic effects in heart cells; however, the subcellular localization of the vitamin D receptor (VDR) in heart has not been studied. Here we show that in adult rat cardiac myocytes the VDR is primarily localized to the t-tubule. Using immunofluorescence and Western blot analysis, we show that the VDR is closely associated with known t-tubule proteins. Radioligand binding assays using (3)H-labeled 1,25(OH)(2)D(3) demonstrate that a t-tubule membrane fraction isolated from homogenized rat ventricles contains a 1,25(OH)(2)D(3)-binding activity similar to the classic VDR. For the first time, we show that cardiac myocytes isolated from VDR knockout mice show accelerated rates of contraction and relaxation as compared with wild type and that 1,25(OH)(2)D(3) directly affects contractility in the wild-type but not the knockout cardiac myocyte. Moreover, we observed that acute (5 min) exposure to 1,25(OH)(2)D(3) altered the rate of relaxation. A receptor localized to t-tubules in the heart is ideally positioned to exert an immediate effect on signal transduction mediators and ion channels. This novel discovery is fundamentally important in understanding 1,25(OH)(2)D(3) signal transduction in heart cells and provides further evidence that the VDR plays a role in heart structure and function.     

Pre-clinical evidence and clinical translation of benign prostatic hyperplasia treatment by the vitamin D receptor agonist BXL-628 (Elocalcitol)

The active form of vitamin D, 1,25-dihydroxyvitamin D3, is a secosteroid hormone that binds to the vitamin D receptor (VDR), a member of the superfamily of nuclear receptors, and exerts a number of diverse biological functions. The natural hormone and synthetic VDR agonists are well known for their capacity to control calcium and bone metabolism, but they also regulate proliferation and differentiation of many cell types, and possess exquisite immunoregulatory properties, mostly by targeting dendritic cells (DC) and T cells. These properties have been clinically exploited in the treatment of different diseases, from secondary hyperparathyroidism to osteoporosis to psoriasis. The VDR is expressed by most cell types, including cells of the urogenital system such as prostate and bladder cells. In particular, the prostate has been recognized as a target organ of VDR agonists and represents an extra-renal synthesis site of 1,25-dihydroxyvitamin D3, but its capacity to respond to VDR agonists has, so far, been probed only for the treatment of prostate cancer. We have taken a different approach, and have analysed the capacity of VDR agonists to treat benign prostatic hyperplasia (BPH), a complex syndrome characterized by a static component related to prostate overgrowth, a dynamic component responsible for urinary irritative symptoms, and a possible inflammatory component. Pre-clinical data reviewed here demonstrate that VDR agonists, and notably BXL-628 (Elocalcitol), reduce the static component of BPH by inhibiting the activity of intra-prostatic growth factors downstream of the androgen receptor, and the dynamic component by targeting bladder cells. These data have led to a proof-of-concept clinical study that has successfully shown arrest of prostate growth in BPH patients treated with BXL-628. Ongoing clinical studies will assess the capacity of this VDR agonist to reduce symptoms and ameliorate flow parameters in BPH-affected individuals. The pronounced effects of BXL-628 on bladder smooth muscle cells and its anti-inflammatory properties indeed anticipate beneficial effects also on BPH-related lower urinary tract symptoms.     

Autocrine control of vitamin D metabolism in synovial cells from arthritic patients

OBJECTIVE: This study was designed to investigate whether 1, 25-dihydroxyvitamin D3 (1,25-(OH)2D3), produced by activated synovial fluid macrophages, promotes its own catabolism by upregulating vitamin D-24-hydroxylase (24-OHase) in synovial fibroblasts through a vitamin D receptor (VDR) mediated mechanism. METHODS: Synovial macrophages and fibroblasts were derived from patients with rheumatoid arthritis. Expression of VDR and 24-OHase mRNAs was determined using in situ hybridisation. Vitamin D hydroxylase activity was determined by incubating cells with [3H]-25-(OH)D3, or [3H]-1,25-(OH)2D3, and metabolite synthesis quantified using high performance liquid chromatography. RESULTS: 1, 25-(OH)2D3 increased expression of mRNA for both VDR and 24-OHase in fibroblasts by approximately threefold over 24 hours. 1,25-(OH)2D3 increased fibroblast 24-OHase activity, yielding 24-hydroxylated, and more polar, metabolites. In co-culture, fibroblasts were able to catabolise macrophage derived 1,25-(OH)2D3. CONCLUSIONS: 1, 25-(OH)2D3 is produced by macrophages in vitro at biologically relevant concentrations and can increase its own catabolism by synovial fibroblasts; this effect is probably mediated via upregulation of both synovial fibroblast VDR and 24-OHase.     

Vitamin D metabolism and function in the skin

The keratinocytes of the skin are unique in being not only the primary source of vitamin D for the body, but in possessing the enzymatic machinery to metabolize vitamin D to its active metabolite 1,25(OH)(2)D. Furthermore, these cells also express the vitamin D receptor (VDR) that enables them to respond to the 1,25(OH)(2)D they produce. Numerous functions of the skin are regulated by 1,25(OH)(2)D and/or its receptor. These include inhibition of proliferation, stimulation of differentiation including formation of the permeability barrier, promotion of innate immunity, and promotion of the hair follicle cycle. Regulation of these actions is exerted by a number of different coregulators including the coactivators DRIP and SRC, the cosuppressor hairless (Hr), and β-catenin. This review will examine the regulation of vitamin D production and metabolism in the skin, and explore the various functions regulated by 1,25(OH)(2)D and its receptor.