The assessment of circulating 25(OH)D and 1,25(OH)2D: where we are and where we are going

The field of Vitamin D assay technology has progressed significantly over the past 4 decades. Further, the clinical utility of these measurements has moved from esoteric into mainstream clinical diagnosis. This movement has been fueled by the realization that Vitamin D is involved in bodily systems beyond skeletal integrity. The clinical assay techniques for circulating 25(OH)D and 1,25(OH)₂D have progressed away from competitive protein binding assay (CPBAs) that utilize tritium reporters to radioimmunoassay (RIAs) that utilize both I¹²⁵ and chemiluminescent reporters. These advances have allowed direct serum analysis of 25(OH)D in an automated format that provides a huge sample throughput. Detection of circulating 25(OH)D can also be achieved utilizing direct high-performance liquid chromatographic (HPLC) or liquid chromatography coupled with mass spectrometry (LC–MS) techniques. These methods are accurate, however, they require expensive equipment and restrict sample throughput in the large clinical laboratory. Direct serum detection of 1,25(OH)₂D is unlikely to occur for many reasons as a sample pre-purification will always be required. However, a semi-automated chemiluminescent detection system with automated sample preparation is in final development for the determination of circulating 1,25(OH)₂D. These advances will allow both 25(OH)D and 1,25(OH)₂D to be detected in an accurate, rapid fashion to meet the clinical demands we see emerging.     

Calciotropic hormones during reproduction

This review summarizes the reported effects of the menstrual cycle, pregnancy and lactation on serum concentration of the calciotropic hormones PTH and 1,25(OH)₂D. A midcycle rise in PTH and 1,25(OH)₂D has been observed, but in the majority of studies there was no change in PTH and 1,25(OH)₂D concentrations throughout the menstrual cycle. Both total and free 1,25(OH)₂D levels are increased during pregnancy. The renal 1,25(OH)₂D production is stimulated, and there is some evidence of 1,25(OH)₂D production by decidua/placenta and fetal kidney in vitro; the decidual/placental production should not be overestimated in vivo. The increased renal 1-hydroxylase activity is possibly mediated by estrogens and PTH, although the effect of pregnancy on PTH remains uncertain. Increased serum 1,25(OH)₂D concentrations probably result in a rise of intestinal calcium absorption during pregnancy. There is a postdelivery drop in PTH and 1,25(OH)₂D levels, but they are increased when lactation is prolonged, or in mothers nursing twins. The 1-hydroxylase activity during lactation may be stimulated by PTH, but also by prolactin.     

Identification of novel mediators of Vitamin D signaling and 1,25(OH)2D3 resistance in mammary cells

Since the discovery of the Vitamin D receptor (VDR) in mammary cells, the role of the Vitamin D signaling pathway in normal glandular function and in breast cancer has been extensively explored. In vitro studies have demonstrated that the VDR ligand, 1,25(OH)₂D₃, modulates key proteins involved in signaling proliferation, differentiation and survival of normal mammary epithelial cells. Anti-proliferative and pro-differentiating effects of 1,25(OH)₂D₃ have also been observed in VDR positive breast cancer cells, indicating that transformation per se does not abolish Vitamin D signaling. However, many breast cancer cell lines are less sensitive to 1,25(OH)₂D₃ than normal mammary epithelial cells. Reduced sensitivity to 1,25(OH)₂D₃ has been linked to alterations in Vitamin D metabolizing enzymes as well as down regulation of VDR expression or function. In this report, we describe results from a proteomics screening approach used to search for proteins involved in dictating sensitivity or resistance to Vitamin D mediated apoptosis in breast cancer cells. Several proteins not previously linked to 1,25(OH)₂D₃ signaling were identified with this approach, and a distinct subset of proteins was linked to 1,25(OH)₂D₃ resistance. Follow-up studies to determine the relevance of these proteins to Vitamin D signaling in general are in progress.     

Impact of the Vitamin D3 receptor on growth-regulatory pathways in mammary gland and breast cancer

1,25-Dihydroxyvitamin D₃ (1,25(OH)₂D₃) interacts with the Vitamin D₃ receptor (VDR) to modulate proliferation and apoptosis in a variety of cell types, including breast cancer cells. In this review, we discuss three issues related to the role of the VDR in growth control: first, whether mammary glands lacking VDR exhibit abnormal growth; second, whether the VDR is essential for induction of apoptosis by 1,25(OH)₂D₃; and third, whether VDR up-regulation can sensitize cells to 1,25(OH)₂D₃. Studies from our laboratory have demonstrated that mammary glands from VDR knockout (VDR KO) mice exhibit accelerated growth and branching during puberty, pregnancy and lactation as compared to wild-type (WT) mice. In addition, involution after weaning, a process driven by epithelial cell apoptosis, proceeds at a slower rate in VDR KO mice compared to WT mice. Using cells isolated from VDR KO and WT mice, we report that both normal and transformed mammary cells derived from WT mice are growth inhibited by 1,25(OH)₂D₃, however, cells derived from VDR KO mice are completely unresponsive to 1,25(OH)₂D₃. In human breast cancer cells, we have identified a variety of agents, including steroid hormones, phytoestrogens and growth factors, that up-regulate VDR expression and enhance sensitivity to 1,25(OH)₂D₃-mediated growth inhibition. Collectively, these studies support a role for 1,25(OH)₂D₃ and the VDR in negative growth regulation of both normal mammary gland and breast cancer cells.     

Inhibition of Vitamin D3 metabolism enhances VDR signalling in androgen-independent prostate cancer cells

Induction of growth arrest and differentiation by 1,25-dihydroxyvitamin D₃ (1,25-(OH)₂D₃) occurs in non-malignant cell types but is often reduced in cancer cells. For example, androgen-independent prostate cancer cells, DU-145 and PC-3, are relatively insensitive to the anti-proliferative action of 1,25-(OH)₂D₃. This appears to be due to increased 1,25-(OH)₂D₃-metabolism, as a result of CYP24 enzyme-induction, which in turn leads to decreased anti-proliferative efficacy. In the in vitro rat kidney mitochondria assay, the 2-(4-hydroxybenzyl)-6-methoxy-3,4-dihydro-2H-naphthalen-1-one (4) was found to be a potent inhibitor of Vitamin D₃ metabolising enzymes (IC₅₀ 3.5 μM), and was shown to be a more potent inhibitor than the broad spectrum P450 inhibitor ketoconazole (IC₅₀ 20 μM). The combination of the inhibitor and 1,25-(OH)₂D₃ caused a greater inhibition of proliferation in DU-145 cells than when treated with both agents alone. Examination of the regulation of VDR target gene mRNA in DU-145 cells revealed that co-treatment of 1,25-(OH)₂D₃ plus inhibitor of Vitamin D₃ metabolising enzymes co-ordinately upregulated CYP24, p21^waf1/cip1 and GADD45.     

Characterization of Vitamin D insensitive prostate cancer cells

The antitumor effects of 1,25-dihydroxyvitamin D₃ (calcitriol) are being exploited for prevention and treatment of prostate cancer (CaP). These studies examined the antiproliferative effects of calcitriol in primary cell cultures derived from transgenic adenocarcinoma of mouse prostate (TRAMP) mice chronically treated with calcitriol (20 μg/kg) or vehicle 3×/week from 4 weeks-of-age until palpable tumors developed. This is a report on the response of two representative control (Vitamin D naïve, naïve) and calcitriol-treated (Vitamin D insensitive, VDI) cells to calcitriol. VDI cells were less sensitive to calcitriol based on less cell growth inhibition and less inhibition of DNA synthesis as measured by MTT and BrdU incorporation assays. Similarly, VDI cells were less sensitive to growth inhibition by the vitamin analog, 19-nor-1,25-dihydroxyvitamin D₂ (paricalcitol). There was no change in apoptosis following treatment of naïve and VDI cells with calcitriol. Vitamin D receptor (VDR) expression was up-regulated by calcitriol in both naïve and VDI cells. In addition, calcitriol induced the Vitamin D metabolizing enzyme, 24-hydroxylase (cyp24) mRNA and enzyme activity similarly in naïve and VDI cells as measured by RT-PCR and HPLC, respectively. In summary, VDI cells are less responsive to the antiproliferative effects of calcitriol. Understanding Vitamin D insensitivity will further clinical development of Vitamin D compounds for prevention and treatment of CaP.     

In vivo relevance for photoprotection by the vitamin D rapid response pathway

Vitamin D is produced by exposure of 7-dehydrocholesterol in the skin to UV irradiation (UVR) and further converted in the skin to the biologically active metabolite, 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) and other compounds. UVR also results in DNA damage producing cyclobutane pyrimidine dimers (CPD). We previously reported that 1,25(OH)₂D₃ at picomolar concentrations, protects human skin cells from UVR-induced apoptosis, and decreases CPD in surviving cells. 1,25(OH)₂D₃ has been shown to generate biological responses via two pathways—the classical steroid receptor/genomic pathway or a rapid, non-genomic pathway mediated by a putative membrane receptor. Whether the rapid response pathway is physiologically relevant is unclear. A cis-locked, rapid-acting agonist 1,25(OH)₂lumisterol₃ (JN), entirely mimicked the actions of 1,25(OH)₂D₃ to reduce fibroblast and keratinocyte loss and CPD damage after UVR. The effects of 1,25(OH)₂D₃ were abolished by a rapid-acting antagonist, but not by a genomic antagonist. Skh:hr1 mice exposed to three times the minimal erythemal dose of solar-simulated UVR and treated topically with 1,25(OH)₂D₃ or JN immediately after UVR showed reduction in UVR-induced UVR-induced sunburn cells (p < 0.01 and <0.05, respectively), CPD (p < 0.01 for both) and immunosuppression (p < 0.001 for both) compared with vehicle-treated mice. These results show for the first time an in vivo biological response mediated by a rapid-acting analog of the vitamin D system. The data support the hypothesis that 1,25(OH)₂D₃ exerts its photoprotective effects via the rapid pathway and raise the possibility that other D compounds produced in skin may contribute to the photoprotective effects.     

Use of genetically modified mice to examine the skeletal anabolic activity of vitamin D

We employed genetically modified mice to examine the role of 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃] on skeletal and calcium homeostasis. In mice expressing the null mutation for 25-hydroxyvitamin D 1 hydroxylase (1OHase^−/−), or the vitamin D receptor (VDR^−/−), 1,25(OH)₂D₃ and calcium were both required for optimal epiphyseal growth plate development, serum calcium and phosphorus alone were sufficient to mineralize skeletal tissue independent of 1,25(OH)₂D₃ and the VDR, and endogenous 1,25(OH)₂D₃ and the VDR were essential for baseline bone formation. In 2-week-old 1OHase^−/− mice and in 2-week-old mice homozygous for the PTH null mutation(PTH^−/−), PTH and 1,25(OH)₂D₃ were each found to exert independent and complementary effects on skeletal anabolism, with PTH predominantly affecting appositional trabecular bone growth and 1,25(OH)₂D₃ influencing both endochondral bone formation and appositional bone growth. Endogenous 1,25(OH)₂D₃ maintained serum calcium homeostasis predominantly by modifying intestinal and renal calcium transporters but not by producing net bone resorption. Administration of exogenous 1,25(OH)₂D₃ to double mutant PTH^−/−1OHase^−/− mice produced skeletal effects consistent with the actions of endogenous 1,25(OH)₂D₃. These studies reveal an important skeletal anabolic role for both endogenous and exogenous 1,25(OH)₂D₃ and point to a potential role for 1,25(OH)₂D₃ analogs in the treatment of disorders of bone loss.     

Clusterin over-expression modulates proapoptotic and antiproliferative effects of 1,25(OH)2D3 in prostate cancer cells in vitro

Prostate cancer is the most commonly diagnosed cancer in the majority of western countries. Due to their antiproliferative and proapoptotic activity, vitamin D analogues have been introduced recently as an experimental therapy for prostate cancer. Clusterin (CLU) is a glycoprotein that has two known isoforms generated in human cells. A nuclear form of CLU protein (nCLU) is pro-apoptotic, and a secretory form (sCLU) is pro-survival. In this study, we analyzed whether proapoptotic and antiproliferative effects of 1,25(OH)₂D₃ on LNCaP prostate cancer cells are modulated by expression of sCLU. Using colony forming assay, we studied the effect of treatment with different doses of 1,25(OH)₂D₃ (10⁻⁶, 10⁻⁷, 10⁻¹⁰ M) on proliferation of LNCaP cells that were stable transfected and over-express sCLU (LNT-1) as compared to empty vector-transfected cells (LN/C). We also measured apoptosis using TUNEL assay. sCLU over-expression protected against both antiproliferative (30%) and proapoptotic (15%) effects of 1,25(OH)₂D₃, although this effect was statistically not significant. In conclusion, our findings demonstrate that expression of sCLU modulates growth regulatory effects of 1,25(OH)₂D₃ in prostate cancer indicating that CLU interferes with vitamin D signalling pathways.     

Regulation of the ferredoxin component of renal hydroxylases at transcriptional and postranslational levels and of the protein inhibitor of cyclic AMP-dependent kinase

We have studied two proteins potentially involved in the regulation of the 25-OH-D-1-hydroxylase, which is located in the renal mitochondria and which is responsible for the production of the steroid hormone 1,25(OH)₂D₃. The endogenous inhibitor of cyclic AMP-dependent protein kinase, PKI, is down regulated by 1,25(OH)₂D₃. Having cloned and sequenced PKI cDNA, we studied its message levels and found them to be regulated by 1,25(OH)₂D₃ tissue specifically in the kidney and in kidney cell culture. In other experiments we over expressed the ferredoxin component of the 1-hydroxylase and found it to be physically and chemically indistinguishable from those of classic steroidogenic tissues. The mRNA encoding the ferredoxin component is up-regulated by chronic vitamin D deficiency, which at the same time leads to sustained elevation in 1-hydroxylase activity; no short term effect of 1,25(OH)₂D₃ on ferredoxin mRNA in kidney cell culture could be demonstrated. Finally, there was an association between decreased phosphorylation of ferredoxin and decreased 1-hydroxylase activity brought about by treatment of cultured kidney cells with TPA. Control of the renal signaling events involved in the production of 1,25(OH)₂D₃ remains a fruitful area of investigation in the field of the metabolism and actions of vitamin D and its metabolites.     

Regulation of the CYP27B1 5'-flanking region by transforming growth factor-beta in ROS 17/2.8 osteoblast-like cells

The biologically active form of vitamin D, 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃), regulates osteoblast proliferation and differentiation. Production of 1,25(OH)₂D₃ is catalysed by the enzyme 25-hydroxyvitamin D₃-1-hydroxylase (CYP27B1). Though highly expressed in the kidney, the CYP27B1 gene is also expressed in non-renal tissues including bone. It is hypothesised that local production of 1,25(OH)₂D₃ by osteoblasts plays an autocrine or paracrine role. The aim of this study was to investigate what factors regulate expression of the CYP27B1 gene in osteoblast cells. ROS 17/2.8 osteoblast cells were transiently transfected with plasmid constructs containing the 5′-flanking sequence of the human CYP27B1 gene fused to a luciferase reporter gene. Cells were treated with either parathyroid hormone (PTH), 1,25(OH)₂D₃, transforming growth factor-beta (TGF-β) or insulin-like growth factor-1 (IGF-1) and luciferase activity was measured 24 h later. The results showed that 1,25(OH)₂D₃ did not alter expression of the reporter construct, however treatment with PTH, IGF-1 and TGF-β decreased expression by 18, 53 and 58% respectively. The repressive action of TGF-β was isolated to the region between −531 and −305 bp. These data suggest that expression of the 5′-flanking region for the CYP27B1 gene in osteoblast cells may be regulated differently to that previously described in kidney cells.     

Differential biological effects of 1,25-dihydroxyVitamin D3 on melanoma cell lines in vitro

1,25-DihydroxyVitamin D(3) and analogs have been shown to inhibit proliferation and to induce differentiation in different cell types, including human melanocytes. However, various tumor cell lines that fail to respond to the antiproliferative effects of Vitamin D analogs have also been reported. Using real-time PCR (LightCycler), we have compared mRNA expression of Vitamin D receptor (VDR), Vitamin D-25-hydroxylase (25-OHase), 25-hydroxyVitamin D-1alpha-hydroxylase (1alpha-OHase), and 1,25-dihydroxyVitamin D-24-hydroxylase (24-OHase) in a melanoma cell line that responds to antiproliferative effects of Vitamin D (MeWo) with a non-responsive melanoma cell line (SkMel5). Additionally, modulation of cell proliferation by calpain inhibitors, as well as regulation of mRNA expression of VDR, 1alpha-OHase, and 24-OHase genes by Vitamin D analogs were assessed in melanoma cell lines in vitro using a WST-1 based colorimetric assay and real-time PCR, respectively. RNA for VDR, 25-OHase, 1alpha-OHase, and 24-OHase was detected in melanoma cell lines. In contrast to SkMel5 cells, treatment of MeWo cells with calcitriol resulted in a dose-dependent increase in mRNA for VDR and 24-OHase as well as in a suppression of cell proliferation (up to approximately 50%). Our findings demonstrate that local synthesis or metabolism of Vitamin D metabolites may be of importance for growth regulation of MM and melanoma cell lines. Additionally, metastasizing MM represents a promising target for palliative treatment with new Vitamin D analogs that exert little calcemic side effects or for pharmacological modulation of calcitriol synthesis/metabolism in these tumors.     

25(OH)D3 and 1,25(OH)2D3 serum concentration and breast tissue expression of 1alpha-hydroxylase, 24-hydroxylase and Vitamin D receptor in women with and without breast cancer

1,25(OH)2D3 is an antiproliferative agent that may inhibit proliferation of breast cancer (BC) cells in vitro and BC development in animals. Epidemiological studies have shown a high incidence of BC in people less exposed to solar rays. To unravel the role of Vitamin D3 in BC patients, we have investigated serum levels of 25(OH)D3 and its active form 1,25(OH)2D3 as well as tissue expression of 1alpha-hydroxylase, 24-hydroxylase, and Vitamin D-receptor (VDR), determined by semiquantitative RT-PCR, in 88 Brazilian BC patients and 35 women without cancer (submitted to mammoplasties or resection of benign lesions). Median age of women with and without cancer was 51 and 46 years, respectively, and the majority of BC patients were classified as clinical stage II (67%). Although no differences in 25(OH)D3 serum concentration were found, 1,25(OH)2D3 (40+/-21 pg/ml) levels in BC patients were lower than in women without cancer (53+/-23). Our results indicate that 24-hydroxylase, VDR and 1alpha-hydroxylase mRNA tissue expression is similar in both groups and no correlation between 24-hydroxylase, 1alpha-hydroxylase, and VDR expression in breast tumors was found. A low 1,25(OH)2D3 serum concentration seems to be associated to breast cancer, however, the mechanism involved in this regulation is still unclear.     

Functional involvement of calcium in the homologous up-regulation of the 1,25-dihydroxyvitamin D3 receptor in osteoblast-like cells

In several cell types 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) causes up-regulation of its receptor. The present study demonstrates that in the osteoblast-like cell line UMR 106 this up-regulation is inhibited by two different calcium channel blockers (nitrendipine, verapamil). Also with chelating extracellular calcium (EGTA) and by inhibition of calcium release from intracellular stores (TMB-8) comparable results were obtained. These findings indicate that calcium is functionally involved in this cellular response to the steroid hormone 1,25(OH)₂D₃. Moreover, data obtained with EGTA show that the 1,25(OH)₂D₃ receptor level is closely regulated by the extracellular calcium concentration.