Plasma membrane requirements for 1alpha,25(OH)2D3 dependent PKC signaling in chondrocytes and osteoblasts

1,25-Dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)] acts on chondrocytes and osteoblasts through traditional nuclear Vitamin D receptor (VDR) mechanisms as well as through rapid actions on plasma membranes that initiate intracellular signaling pathways. We have investigated the mechanisms involved in activation of protein kinase C (PKC) and downstream biological responses that depend on the latter pathway. These studies show that PKC activation depends on presence of a membrane receptor ERp60 and rapid increases in phospholipase A(2) (PLA(2)) activity. Cells that are responsive to 1alpha,25(OH)(2)D(3) express PLA(2) activating protein (PLAA), suggesting a link between ERp60 and PLA(2). Increased PLA(2) results in increased arachidonic acid release and formation of lysophospholipid, which then activates phospholipase C beta (PLCbeta), leading to rapid formation of inositol-trisphosphate (IP3) and diacylglycerol (DAG). PLA(2), PLC, and DAG are all associated with lipid rafts including caveolae in many cells, suggesting that the caveolar environment may be an important mediator of PKC activation by 1alpha,25(OH)(2)D(3). Here, we use the VDR(-/-) mouse costochondral cartilage growth plate to examine the expression of ERp60 and PLAA in vivo in 1alpha,25(OH)(2)D(3)-responsive hypertrophic chondrocytes (growth zone cells) and in resting zone cells that do not respond to this Vitamin D metabolite in vitro. In addition, we determined if intact lipid rafts are required for the response of rat costochondral cartilage growth zone cells to 1alpha,25(OH)(2)D(3). The results show that ERp60 and PLAA are localized to 1alpha,25(OH)(2)D(3)-responsive growth zone cells and metaphyseal osteoblasts, even in VDR(-/-) mice. Disruption of lipid rafts using beta-cyclodextrin blocks the activation of PKC by 1alpha,25(OH)(2)D(3) and reduces the ability of 1alpha,25(OH)(2)D(3) to regulate [(35)S]-sulfate incorporation.     

Phosphate uptake in chick kidney cells: effects of 1,25(OH)2D3 and 24,25(OH)2D3

Phosphate homeostasis is controlled in part by absorption from the intestine, and reabsorption in the kidney. While the effect of Vitamin D metabolites on enterocytes is well documented, in the current study we assess selected responses in primary cultures of kidney cells. Time course studies revealed a rapid stimulation of phosphate uptake in cells treated with 1,25(OH)(2)D(3), relative to controls. Dose-response studies indicated a biphasic curve with optimal stimulation at 300 pM 1,25(OH)(2)D(3) and inhibition at 600 pM seco-steroid. Antibody 099--against the 1,25D(3)-MARRS receptor - abolished stimulation by the steroid hormone. Moreover, phosphate uptake was mediated by the protein kinase C pathway. The metabolite 24,25(OH)(2)D(3), which was found to inhibit the rapid stimulation of phosphate uptake in intestinal cells, had a parallel effect in cultured kidney cells. Finally, the 24,25(OH)(2)D(3) binding protein, catalase, was assessed for longer term down regulation. In both intestinal epithelial cells and kidney cells incubated with 24,25(OH)(2)D(3) for 5-24h, both the specific activity of the enzyme and protein levels were decreased relative to controls, while 1,25(OH)(2)D(3) increased both parameters over the same time periods. We conclude that the Vitamin D metabolites have similar effects in both kidney and intestine, and that 24,25(OH)(2)D(3) may have effects at the level of gene expression.     

History of the development of new vitamin D analogs: studies on 22-oxacalcitriol (OCT) and 2beta-(3-hydroxypropoxy)calcitriol (ED-71)

In 1981 Suda and his colleagues first reported the new activity of calcitriol namely its ability to differentiate the myeloid leukemia cells into normal monocytes-macrophages. However, the possibility of using calcitriol as an antileukemic drug was not feasible because of its potent calcemic effects. Based on these observations, several pharmaceutical companies initiated the synthesis of vitamin D analogs with the aim to separate the calcemic actions of calcitriol from its actions on regulating the cell growth and differentiation. As a result, numerous noncalcemic analogs with a potential for the treatment of leukemia and other cancers were synthesized. The group at Chugai introduced two characteristic analogs of opposite type namely, 22-oxacalcitriol (OCT) and 2beta-(3-hydroxypropoxy)calcitriol (ED-71) which have been shown to have therapeutic value and are already being used clinically. The work on OCT and ED-71 together with the work on calcipotriol and KH-1060 by Leo Laboratories, and 1alpha,25(OH)(2)-16-ene-23-yne-D(3) by Hoffmann-La Roche, vigorously stimulated research world-wide in the development of vitamin D analogs into pharmaceutical products. More recently new impressive vitamin D analogs such as 3-epi analogs, 19-nor analogs, 18-nor analogs, 2-methyl-20-epi-calcitriol, non-steroidal vitamin D analogs are being developed. The authors are convinced that various vitamin D analogs will become highly effective therapeutic agents at the clinical level in the new century, and also that a new theory on the mechanism of vitamin D action will be generated.     

Anti-endothelial properties of 1,25-dihydroxy-3-epi-vitamin D3, a natural metabolite of calcitriol

BACKGROUND: Calcitriol [1,25-(OH)(2)D(3)] is a strong anti-proliferative agent both in vitro and in vivo. Earlier studies have established that calcitriol inhibits the growth factor-stimulated proliferation of endothelial cells (EC) and angiogenesis. However, the lethal calcemic side effects of calcitriol prohibit its use as a therapeutic agent. Several analogs of vitamin D have been developed to minimize these calcemic side effects. 1,25-dihydroxy-3-epi-vitamin D(3) (3-epiD(3)), a naturally formed vitamin D metabolite is one such analog. OBJECTIVE: To demonstrate that 3-epiD(3), a calcitriol analog, inhibits endothelial cell proliferation and induces apoptosis. RESULTS: Treatment of EC with 3-epiD(3) showed 60% inhibition (P < 0.006) of proliferation. Cell viability assays corroborated these results. Pro-apoptotic caspase-3 activity was increased fourfold (P < 0.01) in 3-epiD(3)-treated cells over controls. 3-epiD(3) induced apoptosis in EC as shown by genomic DNA fragmentation. Cell cycle analysis of 3-epiD(3)-treated EC revealed a G0/G1 arrest. CONCLUSIONS: 3-epiD(3), a low-calcemic, natural analog of calcitriol, inhibits EC proliferation by causing a G0/G1 arrest and induces apoptosis more effectively than 1,25-(OH)(2)D(3). These results suggest that 3-epiD(3) is a potent inhibitor of EC growth.     

1,25-(OH)2-16ene-23yne-D3 reduces secondary hyperparathyroidism in uremic rats with little calcemic effect

OBJECTIVES: To compare the effects of vitamin D analogs versus calcitriol on serum levels of Ca, P and parathyroid hormone (PTH). A compound better than calcitriol should increase the Ca x P product less than calcitriol for an equivalent decrease in PTH levels. METHODS: Biological activity of 4 vitamin D analogs, 1,25-(OH)(2)-16ene- D(3) (RO(1)), 1,25-(OH)(2)-16ene-23yne-D(3) (RO(2)), 1,25-(OH)(2)-26,27-hexafluoro-16ene-23yne-D(3) (RO(3)) and 1,25-(OH)(2)-16ene-23yne-26,27-hexafluoro-19nor-D(3) (RO(4)) was tested vs. calcitriol in parathyroidectomized rats. In a second set of experiments, the effects of RO(2), RO(4) and calcitriol were studied in 5/6 nephrectomized rats with secondary hyperparathyroidism. RESULTS: In parathyroidectomized rats, all analogs (250 pmol/day) led calcemia to rise after 7 days. In uremic rats, all treatments reduced PTH levels. RO(4) revealed toxicity. RO(2) was as effective as calcitriol in suppressing PTH in a dose dependent manner. Mean plasma ionized calcium did not change from baseline to day 14 and day 28 on RO(2) (250 or 500 pmol/day) whereas it increased significantly on RO(2) (1,000 pmol/day) and calcitriol (125 or 250 pmol/day). Increasing the dose of calcitriol led Ca x P to rise more dramatically than increasing the dose of RO(2), which appears to have a wider therapeutic window than calcitriol. CONCLUSION: 1,25-(OH)(2)-16ene-23yne-D(3) (RO(2)) may represent a novel candidate for the treatment of renal osteodystrophy in humans.     

Effects of 1,25(OH)2D3, 25OHD3, and EB1089 on cell growth and Vitamin D receptor mRNA and 1alpha-hydroxylase mRNA expression in primary cultures of the canine prostate

The aim of this study was to investigate effects of 1,25(OH)(2)D(3) (calcitriol), 25OHD(3), and EB1089 on cell growth and on Vitamin D receptor (VDR) mRNA and 1alpha-hydroxylase (1alpha-OHase) mRNA expression in normal canine prostatic primary cultures. Canine prostatic epithelial cells were isolated, cultured, and treated with vehicle (ethanol), calcitriol, 25OHD(3), and EB1089 at 10(-9) and 10(-7)M. The VDR was present in epithelial and stromal cells of the canine prostate gland. 1,25(OH)(2)D(3), 25OHD(3), and EB1089 inhibited epithelial cell growth at 10(-7)M compared to vehicle-treated controls [calcitriol (P < 0.01), EB1089 (P < 0.01), and 25OHD(3) (P < 0.05)]. Epithelial cells treated with calcitriol and EB1089 at 10(-7)M had slightly increased VDR mRNA expression (0.2-0.3-fold) at 6 and 12h compared to controls. There was no difference in 1alpha-OHase mRNA expression in epithelial cells treated with these three compounds. 1,25(OH)(2)D(3) and its analogs may be effective antiproliferative agents of epithelial cells in certain types of prostate cancer.     

1alpha,25(OH)2D3 causes a rapid increase in phosphatidylinositol-specific PLC-beta activity via phospholipase A2-dependent production of lysophospholipid

1alpha,25(OH)(2)D(3) activates protein kinase C (PKC) in rat growth plate chondrocytes via mechanisms involving phosphatidylinositol-specific phospholipase C (PI-PLC) and phospholipase A(2) (PLA(2)). The purpose of this study was to determine if 1alpha,25(OH)(2)D(3) activates PI-PLC directly or through a PLA(2)-dependent mechanism. We determined which PLC isoforms are present in the growth plate chondrocytes, and determined which isoform(s) of PLC is(are) regulated by 1alpha,25(OH)(2)D(3). Inhibitors and activators of PLA(2) were used to assess the inter-relationship between these two phospholipid-signaling pathways. PI-PLC activity in lysates of prehypertrophic and upper hypertrophic zone (growth zone) cells that were incubated with 1alpha,25(OH)(2)D(3), was increased within 30s with peak activity at 1-3 min. PI-PLC activity in resting zone cells was unaffected by 1alpha,25(OH)(2)D(3). 1beta,25(OH)(2)D(3), 24R,25(OH)(2)D(3), actinomycin D and cycloheximide had no effect on PLC in lysates of growth zone cells. Thus, 1alpha,25(OH)(2)D(3) regulation of PI-PLC enzyme activity is stereospecific, cell maturation-dependent, and nongenomic. PLA(2)-activation (mastoparan or melittin) increased PI-PLC activity to the same extent as 1alpha,25(OH)(2)D(3); PLA(2)-inhibition (quinacrine, oleyloxyethylphosphorylcholine (OEPC), or AACOCF(3)) reduced the effect of 1alpha,25(OH)(2)D(3). Neither arachidonic acid (AA) nor its metabolites affected PI-PLC. In contrast, lysophosphatidylcholine (LPC) and lysophosphatidylethanolamine (LPE) activated PI-PLC (LPE>LPC). 1alpha,25(OH)(2)D(3) stimulated PI-PLC and PKC activities via Gq; GDPbetaS inhibited activity, but pertussis toxin did not. RT-PCR showed that the cells express PLC-beta1a, PLC-beta1b, PLC-beta3 and PLC-gamma1 mRNA. Antibodies to PLC-beta1 and PLC-beta3 blocked the 1alpha,25(OH)(2)D(3) effect; antibodies to PLC-delta and PLC-gamma did not. Thus, 1alpha,25(OH)(2)D(3) regulates PLC-beta through PLA(2)-dependent production of lysophospholipid.     

C-25 hydroxylation of 1alpha,24(R)-dihydroxyvitamin D3 is catalyzed by 25-hydroxyvitamin D3-24-hydroxylase (CYP24A1): metabolism studies with human keratinocytes and rat recombinant CYP24A1

Recently, 25-hydroxyvitamin D3-24-hydroxylase (CYP24A1) has been shown to catalyze not only hydroxylation at C-24 but also hydroxylations at C-23 and C-26 of the secosteroid hormone 1alpha, 25-dihydroxyvitamin D3 (1alpha,25(OH)2D3). It remains to be determined whether CYP24A1 has the ability to hydroxylate vitamin D3 compounds at C-25. 1alpha,24(R)-dihydroxyvitamin D3 (1alpha,24(R)(OH)2D3) is a non-25-hydroxylated synthetic vitamin D3 analog that is presently being used as an antipsoriatic drug. In the present study, we investigated the metabolism of 1alpha,24(R)(OH)2D3 in human keratinocytes in order to examine the ability of CYP24A1 to hydroxylate 1alpha,24(R)(OH)2D3 at C-25. The results indicated that keratinocytes metabolize 1alpha,24(R)(OH)2D3 into several previously known both 25-hydroxylated and non-25-hydroxylated metabolites along with two new metabolites, namely 1alpha,23,24(OH)3D3 and 1alpha,24(OH)2-23-oxo-D3. Production of the metabolites including the 25-hydroxylated ones was detectable only when CYP24A1 activity was induced in keratinocytes 1alpha,25(OH)2D3. This finding provided indirect evidence to indicate that CYP24A1 catalyzes C-25 hydroxylation of 1alpha,24(R)(OH)2D3. The final proof for this finding was obtained through our metabolism studies using highly purified recombinant rat CYP24A1 in a reconstituted system. Incubation of this system with 1alpha,24(R)(OH)2D3 resulted in the production of both 25-hydroxylated and non-25-hydroxylated metabolites. Thus, in our present study, we identified CYP24A1 as the main enzyme responsible for the metabolism of 1alpha,24(R)(OH)2D3 in human keratinocytes, and provided unequivocal evidence to indicate that the multicatalytic enzyme CYP24A1 has the ability to hydroxylate 1alpha,24(R)(OH)2D3 at C-25.     

Calcidiol and prostate cancer

Epidemiological studies suggest that serum calcidiol (25(OH)-Vitamin D3) seems to be associated with several cancers including prostate cancer. We have made several experimental studies in order to clarify the mechanism(s) involved in the association. Calcidiol has been regarded as an inactive prohormone for calcitriol, which possesses the highest biological activity of the Vitamin D metabolites, when it is evaluated on the basis of bioactivity/nmol. However, we found recently that at the physiological concentration calcidiol (100-200 nM) is an active hormone, whereas calcitriol (1alpha,25(OH)2-Vitamin D3) (100 pM) is inactive in human primary prostate stromal cells. Calcidiol is able to inhibit cell growth and to induce or inhibit several genes including 1alpha-hydroxylase and 24-hydroxylase genes. This suggests that calcidiol might be an independent endocrine system involved in the control of cell differentiation and proliferation, whereas calcitriol might be mainly involved in the regulation of calcium and phosphorous balance. Several mechanisms may mediate the action of Vitamin D in the prostate. This is a review of some recent studies on the role of (1) Vitamin D metabolism, (2) growth factors and (3) fatty acid metabolism.     

Biologic effect of 1,24(R)(OH)2D3 versus 1,25(OH)2D3 administration in chronic renal failure

1,24(R)(OH)2D3 is a synthetic analogue of 1,25(OH)2D3 which binds to the same receptors as the physiologic metabolite with a lower affinity. The aim of the present study was to compare the activity of 1,24(R)(OH)2D3 and 1,25(OH)2D3 on several target organs in patients with chronic renal failure. Treatment with 1,24(R)(OH)2D3 at doses of either 1 or 2 μg daily was carried out in two groups of 9 patients, with serum creatinine of 4.61 ± 1.59 and 4.66 ± 1.46 mg/dl, respectively. Doses of 1,25(OH)2D3 were 0.5 and 1 μg daily and were administered to 9 and 13 patients, serum creatinine of 4.52 ± 1.67 and 4.3 ± 1.16 mg/dl, respectively. Treatment periods were of 2 weeks. Administration of 1,25(OH)2D3, 1 μg, induced significant increments of intestinal calcium absorption (ICA), ionized calcium, osteocalcin, serum creatinine, urine Ca/GFR, and a decrease in iPTH. 1,25(OH)2D3, 0.5 μg, induced a significant increase in ICA and osteocalcin and a decrease in iPTH. Similarly 1,24(OH)2D3, 2 μg daily, significantly stimulated ICA and raised serum levels of osteocalcin and creatinine while lowering serum iPTH. In addition, 1,24(R)(OH)2D3 administration induced a significant fall of serum 1,25(OH)2D3. Following 1 μg, only osteocalcin increased. Therefore, the dose of 2 μg of 1,24(R)(OH)2D3 has biologic activity similar to 0.5 μg 1,25(OH)2D3 (4:1). However the activity ratio on osteocalcin production appears to be 2:1. In addition, 1,24(R)(OH)2D3 is able to inhibit renal tubular 1-hydroxylase. In conclusion 1,24(R)(OH)2D3 may prove to be useful in the treatment of metabolic bone disease.     

Regulation of serum 1,25(OH)2 vitamin D3 levels by fibroblast growth factor 23 is mediated by FGF receptors 3 and 4

Fibroblast growth factor 23 (FGF23) is a phosphaturic hormone implicated in the pathogenesis of several hypophosphatemic disorders. FGF23 causes hypophosphatemia by decreasing the expression of sodium phosphate cotransporters (NaPi-2a and NaPi-2c) and decreasing serum 1,25(OH)(2)Vitamin D(3) levels. We previously showed that FGFR1 is the predominant receptor for the hypophosphatemic actions of FGF23 by decreasing renal NaPi-2a and 2c expression while the receptors regulating 1,25(OH)(2)Vitamin D(3) levels remained elusive. To determine the FGFRs regulating 1,25(OH)(2)Vitamin D(3) levels, we studied FGFR3(-/-)FGFR4(-/-) mice as these mice have shortened life span and are growth retarded similar to FGF23(-/-) and Klotho(-/-) mice. Baseline serum 1,25(OH)(2)Vitamin D(3) levels were elevated in the FGFR3(-/-)FGFR4(-/-) mice compared with wild-type mice (102.2 ± 14.8 vs. 266.0 ± 34.0 pmol/l; P = 0.001) as were the serum levels of FGF23. Administration of recombinant FGF23 had no effect on serum 1,25(OH)(2)Vitamin D(3) in the FGFR3(-/-)FGFR4(-/-) mice (173.4 ± 32.7 vs. 219.7 ± 56.5 pmol/l; vehicle vs. FGF23) while it reduced serum 1,25(OH)(2)Vitamin D(3) levels in wild-type mice. Administration of FGF23 to FGFR3(-/-)FGFR4(-/-) mice resulted in a decrease in serum parathyroid hormone (PTH) levels and an increase in serum phosphorus levels mediated by increased renal phosphate reabsorption. These data indicate that FGFR3 and 4 are the receptors that regulate serum 1,25(OH)(2)Vitamin D(3) levels in response to FGF23. In addition, when 1,25(OH)(2)Vitamin D(3) levels are not affected by FGF23, as in FGFR3(-/-)FGFR4(-/-) mice, a reduction in PTH can override the effects of FGF23 on renal phosphate transport.     

New analogs of 2-methylene-19-nor-(20S)-1,25-dihydroxyvitamin D3 with conformationally restricted side chains: evaluation of biological activity and structural determination of VDR-bound conformations

We have successfully prepared E- and Z- isomers of 17-20 dehydro analogs of 2-methylene-19-nor-(20S)-1alpha,25-dihydroxyvitamin D3 (2MD). Both isomers bind to the recombinant rat vitamin D receptor (VDR) with high affinity. The Z-isomer (Vit-III 17-20Z) displays activity in vivo and in vitro that is similar to 2MD. The in vitro activity of the E-isomer (Vit-III 17-20E) is comparable to the natural hormone, though in vivo this analog is significantly less calcemic. Crystal structures of the rat VDR ligand binding domain complexed with the analogs demonstrate that the Vit-III 17-20Z analog is oriented almost identically to 2MD, with only minor differences induced by the planar configuration around the C17-C20 double bond. The Vit-III 17-20E analog is oriented in a conformation distinct from both 2MD and the natural hormone. The structural comparisons suggest that the position of C21 in the ligand binding site may be an important determinant of biological activity.     

A non-calcemic analog of 1 alpha,25 dihydroxy vitamin D(3) (JKF) upregulates the induction of creatine kinase B by 17 beta estradiol in osteoblast-like ROS 17/2.8 cells and in rat diaphysis.

We have reported that multiple treatments with so-called 'non-hypercalcemic' analogs of 1 alpha,25(OH)(2) vitamin D(3) (1,25(OH)(2)D(3)) stimulate the specific activity of creatine kinase BB (CK) in ROS 17/2.8 osteoblast-like cells, and that pretreatment with these analogs upregulates responsiveness and sensitivity to 17 beta estradiol (E(2)) for the induction of CK. However, since the analogs showed toxicity in vivo, we have now studied the action of a demonstrably non-calcemic hybrid analog of vitamin D in ROS 17/2.8 cells, and prepubertal rats. The analog JKF was designed to separate its calcemic activity from other biological activities by combining a calcemic-lowering 1-hydroxymethyl group with a potentiating C, D-ring side chain modification including 24 difluoronation. Treatment with 1 pM JKF alone significantly stimulated CK specific activity at 4 h by 30+/-10%. However after three daily pretreatments, JKF upregulated the extent of induction by 30 nM E(2) by 33% at 1 pM and by 97% at 1 nM; the E(2) dose needed for a significant stimulation of CK activity was lowered to 30 pM. The action of the SERMS tamoxifen, tamoxifen methiodide and raloxifene, at 3 microM, was also upregulated by three daily pretreatments with 1 nM JKF; unexpectedly, this pretreatment prevented the inhibition of E(2) stimulation by the SERMS. Upregulation of E(2) action by 1 nM JKF was inhibited by 1 nM ZK159222, an inhibitor of the nuclear action of 1,25(OH)(2)D(3). In vivo, three daily injections of 0.05 ng/g body weight of JKF augmented the response of prepubertal female rat diaphysis and epiphysis to E(2). Therefore, demonstrably non-calcemic analogs of 1,25(OH)(2)D(3) may have potential for use in combination with estrogens or SERMS in the prevention and/or treatment of metabolic bone diseases such as postmenopausal osteoporosis.     

Vitamin D compounds in leukemia

The biologically active form of vitamin D, 1,25-dihydroxyvitamin D3 [1,25(OH)(2)D3,] possess in vitro multiple anti-cancer activities including growth arrest, induction of apoptosis and differentiation of a variety of different types of malignant cells. However, its use as a therapeutic agent is hindered by its calcemic effects. Analogs of 1,25(OH)(2)D3 have enhanced anti-tumor activity, with reduced calcemic effects. However, limited clinical studies using vitamin D compounds have not yet achieved major clinical success. Nevertheless, pre-clinical studies suggest that the combination of either 1,25(OH)(2)D3 or its analogs with other agents can have additive or synergistic anti-cancer activities, suggesting future clinical studies.     

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.