Colon cancer and solar ultraviolet B radiation and prevention and treatment of colon cancer in mice with vitamin D and its Gemini analogs

It has been recognized that people who live at higher latitudes and who are vitamin D deficient are at higher risk of dying from many common cancers including colon cancer. To evaluate the role of vitamin D deficiency on colon tumor growth, Balb/c adult male mice were fed either a vitamin D sufficient or vitamin D deficient diet for 10 weeks. Mice were arranged into groups of six and each animal received subcutaneously 10(4) MC-26 cells in the posterior trunk. The tumor size was recorded daily. By day 9 there was a significant difference in tumor volume between the vitamin D sufficient and vitamin D deficient mice. By day 18 the vitamin D deficient animals had a tumor size that was 56% larger compared to the animals that were vitamin D sufficient. To determine whether treatment with active vitamin D analogs could further decrease colon tumor growth in a vitamin D sufficient state, groups of mice were treated with the novel 19-nor-Gemini compounds. The mice were fed a low calcium diet. Twenty-four hours after tumor implantation, the mice received, three times weekly, one of the vitamin D analogs or the vehicle. The group that received Gemini 1,25-dihydroxy-21(3-hydroxy-3-trifluoromethyl-4-trifluoro-butynyl)-19-nor-20S-cholecalciferol (3) showed a dose-dependent decrease in tumor volume. On day 19, at the dose level of 0.02microg molar equivalents (E), the tumor volume was reduced by 41% when compared to the control group. At the same time point, the hexadeuterated analog 1,25-dihydroxy-21(3-hydroxy-3-trifluoromethyl-4-trifluoro-butynyl)-26,27-hexadeutero-19-nor-20S-cholecalciferol (4), administered at the 10-fold lower dose of 0.002microgE, showed a 52% reduction in tumor volume (p<0.05), compared to the control group. Animals that received 1,25(OH)(2)D(3) at 0.002 and 0.02microg showed a trend in tumor volume reduction at the highest dose but the changes were not statistically significant. An evaluation of serum calcium concentrations revealed that the calcium levels were normal in all groups, except the group receiving 0.02microgE of 4. The results from these studies demonstrate that vitamin D deficiency may accelerate colon cancer growth and that novel Gemini analogs of 1,25(OH)(2)D(3) may be an effective new approach for colon cancer treatment.     

Recent advances in our understanding of 1,25-dihydroxyvitamin D(3) regulation of intestinal calcium absorption

Calcium is required for many cellular processes including muscle contraction, nerve pulse transmission, stimulus secretion coupling and bone formation. The principal source of new calcium to meet these essential functions is from the diet. Intestinal absorption of calcium occurs by an active transcellular path and by a non-saturable paracellular path. The major factor influencing intestinal calcium absorption is vitamin D and more specifically the hormonally active form of vitamin D, 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)). This article emphasizes studies that have provided new insight related to the mechanisms involved in the intestinal actions of 1,25(OH)(2)D(3). The following are discussed: recent studies, including those using knock out mice, that suggest that 1,25(OH)(2)D(3) mediated calcium absorption is more complex than the traditional transcellular model; evidence for 1,25(OH)(2)D(3) mediated active transport of calcium by distal as well as proximal segments of the intestine; 1,25(OH)(2)D(3) regulation of paracellular calcium transport and the role of 1,25(OH)(2)D(3) in protection against mucosal injury.     

Regulation of renal vitamin D receptor is an important determinant of 1alpha,25-dihydroxyvitamin D(3) levels in vivo

The synthesis of 1,25-dihydroxyvitamin D(3) (1,25-(OH)(2)D(3)) is most strongly regulated by dietary calcium and the action of parathyroid hormone to increase 1alpha-hydroxylase (1alpha-OHase) and decrease 24-hydroxylase (24-OHase) in kidney proximal tubules. This study examines the hypothesis that 1,25-(OH)(2)D(3) synthesis, induced by dietary calcium restriction, is also the result of negative feedback regulation blockade. Rats fed a low calcium (0.02%, -Ca) diet and given daily oral doses of vitamin D (0, 0.5, 1.0, 2.0, 4.0, 8.0, and 16.0 microg) remained hypocalcemic despite increasing levels of serum calcium in relation to the vitamin D dose. Plasma levels of 1,25-(OH)(2)D(3) rose to high levels (1200 pg/ml) at the high vitamin D dose levels. As expected, thyroparathyroidectomy caused a rapid fall in serum 1,25-(OH)(2)D(3). In rats fed a 0.47% calcium diet (+Ca) supplemented with vitamin D (4 microg/day), exogenous 1,25-(OH)(2)D(3) suppressed renal 1alpha-OHase and stimulated the 24-OHase. In rats fed the -Ca diet, vitamin D was unable to suppress the renal 1alpha-OHase or stimulate the renal 24-OHase. In contrast, vitamin D was fully able to stimulate intestinal 24-OHase. Intestinal vitamin D receptor (VDR) was present under all circumstances, while kidney VDR was absent under hypocalcemic conditions and present under normocalcemic conditions. It appears that tissue-specific down-regulation of VDR by hypocalcemia blocks the 1,25-(OH)(2)D(3) suppression of the 1alpha-OHase and upregulation of the 24-OHase in the kidney, causing a marked accumulation of 1,25-(OH)(2)D(3) in the plasma.     

Novel Gemini vitamin D(3) analogs have potent antitumor activity

The active form of vitamin D(3), 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)], modulates proliferation and induces differentiation of many cancer cells. A new class of analogs of vitamin D(3) has been synthesized, having two side-chains attached to carbon-20 (Gemini) and deuterium substituted on one side-chain. We have examined six of these analogs for their ability to inhibit growth of myeloid leukemia (HL-60), prostate (LNCaP, PC-3, DU145), lung (H520), colon (HT-29), and breast (MCF-7) cancer cell lines. Dose-response clonogenic studies showed that all six analogs had greater antiproliferative activities against cancer cells than 1,25(OH)(2)D(3). Although they had similar potency, the most active of these analogs was BXL-01-0120. BXL-01-0120 was 529-fold more potent than 1,25(OH)(2)D(3) in causing 50% clonal growth inhibition (ED(50)) of HL-60 cells. Pulse-exposure studies demonstrated that exposure to BXL-01-120 (10(-9)M, 48h) resulted in 85% clonal inhibition of HL-60 growth. BXL-01-0120 (10(-11)M, 4 days) induced the differentiation marker, CD11b. Also, morphologically differentiation was more prominent compared to 1,25(OH)(2)D(3). Annexin V assay showed that BXL-01-0120 (10(-10)M, 4 days) induced significantly (p<0.05) more apoptosis than 1,25(OH)(2)D(3). In summary, these analogs have a unique structure resulting in extremely potent inhibition of clonal proliferation of various types of cancer cells, especially HL-60 cells.     

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.     

Targeted delivery of vitamin D to the colon using β-glucuronides of vitamin D: therapeutic effects in a murine model of inflammatory bowel disease

1,25-Dihydroxyvitamin D(3) [1,25(OH)(2)D] has been shown to inhibit development of dextran sodium sulfate (DSS)-induced colitis in mice but can also cause hypercalcemia. The aim of this study was to evaluate whether β-glucuronides of vitamin D could deliver 1,25(OH)(2)D to the colon to ameliorate colitis while reducing the risk of hypercalcemia. Initial studies demonstrated that bacteria residing in the lower intestinal tract were capable of liberating 1,25(OH)(2)D from 1,25-dihydroxyvitamin D(3)-25-β-glucuronide [β-gluc-1,25(OH)(2)D]. We also determined that a much greater upregulation of the vitamin D-dependent 24-hydroxylase gene (Cyp24) was induced in the colon by treatment of mice with an oral dose of β-gluc-1,25(OH)(2)D than 1,25(OH)(2)D, demonstrating targeted delivery of 1,25(OH)(2)D to the colon. We then tested β-glucuronides of vitamin D in the mouse DSS colitis model in two studies. In mice receiving DSS dissolved in distilled water and treated with 1,25(OH)(2)D or β-gluc-1,25(OH)(2)D, severity of colitis was reduced. Combination of β-gluc-1,25(OH)(2)D with 25-hydroxyvitamin D(3)-25-β-glucuronide [β-gluc-25(OH)D] resulted in the greatest reduction of colitis lesions and symptoms in DSS-treated mice. Plasma calcium concentrations were lower in mice treated with β-gluc-1,25(OH)(2)D alone or in combination with β-gluc-25(OH)D than in mice treated with 1,25(OH)(2)D, which were hypercalcemic at the time of death. β-Glucuronides of vitamin D compounds can deliver 1,25(OH)(2)D to the lower intestine and can reduce symptoms and lesions of acute colitis in this model.     

Biological activity of 1 alpha, 25-dihydroxyvitamin D derivatives--24-epi-1 alpha, 25-dihydroxyvitamin D-2 and 1 alpha,25-dihydroxyvitamin D-7

Biological activity of 24-epi-1 alpha,25-dihydroxyvitamin D-2 (24-epi-1,25(OH)2D2) and 1 alpha,25-dihydroxyvitamin D-7 (1,25(OH)2D7), the 22,23-dihydro derivative of the former compound, was investigated. Both of the vitamin D derivatives stimulated intestinal calcium transport and calcium mobilization from bones in rats; however, the effect was about 50% of that of 1 alpha,25-dihydroxyvitamin D-3 (1,25(OH)2D3). On the other hand, 24-epi-1,25(OH)2D2 and 1,25(OH)2D7 inducement of HL-60 human leukemia cell differentiation was comparable to that of 1,25(OH)2D3. Accordingly, the differentiation-inducing activity of 24-epi-1,25(OH)2D2 and 1,25(OH)2D7 was much greater than their ability to stimulate calcium metabolism. In contrast to 1,25(OH)2D3, 24-epi-1,25(OH)2D2 and 1,25(OH)2D7 exerted little hypercalcemic activity in mice. These results suggest that both vitamin D derivatives will be useful as anti-tumor agents.     

Vitamin D analogs: therapeutic applications and mechanisms for selectivity

The vitamin D endocrine system plays a central role in mineral ion homeostasis through the actions of the vitamin D hormone, 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)], on the intestine, bone, parathyroid gland, and kidney. The main function of 1,25(OH)(2)D(3) is to promote the dietary absorption of calcium and phosphate, but effects on bone, kidney and the parathyroids fine-tune the mineral levels. In addition to these classical actions, 1,25(OH)(2)D(3) exerts pleiotropic effects in a wide variety of target tissues and cell types, often in an autocrine/paracrine fashion. These biological activities of 1,25(OH)(2)D(3) have suggested a multitude of potential therapeutic applications of the vitamin D hormone for the treatment of hyperproliferative disorders (e.g. cancer and psoriasis), immune dysfunction (autoimmune diseases), and endocrine disorders (e.g. hyperparathyroidism). Unfortunately, the effective therapeutic doses required to treat these disorders can produce substantial hypercalcemia. This limitation of 1,25(OH)(2)D(3) therapy has spurred the development of vitamin D analogs that retain the therapeutically important properties of 1,25(OH)(2)D(3), but with reduced calcemic activity. Analogs with improved therapeutic indices are now available for treatment of psoriasis and secondary hyperparathyroidism in chronic kidney disease, and research on newer analogs for these indications continues. Other analogs are under development and in clinical trials for treatment of various types of cancer, autoimmune disorders, and many other diseases. Although many new analogs show tremendous promise in cell-based models, this article will limit it focus on the development of analogs currently in use and those that have demonstrated efficacy in animal models or in clinical trials.     

Vitamin D: A millenium perspective

Vitamin D is one of the oldest hormones that have been made in the earliest life forms for over 750 million years. Phytoplankton, zooplankton, and most plants and animals that are exposed to sunlight have the capacity to make vitamin D. Vitamin D is critically important for the development, growth, and maintenance of a healthy skeleton from birth until death. The major function of vitamin D is to maintain calcium homeostasis. It accomplishes this by increasing the efficiency of the intestine to absorb dietary calcium. When there is inadequate calcium in the diet to satisfy the body's calcium requirement, vitamin D communicates to the osteoblasts that signal osteoclast precursors to mature and dissolve the calcium stored in the bone. Vitamin D is metabolized in the liver and then in the kidney to 1,25-dihydroxyvitamin D [1,25(OH)(2)D]. 1,25(OH)(2)D receptors (VDR) are present not only in the intestine and bone, but in a wide variety of other tissues, including the brain, heart, stomach, pancreas, activated T and B lymphocytes, skin, gonads, etc. 1,25(OH)(2)D is one of the most potent substances to inhibit proliferation of both normal and hyperproliferative cells and induce them to mature. It is also recognized that a wide variety of tissues, including colon, prostate, breast, and skin have the enzymatic machinery to produce 1,25(OH)(2)D. 1,25(OH)(2)D and its analogs have been developed for treating the hyperproliferative disease psoriasis. Vitamin D deficiency is a major unrecognized health problem. Not only does it cause rickets in children, osteomalacia and osteoporosis in adults, but may have long lasting effects. Chronic vitamin D deficiency may have serious adverse consequences, including increased risk of hypertension, multiple sclerosis, cancers of the colon, prostate, breast, and ovary, and type 1 diabetes. There needs to be a better appreciation of the importance of vitamin D for overall health and well being.     

A local effect of CYP24 inhibition on lung tumor xenograft exposure to 1,25-dihydroxyvitamin D(3) is revealed using a novel LC-MS/MS assay

The vitamin D(3) catabolizing enzyme, CYP24, is frequently over-expressed in tumors, where it may support proliferation by eliminating the growth suppressive effects of 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)). However, the impact of CYP24 expression in tumors or consequence of CYP24 inhibition on tumor levels of 1,25(OH)(2)D(3)in vivo has not been studied due to the lack of a suitable quantitative method. To address this need, an LC-MS/MS assay that permits absolute quantitation of 1,25(OH)(2)D(3) in plasma and tumor was developed. We applied this assay to the H292 lung tumor xenograft model: H292 cells eliminate 1,25(OH)(2)D(3) by a CYP24-dependent process in vitro, and 1,25(OH)(2)D(3) rapidly induces CYP24 expression in H292 cells in vivo. In tumor-bearing mice, plasma and tumor concentrations of 1,25(OH)(2)D(3) reached a maximum of 21.6 and 1.70ng/mL, respectively, following intraperitoneal dosing (20μg/kg 1,25(OH)(2)D(3)). When co-administered with the CYP24 selective inhibitor CTA091 (250μg/kg), 1,25(OH)(2)D(3) plasma levels increased 1.6-fold, and tumor levels increased 2.6-fold. The tumor/plasma ratio of 1,25(OH)(2)D(3) AUC was increased 1.7-fold by CTA091, suggesting that the inhibitor increased the tumor concentrations of 1,25(OH)(2)D(3) independent of its effects on plasma disposition. Compartmental modeling of 1,25(OH)(2)D(3) concentration versus time data confirmed that: 1,25(OH)(2)D(3) was eliminated from plasma and tumor; CTA091 reduced the elimination from both compartments; and that the effect of CTA091 on tumor exposure was greater than its effect on plasma. These results provide evidence that CYP24-expressing lung tumors eliminate 1,25(OH)(2)D(3) by a CYP24-dependent process in vivo and that CTA091 administration represents a feasible approach to increase tumor exposure to 1,25(OH)(2)D(3).     

Vitamin D and multiple sclerosis

Vitamin D is a principal regulator of calcium homeostasis. However, recent evidence has indicated that vitamin D can have numerous other physiological functions including inhibition of proliferation of a number of malignant cells including breast and prostate cancer cells and protection against certain immune mediated disorders including multiple sclerosis (MS). The geographic incidence of MS indicates an increase in MS with a decrease in sunlight exposure. Since vitamin D is produced in the skin by solar or UV irradiation and high serum levels of 25-hydroxyvitamin D (25(OH)D) have been reported to correlate with a reduced risk of MS, a protective role of vitamin D is suggested. Mechanisms whereby the active form of vitamin D, 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) may act to mediate this protective effect are reviewed. Due to its immunosuppressive actions, it has been suggested that 1,25(OH)(2)D(3) may prevent the induction of MS.     

Altered expression of key players in vitamin D metabolism and signaling in malignant and benign thyroid tumors

1,25-Dihydroxyvitamin D(3) (1,25(OH)(2)D(3)), the active form of vitamin D, mediates antitumor effects in various cancers. The expression of key players in vitamin D signaling in thyroid tumors was investigated. Vitamin D receptor (VDR) and CYP27B1 and CYP24A1 (respectively activating and catabolizing vitamin D) expression was studied (RT-PCR, immunohistochemistry) in normal thyroid, follicular adenoma (FA), differentiated thyroid cancer (DTC) consisting of the papillary (PTC) and follicular (FTC) subtype, and anaplastic thyroid cancer (ATC). VDR, CYP27B1, and CYP24A1 expression was increased in FA and DTC compared with normal thyroid. However, in PTC with lymph node metastasis, VDR and CYP24A1 were decreased compared with non-metastasized PTC. In ATC, VDR expression was often lost, whereas CYP27B1/CYP24A1 expression was comparable to DTC. Moreover, ATC with high Ki67 expression (>30%) or distant metastases at diagnosis was characterized by more negative VDR/CYP24A1/CYP27B1 staining. In conclusion, increased expression of key players involved in local 1,25(OH)(2)D(3) signaling was demonstrated in benign and differentiated malignant thyroid tumors, but a decrease was observed for local nodal and especially distant metastasis, suggesting a local antitumor response of 1,25(OH)(2)D(3) in early cancer stages. These findings advocate further studies with 1,25(OH)(2)D(3) and analogs in persistent and recurrent iodine-refractory DTC.     

Oral administration of 1,25-dihydroxyvitamin D3 completely protects NOD mice from insulin-dependent diabetes mellitus

1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)), the biologically active form of vitamin D, is widely recognized as a modulator of the immune system as well as a regulator of mineral metabolism. The objective of this study was to determine the effects of vitamin D status and treatment with 1,25(OH)(2)D(3) on diabetes onset in non-obese diabetic (NOD) mice, a murine model of human type I diabetes. We have found that vitamin D-deficiency increases the incidence of diabetes in female mice from 46% (n=13) to 88% (n=8) and from 0% (n=10) to 44% (n=9) in male mice as of 200 days of age when compared to vitamin D-sufficient animals. Addition of 50 ng of 1,25(OH)(2)D(3)/day to the diet prevented disease onset as of 200 days and caused a significant rise in serum calcium levels, regardless of gender or vitamin D status. Our results indicate that vitamin D status is a determining factor of disease susceptibility and oral administration of 1,25(OH)(2)D(3) prevents diabetes onset in NOD mice through 200 days of age.     

Vitamin D signaling in immune-mediated disorders: Evolving insights and therapeutic opportunities

1,25(OH)(2)D(3), the active form of vitamin D, is a central player in calcium and bone metabolism. More recently, important immunomodulatory effects have been attributed to this hormone. The widespread presence of the vitamin D receptor (VDR) in the immune system and the expression of the enzymes responsible for the synthesis of the active 1,25(OH)(2)D(3) regulated by specific immune signals, even suggest a paracrine immunomodulatory role for 1,25(OH)(2)D(3). Additionally, the different molecular mechanisms used by 1,25(OH)(2)D(3) to exert its immunomodulatory effects prove of a broad action radius for this compound. Both, the effects of vitamin D deficiency and/or absence of the VDR as well as intervention with pharmacological doses of 1,25(OH)(2)D(3) or one of its less-calcemic analogs, affects immune system behavior in different animal models of immune-mediated disorders, such as type 1 diabetes. This review aims to summarize the data as they stand at the present time on the role of vitamin D in the pathogenesis of immune-mediated disorders, with special focus on type 1 diabetes, and on the therapeutic opportunities for vitamin D in the prevention and treatment of this autoimmune disease in mouse models and humans.     

Gene expression profiles in rat intestine identify pathways for 1,25-dihydroxyvitamin D(3) stimulated calcium absorption and clarify its immunomodulatory properties

Microarray technology has been used to discover 1,25-dihydroxyvitamin D(3) (1,25-(OH)(2)D(3)) induced gene expression changes in rat small intestine in vivo. Here, we report gene expression changes related to intestinal absorption or transport, the immune system and angiogenesis in response to 1,25-(OH)(2)D(3). Vitamin D deficient rats were intrajugularly given vehicle or vehicle containing 730 ng of 1,25-(OH)(2)D(3)/kg of body weight. Intestinal mRNA was harvested from duodenal mucosa at 15 min, 1, 3, and 6 h post-injection and studied by Affymetrix microarrays. Genes significantly affected by 1,25-(OH)(2)D(3) were confirmed by quantitative RT-PCR with remarkable agreement. The most strongly affected gene in intestine was CYP24 with 97-fold increase at 6 h post-1,25-(OH)(2)D(3) treatment. Intestinal calcium absorption genes: TRPV5, TRPV6, calbindin D(9k), and Ca(2+) dependent ATPase all were up-regulated in response to 1,25-(OH)(2)D(3), supporting the currently accepted mechanism of 1,25-(OH)(2)D(3) induced transcellular calcium transport. However, a 1,25-(OH)(2)D(3) suppression of several intra-/intercellular matrix modeling proteins such as sodium/potassium ATPase, claudin 3, aquaporin 8, cadherin 17, and RhoA suggests a vitamin D regulation of tight junction permeability and paracellular calcium transport. Several other genes related to the immune system and angiogenesis whose expression was changed in response to 1,25-(OH)(2)D(3) provided evidence for an immunomodulatory and anti-angiogenic role of 1,25-(OH)(2)D(3).     

Vitamin D regulated keratinocyte differentiation: role of coactivators

1,25 Dihydroxyvitamin D (1,25(OH)(2)D) regulates the differentiation of keratinocytes. 1,25(OH)(2)D raises intracellular free calcium (Cai) as a necessary early step toward stimulating differentiation. 1,25(OH)(2)D induces the calcium sensing receptor (CaR) in keratinocytes and enhances the calcium response of these cells. Activation of the CaR by calcium increases intracellular free calcium by a mechanism involving phospholipase C (PLC) cleavage of phosphatidylinositolbisphosphate into inositoltrisphosphate (IP(3)) and diacylglycerol (DG). 1,25(OH)(2)D induces the family of PLCs. PLC-gamma1 has a DR6 VDRE in its promoter which binds and is activated by VDR/RAR rather than VDR/RXR. The involucrin gene, which encodes a critical component of the cornified envelope, contains a DR3 VDRE in its promoter that acts in conjunction with a nearby AP-1 site. The sequential regulation of these genes is critical for the differentiation process. In undifferentiated keratinocytes, the VDR binds preferentially to the DRIP complex of coactivators. However, with differentiation DRIP 205 is no longer produced, and the VDR switches partners to the SRC family (SRC2 and 3). These studies suggest that at least part of the sequential activation of genes required during keratinocyte differentiation is regulated by the change (availability) of these different coactivator complexes.     

Immunochemical studies on the putative plasmalemmal receptor for 1, 25-dihydroxyvitamin D(3). III. Vitamin D status

The effect of vitamin D status on levels of the putative 1, 25(OH)(2)D(3) membrane receptor (pmVDR) was studied in chick intestine, kidney, and brain. Western analyses and assays for specific [(3)H]1,25(OH)(2)D(3) binding indicated that, in intestine, pmVDR levels were greatest in -D chicks relative to +1,25D and +D animals (P < 0.05). In kidney, protein levels and specific binding followed the order +D > +1,25D, -D. In brain, vitamin D status did not affect protein levels or specific binding levels. In tissue from normal chicks, both protein and specific binding followed the order of intestine > kidney > brain membranes. Intestinal cells were further evaluated for the effect of 1,25(OH)(2)D(3) on selected "rapid responses." Extrusion of (45)Ca in response to 130 pM 1, 25(OH)(2)D(3) in vitro was greater in cells from -D chicks than from +1,25D or normal birds. Analyses of signal transduction events revealed diminished hormone-induced intracellular calcium oscillations (as assessed by fura-2 fluorescence), and lack of steroid-enhanced protein kinase (PK) A activity in intestinal epithelial cells from -D chicks relative to +D chicks. PK C activation by 130 pM 1,25(OH)(2)D(3) was approximately twofold in cells from +D or -D chicks. The combined results indicate that vitamin D status differentially affects the pmVDR in intestine, kidney, and brain. In intestine, vitamin D deficiency differentially affects (45)Ca handling, intracellular calcium oscillations, PK A and PK C activities in response to 1,25(OH)(2)D(3).     

The rapid effects of 1,25-dihydroxyvitamin D3 require the vitamin D receptor and influence 24-hydroxylase activity: studies in human skin fibroblasts bearing vitamin D receptor mutations

If both rapid and genomic pathways may co-exist in the same cell, the involvement of the nuclear vitamin D receptor (VDR) in the rapid effects of 1,25-dihydroxyvitamin D(3) (1,25-(OH)(2)D(3)) remains unclear. We therefore studied rapid and long term effects of 1,25-(OH)(2)D(3) in cultured skin fibroblasts from three patients with severe vitamin D-resistant rickets and one age-matched control. Patients bear homozygous missense VDR mutations that abolished either VDR binding to DNA (patient 1, mutation K45E) or its stable ligand binding (patients 2 and 3, mutation W286R). In patient 1 cells, 1,25-(OH)(2)D(3) (1 pm-10 nm) had no effect on either intracellular calcium or 24-hydroxylase (enzyme activity and mRNA expression). In contrast, cells bearing the W286R mutation had calcium responses to 1,25-(OH)(2)D(3) (profile and magnitude) and 24-hydroxylase responses to low (1 pm-100 pm) 1,25-(OH)(2)D(3) concentrations (activity, CYP24, and ferredoxin mRNAs) similar to those of controls. The blocker of Ca(2+) channels, verapamil, impeded both rapid (calcium) and long term (24-hydroxylase activity, CYP24, and ferredoxin mRNAs) responses in patient and control fibroblasts. The MEK 1/2 kinase inhibitor PD98059 also blocked the CYP24 mRNA response. Taken together, these results suggest that 1,25-(OH)(2)D(3) rapid effects require the presence of VDR and control, in part, the first step of 1,25-(OH)(2)D(3) catabolism via increased mRNA expression of the CYP24 and ferredoxin genes in the 24-hydroxylase complex.