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It is largely through historical accident in the interval of 1920–1940 that vitamin D3 became classified as a vitamin rather than as a steroid hormone. The formal definition of a vitamin is that it is a trace dietary constituent required to produce the normal function of a physiological process or processes. The emphasis here is on trace and the fact that the vitamin must be supplied regularly in the diet; this implies that the body is unable to metabolically synthesize the vitamin in question. However, the ultraviolet exposure of 7-dehydrocholesterol present in the skin results in the photochemical production of vitamin D3. Thus, vitamin D3 becomes a true vitamin only when the animal or human does not have regular access to sunlight or ultraviolet light. Under normal physiological circumstances, all mammals, including humans, can generate, via ultraviolet exposure of 7-dehydrocholesterol present in the skin, adequate quantities of vitamin D3 to meet their nutritionally defined requirements. There is a vibrant historical record beginning in 1650 and culminating in 1963 concerned with the determination of the chemical structures of vitamin D3 and vitamin D2. A surprising aspect concerning vitamin D3 is that it is itself biologically inert. There are no known essential biological actions or contributions that rely specifically on the molecule vitamin D3. While chemists had certainly appreciated the strong structural similarity between the vitamins D and other steroids, this correlation was never widely acknowledged in the biological, clinical, or nutritional sciences until 1965–1970. The biological role of vitamin D3 is to serve as a substrate for the liver 25-hydroxylase which produces 25-hydroxyvitamin D3 [25(OH)D3]. 25(OH)D3 in turn serves as the substrate for the kidney proximal tubule 25(OH)D3-1α-hydroxylase enzyme which produces the steroid hormone 1α,25(OH)2-vitamin D3 [1α,25(OH)2D3].

Exposure to ultraviolet radiation (UVR) can lead to a range of deleterious responses in the skin. An important form of damage is the DNA photolesion cyclobutane pyrimidine dimer (CPD). CPDs can be highly mutagenic if not repaired prior to cell division and can lead to UV-induced immunosuppression, making them potentially carcinogenic. UVR exposure also produces vitamin D, a prehormone. Different shapes of the steroid hormone 1α,25-dihydroxyvitamin D3 [1,25(OH)2D3] can produce biological responses through binding either to its cognate nuclear receptor (VDR) to regulate gene transcription or to the VDR associated with plasma membrane caveolae to produce, via signal transduction, nongenomic physiologic responses. Here, we show that both 1,25(OH)2D3 and 1α,25(OH)2-lumisterol (JN), a conformationally restricted analogue that can generate only nongenomic responses, are effective inhibitors of UV damage in an immunocompetent mouse (Skh:hr1) model susceptible to UV-induced tumors. Both 1,25(OH)2D3 and JN significantly reduced UVR-induced CPD, apoptotic sunburn cells, and immunosuppression. Furthermore, these compounds inhibited skin tumor development, both papillomas and squamous cell carcinomas, in these mice. The observed reduction of these UV-induced effects by 1,25(OH)2D3 and JN suggests a role for these compounds in prevention against skin carcinogenesis. To the best of our knowledge, this is the first comprehensive report of an in vivo long-term biological response generated by chronic dosing with a nongenomic-selective vitamin D steroid. Cancer Prev Res; 4(9); 1485–94. ©2011 AACR.

Steroids exert their actions through several pathways. The classical genomic pathway, which involves binding of steroids to receptors and subsequent modulation of gene expression, is well characterized. Besides this, rapid actions of steroids have been shown to exist. Since 30 years, research on rapid actions of steroids is an emerging field of science. Today, rapid effects of steroids are well established, and are shown to exist for every type of steroid. The classical steroid receptors have been shown to be involved in rapid actions, but there is also strong evidence that unrelated structures mediate these rapid effects. Despite increasing knowledge about the mechanisms and structures which mediate these actions, there is still no unanimous acceptance of this category. This article briefly reviews the history of the field including current controversies and challenges. It is not meant as a broad review of literature, but should increase the awareness of the endocrinology society for rapid responses to steroids. As members of the organizing committee of the VI International Meeting on Rapid Responses to Steroid Hormones 2009, we propose a research agenda focusing on the identification of new receptoral structures and the identification of mechanisms of actions at physiological steroid concentrations. Additionally, efforts for the propagation of translational studies, which should finally lead to clinical benefit in the area of rapid steroid action research, should be intensified.

Sertoli cell secretory activities are highly dependent on ion channel functions and critical to spermatogenesis. The steroid hormone 1alpha,25(OH)2-vitamin D3 (1,25(OH)2-D3) stimulates exocytosis in different cell systems by activating a nongenotropic vitamin D receptor (VDR). Here, we described 1,25(OH)2-D3 stimulation of secretion via Cl(-) channel activation in the mouse immature Sertoli cell line TM4. 1,25(OH)2-D3 potentiation of chloride currents was dependent on hormone concentration, and correlated with a significant increase in whole-cell capacitance within 20-40 min. In addition, Cl(-) currents were potentiated by the nongenomic VDR agonist 1alpha,25(OH)2 lumisterol D3 (JN), while 1,25(OH)2-D3 potentiation of channels was suppressed by nongenomic VDR antagonist 1beta,25(OH)2-vitamin D3 (HL). Treatment of TM4 cells with PKC and PKA activators PMA and forskolin respectively, increased Cl(-) currents significantly, while PKC and PKA inhibitors Go6983 and H-89, respectively, abolished 1,25(OH)2-D3 stimulation of Cl(-) currents, suggesting phosphorylation pathways in 1,25(OH))2-D3 mediated channel responses. RT-PCR demonstrated the expression of outwardly rectifying ClC-3 channels in TM4 cells. Taken together, our results demonstrate a PKA/PKC-dependent 1,25(OH)2-D3/VDR nongenotropic pathway leading to Cl(-) channel and exocytosis activation in Sertoli cells. We conclude that 1,25(OH)2-D3 appears to be a modulator of male reproductive functions at least in part by stimulating Sertoli cell secretory functions.

Transport of 45Ca from the lumen to the venous effluent was studied in duodena of normal, vitamin D3-replete (+D) chicks perfused through the celiac artery with 130 pM 1,25(OH)2D3 or vehicle. Administration of actinomycin D 3 h prior to perfusion did not alter the unstimulated transport rate or diminish the response to exogenous 1,25(OH)2D3: After 40 min exposure to the seco-steroid, 45Ca in the vascular effluent was 140% of control levels. The anti-microfilament agent cytochalasin b and the ionophore monensin, an inhibitor of Golgi function, similarly failed to suppress 1,25(OH)2D3-stimulated calcium transport. In pilot studies, Golgi and basal-lateral membrane fractions were prepared from duodenal epithelium of vitamin D-deficient (-D) chicks treated with vehicle or 650 pmol of 1,25(OH)2D3in vivo 2 h, 10 h, or 15 h before sacrifice, as well as from +D birds. Analyses of Golgi fractions for cathepsin B (CB) activity revealed a biphasic response with time, increasing to 200% of -D levels 2 h after 1,25(OH)2D3 administration and in equivalent preparations from +D birds. Less pronounced increases in acid phosphatase activity were observed in the same membrane fractions. In basal-lateral membranes, enhanced CB activity was detectable 10 h after 1,25(OH)2D3in vivo, rose to 155% of -D levels at 15 h, and to 245% of controls in fractions from +D birds, whereas acid phosphatase was 75%, 81%, and 125% of controls, respectively, at these times. Levels of CB in whole homogenates from which membranes were isolated rose to 200% of controls at 2 h and 10 h after 1,25(OH)2D3, but declined to 112% of controls in +D chicks. Acid phosphatase levels in the same homogenates were lower than or equivalent to control levels. These findings suggested additional perfusion studies in normal birds. Although vascular perfusion with iodoacetate and 1,25(OH)2D3, or leupeptin and 1,25(OH)2D3, failed to inhibit the effect of the vitamin D metabolite, 1,25(OH)2D3-enhanced calcium transport was abolished by addition of 1 mM leupeptin to the lumenal medium, but not by 1 mM pepstatin. The combined data suggest that lysosomes or their hydrolases may be involved in 1,25(OH)2D3-mediated transport of calcium across intestinal epithelial cells.

The steroid hormone 1alpha,25(OH)(2)-Vitamin D(3) (1,25D) activates both genomic and non-genomic intracellular signaling cascades. It is also well recognized that co-incubation of 1,25D with its C-1 epimer, 1beta,25D (HL), suppresses the efficiency of the non-genomic signal activated by 1,25D alone and that its C-3 epimer, 3alpha-1,25D (HJ) is nearly as potent as 1,25D in suppressing PTH secretion, believed to be propagated by 1,25D's genomic signaling. Both these sterols lack the hypercalcemic effect induced by pharmacological doses of 1,25D and have reduced VDR affinity compared to 1,25D, as measured in a steroid competition assay. Recent functional studies suggest that the VDR is required for both non-genomic and genomic signaling. Along these lines we have recently proposed a Vitamin D sterol/VDR conformational ensemble model that posits the VDR contains two distinct, yet overlapping ligand binding sites, and that the potential differential stabilities of 1,25D and HL in these two pockets can be used to explain their different non-genomic signaling properties. The overlapping region is predominantly occupied by the sterol's A-ring when it is bound to either the genomic ligand binding pocket (G-pocket), defined by X-ray crystallography, or the alternative ligand binding pocket (A-pocket), discovered using in silico techniques (directed docking). Therefore, to gain further insight into the potential application of this model we docked the other A-ring diastereomer [(1beta,3alpha)=HH] of 1,25D and its 1- and 3-deoxy forms (25D and CF, respectively) to the A- and G-pockets to assess their potential stabilities in the pockets, relative to 1,25D. The models were then used to provide putative mechanistic arguments for their known structure-function experimental results. This model may provide new insights into how Vitamin D sterols that uncouple the unwanted hypercalcemic effect from attractive growth inhibitory/differentiation properties can do so by differentially stabilizing different subpopulations of VDR conformational ensemble members.

The hormonal form of vitamin D, 1α,25-dihydroxyvitamin D 3 (1,25D), generates many biological actions by interactions with its nuclear receptor (VDR). The presence of a carbon-25 hydroxyl group is necessary for optimizing binding to the VDR. To examine the effect of spatial orientation of the 25-hydroxyl, two pairs of 22,23-allene sidechain analogs were studied. The 22R orientation in analogs HR (52 ± 2%) and LA (154 ± 19%) resulted in higher affinity binding than the 22S orientation of analogs HQ (21 ± 3%) and LB (3.5 ± 1.3%; 1,25D = 100%). Limited trypsin proteolysis showed that 22R analogs induced VDR conformational changes better able to protect VDR from digestion than 22S analogs. 22R analogs were also able to induce gene transcription at 10–100-fold lower concentrations than 1,25D; 22S analogs were less effective. Analog LA was at least 10-fold more potent than 1,25D at inducing differentiation, while the other analogs were less potent. None of the analogs were as potent as 1,25D in promoting in vivo intestinal calcium absorption or bone calcium mobilization. LA was the most potent of the analogs but required 20–30-fold higher doses than 1,25D. The 25-hydroxyl orientation combined with the 16,17-ene functionality of analog LA enhances its ability to interact with VDR and induce biological actions.

The steroid hormone 1α,25(OH)2-vitamin D3 (1,25D) regulates gene transcription through a nuclear receptor [vitamin D receptor (VDR)] and initiation of rapid cellular responses through a putative plasma membrane-associated receptor (VDRmem). This study characterized the VDRmem present in a caveolae-enriched membrane fraction (CMF), a site of accumulation of signal transduction agents. Saturable and specific [3H]-1,25D binding in vitro was found in CMF of chick, rat, and mouse intestine; mouse lung and kidney; and human NB4 leukemia and rat ROS 17/2.8 osteoblast-like cells; in all cases the 1,25D KD binding dissociation constant = 1–3 nm. Our data collectively support the classical VDR being the VDRmem in caveolae: 1) VDR antibody immunoreactivity was detected in CMF of all tissues tested; 2) competitive binding of [3H]-1,25D by eight analogs of 1,25D was significantly correlated between nuclei and CMF (r2 = 0.95) but not between vitamin D binding protein (has a different ligand binding specificity) and CMF; 3) confocal immunofluorescence microscopy of ROS 17/2.8 cells showed VDR in close association with the caveolae marker protein, caveolin-1, in the plasma membrane region; 4) in vivo 1,25D pretreatment reduced in vitro [3H]-1,25D binding by 30% in chick and rat intestinal CMF demonstrating in vivo occupancy of the CMF receptor by 1,25D; and 5) comparison of [3H]-1,25D binding in VDR KO and WT mouse kidney tissue showed 85% reduction in VDR KO CMF and 95% reduction in VDR KO nuclear fraction. This study supports the presence of VDR as the 1,25D-binding protein associated with plasma membrane caveolae.

Rapid nongenomic responses to steroids include modulation of ion channel activities on the cell membrane of target cells, but little is known about the molecular mechanisms involved. In this paper we investigate the mechanisms underlying the combined action of the secosteroid hormone 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)(2)D3] on three different ion channel types in rat osteoblasts, which include a voltage-gated L-type Ca(2+) channel, a mechanosensitive Cl(-) channel, and a stretch-activated cation (SA-Cat) channel. We found that physiological nanomolar concentrations of 1alpha,25(OH)(2)D3 rapidly modify the overall electrical activity of the membrane in ROS 17/2.8 cells. 1alpha,25(OH)(2)D3 increases the osteoblast L-type Ca(2+) channel activity at low depolarizing voltages in a fashion similar to the 1,4-dihydropyridine (DHP) agonist Bay K8644. At highly depolarizing potentials 1alpha,25(OH)(2)D3 potentiates volume-sensitive Cl(-) currents through mechanisms that may involve a putative membrane receptor. We show for the first time that 1alpha,25(OH)(2)D3 also increases inward currents through SA-Cat channels at positive membrane voltages in a dose-dependent manner. Contrary to our expectations, the stereoisomer 1beta,25(OH)(2)D3, which suppresses 1alpha,25(OH)(2)D3 activation of osteoblast Cl(-) currents, mimicked 1alpha,25(OH)(2)D3 agonist effects on Ca(2+) and SA-Cat channel activities. Cyclic AMP is involved in 1alpha,25(OH)(2)D3 effects on both Ca(2+) and SA-Cat channels, but not in Cl(-) channels. We conclude that 1alpha,25(OH)(2)D3 rapid effects on ion channel activities in ROS 17/2.8 cells occur through multiple mechanisms that, on the one hand, involve a possible direct interaction with the L-type Ca(2+) channel molecule and, on the other hand, molecular pathways that may include a putative membrane receptor.

The steroid hormone 1alpha,25-dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)] produces biological responses by interaction with both a well-characterized nuclear receptor (VDR(nuc)) to regulate gene transcription and with an as-yet uncharacterized membrane-associated protein/receptor (VDR(mem)) to generate a variety of rapid, non-genotropic responses. We report for the first time that [3H]1alpha,25(OH)(2)D(3) binds with high affinity to a chick duodenal caveolae-enriched membrane fraction (CMF) isolated without the use of detergents. Caveolae are plasma membrane invaginations implicated in signal transduction and molecular transport processes. Using the CMF fraction as a possible source of VDR(mem), we found that the in vitro binding of [3H]1alpha,25(OH)(2)D(3) was ligand dependent and saturable; the K(D) and B(max) were 1.3+/-0.6nM and 29+/-11fmol 1,25(OH)(2)D(3)/mg protein (n=17), respectively. Immunoblot analysis of the CMF confirms the presence of caveolin-1, a marker protein for membranes with caveolae. Therefore, chick CMF may represent a good source for isolation and characterization of the putative VDR(mem) for 1alpha,25(OH)(2)D(3).

The steroid hormone 1α,25-dihydroxyvitamin D 3 [1α, 25-(OH) 2 D 3 ] promotes vascular smooth muscle cell (VSMC) growth and calcification, but the precise mechanism by which 1α, 25-(OH) 2 D 3 regulates VSMC migration is unknown. In rat aortic SMCs, we found that 1α, 25-(OH) 2 D 3 (0.1 to 100 nmol/L) induced a dose-dependent increase in VSMC migration. This response required the activation of phosphatidylinositol 3-kinase (PI3 kinase) because 1α, 25-(OH) 2 D 3 -induced migration was completely abolished by the PI3 kinase inhibitors, LY294002 (10 μmol/L) or wortmannin (30 nmol/L). Furthermore, the RNA polymerase inhibitor, 5,6-dichlorobenzimidazole riboside (50 μmol/L), did not affect 1α, 25-(OH) 2 D 3 -induced VSMC migration, suggesting that gene transcription is not involved in this rapid response. Using analogs of 1α, 25-(OH) 2 D 3 , which have been characterized for their abilities to induce either transcriptional or nontranscriptional responses of 1α, 25-(OH) 2 D 3 , we found that 1α,25-dihydroxylumisterol, which is a potent agonist of the rapid, nongenomic responses, was equipotent with 1α, 25-(OH) 2 D 3 in inducing PI3 kinase activity and VSMC migration. Moreover, 1β, 25-(OH) 2 D 3 , which specifically antagonizes the nongenomic actions of 1α, 25-(OH) 2 D 3 , abolished 1α, 25-(OH) 2 D 3 -induced PI3 kinase activity and VSMC migration, whereas the inhibitor of the genomic actions of vitamin D, (23S)-25-dehydro-1α-OH-D 3 -26,23-lactone, did not affect these responses. These results indicate that 1α, 25-(OH) 2 D 3 induces VSMC migration independent of gene transcription via PI3 kinase pathway, and suggest a possible mechanism by which 1α, 25-(OH) 2 D 3 may contribute to neointima formation in atherosclerosis and vascular remodeling.

1 alpha,25-(OH)(2)D(3) exerts its effects on chondrocytes and enterocytes via nuclear receptors (1,25-nVDR) and a separate membrane receptor (1,25-mVDR) that activates protein kinase C (PKC). 24R,25-(OH)(2)D(3) also stimulates PKC in chondrocytes, but through other membrane mechanisms. This study examined the hypothesis that osteoblasts possess distinct membrane receptors for 1 alpha,25-(OH)(2)D(3) and 24R,25-(OH)(2)D(3) that are involved in the activation of PKC and that receptor expression varies as a function of cell maturation state. 1 alpha,25-(OH)(2)D(3) stimulated PKC in well differentiated (UMR-106, MC-3T3-E1) and moderately differentiated (ROS 17/2.8) osteoblast-like cells, and in cultures of fetal rat calvarial (FRC) cells and 2T3 cells treated with rhBMP-2 to promote differentiation. 24R,25-(OH)(2)D(3) stimulated PKC in FRC and 2T3 cultures that had not been treated to induce differentiation, and in ROS 17/2.8 cells. MG63 cells, a relatively undifferentiated osteoblast-like cell line, had no response to either metabolite. Ab99, a polyclonal antibody generated to the chick enterocyte 1,25-mVDR, but not a specific antibody to the 1,25-nVDR, inhibited response to 1 alpha,25-(OH)(2)D(3). 1 alpha,25-(OH)(2)D(3) exhibited specific binding to plasma membrane preparations from cells demonstrating a PKC response to this metabolite that is typical of positive cooperativity. Western blots of these membrane proteins reacted with Ab99, and the Ab99-positive protein had an Mr of 64 kDa. There was no cross-reaction with antibodies to the C- or N-terminus of annexin II. The effect of 24,25-(OH)(2)D(3) on PKC was stereospecific; 24S,25-(OH)(2)D(3) had no effect. These results demonstrate that response to 1 alpha,25-(OH)(2)D(3) and 24R,25-(OH)(2)D(3) depends on osteoblast maturation state and suggest that specific and distinct membrane receptors are involved.

Drug developments in the vitamin D field have continued to focus on structure-function studies of analogs produced by chemically modifying the structure of 1α,25-dihydroxyvitamin D 3 (1,25-D3) and its metabolites. Direct structural information gleaned from X-ray crystallographic or NMR studies regarding the ligand-receptor complex and other guest-host systems, which are likely involved in initiating biologic responses, also offers potential insight into drug design. Evidence has accrued suggesting that topologically different conformers of 1,25-D3 may bind to proteins in different ways, including the induction of different conformations of protein. This paper concerns our progress on the chemical synthesis of analogs (e.g. ansa-steroids, suprasterols, vinylallenes and other analogs) conformationally locked or at least rotationally restricted to mimic higher energy conformers of 1,25-D3.

The integrated operation of the vitamin D endocrine system which produces the steroid hormone 1alpha,25(OH)(2)-vitamin D(3) (1alpha,25(OH)(2)D(3)) is dependent on four classes of proteins each of which have inherent in their secondary and tertiary structure a ligand binding domain (LBD) that allows the stereospecific binding of 1alpha,25(OH)(2)D(3) or related analogs as a substrate or ligand. These LBDs include: (a) the cytochrome P450 enzymes in the liver, kidney, and other tissues which metabolize vitamin D(3) into biologically active metabolites; (b) the plasma vitamin D binding protein (DBP) which selectively transports these hydrophobic molecules to the various target organs of the vitamin D endocrine system; (c) the nuclear receptor VDR(nuc) that is involved in regulation of gene transcription in over 30 cell types which possess this receptor; and (d) a plasma membrane receptor, VDR(mem), that is involved in initiation of signal transduction pathways which generate rapid biological responses. This article reviews the evidence that supports the conclusions that the LBD of the DBP, VDR(mem) and VDR(nuc) each select as their preferred ligand a unique shape of the conformationally flexible 1alpha,25(OH)(2)D(3). Two critical aspects of the conformationally flexible 1alpha,25(OH)(2)D(3) molecule which defines the optimum ligand shape are (a) the orientation and relative rigidity of the flexible 8 carbon side chain and (b) the position of the A ring in relation to the C/D rings as determined by the extent of rotation around the 6,7 single carbon bond of the seco B ring. These conclusions are based on consideration of structure-function studies of over 300 analogs of 1alpha,25(OH)(2)D(3), of these, 22 analogs are highlighted in this presentation.

(23S)-25-dehydro-1α-Dihydroxyvitamin D3-26,23-lactone (TEI-9647; MK) has been reported to antagonize the 1α,25-dihydroxyvitamin D3 nuclear receptor (VDR)- mediated increase in transcriptional activity. Using a transient transfection system incorporating the osteocalcin VDRE (vitamin D response element) in Cos-1 cells, we found that 20 nm MK antagonizes VDR-mediated transcription by 50% when driven by 1 nm 1α,25(OH)2D3. Four analogs of 1α,25(OH)2D3, also at 1 nm, were antagonized 25 to 39% by 20 nm MK. However, analogs with 16-ene/23-yne or 20-epi modifications, which have a significantly lower agonist ED50 for the VDR than 1α,25(OH)2D3, were antagonized by 20 nm MK only at 100 pm or 10 pm, respectively. One possible mechanism for antagonism is that the 25-dehydro alkene of MK might covalently bind the ligand-binding site of the VDR rendering it inactive. Utilization of a ligand exchange assay, however, demonstrated that MK bound to VDR is freely exchanged with 1α,25(OH)2D3 in vitro. These data support the apparent correlation between VDR transcriptional activation by agonists and the effective range of MK antagonism by competition. Furthermore, protease sensitivity analysis of MK bound to VDR indicates the presence of a unique conformational change in the VDR ligand-binding domain, showing a novel doublet of VDR fragments centered at 34 kDa, whereas 1α,25(OH)2D3 as a ligand produces only a single 34-kDa fragment. In comparison, the natural metabolite 1α,25dihydroxyvitamin D3-26,23-lactone yields only the 30-kDa fragment that is produced by all ligands to varying degrees. Collectively, these results support that MK is a potent partial antagonist of the VDR for 1α,25(OH)2D3 and its analogs when in appropriate excess of the agonist.

The hormone 1alpha,25(OH)(2)-vitamin D(3) (125D) binds to its nuclear receptor (VDR) to stimulate gene transcription activity. Inversion of configuration at C-20 of the side chain to generate 20-epi-1alpha,25(OH)(2)D(3) (20E-125D) increases transcription 200-5000-fold over 125D with its 20-normal (20N) side chain. This enhancement has been attributed to the VDR ligand-binding domain (LBD) having different contact sites for 20N and 20E side chains that generate different VDR conformations. We synthesized 1alpha, 25-dihydroxy-21-(3-hydroxy-3-methylbutyl)vitamin D(3) (Gemini) with two six-carbon side chains (both 20N and 20E orientations). Energy minimization calculations indicate the Gemini side chain possesses significantly more energy minima than either 125D or 20E-125D (2346, 207, and 127 minima, respectively). We compared activities of 125D, 20E-125D, and Gemini, respectively, in several assays: binding to wild-type (100%, 147%, and 38%) and C-terminal-truncated mutant VDR; transcriptional activity (of the transfected osteopontin promoter in ROS 17/2.8 cells: ED(50) 10, 0.005, and 1.0 nM); mediation of conformational changes in VDR assessed by protease clipping (major trypsin-resistant fragment of 34, 34, and 28 kDa). For inhibition of cellular clonal growth of human leukemia (HL-60) and breast cancer (MCF7) cell lines, the ED(50)(125D)/ED(50)(Gem) was respectively 380 and 316. We conclude that while Gemini readily binds to the VDR and generates unique conformational changes, none of them is able to permit a superior gene transcription activity despite the presence of a 20E side chain.

We examined the effects of two novel 1alpha,25-dihydroxyvitamin D3-26,23-lactone (1alpha,25-lactone) analogues on human promyelocytic leukemia cell (HL-60) differentiation using the evaluation system of the vitamin D nuclear receptor (VDR)/vitamin D-responsive element (DRE)-mediated genomic action stimulated by 1alpha,25-dihydroxyvitamin D3 (1alpha,25(OH)2D3) and its analogues. We found that the 1alpha,25-lactone analogues (23S)-25-dehydro-1alpha-hydroxyvitamin-D3-26,23-lactone (TEI-9647), and (23R)-25-dehydro-1alpha-hydroxyvitamin-D3-26,23-lactone (TEI-9648) bound much more strongly to the VDR than the natural (23S, 25R)-1alpha,25(OH)2D3-26,23-lactone, but did not induce cell differentiation even at high concentrations (10(-6) M). Intriguingly, the differentiation of HL-60 cells induced by 1alpha,25(OH)2D3 was inhibited by either TEI-9647 or TEI-9648 but not by the natural lactone. In contrast, retinoic acid or 12-O-tetradecanoylphorbol-13-acetate-induced HL-60 cell differentiation was not blocked by TEI-9647 or TEI-9648. In separate studies, TEI-9647 (10(-7) M) was found to be an effective antagonist of both 1alpha,25(OH)2D3 (10(-8) M) mediated induction of p21(WAF1, CIP1) in HL-60 cells and activation of the luciferase reporter assay in COS-7 cells transfected with cDNA containing the DRE of the rat 25(OH)D3-24-hydroxylase gene and cDNA of the human VDR. Collectively the results strongly suggest that our novel 1alpha,25-lactone analogues, TEI-9647 and TEI-9648, are specific antagonists of 1alpha, 25(OH)2D3 action, specifically VDR/DRE-mediated genomic action. As such, they represent the first examples of antagonists, which act on the nuclear VDR.

The secosteroid hormone 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3] is metabolized into calcitroic acid through the carbon 24 (C-24) oxidation pathway. It is now well established that the C-24 oxidation pathway plays an important role in the target tissue inactivation of 1alpha,25(OH)2D3. Recently, we reported that 1alpha,25(OH)2D3 is also metabolized into 1alpha,25-dihydroxy-3-epi-vitamin D3 [1alpha,25(OH)2-3-epi-D3] through the carbon 3 (C-3) epimerization pathway in human keratinocytes, human colon carcinoma cells (Caco-2), and bovine parathyroid cells. In a previous study, it was demonstrated that 1alpha,25(OH)2-3-epi-D3 when compared to 1alpha,25(OH)2D3 was less active in stimulating intestinal calcium absorption, calcium mobilization from bone, and induction of calbindin D28k. These findings suggest that the C-3 epimerization pathway, like the C-24 oxidation pathway, may play a role in the target tissue inactivation of 1alpha,25(OH)2D3. In this study, we determined the relationship between the C-24 oxidation and the C-3 epimerization pathways by investigating the metabolism of 1alpha,25(OH)2D3 in two rat osteosarcoma cell lines (UMR 106 and ROS 17/2.8). These two cell lines differ from each other in their ability to metabolize 1alpha,25(OH)2D3 through the C-24 oxidation pathway. It has been previously reported that the C-24 oxidation pathway is expressed only in UMR 106 cells but not in ROS 17/2.8 cells. The results of our present study provide new evidence that both cell lines possess the ability to metabolize 1alpha,25(OH)2D3 into 1alpha,25(OH)2-3-epi-D3 through the C-3 epimerization pathway. Our results also reconfirm the findings of previous studies indicating that UMR 106 cells are the only ones which express the C-24 oxidation pathway out of the two cell lines studied. Furthermore, this study reveals for the first time that the C-3 epimerization pathway may become an alternate metabolic pathway for the target tissue inactivation of 1alpha,25(OH)2D3 in some cells, such as ROS 17/2.8, in which the C-24 oxidation pathway is not expressed.

The hormone 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3] generates biological responses via both genomic and rapid, nongenomic mechanisms. The genomic responses utilize signal transduction pathways linked to a nuclear receptor (VDRnuc) for 1alpha,25(OH)2D3, while the rapid responses are believed to utilize other signal transduction pathways that may be linked to a putative membrane receptor for 1alpha,25(OH)2D3. The natural seco steroid is capable of facile rotation about its 6,7 single carbon bond, which permits generation of a continuum of potential ligand shapes extending from the 6-s-cis (steroid like) to the 6-s-trans (extended). To identify the shape of conformer(s) that can serve as agonists for the genomic and rapid biological responses, we measured multiple known agonist activities of two families of chemically synthesized analogs that were either locked in the 6-s-cis (6C) or 6-s-trans (6T) conformation. We found that 6T locked analogs were inactive or significantly less active than 1alpha,25(OH)2D3 in both rapid responses (transcaltachia in perfused chick intestine, 45Ca2+ influx in ROS 17/2.8 cells) and genomic (osteocalcin induction in MG-63 cells, differentiation of HL-60 cells, growth arrest of MCF-7 cells, promoter transfection in COS-7 cells) assays. In genomic assays, 6C locked analogs bound poorly to the VDRnuc and were significantly less effective than 1alpha,25(OH)2D3 in the same series of assays designed to measure genomic responses. In contrast, the 6C locked analogs were potent agonists of both rapid response pathways and had activities equivalent to the conformationally flexibile 1alpha,25(OH)2D3; this represents the first demonstration that 6-s-cis locked analogs can function as agonists for vitamin D responses.

Six A-ring analogs of 1alpha,25-dihydroxyvitamin D3 (1, 1alpha,25-(OH)2-D3) 3-deoxy-3-thia-1alpha,25-(OH)2-D3 (3), 3-deoxy-3-thia-1alpha,25-(OH)2-D3-3alpha-oxide (6), 3-deoxy-3-thia-1alpha,25-(OH)2-D3-3beta-oxide (7) and the 5,6-trans counterparts 5, 8, and 9, respectively--were tested for their ability to inhibit 25-hydroxy-D3-1alpha-hydroxylase (1-OH-ase) in vitro in mitochondria isolated from kidneys of vitamin D deficient chicks. The six analogs were also evaluated in terms of their ability to bind to the chicken intestinal nuclear receptor (VDR) in comparison to the natural hormone 1alpha,25-(OH)2-D3. Analog 7 is not only the best inhibitor of the 1-OH-ase but it also binds effectively to the chick intestinal receptor. It is established that vitamin D analogs must have a 1alpha oxygen group for effective inhibition of the 1-OH-ase. This functional group is also needed for effective binding to the chick intestinal VDR.

Muscle has long been recognized as a target tissue for 1,25-dihydroxy-vitamin D3 (1,25[OH]2D3). Evidence of the presence of VDR is provided here, thus supporting the existence of a receptor-mediated mechanism of action of 1,25(OH)2D3. Vitamin D receptor (VDR) expression is evidenced by detection of VDR-mRNA, through reverse transcription and polymerase chain reaction (RT/PCR), in chicken muscle and muscle cells (myoblasts) as well as in a variety of tissues such as intestine, kidney, heart and brain. VDR presence is also demonstrated by Southern blot of PCR products with a specific VDR-cDNA probe and by immunocytochemistry carried out on myoblasts and cardiac myocytes. Localization of VDR is mainly nuclear and more faintly detected in the cytosol.

1 alpha,25-dihydroxyvitamin-D3 [1 alpha,25(OH)2D3] is known to potentiate in osteoblast-like rat osteosarcoma cells (ROS 17/2.8) the influx of Ca2+ through voltage-activated L-type Ca2+ channels. In the present study, by means of the whole-cell patch-clamp technique, we found that physiological concentrations of 1 alpha,25(OH)2D3 (5-5000 x 10(-11) M) also stimulate, in a dose-dependent manner, outwardly rectifying anionic currents over the course of 1-5 minutes. These currents, recorded in the presence of 130 mM glutamate or approximately 140 mM C1- in the external solution, were blocked by 200 microM DIDS, a stilbene derivative selective for C1- channels. We conclude that, in addition to the already known effects of 1 alpha,25(OH)2D3 on Ca2+ channels, the hormone also enhances in ROS 17/2.8 cells an outward anion conductance. This is the first report concerning activation of anion channels by 1 alpha,25(OH)2D3.

The metabolism of 1alpha,25(OH)2D3 and 1alpha,25(OH)2-l6-ene-23-yne-D3 is examined by in vitro incubation of the steroid hormones with WEHI-3 myeloid leukemia cells. Two experimental systems were used to evaluate the metabolism of each compound: (a) a double label incubation was performed to determine both the propensity for metabolism of each analog and the in vitro half-life of the analogs; and (b) separate single label incubations were performed to determine the metabolite profile derived from each of the analogs. The double label WEHI-3 cell incubation with 25 nM [23,24(3)H]1alpha,25(OH)2D3 and 25 nM [25(14)C]1alpha,25(OH)2-16-ene-23-yne-D3 produced a single comigrating metabolite of higher polarity from each of the parent compounds in a gradient HPLC chromatogram. The half-life of each analog determined from this in vitro incubation was 6.9 and 6.7 h for 1alpha,25(OH)2D3 and 1alpha,25(OH)2-16-ene-23-yne-D3, respectively. Individual incubations of 1alpha,25(OH)2D3 yielded a metabolite that comigrates with 1alpha,24,25(OH)2D3 standard in each of three independent HPLC separations. Individual incubations with [25(14)C]1alpha,25(OH)2-16-ene-23-yne-D3 generated multiple metabolites, which are resistant to degradation as evaluated by intermediate build-up, are of increasing polarity and do not comigrate with the 1alpha,24,25(OH)3D3 standard. From these studies it is determined that 1alpha,25(OH)2-l6-ene-23-yne-D3 is metabolized differently but not preferentially compared to 1,25(OH)2D3 in myelogenous leukemia cells.

Vitamin D is responsible, through the actions of its metabolite, 1 alpha,25-dihydroxyvitamin D3 [1 alpha,25-(OH)2D3], for the generation of a wide array of biological responses, particularly in the intestine, kidney, and bone. 1 alpha,25-(OH)2D3 is known to interact with its nuclear receptor to mediate the regulation of gene transcription. Although many genes and gene products have been shown to be regulated by 1 alpha,25-(OH)2D3 (e.g. calbindin-D28K in the intestine and kidney; collagen, osteocalcin,and osteopontin in bone), their recognition has been largely the result of empirical testing. In this report we have used subtractive hybridization analysis of complementary DNA libraries prepared from messenger RNA (mRNA) isolated from the intestine and kidney of vitamin D-replete or vitamin D-deficient chicks to identify genes for novel proteins whose steady state mRNA levels are regulated by dietary vitamin D status. In the kidney we observed the down-regulated expression of at least seven mitochondrially encoded transcripts and the up-regulated expression of five nuclear encoded genes, two of which are metallothionein and the beta-subunit of aldolase. In the intestine, six mitochondrially encoded transcripts are up-regulated, and seven nuclear encoded transcripts were either up- or down-regulated. Thus, in addition to identifying new nuclear encoded genes whose mRNAs are regulated by vitamin D status, our approach has demonstrated the tissue-specific regulation of mitochondrial gene expression in the intestine and kidney.

1,25-dihydroxy-vitamin D3 [1,25(OH)2D3] is a steroid hormone that modulates the expression of specific proteins by a genomic mechanism of action. Calbindin D-9K is a calcium-binding protein that heretofore has only been found in mammalian tissues and whose gene expression is regulated by 1,25(OH)2D3 in a tissue specific fashion. By combined reverse transcription and polymerase chain reaction, calbindin D-9K gene expression was demonstrated for the first time to be present in several chicken tissues. Subcloning and sequencing of a partial 160 bp-cDNA PCR product revealed that the cDNA corresponds to calbindin D-9K-cDNA. This constitutes the first evidence of calbindin D-9K gene presence and expression in the avian class.

1 alpha,25-dihydroxyvitamin D3 [1 alpha,25(OH)2D3] has been shown to generate biological responses via both genomic and nongenomic pathways. In previous studies concerning the mechanism of 1 alpha,25(OH)2D3-stimulated rapid intestinal transport of Ca2+, a process termed transcaltachia, we assessed the ability of seven analogs of 1 alpha,25(OH)2D3 to initiate transcaltachia in the vitamin D-replete chick and bind in vitro to the classical nuclear chick intestinal receptor for 1 alpha,25(OH)2D3 (VDR). Two groups of analogs were found, one able to stimulate transcaltachia and the other able to bind to the VDR. In the present study, we have examined both the genomic effect of these analogs in the intestine of vitamin D-deficient chicks and their in vitro binding to vitamin D binding protein. It was found that analogs known to bind effectively to the nuclear receptor in vitro could achieve a significant occupancy of the VDR in vivo and stimulated calbindin-D28K messenger RNA and protein synthesis. In contrast, those analogs that were good agonists of transcaltachia were ineffective either in occupying the VDR in vitro or in stimulating in vivo calbindin-D28K messenger RNA and protein synthesis. These differences are consistent with our hypothesis that there may be two receptors for 1,25-(OH)2D3 with differing ligand binding domains, one being the classical nuclear VDR and the second a membrane receptor associated with transcaltachia.

Biological responses mediated by vitamin D occur as a consequence of the integrated actions of the vitamin D endocrine system. The vitamin D endocrine system is characterized by the sequential two-step metabolism of vitamin D to 1α,25(OH)2D3 by the liver and kidney, and by the ability to generate biological responses in over 30 target tissues through nuclear receptor (nVDR) regulation of gene transcription and nongenomic pathways. It is now clear that the vitamin D endocrine system embraces many more target tissues than simply the intestine, bone and kidney. Notable additions to this list of tissues containing the nVDR include pancreatic B cells, pituitary gland, breast tissue, placenta, lymphocytes, keratinocytes, colon, and prostate, as well as many cancer cell lines. In addition to the classical actions of 1α,25(OH)2D3 on mediating calcium homeostasis, this seco steroid has been identified as a potent stimulator of cell differentiation as well as an inhibitor of proliferation. Over the past decade at least 400 analogs of 1α,25(OH)2D3 have been chemically synthesized and their biological properties systematically explored in a variety of assays which quantified both their calcemic effects and cell differentiating potential. The objective has been to identify new analogs devoid of the classical calcemic consequences of high does of 1α,25(OH)2D3, namely hypercalcemia, soft tissue calcification and nephrocalcinosis. As a consequence, several analogs of 1α,25(OH)2D3 have recently been identified and are discussed in this paper for consideration as possible chemotherapeutic agents for acute promyelocytic leukemia, breast, colon, and prostate cancer, or as immunosuppressive agents with possible beneficial structure-activity profiles for use in cardiac allografts, autommune graft rejection, lupus erthematosus and psoriasis.

Computer simulation of calcium homeostasis in chicks predicted an oscillatory behavior of bone calcium flow and kidney 25-hydroxyvitamin D3-1-hydroxylase with a periodicity of 56 h and a 9 h phase difference between the two signals. In growing chickens subjected to a light: dark cycle of 22:2 h, and intravenously dosed with 45Ca, the temporal changes in plasma 45Ca could be described by an exponential decline with superimposed diurnal oscillations. The activity of the renal 25-hydroxyvitamin D3-1-hydroxylase in chicks subjected to a 12:12 h light: dark cycle ALSO followed diurnal oscillations, with a nadir at the beginning of the light period and a peak 12 h later. The production of 1,25-dihydroxyvitamin D3 by primary cultures of chicken kidney cells oscillated with a periodicity of 5.6 h or shorter. It is suggested that despite the differences in phase and periodicity between the simulation predictions and actual results, the oscillations in both 1-hydroxylase and bone calcium flow could be coupled through the hormonal systems involved in regulation of plasma calcium.

The steroid hormone 1α,25-dihydroxyvitamin D 3 [1α,25(OH) 2 D 3 ] stimulates biological responses via both genomic mechanisms and nongenomic mechanisms (opening of voltage-gated Ca 2+ channels). We report here that the two closed B-ring steroid analogs of 1α,25(OH) 2 D 3 , 1α,25(OH) 2 -7-dehydrocholesterol and 1α,25(OH) 2 -lumisterol 3 , are able to generate the nongenomic response, transcaltachia, without the ability to compete with the natural metabolite for binding to its nuclear receptor. We propose that the nongenomic membrane associated receptor can accept the ligand in its closed "6-s- cis " conformation whereas the nuclear receptor prefers the extended "6-s- trans " conformer.

The conformationally flexible secosteroid, 1α,25(OH)₂vitamin D₃ (1α,25(OH)₂D₃) initiates biological responses via binding to the vitamin D receptor (VDR). The VDR contains two overlapping ligand binding sites, a genomic pocket (VDR-GP) and an alternative pocket (VDR-AP), that respectively bind a bowl-like ligand configuration (gene transcription) or a planar-like ligand shape (rapid responses). When occupied by 1α,25(OH)₂D₃, the VDR-GP interacts with the retinoid X receptor to form a heterodimer that binds to vitamin D responsive elements in the region of genes directly controlled by 1α,25(OH)₂D₃. By recruiting complexes of either coactivators or corepressors, activated VDR modulates the transcription of genes encoding proteins that promulgate the traditional genomic functions of vitamin D, including signaling intestinal calcium and phosphate absorption to effect skeletal and calcium homeostasis. 1α,25(OH)₂D₃/VDR control of gene expression and rapid responses also delays chronic diseases of aging such as osteoporosis, cancer, type-1 and -2 diabetes, arteriosclerosis, vascular disease, and infection.

We have postulated that the vitamin D receptor (VDR) contains two overlapping ligand binding sites, a genomic pocket and an alternative pocket (AP), that mediate regulation of gene transcription and rapid responses, respectively. Flexible VDR + ligand docking calculations predict that the major blood metabolite, 25(OH)-vitamin D3 (25D3), and curcumin (CM) bind more selectively to the VDR-AP when compared with the seco-steroid hormone 1α,25(OH)2-vitamin D3 (1,25D3). In VDR wild-type-transfected COS-1 cells and TM4 Sertoli cells, 1,25D3, 25D3, and CM each trigger voltage-gated, outwardly rectifying chloride channel (ORCC) currents that can be blocked by the VDR antagonist 1β,25(OH)2-vitamin D3 and the chloride channel antagonist (4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid). VDR mutational analysis in transfected COS-1 cells demonstrate the DNA-binding domain is not, but the ligand binding and hinge domains of the VDR are, required for 1,25D3 and 25D3 to activate the ORCC. Dose-response studies demonstrate that 25D3 and 1,25D3 are approximately equipotent in stimulating ORCC rapid responses, whereas 1 nm 1,25D3 was 1000-fold more potent than 25D3 and CM in stimulating gene expression. The VDR-AP agonist effects of 1,25D3, 25D3, and low-dose CM are lost after pretreatment of TM4 cells with VDR small interfering RNA. Collectively, these results are consistent with an essential role for the VDR-AP in initiating the signaling required for rapid opening of ORCC. The fact that 25D3 is equipotent to 1,25D3 in opening ORCC suggests that reconsideration of the ability of 25D3 to generate biological responses in vivo may be in order.

The role of the vitamin D-induced calcium binding protein termed calbindin-D (CaBP) in the biological response to 1,25-dihydroxyvitamin D3 was assessed by photoaffinity labeling techniques. The heterobifunctional cross-linking reagent methyl-4-azidobenzoimidate was employed for studies with the 28 KD chick intestinal calbindin-D28K. Calcium-dependent interactions were evident with purified chick intestinal CaBP-immunoglobulins and bovine intestinal alkaline phosphatase; in the absence of Ca2+ there was a greatly diminished crosslinking process. There were also at least two membrane components of chick intestinal brush border membranes, with M(R) = 60,000 and 130,000, which were photoaffinity cross-linked with CaBP in a calcium-dependent manner. Similar interactions were demonstrated following incubations of CaBP with phosphatidylinositol-specific phospholipase C (PI-PLC)-treated supernatant fractions from chick intestinal brush borders. PI-PLC was shown to release 14% of the alkaline phosphatase from chick intestinal brush borders compared to greater than 80% for rabbit and chick kidney BBM preparations. Specific interactions between CaBP and brush border membrane proteins could also be demonstrated in the absence of photoaffinity labeling by Sephadex G-150 chromatography of Triton X-100 solubilized incubations between calbindin-D28K and chick intestinal BBMS, with 17% of the radiolabelled CaBP comigrating with alkaline phosphatase activity. These studies collectively demonstrate that calbindin-D28K undergoes calcium-dependent conformational changes which alter its subsequent interactions with cellular proteins in a way consistent with other calcium-binding proteins such as calmodulin or troponin C.

The steroid hormone 1α,25-dihydroxyvitamin-D 3 [1α,25(OH) 2 D 3 ] stimulates biological responses via both genomic mechanisms and nongenomic mechanisms (opening of voltage-gated Ca 2+ channels). We report here that 1β,25(OH) 2 -vitamin-D 3 (a) is devoid of activity as an agonist for transcaltachia, (b) is a potent stereospecific antagonist of 1α,25(OH) 2 D 3 stimulation of the nongenomic transcaltachia response and also (c) has less than 1% the ability of 1α,25(OH) 2 D 3 to bind to the chick intestinal nuclear 1β,25(OH) 2 D 3 receptor. We conclude that the membrane response element(s) which generates the nongenomic response of transcaltachia has a different ligand specificity than the classic nuclear 1α,25(OH) 2 D 3 receptor.

Physiological studies of the Atlantic cod, Gadus morhua, have suggested a role for the vitamin D3 system in this marine teleost similar to that in other vertebrates. Accordingly, the present study was carried out to assess the plasma concentrations of vitamin D3, 25-hydroxyvitamin D3 (25OHD3), and 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] in this fish. Additionally, the presence of binding proteins in plasma and target-specific tissue receptors for these vitamin D3 metabolites was studied in organs normally associated with calcium regulation. Plasma levels of 25-OHD3 (undetectable to 148 pg/ml; n = 5) were comparatively low (20-30 ng/ml), whereas the levels of vitamin D3 (approximately 30 ng/ml) and 1,25-(OH)2D3 (approximately 50 pg/ml) were comparable to levels reported in higher vertebrates. Cod plasma contained a protein that bound both 25OHD3 and 1,25-(OH)2D3. This plasma binding protein revealed low affinity for 25OHD3, did not bind G-actin, and had a sedimentation coefficient of 3.4S. High affinity 1,25-(OH)2D3 receptors [Kd, 1.02 +/- 0.36 (n = 6), 1.02 +/- 0.3 (n = 5), and 0.95 +/- 0.51 (n = 5) nM; mean +/- SEM] were found in high salt cytosols from intestine, liver, and gills, respectively, and had sedimentation coefficients (3.6-3.8S in 0.3 M KCl sucrose gradients) similar to those in higher vertebrates. No specific 1,25-(OH)2D3 binding was found in kidney, ultimobranchial glands, corpuscles of Stannius, or bone. The finding of significant quantities of 1,25-(OH)2D3 in the plasma, the presence of plasma binding proteins that bind this seco-steroid, and the localization of specific high affinity receptors for this metabolite in calcium regulatory tissues in teleosts are all consistent with a physiological role for the vitamin D3 system in the calcium regulation of the cod.

The high affinity calcium-binding protein calbindin-D28K is one of the known proteins transcriptionally up-regulated by the hormonally active form of vitamin D3, 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]. This regulation is tissue specific, since in the absence of 1,25-(OH)2D3, the expression of calbindin-D28K is virtually abolished in intestine, whereas it is decreased, but clearly detectable, in kidney, and it remains present at its highest level in cerebellum. Several studies have shown that there is a strong correlation between an increase in the sensitivity to nuclease digestion of a given gene locus and its potential for transcription. Furthermore, hypersensitive sites have often been mapped to regions of DNA including or surrounding sequences known to be important for the regulation of gene transcription. In this study we have scanned the 5'-end and flanking DNA of the calbindin-D28K gene for the presence of DNase-I-hypersensitive (DH) sites in order to localize possible regulatory regions involved in the tissue-specific and hormone-dependent regulation of this gene. We have found that in tissues where calbindin is not expressed, such as liver, no DH sites could be detected. In cerebellum, the same set of DH sites was observed in the presence or absence of 1,25-(OH)2D3 treatment, reflecting the vitamin D-independent expression of the calbindin gene in this tissue. A more complex pattern of DH sites was found in intestine, independently of the vitamin D status of the animal.(ABSTRACT TRUNCATED AT 250 WORDS)

Ultraviolet (UV) irradiation causes DNA damage in skin cells, immunosuppression and photocarcinogenesis. 1alpha,25-dihydroxyvitamin D3 (1,25D) reduces UV-induced DNA damage in the form of cyclobutane pyrimidine dimers (CPD) in human keratinocytes in culture and in mouse and human skin. UV-induced immunosuppression is also reduced in mice by 1,25D, in part due to the reduction in CPD and a reduction in interleukin (IL-6. The cis-locked analog, 1alpha,25-dihydroxylumisterol3 (JN), which has almost no transactivating activity, reduces UV-induced DNA damage, apoptosis and immunosuppression with similar potency to 1,25D, consistent with a non-genomic signalling mechanism. The mechanism of the reduction in DNA damage in the form of CPD is unclear. 1,25D doubles nuclear expression of p53 compared to UV alone, which suggests that 1,25D facilitates DNA repair. Yet expression of a key DNA repair gene, XPG is not affected by 1,25D. Chemical production of CPD has been described. Incubation of keratinocytes with a nitric oxide donor, SNP, induces CPD in the dark. We previously reported that 1,25D reduced UV-induced nitrite in keratinocytes, similar to aminoguanidine, an inhibitor of nitric oxide synthase. A reduction in reactive nitrogen species has been shown to facilitate DNA repair, but in view of these findings may also reduce CPD formation via a novel mechanism.

Steroid hormones serve as chemical messengers in a wide number of species and target tissues by transmitting signals that result in both genomic and nongenomic responses. Genomic responses are mediated by the formation of a ligand-receptor complex with its cognate steroid hormone nuclear receptor (NR). Nongenomic responses can be mediated at the plasma membrane by a membrane-localized NR. The focus of this Review is on the structural attributes and molecular mechanisms underlying vitamin D sterol (VDS)-vitamin D receptor (VDR) selective and stereospecific regulation of nongenomic and genomic signaling. The VDS-VDR conformational ensemble model describes how VDSs can selectively initiate or block either nongenomic or genomic biological responses by interacting with two VDR ligand-binding pockets, one kinetically favored by 1alpha,25(OH)(2)D(3) (1,25D) and the other thermodynamically favored. We describe the variables that affect the three major elements of the model: the conformational flexibility of the unliganded (apo) protein, the flexibility of the VDS, and the physicochemical selectivity of the VDR genomic pocket (VDR-GP) and alternative pocket (VDR-AP). We also discuss how these three factors collectively provide a rational explanation for the complexities of VDS regulation of cell biology and highlight the current limitations of the model.

Recent work has indicated that vectorial Ca2+ transport across the intestinal epithelium occurs in vesicles and may involve the participation of microtubules [Nemere et al., 1986]. Since 1,25 dihydroxyvitamin D3 (1,25(OH)2D3) stimulates this Ca2+ transport process, microtubule (MT) isotypes were studied as a potential regulatory point. The effect of 1,25(OH)2D3 status on tubulin isotypes was analyzed by isoelectric focusing (IEF) gels of taxol stabilized MTs prepared from intestinal epithelium of vitamin D-deficient chicks dosed with vehicle (-D) or 1.3 nmoles of 1,25(OH)2D3 (+D) 2.5, 5, 10, 15, or 43 h prior to sacrifice. Four bands, one of which was identified as alpha-tubulin on the basis of Western analysis, increased in Coomassie Blue staining intensity 5-15 h after 1,25(OH)2D3, corresponding to the time course of augmented vesicular Ca2+ transport. Dose-response studies revealed similar changes in tubulin isotype profiles in IEF gels, again corresponding to doses known to elicit enhanced Ca2+ absorption (52-6,500 pmoles of hormone). The role of Ca2+ transport was also examined. Isoelectrically focused intestinal epithelial tubulin from -D chicks allowed to transport Ca2+ for 30 min revealed increased staining of bands relative to nonabsorbing -D controls. By comparison, Ca2+ transport in +D chicks resulted in fainter bands relative to nonabsorbing, +D controls. MTs prepared from fasted or fed chicks revealed similar changes upon IEF, but of much smaller magnitude. Enhanced phosphorylation did not account for the appearance of the more acidic bands, although 1,25(OH)2D3 treatment resulted in decreased 32P content of a presumptive non-tubulin component, relative to preparations from -D controls. Glucocorticoids, which are known to suppress 1,25(OH)2D3-stimulated Ca2+ transport, led to severely diminished levels of total tubulin, as judged by SDS-PAGE, rather than altered tubulin isotypes. Thus, MTs of intestine are subject to regulation by hormonal status, as well as by the amount of Ca2+ available for transepithelial transport.

Vitamin D and its hormonally active metabolite 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] are known to alter several parameters associated with stimulated intestinal Ca2+ transport: levels of calbindin-D28K, tubulin, and endosomal-lysosomal organelles containing Ca2+, and calbindin-D28K. In the present study the as yet unexamined relationship among Ca2+ transport, calbindin-D28K, and microtubules was studied by immunofluorescence microscopy. In vitamin D3-treated or 1,25-(OH)2D3-treated chicks, in the absence of Ca2+ transport, immunofluorescence microscopy of intestinal tissue fixed at 25 C indicated a colocalization of calbindin-D28K and tubulin along epithelial cell brush border and basal-lateral membranes. Initiation of in situ Ca2+ absorption for 10, 20, or 30 min before tissue fixation resulted first in increased punctate calbindin-D28K staining and then in a progressive decrease in intestinal cell- and microtubule-associated calbindin-D28K, with a concomitant increase in calbindin-D28K labeling in the villus core. When intestinal tissue from 1,25-(OH)2D3-treated chicks was chilled to 4 C before fixation (a procedure shown by others to cause microtubule depolymerization), evaluation by immunofluorescence microscopy revealed diffuse cytoplasmic staining of both the immunoreactive tubulin and its associated calbindin-D28K. These results indicate the possible involvement of calbindin-D28K with tubulin during the process of Ca2+ transport and the secretion of the calbindin-D28K as a consequence of the overall transport process. Electron microscopy with immunogold labeling revealed intestinal epithelial calbindin-D28K to be localized inside of small vesicles and lysosome-like structures, with sparse cytoplasmic labeling. Subsequent electron microscopic analysis of intestinal epithelial microtubules prepared by polymerization and depolymerization revealed immunogold labeling in coprecipitated vesicular remnants, with consistently light staining of filaments traversing segments of the microtubules. In biochemical studies, isolation of intestinal microtubules or tubulin by three distinct procedures revealed increasing levels of associated calbindin-D28K as a function of time after 1,25-(OH)2D3 repletion of vitamin D-deficient chicks. Addition of calbindin-D28K to intestinal microtubules isolated from vitamin D-deficient chicks exhibited saturable binding when exogenous calbindin-D28K reached levels comparable to those present in vitamin D-replete chick intestine. Collectively, these results suggest that calbindin-D28K is predominantly located in membrane-delimited vesicles, with a very minor component associated with filamentous elements that can be isolated with tubulin and microtubules. Additionally, calbindin-D28K is dynamically involved in Ca2+ transport in the intestine.

Osteoblast-like osteosarcoma cells (ROS 17/2.8) display a rapid transmembrane influx of extracellular calcium after stimulation by 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] that is mediated largely by the opening of voltage-gated calcium channels. These cells also constitutively express high numbers (greater than 18,000/cell) of nuclear receptors for this seco-steroid hormone that are involved in the modulation of genomic activity in the osteoblast and in the up-regulation of transcript ion of osteoblast-specific genes such as osteocalcin. The objective of this study was to determine the structural hierarchy of vitamin D3 analogs with regard to their efficacy as molecular transducers of the genomic and nongenomic pathways that are activated upon treatment of osteoblasts with 1,25-(OH)2D3. To test the structural features of the agonist required for initiation of these distinct pathways, a series of ligand analogs and naturally occurring metabolites of 1,25-(OH)2D3 were used that contain A-ring, D-ring, and side-chain modifications. The abilities of these analogs/metabolites to 1) bind to nuclear receptors and 2) stimulate transmembrane calcium influx were measured. Several analogs (25-hydroxy-16-ene-23-yne-D3 and 25-hydroxy-23-yne D3) were found to stimulate Ca2+ channel opening, but bind only poorly to the 1,25-(OH)2D3 nuclear receptor. Conversely, other analogs (1,24-dihydroxy-22-ene-24-cyclopropyl D3 and 1,25-dihydroxy-16-ene-23-yne,26,27 F6-D3) were found to bind very well to the nuclear receptor, but displayed little or no activity in opening Ca2+ channels. Pertussis toxin, which interferes with coupling of certain ligand-gated receptors to ion channels, failed to block the activation of calcium channels by 1,25-(OH)2D3 or active agonist analogs. Our results indicate that there are likely to be distinct nuclear and plasma membrane-associated forms of the 1,25-(OH)2D3 receptor that are involved in genomic and nongenomic activation of osteoblast activity, respectively. The membrane-associated receptors do not appear to be coupled to pertussis toxin-sensitive G-proteins.

Calcium as an intracellular second messenger is not a new concept, although the subject is undergoing a resurgence in interest, in part reflecting our current appreciation of calcium channels and their role in Ca2+ influx, as well as the regulation of cytoplasmic Ca2+ movements by phosphoinositide metabolism (Michell, 1986). Perhaps the most intriguing aspect of the topic is that the agonists are not restricted to a single structural class, but include neurotransmitters, peptide hormones, and steroid hormones (see Table 1). Parallels in signal transduction between divergent structural classes of regulatory substances have been proposed before (Szego, 1978). The current work will attempt to focus more closely on systems where agonist-mediated changes in cellular calcium culminate in an exocytotic event.

Expression of the vitamin D induced calbindin-D28K protein is transcriptionally controlled by the steroid hormone 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) in a tissue-specific manner in the intestine and kidney. In order to examine the cis-acting elements of the calbindin-D28K promoter and its modulation by 1,25-dihydroxyvitamin D3, chimeric plasmids containing 2.1 kb of 5' flanking region linked to the reporter gene chloramphenicol acetyl transferase (CAT) were transfected by lipofection into primary cultures of chick kidney cells. Transfected chick kidney cells exhibited a high basal expression of the chloramphenicol acetyl transferase gene, reflecting the strong activity of the calbindin-D28K promoter. Expression of the pCaBP2.1 reporter gene was increased 2-fold in the presence of the hormone 1,25(OH)2D3 in the primary kidney cells. Deletion of a 1.42 kb fragment ending -679 base pairs upstream from the transcription start site led to a 2-fold repression in the reporter gene activity by the hormone 1,25(OH)2D3 in primary chick kidney cultures. These preliminary results suggest that both positive and negative elements normally act to regulate the expression of the calbindin-D28K gene in primary chick kidney cells.

We have compared the relative responsiveness of pancreatic, intestinal and renal tissue calbindin-D28K protein content to the stimulatory actions of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] in vitamin D-deficient (-D) chicks. Tissue concentrations of calbindin-D28K were undetectable in the -D chick intestine but present, albeit at low concentrations (less than 1 microgram CaBP/mg protein) in the -D kidney and pancreas. Intestinal, pancreatic and renal calbindin-D28K content was stimulated 318, 9.8 and 2.9 fold respectively, 48 hours after -D chicks received a single dose of 1,25(OH)2D3 [6.5 nmol/animal]. The pancreatic calbindin-D28K content could be significantly stimulated as early as 5 hours after 1,25(OH)2D3 administrations in vivo. These findings support the contention that the pancreas is a target for vitamin D, and is consistent with the view that calbindin-D28K plays a role in normal pancreatic functions.

Previous studies have demonstrated that human alveolar and bone marrow macrophages when activated in vitro can metabolize 25-hydroxyvitamin[3H]D3 to 1 alpha,25-dihydroxyvitamin[3H]D3; however, to date no animal models to study this system have been available. In the present study, cultured porcine pulmonary alveolar macrophages from two animals were assayed for their capability for metabolism of 25-hydroxyvitamin[3H]D3. The porcine alveolar macrophages constitutively produced a metabolite of 25-hydroxyvitamin[3H]D3 which was identified as 1 alpha,25-dihydroxyvitamin[3H]D3 by high performance liquid chromatography. The apparent KM was in the range of 300 nM. Unlike human macrophages, treatment of porcine alveolar macrophages with lipopolysaccharide did not stimulate 1 alpha,25-dihydroxyvitamin[3H]D3 production. Addition of 1 alpha,25-dihydroxyvitamin-D3 to macrophages cultures led to a sensitive proportional inhibition of 1 alpha,25-dihydroxyvitamin[3H]D3 synthesis.

Based on their content of prolactin receptors, osteosarcoma cells were predicted to be responsive to prolactin (PRL), but whether PRL would be beneficial or contribute to pathogenesis was unclear. 1,25(OH)(2) vitamin D(3) [1alpha,25(OH)(2)D(3)] has antiproliferative effects on osteosarcoma cells, and a complex interregulatory situation exists between PRL and 1alpha,25(OH)(2)D(3). Using osteosarcoma cells, Western blot, real time RT-PCR, and promoter-luciferase assays, we have examined the interaction between PRL and 1alpha,25(OH)(2)D(3) and demonstrated that physiological concentrations of PRL block increased osteocalcin and vitamin D receptor (VDR) expression in response to 1alpha,25(OH)(2)D(3.) This blockade was shown to be the result of lack of nuclear accumulation of the VDR in response to 1alpha,25(OH)(2)D(3). Although inhibition of proteasomic degradation with MG132 had no effect on the VDR itself in a 30-min time frame, it relieved the blockade by PRL. Analysis of ubiquitinated proteins brought down by immunoprecipitation with anti-VDR showed PRL regulation of a 250-kDa protein-VDR complex. P250 was identified as the breast cancer tumor suppressor gene product, BRCA1, by Western blot of the VDR immunoprecipitate and confirmed by immunoprecipitation with anti-BRCA1 and blotting for the VDR in the absence and presence of PRL. Knockdown of BRCA1 inhibited nuclear translocation of the VDR and the ability of 1alpha,25(OH)(2)D(3) to induce the VDR. This, to our knowledge, is the first demonstration of a role for BRCA1 in nuclear accumulation of a steroid hormone and the first demonstration that PRL has the potential to affect the cell cycle through effects on BRCA1.

We investigated the role of extracellular Ca2+ in 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] rapid stimulation of intestinal Ca2+ transport (termed transcaltachia) in the perfused duodenal of vitamin D-replete chicks. The carboxylic ionophore ionomycin (2 microM) was found to stimulate 45Ca2+ transport from the lumen to the vascular effluent to the same extent as physiological levels of 1,25-(OH)2D3. The increase in duodenal 45Ca2+ transport caused by 1,25-(OH)2D3 was dependent on the presence of medium Ca2+, since it was abolished by prior addition of EGTA and was restored upon the addition of Ca2+. Depolarization of the basal lateral membrane of intestinal epithelial cells with 70 mM K+ caused a rapid increase in 45Ca2+ transport (30% above control values within 2 min and 250% after 20 min of vascular perfusion). The rise was also abolished by prior addition of EGTA. Intracellular calcium concentrations ([Ca2+]i) were measured in isolated duodenal cells from vitamin D-replete chicks using the fluorescent dye fura 2. A 1-min incubation with physiological concentrations of 1,25-(OH)2D3 (130 pM) caused an increase in [Ca2+]i from a basal level of 168 +/- 23 nM to 363 +/- 44 nM. Pretreatment of intestinal epithelial cells with the protein kinase-C activator tetradeconyl-phorbol acetate (100 nM) or the adenylate cyclase activator forskolin (10 microM), both shown to induce acute stimulation of intestinal 45Ca2+ transport in the perfused duodenum, also mimicked the stimulatory effect of 1,25-(OH)2D3 on [Ca2+]i. The increase in [Ca2+]i elicited by the 1,25-(OH)2D3 was due to Ca2+ influx from the extracellular medium, since it was blocked by the Ca2+ chelator EGTA (5 mM) and the Ca2+ channel antagonist nifedipine (1 microM). These results suggest that the acute effects of 1,25-(OH)2D3 on duodenal 45Ca2+ transport are triggered by the influx of Ca2+ through voltage-operated Ca2+ channels and that both protein kinase-C and protein kinase-A play an important role in mediating or modulating 1,25-(OH)2D3 effects on transcaltachia.

We have produced 25 clones secreting antibodies directed against chicken Calbindin D-28k. Two of them, 300 and 318, recognize determinants conserved in fish, chicken, mouse, rat, rabbit, monkey and human Calbindin D-28k. We demonstrate their use in the immunohistochemical localization of Calbindin D-28k, and in the detection of Calbindin D-28k on immunoblots.