Effects of the vitamin D3 analog 1α,25-dihydroxyvitamin D3-3β-bromoacetate on rat osteosarcoma cells: Comparison with 1α,25-dihydroxyvitamin D3

The actions of the hormonal form of vitamin D, 1α,25-dihydroxyvitamin D₃ [1α,25-(OH)₂D₃], are mediated by both genomic and nongenomic mechanisms. Several vitamin D synthetic analogs have been developed in order to identify and characterize the site(s) of action of 1α,25-(OH)₂D₃ in many cell types including osteoblastic cells. We have compared the effects of 1α,25-(OH)₂D₃ and a novel 1α,25-(OH)₂D₃ bromoester analog (1,25-(OH)₂-BE) that covalently binds to vitamin D receptors. Rat osteosarcoma cells that possess (ROS 17/2.8) or lack (ROS 24/1) the classic intracellular vitamin D receptor were studied to investigate genomic and nongenomic actions. In ROS 17/2.8 cells plated at low density, the two vitamin D compounds (1 × 10⁻⁸ M) caused increased cell proliferation, as assessed by DNA synthesis and total cell counts. Northern blot analysis revealed that the mitogenic effect of both agents was accompanied by an increase in steady-state osteocalcin mRNA levels, but neither agent altered alkaline phosphatase mRNA levels in ROS 17/2.8 cells. ROS 17/2.8 cells responded to 1,25-(OH)₂-BE but not the natural ligand with a significant increase in osteocalcin secretion after 72, 96, 120, and 144 hr of treatment. Treatment of ROS 17/2.8 cells with the bromoester analog also resulted in a significant decrease in alkaline phosphatase-specific activity. To compare the nongenomic effects of 1α,25-(OH)₂D₃ and 1,25-(OH)₂-BE, intracellular calcium was measured in ROS 24/1 cells loaded with the fluorescent calcium indicator Quin 2. At 2 × 10⁻⁸ M, both 1α,25-(OH)₂D₃ and 1,25-(OH)₂-BE increased intracellular calcium within 5 min. Both the genomic and nongenomic actions of 1,25-(OH)₂-BE are similar to those of 1α,25-(OH)₂D₃, and since 1,25-(OH)₂-BE has more potent effects on osteoblast function than the naturally occurring ligand due to more stable binding, this novel vitamin D analog may be useful in elucidating the structure and function of cellular vitamin D receptors. © 1996 Wiley-Liss, Inc.     

1α,25-Dihydroxyvitamin D3 rapidly increases nuclear calcium levels in rat osteosarcoma cells

1α,25-Dihydroxyvitamin D₃ increases intracellular calcium in rat osteoblast-like cells that possess the classic receptor (ROS 17/2.8) as well as those that lack the classic receptor (ROS 24/1), indicating that a separate signalling system mediates this rapid nongenomic action. To determine the intracellular sites of this calcium increase, cytosolic and nuclear fluorescence (340 nm/380 nm ratio) were measured in Fura 2AM loaded ROS 17/2.8 cells using digital microscopy. Within 5 min, cytosolic fluorescence increased by 29% (P < 0.05) and nuclear fluorescence by 30% (P < 0.01) after exposure to 1α,25-dihydroxyvitamin D₃ (20 nM). This effect was blocked by the inactive epimer 1β,25-dihydroxyvitamin D₃. In an individual cell, cytosolic and nuclear fluorescence increased gradually after 1, 3, and 5 min exposure to vitamin D. Nuclei were then isolated from ROS 17/2.8 cells to directly measure the hormone's effect on nuclear calcium. The calcium content of Fura 2AM loaded nuclei was not affected by increasing the calcium concentration in the incubation buffer from 50 nM to 200 nM. After 5 min, 1α,25-dihydroxyvitamin D₃, 20 nM, increased the calcium of isolated nuclei in medium containing 50 nM calcium and 200 nM calcium. 1β,25-dihydroxyvitamin D₃, 20 nM, had no effect on nuclear calcium but blocked the 1α,25-dihydroxyvitamin D₃ induced rise in the isolated nuclei. The results indicate that the nuclear membrane of the ROS 17/2.8 cells contain calcium permeability barriers and transport systems that are sensitive to and specific for 1α,25-dihydroxyvitamin D₃. 1α,25-Dihydroxyvitamin D₃ rapidly increases nuclear calcium levels in both intact cells and isolated nuclei suggesting that rapid nongenomic activation of nuclear calcium may play a functional role in osteoblastic activity.     

Binding characteristics of a membrane receptor that recognizes 1α25-dihydroxyvitamin D3 and its epimer, 1β,25-dihydroxyvitamin D3

The Steroid hormon 1α, @5-Dihydroxyvitamin D₃ has been shown to expert rapid effect (15 s to 5 min) in osteoblast. These occur in osteoblast-like cells lacking the nuclear vitamin D receptor, ROS 24/1, suggesting that a separate signalling system mediates the rapid action. These non-genomic action include rapid activation of phospholipase C and opening of calcium channels, pointing to a membrane localization of this signalling system. Previous studies have shown that the 1β epimer of 1α25-dihydroxyvitamina D₃ can block these rapid action, indicating that the 1β epimer may bind to the recptor responsible for the rapid action sin a competative manner. We have assessed the displacement of ³H-1α,25dihydroxyvitamin D₃ by vitamin D compounds, as well as the apparent dissociation constant of 1α25-dihydroxyvitamin D₃ and its 1β epimer for the memberane receptor in membrane prepration from ROS 24/1 cells. Increasing concentrations of 1α25-dihydroxyvitamin D₃, 7.25 nM to 725 nM, displaced ³H-1α25-dihydrxyvitamin D₃ from the membranes with 725 nM of the hormone displacing 40–49% of the radioactivity. Similarly, 1β,25-dihydroxyvitamin D₃, 7.25 nM and 72.5 nM, displaced 1α25-dihydroxyvitamin D₃ binding while 25-hydroxyvitamin D₃, 7.25 nM, did not. The apparent dissociation constant (K_D) for 1α25-dihydroxyvitamin D₃ was detrermined from displacement of ³H-1α25-dihydroxyvitamin D₃ yielding a value of 8.1 × 10^?7 M by Scatchard analysis. The K_D for the 1β epimer determine from displacement of ³H-1α25-dihydroxyvitamin D₃ was 4.8 × 10^?7 M. The data suggest the presence of a receptor on the membrane of ROS 24/1 cells that reconize 1α25-dihydroxyvitamin D₃ and its 1β epimer, but not 25-dihydroxyvitamin D₃. Its ability to reconize the 1β epimer which appears to be a specific anagonist of the rapid effect of the hormone suggests that these studies may be the initial steps in the isolation and characterization of the signalling system mediating the rapid action of vitamin D.     

1α,25-Dihydroxyvitamin D3-induced changes in intracellular pH in osteoblast-like cells modulate gene expression

1α,25-Dihydroxyvitamin D₃ exerts rapid nongenomic effects on rat osteoblast-like cells independent of the classic nuclear receptor. These effects include changes in phospholipid metabolism and cell calcium. Intracellular calcium itself has been proposed to regulate intracellular pH in osteoblast cell lines. The purpose of this study was to determine the effect of 1α,25-dihydroxyvitamin D₃ on intracellular pH, the relationship of changes in calcium to changes in pH, and the role of pH changes in genomic activation. 1α,25-Dihydroxyvitamin D₃ increased intracellular pH within 10 min in rat osteoblast-like cells, an effect that was inhibited by removal of extracellular sodium and by the biologically inactive epimer 1β,25-dihydroxyvitamin D₃. The hormone increased intracellular calcium in Quin 2 loaded cells in the presence and absence of extracellular sodium. The 1α,25-dihydroxyvitamin D₃-induced increments in osteocalcin and osteopontin mRNA levels were abolished in sodium-free medium. The results indicate that 1α,25-dihydroxyvitamin D₃-induced increments in cellular calcium precede cell alkalinization and that these changes in intracellular pH may modulate steady-state mRNA levels of genes induced by vitamin D.     

1α,25(OH)2-vitamin D3 membrane-initiated calcium signaling modulates exocytosis and cell survival

1α,25(OH)₂-vitamin D₃ (1,25D) is considered a bone anabolic hormone. 1,25D actions leading to bone formation involve gene transactivation, on one hand, and modulation of cytoplasmic signaling, on the other. In both cases, a functional vitamin D receptor (VDR) appears to be required. Here we study 1,25D-stimulated calcium signaling that initiates at the cell membrane and leads to exocytosis of bone materials and increased osteoblast survival. We found that rapid 1,25D-induction of exocytosis couples to cytoplasmic calcium increase in osteoblastic ROS 17/2.8 cells. In addition, we found that elevation of cytoplasmic calcium concentration is involved in 1,25D anti-apoptotic effects via Akt activation in ROS 17/2.8 cells and non-osteoblastic CV-1 cells. In both cases, 1,25D-stimulated elevation of intracellular calcium is due in part to activation of L-type Ca²⁺ channels. We conclude that 1,25D bone anabolic effects that involve increased intracellular Ca²⁺ concentration in osteoblasts can be explained at two levels. At the single-cell level, 1,25D promotes Ca²⁺-dependent exocytotic activities. At the tissue level, 1,25D protects osteoblasts from apoptosis via a Ca²⁺-dependent Akt pathway. Our studies contribute to the understanding of the molecular basis of bone diseases characterized by decreased bone formation and mineralization.     

Nongenomic regulation of protein kinase C isoforms by the vitamin D metabolites 1α,25-(OH)2D3 and 24R,25-(OH)2D3

Prior studies have shown that vitamin D regulation of protein kinase C activity (PKC) in the cell layer of chondrocyte cultures is cell maturation-dependent. In the present study, we examined the membrane distribution of PKC and whether 1α,25-(OH)₂D₃ and 24R,25-(OH)₂D₃ can directly regulate enzyme activity in isolated plasma membranes and extracellular matrix vesicles. Matrix vesicle PKC was activated by bryostatin-1 and inhibited by a PKC-specific pseudosubstrate inhibitor peptide. Depletion of membrane PKC activity using isoform-specific anti-PKC antibodies suggested that PKCα is the major isoform in cell layer lysates as well as in plasma membranes isolated from both cell types; PKCζ is the predominant form in matrix vesicles. This was confirmed in Western blots of immunoprecipitates as well as in studies using control peptides to block binding of the isoform specific antibody to the enzyme and using a PKCζ-specific pseudosubstrate inhibitor peptide. The presence of PKCζ in matrix vesicles was further verified by immunoelectron microscopy. Enzyme activity in the matrix vesicle was insensitive to exogenous lipid, whereas that in the plasma membrane required lipid for full activity. 1,25-(OH)₂D₃ and 24,25-(OH)₂D₃ inhibited matrix vesicle PKC, but stimulated plasma membrane PKC when added directly to the isolated membrane fractions. PKC activity in the matrix vesicle was calcium-independent, whereas that in the plasma membrane required calcium. Moreover, the vitamin D-sensitive PKC in matrix vesicles was not dependent on calcium, whereas the vitamin D-sensitive enzyme in plasma membranes was calcium-dependent. It is concluded that PKC isoforms are differentially distributed between matrix vesicles and plasma membranes and that enzyme activity is regulated in a membrane-specific manner. This suggests the existence of a nongenomic mechanism whereby the effects of 1,25-(OH)₂D₃ and 24,25-(OH)₂D₃ may be mediated via PKC. Further, PKCζ may be important in nongenomic, autocrine signal transduction at sites distal from the cell. © 1996 Wiley-Liss, Inc.     

A-ring analogues of 1,25-(OH)2D3 with low affinity for the vitamin D receptor modulate chondrocytes via membrane effects that are dependent on cell maturation

1,25-(OH)₂D₃ (1,25) and 24,25-(OH)₂D₃ (24,25) mediate their effects on chondrocytes through the classic vitamin D receptor (VDR) as well as through rapid membrane-mediated mechanisms, which result in both nongenomic and genomic effects. In intact cells, it is difficult to distinguish between genomic responses via the VDR and genomic and nongenomic responses via membrane-mediated pathways. In this study, we used two analogues of 1,25 that have been modified on the A-ring (2a, 2b) and are only 0.1% as effective in binding to the VDR as 1,25, to examine the role of the VDR in the response of rat costochondral resting zone (RC) and growth zone (GC) chondrocytes to 1,25 and 24,25. Chondrocyte proliferation ([³H]-thymidine incorporation), proteoglycan production ([³⁵S]-sulfate incorporation), and second messenger activation (activity of protein kinase C) were measured after treatment with 10^-8 M 1,25, 10^-7 M 24,25, or the analogues at 10^-9–10^-6 M. Both analogues inhibited proliferation of both cell types, as did 1,25 and 24,25. Neither 2a nor 2b had an effect on proteoglycan production by GCs or RCs. 2a caused a dose-dependent stimulation of protein kinase C (PKC) that was not inhibited by cycloheximide or actinomycin D in either GC or RC cells. 2b, on the other hand, had no effect on PKC activity in RCs and only a slight stimulatory effect in GCs. Both cells produce matrix vesicles, extracellular organelles associated with the initial stages of calcification, in culture that are regulated by vitamin D metabolites. Since these organelles contain no DNA or RNA, they provide an excellent model for studying the mechanisms used by vitamin D metabolites to mediate their nongenomic effects. When matrix vesicles were isolated from naive cultures of growth zone cells and treated with 2a, a dose-dependent inhibition of PKC activity was observed that was similar to that found with 1,25-(OH)₂D₃. Plasma membranes contained increased PKC activity after treatment with 2a, but the magnitude of the effect was less than that seen with 1,25-(OH)₂D₃. Analogue 2b had no affect on PKC activity in either membrane fraction. When matrix vesicles from resting zone chondrocyte cultures were treated with 24,25-(OH)₂D₃, a significant decrease in PKC activity was observed. No change in enzyme activity was found for either 1,25-(OH)₂D₃ or the analogues. PKC activity in the plasma membrane fraction, however, was increased by 24,25-(OH)₂D₃ as well as by analogue 2a. This study shows that these analogues, with little or no binding to the vitamin D receptor, can affect cell proliferation and PKC activity, but not proteoglycan production. The direct membrane effect is analogue specific and cell maturation dependent. Further, by eliminating the VDR-mediated component of the cellular response, we have provided further evidence for the existence of a membrane receptor(s) involved in mediating nongenomic effects of vitamin D metabolites. J. Cell. Physiol. 171:357–367, 1997. © 1997 Wiley-Liss, Inc.     

Increased PKC activity in cultured human keratinocytes and fibroblasts after treatment with 1α,25-dihydroxyvitamin D3

1α,25-Dihydroxyvitamin D₃ (10^?12 M to 10^?8 M) caused a dose dependent increase in PKC activity in the solubilized membrane fractions of cultured human keratinocytes and in the cytosolic fractions of cultured human fibroblasts. Maximum activity was induced by 1α,25-dihydroxyvitamin D₃ at 24 h. Sphingosine, which is believed to inhibit PKC mediated biological responses, blunted 1α,25(OH)₂D₃′s inducement of PKC activity in both keratinocytes and fibroblasts. Identical hormone treatment of vitamin D receptor deficient fibroblasts did not increase PKC activity. Treatment of keratinocytes and fibroblasts with 1β,25-dihydroxyvitamin D₃, which is believed to be ineffective in inducing genomic responses, did not induce PKC activity.     

Plasma membrane Pdia3 and VDR interact to elicit rapid responses to 1α,25(OH)2D3

1α,25-Dihydroxyvitamin D3 (1α,25(OH)₂D₃) regulates osteoblasts through genomic and rapid membrane-mediated responses. Here we examined the interaction of protein disulfide isomerase family A, member 3 (Pdia3) and the traditional vitamin D receptor (VDR) in plasma membrane-associated responses to 1α,25(OH)₂D₃. We found that Pdia3 co-localized with VDR and the caveolae scaffolding protein, caveolin-1 on the surface of MC3T3-E1 osteoblasts. Immunoprecipitation showed that both Pdia3 and VDR interacted with caveolin-1. Pdia3 further interacted with phospholipase A2 activating protein (PLAA), whereas VDR interacted with c-Src. 1α,25(OH)₂D₃ changed the interactions and transport of the two receptors and rapidly activated phospholipase A2 (PLA2) and c-Src. Silencing either receptor or caveolin-1 inhibited both PLA2 and c-Src, indicating that the two receptors function interdependently. These two receptor dependent rapid responses to 1α,25(OH)₂D₃ regulated gene expression, proliferation and apoptosis of MC3T3-E1 cells. These data demonstrate the importance of both receptors and caveolin-1 in mediating membrane responses to 1α,25(OH)₂D₃ and subsequently regulating osteoblast biology.     

Vitamin D3 regulation of stromelysin-1 (MMP-3) in chondrocyte cultures is mediated by protein kinase C

Matrix metalloproteinases (MMPs) are a group of enzymes with the potential to degrade extracellular matrix proteins. One of the MMPs, stromelysin-1 (MMP-3) has been localized to extracellular matrix vesicles in growth plate chondrocyte cultures, suggesting involvement of this enzyme in remodeling of the extracellular matrix during endochondral development, a process which is regulated by the vitamin D metabolites, 1,25-(OH)₂D₃ and 24,25-(OH)₂D₃. To determine whether stromelysin-1 is regulated by vitamin D as well, confluent cultures of cells derived from growth zone (GC) and resting zone (RC) rat costochondral cartilage were treated with 1α,25-(OH)₂D₃ (1,25) and 24R,25-(OH)₂D₃ (24,25), respectively, and the effect on stromelysin-1 assessed by casein gel zymography and Western blots. Although stromelysin-1 activity was enriched in the matrix vesicle fraction, only the plasma membrane enzyme was affected by the treatment; 1,25 and 24,25 caused a marked decrease in plasma membrane stromelysin-1 activity in their target cells. Since plasma membrane protein kinase C (PKC) activity is stimulated by 1,25 and 24,25, we hypothesized that stromelysin-1 activity was regulated by the vitamin D metabolites via PKC-dependent phosphorylation. To test this, membrane fractions (containing endogenous PKCα and ζ as well as stromelysin-1) were incubated in the presence of purified rat brain PKC and/or recombinant human (rh) stromelysin-1 and [γ³²P]-ATP and anti-stromelysin-1 immunoprecipitates were analyzed by autoradiography and Western blots. Immuno-phospho-stromelysin-1 was localized to a 52-kDa band in the plasma membrane fraction only; no phosphorylation was observed in the matrix vesicle fraction. Selective inhibitors of PKC activity demonstrated that phosphorylation was inhibited by H7 and low concentrations of H8, but not by HA1004, indicating that PKC, not PKA, was responsible. Protein phosphatase 2A, (PP2A), a serine/threonine-specific phosphatase, selectively removed the radiolabel in a time-dependent manner, providing further support for a PKC-dependent phosphorylation mechanism. Incubation of resting zone cell plasma membranes with 24,25, but not 1,25, resulted in phosphorylation of stromelysin-1, demonstrating that the nongenomic effect was metabolite-specific. This suggests that this may be one mechanism by which vitamin D metabolites regulate stromelysin-1 activity and that PKC-dependent phosphorylation inhibits the metalloproteinase. © 1996 Wiley-Liss, Inc.     

Nongenomic regulation of extracellular matrix events by vitamin D metabolites

Vitamin D metabolites appear to regulate chondrocytes and osteoblasts via a combination of genomic and nongenomic mechanisms. Specificity of the nongenomic response to either 1,25-(OH)₂D₃ or 24, 25-(OH)₂D₃ may be conferred by the chemical composition of the target membrane and its fluid mosaic structure, by the presence of specific membrane receptors, or by the interaction with classic Vitamin D receptors. Nongenomic effects have been shown to include changes in membrane fluidity, fatty acid acylation and reacylation, arachidonic acid metabolism and prostaglandin production, calcium ion flux, and protein kinaase C activity. Chondrocytes metabolize 25-(OH)D₃ to 1,25-(OH)₂D₃ and 24,25-(OH)₂D₃; production of these metabolites is regulated by both growth factors and hormones and is dependent on the state of cell maturation. 1,25-(OH)₂D₃ and 24,25-(OH)₂D₃ may interact directly with extracellular matix vesicles to regulate their function in the matrix, including protease activity, resulting in matrix modefication and calcification. Isolated matrix vesicles, produced by growth zone chondrocytes, can activate latent transforming growth factor-β when incubated with exogenous 1,25-(OH)₂D₃. These observations suggest that nongenomic regulation of martix vesicle structure and function may be a mechanism by which mesenchymal cells, like osteoblasts and chndrocytes, may modulate events in the extracellular matrix at sites distant from the cell surace.     

Hybrid structural analogues of 1,25-(OH)2D3 regulate chondrocyte proliferation and proteoglycan production as well as protein kinase C through a nongenomic pathway

1,25-(OH)₂D₃ and 24,25-(OH)₂D₃ mediate their effects on chondrocytes through the classic vitamin D receptor (VDR) as well as through rapid membrane-mediated mechanisms which result in both nongenomic and genomic effects. In intact cells, it is difficult to distinguish between genomic responses via the VDR and genomic and nongenomic responses via membrane-mediated pathways. In this study, we used two hybrid analogues of 1,25-(OH)₂D₃ which have been modified on the A-ring and C,D-ring side chain (1α-(hydroxymethyl)-3β-hydroxy-20-epi-22-oxa-26,27-dihomo vitamin D₃ (analogue MCW-YA = 3a) and 1β-(hydroxymethyl)-3α-hydroxy-20-epi-22-oxa-26,27-dihomo vitamin D₃ (analogue MCW-YB = 3b) to examine the role of the VDR in response of rat costochondral resting zone (RC) and growth zone (GC) chondrocytes to 1,25-(OH)₂D₃ and 24,25-(OH)₂D₃. These hybrid analogues are only 0.1% as effective in binding to the VDR from calf thymus as 1,25-(OH)₂D₃. Chondrocyte proliferation ([³H]-thymidine incorporation), proteoglycan production ([³⁵S]-sulfate incorporation), and activity of protein kinase C (PKC) were measured after treatment with 1,25-(OH)₂D₃, 24,25-(OH)₂D₃, or the analogues. Both analogues inhibited proliferation of both cell types, as did 1,25-(OH)₂D₃ and 24,25-(OH)₂D₃. Analogue 3a had no effect on proteoglycan production by GCs but increased that by RCs. Analogue 3b increased proteoglycan production in both GC and RC cultures. Both analogues stimulated PKC in GC cells; however, neither 3a nor 3b had an effect on PKC activity in RC cells. 1,25-(OH)₂D₃ and 3a decreased PKC in matrix vesicles from GC cultures, whereas plasma membrane PKC activity was increased, with 1,25-(OH)₂D₃ having a greater effect. 24,25-(OH)₂D₃ caused a significant decrease in PKC activity in matrix vesicles from RC cultures; 24,25-(OH)₂D₃, 3a, and 3b increased PKC activity in the plasma membrane fraction, however. Thus, with little or no binding to calf thymus VDR, 3a and 3b can affect cell proliferation, proteoglycan production, and PKC activity. The direct membrane effect is analogue-specific and cell maturation–dependent. By studying analogues with greatly reduced affinity for the VDR, we have provided further evidence for the existence of a membrane receptor(s) involved in mediating nongenomic effects of vitamin D metabolites. J. Cell. Biochem. 66:457–470, 1997. © 1997 Wiley-Liss, Inc.     

1α,25(OH)2-Vitamin D3 signaling in chick enterocytes: Enhancement of tyrosine phosphorylation and rapid stimulation of mitogen-activated protein (MAP) kinase

The steroid hormone 1α,25(OH)₂–vitamin D₃ (1α,25(OH)₂D₃) generates biological responses in intestinal and other cells via both genomic and rapid, nongenomic signal transduction pathways. We examined the hypothesis that 1α,25(OH)₂D₃ action in chick enterocytes may be linked to pathways involving tyrosine phosphorylation. Brief exposure of isolated chick enterocytes to 1α,25(OH)₂D₃ demonstrated increased tyrosine phosphorylation of several cellular proteins (antiphosphotyrosine immunoblots of whole cell lysates) with prominent bands at 42–44, 55–60, and 105–120 Kda. The 42–44 Kda bands comigrated with mitogen-activated protein (MAP) kinase (immunoblotting with anti-MAP kinase antibody) The response occurred within 30 s, peaked at 1 min, and was dose-dependent (0.01–10 nM), with maximal stimulation at 1 nM (three- to fivefold). This effect was specific for 1α,25(OH)₂D₃ since its metabolic precursors 25(OH)D₃and vitamin D₃ did not increase MAP kinase tyrosine phosphorylation. The tyrosine kinase inhibitor, genistein, blocked 1α,25(OH)₂D₃-induced tyrosine phosphorylation of MAP kinase, while staurosporine, a PKC inhibitor, attenuated the hormone's effects by 30%. We have evaluated the ability of 1α,25(OH)₂D₃ analogs, which have complete flexibility around the 6,7 carbon-carbon bond (6F) or which are locked in either the 6-s-cis (6C) or the 6-s-trans(6T) shape(s), to activate MAP kinase. Thus, two 6F and one 6C analog stimulated while one 6T analog did not stimulate MAP kinase tyrosine phosphorylation. In addition, 1β,25(OH)₂D₃, a known antagonist of 1α,25(OH)₂D₃-mediated rapid responses, blocked the hormone effects on MAP kinase. We conclude that 1α,25(OH)₂D₃ and analogs which can achieve the 6-s-cis shape (6F and 6C) can increase tyrosine phosphorylation and activation of MAP kinase in chick enterocytes. J. Cell. Biochem. 69:470–482, 1998. © 1998 Wiley-Liss, Inc.     

25-Hydroxyvitamin D3 is an agonistic vitamin D receptor ligand

25-Hydroxyvitamin D₃ 1α-hydroxylase encoded by CYP27B1 converts 25-hydroxyvitamin D₃ into 1α,25-dihydroxyvitamin D₃, a vitamin D receptor ligand. 25-Hydroxyvitamin D₃ has been regarded as a prohormone. Using Cyp27b1 knockout cells and a 1α-hydroxylase-specific inhibitor we provide in four cellular systems, primary mouse kidney, skin, prostate cells and human MCF-7 breast cancer cells, evidence that 25-hydroxyvitamin D₃ has direct gene regulatory properties. The high expression of megalin, involved in 25-hydroxyvitamin D₃ internalisation, in Cyp27b1^?/? cells explains their higher sensitivity to 25-hydroxyvitamin D₃. 25-Hydroxyvitamin D₃ action depends on the vitamin D receptor signalling supported by the unresponsiveness of the vitamin D receptor knockout cells. Molecular dynamics simulations show the identical binding mode for both 25-hydroxyvitamin D₃ and 1α,25-dihydroxyvitamin D₃ with the larger volume of the ligand-binding pocket for 25-hydroxyvitamin D₃. Furthermore, we demonstrate direct anti-proliferative effects of 25-hydroxyvitamin D₃ in human LNCaP prostate cancer cells. The synergistic effect of 25-hydroxyvitamin D₃ with 1α,25-dihydroxyvitamin D₃ in Cyp27b1^?/? cells further demonstrates the agonistic action of 25-hydroxyvitamin D₃ and suggests that a synergism between 25-hydroxyvitamin D₃ and 1α,25-dihydroxyvitamin D₃ might be physiologically important. In conclusion, 25-hydroxyvitamin D₃ is an agonistic vitamin D receptor ligand with gene regulatory and anti-proliferative properties.     

The classic receptor for 1alpha,25-dihydroxy vitamin D3 is required for non-genomic actions of 1alpha,25-dihydroxy vitamin D3 in osteosarcoma cells

1alpha,25-dihydroxy vitamin D3 has a major role in the regulation of the bone metabolism as it promotes the expression of key bone-related proteins in osteoblastic cells. In recent years it has become increasingly evident that in addition to its well-established genomic actions, 1alpha,25-dihydroxy vitamin D3 induces non-genomic responses by acting through a specific plasma membrane-associated receptor. Results from several groups suggest that the classical nuclear 1alpha,25-dihydroxy vitamin D3 receptor (VDR) is also responsible for these non-genomic actions of 1alpha,25-dihydroxy vitamin D3. Here, we have used siRNA to suppress the expression of VDR in osteoblastic cells and assessed the role of VDR in the non-genomic response to 1alpha,25-dihydroxy vitamin D3. We report that expression of the classic VDR in osteoblasts is required to generate a rapid 1alpha,25-dihydroxy vitamin D3-mediated increase in the intracellular Ca(2+) concentration, a hallmark of the non-genomic actions of 1alpha,25-dihydroxy vitamin D3 in these cells.     

Synthesis and biological activity of 22-iodo- and (E)-20(22)-dehydro analogues of 1α,25-dihydroxyvitamin D3

Construction of 25-hydroxy-steroidal side chain substituted with iodine at C-22 was elaborated on a model PTAD-protected steroidal 5,7-diene and applied to a synthesis of (22R)- and (22S)-22-iodo-1α,25-dihydroxyvitamin D₃. Configuration at C-22 in the iodinated vitamins, obtained by nucleophilic substitution of the corresponding 22S-tosylates with sodium iodide, was determined by comparison of their iodine-displacement processes and cyclizations leading to isomeric five-membered (22,25)-epoxy-1α-hydroxyvitamin D₃ compounds. Also, 20(22)-dehydrosteroids have been obtained and their structures established by ¹H NMR spectroscopy. When compared to the natural hormone, (E)-20(22)-dehydro-1α,25-dihydroxyvitamin D₃ was found 4 times less potent in binding to the porcine intestinal vitamin D receptor (VDR) and 2 times less effective in differentiation of HL-60 cells. 22-Iodinated vitamin D analogues showed somewhat lower in vitro activity, whereas (22,25)-epoxy analogues were inactive. Interestingly, it was established that (22S)-22-iodo-1α,25-dihydroxyvitamin D₃ was 3 times more potent than its (22R)-isomer in binding to VDR and four times more effective in HL-60 cell differentiation assay. The restricted mobility of the side chain of both 22-iodinated vitamin D compounds was analyzed by a systematic conformational search indicating different spatial regions occupied by their 25-oxygen atoms. Preliminary data on the in vivo calcemic activity of the synthesized vitamin D analogues indicate that (E)-20(22)-dehydro-1α,25-dihydroxyvitamin D₃ and 22-iodo-1α,25-dihydroxyvitamin D₃ isomers were ca. ten times less potent than the natural hormone 1α,25-(OH)₂D₃ both in intestinal calcium transport and bone calcium mobilization.