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1.
The mechanisms that control fibroproliferation and matrix deposition in lung fibrosis remain unclear. We speculate that vitamin D deficiency may contribute to pulmonary fibrosis since vitamin D deficiency has been implicated in several diseases. First, we confirmed the presence of vitamin D receptors (VDRs) in cultured NIH/3T3 and lung fibroblasts. Fibroblasts transfected with a vitamin D response element–reporter construct and exposed to the active vitamin D metabolite, 1,25(OH)2D3, showed increased promoter activity indicating VDR functionality in these cells. Testing the effects of 1,25(OH)2D3 on fibroblasts treated with transforming growth factor β1 (TGFβ1), considered a driver of many fibrotic disorders, we found that 1,25(OH)2D3 inhibited TGFβ1-induced fibroblast proliferation in a dose-dependent fashion. 1,25(OH)2D3 also inhibited TGFβ1 stimulation of α-smooth muscle actin expression and polymerization and prevented the upregulation of fibronectin and collagen in TGFβ1-treated fibroblasts. Finally, we examined how 1,25(OH)2D3 affects epithelial–mesenchymal transformation of lung epithelial cells upon exposure to TGFβ1. We showed that the TGFβ1-induced upregulation of mesenchymal cell markers and abnormal expression of epithelial cell markers were blunted by 1,25(OH)2D3. These observations suggest that under TGFβ1 stimulation, 1,25(OH)2D3 inhibits the pro-fibrotic phenotype of lung fibroblasts and epithelial cells.  相似文献   

2.
Breast cancer is the most common non-cutaneous malignancy in American women, and better preventative strategies are needed. Epidemiological and laboratory studies point to vitamin D3 as a promising chemopreventative agent for breast cancer. Vitamin D3 metabolites induce anti-proliferative effects in breast cancer cells in vitro and in vivo, but few studies have investigated their effects in normal mammary epithelial cells. We hypothesized that 1,25(OH)2D3, the metabolically active form of vitamin D3, is growth suppressive in normal mouse mammary epithelial cells. In addition, we have previously established a role for the cytokine interleukin-1 alpha (IL1α) in the anti-proliferative effects of 1,25(OH)2D3 in normal prostate cells, and so we hypothesized that IL1α is involved in the 1,25(OH)2D3 response in mammary cells. Evaluation of cell viability, clonogenicity, senescence, and induction of cell cycle regulators p21 and p27 supported an anti-proliferative role for 1,25(OH)2D3 in mammary epithelial cells. Furthermore, 1,25(OH)2D3 increased the intracellular expression of IL1α, which was necessary for the anti-proliferative effects of 1,25(OH)2D3 in mammary cells. Together, these findings support the chemopreventative potential of vitamin D3 in the mammary gland and present a role for IL1α in regulation of mammary cell proliferation by 1,25(OH)2D3.  相似文献   

3.
Summary After injection of radiolabeled 1,25 (OH)2 vitamin D3, nuclear concentration of radioactivity is observed in parenchymal cells of the parathyroid gland in pregnant, adult male, and 10-day male neonatal rats. In competition studies with unlabeled 1,25 (OH)2 vitamin D3, but not with 25 (OH) vitamin D3, nuclear uptake is prevented. Experiments with 3H 25 (OH) vitamin D3, in contrast to 3H 1,25 (OH)2 vitamin D3, do not show nuclear concentration in cells of the parathyroid. The results of the autoradiographic studies suggest the presence of receptors for a direct effect of 1,25 (OH)2 vitamin D3 on the parathyroid gland for modulation of parathyroid hormone secretion.  相似文献   

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Understanding the regulation of airway epithelial barrier function is a new frontier in asthma and respiratory viral infections. Despite recent progress, little is known about how respiratory syncytial virus (RSV) acts at mucosal sites, and very little is known about its ability to influence airway epithelial barrier function. Here, we studied the effect of RSV infection on the airway epithelial barrier using model epithelia. 16HBE14o- bronchial epithelial cells were grown on Transwell inserts and infected with RSV strain A2. We analyzed (i) epithelial apical junction complex (AJC) function, measuring transepithelial electrical resistance (TEER) and permeability to fluorescein isothiocyanate (FITC)-conjugated dextran, and (ii) AJC structure using immunofluorescent staining. Cells were pretreated or not with protein kinase D (PKD) inhibitors. UV-irradiated RSV served as a negative control. RSV infection led to a significant reduction in TEER and increase in permeability. Additionally it caused disruption of the AJC and remodeling of the apical actin cytoskeleton. Pretreatment with two structurally unrelated PKD inhibitors markedly attenuated RSV-induced effects. RSV induced phosphorylation of the actin binding protein cortactin in a PKD-dependent manner. UV-inactivated RSV had no effect on AJC function or structure. Our results suggest that RSV-induced airway epithelial barrier disruption involves PKD-dependent actin cytoskeletal remodeling, possibly dependent on cortactin activation. Defining the mechanisms by which RSV disrupts epithelial structure and function should enhance our understanding of the association between respiratory viral infections, airway inflammation, and allergen sensitization. Impaired barrier function may open a potential new therapeutic target for RSV-mediated lung diseases.  相似文献   

7.
Summary Autoradiographic studies were conducted to identify and characterize target cells for 1,25(OH)2 vitamin D3 in the pyloric region of rats and mice. After injection of 3H 1,25(OH)2 vitamin D3, nuclear concentration of radioactivity was observed in nuclei of duodenal epithelium and certain cells of pyloric glands, while most of the epithelial cells in the pyloric and gastric glands did not show nuclear labeling. In combined immunohistochemical studies, cells in the pyloric glands that showed nuclear concentration of radioactivity, were stained in their cytoplasm with antibodies to gastrin. Also, cells of the pyloric sphincter muscle showed nuclear labeling, in contrast to cells of the duodenal muscularis, which remained unlabeled under the conditions of the experiments. The results indicate that the cells with nuclear radioactivity contain receptors for 1,25(OH)2 vitamin D3 and suggest that gastrin secretion and pyloric muscle functions are regulated by a direct action of 1,25(OH)2 vitamin D3 on these cells.  相似文献   

8.
Maintaining blood-brain barrier integrity and minimizing neuronal injury are critical components of any therapeutic intervention following ischemic stroke. However, a low level of vitamin D hormone is a risk factor for many vascular diseases including stroke. The neuroprotective effects of 1,25(OH)2D3 (vitamin D) after ischemic stroke have been studied, but it is not known whether it prevents ischemic injury to brain endothelial cells, a key component of the neurovascular unit. We analyzed the effect of 1,25(OH)2D3 on brain endothelial cell barrier integrity and tight junction proteins after hypoxia/reoxygenation in a mouse brain endothelial cell culture model that closely mimics many of the features of the blood-brain barrier in vitro. Following hypoxic injury in bEnd.3 cells, 1,25(OH)2D3 treatment prevented the decrease in barrier function as measured by transendothelial electrical resistance and permeability of FITC-dextran (40 kDa), the decrease in the expression of the tight junction proteins zonula occludin-1, claudin-5, and occludin, the activation of NF—kB, and the increase in matrix metalloproteinase-9 expression. These responses were blocked when the interaction of 1,25(OH) )2D3 with the vitamin D receptor (VDR) was inhibited by pyridoxal 5’-phosphate treatment. Our findings show a direct, VDR-mediated, protective effect of 1,25(OH) )2D3 against ischemic injury-induced blood-brain barrier dysfunction in cerebral endothelial cells.  相似文献   

9.
Summary After injection of 3H 1,25(OH)2 vitamin D3 to rats fed a vitamin D-deficient diet, nuclear concentration and retention of radioactivity exists in reticular cells of the thymus medulla and cortex, as well as outer cells of developing Hassal's corpuscles. Lymphocytes do not show nuclear concentration of radioactivity. Nuclear concentration in reticular cells is prevented by prior injection of excess 1,25(OH)2 vitamin D3. The results indicate that reticular-endothelial cells contain nuclear receptors for 1,25(OH)2 vitamin D3 and suggest that effects of 1,25(OH)2 vitamin D3 on immune response and lymphocyte differentiation are indirect and mediated through genomic modulation of reticular cell functions such as messenger secretion.  相似文献   

10.
Summary Rats and mice fed a vitamin D-deficient or vitamin D-complete diet were injected with 3H 1,25 (OH)2 vitamin D3. Autoradiograms prepared from cross sections through the neck region revealed nuclear concentration of radioactivity strongest in parathyroid chief cells, occasionally in thyroid follicular epithelial and interfollicular cells, in the epithelium of tubular remnants of the ultimobranchial body, in epithelium of the esophagus, in chondrocytes of tracheal cartilage, and in myoepithelial cells of tracheal glands. In the thyroid, most of the follicle epithelial cells did not show nuclear concentration of radioactivity which occurred only occasionally and predominantly in follicles located in marginal positions. Thyroglobulin in lumina of thyroid follicles contained varying amounts of radioactivity that correspond to the diameter of the follicles, with relatively high amounts in large follicles and little or no radioactivity in small follicles. Competition with excess of unlabeled 1,25 (OH)2 vitamin D3 abolished nuclear radioactivity, but not the radioactivity in the colloid, while 25 (OH) vitamin D3 did not affect either. When a combination of autoradiography and immunohistochemistry was applied, follicular and parafollicular C-cells positive for calcitonin antibodies, did not show nuclear concentration of radioactivity. Tubular remnants of ultimobranchial bodies, however, showed distinct nuclear labeling, but did not stain, or only weakly stain, with antibodies to calcitonin. When 3H 25 (OH) vitamin D3 was injected, no nuclear concentration of radioactivity was noted in any of the tissues.The results from these histochemical studies suggest the existence of nuclear receptors and direct genomic effects of 1,25 (OH)2 vitamin D3 in heterogeneous tissues of the neck region, which include parathyroid chief cells, myoepithelial cells of tracheal glands, chondrocytes of tracheal cartilage, epithelial cells of esophagus, and certain thyroid follicle epithelial cells. No evidence could be obtained for nuclear receptors in C-cells and cells of striated muscle.  相似文献   

11.
Summary Evidence from results of autoradiographic studies in mice indicates that nuclei of Sertoli cells and of epithelial cells in the ductuli efferentes contain receptors for 1,25(OH)2 vitamin D3.  相似文献   

12.
In addition to classical roles in calcium homeostasis and bone development, 1,25 dihydroxyvitamin D3 [1,25(OH)2D3] inhibits the growth of several cancer types, including breast cancer. Although cellular effects of 1,25(OH)2D3 traditionally have been attributed to activation of a nuclear vitamin D receptor (VDR), a novel receptor for 1,25(OH)2D3 called 1,25D3-MARRS (membrane-associated, rapid response steroid-binding) protein was identified recently. The purpose of this study was to determine if the level of 1,25D3-MARRS expression modulates 1,25(OH)2D3 activity in breast cancer cells.Relative levels of 1,25D3-MARRS protein in MCF-7, MDA MB 231, and MCF-10A cells were estimated by real-time RT-PCR and Western blotting. To determine if 1,25D3-MARRS receptor was involved in the growth inhibitory effects of 1,25(OH)2D3 in MCF-7 cells, a ribozyme construct designed to knock down 1,25D3-MARRS mRNA was stably transfected into MCF-7 cells. MCF-7 clones in which 1,25D3-MARRS receptor expression was reduced showed increased sensitivity to 1,25(OH)2D3 ( IC50 56 ± 24 nM) compared to controls (319 ± 181 nM; P < 0.05). Reduction in 1,25D3-MARRS receptor lengthened the doubling time in transfectants treated with 1,25(OH)2D3. Knockdown of 1,25D3-MARRS receptor also increased the sensitivity of MCF-7 cells to the vitamin D analogs KH1060 and MC903, but not to unrelated agents (all-trans retinoic acid, paclitaxel, serum/glucose starvation, or the isoflavone, pomiferin). These results suggest that 1,25D3-MARRS receptor expression interferes with the growth inhibitory activity of 1,25(OH)2D3 in breast cancer cells, possibly through the nuclear VDR. Further research should examine the potential for pharmacological or natural agents that modify 1,25D3-MARRS expression or activity as anticancer agents.  相似文献   

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The role of 1,25(OH)2D3 on the intestinal NCX activity was studied in vitamin D-deficient chicks (-D) as well as the hormone effect on NCX1 protein and gene expression and the potential molecular mechanisms underlying the responses. Normal, -D and -D chicks treated with cholecalciferol or 1,25(OH)2D3 were employed. In some experiments, -D chicks were injected with cycloheximide or with cycloheximide and 1,25(OH)2D3 simultaneously. NCX activity was decreased by -D diet, returning to normal values after 50 IU daily of cholecalciferol/10 days or a dose of 1 μg calcitriol/kg of b.w. for 15 h. Cycloheximide blocked NCX activity enhancement produced by 1,25(OH)2D3. NCX1 protein and gene expression were diminished by -D diet and enhanced by 1,25(OH)2D3. Vitamin D receptor expression was decreased by -D diet, effect that disappeared after 1,25(OH)2D3 treatment. Rapid effects of 1,25(OH)2D3 on intestinal NCX activity were also demonstrated. The abolition of the rapid effects through addition of Rp-cAMPS and staurosporine suggests that non genomic effects of 1,25(OH)2D3 on NCX activity are mediated by activation of PKA and PKC pathways. In conclusion, 1,25(OH)2D3 enhances the intestinal NCX activity in -D chicks through genomic and non genomic mechanisms.  相似文献   

15.
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.  相似文献   

16.
We have crossed ERp57flx/flx mice with commercially available mice expressing villin-driven cre-recombinase. Lysates of intestinal epithelial cells were prepared from knock-out (KO) mice and littermates (LM) and used in Western blot analyses with Ab099 against the N terminus of the 1,25D3-MARRS (membrane-associated, rapid response steroid-binding) receptor: LM mice exhibited one positive band, which was absent in preparations from KO mice. Saturation analyses of cell lysates with [3H]1,25D3 revealed negligible binding in preparations from either female or male KOs. Lysates from female and male LM mice had similar affinities but different numbers of binding sites. Isolated enterocytes were tested for steroid-stimulated calcium uptake. Treatment of cells from female or male LM mice with 1,25D3 elicited enhanced calcium uptake in females and males within 5 min. Intestinal cells from KO mice exhibited a severely blunted or completely absent response to hormone. Confocal microscopy of intestinal cells revealed the presence of cell surface vitamin D receptors. However, antibodies to the vitamin D receptor failed to block 1,25D3-stimulated calcium uptake. In chick enterocytes we have found that the PKA pathway mediates calcium uptake. The time course for activation of PKA in mouse enterocytes paralleled that for enhanced calcium uptake and for LM females reached 250% of controls within 5 min, and 150% of controls in cells prepared from LM males. Enterocytes from female or male KO mice failed to exhibit steroid hormone-stimulated PKA activity, but did respond to forskolin with enhanced calcium uptake. We conclude that the 1,25D3-MARRS receptor is of central importance to steroid hormone-stimulated calcium uptake in mammalian intestinal cells.  相似文献   

17.
In mice, respiratory syncytial virus (RSV) infection enhances allergic airway sensitization, resulting in lung eosinophilia and in airway hyperresponsiveness (AHR). The mechanisms by which RSV contributes to development of asthma and its effects on allergic airway sensitization in mice are not known. We tested whether these consequences of RSV infection can be adoptively transferred by T cells and whether depletion of T cell subsets prevents the effects of RSV infection on subsequent airway sensitization. Mononuclear cells, T lymphocytes, or CD4 or CD8 T cells from peribronchial lymph nodes (PBLN) of RSV-infected mice were transferred into naive BALB/c mice which were then exposed to OVA via the airways. Additionally, RSV-infected mice were depleted of CD4 or CD8 T cells following acute RSV infection but prior to airway sensitization. Following sensitization, airway responsiveness to inhaled methacholine, numbers of lung eosinophils, and levels of IFN-gamma, IL-4, and IL-5 in PBLN cell cultures were monitored. Transfer of T cells from RSV-infected mice resulted in increased eosinophil influx into the lungs, increased IL-5 production, and development of AHR following airway sensitization to allergen. Transfer of CD8 but not CD4 T cells from the PBLN of RSV-infected mice also resulted in AHR following 10 days of OVA exposure. Further, depletion of CD8 T cells prevented these consequences of RSV infection while CD4 T cell depletion reduced them. We conclude that T cells, in particular CD8 T cells, are critical in mediating RSV-induced development of lung eosinophilia and AHR following allergic airway sensitization.  相似文献   

18.
The active form of vitamin D3, 1,25(OH)2D3, has significant immunomodulatory properties and is an important determinant in the differentiation of CD4+ effector T cells. The biological actions of 1,25(OH)2D3 are mediated by the vitamin D receptor (VDR) and are believed to correlate with the VDR protein expression level in a given cell. The aim of this study was to determine if and how 1,25(OH)2D3 by itself regulates VDR expression in human CD4+ T cells. We found that activated CD4+ T cells have the capacity to convert the inactive 25(OH)D3 to the active 1,25(OH)2D3 that subsequently up-regulates VDR protein expression approximately 2-fold. 1,25(OH)2D3 does not increase VDR mRNA expression but increases the half-life of the VDR protein in activated CD4+ T cells. Furthermore, 1,25(OH)2D3 induces a significant intracellular redistribution of the VDR. We show that 1,25(OH)2D3 stabilizes the VDR by protecting it from proteasomal degradation. Finally, we demonstrate that proteasome inhibition leads to up-regulation of VDR protein expression and increases 1,25(OH)2D3-induced gene activation. In conclusion, our study shows that activated CD4+ T cells can produce 1,25(OH)2D3, and that 1,25(OH)2D3 induces a 2-fold up-regulation of the VDR protein expression in activated CD4+ T cells by protecting the VDR against proteasomal degradation.  相似文献   

19.
Our laboratory has recently demonstrated that 1,25-dihydroxyvitamin D3(1,25(OH)2D3) rapidly stimulated membrane polyphosphoinositide breakdown and increased intracellular calcium, as well as activated protein kinase C (PKC) in vitamin D-sufficient rat colonocytes. These effects of 1,25(OH)2D3 were, however, lost in vitamin D-insufficient rats and restored by the in vivo repletion of 1,25(OH)2D3. In the present studies we have examined the ability of 1,25(OH)2D3 to stimulate the phosphorylation of colonic membrane proteins in intact D-sufficient cells. In addition, we investigated the effects of vitamin D status on the phosphorylation of these membrane proteins in broken cell preparations. These studies demonstrated that 1,25(OH)2D3 increased the phosphorylation of at least two colonic membrane proteins with apparent molecular weights of 42,000 (pp42) and 48,000 (pp48) in intact cells of vitamin D-sufficient rats. Moreover, in vitamin D-sufficient rats, treatment of colonocytes with 1,25(OH)2D3 or 12-Otertradecanoyl phorbol 13-acetate (TPA), a known activator of PKC, significantly increased the phosphorylation of pp42 and pp48 in broken cell preparations. The kinetics of these phosphorylations in response to 1,25(OH)2D3 were both rapid and transient. In addition, PKC19–36, a specific PKC inhibitor, decreased the phosphorylation of pp42 and pp48, whereas okadaic acid (OA), a type 1 and 2A protein phosphatase inhibitor, further augmented their phosphorylation in response to 1,25(OH)2D3. The isoelectric points of pp42 and pp48 were 5.79 and 5.97, respectively, and both were predominantly phosphorylated on threonine residues. In contrast to our findings in colonocytes from vitamin D-sufficient animals, basal phosphorylation of pp42 and pp48 were increased in membranes prepared from vitamin D-insufficient rats. Moreover, these phosphorylations failed to change in response to 1,25(OH)2D3-treatment of colonocytes from vitamin D-insufficient rats. The basal phosphorylation of each of these proteins was restored to control levels, as was their ability to respond to the direct addition of 1,25(OH)2D3 following the in vivo repletion of vitamin D-insufficient rats with this secosteroid. In summary, we have identified two acidic membrane proteins from rat colonocytes that are phosphorylated in both intact and broken cell preparations in response to 1,25(OH)2D3 treatment, an event modulated by vitamin D status and mediated, at least in part, by PKC. © 1995 Wiley-Liss, Inc.  相似文献   

20.
Of the various risk factors contributing to osteoporosis, dietary/lifestyle factors are important. In a clinical study we reported that women with caffeine intakes >300 mg/day had higher bone loss and women with vitamin D receptor (VDR) variant, tt were at a greater risk for this deleterious effect of caffeine. However, the mechanism of how caffeine effects bone metabolism is not clear. 1,25-Dihydroxy vitamin D3 (1,25(OH)2D3) plays a critical role in regulating bone metabolism. The receptor for 1,25(OH)2D3, VDR has been demonstrated in osteoblast cells and it belongs to the superfamily of nuclear hormone receptors. To understand the molecular mechanism of the role of caffeine in relation to bone, we tested the effect of caffeine on VDR expression and 1,25(OH)2D3 mediated actions in bone. We therefore examined the effect of different doses of caffeine (0.2, 0.5, 1.0 and 10 mM) on 1,25(OH)2D3 induced VDR protein expression in human osteoblast cells. We also tested the effect of different doses of caffeine on 1,25(OH)2D3 induced alkaline phosphatase (ALP) activity, a widely used marker of osteoblastic activity. Caffeine dose dependently decreased the 1,25(OH)2D3 induced VDR expression and at concentrations of 1 and 10 mM, VDR expression was decreased by about 50–70%, respectively. In addition, the 1,25(OH)2D3 induced alkaline phosphatase activity was also reduced at similar doses thus affecting the osteoblastic function. The basal ALP activity was not affected with increasing doses of caffeine. Overall, our results suggest that caffeine affects 1,25(OH)2D3 stimulated VDR protein expression and 1,25(OH)2D3 mediated actions in human osteoblast cells.  相似文献   

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