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PubMed1990

1,25(OH)2 vitamin D3 stimulates membrane phosphoinositide turnover, activates protein kinase C, and increases cytosolic calcium in rat colonic epithelium.

The hormonal form of vitamin D, 1,25(OH)2 vitamin D3 [1,25(OH)2D3], regulates colonic calcium absorption and colonocyte proliferation and differentiation. In this study, we have examined the effect of 1,25(OH)2D3 on membrane phosphoinositide turnover, protein kinase C activation, and regulation of intracellular calcium concentration [( Ca+2]i) in isolated rat colonic epithelium. In a concentration-dependent manner, 1,25(OH)2D3 stimulated breakdown of membrane phosphoinositides within 15 s, generating diacylglycerol and inositol 1,4,5-triphosphate (IP3). 1,25(OH)2D3 rapidly activated colonic protein kinase C, with maximal translocation of activity from the cytosol to the membrane occurring within 1 min of exposure to the secosteroid. Studies performed in isolated colonocytes with the fluorescent dye fura-2 demonstrated that 10(-8) M 1,25(OH)2D3 caused a rapid rise in [Ca+2]i which then transiently decreased before rising to a new plateau value. When these experiments were performed in a calcium-free buffer, an increase in [Ca+2]i was observed, but both the transient and secondary rise were diminished in magnitude, suggesting that 1,25(OH)2D3 may stimulate both release of intracellular calcium stores and calcium influx. 1,25(OH)2D3 stimulated [3H]thymidine uptake in rat colonocytes, 4 h after an in vivo injection. These studies indicate that 1,25(OH)2D3 exerts a rapid influence on membrane phosphoinositide metabolism which may mediate certain of the secosteroid's effects on colonocyte calcium transport and proliferation.

Animals↗
PubMed2001

Activation of latent transforming growth factor beta1 by stromelysin 1 in extracts of growth plate chondrocyte-derived matrix vesicles.

Previous studies have shown that matrix vesicles isolated from cultures of costochondral growth zone chondrocytes and treated with 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3] can activate recombinant human latent transforming growth factor beta1 (rhTGF-beta1). It is unknown what enzyme or other factor in the extracellular organelles is responsible for the activation. This study tested the hypothesis that enzymes present in matrix vesicles can activate latent TGF-beta1 and that this is regulated by 1alpha,25(OH)2D3. To do this, we examined the ability of matrix vesicle extracts to activate small latent rhTGF-beta1. In addition, enzymes previously determined to be present in matrix vesicles were screened for their ability to activate small latent rhTGF-beta1. Recombinant human matrix metalloproteinase 2 (rhMMP-2; 72 kDa gelatinase), rhMMP-3 (stromelysin 1), purified human plasminogen, and purified urokinase (plasminogen activator) were each tested at varying concentrations. To assess the role of cell maturation, we used a cell culture model in which chondrocytes are derived from two distinct zones of rat costochondral cartilage, the resting zone and the growth zone. Matrix vesicles were isolated from these cultures and then tested. The results showed that extracts of matrix vesicles produced by both growth zone and resting zone chondrocytes were able to activate small latent rhTGF-beta1. The effects were dose and time dependent, with greater activity being found in extracts of matrix vesicles from the growth zone chondrocyte cultures. Only rhMMP-3 was able to activate small latent rhTGF-beta1, indicating that stromelysin-1, but not MMP-2, plasminogen, or urokinase, was involved. As observed in the extracts, the effect of rhMMP-3 was time and dose dependent. When anti-MMP-3 antibody was added to matrix vesicle extracts from both cell types, activation of small latent rhTGF-beta1 was dose-dependently blocked. Neither 1alpha,25(OH)2D3 nor 24R,25(OH)2D3 had a direct effect on activation of small latent rhTGF-beta1 by the extracts. However, when intact matrix vesicles were treated with 1alpha,25(OH)2D3, their ability to activate small latent rhTGF-beta1 was increased. Inhibition of phospholipase A2 with quinacrine blocked the 1alpha,25(OH)2D3-dependent effect. These results suggest that the ability of 1alpha,25(OH)2D3-treated matrix vesicles to activate small latent TGF-beta1 is via action of the secosteroid on the matrix vesicle membrane, not on the enzymes responsible for activating latent TGF-beta1. Because matrix vesicles isolated from growth zone chondrocytes have been shown to contain increased phospholipase A2 activity after treatment with 1alpha,25(OH)2D3, it is likely that this secosteroid promotes loss of membrane integrity through phospholipase A2-dependent formation of lysophospholipids, resulting in the release of MMP-3 into the matrix, where latent TGF-beta1 is stored. Taken together, the results of the current study show that matrix vesicles produced by growth plate chondrocytes contain MMP-3, that this enzyme is at least partially responsible for activation of small latent TGF-beta1 in the matrix, and that 1alpha,25(OH)2D3 regulates MMP release from matrix vesicles.

Animals↗
PubMed2005

Sphingosine 1-phosphate is involved in cytoprotective actions of calcitriol in human fibroblasts and enhances the intracellular Bcl-2/Bax rheostat.

Calcitriol is originally known to decrease proliferation rates of several carcinoma cells, partly via induction of apoptosis. On the other hand, the secosteroid is revealed to protect some cell types like thyrocytes, HL-60 cells and melanocytes against programmed cell death. Here we report that calcitriol despite its strong antiproliferative effect on human dermal fibroblasts did not induce apoptosis in these cells. In contrast, calcitriol possessed an antiapoptotic action in dermal fibroblasts. Thus, the ability of the apoptotic stimuli TNFalpha/actinomycin and C2-ceramides (C2-Cer) to induce programmed cell death was drastically diminished in the presence of calcitriol. Moreover, we identified sphingosine 1-phosphate (S1P) as a downstream mediator of calcitriol for its cytoprotective property. Thus, the secosteroid could not protect fibroblasts from apoptosis in the presence of N,N-dimethylsphingosine (DMS), which inhibits sphingosine kinase, the crucial enzyme to form S1P. Like calcitriol, S1P in different concentrations did not induce fibroblast apoptosis and moreover drastically decreased the rates of apoptotic cells after treatment with TNFalpha1/actinomycin. As S1P has been identified to modify the Bcl-2/ Bax ratio in epithelial cells and keratinocytes, we also measured the expression of these proteins in dermal fibroblasts revealing an increased Bcl-2 level after stimulation with S1P while the Bax protein expression was not modified. In conclusion, calcitriol H was revealed to protect human fibroblasts from apoptosis by formation of S1P resulting in a changed Bcl-2/Bax ratio.

Annexin A5↗
PubMed1988

Liver extraction of vitamin D3 is independent of its hepatic venous or arterial route of delivery. Studies in isolated-perfused rat liver preparations.

The hepatic extraction of a naturally occurring secosteroid, vitamin D3 (D3), in relation to its hepatic arterial or portal venous route of delivery has been studied in isolated rat liver preparations perfused at an arterial/venous flow ratio of 1:4. No significant difference in the fractional hepatic extraction of D3 was observed when the vitamin was administered via the portal venous route compared to when it was administered via the hepatic arterial route. Estimation of the uptake and clearance of D3 in relation to its route of delivery revealed, however, that due to the higher perfusion flow through the portal venous than through the arterial route, both the hepatic uptake and clearance of D3 were significantly higher after portal vein than after hepatic artery delivery. Moreover, calculation of the uptake of D3 after delivery through the portal venous route also revealed that it was not significantly different from that of the total hepatic uptake (uptake following portal vein + hepatic artery delivery). The data obtained during the present studies indicate, then, that the fractional hepatic extraction of D3 is not dependent on its route of entry into the liver; it also points out that, in experimental models such as in isolated-perfused liver preparations, the portal vein administration of D3 should represent adequately the total hepatic handling of the secosteroid by the normal rat liver.

Animals↗
PubMed1991

Gas chromatography-mass spectrometry in the investigation of on-column dehydration of steroid hormones during gas-liquid chromatography.

Some underivatized steroids when injected onto conventional packed columns for gas-liquid chromatography underwent varying degrees of dehydration. This problem was traced to the presence of small pieces of broken glass on the top of the column at the point of injection. This observation provoked an examination of the effect of pre-column dehydration on a number of different types of steroids. Powdered aluminium was placed in the injection liner of a Hewlett-Packard gas chromatograph fitted with an HP1 capillary column connected to a mass selective detector, and injections were made using a new high temperature septumless injection system at temperatures between 200 and 400 degrees C. 5 alpha-androstan-3 alpha-ol, a simple monofunctional C19 steroid chosen as a model to establish optimum conditions, underwent dehydration at injection temperatures greater than 250 degrees C and the product reached a maximum at 400 degrees C when no unchanged steroid was present. Monohydroxylated androgens and oestrogens underwent dehydration at 400 degrees C producing products whose mass spectra indicated they were monenes, although the position of the double bond could not be assigned. Polyfunctional androgens and oestrogens and corticosteroids underwent complex changes producing a number of products some of whose structures could not be determined. The dehydration products had the advantage that they had relatively intense high mass ions and for suitable steroids this might provide enhanced sensitivity of detection during mass fragmentography. In such cases dehydration was reproducible and straight line standard curves were obtained. C27 and C28 secosteroids (vitamins D2 and D3) and some of their metabolites (e.g. 25-hydroxyvitamin D) underwent efficient dehydration, again producing products with intense molecular ions. In the case of 24,25-dihydroxyvitamin D3 and 25,26-dihydroxyvitamin D3, dehydration produced different products which were easily resolved in the chromatographic system used. Dehydration of vitamin D metabolites eliminates the need for derivatization and gives enhanced sensitivity of measurement by gas chromatography-mass spectrometry.

Androgens↗
PubMed2005

Prevention of chronic allograft rejection by Vitamin D receptor agonists.

While immunosuppressive drugs now permit a good control of acute allograft rejection, chronic rejection remains an important unmet medical problem. We propose that Vitamin D receptor (VDR) agonists, secosteroid hormones that control cell proliferation and differentiation and exert immunoregulatory activities, in addition to regulate calcium and bone metabolism, have the potential to contribute to the management of chronic allograft rejection. Recent advances in understanding the immunomodulatory and growth-regulating properties of VDR agonists indicate the clinical applicability of these hormones in transplantation, with the aim of facilitating tolerance induction and preventing chronic graft rejection.

Animals↗
PubMed2004

Synthesis of 13,14-secotestosterone derivatives.

A number of testosterone analogs with a 13,14-secosteroidal fragment have been prepared from (13S)-13-iodo-6beta-methoxy-3alpha, 5-cyclo-13,14-seco-5alpha-androstan-14,17-dione. The key steps involved stereoselective deiodination of the starting compound with triphenylphosphine and selective protection of the 17-keto group with trimethylsilylcyanide. Removal of iodine at C-13 proceeded with inversion of the configuration at C-13, which has been established by X-ray crystallography. 13,14-Secotestosterone analogues substituted and non-substituted at C-14 have been prepared. The obtained compounds containing flexible CD ring fragments are of great interest for comparative studies in biological tests together with testosterone and other steroids with a rigid tetracyclic skeleton.

Models, Molecular↗
PubMed2006

High-throughput bioluminescence screening of ubiquitin-proteasome pathway inhibitors from chemical and natural sources.

To discover original inhibitors of the ubiquitin-proteasome pathway, the authors have developed a cell-based bioluminescent assay and used it to screen collections of plant extracts and chemical compounds. They first established a DLD-1 human colon cancer cell line that stably expresses a 4Ubiquitin-Luciferase (4Ub-Luc) reporter protein, efficiently targeted to the ubiquitin-proteasome degradation pathway. The assay was then adapted to 96- and 384-well plate formats and calibrated with reference proteasome inhibitors. Assay robustness was carefully assessed, particularly cell toxicity, and the statistical Z factor value was calculated to 0.83, demonstrating a good performance level of the assay. A total of 18,239 molecules and 15,744 plant extracts and fractions thereof were screened for their capacity to increase the luciferase activity in DLD-1 4Ub-Luc cells, and 21 molecules and 66 extracts inhibiting the ubiquitin-proteasome pathway were identified. The fractionation of an active methanol extract of Physalis angulata L. aerial parts was performed to isolate 2 secosteroids known as physalin B and C. In a cell-based Western blot assay, the ubiquitinated protein accumulation was confirmed after a physalin treatment confirming the accuracy of the screening process. The method reported here thus provides a robust approach to identify novel ubiquitin-proteasome pathway inhibitors in large collections of chemical compounds and natural products.

Cell Count↗
PubMed1992

1,25-Dihydroxyvitamin D3 analog structure-function assessment of the rapid stimulation of intestinal calcium absorption (transcaltachia).

The possibility is now emerging that 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] can mediate biologic responses via both genomic and nongenomic pathways. To understand the molecular basis of the nongenomic response of transcaltachia, defined as the 1,25-(OH)2D3-mediated rapid (2-10 minutes) stimulation of calcium transport from the brush border to the basal lateral membrane of the epithelial cell in vitamin D-replete chick intestine, and to address the issue of whether the same receptor for the secosteroid serves as the signal transducer for both genomic and nongenomic pathways, we carried out structure-function studies using seven analogs of 1,25-(OH)2D3 with different affinities for the classic nuclear 1,25-(OH)2D3 receptor as measured by determination in a steroid competition assay of the relative competitive index (RCI). The RCI of 1,25-(OH)2D3 is by definition 100. 1,25-(OH)2D3 initiates transcaltachia within 2-10 minutes of vascular perfusion and yields a biphase response curve. Dose-dependent stimulations of Ca2+ transport by the seven analogs indicates that different structural features are essential for initiating the transcaltachic response as contrasted with binding to the classic nuclear receptor. Vascular perfusion with analogs AT (25-OH-16-ene-23-yne-D3) and Y (25-OH-23-yne-D3), which are known to activate Ca2+ channels but bind very poorly to the classic receptor (RCI less than 0.5), are efficient in stimulating Ca2+ transport. By comparison, compounds BT [1 alpha,24S-(OH)2-22-en-26,27-dehydrovitamin D3] and V (1,25-(OH)2-16-ene-23-yne-D3], which bind very well to the classic nuclear receptor (RCI 75-111) but do not activate Ca2+ channels, are inefficient in stimulating Ca2+ transport. These results indicate that the membrane components that respond to the analogs of 1,25-(OH)2D3 with activation of Ca2+ channels have a different ligand specificity than the classic nuclear receptor.

Animals↗
PubMed1995

Expression of the 1,25-dihydroxyvitamin D3-24-hydroxylase gene in rat intestine: response to calcium, vitamin D3 and calcitriol administration in vivo.

The 25(OH)D3/1,25(OH)2D3 24-hydroxylase (24-hydroxylase) displays an induction profile responsive to vitamin D (D) abundance and is hence only observed in normal extracellular Ca2+ concentrations. However, the participation of Ca2+ in the expression of the 24-hydroxylase gene in vivo is not known. The present studies investigate the role played by the circulating Ca2+ and the D3 and/or 1,25(OH)2D3 status on the 1,25(OH)2D3-mediated inducibility of the 24-hydroxylase gene in rat duodenum. Hypocalcemic D-depleted rats were supplemented with calcium alone to normalize serum Ca2+ without normalizing the D3 status or were acutely or chronically supplemented with D3 or 1,25(OH)2D3. Messenger RNA for the 24-hydroxylase was undetectable in the intestine of hypocalcemic D-depleted rats, and short- or long-term calcium supplementation was completely unsuccessful in inducing its expression. By contrast, acute 1,25(OH)2D3 administration led to significant increases in the levels of expression of the gene which was independent of the calcium intake, the prevailing circulating Ca2+, and the D3 or 1,25(OH)2D3 status. Moreover, 24-hydroxylase gene expression was only found to respond to acutely administered 1,25(OH)2D3, the mRNA levels being unaltered following continuous exposure to physiological or pharmacological doses of the hormone for 7 days. Time-course studies revealed, however, that induction of the gene was clearly apparent early in the 1,25(OH)2D3 supplementation course but gradually faded by 3 days to return to basal uninduced levels by 7 days, suggesting the presence of intestinal adaptation mechanism(s) which down-regulated the responsiveness in the continuous presence of 1,25(OH)2D3. Our data show the lack of effect of calcium alone or in combination with 1,25(OH)2D3 on the in vivo induction of the 24-hydroxylase gene expression in rat intestine. By rapidly reacting to surges in 1,25(OH)2D3 concentrations, the 24-hydroxylase efficiently controls the amount of 1,25(OH)2D3 in intestine as the first step in the biotransformation pathway aimed at the irreversible clearance of the secosteroid hormone.

Analysis of Variance↗
PubMed2003

New insights into the mechanisms of vitamin D action.

The biologically active metabolite of vitamin D, 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) is a secosteroid whose genomic mechanism of action is similar to that of other steroid hormones and is mediated by stereospecific interaction of 1,25(OH)(2)D(3) with the vitamin D receptor (VDR) which heterodimerizes with the retinoid X receptor (RXR). After interaction with the vitamin D response element (VDRE) in the promoter of target genes, transcription proceeds through the interaction of VDR with coactivators and with the transcription machinery. The identification of the steps involved in this process has been a major focus of recent research in the field. However, the functional significance of target proteins as well as the functional significance of proteins involved in the transport and metabolism of vitamin D is also of major importance. Within the past few years much new information has been obtained from studies using knockout and transgenic mice. New insight has been obtained using this technology related to the physiological significance of the vitamin D binding protein (DBP), used to transport vitamin D metabolites, as well as the physiological significance of target proteins including 25-hydroxyvitamin D(3) 24-hydroxylase (24(OH)ase), 25-hydroxyvitamin D(3)-1 alpha-hydroxylase (1 alpha-(OH)ase), VDR, and osteopontin. The crystal structure of the DBP and the ligand binding domain of the VDR have recently been reported, explaining, in part, the unique properties of these proteins. In addition novel 1,25(OH)(2)D(3) target genes have been identified including the epithelial calcium channel, present in the proximal intestine and in the distal nephron. Thus in recent years a number of exciting discoveries have been made that have enhanced our understanding of mechanisms involved in the pleiotropic actions of 1,25(OH)(2)D(3).

Animals↗
PubMed2003

Vitamin D receptor is not required for the rapid actions of 1,25-dihydroxyvitamin D3 to increase intracellular calcium and activate protein kinase C in mouse osteoblasts.

The rapid, non-genomic actions of 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] have been well described, however, the role of the nuclear vitamin D receptor (VDR) in this pathway remains unclear. To address this question, we used VDR(+/+) and VDR(-/-) osteoblasts isolated from wild-type and VDR null mice to study the increase in intracellular calcium ([Ca(2+)](i)) and activation of protein kinase C (PKC) induced by 1,25(OH)(2)D(3). Within 1 min of 1,25(OH)(2)D(3) (100 nM) treatment, an increase of 58 and 53 nM in [Ca(2+)](i) (n = 3) was detected in VDR(+/+) and VDR(-/-) cells, respectively. By 5 min, 1,25(OH)(2)D(3) caused a 2.1- and 1.9-fold increase (n = 6) in the phosphorylation of PKC substrate peptide acetylated-MBP(4-14) in VDR(+/+) and VDR(-/-) osteoblasts. The 1,25(OH)(2)D(3)-induced phosphorylation was abolished by GF109203X, a general PKC inhibitor, in both cell types, confirming that the secosteroid induced PKC activity. Moreover, 1,25(OH)(2)D(3) treatment resulted in the same degree of translocation of PKC-alpha and PKC-delta, but not of PKC-zeta, from cytosol to plasma membrane in both VDR(+/+) and VDR(-/-) cells. These experiments demonstrate that the 1,25(OH)(2)D(3)-induced rapid increases in [Ca(2+)](i) and PKC activity are neither mediated by, nor dependent upon, a functional nuclear VDR in mouse osteoblasts. Thus, VDR is not essential for these rapid actions of 1,25(OH)(2)D(3) in osteoblasts.

Animals↗
PubMed2003

TRPC3-like protein is involved in the capacitative cation entry induced by 1alpha,25-dihydroxy-vitamin D3 in ROS 17/2.8 osteoblastic cells.

In ROS 17/2.8 rat osteoblastic-like cells a capacitative Ca(2+) entry (CCE) pathway operates which is activated by either 1alpha,25-dihydroxy-vitamin D3 (1alpha,25(OH)(2)D3 or thapsigargin (Tpg)-induced depletion of Ca(2+) stores. In view of recent evidence favoring a role for transient receptor potential (TRP) proteins in mediating CCE, we investigated if channels involved in the 1alpha,25(OH)(2)D3-sensitive CCE in rat osteoblasts were related to an endogenous TRP-canonical (TRPC) isoform homologue. By reverse transcription (RT)-PCR using mRNA from ROS 17/2.8 cells and primers based on conserved regions within the mammalian TRPC3/6/7 subfamily, two fragments were amplified of 390 and 201 bp with 100 and 94% sequence identity, respectively, with human TRPC3. Northern blot analysis showed the presence of a 3.5 kb transcript and both immunobloting and immunocytochemistry using a specific anti-TRPC3 antibody confirmed endogenous expression of a TRPC3-like protein ( approximately 110 kDa) with membrane localization. In ROS 17/2.8 cells intranuclearly microinjected with anti-TRPC3 antisense oligodeoxynucleotides (ODN), both the initial rate and magnitude of CCE activated by either 1alpha,25(OH)(2)D3 or Tpg were markedly reduced, whereas no changes were detected in control-injected cells. The present findings constitute the first evidence to date suggesting that an endogenous TRPC3-like protein is functionally involved in the CCE route activated by 1alpha,25(OH)(2)D3 in a secosteroid target cell. We anticipate TRPC3 as a candidate for mediating store-operated non-selective cation entry into osteoblasts.

Animals↗
PubMed1995

1,25-Dihydroxyvitamin D3 or dexamethasone modulate arachidonic acid uptake and distribution into glycerophospholipids by normal adult human osteoblast-like cells.

The effects of treatment with the osteotropic steroids 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), 17 beta-estradiol, or dexamethasone on [1-14C]arachidonic acid (AA) uptake and distribution into glycerophospholipid classes by normal adult human osteoblast-like (hOB) cells were investigated. Total uptake of [1-14C]AA was decreased in cells treated with dexamethasone when assayed after a 24-, 48-, or 96-h exposure to the hormone. Specific radiolabel incorporation into phosphatidylcholine was reduced by a 48-h treatment with dexamethasone with a concurrent increase in the radiolabeling of phosphatidylethanolamine. However, these changes were transient, and by 96 h of dexamethasone treatment the distribution of the radiolabeled fatty acid had reequilibrated to resemble the pattern found for vehicle treated samples. Total uptake of [1-14C]AA was diminished by 96-h treatment with 1,25(OH)2D3 (79 +/- 3% of control, P < 0.01); at that time point, a significant decrease in the proportional radiolabeling of the phosphatidylinositol pool was identified (92 +/- 2% of control, P < 0.05). The 1,25(OH)2D3-dependent decrease in total uptake and in phosphatidylinositol incorporation of [1-14C]AA were found to be hormone dose dependent. Treatment with 24,25(OH)2D3 was without effect on either total [1-14C]AA uptake or the specific [1-14C]AA radiolabeling of the phosphatidylinositol pool. 1,25(OH)2D3 treatment decreased hOB cell uptake of [1-14C]oleic acid and decreased its proportional incorporation into the phosphatidylinositol pool. Gas chromatographic analyses revealed no 1,25(OH)2D3-dependent effects on total phosphatidylinositol lipid mass or on the mole percent of arachidonic acid within the phosphatidylinositol pool, leaving the mechanism of the effects of the secosteroid on hOB cell AA metabolism unexplained. 17 beta-Estradiol had no effects on the parameters of AA metabolism measured. As a consequence of their modulation of arachidonic acid uptake and its distribution into hOB cellular phospholipids, steroids might alter the biological effects of other hormones whose actions include the stimulated production of bioactive AA metabolites, such as prostaglandins or the various lipoxygenase products.

Adolescent↗
PubMed1995

1,25-Dihydroxyvitamin D3 stimulates the phosphorylation of two acidic membrane proteins of 42,000 and 48,000 daltons in rat colonocytes: an effect modulated by vitamin D status.

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-O-tetradecanoyl 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.

Animals↗
PubMed2006

Prostate derived factor in human prostate cancer cells: gene induction by vitamin D via a p53-dependent mechanism and inhibition of prostate cancer cell growth.

The secosteroid hormone 1alpha, 25-dihydroxyvitamin D3 (1,25D) has been shown to regulate the growth and differentiation of human prostate cancer (PCa) cells, although the precise molecular mechanisms mediating these effects have not been defined. Previous studies in our laboratory demonstrated that the antiproliferative effects of 1,25D on PCa cells are mediated through the nuclear vitamin D receptor (VDR). In the present study, we performed gene profiling of LNCaP human PCa cells following 1,25D treatment and identified the antitumorigenic gene, prostate derived factor (PDF), as being highly induced by 1,25D. PDF is a member of the TGF-beta superfamily and has been implicated in a variety of functions directly related totumorigenicity including antiproliferative and pro-apoptotic effects. Gene expression studies using 1,25D analogs and a VDR antagonist demonstrate that 1,25D-mediated induction of PDF message and protein in PCa cells is dependent on VDR action. PDF is a transcriptional target of the tumor suppressor, p53. Here we show that the expression of PDF in nine PCa cell lines is dependent on functional p53. Additionally, transfection of p53-null ALVA-31 PCa cells with a p53 expression plasmid, and expression of dominant negative p53 in LNCaP PCa cells, show that the ability of VDR to induce PDF requires functional p53. Importantly, forced PDF expression in PC-3 cells results in decreased cell proliferation, soft agar cloning, and xenograft tumor size. These data demonstrate that PDF exerts antitumorigenic properties on PCa cells and its regulation by 1,25D may provide insights into the action of 1,25D in PCa.

Animals↗
PubMed1994

Induction of glioma cell death by 1,25(OH)2 vitamin D3: towards an endocrine therapy of brain tumors?

The secosteroid 1,25-dihydroxyvitamin D3 (1,25 (OH)2D3) is the major biologically active metabolite of vitamin D. Antitumor activity of this hormone has been observed on several cell lines and on breast cancer in vivo. The purpose of this in vitro study was to determine the possible effect of 1,25(OH)2D3 on glioma cells. Two glioma cell lines from rat (C6) or human (GHD) origin were cultured in the presence of 1,25(OH)2D3. The sensitivity of these cells to 1,25 (OH)2D3 was assessed with a colorimetric MTT assay. A cytotoxic effect of 1,25(OH)2D3 was detected at concentrations around 10(-8) M. A lag period of 3 days was required between the onset of the treatment and the observation of the effects. However, the continuous presence of 1,25(OH)2D3 is not required since cell death occurred even when C6 cells were challenged for 24 hr with 1,25(OH)2D3 and then cultured in the absence of the hormone. In addition, 1,25(OH)2D3 regulates the expression of its own receptors in C6 glioma. These results provide to our knowledge the first evidence for a cytotoxic effect of 1,25(OH)2D3 on rat and human glioma cells and could offer both an experimental model to study a programmed cell death in a brain-derived cell line and a new strategy for the inhibition of glioma growth in vivo.

Animals↗
PubMed1978

Total synthesis of 1,11- and 3,11-diazasteroids.

The 2- and 6-methoxy derivatives of 8-aminoquinoline, as well as 5-aminoisoquinoline, condensed with 2-ethoxycarbonylcyclopentanone to give tricyclic intermediates in 86-90% yield. These secosteroids were cyclized in 35-69% yield with polyphosphoric acid to 2-methoxy-1,11-diaza-, 7-methoxy-1,11-diaza-, and 3,11-diaza-1,3,5,7,9,13-gonahexaen-12-ones, respectively. The latter exhibits slight antileukemic activity.

Animals↗