Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Secosteroids”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 127 records · Page 7Linked to original sources
PubMed2000

Anticlastogenic potential of 1alpha,25-dihydroxyvitamin D3 in murine lymphoma.

Vitamin D3, having gained scientific interest for so long because of its role in mineral homeostasis, has now received great importance as a possible antitumor agent. This study was undertaken in an attempt to visualize the possible anticlastogenic potential of the vitamin in an ascitic mouse lymphoma model namely, Dalton's lymphoma. Frequencies of structural type chromosomal aberrations, sister chromatid exchanges and micronucleus assays have been chosen as the genotoxic endpoints in the proposed investigation. All these cytogenetic markers have been found to be markedly elevated during the progression of lymphoma in bone marrow cells. Vitamin D3 effectively suppressed the frequencies of chromosomal aberrations and sister chromatid exchanges in the lymphoma-bearing mice during the entire phase of tumor growth that significantly coupled with almost two-fold increase in survival time (37 +/- 2 and 68 +/- 2 days in lymphoma controls and vitamin D3-treated lymphoma-bearing mice, respectively), thus substantiating the antineoplastic efficacy of this secosteroid. The outcome of this study also is clearly reflected in the depletion of circulating (serum) vitamin D3 levels in the lymphoma control mice compared with normal (vehicle) controls while a still higher level was maintained in the VD3-treated lymphoma mice. This anticlastogenic property of the vitamin has so far been neglected and this is the first attempt to unravel the vitamin D3's effect in combating tumor development in vivo by limiting the frequencies of chromosomal aberrations, sister chromatid exchanges and micronuclei at least in transplantable murine model studied herein.

Animals↗
PubMed2003

Novel high energy intermediate analogues with triazasterol-related structures as inhibitors of ergosterol biosynthesis. III. Synthesis and antifungal activity of N4-alkyl-1,6,7,11b-tetrahydro-2H-pyrimido[4,3-a]isoquinolin-4-amine salts.

A series of N4-alkyl-1,6,7,11b-tetrahydro-2H-pyrimido[4,3-a]isoquinolinamine hydroiodides with triazasterol-related structures was designed and synthesized to mimic, as stable analogues, native high energy intermediates (HEI) of ergosterol biosynthesis. The title compounds can be regarded as 8,13,15-triaza-13,17-secosteroids with aromatic ring A bearing the positive charge in the guanidinium moiety. Hence, these compounds present structural similarities with corresponding carbocationic intermediates occurring during the enzyme catalyzed transformation of squalene into ergosterol. The N4-alkylaminopyrimidoisoquinolinium salts were prepared by reaction of respective S-methylthiotetrahydropyrimidoisoquinoline hydroiodides with octylamine, and appropriately methyl-branched alkyl- and alkenylamines. In order to prepare (3R)-6-isopropyl-3-methyl-6-hepten-1-amine several synthetic routes were investigated. The structures of all reported compounds were proved and completely assigned on the basis of homo- and heteronuclear correlated 1D and 2D NMR spectroscopy. The in vitro antifungal susceptibility tests of the title compounds with a standard panel of eight pathogenic fungi revealed especially against the used dermatophytes and yeasts with MICs in the range of 1-32 microg/ml moderate to good antimycotic effects. Depending on the nature of the N4-alkyl substituents structure-activity relationships were found with a maximum of antifungal efficacy of the N4-3,7-dimethyloctylaminopyrimidoisoquinolinium iodide.

Antifungal Agents↗
PubMed2001

Synthesis and biological evaluation of all A-ring stereoisomers of 5,6-trans-2-methyl-1,25-dihydroxyvitamin D(3) and their 20-epimers: possible binding modes of potent A-ring analogues to vitamin D receptor.

BACKGROUND: The secosteroid 1 alpha,25-dihydroxyvitamin D(3) (1) has a wide variety of biological activities, which makes it a promising therapeutic agent for the treatment of cancer, psoriasis and osteoporosis. Insight into the structure-activity relationships of the A-ring of 1 is still needed to assist the development of more potent and selective analogues as candidate chemotherapeutic agents, as well as to define the molecular mode of action. RESULTS: All possible A-ring stereoisomers of 5,6-trans-2-methyl-1,25-dihydroxyvitamin D(3) (6a-h) and their 20-epimers (7a-h) were designed and efficiently synthesized. The dependence of the affinities for vitamin D receptor (VDR) and vitamin D binding protein (DBP), as well as the HL-60 cell differentiation-inducing activity, upon the stereochemistry of the A-ring and at C20 in the side chain was evaluated. CONCLUSIONS: The binding affinities and potency of the 5,6-trans and 5,6-cis analogues were enhanced by a 2-methyl substituent in a certain orientation. Molecular docking studies based upon the X-ray crystal structure of VDR suggested that the axial 2-methyl group would be accommodated in a pocket surrounded by hydrophobic amino acid residues in the ligand binding domain, resulting in enhanced interaction.

Animals↗
PubMed2002

1Alpha,25-dihydroxy-3-epi-vitamin D3, a natural metabolite of 1alpha,25-dihydroxy vitamin D3: production and biological activity studies in pulmonary alveolar type II cells.

Pulmonary alveolar type II cells have been shown to be a possible target for the secosteroid hormone, 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3], during perinatal transition. At present, there is great interest to isolate and identify the metabolites of 1alpha,25(OH)2D3 produced in its target tissues and to determine the contribution of each individual metabolite of 1alpha,25(OH)2D3 to the final expression of the pleiotropic actions attributed to 1alpha,25(OH)2D3. Of all the known metabolites of 1alpha,25(OH)2D3, 1alpha,25(OH)2-3-epi-D3 has gained most attention as it is produced only in specific tissues and possesses significant activity in tissues in which it is produced. Furthermore, in vivo studies indicate that this metabolite when compared to 1alpha,25(OH)2D3 is less calcemic. Therefore, we performed the present study to identify production of 1alpha,25(OH)2-3-epi-D3 in alveolar type II cells, and to evaluate its effect on surfactant synthesis. We incubated NCI-H441 cells, an alveolar type II cell line, with 1alpha,25(OH)2D3 and demonstrated that these cells metabolize 1alpha,25(OH)2D3 to various previously well-characterized polar metabolites, and to a less polar metabolite which was unequivocally identified as 1alpha,25(OH)2-3-epi-D3 by GC/MS and HPLC analysis. Further, biological activity studies in H441 cells indicated that 1alpha,25(OH)2-3-epi-D3 possesses significant activity in terms of its ability: (i) to increase surfactant phospholipid synthesis, (ii) to induce surfactant SP-B mRNA gene expression, and (iii) to increase surfactant SP-B protein synthesis. However, the activity of 1alpha,25(OH)2-3-epi-D3 when compared to 1alpha,25(OH)2D3 in generating VDR-mediated transcriptional activity in ROS 17/2.8 cells transfected with human osteocalcin VDRE/growth hormone gene construct, was significantly reduced. The high metabolic stability of 1alpha,25(OH)2-3-epi-D3, as previously proposed by us, may be a possible explanation for the high in vitro activity in spite of the reduced VDR-mediated transcriptional activity. In summary, we report for the first time the pathways of 1alpha,25(OH)2D3 metabolism in pulmonary alveolar type II cells and indicate that 1alpha,25(OH)2-3-epi-D3, a natural intermediary metabolite of 1alpha,25(OH)2D3 possesses significant activity in stimulating surfactant synthesis in alveolar type II cells.

Calcitriol↗
PubMed2002

The coming of age of 1,25-dihydroxyvitamin D(3) analogs as immunomodulatory agents.

The active form of vitamin D, 1,25-dihydroxyvitamin D(3)[1,25(OH)(2)D(3)], is a secosteroid hormone that regulates calcium and bone metabolism, controls cell proliferation and differentiation, and exerts immunoregulatory activities. This range of functions has been exploited clinically to treat a variety of conditions, from secondary hyperparathyroidism to osteoporosis, to autoimmune diseases such as psoriasis. Recent advances in understanding 1,25(OH)(2)D(3) functions and novel insights into the mechanisms of its immunomodulatory properties suggest a wider applicability of this hormone in the treatment of autoimmune diseases and allograft rejection.

Adjuvants, Immunologic↗
PubMed2002

Immunomodulatory effects of vitamin D receptor ligands in autoimmune diseases.

The active form of vitamin D, 1,25-Dihydroxyvitamin D3 [l,25(OH)2D3], is a secosteroid hormone that binds to the vitamin D receptor (VDR), a member of the superfamily of nuclear receptors for steroid hormones, thyroid hormone, and retinoic acid. VDR ligands regulate calcium and bone metabolism, control cell proliferation and differentiation, and exert immunoregulatory activities. The immunoregulatory properties of VDR ligands are currently exploited clinically for the topical treatment of psoriasis, a Th1 cell-mediated autoimmune disease of the skin, but recent advances in understanding their functions and novel insights into the immunomodulatory mechanisms they control suggest a wider applicability in the treatment of autoimmune diseases. In addition to direct effects on T cell activation, VDR ligands modulate with different mechanisms the phenotype and function of antigen-presenting cells (APCs), and, in particular, of dendritic cells (DCs). In vitro and in vivo experiments have shown that VDR ligands induce DCs to acquire tolerogenic properties that favor the induction of regulatory rather than effector T cells. These intriguing actions of VDR ligands have been demonstrated in several experimental models and could be exploited, in principle, to treat a variety of human autoimmune diseases.

Adjuvants, Immunologic↗
PubMed2003

Multiple molecular mechanisms of 1 alpha,25(OH)2-vitamin D3 rapid modulation of three ion channel activities in osteoblasts.

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.

Animals↗
PubMed1999

Production of 1alpha,25-dihydroxy-3-epi-vitamin D3 in two rat osteosarcoma cell lines (UMR 106 and ROS 17/2.8): existence of the C-3 epimerization pathway in ROS 17/2.8 cells in which the C-24 oxidation pathway is not expressed.

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.

Animals↗
PubMed1997

Synthesis, stereochemistry, and biological activity of 1alpha,23,25-trihydroxy-24-oxovitamin D3, a major natural metabolite of 1alpha,25-dihydroxyvitamin D3.

The C(23) epimers of 1alpha,23,25(OH)3-24-oxovitamin D3, a major natural metabolite of the secosteroid hormone, 1alpha,25(OH)2D3, were chemically synthesized for the first time. The metabolite was synthesized by palladium coupling of the appropriate CD ring analog with an A ring enyne. Various approaches from quinic acid to the A ring precursors were explored, and a new route to the A ring enyne from quinic acid was developed. The C(23) stereochemistry of the natural 1alpha,23,25(OH)3-24-oxovitamin D3 produced in neonatal human keratinocytes was determined to be S on the basis of the 1H NMR and the HPLC data. The biological activity of 1alpha,23(S), 25(OH)3-24-oxovitamin D3 in primary cultures of bovine parathyroid cells was determined by comparing the potency of this metabolite to that of 1alpha,25(OH)2D3 in suppression of parathyroid hormone (PTH) secretion. The results indicate that 1alpha,23(S), 25(OH)3-24-oxovitamin D3 potently suppressed PTH secretion even at concentrations as low as 10(-)12 M and is equipotent with 1alpha, 25(OH)2D3. The high activity of 1alpha,23(S),25(OH)3-24-oxovitamin D3 cannot be explained on the basis of its affinity for the vitamin D receptor as this metabolite was found to be 10 times less effective than radioinert 1alpha,25(OH)2D3 in blocking the uptake and receptor binding of [3H]-1alpha,25(OH)2D3 in intact parathyroid cells. Further studies are required to explain the molecular basis for the activity of 1alpha,23(S),25(OH)3-24-oxovitamin D3 in its ability to suppress PTH secretion. In summary, our present study indicates that the C(23) stereochemistry of the natural 1alpha,23, 25(OH)3-24-oxovitamin D3 is S and this metabolite is equipotent to 1alpha,25(OH)2D3 in suppressing PTH secretion.

Animals↗
PubMed2001

Synthesis of (-)-astrogorgiadiol.

Reaction of Rh2(S)-PTPA4 with the (R)-citronellol-derived alpha-diazo-beta-ketoester 1 led to the formation of cyclic beta-ketoester 2 in 95% yield and 48% diastereomeric excess. The purity of 2 was increased to > 99% de after one crystallization. To demonstrate its utility in steroid total synthesis, the beta-ketoester 2 was carried on to secosteroid (-)-astrogorgiadiol (3), a naturally occurring vitamin D analogue with antiproliferative properties.

Catalysis↗
PubMed2006

9,11-Secosterols from the soft corals Sinularia lochmodes and Sinularia leptoclados.

Chemical investigations on the EtOAc-soluble fractions from the EtOH extract of two Formosan soft corals afforded two new 9,11-secosteroids, 3beta,11-dihydroxy-5beta,6beta-epoxy-24-methylene-9,11-secocholestan-9-one (1) and 3beta,11-dihydroxy-24-methylene-9,11-secocholestan-9-one (2), from Sinularia lochmodes and Sinularia leptoclados, respectively, along with two known analogues (3 and 4) from S. leptoclados. The structures of the new metabolites were elucidated on the basis of extensive spectroscopic analysis and by comparison of their NMR data with those of the known compound 3. The cytotoxicity of 2-4 toward a limited panel of cancer cell lines is also reported.

Animals↗
PubMed1999

A new cyclized 9,11-secosterol enol-ether from the australian sponge euryspongia arenaria

The polar fraction of the crude extract from the sponge Euryspongia arenaria was separated by chromatography. Structure elucidation by spectrometric methods allowed the identification of a new steroid, stellattasterenol (2), containing an unprecedented seven-membered cyclic enol-ether in ring C. A related known compound, stellettasterol (3), a pentahydroxy-9,11-secosteroid, was also identified.

Journal Article↗
PubMed1999

Synergistic inhibition of prostate cancer cell lines by a 19-nor hexafluoride vitamin D3 analogue and anti-activator protein 1 retinoid.

The secosteroid hormones, all-trans- and 9-cis-retinoic acid and vitamin D3, have demonstrated significant capacity to control proliferation in vitro of many solid tumour cell lines. Cooperative synergistic effects by these two ligands have been reported, and it is, therefore, possible that greater therapeutic effects could be achieved if these compounds were administered together. The role of retinoid-dependent anti-activator protein 1 (anti-AP-1) effects in controlling cancer cell proliferation appears significant. We have utilized an anti-AP-1 retinoid [2-(4,4-dimethyl-3,4-dihydro-2H-1 benzopyran-6-yl)carbonyl-2-(4-carboxyphenyl)-1,3,-dithiane; SR11238], which does not transactivate through a retinoic acid response element (RARE), and a potent vitamin D3 analogue [1alpha,25(OH)2-16-ene-23-yne-26,27-F6-19-nor-D3, code name LH] together at low, physiologically safer doses against a panel of prostate cancer cell lines that represent progressively more transformed phenotypes. The LNCaP (least transformed) and PC-3 (intermediately transformed) cell lines were synergistically inhibited in their clonal growth by the combination of LH and SR11238, whereas SR11238 alone was essentially inactive. DU-145 cells (most transformed) were completely insensitive to these analogues. LNCaP cells, but neither PC-3 nor DU-145, underwent apoptosis in the presence of LH and SR11238. Transactivation of the human osteocalcin vitamin D response element (VDRE) by LH was not enhanced in the presence of SR11238, although the expression of E-cadherin in these cells was additively up-regulated in the presence of both compounds. These data suggest the anti-AP-1 retinoid and the vitamin D3 analogue may naturally act synergistically to control cell proliferation, a process that is interrupted during transformation, and that this combination may form the basis for treatment of some androgen-independent prostate cancer.

Antineoplastic Agents↗
PubMed1999

Rapid actions of calcitriol and its side chain analogues CB1093 and GS1500 on intracellular calcium levels in skeletal muscle cells: a comparative study.

1. The ability of synthetic analogues of the secosteroid hormone 1alpha,25-dihydroxy-vitamin-D3 [calcitriol, CT; 1,25(OH)2D3] to exert non-genomic (rapid) effects on target cells has been scarcely studied. To evaluate the pharmacological potential of the CT side-chain analogues CB1093 and GS1500, we compared their fast effects on intracellular calcium concentration ([Ca2+]i) in chick skeletal muscle cells with those elicited by the natural hormone. 2. Both analogues, similarly to CT, specifically induced rapid (30-60 s) and sustained rises in [Ca2+]i levels. CB1093 and GS1500 were more potent than the natural hormone at concentrations as low as 10(-13) M (4.5 fold stimulation) and 10(-12) M (2.5 fold), respectively, whereas higher concentrations (10(-9)- 10(-8) M) of CT were more effective than the analogues in elevating [Ca2+]i. Cyclic AMP was markedly increased by both analogues pointing for a role of this messenger in the fast actions of the synthetic compounds. 3. In Ca2+ free medium CT and analogues elicited a transient elevation in [Ca2+]i. The PLC inhibitors U73122 (2 microM) and neomycin (0.5 mM), as well as depletion of intracellular stores with thapsigargin (1 microM), completely prevented CB1093/GS1500-dependent changes in [Ca2+]i suggesting that, similarly to CT, these analogues mobilized Ca2+ from an IP3/thapsigargin-sensitive store. 4. The voltage-dependent calcium channel (VDCC) blocker nifedipine (2 microM) reduced by 50-60% the influx phase of the [Ca2+]i response to CB1093 and GS1500, indicating that VDCC contributed partially to Ca2+ entry. The Ca2+ readdition protocol suggested that analogue-dependent activation of a SOC entry pathway accounted, to the same extent as for CT, for the remaining non-VDCC mediated Ca2+ influx.

Animals↗
PubMed1996

Genomic mechanisms involved in the pleiotropic actions of 1,25-dihydroxyvitamin D3.

The biologically active metabolite of vitamin D (cholecalciferol), i.e. 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], is a secosteroid hormone whose mode of action involves stereospecific interaction with an intracellular receptor protein (vitamin D receptor; VDR). 1,25(OH)2D3 is known to be a principal regulator of calcium homeostasis, and it has numerous other physiological functions including inhibition of proliferation of cancer cells, effects on hormone secretion and suppression of T-cell proliferation and cytokine production. Although the exact mechanisms involved in mediating many of the different effects of 1,25(OH)2D3 are not completely defined, genomic actions involving the VDR are clearly of major importance. Similar to other steroid receptors, the VDR is phosphorylated; however, the exact functional role of the phosphorylation of the VDR remains to be determined. The VDR has been reported to be regulated by 1,25(OH)2D3 and also by activation of protein kinases A and C, suggesting co-operativity between signal transduction pathways and 1,25(OH)2D3 action. The VDR binds to vitamin D-responsive elements (VDREs) in the 5' flanking region of target genes. It has been suggested that VDR homodimerization can occur upon binding to certain VDREs but that the VDR/retinoid X receptor (RXR) heterodimer is the functional transactivating species. Other factors reported to be involved in VDR-mediated transcription include chicken ovalbumin upstream promoter (COUP) transcription factor, which is involved in active silencing of transcription, and transcription factor IIB, which has been suggested to play a major role following VDR/RXR heterodimerization. Newly identified vitamin D-dependent target genes include those for Ca2+/Mg(2+)-ATPase in the intestine and p21 in the myelomonocytic U937 cell line. Elucidation of the mechanisms involved in the multiple actions of 1,25(OH)2D3 will be an active area of future research.

Amino Acid Sequence↗
PubMed2002

1alpha,25(OH)2D3 regulates chondrocyte matrix vesicle protein kinase C (PKC) directly via G-protein-dependent mechanisms and indirectly via incorporation of PKC during matrix vesicle biogenesis.

Matrix vesicles are extracellular organelles involved in mineral formation that are regulated by 1alpha,25(OH)(2)D(3). Prior studies have shown that protein kinase C (PKC) activity is involved in mediating the effects of 1alpha,25(OH)(2)D(3) in both matrix vesicles and plasma membranes. Here, we examined the regulation of matrix vesicle PKC by 1alpha,25(OH)(2)D(3) during biogenesis and after deposition in the matrix. When growth zone costochondral chondrocytes were treated for 9 min with 1alpha,25(OH)(2)D(3), PKCzeta in matrix vesicles was inhibited, while PKCalpha in plasma membranes was increased. In contrast, after treatment for 12 or 24 h, PKCzeta in matrix vesicles was increased, while PKCalpha in plasma membranes was unchanged. The effect of 1alpha,25(OH)(2)D(3) was stereospecific and metabolite-specific. Monensin blocked the increase in matrix vesicle PKC after 24 h, suggesting the secosteroid-regulated packaging of PKC. In addition, the 1alpha,25(OH)(2)D(3) membrane vitamin D receptor (1,25-mVDR) was involved, since a specific antibody blocked the 1alpha,25(OH)(2)D(3)-dependent changes in PKC after both long and short treatment times. In contrast, antibodies to annexin II had no effect, and there was no evidence for the presence of the nuclear VDR on Western blots. To investigate the signaling pathways involved in regulating matrix vesicle PKC activity after biosynthesis, matrix vesicles were isolated and then treated for 9 min with 1alpha,25(OH)(2)D(3) in the presence and absence of specific inhibitors. Inhibition of phosphatidylinositol-phospholipase C, phospholipase D, or G(i)/G(s) had no effect. However, inhibition of G(q) blocked the effect of 1alpha,25(OH)(2)D(3). The rapid effect of 1alpha,25(OH)(2)D(3) also involved the 1,25-mVDR. Moreover, arachidonic acid was found to stimulate PKC when added directly to isolated matrix vesicles. These results indicate that matrix vesicle PKC is regulated by 1alpha,25(OH)(2)D(3) at three levels: 1) during matrix vesicle biogenesis; 2) through direct action on the membrane; and 3) through production of other factors such as arachidonic acid.

Animals↗
PubMed2002

Role of MAP kinases in the 1,25-dihydroxyvitamin D3-induced transactivation of the rat cytochrome P450C24 (CYP24) promoter. Specific functions for ERK1/ERK2 and ERK5.

The current study investigated the action of 1,25-dihydroxyvitamin D(3) (1,25D) at the genomic and signal transduction levels to induce rat cytochrome P450C24 (CYP24) gene expression. A rat CYP24 promoter containing two vitamin D response elements and an Ets-1 binding site was used to characterize the mechanism of actions for the 1,25D secosteroid hormone. The Ets-1 binding site was determined to function cooperatively with the most proximal vitamin D response element in a hormone-dependent fashion. Evidence was obtained for distinct roles of ERK1/ERK2 and ERK5 in the 1,25D-inductive actions. Specifically, 1,25D stimulated the activities of ERK1/ERK2 and ERK5 in a Ras-dependent manner. Promoter induction was inhibited by mitogen-activated protein (MAP) kinase inhibitors (PD98059 and U0126) and a dominant-negative Ras mutant (Ras17N). Induction of CYP24 by 1,25D was also inhibited by overexpression of dominant-negative mutants of ERK1 and MEK5 (ERK1K71R and MEK5(A)). The p38 and JNK MAP kinases were not required for the action of 1,25D. 9-cis retinoid X receptor alpha (RXR alpha) interacted with ERK2 but not ERK5 in intact cells, whereas Ets-1 interacted preferentially with ERK5. Increased phosphorylation of RXR alpha and Ets-1 was detected in response to 1,25D. Activated ERK2 and ERK5 specifically phosphorylated RXR alpha and Ets-1, respectively. Mutagenesis of Ets-1 (T38A) reduced CYP24 promoter activity to levels observed with the dominant-negative MEK5(A) and inhibited ERK5-directed phosphorylation. Mutated RXR alpha (S260A) inhibited 1,25D-induced CYP24 promoter activity and abolished phosphorylation by activated ERK2. The 1,25D-inductive action through ERK5 involved Ets-1 phosphorylation at threonine 38, whereas hormone stimulation of ERK1/ERK2 required RXR alpha phosphorylation on serine 260. The ERK1/ERK2 and ERK5 modules provide a novel mechanism for linking the rapid signal transduction and slower transcription actions of 1,25D to induce CYP24 gene expression.

Animals↗
PubMed2003

De-orphanization of cytochrome P450 2R1: a microsomal vitamin D 25-hydroxilase.

The conversion of vitamin D into an active ligand for the vitamin D receptor requires 25-hydroxylation in the liver and 1alpha-hydroxylation in the kidney. Mitochondrial and microsomal vitamin D 25-hydroxylase enzymes catalyze the first reaction. The mitochondrial activity is associated with sterol 27-hydroxylase, a cytochrome P450 (CYP27A1); however, the identity of the microsomal enzyme has remained elusive. A cDNA library prepared from hepatic mRNA of sterol 27-hydroxylase-deficient mice was screened with a ligand activation assay to identify an evolutionarily conserved microsomal cytochrome P450 (CYP2R1) with vitamin D 25-hydroxylase activity. Expression of CYP2R1 in cells led to the transcriptional activation of the vitamin D receptor when either vitamin D2 or D3 was added to the medium. Thin layer chromatography and radioimmunoassays indicated that the secosteroid product of CYP2R1 was 25-hydroxyvitamin D3. Co-expression of CYP2R1 with vitamin D 1alpha-hydroxylase (CYP27B1) elicited additive activation of vitamin D3, whereas co-expression with vitamin D 24-hydroxylase (CYP24A1) caused inactivation. CYP2R1 mRNA is abundant in the liver and testis, and present at lower levels in other tissues. The data suggest that CYP2R1 is a strong candidate for the microsomal vitamin D 25-hydroxylase.

25-Hydroxyvitamin D3 1-alpha-Hydroxylase↗