Biomedical subjects

C Carlberg

Publications and source records attributed to C Carlberg.

At least 37 records · Page 2Linked to original sources

PubMed2000

Gene regulation by melatonin.

The physiological and neuroendocrine functions of the pineal gland hormone, melatonin, and its therapeutic potential critically depend on the understanding of its target sites and its mechanisms of action. This has progressed considerably in the last few years through the cloning of G protein-coupled seven-transmembrane melatonin receptors (Mel1a and Mel1b) as well as of nuclear receptors (RZR/ROR alpha and RZR beta) that are associated with melatonin signaling. The transcription factor RZR/ROR alpha appears to mediate a direct gene regulatory action of the hormone, and specific binding sites have been identified in promoter regions of a variety of genes, such as 5-lipoxygenase (5-LO), p21WAF1/CIP1, and bone sialoprotein (BSP). The membrane signaling pathway clearly shows higher ligand sensitivity than the nuclear signaling pathway, but details of its signal transduction cascade, and target genes are presently unknown. Membrane melatonin receptors are expressed mainly in the central nervous system, whereas RZR/ROR alpha is prominently expressed both in the periphery and the brain. The action of membrane melatonin receptors and their specific agonists have been associated with circadian rhythmicity, whereas direct effects of melatonin in the periphery, such as immunomodulation, cellular growth, and bone differentiation, mainly appear to be mediated by RZR/ROR alpha. It is hypothesized in this review that, in some cases, RZR/ROR alpha may be a primary target of membrane melatonin receptors.

Animals↗

PubMed2000

Structure activity relationship of carboxylic ester antagonists of the vitamin D(3) receptor.

A 25-carboxylic ester analog of 1alpha,25-dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)], ZK159222 (compound 1), was recently described as a novel type of antagonist of 1alpha,25(OH)(2)D(3) signaling. In this study five derivatives of compound 1 (compounds 2-6) were selected because of their sensitivity in facilitating complex formation between the 1alpha,25(OH)(2)D(3) receptor (VDR) and the retinoid X receptor on a 1alpha,25(OH)(2)D(3) response element that was comparable to that of the natural hormone (0.2-0.9 nM). Most derivatives of compound 1 reacted as typical agonists, because they were able to promote ligand-dependent interaction of the VDR with the coactivator TIF2, stabilized the VDR preferentially in its agonistic conformation c1(LPD), and stimulated VDR-dependent gene activity with a potency similar to 1alpha,25(OH)(2)D(3). In contrast, only compound 2 showed the antagonistic profile of compound 1, which includes the incompetence to induce a VDR-TIF2 contact, the stabilization of the antagonistic conformation c2(LPD), and only a very weak and insensitive functional activity. Accordingly, only compounds 1 and 2, but not compounds 3 to 6, showed prominent antagonistic effects in cellular systems. The comparison of the structures of the compounds indicates that the essential requirements for an antagonistic function are a cyclopropyl ring at carbon 25, a hydroxy group at carbon 24, and at least a butylester. Interestingly, compound 2 was an approximately 3 times more sensitive antagonist than compound 1 and even displayed a lower residual agonistic activity. In conclusion, only a very limited number of structural variations of compound 1 are possible to keep its antagonistic profile, but the tools presented here for their in vitro evaluation allow an accurate prediction of the effects and are suited to screening for even more potent 1alpha, 25(OH)(2)D(3) antagonists.

Calcitriol↗

PubMed2000

The impact of functional vitamin D(3) receptor conformations on DNA-dependent vitamin D(3) signaling.

The vitamin D(3) receptor (VDR) is the nuclear receptor for 1alpha, 25-dihydroxyvitamin D(3) (VD) that acts primarily as a heterodimer with the retinoid X receptor (RXR) on different types of VD response elements, i.e., DNA-bound VDR-RXR heterodimers are the molecular switches in nuclear VD signaling pathways. In this study, DNA-dependent limited protease digestion assays and gel shift clipping assays were used for the analysis of VDR conformations and showed the same high ligand sensitivity for VD response element-bound VDR-RXR heterodimers (EC(50) of 0.1 nM for VD). In contrast, DNA-independent limited protease digestion assays clearly demonstrated a reduced ligand sensitivity for monomeric VDR in solution. Interestingly, the relative amount of reduction was found to be specific for each VDR agonist. Moreover, complex formation of the VDR on DNA resulted in a shift from the receptor's low-affinity ligand binding conformation (c3(LPD)) to its high affinity conformation (c1(LPD)). Finally, the characterization of the conformations of N- and C-terminally truncated VDR proteins defined the high-affinity ligand binding domain of the VDR as being positioned between amino acids 128 and 427. Taken together, the analysis of VDR conformations in solution in comparison to those of DNA-complexed VDR-RXR heterodimers allows a differentiation to be drawn between DNA-dependent and DNA-independent VD signaling pathways that can in turn be used for the identification of pathway selective VDR agonists.

Binding, Competitive↗

PubMed2000

Response element and coactivator-mediated conformational change of the vitamin D(3) receptor permits sensitive interaction with agonists.

The vitamin D receptor (VDR) is the nuclear receptor for 1, 25-dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)] that acts as a ligand-dependent transcription factor via combined contact with coactivator proteins (steroid receptor coactivator-1, transcriptional intermediary factor 2, and receptor associated coactivator 3) and specific DNA binding sites [vitamin D response elements (VDREs)]. Ligand-mediated conformational changes of the VDR contribute to the key mechanisms in this nuclear hormone signaling process. 1alpha,25(OH)(2)D(3), MC1288 [20-epi-1alpha,25(OH)(2)D(3)], ZK161422 [20-methyl-1alpha,25(OH)(2)D(3)], and Ro27-2310 (also called Gemini, having two side chains at carbon 20) were used as model VDR agonists. The analysis of agonist-induced VDR conformations and coactivator interactions were found to be insufficient for extrapolating in vivo activities. In DNA-independent assays, such as classical limited protease digestions and glutathione S-transferase pull downs, Gemini seemed to be up to 10,000-fold and the other VDR agonists 10- to 100-fold weaker than in functional in vivo assays. A more accurate description of the gene regulatory potential of VDR agonists was obtained with all tested VDR agonists by analyzing VDR conformations in the context of VDRE-bound VDR-retinoid X receptor heterodimers, in such assays as gel supershift, gel shift clipping, and limited protease digestion in the presence of DNA and cofactor. Coactivators were found to shift the ligand sensitivity (by a factor of 4 for Gemini) and the ratio of VDR conformations in the presence of DNA toward the high-affinity ligand binding conformation (c1(LPD)). In conclusion, the induction of response element- and coactivator-modulated VDR conformations appears to be a key step for the gene regulatory function of a VDR agonist. The quantification of these effects would be of central importance for the evaluation of the cell-specific efficacy of systemically applied 1alpha, 25(OH)(2)D(3) analogs.

Animals↗

PubMed1999

Inhibitory effect of uremic solutions on protein-DNA-complex formation of the vitamin D receptor and other members of the nuclear receptor superfamily.

Chronic renal failure is often associated with a resistance to the biologically active form of vitamin D(3), the nuclear hormone 1alpha, 25-dihydroxyvitamin D(3) (VD). The actions of VD are mediated by the vitamin D receptor (VDR), a ligand-dependent transcription factor that binds as a dimeric complex with the retinoid X receptor (RXR) to specific DNA binding sites in the promoter regions of primary VD responding genes, referred to as VD response elements (VDREs). It could be shown in this study that uremic solutions derived from ultrafiltrate from hemodialysis patients and dialysate from peritoneal dialysis patients had an inhibitory effect on the complex formation and ligand inducibility of VDR-RXR heterodimers on different VDRE types. This inhibition was attributed to the formation of Schiff bases between "reactive aldehydes" and lysine residues of the DNA binding domain (DBD) of the VDR, but point mutagenesis data of different lysine residues in this study could not confirm this idea. However, the inhibitory effect of uremic solutions could also be observed for the complex formation of other homo- or heterodimer forming nuclear receptors, whereas an as a monomer binding nuclear receptor did not appear to be affected. These results indicate that VDR is a target of substances in uremic solutions in vitro, but also to some extent other nuclear receptors (i.e., other endocrine signaling systems) may be affected by renal failure.

DNA-Binding Proteins↗

PubMed1999

Positive and negative interaction of 1,25-dihydroxyvitamin D3 and the retinoid CD437 in the induction of human melanoma cell apoptosis.

The natural ligands of the nuclear receptors vitamin D receptor (VDR) and retinoic acid receptor (RAR), i.e., 1alpha,25-dihydroxyvitamin D3 (VD) and all-trans retinoic acid, have important effects on the proliferation, differentiation and apoptosis of a variety of malignant cells, including melanoma. The therapeutic potential of the 2 nuclear hormones can be enhanced by the use of synthetic analogues. In this study, the 2 human melanoma cell lines WM1341 and MeWo were compared for the combined effect of VD and synthetic retinoids. Both cell lines expressed reasonable amounts of VDR, RARgamma and retinoid X receptor alpha and differed only in the relative expression of RARalpha and beta. From 9 functional variants of retinoids, only the RARgamma-selective retinoid CD437 showed, in both cell lines, a significant anti-proliferative effect. In MeWo cells, but not in WM1341 cells, VD induced growth arrest but showed no synergistic interaction with the effects of CD437. In contrast, VD induced apoptosis in WM1341, but not in MeWo, cells. CD437 was a strong inducer of apoptosis in both melanoma cell lines. Parallel treatment with CD437 and VD resulted in synergistic enhancement of apoptosis in WM1341 cells, whereas a clear decrease in induction of apoptosis in MeWo cells occurred. Our results indicate that a combined treatment of melanoma with VD and selected retinoids is promising but should be adapted to individual types of tumor.

Antineoplastic Agents↗

PubMed1999

Lipid soluble vitamins in gene regulation.

Vitamin A (retinol) and vitamin D are lipid soluble vitamins that are precursors of the nuclear hormones all-trans retinoic acid (RA) and 1alpha,25-dihydroxyvitamin D3 (VD) that bind with high affinity to their cognate nuclear receptors, referred to as retinoic acid receptor (RAR) and vitamin D receptor (VDR). Both types of nuclear receptors are structurally related and belong to the same subclass of the nuclear receptor superfamily, a large family of ligand-inducible transcription factors. Both RAR and VDR form heterodimers preferentially with the nuclear receptor for 9-cis RA, referred to as the retinoid X receptor (RXR), but functional RAR-VDR heterodimers have also been observed. Moreover, both types of nuclear receptors interact in a ligand-dependent fashion with members of the same class of co-activator, co-repressor and co-integrator proteins. These similar molecular mechanisms of action provide several possibilities for an interaction of RARs with VDR that are all based on allosteric protein-protein interactions. These interactions can result in either an additive or a transrepressive functional interference between RA and VD. The two remaining lipid soluble vitamins, vitamins E and K, are not known to interact with nuclear receptors, but their structure does not exclude this possibility. Moreover, for vitamin E modulatory effects on transcription factors, such as AP-1, have been described. This review will discuss briefly gene regulation by the four lipid soluble vitamins.

Animals↗

PubMed1999

Potentiation by vitamin D analogs of TNFalpha and ceramide-induced apoptosis in MCF-7 cells is associated with activation of cytosolic phospholipase A2.

Synthetic analogs of vitamin D induce apoptosis in cultured breast cancer cells and cause regression of experimentally-induced rat mammary tumors. To further elucidate the mechanisms involved, we have examined interactions between two vitamin D analogs (CB1093 and EB1089) and known mediators of apoptosis, TNFalpha and ceramide. Pretreatment of MCF-7 breast cancer cells with CB1093 and EB1089 substantially potentiated cytotoxic effects of TNFalpha as assessed by cell viability assay, DNA fragmentation and videomicroscopy. No significant changes in the levels of TNFalpha or TNF-RI transcripts were detected. CB1093 primed cells demonstrated enhanced responsiveness to cell permeable C2-ceramide in terms of increased DNA fragmentation and loss of cell viability. Activation of cytosolic phospholipase A2 (cPLA2) has been implicated in TNFalpha-mediated apoptosis. As assessed by [3H]-arachidonic acid release, cells primed for 48 h with CB1093 (50 nM) showed enhanced cPLA2 activation in response to TNFalpha or ceramide. CB1093 treatment alone led to cPLA2 activation and loss of cell viability which was inhibited by the specific inhibitor AACOCF3. These results suggest that TNFalpha and vitamin D analogs share a common pathway leading to apoptosis involving cPLA2 activation and/or ceramide generation.

Antigens, CD↗

PubMed1999

Selective recognition of vitamin D receptor conformations mediates promoter selectivity of vitamin D analogs.

The transcription factor VDR is the nuclear receptor for 1alpha, 25-dihydroxyvitamin D3 (VD) and the mediator of all genomic actions of the nuclear hormone and its synthetic analogs. The sharp biological profile of the model VD analog 1(S), 3(R)-dihydroxy-20(R)-(5'-ethyl-5'-hydroxy-hepta-1'(E), 3'(E)-dien-1'-yl)-9,10-secopregna-5(Z),7(E),10(19)-triene (EB1089) (i.e., its high antiproliferative effect combined with low calcemic actions) has been correlated with the selectivity of EB1089 to activate heterodimeric complexes of VDR with its partner retinoid X receptor (RXR) on VD response elements (VDREs). These VDREs are formed by an inverted palindromic arrangement of two hexameric core binding motifs spaced by nine nucleotides (IP9) rather than VDREs that are formed by direct repeats with three intervening nucleotides (DR3). In this report, ligand-dependent gel-shift assays were used for a comparison of the ability of VD and EB1089 to stabilize VDR-RXR heterodimers on these two VDRE types. The gel-shift assays revealed EB1089 to be more sensitive for complexes on IP9-type VDREs than on DR3-type VDREs. In addition, a gel-shift clipping method was established to identify and compare complexes of ligand-stabilized VDR-RXR heterodimers on different VDREs. On each VDRE, two complexes could be discriminated that seemed to contain different functional conformations of the VDR and allowed a more differential view on DNA-complexed VDR-RXR heterodimers. The VDR-RXR conformation (which was more ligand-sensitive) gained through EB1089 a higher affinity (7-fold) for DNA binding and a more sensitive (9-fold) activation of an IP9-type VDRE than of a DR3-type VDRE, whereas with the natural hormone VD, no VDRE-type preference could be observed. This indicates that promoter selectivity of VDR ligands is based on their property to selectively increase affinity for VDREs and very sensitively stabilize VDR conformations in VDR-RXR-VDRE complexes.

Calcitriol↗

PubMed1999

Zinc finger transcription factors as molecular targets for nitric oxide-mediated immunosuppression: inhibition of IL-2 gene expression in murine lymphocytes.

BACKGROUND: Nitric oxide (NO) has frequently been shown to display immunosuppressive activities. We describe here a molecular mechanism contributing to this effect. MATERIALS AND METHODS: Murine T cell lymphoma EL4-6.1 cells were activated with the physiological stimulus interleukin (IL)-1beta to express IL-2 mRNA in the presence or absence of subtoxic concentrations of the physiological spontaneous NO donor S-nitrosocysteine (SNOC). Subsequently, semiquantitative RT-PCR and gel shift assays with nuclear extracts were performed to analyze the effects of NO on IL-2 mRNA expression and on the activity of the dominant regulating transcription factors Sp1, EGR-1, and NFATc. RESULTS: NO inhibits IL-1beta-induced IL-2 mRNA expression in EL4-6.1 cells. The suppressive activity of NO was concentration dependent and found to be completely reversible. Importantly, NO at the concentrations used induced neither apoptosis nor necrosis. Dominant regulation of IL-2 gene expression is known to reside in the zinc finger transcription factors Sp1 or EGR-1 and in the non-zinc finger protein NFAT. NO abrogates the DNA binding activities of recombinant Sp1 and EGR-1. More importantly, gel shift assays also showed a lack of DNA binding of native Sp1 derived from NO-treated nuclear extracts and that from NO-treated viable lymphocytes. This effect is selective, as the DNA binding activity of recombinant NFATc was not affected by NO. CONCLUSION: Inactivation of zinc finger transcription factors by NO appears to be a molecular mechanism in the immunosuppressive activity of NO in mammals, thus contributing to NO-mediated inhibition of IL-2 gene expression after physiological stimuli. The exact understanding of the molecular mechanism leading to NO-mediated, fully reversible suppression of immune reactions may lead to use of this naturally occurring tool as an aid in inflammatory diseases.

Animals↗

PubMed1998

The role of the T-box for the function of the vitamin D receptor on different types of response elements.

The nuclear hormone 1alpha,25-dihydroxyvitamin D3(VD) mainly functions through a heterodimer formed between the VD receptor (VDR) and the retinoid X receptor (RXR). This transcription factor complex specifically recognizes DNA sequences, referred to as VD response elements (VDREs), that are formed by two hexameric core binding motifs arranged either as direct repeats spaced by 3 nt (DR3) or inverted palindromes with nine intervening nucleotides (IP9). Gel shift clipping assays provided the first evidence that VDR-RXR heterodimers form different conformations on these two types of VDREs. Since the T-box within the C-terminal extension of the receptor DNA binding domain (DBD) was previously shown to form a dimerization interface with the partner receptor DBD when bound to DR-type response elements, all six amino acid residues of the VDR T-box were investigated for their role in VDR-RXR heterodimer complex formation on DR3- and IP9-type VDREs. Interestingly, the residue Phe93 (F93) was found to be critical on both types of VDREs, whereas the role of the residue Ile94 (I94) was found to depend on ionic strength of the binding reaction and the nature of the VDRE. However, under physiological conditions I94 was also shown to be critical on both VDRE types. The monitored differences between the two VDR-containing protein-DNA complexes helps in an understanding of the differential action of the nuclear hormone VD and its therapeutically important analogues.

Amino Acid Sequence↗

PubMed1998

Structural variants of the vitamin D analogue EB1089 reduce its ligand sensitivity and promoter selectivity.

The nuclear hormone 1alpha,25-dihydroxyvitamin D3 (VD) has important cell-regulatory functions but also a strong calcemic effect. Therefore, various VD analogues have been synthesized and screened for their biological profile. In order to gain more insight into the molecular basis of the high antiproliferative but low calcemic action of the VD analogue EB1089, we characterized this compound in comparison to five structurally related VD analogues. The activities of the six VD analogues in in vitro assays (limited protease digestion assays for determining interaction with monomeric vitamin D receptor (VDR), ligand-dependent gel shift assays for showing the increase of DNA binding of VDR-retinoid X receptor (RXR) heterodimers, and reporter gene assays on different types of VD response elements for demonstrating the efficacy in nuclear VD signalling) were found to represent their biological potency (antiproliferative effect on different malignant cell lines). In this series, EB1089 proved to be the most potent VD analogue; that is, every structural modification (20-epi configuration, cis-configuration at position C24, or changes at the ethyl groups at position C25) appeared to reduce the determined activities mediated through the VDR of these analogues. Moreover, the modifications of EB1089 resulted in a loss of VD response element selectivity, suggesting that this parameter is very critical for the biological profile of this VD analogue.

Calcitriol↗

PubMed1998

Identification of two activating elements in the proximal promoter region of the human glutathione transferase-A1 and -A2 genes.

Promoter regions derived from the human glutathione S-transferase (GST) alpha gene cluster located on chromosome 6p12 were studied: the identical proximal promoters of the GST A1 and GST A2 genes and a proximal promoter of a pseudogene of this class. The sequence of the pseudogene promoter differs in four single nucleotides at positions -86, -66, -41, and -13, and a noncritical TTT insertion at positions -71 to -69 from the GST A1/A2 promoter. Here, it was shown that the GST A1/A2 proximal promoters differed by a factor of 3.4 in their activity from the proximal pseudogene promoter. Therefore, the functional significance of single base exchanges was examined by introducing individual point mutations at the four positions within the proximal GST A1/A2 promoter. In functional tests in transiently transfected human hepatoblastoma HepG2 cells the base exchange at position -13 showed no effect, whereas mutations at position -41 or -86 diminished the promoter activity to a level comparable to the pseudogene promoter. Promoter fragments of both genes spanning over these four sites were analyzed in a heterologous promoter context for their functionality in HepG2 cells. Moreover, gel shift experiments showed specific binding of nuclear proteins to these promoter fragments. The results show that in the proximal GST A1/A2 promoter the sites at position -41 or -86 are essential for the binding of activating transcription factor complexes.

Base Sequence↗

PubMed1998

Metabolism of the vitamin D3 analogue EB1089 alters receptor complex formation and reduces promoter selectivity.

1. 1alpha,25-dihydroxyvitamin3 (VD) is a nuclear hormone that has important cell regulatory functions but also a strong calcemic effect. EB1089 is a potent antiproliferative VD analogue, which has a modified side chain resulting in increased metabolic stability and a selective functional profile. Since EB1089 is considered for potential systemic application, it will be investigated to what extent its recently identified metabolites (hydroxylated at positions C26 and C26a) contribute to biological profile of the VD analogue. 2. Limited protease digestion analysis demonstrated that EB1089 is able to stabilize the high affinity ligand binding conformation of the VDR, starting at concentrations of 0.1 nM and affecting up to 80% of all receptor molecules. The metabolites EB1445 and EB1470 showed to be 100 fold less potent than EB1089, whereas the remaining three metabolites (EB1435, EB1436 and EB1446) showed a clearly reduced ability to stabilize the high affinity ligand binding conformation. Interestingly, at pharmacological concentrations all EB1089 metabolites stabilized a second, apparently lower affinity conformation to a much higher extent than EB1089. 3. In reporter gene assays all metabolites showed lower potency than EB1089. Moreover, the preference of EB1089 for activation of VDR binding to sites formed by inverted palindromic arrangements spaced by nine nucleotide (IP9-type VD response elements) appeared to be reduced (with EB1445 and EB1470) or completely lost (with EB1435, EB1436 and EB1446). The ranking of EB1089 and its metabolites that was obtained by limited protease digestion and reporter gene assays was confirmed by an analysis of their antiproliferative effect in breast cancer cells. . The potency and selectivity of the EB1089 metabolites in mediating gene regulatory effects was found to be drastically reduced in comparison to the parent compound suggesting that the contribution of the metabolites to the biological effect of EB1089 is minor. However, the compounds showed to be interesting tools for understanding the selective biological profile of EB1089.

Antineoplastic Agents↗

PubMed1998

Differential apoptotic response of human melanoma cells to 1 alpha,25-dihydroxyvitamin D3 and its analogues.

Pleiotropic actions of the biologically active form of vitamin D3, 1 alpha,25-dihydroxyvitamin D3 (VD), include antiproliferative effects in both normal human melanocytes and malignant melanoma cell lines. In this study the actions of VD and its low calcemic analogues EB1089 and CB1093, have been examined in two human melanoma cell lines MeWo and WM1341. Both cell lines express similar amounts of vitamin D receptor mRNA and show functional gene regulatory effects in response to VD and its analogues. VD, EB1089 and CB1093 induced apoptosis only in WM1341 cells and not in MeWo cells, even though both cell lines responded well to etoposide, a strong inducer of apoptosis. Additionally, these results were confirmed by analysis of cell morphology. Interestingly in WM1341 cells, CB1093 was found to be more potent in inducing apoptosis than EB1089 and the natural hormone. Moreover, CB1093 appeared to induce apoptosis at a relatively low concentration of 0.1 nM, whereas greater than tenfold higher concentrations of VD and EB1089 were needed to obtain comparable effects. These observations highlight CB1093 as a promising drug for a future treatment against specific types of melanoma.

Antineoplastic Agents↗

PubMed1998

Gene regulation by vitamin D3.

The physiologically active form of vitamin D3, 1 alpha,25-dihydroxyvitamin D3 (VD), is a nuclear hormone with pleiotropic action on the control of calcium homeostasis and bone formation, induction of cellular differentiation and apoptosis, inhibition of cellular proliferation, and other cellular signaling processes. The actions of the hormone are mediated by the vitamin D receptor (VDR), a transcription factor that is a nuclear receptor for VD and a member of the nuclear receptor superfamily. The structural relationship between the members of this transcription factor family suggests similar function in DNA binding, transactivation, and contact to other nuclear proteins. However, each nuclear receptor also demonstrates individual properties that are characteristic and not shared by its respective relatives. In this review, both common as well as individual characteristics of VDR-mediated transcriptional regulation are critically discussed.

Animals↗

PubMed1998

The potential role of the transcription factor RZR/ROR as a mediator of nuclear melatonin signaling.

The pineal gland hormone melatonin is well known as a regulator of circadian rhythmicity, but has also other functions in the central nervous system as well as in the periphery including the maturation of neurons and the regulation of cellular growth and differentiation. Three mechanisms of the hormone's action are currently discussed: a membrane signaling pathway, a nuclear signaling pathway and a receptor-independent radical scavenging function. Melatonin membrane receptors are seven transmembrane receptors and mediate their functions through a G-protein-coupled second messenger pathway. Nuclear melatonin signaling seems to be mediated via the transcription factor RZR/ROR, which is an orphan member of the nuclear receptor superfamily. The widespread distribution of the alpha-subtype of RZR/ROR suggests that this receptor may be an important mediator of those effects of melatonin that can not be explained by membrane receptors or radical scavenging. Interestingly, natural RZR/RORalpha "knock-out" mice (staggerer) show severe defects in the development of cerebellar Purkinje cells, a reduced body weight and immunological defects. RZR/ROR binds as a monomer to DNA, but also forms homodimers on appropriate binding sites. Natural RZR/ROR binding sites have been identified in the regulatory regions of many genes. 5-lipoxygenase, p21WAF1/CIP1, apolipoprotein A-1, N-myc and Purkinje cell protein 2 may be functionally important target genes. On some of these binding sites RZR/ROR competes with other members of the nuclear receptor superfamily (e.g., COUP-TF, RAR and Rev-ErbA) indicating a cross-talk between these transcription factors RZR/ROR often shows in transient transfection assays a high constitutive, i.e. ligand-independent activity. However, under conditions of low constitutive activity a significant and specific stimulation of RZR/ROR by low nanomolar concentrations of melatonin and structurally novel class of thiazolidinediones (lead structure: CGP52608) has been observed. Taken together, the effects of melatonin on transcriptional regulation clearly depend on the expression fo RZR/ROR and support the concept that the receptor is a mediator of nuclear melatonin signaling.

Journal Article↗

PubMed1998

Specific activation of the nuclear receptors PPARgamma and RORA by the antidiabetic thiazolidinedione BRL 49653 and the antiarthritic thiazolidinedione derivative CGP 52608.

The thiazolidinedione BRL 49653 and the thiazolidinedione derivative CGP 52608 are lead compounds of two pharmacologically different classes of compounds. BRL 49653 is a high affinity ligand of peroxisome proliferator-activated receptor gamma (PPARgamma) and a prototype of novel antidiabetic agents, whereas CGP 52608 activates retinoic acid receptor-related orphan receptor alpha (RORA) and exhibits potent antiarthritic activity. Both receptors belong to the superfamily of nuclear receptors and are structurally related transcription factors. We tested BRL 49653 and CGP 52608 for receptor specificity on PPARgamma, RORA, and retinoic acid receptor alpha, a closely related receptor to RORA, and compared their pharmacological properties in in vitro and in vivo models in which these compounds have shown typical effects. BRL 49653 specifically induced PPARgamma-mediated gene activation, whereas CGP 52608 specifically activated RORA in transiently transfected cells. Both compounds were active in nanomolar concentrations. Leptin production in differentiated adipocytes was inhibited by nanomolar concentrations of BRL 49653 but not by CGP 52608. BRL 49653 antagonized weight loss, elevated blood glucose levels, and elevated plasma triglyceride levels in an in vivo model of glucocorticoid-induced insulin resistance in rats, whereas CGP 52608 exhibited steroid-like effects on triglyceride levels and body weight in this model. In contrast, potent antiarthritic activity in rat adjuvant arthritis was shown for CGP 52608, whereas BRL 49653 was nearly inactive. Our results support the concept that transcriptional control mechanisms via the nuclear receptors PPARgamma and RORA are responsible at least in part for the different pharmacological properties of BRL 49653 and CGP 52608. Both compounds are prototypes of interesting novel therapeutic agents for the treatment of non-insulin-dependent diabetes mellitus and rheumatoid arthritis.

3T3 Cells↗