The anti-inflammatory action of glucocorticoid hormones.
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Animals↗
Biomedical subjects
M Göttlicher
Publications and source records attributed to M Göttlicher.
Valproic acid defines a novel class of HDAC inhibitors inducing differentiation of transformed cells.
Histone deacetylases (HDACs) play important roles in transcriptional regulation and pathogenesis of cancer. Thus, HDAC inhibitors are candidate drugs for differentiation therapy of cancer. Here, we show that the well-tolerated antiepileptic drug valproic acid is a powerful HDAC inhibitor. Valproic acid relieves HDAC-dependent transcriptional repression and causes hyperacetylation of histones in cultured cells and in vivo. Valproic acid inhibits HDAC activity in vitro, most probably by binding to the catalytic center of HDACs. Most importantly, valproic acid induces differentiation of carcinoma cells, transformed hematopoietic progenitor cells and leukemic blasts from acute myeloid leukemia patients. More over, tumor growth and metastasis formation are significantly reduced in animal experiments. Therefore, valproic acid might serve as an effective drug for cancer therapy.
Animals↗
Repression of inflammatory responses in the absence of DNA binding by the glucocorticoid receptor.
The glucocorticoid receptor (GR) acts both as a transcription factor itself on genes carrying GR response elements (GREs) and as a modulator of other transcription factors. Using mice with a mutation in the GR, which cannot activate GRE promoters, we examine whether the important anti-inflammatory and immune suppressive functions of glucocorticoids (GCs) can be established in this in vivo animal model. We find that most actions are indeed exerted in the absence of the DNA-binding ability of the GR: inhibition of the inflammatory response of locally irritated skin and of the systemic response to lipopolysaccharides. GCs repress the expression and release of numerous cytokines both in vivo and in isolated primary macrophages, thymocytes and CD4(+) splenocytes. A transgenic reporter gene controlled by NF-kappa B exclusively is also repressed, suggesting that protein- protein interaction with other transcription factors such as NF-kappa B forms the basis of the anti-inflammatory activity of GR. The only defect of immune suppression detected so far concerns the induced apoptosis of thymocytes and T lymphocytes.
Animals↗
Novel target genes of the Ah (dioxin) receptor: transcriptional induction of N-myristoyltransferase 2.
Dioxins are potent mammalian carcinogens and toxins affecting liver, skin, and immune and reproductive systems. The intracellular Ah receptor, a ligand-dependent transcription factor of the basic region/helix-loop-helix/Per-Ahr/Arnt-Sim homology domain (bHLH-PAS) protein family, mediates responses to dioxins. Target genes of the Ah receptor that mediate dioxin toxicity and carcinogenicity are, however, mostly unknown. We used 5L rat hepatoma cells to identify dioxin-inducible genes by suppression subtractive hybridization. Eleven cDNA fragments were identified that represented novel sequences or genes for which induction by dioxins had not been known. N-myristoyltransferase 2 (NMT2) is one of the later dioxin-inducible genes. Induction of NMT2 was confirmed in livers of mice in vivo. NMT2 induction was a direct consequence of Ah receptor activation in 5L cells. [(3)H]myristic acid incorporation into 5L cell proteins was inducible by dioxins, indicating that protein myristoylation is a regulated rather than a housekeeping function and that NMT activity is limiting in noninduced 5L cells. Here we show for the first time that expression of NMT2 and induced protein myristoyltransferase activity are direct responses to carcinogen exposure. Because inappropriate protein NH(2)-terminal myristoylation appears to play a role in carcinogenesis, induction of NMT2 may play a central role in dioxin carcinogenicity.
Acyltransferases↗
Histone deacetylase as a therapeutic target.
The maintenance of health depends on the coordinated and tightly regulated expression of genetic information. Certain forms of leukemia have become paradigms for the pathogenic role of aberrant repression of differentiation genes. In these acute leukemias, fusion proteins generated by chromosomal translocations no longer function as transcriptional activators, but instead repress target genes by recruiting histone deacetylases (HDACs). The potential benefit of HDAC inhibition has been established by the use of enzyme inhibitors in vitro and in a single reported case of experimental therapy. Because recently identified HDAC inhibitors appear to overcome many drawbacks of early inhibitory compounds in clinical use, the stage is set to test the therapeutic value of HDAC inhibition in leukemias and in other diseases, including solid tumors and aberrant hormonal signaling. This review summarizes the range of diseases expected to respond to HDAC inhibition.
Acute Disease↗
Redox regulation in mammalian signal transduction.
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Animals↗
Induction of differentiation in F9 cells and activation of peroxisome proliferator-activated receptor delta by valproic acid and its teratogenic derivatives.
The antiepileptic drug valproic acid (VPA) is teratogenic, because it induces birth defects in some children of mothers treated for epilepsy. Cellular and molecular actions associated with teratogenicity were identified by testing differentiation of F9 embryocarcinoma cells. VPA altered cell morphology and delayed proliferation. Specific differentiation markers (e.g., c-fos and keratin 18 mRNA and particularly the activating protein-2 transcription factor protein) were induced. This pattern differs from the pattern induced by other teratogens or F9 cell-differentiating agents. Induction of differentiation correlated with teratogenicity because teratogenic derivatives of VPA, such as (S)-4-yn-VPA, induced differentiation, whereas closely related nonteratogenic compounds, such as (R)-4-yn-VPA, 2-en-VPA, and 4-methyl-VPA, did not. In the cellular signaling network, the peroxisome proliferator-activated receptor delta (PPARdelta) was activated selectively by VPA and teratogenic derivatives. Depletion of PPARdelta by antisense RNA expression precluded the response of F9 cells to VPA. In conclusion, our data show that VPA and its teratogenic derivatives induce a specific type of F9 cell differentiation and that PPARdelta is a limiting factor in the control of differentiation.
Animals↗
Prediction of embryotoxic effects of valproic acid-derivatives with molecular in vitro methods.
Therapy with the antiepileptic drug valproic acid (2-propylpentanoic acid, VPA) during early pregnancy can cause similar teratogenic effects (neural tube defects) in human and mice. In this study a new molecular bioassay is presented using following endpoints: differentiation of F9 teratocarcinoma cells, altered cell morphology, induction of possible targeted genes, and the induction of viral RSV-promoter. The induction of a transiently transfected viral (RSV) promoter driven luciferase gene by VPA was used to screen a set of VPA-derivatives. Structure-activity investigations showed: the longer the aliphatic side chain the more the induction of the RSV-reporter gene. The specific induction was stereoseletive. The teratogenic enantiomer S-4-yn-VPA (2-propyl-4-pentynoic acid) induced the RSV-driven reporter gene while the non teratogenic R-4-yn-VPA does not. Heptyl-4-yn-VPA was the most potent teratogen in vitro and in vivo. Non teratogenic VPA-derivatives like R-4-yn-VPA and 2-en-VPA (2-propyl-2-pentenoic acid) were ineffective in this system. Thus, the teratogenic effect of VPA and VPA-derivatives in the mouse correlated with the specific induction of the viral RSV-promoter controlled reporter in F9-cells. Acid compounds such as fatty acids are known to interact with peroxisome proliferator-activated receptors (PPARs). To test structure-activity relationships by VPA or its derivatives we used CHO cells stably expressing hybrid proteins of the ligand-binding domain of either of the PPARs. The teratogen VPA and the teratogenic derivatives of VPA activated the PPAR-delta construct in a very specific structure- and stereoselective way which correlated well with the activities in the reporter gene assay (bioassay) and those in vivo. No such correlation was found with respect to activation of PPAR-alpha or PPAR-gamma. These structure-activity relationships indicate that PPAR-delta may be a potential mediator of VPA-induced differentiation of F9 cells and may possibly be involved in the mechanism of teratogenicity of VPA in vivo. Furthermore two bioassays were designed with clearly defined endoints, amenable to automation and screening of great number of compounds. The test system allows to replace animal experiments in the preclinical development of new antiepileptics drugs with reduced teratogenic risk. Supported by BgVV-ZEBET (Berlin).
Animals↗
New molecular bioassays for the estimation of the teratogenic potency of valproic acid derivatives in vitro: activation of the peroxisomal proliferator-activated receptor (PPARdelta).
Therapy with the antiepileptic drug valproic acid (2-propylpentanoic acid, VPA) during early pregnancy can cause teratogenic effects (neural tube defects) in humans and in mice. VPA and a teratogenic derivative specifically induce differentiation of F9 teratocarcinoma cells and activate PPARdelta. We have now studied structure-activity relationships of 11 VPA-related compounds by quantitatively comparing their teratogenic potency with their effects in the two novel in vitro systems. Based on the induction of a Rous sarcoma virus (RSV) promoter-driven reporter gene, which is associated with the differentiation of F9 cells, a system suitable for high-throughput and quantitative screening was established. Structure-activity investigations showed that only teratogenic derivatives of VPA induced the response in F9 cells as well as activated the PPARdelta-dependent reporter system in Chinese hamster ovary (CHO) cells. Increases in the length of the side chain in the VPA-related 2-alkyl-pentynoic acid generate more potent inducers in the cell-culture-based assays, which also show higher teratogenicity and embryonic lethality rates. Activation of PPARdelta correlated well with the effects in the F9 cell assay and with teratogenic potency in vivo (p < 0.007). Evaluation of the effects of the presented set of compounds allows the conclusion that the in vitro systems faithfully reflect teratogenicity of VPA-related compounds. Whether the activation of PPARdelta is causally related to the disruption of proper embryonic development or whether it reflects other yet unknown VPA-induced events remains to be established.
Animals↗
In contrast with docosahexaenoic acid, eicosapentaenoic acid and hypolipidaemic derivatives decrease hepatic synthesis and secretion of triacylglycerol by decreased diacylglycerol acyltransferase activity and stimulation of fatty acid oxidation.
Hypolipidaemic fatty acid derivatives and polyunsaturated fatty acids decrease concentrations of plasma triacylglycerol by mechanisms that are not fully understood. Because poor susceptibility to beta- and/or omega-oxidation is apparently a determinant of the peroxisome proliferating and hypolipidaemic capacity of fatty acids and derivatives, the relative importance of activation of the peroxisome-proliferator-activated receptor alpha (PPARalpha), fatty acid oxidation and triacylglycerol synthesis were examined. We have compared the effects of differentially beta-oxidizable fatty acids on these parameters in primary cultures of rat hepatocytes. Tetradecylthioacetic acid (TTA), 2-methyleicosapentaenoic acid and 3-thia-octadecatetraenoic acid, which are non-beta-oxidizable fatty acid derivatives, were potent activators of a glucocorticoid receptor (GR)-PPARalpha chimaera. This activation was paradoxically reflected in an substantially increased oxidation of [1-(14)C]palmitic acid and/or oleic acid. The incorporation of [1-(14)C]palmitic acid and/or oleic acid into cell-associated and secreted triacylglycerol was decreased by 15-20% and 30% respectively with these non-beta-oxidizable fatty acid derivatives. The CoA ester of TTA inhibited the esterification of 1, 2-diacylglycerol in rat liver microsomes. Both eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) activated GR-PPARalpha. EPA increased the oxidation of [1-(14)C]palmitic acid but DHA had no effect. The CoA ester of EPA inhibited the esterification of 1, 2-diacylglycerol, whereas DHA-CoA had no effect. The ratio between synthesized triacylglycerol and diacylglycerol was lower in hepatocytes cultured with EPA in the medium compared with DHA or oleic acid, indicating a decreased conversion of diacylglycerol to triacylglycerol. Indeed, the incorporation of [1-(14)C]oleic acid into secreted triacylglycerol was decreased by 20% in the presence of EPA. In conclusion, a decreased availability of fatty acids for triacylglycerol synthesis by increased mitochondrial beta-oxidation and decreased triacylglycerol formation caused by inhibition of diacylglycerol acyltransferase might explain the hypolipidaemic effect of TTA and EPA.
Acyltransferases↗
p27(Kip1) induction and inhibition of proliferation by the intracellular Ah receptor in developing thymus and hepatoma cells.
The Ah receptor (AhR), a bHLH/PAS transcription factor, mediates dioxin toxicity in the immune system, skin, testis and liver. Toxic phenomena are associated with altered cell proliferation or differentiation, but signaling pathways of AhR in cell cycle regulation are poorly understood. Here we show that AhR induces the p27(Kip1) cyclin/cdk inhibitor by altering Kip1 transcription in a direct mode without the need for ongoing protein synthesis or cell proliferation. This is the first example of Kip1 being a direct transcriptional target of a toxic agent that affects cell proliferation. Kip1 causes dioxin-induced suppression of 5L hepatoma cell proliferation because Kip1 antisense-expressing cells are resistant to dioxins. Kip1 is also induced by dioxins in cultures of fetal thymus glands concomitant with inhibition of proliferation and severe reduction of thymocyte recovery. Kip1 expression is likely to mediate these effects as thymic glands of Kip1-deficient mice (Kip1(Delta51)) are largely, though not completely, resistant.
Animals↗
Sequential DNA damage-independent and -dependent activation of NF-kappaB by UV.
NF-kappaB activation in response to UV irradiation of HeLa cells or of primary human skin fibroblasts occurs with two overlapping kinetics but totally different mechanisms. Although both mechanisms involve induced dissociation of NF-kappaB from IkappaBalpha and degradation of IkappaBalpha, targeting for degradation and signaling are different. Early IkappaBalpha degradation at 30 min to approximately 6 h is not initiated by UV-induced DNA damage. It does not require IkappaB kinase (IKK), as shown by introduction of a dominant-negative kinase subunit, and does not depend on the presence of the phosphorylatable substrate, IkappaBalpha, carrying serines at positions 32 and 36. Induced IkappaBalpha degradation requires, however, intact N- (positions 1-36) and C-terminal (positions 277-287) sequences. IkappaB degradation and NF-kappaB activation at late time points, 15-20 h after UV irradiation, is mediated through DNA damage-induced cleavage of IL-1alpha precursor, release of IL-1alpha and autocrine/paracrine action of IL-1alpha. Late-induced IkappaBalpha requires the presence of Ser32 and Ser36. The late mechanism indicates the existence of signal transfer from photoproducts in the nucleus to the cytoplasm. The release of the 'alarmone' IL-1alpha may account for some of the systemic effects of sunlight exposure.
Autocrine Communication↗
Transcriptional cross-talk, the second mode of steroid hormone receptor action.
Physiological and therapeutic activities of glucocorticoids and other steroid hormones are mediated by the family of steroid hormone receptors. In addition to the classical mode of receptor action which involves binding as a dimer to regulatory sequences in target gene promoters and subsequent activation of transcription, a second mode of action is based predominantly on protein-protein interactions. As the paradigm of this so-called transcriptional cross-talk, the glucocorticoid receptor (GR) and the AP-1 transcription factor interact on target gene promoters which contain only a binding site for either one of the two transcription factors. Most frequently negative interference of both factors with each other's activity has been observed, for example, when AP-1 is composed of c-Fos and c-Jun; however, synergism is also possible under cell-specific conditions and when AP-1 is a homodimer of c-Jun. Since the detection of the GR/AP-1 cross-talk numerous other examples of transcription factor interactions have been described. Many members of the nuclear hormone receptor superfamily, including class II receptors, have been shown to participate in such cross-talk. Moreover, the transcription factor families of NF-kappaB/Rel as well as Stat, Oct, and C/EBP are engaged in cross-talk with steroid receptors. Despite the identification of a multitude of target genes which appear to be regulated by this type of transcription factor interaction, the exact molecular mechanism of the cross-talk has not yet been elucidated. This review discusses the current models to explain the molecular events of transcription factor cross-talk. Concepts are emphasized which suggest that the classical and the cross-talk mode of steroid receptor action can be triggered separately by the choice of specific ligands. A final section summarizes the partially contradictory data which assign a certain type of receptor action to a biological response particularly in the immune system.
Animals↗
Identification of IHABP, a 95 kDa intracellular hyaluronate binding protein.
The extracellular matrix component hyaluronan is believed to play important roles in various processes of organogenesis, cell migration and cancer. Recognition of and binding to hyaluronan is mediated by cell surface receptors. Three of them, CD44, ICAM-1 and RHAMM (receptor for hyaluronic acid mediated motility), have been identified. A cDNA clone designated RHAMM turned out to possess transforming capacity. Based on this published sequence, we isolated the complete cDNA of the murine gene. The cDNA comprises an open reading frame of 2.3 kb and encodes a 95 kDa protein. The protein carries a hyaluronan binding motif which binds to hyaluronan in vitro but not to heparin or chondroitin sulphate. It is ubiquitously expressed in normal cells and in all tumour cell lines irrespective of their metastatic properties. One tumour cell line, the metastatic Lewis lung carcinoma, expresses a larger 105 kDa variant form of the protein due to a genomic rearrangement. Antibodies raised against the 95 kDa protein were used for subcellular localization studies. The hyaluronan binding protein is not detectable at the cell surface but is rather localized exclusively intracellularly. Clearly, the sequence we have identified encodes a protein with properties substantially different to the RHAMM protein. We tentatively name the protein intracellular hyaluronic acid binding protein, IHABP.
3T3 Cells↗
I kappaB alpha-independent downregulation of NF-kappaB activity by glucocorticoid receptor.
I kappaB alpha is an inhibitor protein that prevents nuclear transport-and activation of the transcription factor NF-kappaB. In acute inflammation, NF-kappaB is activated and increases the expression of several pro-inflammatory cytokine and chemokine genes. Glucocorticoids counteract this process. It has been proposed that the glucocorticoid-dependent inhibition of NF-kappaB activity is mediated by increased synthesis of I kappaB alpha which should then sequester NF-kappaB in an inactive cytoplasmic form. Here, we show by the use of a mutant glucocorticoid receptor and steroidal ligands that hormone-induced I kappaB alpha synthesis and inhibition of NF-kappaB activity are separable biochemical processes. A dimerization-defective glucocorticoid receptor mutant that does not enhance the I kappaB alpha level is still able to repress NF-kappaB activity. Conversely, glucocorticoid analogues competent in enhancing I kappaB alpha synthesis do not repress NF-kappaB activity. These results demonstrate that increased synthesis of I kappaB alpha is neither required nor sufficient for the hormone-mediated downmodulation of NF-kappaB activity.
Animals↗
Complementation of Ah receptor deficiency in hepatoma cells: negative feedback regulation and cell cycle control by the Ah receptor.
The Ah receptor (AhR) is a ligand-dependent transcription factor subunit that heterodimerizes with the AhR nuclear translocator (Arnt) and mediates the predominant biological effects of 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD). TCDD activates target genes in xenobiotica metabolism in many cell lines and, more specifically, delays G1-S progression of 5L hepatoma cells. Here we describe transient and stable AhR-expression analysis in AhR-deficient subclones of the TCDD-sensitive 5L cells. We tested the integrity of the AhR-signaling system beyond the lack of the receptor in the variant subclone and analyzed the role of AhR in cell cycle regulation. Transiently expressed AhR has a high basal activity on promoters containing AhR-binding sites, so-called XREs, when transfected into receptor-deficient variant cells compared to wild-type cells. Single- and double-hybrid analysis dissociates AhR ligand responsiveness, transactivation, and heterodimerization with Arnt from receptor binding to an XRE. Hybrid receptors also show the high basal activity in the absence of exogenous TCDD in AhR-deficient variant cells, indicating that the endogenous AhR-activating signal acts directly on the receptor rather than XRE-dependent promoters or DNA binding of the receptor. Stable expression of AhR in variant cell clones by retroviral infection fully reconstitutes TCDD responsiveness, including target-gene induction and delay of cell cycle progression. These AhR-reconstituted cells, like AhR-containing wild-type cells, show low basal activity of the transiently expressed AhR hybrid. Thus, the increased basal activity in AhR-deficient cells suggests a negative feedback control of AhR activity. In vitro ligand-binding assays are compatible with the idea that the increased basal activity is due to the accumulation of an AhR-binding endogenous ligand. In conclusion, AhR is causally responsible for TCDD-dependent cell cycle regulation and feedback control of AhR activity.
Animals↗
Interaction of the Ubc9 human homologue with c-Jun and with the glucocorticoid receptor.
Glucocorticoid hormones convert the glucocorticoid receptor (GR) from an inactive cytosolic complex to a nuclear form that regulates transcription. Binding of GR to palindromic DNA-recognition sites (hormone response elements) leads to activated target gene transcription. GR also exerts negative actions on transcription, e.g., by interfering with the function of several other transcription factors such as AP-1, NK-kappa B, CREB, and Oct-1. Physical interactions of GR with AP-1 subunits are readily detectable but do not seem sufficient since nonrepressing GR mutants still interact in vitro, so that specific conformational changes and/or interactions with additional partner proteins may be required for negative action. In an attempt to find such partner proteins, we defined regions of c-Jun and GR essential for mutual interference and used in those a yeast two-hybrid screen for interacting proteins. Repeatedly we isolated overlapping cDNA sequences of one protein interaction with both c-Jun and GR. This protein does not interact with c-Fos or a non-repressing GR mutant and expressed in mammalian cells does not substantially affect AP-1 or GR activity. Interestingly, however, the protein rescues yeast cells from the toxic effects of the GR fragment used for screening. The protein represents the human homologue of the yeast E2 ubiquitin-conjugating enzyme, Ubc9; its specific interactions with both GR and c-Jun, but not mutant GR, suggest that it may exert physiologic regulatory functions.
Animals↗
Fatty acid activation of peroxisome proliferator-activated receptor (PPAR).
Peroxisome proliferators such as clofibric acid, nafenopin, and WY-14,643 have been shown to activate peroxisome proliferator-activated receptor (PPAR), a member of the steroid nuclear receptor superfamily. We have cloned the cDNA from rat that is homologous to that from mouse, which encodes a 97% similar protein. To search for physiologically occurring activators, we established a transcriptional transactivation assay by stably expressing in CHO cells a chimera of rat PPAR and the human glucocorticoid receptor that activates expression of the placental alkaline phosphatase reporter gene under the control of the mouse mammary tumor virus promoter. 150 microM concentrations of arachidonic or linoleic acid but not of dehydroepiandrosterone, cholesterol, or 25-hydroxy-cholesterol, activated the receptor chimera. In addition, saturated fatty acids induced the reporter gene. Shortening the chain length to n = 6 or introduction of an omega-terminal carboxylic group abolished the activation potential of the fatty acid. To test whether a common PPAR binding metabolite might be formed from free fatty acids we tested the effects of differentially beta-oxidizable fatty acids and inhibitors of fatty acid metabolism. The peroxisomal proliferation-inducing, non-beta-oxidizable, tetradecylthioacetic acid activated PPAR to the same extent as the strong peroxisomal proliferator WY-14,643, whereas the homologous beta-oxidizable tetradecylthiopropionic acid was only as potent as a non-substituted fatty acid. Cyclooxygenase inhibitors, radical scavengers or cytochrome P450 inhibitors did not affect activation of PPAR. In conclusion, beta-oxidation is apparently not required for the formation of the PPAR-activating molecule and this moiety might be a fatty acid, its ester with CoA, or a further derivative of the activated fatty acid prior to beta-oxidation of the acyl-CoA ester.
Animals↗