Left ventricular remodeling in swine after myocardial infarction: a transcriptional genomics approach

Despite the apparent appropriateness of left ventricular (LV) remodeling following myocardial infarction (MI), it poses an independent risk factor for development of heart failure. There is a paucity of studies into the molecular mechanisms of LV remodeling in large animal species. We took an unbiased molecular approach to identify candidate transcription factors (TFs) mediating the genetic reprogramming involved in post-MI LV remodeling in swine. Left ventricular tissue was collected from remote, non-infarcted myocardium, 3 weeks after MI-induction or sham-surgery. Microarray analysis identified 285 upregulated and 278 downregulated genes (FDR < 0.05). Of these differentially expressed genes, the promoter regions of the human homologs were searched for common TF binding sites (TFBS). Eighteen TFBS were overrepresented >two-fold (p < 0.01) in upregulated and 13 in downregulated genes. Left ventricular nuclear protein extracts were assayed for DNA-binding activity by protein/DNA array. Out of 345 DNA probes, 30 showed signal intensity changes >two-fold. Five TFs were identified in both TFBS and protein/DNA array analyses, which showed matching changes for COUP-TFII and glucocorticoid receptor (GR) only. Treatment of swine with the GR antagonist mifepristone after MI reduced the post-MI increase in LV mass, but LV dilation remained unaffected. Thus, using an unbiased approach to study post-MI LV remodeling in a physiologically relevant large animal model, we identified COUP-TFII and GR as potential key mediators of post-MI remodeling

Pro-asthmatic cytokines regulate unliganded and ligand-dependent glucocorticoid receptor signaling in airway smooth muscle

To elucidate the regulation of glucocorticoid receptor (GR) signaling under pro-asthmatic conditions, cultured human airway smooth muscle (HASM) cells were treated with proinflammatory cytokines or GR ligands alone and in combination, and then examined for induced changes in ligand-dependent and -independent GR activation and downstream signaling events. Ligand stimulation with either cortisone or dexamethsone (DEX) acutely elicited GR translocation to the nucleus and, comparably, ligand-independent stimulation either with the Th2 cytokine, IL-13, or the pleiotropic cytokine combination, IL-1β/TNFα, also acutely evoked GR translocation. The latter response was potentiated by combined exposure of cells to GR ligand and cytokine. Similarly, treatment with either DEX or IL-13 alone induced GR phosphorylation at its serine-211 residue (GRSer211), denoting its activated state, and combined treatment with DEX+IL-13 elicited heightened and sustained GRSer211phosphorylation. Interestingly, the above ligand-independent GR responses to IL-13 alone were not associated with downstream GR binding to its consensus DNA sequence or GR transactivation, whereas both DEX-induced GR:DNA binding and transcriptional activity were significantly heightened in the presence of IL-13, coupled to increased recruitment of the transcriptional co-factor, MED14. The stimulated GR signaling responses to DEX were prevented in IL-13-exposed cells wherein GRSer211 phosphorylation was suppressed either by transfection with specific serine phosphorylation-deficient mutant GRs or treatment with inhibitors of the MAPKs, ERK1/2 and JNK. Collectively, these novel data highlight a heretofore-unidentified homeostatic mechanism in HASM cells that involves pro-asthmatic cytokine-driven, MAPK-mediated, non-ligand-dependent GR activation that confers heightened glucocorticoid ligand-stimulated GR signaling. These findings raise the consideration that perturbations in this homeostatic cytokine-driven GR signaling mechanism may be responsible, at least in part, for the insensirtivity to glucocorticoid therapy that is commonly seen in individuals with severe asthma

Interaction fonctionnelle de l’Hsp90 avec les récepteurs des stéroïdes

L’Hsp90 (Heat Shock Protein 90) fait partie de la structure hétéro-oligomérique métastable et inactive des récepteurs des stéroïdes. Les données récentes sur ses propriétés de protéine de stress, de chaperon moléculaire spécialisé ainsi que les acquisitions sur sa structure tridimensionnelle sont ici exposées avec une focalisation particulière sur son impact dans la signalisation par les stéroïdes. En particulier, son double rôle de modulateur positif et négatif vis-à-vis de la fonction des récepteurs est analysée tout au long du processus d’activation-désactivation par les ligands stéroïdiens. Il est proposé que la machinerie de chaperonnage de l’Hsp90 assiste le récepteur dès sa synthèse en évitant son collapsus et en facilitant sa structure ouverte, apte à lier l’hormone. De plus, l’Hsp90 pourrait intervenir au cours de la structuration du core hydrophobe du récepteur autour de l’hormone, mais également prendre en charge le récepteur après la dissociation du ligand

A natural sequence consisting of overlapping glucocorticoid-responsive elements mediates glucocorticoid, but not androgen, regulation of gene expression.

Cytosolic aspartate aminotransferase (cAspAT) is regulated by glucocorticoids in rat liver and kidney. Part of this regulation is mediated by an unusual glucocorticoid-responsive element (GRE)-like sequence called GRE A. GRE A is composed of two overlapping imperfect GREs, each comprising a conserved half-site (half-sites 1 and 4 respectively) and a poorly conserved half-site (half-sites 2 and 3 respectively). The sequence binds co-operatively two dimers of the glucocorticoid receptor (GR) and mediates efficient glucocorticoid regulation of gene expression. Analysis of deletions of the cAspAT gene promoter and subcloning of GRE A upstream of the thymidine kinase promoter indicate that this sequence is responsive to glucocorticoids, but not to androgens. Electrophoretic mobility shift assays indicate that the GRE A unit does not bind the androgen receptor (AR). The modification of three nucleotides in the poorly conserved half-sites 2 and 3, converting GRE A into two overlapping high-affinity GREs (ov-cGRE), resulted in co-operative binding of the AR. Furthermore, ov-cGRE efficiently mediated androgen regulation of the thymidine kinase promoter. A single base modification in half-site 2 or 3 in GRE A allowed the binding of the AR as one or two dimers respectively, and restored transcriptional activation by androgens only in the latter case. Thus the poor affinity of the AR for half-sites 2 and 3 prevented its binding to GRE A, indicating that the overlapping GRE A sequence of the cAspAT gene promoter discriminates a glucocorticoid-mediated from an androgen-mediated response