Mikael Brönnegård

EVIDENCE THAT THE BETA -ISOFORM OF THE HUMAN GLUCOCORTICOID RECEPTOR DOES NOT ACT AS A PHYSIOLOGICALLY SIGNIFICANT REPRESSOR

Alternative splicing of the human glucocorticoid receptor (hGR) primary transcript generates two receptor isoforms, hGRα and hGRβ, with different carboxyl termini diverging at amino acid 727. By reverse transcriptase-polymerase chain reactions it was previously demonstrated that the hGRβ message had a widespread tissue distribution. To demonstrate the presence of hGRβ as protein we produced specific rabbit antisera to hGRβ, as well as a hGRβ-specific mouse monoclonal IgM antibody, by peptide immunizations. By SDS-polyacrylamide gel electrophoresis and Western immunoblotting we showed that hGRβ is endogenously expressed at the protein level in HeLa cells and human lymphatic leukemia cells. Using an antibody directed against an epitope shared by both isoforms we showed a relatively lower expression of the hGRβ form. We also showed that hGRβ bound to hsp90 by immunoprecipitation of in vitro translated hGRβ in reticulocyte lysate with hsp90-specific antibodies, a coprecipitation occurring also in the presence of dexamethasone. We could not demonstrate that hGRβ inhibited the effects of dexamethasone-activated hGRα on a glucocorticoid-responsive reporter gene. In conclusion, low hGRβ expression levels and hGRβ-hsp90 interaction maintained in the presence of ligand and lack of inhibition of hormone-activated hGRα effects challenge the concept of the hGRβ isoform as a proposed dominant negative inhibitor of hGRα activity.

Glucocorticoid Resistant Syndromes—Molecular Basis andClinical Presentations

The mechanisms of action of glucocorticoid hormones are mediated via specific intracellular receptor proteins. The glucocorticoid receptor (GR) regulates expression of specific target genes or gene networks by ligand‐dependent transcriptional activation, i.e. ligand‐dependent activation of the receptor with subsequent dimer formation and DNA binding. There are a number of factors, such as the receptor concentration, receptor associated proteins, receptor alterations and the effects on the gene network including hormonal regulation of transcription, mRNA splicing and translation, that might influence glucocorticoid responsiveness in a normal and healthy population as well as in different diseases. Several categories of glucocorticoid resistance have been described including inherited GR resistance which has been explained in terms of specific mutations and offers an important model for genetic and clinical studies of steroid sensitivity, and relative glucocorticoid resistance, which occurs naturally in the course of cellular differentiation, cell to cell or tissue to tissue, since all cells possess receptors for glucocorticoids but do not show the same response to them. From a clinical point of view, it is also interesting to consider preexisting genetic susceptibility to glucocorticoids, acquired changes in the GR gene structure and organization, including alterations of noncoding sequences, and the importance of mutations, deletions and other changes in the GR gene affecting receptor function. Analysis of mutations within the receptor resulting in relative glucocorticoid resistance, both generalized inherited glucocorticoid resistance (GIGR) and directed mutagenesis, has identified two regions of clustered mutations in the proximity of previosuly identified affinity labeled residues directly affecting the steroid binding function. Finally, studies of New Words primates and cell lines derived from hematologic malignancies constitute animal and human models for the molecular basis of glucocorticoid resistance where a number of inherited and aquired mutations in the GR gene have been demonstrated.

Growth hormone increases the lipolytic sensitivity for catecholamines in adipocytes from healthy adults

The lipolytic effect of growth hormone (GH) was investigated in adipocytes obtained during elective surgery from otherwise healthy adults, 18-40 years old. No lipolytic or antilipolytic effect of GH was found when the cells were incubated with GH alone during 30min-6h. When the cells were preincubated with GH during 3h, the lipolytic sensitivity for isoprenaline increased markedly without any change in maximal lipolysis. However, a full effect was only obtained if GH was also present during the incubation with isoprenaline. GH did not alter DB-CAMP, enprophylline, or forskolin-induced lipolysis in human fat cells. In conclusion, GH had no direct lipolytic effect on human fat cells but GH markedly increased the catecholamine sensitivity. The site of the GH effect seems to be in the beta-adrenoceptors or in the Gs coupling protein.

The Relationship of Nasal Polyps, Infection, and Inflammation:

The role of infection as cause or effect in nasal polyps is debated. In experimentally induced sinusitis in rabbits, polyps are frequent. The initial polyp formation sequence involves multiple epithelial disruptions with proliferating granulation tissue. Regenerating epithelial branches spread into the underlying connective tissue, where intraepithelial microcavities give rise to a polyp body from the adjacent mucosa. Clinical as well as experimental studies indicate that nasal polyp formation and growth are activated and perpetuated by an integrated process of mucosal epithelium, matrix, and inflammatory cells, which in turn may be initiated by both infectious and noninfectious inflammation. The complexity of the pathophysiologic events in nasal polyposis is reinforced by the finding that epithelial desquamation, combined with infection or inflammation, will initiate polyp formation. Systemic glucocorticosteroids inhibit polyp formation as well as growth of pathogenic bacteria in the sinuses of rabbits with experimental infection. Therapeutic use of corticosteroids in polyp disease, combined with antibiotics or surgery, should be modified in relation to long-term progression, intensity variations, and predisposing conditions.

Molecular basis of glucocorticoid-resistant syndromes

Generalized inherited glucocorticoid resistance (GIGR) is a rare syndrome characterized by elevated levels of plasma cortisol but lacking the symptoms of Cushings syndrome. Biochemically, the condition is characterized by a relative resistance to glucocorticoids that can be compensated for by the elevated levels of cortisol. The inheritance pattern of GIGR is incompletely understood, and one of the central questions is whether there is a correlation between genotype and phenotype. Analysis of mutations within the receptor resulting in relative glucocorticoid resistance has identified two regions of clustered mutations in the proximity of previously identified affinity-labeled residues, the putative steroid-binding site. In the majority of cases, the mutation affects steroid binding and transactivation to the same degree, with the exceptions suggesting an explanation for the variability of the clinical manifestations. From a clinical point of view, in addition to preexisting genetic resistance to glucocorticoids, it is important to consider acquired changes in glucocorticoid receptor (GR) gene structure and organization, including alterations of noncoding sequences, and the importance of the resultant mutations, deletions, and other changes affecting receptor function. Finally, studies of New World primates and cell lines derived from hematologic malignancies constitute animal and human models for the molecular basis of glucocorticoid resistance where a number of inherited and acquired mutations in the GR gene have been demonstrated.

Cardiac nuclear hormone receptor mRNA in heart failure in man

Eight donor hearts and six explanted hearts due to dilated cardiomyopathy, normal skeletal muscle and liver were analysed. Glucocorticoid receptor (GR) and thyroid hormone receptor (T3R) isoforms beta 1, beta 2, alpha 1 and alpha 2 mRNA abundance were determined by solution hybridization. Both GR and T3R receptor mRNA isoforms were lower in the myocardium as compared to skeletal muscle and in particular to liver. GR mRNA abundance was higher than that of any T3R isoform while the sum of ligand-binding isoforms (beta 1, beta 2 and alpha 1) were similar in the myocardium and in skeletal muscle as opposed to the liver where GR mRNA was higher. GR mRNA abundance was similar in right and left ventricles from donor hearts and in cardiomyopathy. T3R beta 1 showed higher levels in the right ventricle with higher levels in cardiomyopathy as compared to donor heart. T3R isoform alpha 1 and especially the alpha 1/alpha 2 ratio were lower in left ventricle in cardiomyopathy compared to donor hearts. In conclusion, GR and T3R isoforms mRNA abundance are low in the human myocardium. In the failing myocardium GR and T3R beta 2 and alpha 2 show no signs of reactivity while T3R beta 1 and alpha 1 mRNA adapt.

Regulation of glucocorticoid receptor mRNA in nasal mucosa by local administration of fluticasone and budesonide

The glucocorticoid receptor (GR) is downregulated by glucocorticoids (autoregulation). In contrast, the metallothionein gene (MTIIa) is positively regulated by glucocorticoids, which requires a functional receptor protein. We have investigated the expression of GR and MTIIa mRNA in nasal mucosal biopsy specimens, nasal brush-lavage samples, and peripheral blood lymphocytes from 14 healthy volunteers after local treatment with one of two different glucocorticoids: fluticasone propionate or budesonide. In nasal mucosal biopsy specimens, a significant decrease in GR mRNA occurred with increasing doses of both steroids, whereas a significant and parallel increase in MTIIa mRNA was observed. We found nasal brush-lavage less suitable for studies of GR mRNA and MTIIa mRNA regulation by locally administered glucocorticoids. In mucosal biopsy specimens, but not in peripheral blood lymphocytes, we found a correlation between basal GR mRNA and MTIIa mRNA levels, where low GR mRNA levels were associated with low MTIIa mRNA levels, and vice versa. In conclusion, this study shows that locally administered glucocorticoids significantly affect the expression of specific genes and that there is an interindividual and tissue-specific variation in GR mRNA and MTIIa mRNA expression, which may be used in studies of variations in clinical responses to nasal glucocorticoids.

The genetic basis of glucocorticoid resistance

Familial glucocorticoid resistance is a rare syndrome characterized by elevated levels of plasma cortisol but lacking the symptoms of Cushings syndrome. Biochemically, the condition is characterized by a relative resistance to glucocorticoids that can be compensated for by the elevated levels of cortisol. Analysis of mutations within the receptor resulting in relative glucocorticoid resistance, both familial glucocorticoid resistance and directed mutagenesis, has identified two regions of clustered mutations in the proximity of previously identified affinity-labeled residues. In the majority of cases, the mutation affects steroid binding and transactivation to the same degree, but this is not always the case.

Growth Hormone (GH) Treatment Up-Regulates GH Receptor mRNA Levels in Adipocytes from Patients with GH Deficiency and Prader-Willi Syndrome

ABSTRACT: We have investigated the effect of growth hormone (GH) treatment on GH receptor mRNA expression in five prepubertal children with Prader-Willi syndrome and in eight patients with GH deficiency. An adipose tissue needle biopsy was taken before and after 2–4 mo of GH treatment, and RNA was isolated from adipose tissue and from adipocytes. GH receptor mRNA levels were determined by an RNase protection/solution hybridization assay. To further assess the specificity of the assay for GH receptor mRNAs, RNA extracted from human adipose tissue was subjected to Northern blot analysis. GH treatment significantly increased GH receptor mRNA levels in adipose tissue and isolated adipocytes. Our results indicate that GH may have an important role in regulating the GH receptor in humans.

Effects of growth hormone on lipolysis in humans

The ability to change the rate of lipolysis rapidly is one of the key functions of adipocytes. It is well established that adipose tissue is a target for growth hormone (GH), and many studies have been performed to determine the role of GH in the regulation of lipolysis since it was shown that growth-promoting extracts given to rats reduced the fat mass (1) and that plasma fatty acids were affected by GH (2). The results of these studies, however, are conflicting, and GH apparently has very complex effects on lipolysis. In this review, several aspects of the effects of GH on lipolysis in humans are discussed.