Johan Liden

A New Function for the C-terminal Zinc Finger of the Glucocorticoid Receptor REPRESSION OF RelA TRANSACTIVATION

Glucocorticoids inhibit NF-κB signaling by interfering with the NF-κB transcription factor RelA. Previous studies have identified the DNA-binding domain (DBD) in the glucocorticoid receptor (GR) as the major region responsible for this repressive activity. Using GR mutants with chimeric DBDs the repressive function was found to be located in the C-terminal zinc finger. As predicted from these results the mineralocorticoid receptor that contains a C-terminal zinc finger identical to that of the GR was also able to repress RelA-dependent transcription. Mutation of a conserved arginine or a lysine in the second zinc finger of the GR DBD (Arg-488 or Lys-490 in the rat GR) abolished the ability of GR to inhibit RelA activity. In contrast, C-terminal zinc finger GR mutants with mutations in the dimerization box or mutations necessary for full transcriptional GR activity were still able to repress RelA-dependent transcription. In addition, we found that the steroid analog ZK98299 known to induce GR transrepression of AP-1 had no inhibitory effect on RelA activity. In summary, these results demonstrate that the inhibition of NF-κB by glucocorticoids involves two critical amino acids in the C-terminal zinc finger of the GR. Furthermore, the results from the use of mineralocorticoid receptor and anti-glucocorticoids suggest that the mechanisms for GR-mediated repression of NF-κB and AP-1 are different.

Expression profiling reveals a distinct transcription signature in follicular thyroid carcinomas with a PAX8-PPAR|[gamma]| fusion oncogene

The demonstration of the PAX8-PPARγ fusion oncogene in a subset of follicular thyroid tumors provides a new and promising starting point to dissect the molecular genetic events involved in the development of this tumor form. In the present study, we compared the gene expression profiles of follicular thyroid carcinomas (FTCs) bearing a PAX8-PPARγ fusion against FTCs that lack this fusion. Using unsupervised clustering and multidimensional scaling analyses, we show that FTCs possessing a PAX8-PPARγ fusion have a highly uniform and distinct gene expression signature that clearly distinguishes them from FTCs without the fusion. The PAX8-PPARγ(+) FTCs grouped in a defined cluster, where highly ranked genes were mostly associated with signal transduction, cell growth and translation control. Notably, a large number of ribosomal protein and translation-associated genes were concurrently underexpressed in the FTCs with the fusion. Taken together, our findings further support that follicular carcinomas with a PAX8-PPARγ rearrangement constitute a distinct biological entity. The current data represent one step to elucidate the molecular pathways in the development of FTCs with the specific PAX8-PPARγ fusion.

Expression Level-Dependent Contribution of Glucocorticoid Receptor Domains for Functional Interaction with STAT5

ABSTRACT The action of the glucocorticoid receptor (GR) on β-casein gene transcription serves as a well-studied example of a case where the action of the GR is dependent on the activity of another transcription factor, STAT5. We have investigated the domain-requirement of the GR for this synergistic response in transfection experiments employing GR mutants and CV-1 or COS-7 cells. The results were influenced by the expression levels of the GR constructs. At low expression, STAT5-dependent transactivation by mutants of the GR DNA binding domain or N-terminal transactivation domain was impaired and the antiglucocorticoid RU486 exhibited a weak agonistic activity. When the N-terminal region of the GR was exchanged with the respective domain of the progesterone receptor, STAT5-dependent transactivation was reduced at low and high expression levels. Only at high expression levels did the GR exhibit the properties of a coactivator and enhanced STAT5 activity in the absence of a functional DNA binding domain and of GR binding sites in the proximal region of the β-casein gene promoter. Furthermore, at high GR expression levels RU486 was nearly as efficient as dexamethasone in activating transcription via the STAT5 dependent β-casein gene promoter. The results reconcile the controversial issue regarding the DNA binding-independent action of the GR together with STAT5 and provide evidence that the mode of action of the GR depends not only on the type of the particular promoter at which it acts but also on the concentration of the GR. GR DNA binding function appears to be mandatory for β-casein gene expression in mammary epithelial cells, since the promoter function is completely dependent on the integrity of GR binding sites in the promoter.

Nuclear factor-κ B repression in antiinflammation and immunosuppression by glucocorticoids

The transcription factor nuclear factor-kappaB (NF-kappaB) directs transcription of a large number of key molecules in immunological and inflammatory responses. The recently discovered inhibition of transcriptional activity of this factor by the activated glucocorticoid receptor (GR) provides a molecular basis for the potent antiinflammatory and immunosuppressive properties of glucocorticoids. This repressive activity of the GR can function independently of transcriptional activity. Because it has been shown that certain steroid receptor ligands can differentially address transactivation and transrepression functions, it may be possible to develop ligands that specifically suppress NF-kappaB activity and, as a result, are more efficient in treatment of a variety of important chronic inflammatory diseases with less severe side effects than those of currently available drugs.

Molecular characterization of tumour heterogeneity and malignant mesothelioma cell differentiation by gene profiling.

Malignant mesothelioma is an aggressive tumour, characterized by a variable differentiation pattern and poor prognosis. At present, the clinical outcome in patients with malignant mesothelioma is mainly predicted by the morphological phenotype of the tumour. However, this conventional clinicopathological parameter is of limited value, partly because of the biological heterogeneity of this tumour and poor understanding of the regulatory mechanisms underlying the various patterns of growth. To elucidate the intrinsic molecular programmes that determine tumour differentiation, oligonucleotide arrays were used in an in vitro model of mesothelioma differentiation. The analysis of 2059 genes detected 102 genes that were significantly deregulated. Clustering of these genes into functional categories showed distinctive patterns for the two phenotypes, namely epithelioid and sarcomatoid. The molecular fingerprint of the sarcomatoid tumour component indicates overrepresentation of growth factor receptors and growth factor binding proteins, whereas epithelioid mesothelioma cells express other tumour‐promoting factors involved in differentiation, metabolism, and regulation of apoptosis. These differences in the molecular phenotype may give a better basis for diagnosis and for designing novel therapies. Copyright

Organic dust activates NF-κB in lung epithelial cells

Exposure in swine confinement facilities induces airway inflammation in healthy subjects. The aim of the present study was to elucidate the role of nuclear factor (NF)-kappaB in the inflammatory response induced by organic dust. A human lung epithelial carcinoma cell line (A549) was transfected with reporter genes of the human IL-6 promoter or the NF-kappaB binding site fused to the luciferase reporter gene and stimulated with dust from a swine confinement building. Cytokine release in cell culture supernatants and luciferase activity was measured. The dust-induced the activities of the IL-6 promoter reporter gene and the NF-kappaB reporter gene in parallel with an increase in IL-6 and IL-8 release. The addition of pyrrolidinedithiocarbamate, a chemical NF-kappaB blocking agent, inhibited IL-6 and IL-8 secretion as well as the NF-kappaB reporter gene activity. Increasing the amount of IkappaB alpha led to inhibition of organic dust-induced IL-6 promoter and NF-kappaB reporter gene activities. Fluticasone inhibited the organic dust-induced NF-kappaB activation and IL-6 and IL-8 secretion. Finally, swine dust incubation of A549 cells resulted in a NF-kappaB DNA binding, which is composed of the NF-kappaB1 and RelA proteins. In conclusion, by interference at various levels we have shown that NF-kappaB plays a key role in the inflammatory response to organic dust.