Martin Eggert

Alien, a Highly Conserved Protein with Characteristics of a Corepressor for Members of the Nuclear Hormone Receptor Superfamily

ABSTRACT Some members of nuclear hormone receptors, such as the thyroid hormone receptor (TR), silence gene expression in the absence of the hormone. Corepressors, which bind to the receptor’s silencing domain, are involved in this repression. Hormone binding leads to dissociation of corepressors and binding of coactivators, which in turn mediate gene activation. Here, we describe the characteristics of Alien, a novel corepressor. Alien interacts with TR only in the absence of hormone. Addition of thyroid hormone leads to dissociation of Alien from the receptor, as shown by the yeast two-hybrid system, glutathioneS-transferase pull-down, and coimmunoprecipitation experiments. Reporter assays indicate that Alien increases receptor-mediated silencing and that it harbors an autonomous silencing function. Immune staining shows that Alien is localized in the cell nucleus. Alien is a highly conserved protein showing 90% identity between human and Drosophila. Drosophila Alien shows similar activities in that it interacts in a hormone-sensitive manner with TR and harbors an autonomous silencing function. Specific interaction of Alien is seen with Drosophila nuclear hormone receptors, such as the ecdysone receptor and Seven-up, the Drosophila homologue of COUP-TF1, but not with retinoic acid receptor, RXR/USP, DHR 3, DHR 38, DHR 78, or DHR 96. These properties, taken together, show that Alien has the characteristics of a corepressor. Thus, Alien represents a member of a novel class of corepressors specific for selected members of the nuclear hormone receptor superfamily.

The glucocorticoid receptor is associated with the RNA-binding nuclear matrix protein hnRNP U.

The glucocorticoid receptor (GR) is a ligand-dependent transcription factor that is able to modulate gene activity by binding to its response element, interacting with other transcription factors, and contacting several accessory proteins such as coactivators. Here we show that GRIP120, one of the factors we have identified to interact with the glucocorticoid receptor, is identical to the heterogeneous nuclear ribonucleoprotein U (hnRNP U), a nuclear matrix protein binding to RNA as well as to scaffold attachment regions. GR·hnRNP U complexes were identified by blotting and coimmunoprecipitation. The subnuclear distribution of GR and hnRNP U was characterized by indirect immunofluorescent labeling and confocal laser microscopy demonstrating a colocalization of both proteins. Using a nuclear transport-deficient deletion of hnRNP U, nuclear translocation was seen to be dependent on GR and dexamethasone. Transient transfections were used to identify possible interaction domains. Overexpressed hnRNP U interfered with glucocorticoid induction, and the COOH-terminal domains of both proteins were sufficient in mediating the transcriptional interference. A possible functional role for this GR binding-protein in addition to its binding to the nuclear matrix, to RNA, and to scaffold attachment regions is discussed.

Involvement of the Glucocorticoid Receptor in the Pathogenesis of Rheumatoid Arthritis

Abstract: The glucocorticoid receptor (GR) is a ligand‐inducible transcription factor which controls the expression of several genes. Its cognate ligand, the glucocorticoids, induces receptor activation by binding to the cytoplasmic located receptor, ultimately leading to translocation of the receptor/hormone complex into the nucleus and the regulation of gene activity. Because glucocorticoids are widely used for suppression of inflammation in rheumatoid arthritis (RA), we investigated whether the expression level of GR is correlated with RA. We designed a study to detect the total amount of GR in lymphocytes of untreated RA patients, glucocorticoid‐treated RA patients, and healthy controls. We observed a significant change in the expression levels of GR. Untreated RA patients exhibited a significantly higher amount of GR than the healthy controls, whereas glucocorticoid‐treated RA patients showed a strongly decreased receptor density. These results seem to reflect a functional dysregulation of the HPA axis and may lead to a better understanding of the pathogenesis of RA.

Molecular aspects of glucocorticoid hormone action in rheumatoid arthritis

Glucocorticoids (GC) are the most powerful anti-inflammatory drugs used in the treatment of autoimmune diseases such as rheumatoid arthritis. In addition, endogenous GC are involved in numerous physiological processes. Most of their effects are mediated by the glucocorticoid receptor (GR) via activation or repression of gene expression. Whereas activation requires DNA binding of the receptor, repression is mediated by protein-protein interactions with other transcription factors. In particular, most immunosuppressive and anti-inflammatory effects are exerted by an interaction of GR with the activating protein 1 (AP-1) and nuclear factor kappaB (NF-kappaB) families of transcription factors without DNA binding. Cytokines such as tumor necrosis factor alpha (TNF-alpha) and interleukin 1 (IL-1) activate the hypothalamus pituitary adrenal (HPA) axis, whereas GC inhibit IL-1 and TNF-alpha forming a cytokine-HPA axis feedback circuit. The high effectiveness of cytokine-antagonists blocking TNF-alpha or IL-1 in RA and the understanding of the precise molecular mechanisms of GC function will enhance our understanding of autoimmune diseases, such as RA, and could suggest new beneficial therapeutic approaches with fewer side-effects.

Effects of the heterogeneous nuclear ribonucleoprotein U (hnRNP U/SAF-A) on glucocorticoid-dependent transcription in vivo.

The glucocorticoid receptor (GR) is a ligand dependent transcription factor, which regulates the transcription of multiple hormone-dependent genes. The transcriptional regulation by GR takes place by interaction of GR with the basal transcription machinery and by recruiting glucocorticoid receptor interacting proteins (GRIPs). Previously we identified hnRNP U/SAF-A as a factor interfering with GR-dependent transcription by repressing glucocorticoid induced activation. To gain insight into the mechanisms that govern this interference, we have now investigated the transcription of GR-dependent reporter genes in Ltk(-) cells transiently transfected with a variety of hnRNP U constructs. We demonstrate that a hnRNP U construct lacking the GR-binding domain acts as a dominant negative factor that now enhances GR-driven transcription. In addition, hnRNP U repression of glucocorticoid induced transcription was found to be dependent on the amount of cotransfected GR, where a high amount of GR leads to ligand-inducible repression of GR-dependent reporter gene activity by hnRNP U, whereas low amounts of GR showed nearly no effect. The relative concentrations of GR, hnRNP U and DNA-binding sites for GR are important for the effect of hnRNP U on transcription, suggesting a model where hnRNP-U acts as a storage site for intranuclear GR.

Identification of phosphorylation sites on murine nuclear lamin C by RP-HPLC and microsequencing

Isolated interphase lamin C, obtained from Ehrlich ascites tumor cells, was digested by Lys‐C endoproteinase, the resulting peptides separated by reversed‐phase HPLC and subjected to microsequencing in order to identify phosphorylation sites in interphase and following phosphorylation in vitro by cdc2‐kinase, protein kinase C (PKC) and protein kinase A (PKA), respectively. Nuclear lamin C showed partial phosphorylation of Ser392 and Ser409, and possibly Ser407 in interphase. Phosphorylation was increased in response to cdc2‐kinase at Ser390 and Ser392 and to PKC at Ser572. The N‐terminal peptide (aa 1–32) containing consensus sequences for the 3 kinases was phosphorylated by cdc2‐kinase, PKC and PKA. The sequence data suggests that multiple molecular switches via lamina modification control the dynamic behaviour of the nucleoskeleton during the cell cycle.

Transcription Factors in Autoimmune Diseases

The analysis of the molecular basis of autoimmune diseases is currently under intense investigation. The identification of novel mechanisms underlying the pathogenesis of these diseases generates the possibility for the development of new therapeutic agents. In this review we summarize the results leading to novel insights concerning the molecular processes involved in the pathogenesis of rheumatoid arthritis, systemic lupus erythematodes, multiple sclerosis and diabetes type 1. We focus on the role of transcription factors such as nuclear factor kappa B, activator protein 1, peroxisome proliferator-activated receptor, vitamin D receptor and the glucocorticoid receptor that mediate pro- and anti-inflammatory effects and therefore represent direct or indirect targets for therapeutic intervention.

Changes in the activation level of NF-kappa B in lymphocytes of MS patients during glucocorticoid pulse therapy.

Nuclear factor-kappaB activity was analyzed in multiple sclerosis (MS) patients during the course of a methylprednisolone pulse therapy. Molecular effects were evaluated using lymphocytes derived from 20 MS patients before and after therapy and 24 healthy individuals. All patients responded to treatment clinically. The mean level of DNA-binding p65 in MS was proportionate to that of healthy controls, but was significantly decreased directly after therapy whereas the level of DNA-binding p50 was significantly elevated prior to therapy and remained unchanged. In summary, pulse therapy resulted in a decreased level of activated p65 NF-kappaB subunits leading to decreased levels of transcriptionally active pro-inflammatory NF-kappaB.

The Anti-mutated Citrullinated Vimentin Response Classifies Patients with Rheumatoid Arthritis into Broad and Narrow Responders

Objective. Autoantibodies against citrullinated peptide antigens (ACPA) are routinely determined to diagnose rheumatoid arthritis (RA) and are predictive of a more severe course of the disease. We here set out to address an involvement of ACPA in the pathogenesis of RA and investigated the recognition pattern of antibodies against 2 citrullinated antigens in more detail. Methods. The sera of 77 patients fulfilling the American College of Rheumatology criteria for RA were analyzed for subclass titers of anti-mutated citrullinated vimentin (MCV) and anticyclic citrullinated peptide (CCP) antibodies by combining subclass specific detection antibodies with commercially available CCP and MCV ELISA plates. Cross-reactivities between anti-MCV and anti-CCP antibodies were detected using a sequential ELISA system. Results. IgG1, IgG3, and IgG4 titers among anti-MCV and anti-CCP antibodies correlated significantly. Cross-reactivity of MCV-specific antibodies against CCP could be detected in 8 of 16 patients’ sera; however, cross-binding of MCV-specific IgG4 was weaker compared to total IgG. Conclusion. The inherent capacity of IgG4 to exchange F(ab) arms provides insight into the anti-MCV antibody diversity and suggests a classification of ACPA positive patients into broad and narrow responders.

Enhancement of nuclear receptor transcriptional signalling

Glucocorticoids and thyroid hormones induce complex responses in about every mammalian tissue. These effects are mediated by the transcription factor function of the corresponding nuclear receptors, which in most cases achieve the observed regulatory strength in synergy with other factors. Here we describe the functional interaction of the glucocorticoid receptor (GR) with liver-specific transcription factors, the functional synergy of GR with the thyroid hormone receptor (TR), the synergizing sub-domains of the TR, and finally the direct interaction of the GR with other proteins.