Regulation des Glucocorticoid-induced leucine zipper (GILZ) in Entzündungsprozessen: Mechanismen und funktionelle Bedeutung

Abstract Glucocortcoid-induced leucine zipper (GILZ) is an anti-inflammatory mediator which is intimately involved in the effects of glucocorticoids. The aim of this work was to examine the regulation mechanisms of GILZ in inflammation. Because of the great importance of TLR-stimulation in inflammatory reactions, we analysed the hypothesis that GILZ levels are regulated in TLR-mediated inflammatory processes. We indicated a regulation of GILZ via the mRNA-BP ZFP36 upon treatment of macrophages and endothelial cells with the inflammatory cytokine TNF-α. However, in case of the GILZ regulation after stimulation of the TRIF- dependent pathway, our results suggest that the expression of GILZ-regulating miRNAs plays a role. They probably have an inhibiting effect on GILZ mRNA translation. Anti-inflammatory processes regarding GILZ regulation were analysed as well. We could demonstrate that the natural compound curcumin can upregulate GILZ, independently of a GR-activation as well as a transcriptionell induction or protein- or mRNA stabilisation. GILZ induction is mediated by an induced GILZ mRNA translation through the mRNA-BP HuR instead. All these investigations to clarify the GILZ regulation pathways are highly important for the understanding and treatment of inflammatory diseases, where GILZ could act as a novel target.Zusammenfassung Glucocorticoid-induced Leucine Zipper (GILZ) ist ein anti-inflammatorischer Mediator, der wesentlich an den Wirkungen von Glukokortikoiden beteiligt ist. Das Ziel dieser Arbeit war die Regulation von GILZ in Entzündungsmodellen zu untersuchen. Aufgrund der zentralen Rolle einer Toll-like receptor (TLR)-Aktivierung in Entzündungsprozessen wurde die Hypothese überprüft, dass GILZ-Spiegel in TLR- vermittelten Entzündungsreaktionen reguliert werden. Es wurde gezeigt, dass in Makrophagen und Endothelzellen GILZ nach Behandlung mit dem Zytokin TNF-α über das mRNA-BP ZFP36 reguliert wird. In dem Prozess der GILZ-Regulation nach Anstoß des TRIF-abhängigen Signalweges hingegen weisen unsere Ergebnisse darauf hin, dass die Expression von GILZ-regulierenden miRNAs, die vermutlich einen Translations-hemmenden Effekt auf die GILZ mRNA ausüben, eine Rolle spielt. Daneben wurden auch anti-inflammatorische Prozesse im Hinblick auf die GILZ- Regulation untersucht. Es wurde gezeigt, dass durch den Naturstoff Curcumin GILZ hochreguliert wird, was weder abhängig von einer GR-Aktivierung, noch von einer transkriptionellen Induktion oder einer Protein- oder mRNA-Stabilisierung verläuft. Stattdessen wird die GILZ-Induktion über das mRNA-BP HuR mittels einer vermehrten GILZ mRNA-Translation vermittelt. Diese Untersuchungen zur Aufklärung der Regulationsmechanismen von GILZ sind von großer Bedeutung für das Verständnis und die Behandlung von entzündlichen Erkrankungen, in denen GILZ als neues Target dienen könnte

The long non-coding {RNA} {H19} suppresses carcinogenesis and chemoresistance in hepatocellular carcinoma

The long non-coding RNA (lncRNA) H19 represents a maternally expressed and epigenetically regulated imprinted gene product and is discussed to have either tumor-promoting or tumor-suppressive actions. Recently, H19 was shown to be regulated under inflammatory conditions. Therefore, aim of this study was to determine the function of H19 in hepatocellular carcinoma (HCC), an inflammation-associated type of tumor. In four different human HCC patient cohorts H19 was distinctly downregulated in tumor tissue compared to normal or non-tumorous adjacent tissue. We therefore determined the action of H19 in three different human hepatoma cell lines (HepG2, Plc/Prf5, and Huh7). Clonogenicity and proliferation assays showed that H19 overexpression could suppress tumor cell survival and proliferation after treatment with either sorafenib or doxorubicin, suggesting chemosensitizing actions of H19. Since HCC displays a highly chemoresistant tumor entity, cell lines resistant to doxorubicin or sorafenib were established. In all six chemoresistant cell lines H19 expression was significantly downregulated. The promoter methylation of the H19 gene was significantly different in chemoresistant cell lines compared to their sensitive counterparts. Chemoresistant cells were sensitized after H19 overexpression by either increasing the cytotoxic action of doxorubicin or decreasing cell proliferation upon sorafenib treatment. An H19 knockout mouse model (H19Δ3) showed increased tumor development and tumor cell proliferation after treatment with the carcinogen diethylnitrosamine (DEN) independent of the reciprocally imprinted insulin-like growth factor 2 (IGF2). In conclusion, H19 suppresses hepatocarcinogenesis, hepatoma cell growth, and HCC chemoresistance. Thus, mimicking H19 action might be a potential target to overcome chemoresistance in future HCC therapy

Amplified Host Defense by Toll-Like Receptor-Mediated Downregulation of the Glucocorticoid-Induced Leucine Zipper (GILZ) in Macrophages

Activation of toll-like receptors (TLRs) plays a pivotal role in the host defense against bacteria and results in the activation of NF-κB-mediated transcription of proinflammatory mediators. Glucocorticoid-induced leucine zipper (GILZ) is an anti-inflammatory mediator, which inhibits NF-κB activity in macrophages. Thus, we aimed to investigate the regulation and role of GILZ expression in primary human and murine macrophages upon TLR activation. Treatment with TLR agonists, e.g., Pam3CSK4 (TLR1/2) or LPS (TLR4) rapidly decreased GILZ mRNA and protein levels. In consequence, GILZ downregulation led to enhanced induction of pro-inflammatory mediators, increased phagocytic activity, and a higher capacity to kill intracellular bacteria (Salmonella enterica serovar typhimurium), as shown in GILZ knockout macrophages. Treatment with the TLR3 ligand polyinosinic: polycytidylic acid [Poly(I:C)] did not affect GILZ mRNA levels, although GILZ protein expression was decreased. This effect was paralleled by sensitization toward TLR1/2- and TLR4-agonists. A bioinformatics approach implicated more than 250 miRNAs as potential GILZ regulators. Microarray analysis revealed that the expression of several potentially GILZ-targeting miRNAs was increased after Poly(I:C) treatment in primary human macrophages. We tested the ability of 11 of these miRNAs to target GILZ by luciferase reporter gene assays. Within this small set, four miRNAs (hsa-miR-34b*,−222,−320d,−484) were confirmed as GILZ regulators, suggesting that GILZ downregulation upon TLR3 activation is a consequence of the synergistic actions of multiple miRNAs. In summary, our data show that GILZ downregulation promotes macrophage activation. GILZ downregulation occurs both via MyD88-dependent and -independent mechanisms and can involve decreased mRNA or protein stability and an attenuated translation