Publikationsserver der RWTH Aachen University
Regulation der Genexpression und Promotoraktivität von RGS4 durch Leptin
Abstract
dc:descriptionProduced by white adipose tissue, leptin regulates bodyweight by reducing food intake and energy expenditure. Leptin exerts its main effects on the hypothalamus but also acts on peripheral cell types like pancreatic b-cells, where it inhibits insulin secretion. The leptin receptor (LepRb) belongs to the class I cytokine receptor family. Upon leptin binding, receptor-associated Janus kinases (Jaks) phosphorylate transcription factors of the STAT-family (signal transducers and activators of transcription), which then dimerize and translocate to the cell nucleus where they regulate gene expression. This Jak/STAT pathway is also activated by the cytokines interferon g (IFN-g) and interleukin-6 (IL-6) while IL-1b mediates its signal transduction using the transcription factor NFkB (nuclear transcription factor kB). This work first describes the regulatory action on leptin target genes. By Western blot analysis using phosphospecific antibodies in rat RINm5F insulinoma cells, it was shown that leptin is able to activate both transcription factor STAT1 and STAT3. In contrast, IFN-g specifically activates STAT1 while IL-6 activates only STAT3. Subsequently, the role of STAT1 and STAT3 in the regulation of known leptin target genes (Fibrinogen b, RGS4, SOCS3, PAP1, tPA, Ppap2a, SOD2, Tachykinin, LCN2) was investigated in Northern blots using different cytokines. None of these genes was activated by IFN-g, while stimulation of the IL-6 receptor subunit gp130 showed effects analogous to those elicited by stimulation of the leptin receptor. Thus STAT1 alone cannot induce gene expression while STAT3 plays a pivotal role. In addition, leptin increased the stimulatory effect of IL-1b, an important mediator of islet cell inflammation. In the second part, the regulation by leptin of one of these target genes (RGS4, regulator of G-protein signaling) was investigated in more detail. RGS proteins inhibit signaling pathways of heterorimeric G proteins by increasing the intrinsic GTPase activity of tha alpha subunit. The mRNA of RGS4 in RINm5F cells showed a rapid upregulation within 30 minutes after leptin stimulation and a short half-life of 3 h. Both is necessary for a protein with rapid regulation of cellular functions. The leptin receptor comprises three intracellular tyrosine residues (Tyr985, Tyr1077 and Tyr1138) which activate different pathways in signal transduction. In order to study the influence of these signal pathways, the requirement of individual tyrosine residues for activation of the RGS4 promoter was tested in HepG2 cells. To this end, the rat RGS4 promoter was cloned into a reporter gene plasmid. Luciferase assays showed that promoter induction was depending on Tyr1138, which is responsible for recruiting STAT1 and STAT3. In line with this result, the expression of RGS4 mRNA was induced by gp130 stimulation, which also leads to activation of STAT3. Moreover, overexpression of a dominant negative mutant of STAT3 abolished the activation of the RGS4 promoter by leptin on promoter level. These results provide evidence for an essential role of STAT3 on RGS4 regulation by leptin on promoter level. Furthermore, it was shown via RNA interference that activation of the RGS4 promoter upon stimulation of the wild type leptin receptor was downregulated by the feedback inhibitor SOCS3 (suppressor of cytokine signaling). In the RGS4 promoter, 63 bases upstream of the transcription start a CCAAT box, matching the consensus binding sequence of NF-Y (nuclear factor-Y, a CCAAT box binding factor) is located close to the TATA box. This part of the rat promoter bears a striking sequence similarity with the RGS4 promoter of other species. A RGS4 promoter contsruct with a mutation of a single base of the CCAAT box exhibited a dramatically decreased basal activity and was no longer stimulated by leptin in reporter gene assays of HepG2 cells. Thus, the CCAAT box plays an essential role in the regulation of promoter activity by leptin, possibly via action of the transcription factor NF-y. The finding that the expression of RGS4 is controlled by STAT3 reveals a new mechanism of cross-talk by which cytokines may interfere with the function of G protein-coupled receptors.
Degree
thesis:*- Grantor dc:publisher
- Publikationsserver der RWTH Aachen University
- Year dc:date
- 2008
Author and committee
dc:creator, dc:contributor.*- Author dc:creator
-
- Korfmacher, Stefanie
- Contributors dc:contributor
-
- Becker, Walter
Subjects
dc:subject × 12Rights
dc:rights- Statement dc:rights
-
- info:eu-repo/semantics/openAccess
- Language dc:language
- ger