Differentielle Genexpression in der Insulinoma-Zelllinie RINm5F nach Stimulation mit Leptin

Leptin, a cytokine secreted by the white adipose tissue, decreases food intake via hypothalamic pathways and is a major regulator of energy homoestasis. Leptin has also important peripheral effects, such as the attenuation of insulin secre-tion from pancreatic islets. Leptin signals through the JAK/STAT pathway which is also used by other cytokines such as interleukin-6 (IL-6) or growth hormone (GH). Activation of this pathway leads to phosphorylation of STAT transcription factors which migrate into the nucleus and regulate gene expression. The aim of this study was to compare the leptin signalling pathway with that of IL-6 and GH and to identify leptin-regulated genes. I have used the pancreatic beta cell line RINm5F, which I transfected stably with the leptin receptor (LepRb) cDNA using a retroviral expression system. These cells were stimulated with leptin, IL-6 or GH, and STAT factor phosphorylation was analysed. Leptin stimulated tyrosine phosphorylation of a broad range of STAT factors (STAT1, STAT3, STAT5 and STAT6). In contrast, IL6 and GH only induced tyrosine phosphorylation of a smaller subset of STAT factors (STAT5 and STAT6 with GH; STAT1 and STAT3 with IL-6). Leptin treatment also led to the phosphorylation of the serine residue 727 in STAT3 and to the activation of the ERK pathway. In order to identify leptin-induced genes, a filter array containing 27,648 different cDNAs was screened by differential hybridization with a complex cDNA probe isolated from leptin-stimulated or non-stimulated RINm5F-LepRb cells. 35 transcripts were found to be upregulated after 16 h of leptin stimulation and 21 to be upregulated after 1 h of leptin stimulation. Northern blot analysis allowed to distinguish immediate early, delayed early and late target genes, which had their expression maximum at 1 h, 7 h and 16 h of leptin stimulation, respectively. The following genes were assigned to the first group: SOCS3 (suppressor of cytokine signalling 3), CISH (cytokine-inducible SH2-containing protein), fibrinogen-beta and RGS4 (regulator of Gprotein signalling 4). To the second group was assigned: PAP1 (pancreatitis associated protein 1), and to the third group tPA (tissue plasminogen activator), Ppap2a (phosphatidate phosphohydrolase type 2a), SOD2 (superoxide dismutase 2), lipocalin-2 and tachykinin. Several of those genes encoded proteins which are secreted during inflammatory processes (fibrinogen-beta, PAP1, lipocalin-2 and tachykinin), whereas others play roles in intracellular signalling pathways (SOCS3, CISH, RGS4 and Ppap2a). The effect of the intracellular leptin receptor tyrosine residues 985, 1077 and 1138 on the expression of RGS4 and PAP1 was also evaluated using point mutants of the leptin receptor stably transfected into RINm5F cells. This experiment revealed that the expression of RGS4 and PAP1 mainly depended on Tyr1138, the known STAT3 binding site.

Grantor dc:publisher

Publikationsserver der RWTH Aachen University

Year dc:date

2004

Author dc:creator

• Hekerman, Paul

Contributors dc:contributor

• Becker, Walter

• info:eu-repo/classification/ddc/570
• Säugetiere
• Bauchspeicheldrüsentumor
• Zelllinie
• Differentielle Genexpression
• Leptine
• Biowissenschaften, Biologie
• Leptin
• STAT
• Genexpression
• Pankreas
• Insel
• RINm5F array

Statement dc:rights

• info:eu-repo/semantics/openAccess

Language dc:language

ger

Identifier

https://publications.rwth-aachen.de/search?p=id:%22RWTH-CONV-121275%22

OAI identifier oai:identifier

oai:publications.rwth-aachen.de:59496