27: IL-6 possibly modulates ghrelin expression through MEK1/p90RSK signaling cascade in pancreatic cell lines

Abstract

Ghrelin is a novel acylated 28-amino acid peptide involved in many physiological and biological roles, i.e. stimulating appetite and increasing food intake to exerting anti-inflammatory effects. The purpose of this study was to investigate the effect of pro-inflammatory cytokines on ghrelin expression, and to map the signaling transduction pathway undertaken by the cytokine to modulate ghrelin levels by using pancreatic cancer cell lines as model system. AR42J pancreatic cell line (ATCC) was cultured in F-12 Ham (F12K) medium (Sigma Aldrich, USA) supplemented with 20% (v/v) of fetal bovine serum (FBS). Dose- and time-response tests with TNF- α , IL-1 β , IL-1 α , IFN- γ , and IL-6 were carried out, and the total RNA and protein were extracted. Real-time RT-PCR and western blot were then carried out to investigate the effect on the cytokines on ghrelin expression using Quantifast SYBR Green RT-PCR Kit (QIAGEN, Germany) and anti-ghrelin antibody (Santa Cruz Biotechnology, USA). The cells were stimulated in the presence or absence of inhibitor to specific signaling pathways prior to cytokine treatment to map the pathway undertaken by the cytokine to influence ghrelin expression by western blot using total and phosphorylated antibodies of Raf/MEK/ERK/ p90RSK (Cell Signaling Technology Inc). β -actin was used as the housekeeping gene. TNF- α , IL-1 β IFN- γ , and IL-6, but not IL-1 α , down-regulated ghrelin expression significantly. Time course experiments showed that ghrelin was increased significantly in 2-h, but was reduced significantly at 16-h in the presence of IL-6. Out of the 9 inhibitors used to investigate the signaling pathway undertaken by IL-6, only rapamycin, U0126 and PD98059 abolished the IL-6 inhibitory effect on ghrelin expression suggesting the involvements of Akt/Raf/MEK1/ERK/p90RSK cascade. Further experiments confirmed that the phosphorylation of MEK1 at serine 298 and p90RSK at serine 380 play crucial roles in IL-6 regulation of ghrelin expression. IL-6 affected the expression of ghrelin significantly in dose- and time-dependent manner, and may possibly exert its effects via the signaling cascade of Akt/Raf/MEK/ERK/p90RSK.