Experimental Study Of The Molecular Mechanism Of 67-kDa LNR-induced FasL Upregulation In QBC-939 Cells

Backbround: Fas ligand (FasL) is an approximately 40-kDa transmembrane proteinbelongings to the tumor necrosis factor(TNF) superfamily that can trigger apoptotic celldeath by binding to its receptor,Fas (CD95/APO-1) . FasL is expressed not only byactivated T cells but also by various tumors . We have found that a humancholangiocarcinoma cell line (QBC-939 cells) expresses various levels of functional FasL.We have also observed that higher expression levels of FasL in QBC-939 cells resulted inincreased apoptosis of T lymphocytes when both cell types were co-cultured. This result isconsistent with the hypothesis that tumors gain immune privilege by upregulating FasLexpression in order to"counterattack"and kill Fas-expressing infiltrating lymphocytes.Moreover,our previous study showed that 67-kDa laminin receptor(67-kDa LNR) andLn-1 play a definite role in the upregulation of FasL in QBC-939 cells.67-kDa laminin receptor (67-kDa LNR) is thought to be a major laminin receptor thatis upregulated in neoplastic cells compared to their normal counterparts. Our previous studyshowed that human cholangiocarcinoma cells expressed a higher level of 67-kDa LNR thannormal epithelial cells. It has been shown that a cell-laminin interaction via the 67-kDaLNR is an important step in signal transduction pathways and that laminin and 67-kDa LNRare likely involved in MAPK (mitogen-activated protein kinase)- and DUSP(dual-specificity MAPK phosphatase)-mediated laminin signaling in human melanomacells . In human glioma cell lines,MEK inhibitors have been shown to decreasephosphorylated extracellular signal-regulated kinase (pERK) levels caused by stimulationwith either ligand or activating antibody to Fas. Based on our previous studies and the workof other researchers,we investigated the effect of the MAPK-ERK cascade on 67-kDaLNR- and Ln-1-induced FasL upregulation in QBC-939 cells.FasL gene expression is controlled by transcription factor–DNA interactions at the FasL promoter. c-Myc belongs to the Myc family of transcription factors and c-Myc mRNAand protein are overexpressed in many cancer cells,including human cholangiocarcinomacells. Numerous signal transduction pathways are involved in the control of c-Myctranscription. Bermudez et al. reported that EGF activates c-Myc transcription via theRas/MEK/ERK pathway. Because relatively little is known about c-Myc regulation of FasLexpression outside of the immune system,we focused on the role of c-Myc in 67-kDaLNR and laminin-induced FasL upregulation in QBC-939 cells. And in other aspect,weset out to investigate the role of MAPK-ERK pathway in 67-kDa LNR induced FasLexpression and FasL-mediated apoptosis in human cholangiocarcinoma cells.MethodsMethods: (1) The parent QBC-939 cells were pretreated with or without PD98059 (20μM) or DMSO (Vehicle) for 4 h and then incubated with Ln-1 (100μg/ml) for 24 h,theexpression of c-Myc and FasL were analyzed by immunohistochemistry,RT-PCR andWestern blotting. (2) To assess FasL promoter activity,the construct of pGL3-FasL-Prluciferase reporter gene vector and mutant construct were generated and wereco-transfected into QBC-939 cells with pRL-TK plasmid for 24h respectively. After that,the luciferase activity were measured using a luminometer. (3) Chromatinimmunoprecipitation assays was used to show the binding of phosphorylated c-Myc to thehuman FasL promoter in vivo. (4) We also examined the human cholangiocarcinomacells-induced apoptosis of Fas-sensitive Jurkat T cells using TUNEL assay after the parentQBC-939 cells were pretreated with or without PD98059 (20μM) or DMSO (Vehicle) for 4h and then incubated with Ln-1 (100μg/ml) for 24 h.Results : (1) PD98059 significantly attenuated phosphorylation of c-Myc on Ser-62and FasL upregulation in QBC-939 cells and these cells showed decreased cytotoxicityagainst Fas-sensitive Jurkat T cells. (2) A luciferase reporter assay revealed that levels ofFasL promoter activity were significantly reduced after the parent QBC-939 cells werepretreated with PD98059 (20μM) . (3) Furthermore,mutational analysis of the c-Mycsites located on FasL promoter suggested that these sites are required for FasL promoteractivity in QBC-939 cells. (4) In addition,chromatin immunoprecipitation assays showedthe binding of phosphorylated c-Myc to the human FasL promoter in vivo. ConclusionsConclusions: Based on these results,we conclude that:1. MAPK-ERK pathway in 67-kDa LNR induced FasL expression and FasL-mediatedapoptosis take an important role in human cholangiocarcinoma cells.2. 67-kDa LNR may increase the activation of the FasL promoter by phosphorylatingof c-Myc on Ser-62 to bind to the human FasL promoter in vivo in humancholangiocarcinoma cells.