GSK3 Attenuates TNFα-induced GM-CSF Via Inhibition Of ERK Signaling In Nasopharyngeal Carcinoma(NPC)

Objective:Granulocyte-macrophage colony stimulating factor(GM-CSF)functions to drive nasopharyngeal cancer(NPC)metastasis via recruitment and activation of macrophages.But the source and the regulation of GM-CSF in tumor microenviroment of NPC is not fully understood.The purpose of this study was to analyze whether nasopharyngeal carcinoma cells secrete GM-CSF,to investigate whether there is tolerance in their expression,and to investigate whether the GSK3 pathway is involved in the regulation of GM-CSF expression.By exploring the reason of GM-CSF production tolerance induced by tumor necrosis factor(TNFα),to elucidate the mechanism by which GSK3 pathway regulates TNFα-induced GM-CSF expression in tumor microenvironment.And finally to provide theoretical basis for clinical intervention of tumor development by searching for therapeutic targets.Methods:1.ELISA was used to detect the protein levels of GM-CSF cytokines in NPC cells.2.QRT-PCR was used to detect GM-CSF and A20 mRNA levels in NPC cells.3.The expression of p-GSK3α(T279),p-GSK3β(T216),A20,p-ERK,ERK,p-JNK,JNK,p-P38,P38,p-P65,P65,GAPDH in NPC cells was detected by Western Blot.4.Lipofection was used to transfect NPC cells with plasmid containing sequence coding for human A20 protein.Results:In this study,we found that TNFα induced a time-and dose-dependent production of GM-CSF in NPC CNE1,CNE2,and 5-8F cells.GM-CSF production was tolerant,as the pretreatment of NPC cells with TNFα down-regulated the GM-CSF production induced by TNF-α re-treatment.TNFα activated glycogen synthase kinase-3(GSK-3),which is an enzyme that regulates glycogen synthesis,and is a critical downstream element of the PI3K/Akt to regulate cell survival.The activation of GSK3 down-regulated GM-CSF production.Pharmacological inhibition of GSK3 signaling up-regulated GM-CSF,and reversed GM-CSF tolerance induced by TNFα re-treatment.GM-CSF tolerant was not related to ubiquitin-editing enzyme A20.The over-expression of A20 did not regulate GM-CSF production induced by TNFα.However,GSK3 inhibition up-regulated ERK activation,which contributed to the production of GM-CSF induced by TNFα,suggesting that GSK3 negatively regulated TNFαinduced GM-CSF via dwon-regulation of ERK signaling.Taken together,these results suggested that GSK3 pathway may be a target for the regulation of TNFα-induced GM-CSF in tumor microenviroment.Conclusion:1.TNFα induces tolerant production of GM-CSF in NPC cells.2.A20 does not regulate TNFα-induced GM-CSF.3.ERK pathway contributes to TNFα-induced GM-CSF.4.GSK3 attenuates TNFα-induced GM-CSF via inhibition of ERK signaling.