The Role Of ETV5 In The Synthesis And Secretion Of Ghrelin

Background and Objective: ETV5 is an E-twenty-six(ETS)transcription factor ubiquitously expressed and its biological functions have been implicated in multiple areas.Genome-wide association study(GWAS)have revealed an association between ETV5 and human obesity.Etv5 knockout mice also have reduced body weight and less fat mass,indicating that ETV5 is involved in the control of energy homeostasis.Ghrelin,a 28 amino acid peptide hormone,regulates multiple important metabolic functions.Its acylation by ghrelin O-acyl-transferase enzyme(GOAT)is required for developing into active ghrelin and playing its biological function.However,mechanism underlying the regulation of GOAT and acyl ghrelin remains unclear.In the present study,we examined the role of ETV5 in GOAT/ghrelin system using the CLU122 cells,a murine hypothalamic cell line,as well as in mice.So as to provide a new therapeutic medical target for appetite control and obesity treatment.Methods: 1.Immunohistochemical staining on serial sections of mouse gastric tissues,use ETV5 and ghrelin antibodys respectively.2.We altered ETV5's gene expression level through transiently transfecting the CLU122 cells.Ghrelin was measured by active and total ghrelin ELISA kit.Western blotting analysis for GOAT protein expression.qPCR analysis for GOAT mRNA expression.3.GOAT promoter luciferase reporter vectors were constructed,and transfected together with ETV5 or vector control into HEK293 T cells.Dual-glo luciferase assay system was used to detect the effects of transcription factor ETV5 on the activity of GOAT promoter.4.CLU122 cells were transiently transfected plasmid or treated with mTORC1 pathway inhibitor rapamycin to alter ETV5 expression.Real-time qPCR and Western blotting were performed to observe ETV5 expression.5.We injected C57 male mice with rapamycin and obtained the gastric tissues from mice with specific deletion of mTOR in ghrelin positive cells to verified the in vivo effects of mTORC1 activity on ETV5's expression.6.CLU122 cells were transfected with pCMV-ETV5 or siRNA against ETV5,then treatedwith rapamycin to alter the mTORC1 activity.The cell lysates were collected for active ghrelin ELISA measurement.Result: 1.ETV5 and ghrelin have a colocalization in the X/A like cells.2.Overexpression of ETV5 increased the protein level of GOAT and stimulated the secretion of active ghrelin.Whereas the mRNA level of GOAT and the secretion of total ghrelin remained unaltered.3.ETV5 markedly enhanced GOAT's promoter activity.4.In vitro,mTORC1 activity negatively regulated ETV5's protein level,but has no effect on the level of mRNA.5.Inhibition of mTORC1 signaling profoundly increased gastric ETV5's protein level but not the mRNA level.6.The effect of mTORC1 on active ghrelin was,at least partially,acted through ETV5's gene level.Conclusion: The present study demonstrated that ETV5 could transactivate GOAT promoter region and increase its expression,leading to subsequent increase in the production of active ghrelin.The activity of mTORC1 signaling pathway modulated ETV5 expression.Moreover,ETV5 mediated the effects of mTORC1 signaling on the expression level of active ghrelin.ETV5 may be a key regulator of mTORC1-GOAT/ghrelin axis in ghrelin producing cells and a potential therapeutic target for organism energy imbalance.