Glucocorticoid Inhibits WNK4 Ubiquitination By Regulating KLHL3

Introduction:Glucocorticoids(Gc)are steroid hormones secreted by the adrenal cortex,Gc has a variety of physiological and pharmacological effects such as regulation of immunity,metabolism,osmotic pressure,growth and development.It is generally believed that Gc functions through a classical genomic mechanism,that is,a lipid-soluble Gc Can bind to the glucocorticoid receptor present in the cytoplasm via the cell membrane of various tissues,either freely or through the help of transport proteins,and subsequently bind to the glucocorticoid response element in the vicinity of the target gene promoter,regulating the transcription of the gene,Inhibiting or enhancing the expression of the gene,exerting its physiological and / or pharmacological effects.Recent studies have found that there are other Gc-independent genomic pathways,called "non-genomic effects",having effects in red blood cells,platelets as well as eukaryotic cells.The molecular mechanism of Gc's non-genomic effects is not yet clear.However,studies have shown that PI3 K,protein kinase AKT,p38,PKC,ion channels,cAMP,IP3 and so on are involved in its signal pathway.WNK(With-No-Lysine(K))kinases are serine-threonine kinases characterized by an atypical placement of a catalytic lysine within the kinase domain.There are currently 4 family members,namely WNK1-4.Mutations in human WNK1 or WNK4 cause an autosomal dominant syndrome,type II pseudohyphalonism(PHAII),caused by kidney abnormal ion transportation.Experimental study confirmed that WNK4 can inhibit activity and expression of distal nephron channel protein such as NCCT,ENaC and ROMK.In 2012,two other PHAII pathogenic genes,KLHL3 and CUL3,were cloned by exome sequencing.They are components of the E3 ligase ubiquitin-degrading complex that specifically binds and degrades WNK4.Studies have shown that pathogenic mutations of WNK4 is located on the Acidic sequence of WNK4.KLHL3 is mainly a missense mutation at S433,which directly inhibits the binding of KLHL3 and WNK4 and thus inhibits the degradation of WNK4,resulting in clinical Symptoms.Gc has effects on the distal nephrons,promoting sodium and water retention,as aldosterone does.Our previous work identified the glucocorticoid response element in the promoter region of WNK4 through which Gc down-regulates WNK4 gene expression.However,it remains unclear whether glucocorticoids has effects on degradation of WNK4.In this study,HEK293 cells were stimulated with glucocorticoid and the expression of WNK4,KLHL3 and CUL3 genes were detected by Realtime PCR and Western blot.The Binding force of KLHL3 and WNK4 was detected by CO-IP,under the action of a kinase inhibitor,revealing the mechanism and function of glucocorticoids on WNK4.Materials and methods:1.Materials: human embryo kidney cell line HEK293,mouse kidney cell line M1.2.Methods: cell culture,transient gene transfection method,Real-time PCR assay,Western blot detection,co-immunoprecipitation and statistical analysis.Results:1.HA-WNK4 expression vector was transfected into HEK293 cells,The results of IP showed that the hybridization signal of ubiquitin antibody was weakened after glucocorticoid stimulation(P <0.05),suggesting that glucocorticoids have effects on ubiquitination degradation of WNK4.2.Real-time PCR and Western blot results showed that glucocorticoid significantly down-regulated the mRNA and protein expression of WNK4(P <0.05),and down-regulated the mRNA and protein expression of KLHL3(P <0.05),while CUL3 mRNA and protein expression have no significant changes.3.The addition of proteasome inhibitor MG-132 and glucocorticoid receptor antagonist RU486 increased the amount of WNK4 protein,suggesting that glucocorticoid non-genomic effects inhibit WNK4 degradation.4.Western blot results showed that the signal of WNK4 hybridization was enhanced after addition of PKC inhibitor(P <0.05),indicating that PKC inhibitor enhanced the binding ability of KLHL3 and WNK4.Conclusions:1.Glucocorticoids alter WNK4 ubiquitination status.2.Glucocorticoid down-regulates the expression of KLHL3 through the genomic mechanism,and changes the binding of KLHL3 to WNK4 through the PKC pathway,inhibiting the degradation of WNK4.