MEKK1-MKK4-JNK-AP1 Pathway Negatively Regulates Rgs4 Expression in Colonic Smooth Muscle Cells 英文参考文献.docVIP

MEKK1-MKK4-JNK-AP1PathwayNegativelyRegulates Rgs4ExpressioninColonicSmoothMuscleCells YonggangZhang1,FangLi1,ShuLiu1,HongWang1,SunilaMahavadi2,KarnamS.Murthy2, KamelKhalili1,WenhuiHu1* 1DepartmentofNeuroscience,TempleUniversitySchoolofMedicine,Philadelphia,Pennsylvania,UnitedStatesofAmerica,2DepartmentofPhysiologyandBiophysics, MedicalCollegeofVirginiaCampus,VirginiaCommonwealthUniversity,Richmond,Virginia,UnitedStatesofAmerica Abstract Background:RegulatorofG-proteinSignaling4(RGS4)playsanimportantroleinregulatingsmoothmusclecontraction, cardiac development, neural plasticity and psychiatric disorder. However, the underlying regulatory mechanisms remain elusive. Our recent studies have shown that upregulation of Rgs4 by interleukin (IL)-1b is mediated by the activation of NFkB signaling and modulated by extracellular signal-regulated kinases, p38 mitogen-activated protein kinase, and phosphoinositide-3kinase.Hereweinvestigatetheeffectofthec-JunN-terminalkinase(JNK)pathwayonRgs4expression inrabbitcolonicsmoothmusclecells. Methodology/Principal Findings: Cultured cells at first passage were treated with or without IL-1b (10ng/ml) in the presenceorabsenceoftheselectiveJNKinhibitor(SP600125)orJNKsmallhairpinRNA(shRNA).Theexpressionlevelsof Rgs4 mRNA and protein were determined by real-time RT-PCR and Western blot respectively. SP600125 or JNK shRNA increased Rgs4 expression in the absence or presence of IL-1b stimulation. Overexpression of MEKK1, the key upstream kinaseofJNK,inhibitedRgs4expression,whichwasreversedbyco-expressionofJNKshRNAordominant-negativemutants forMKK4orJNK.BothconstitutiveandinducibleupregulationofRgs4expressionbySP600125wassignificantlyinhibited by pretreatment with the transcription inhibitor, actinomycin D. Dual reporter assay showed that pretreatment with SP600125 sensitized the promoter activity of Rgs4 in response to IL-1b. Mutation of the AP1-binding site within Rgs4 promoter increased the promoter activity. Western blot analysis confir