CONICET | Buscador de Institutos y Recursos Humanos

Tonicity-responsive enhancer binding protein (TonEBP) belongs tothe Rel superfamily of transcription factors (TF) and is consideredthe master regulator of osmoprotective gene expression. TonEBPexists as a dimer that completely encircles its DNA target (TonE).Nuclear factor kappa B (NFkB) also belongs to the Rel family andalso forms dimers when it binds to its response elements (kB) onits target genes. In renal cells, TonEBP and NFkB, are activated byhyperosmotic environment and regulate COX2 expression, whichis considered an osmoprotective gene. The present work exploreswhether TonEBP and NFkB act together or as individual TFs intheir regulatory action on osmoprotective gene expression underhyperosmotic conditions. For this purpose, we blocked the activityof TonEBP and NFkB, individually or together and then, evaluatedthe expression of their reporter genes (MCP1, IkB for NFkB andBGT1, AR, SMIT for TonEBP) as well as COX2. To do this, culturesof the renal epithelial cell line MDCK were grown in isoosmotic orhyperosmotic (298 or 500 mOsm/Kg H2O) media in the absence orpresence of TonEBP siRNA, a NFkB inhibitor (parthenolide, Parthe)or both. After 24 h, osmoprotective gene expression was evaluatedby RT-PCR. The possible interaction between both TFs was studiedby co-immunoprecipitation and immunofluorescence microscopy.Parthe downregulated COX2 and unexpectedly, TonEBP reportergenes. TonEBP-silencing knocked down COX2 and NFkB reportergenes. Parthe treatment blocked TonEBP expression but, TonEBPsilencing did not affect NFkB. These results show that both TFs areneeded to the expression of their reporter genes, and suggest asequential action where NFkB modulates TonEBP expression andthen, both together activate gene transcription. Immunofluorescenceshows the possible colocalization of both TFs in the nucleus. Suchinteraction was confirmed by co-immunoprecipitation. This findingindicates that both TFs act together to regulates gene expression

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