SEQUENTIAL AND COOPERATIVE ACTION OF TonEBP AND NFkB TRANSCRIPTION FACTORS IS NEEDED FOR OSMOPROTECTIVE GENE EXPRESSION.

ERJAVEC, LUCIANA; FERNANDEZ TOME, MARÍA DEL CARMEN; CASALI, CECILIA IRENE

Ciudad Autónoma de Buenos Aires

Congreso; Reunión conjunta de las Sociedades de BioCiencias; 2017

Sociedades participantes

Tonicity-responsive enhancer binding protein (TonEBP) belongs to the Rel superfamily of transcription factors (TF) and is considered the master regulator of osmoprotective gene expression. TonEBP exists as a dimer that completely encircles its DNA target (TonE). Nuclear factor kappa B (NFkB) also belongs to the Rel family and also forms dimers when it binds to its response elements (kB) on its target genes. In renal cells, TonEBP and NFkB, are activated by hyperosmotic environment and regulate COX2 expression, which is considered an osmoprotective gene. The present work explores whether TonEBP and NFkB act together or as individual TFs in their regulatory action on osmoprotective gene expression under hyperosmotic conditions. For this purpose, we blocked the activity of TonEBP and NFkB, individually or together and then, evaluated the expression of their reporter genes (MCP1, IkB for NFkB and BGT1, AR, SMIT for TonEBP) as well as COX2. To do this, cultures of the renal epithelial cell line MDCK were grown in isoosmotic or hyperosmotic (298 or 500 mOsm/Kg H2O) media in the absence or presence of TonEBP siRNA, a NFkB inhibitor (parthenolide, Parthe) or both. After 24 h, osmoprotective gene expression was evaluated by RT-PCR. The possible interaction between both TFs was studied by co-immunoprecipitation and immunofluorescence microscopy. Parthe downregulated COX2 and unexpectedly, TonEBP reporter genes. TonEBP-silencing knocked down COX2 and NFkB reporter genes. Parthe treatment blocked TonEBP expression but, TonEBP silencing did not affect NFkB. These results show that both TFs are needed to the expression of their reporter genes, and suggest a sequential action where NFkB modulates TonEBP expression and then, both together activate gene transcription. Immunofluorescence shows the possible colocalization of both TFs in the nucleus. Such interaction was confirmed by co-immunoprecipitation. This finding indicates that both TFs act together to regulates gene expression.