IN VITRO BINDING AND TRANSCRIPTIONAL EFFECT OF ZEB-1a AND ZEB-1b IS REVERTED BY TPA TREATMENT
Cabanillas, Ana María, Arroyo Daniela and Knubel Carolina. CIBICI-CONICET, Fac CC QQ, Universidad Nacional de Córdoba.
ZEB1/ZEB1 (Zinc Finger Homeodomain Enhancer-binding Protein) is involved in lymphopoiesis, neurogenesis and myogenesis and it is expressed as two isoforms, ZEB1-a and ZEB1-b. ZEB1 also exists as two phosphorylated forms. Our goal was to examine the effect of phosphorylation on the regulation of ZEB1 transcriptional effect. ZEB1 expressing cell lines were treated with 10 ng/ml phorbol esters (PMA) and 500 ng/ml ionomycin (IO) for 30 min. Nuclear extracts (NE) from untreated cells were incubated with phosphatase (CIP) or with CIP+phosphate. EMSAs were performed with Jurkat/CHO-K1/COS-7 NE (CIP, CIP+phosphate or PMA/IO treated) and ZEB1-b programmed rabbit reticulocyte lysates in the presence of [32P]-labeled oligonucleotides harboring ZEB1 binding sites from ZEB1, a4integrin, CD4 and p73 promoters. CIP-treated samples increased ZEB1 binding capacity to all the probes assayed. ZEB1-a and ZEB1-b showed similar results. Retardation complexes were competed by either anti-ZEB1 antibodies or an excess of cold oligonucleotides. PMA/IO- treated cells shown no band of retardation. Same cell lines were transfected with ZEB1 expression vector and CD4, p73 y ZEB1/luciferase promoters by O/N Ca-P precipitation. Then, cells were incubated with PMA/IO for 8 h. As expected, luciferase activities (normalized by bgal) were diminished by ZEB1. PMA/IO treatment reverted ZEB1 repression to its target genes. The results show that phosphorylation through protein kinase C (PKC) is one of the mechanisms of regulation of the transcriptional activity of ZEB1. ZEB1 mediates TGF-b dependent gene activation in myogenesis. The status of phosphorylation of ZEB1 would provide a way to modulate the activity of TGF-b signaling pathway which is also regulated by PKC pathway.
