ROLE OF KINASES EGFR AND SRC IN ESTRADIOL 17b-GLUCURONIDE (E17G) INDUCED CHOLESTASIS IN ISOLATED RAT HEPATOCYTES COUPLETS (IRHC)

BAROSSO, ISMAEL R.; ZUCCHETTI, ANDRÉS E.; MISZCZUK, GISEL S.; ROMA, MARCELO G.; CROCENZI, FERNANDO A.; SANCHEZ POZZI, ENRIQUE J.

Londres

Congreso; 49th annual meeting of the European Association for the Study of the Liver; 2014

European Association for the Study of the Liver

Background and Aims: E17G internalizes canalicular transporters Bsep and Mrp2. Estrogen receptors (ERa and GPR30) are implied in E17G actions, activating different pathways. We described a role of EGFR in this model of cholestasis independent of GPR30 (Hepatology 2013 doi:10.1002/hep.26752). Since EGFR can interact with SRC and ERa, we evaluated whether these kinases are activated by E17G sharing the same pathway. Methods: IRHC were exposed 15min to either: EGFR inhibitors: Tyrphostin AG1478 (Tyr, 150 nM), or Cl-7387785 (Cl, 1mM), SRC inhibitors: PP2 (5mM) and SRC inhibitor 1 (IS, 1mM), ERa inhibitor: ICI 182,780 (ICI, 1mM) and then incubated with E17G (100mM) 20min.We assessed the canalicular vacuolar accumulation (cVA) of cholyl-glysylamidofluorescein (CGamF, Bsep substrate) or glutathion-methylfluorescein (GS-MF, Mrp2 substrate). Activation of EGFR, SRC and ERa were assessed by western blot, determining their relative amount of phosphorylation (pEGFR tyr1156, p-SRC tyr416, pER ser118). Results: See the table. Western blot studies (n = 3): E17G induced protein phosphorylation (pEGFR: 325±17%, pSRC: 201±31%, pER: 233±29%, vs control), ICI partially blocked EGFR phosphorylation (195±34%, p < 0.05) whereas IS did not (272±56%). Neither Tyr nor IS affected ERa phosphorylation (Tyr: 250±38%, IS: 189±30%). Finally, SRC phosphorylation was not affected by Tyr (175±26%) or ICI (144±26%).