Role of kinases EGFR and SRC in estradiol 17ß-glucuronide (E17G)-induced cholestasis in isolated rat hepatocytes couplets (IRHC)

BAROSSO, ISMAEL R.; ZUCCHETTI, ANDRÉS ERNESTO; MISZCZUK, GISEL SABRINA; ROMA, MARCELO G; CROCENZI, FERNANDO A.; SÁNCHEZ POZZI, ENRIQUE J

Londres

Congreso; 49th Annual Meeting of the European Association for the Study of the Liver (EASL); 2014

EASL

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 for 15 min to either, the EGFR inhibitors Tyrphostin AG1478 (Tyr, 150 nM) and Cl-7387785 (Cl, 1mM), the SRC inhibitors, PP2 (5 mM) and SRC inhibitor 1 (IS, 1 mM), or the ERa inhibitor: ICI 182,780 (ICI, 1 mM), and then incubated with E17G (100 mM) for 20 min. 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, by determining their relative amount of phosphorylation (pEGFR tyr1156, p-SRC tyr416, pER ser118).   Results: See the table.                         Control   E17G   E17G+ICI   E17G+Tyr   E17G+Cl   E17G+IS   E17G+PP2   E17G+Tyr+ICI   E17G+IS+ICI cVA GS-MF      100±0    52±3a    70±5b        70±9b         74±2b      71±5b         69±7b            72±7b               69±4b cVA CGam       100±0    51±4a     66±2b       66±3b          64±5b     67±3b         67±3b            65±3b               67±3b 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%). Conclusions: EGFR and SRC participate in E17G-induced alterations of canalicular transporters. The lack of additive effect of inhibitors indicates that they share the same pathway as ERa. EGFR activation is downstream of ERa . The location of SRC in the pathway is not clear based on our evidences.