INVESTIGADORES
ROMA Marcelo Gabriel
congresos y reuniones científicas
Título:
Role of kinases EGFR and SRC in estradiol 17ß-glucuronide (E17G)-induced cholestasis in isolated rat hepatocytes couplets (IRHC)
Autor/es:
BAROSSO I.R.; ZUCCHETTI A.E.; MISZCZUK G.S.; ROMA M.G.; CROCENZI F.A.; SÁNCHEZ POZZI E.J.
Lugar:
Londres
Reunión:
Congreso; The International Liver Congress - 47th Annual Meeting of the European Association for the Study of the Liver (EASL); 2014
Institución organizadora:
European Association for the Study of the Liver (EASL)
Resumen:
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.