Glycogen synthase kinase 3ß participates in estradiol-17ß-D-glucuronide induced impairment of Mrp2 function in isolated rat hepatocyte couplets

BAROSSO, ISMAEL RICARDO; ANDERMATTEN, ROMINA BELÉN; SCHUCK, VIRGINIA SOLEDAD; CIRIACI, NADIA; TABORDA, DIEGO; CROCENZI, FERNANDO ARIEL; SANCHEZ POZZI, ENRIQUE J.

Mar del Plata

Congreso; LXIV Reunión Anual de la SAIC; 2019

Sociedad Argentina de Investigación Clínica

Introduction. E17G induces acute cholestasis in rat with endocytic internalization of the canalicular transporter Mrp2, activating two different pathways, each one involving an estrogen receptor: ERalpha or GPR30. cPKC activation precedes that of ERalpha. cPKC probably uses intermediaries to phosphorylate ERalpha. Several kinases can be phosphorylated by cPKC and are able to phosphorylate the ERalpha, among them we find GSK3ß. Aim. To evaluate the role of GSK3ß in the E17G-induced alteration of Mrp2 activity.Methods: IRHC were treated with GSK3ß inhibitors Li (3mM) or BIO (1µM) and then exposed to E17G (100µM). To investigate in which pathway GSK3ß participates, IRHC were exposed to BIO and inhibitors of ERalpha (ICI182,780, ICI, 1µM), cPKC (Gö6976, Gö, 1µM) or PI3K (Wortmannin, W, 100nM) before exposure to E17G. All preparations were incubated with CMFDA (intracellularly converted in gluthation-methylfluorescein [GMF], substrate of Mrp2). IHRC accumulating GMF in their canalicular vacuoles (cVA) were counted and compared to control IHRC. Results (% Control): GSK3ß inhibition (Li+E17G: 71±7b; BIO+E17G: 70±5b) partially prevented the effect of E17G (48±4a) on cVA of GMF. The preventive effects of W (W+E17G: 75±5b) and BIO on the decrease in cVA induced by E17G were additive (BIO+W+E17G: 91±1a,c). Contrarily, the preventive effects of ICI (ICI+E17G: 69±3b) or Gö (Gö+E17G: 77±3b) did not modified BIO protective effects (BIO+ICI+E17G: 72±2b) and (BIO+Gö+E17G: 77±10b). a significantly different from Control; b significantly different from E17G and Control. c significantly different from E17G+BIO and E17G+W. BIO, Li, W, Gö, and ICI did not affect % cVA. P < 0.05, n=3.Conclusions: GSK3ß inhibition protects against E17G-induced impairment of Mrp2 transport, indicating a role of the kinase in estrogen cholestasis. Co-inhibition studies suggest that GSK3ß participates in the same pathway of ERalpha and cPKC and in different pathway of PI3K (downstream of GPR30).