Tauroursodesoxicolate prevents activation of pro-cholestatic signalling pathways in estradiol 17ß-D-glucuronide-induced cholestasis independently of protein phosphatases and the integrin receptor.

MEDEOT A.; MAIDAGAN P.M.; BOAGLIO A.C.; RAZORI M.V.; ANDERMATTEN R.B.; CIRIACI N.; CROCENZI F.A.; ROMA M.G.

Rosario, Santa Fe

Congreso; Reunión Anual de la Sociedad Argentina de Fisiología (SAFIS) 2019; 2019

Sociedad Argentina de Fisiología (SAFIS) 2019

Estradiol 17ß-D-glucuronide (E217G), a main etiologic agent of pregnancy-induced cholestasis, induces bile flow drop by endocytosis and intracellular retention of the main canalicular transporters involved in bile secretion, Mrp2 and Bsep, via activation of the pro-cholestatic signaling pathways cPKC and PI3K, respectively. The most effective medication for pregnancy-induced cholestasis is ursodeoxycholic acid (UDCA), but its action mechanisms are still unknown. We tested here whether the main active metabolite of UDCA, tauroursodeoxycholate (TUDC), prevents the secretory failure induced by E217G by inhibiting the activation of cPKC and PI3K/Akt signaling pathways, and whether protein phosphatases (PPs) or integrin receptor are involved in this mechanism. Activation of cPKC and Akt was quantified by assessing the plasma membrane amount and the phosphorylated amount of these proteins, respectively, in isolated rat hepatocytes by Western Blot. Pretreatment with TUDC (100 µM), followed by exposure to E217G (100 µM), prevented activation of cPKC (-34±4%) and Akt (-37±2%); p<0.05 vs. E217G. Mrp2 and Bsep transport activity was assessed in rat hepatocyte couplets (RHC) by quantifying the % of RHC displaying canalicular vacuolar accumulation (CVA) of fluorescent substrates GSH-S-methylflourescein (GS-MF) and cholyl-lysilfluorecein (CLF), respectively. E217G decreased CVA of CLF by 41±3% (p<0.05 vs. control), while TUDC partially prevented this alteration by 79±7% (p<0.05 vs. E217G). E217G-induced impairment in CVA of the Mrp2 substrate GS-MF was similarly prevented by TUDC. The pretreatments with the PP1/PP2A inhibitors, tautomycin (1 nM) and okadaic acid (5 nM), the PP2B inhibitor tacrolimus (1 µM), or the integrin receptor inhibitor RGD (20 and 30 µM) did not prevent the CVA improvement afforded by TUDC. We conclude that TUDC-induced preservation of Bsep and Mrp2 function and location in E217G-induced cholestasis involves halting of cPKC and PI3K activation by mechanisms independent of protein phosphatase-induced dephosphorylation or integrin-mediated signaling, but rather by counteracting a yet not explored E217G-activated cholestatic signaling pathways upstream of cPKC and PI3K.