Tauroursodeoxycholate prevents estradiol 17-β D-glucuronide-mediated cholestasis by inhibiting the phosphorylative activation of pro-cholestatic protein kinases, independently of protein phosphatases

MAIDAGAN P.M.; BOAGLIO A.C.; RAZORI M.V.; CIRIACI N.; MISZCZUK G.S.; CROCENZI F.A.; ROMA M.G.

Mar del Plata

Congreso; LXI Reunión Científica Anual de la Sociedad Argentina de Investigación Clínica (SAIC); 2016

Sociedad Argentina de Investigación Clínica (SAIC)

Wehave previously shown that estradiol 17-β D-glucuronide (E217G), etiologic agent of pregnancy-induced cholestasis, altersfunction and location of both Mrp2 and Bsep, two key canalicular transporters, byenhancing phosphorylative activation of "classical" protein kinase C (PKCc) and phosphatidylinositol 3kinase (PI3K)/Akt-dependent signaling pathways. Pregnancy-induced cholestasisis treated with ursodeoxycholate, but its therapeutic mechanisms are stillunknown. To address this question, we evaluated whether its active metabolite, tauroursodeoxycholate(TUDC), prevents E217G-induced canalicular secretory failure byinhibiting the phosphorylative activation of PKCc and PI3K/Akt, and whether proteínphosphatases (PPs) are involved in this phenomenon. For this purpose, westudied by Western Blot, in isolated rat hepatocytes, the effect of TUDC on activationof PKCc (% of membrane translocation) and Akt (% of phosphorylated form). Pretreatmentwith TUDC (100 µM), followed by exposure to E217G (100 µM), preventedactivation of PKCc (-34±4%) and Akt (-37±2%); p<0.05 vs. E217G. Canalicular transport function was assessedin rat hepatocyte couplets by quantifying the % of couplets displaying canalicularvacuolar accumulation (CVA) of the fluorescent Bsep and Mrp2 substrates cholyl-lysilfluorescein(CLF) and GSH-S-methylfluorescein (GS-MF), respectively. E217Gdecreased CVA of CLF by 41±3% (p<0.05 vs. control), while TUDC partially prevented this alteration by79±7% (p<0.05 vs. E217G).E217G-induced impairment in CVA of GS-MF was similarly prevented by TUDC. Neither the PP1/PP2Ainhibitors, tautomycin (1 nM)and okadaic acid (5 nM), nor the PP2B inhibitor tacrolimus (1 µM) affected the preventiveeffect of TUDC. We conclude that TUDC preserves function and location of Bsepand Mrp2 in E217G-induced cholestasis by preventing activation of upstreamsignaling pathways that activate PKCc and PI3K/Akt rather than by increasing theirdephosphorylation via PPs.