Sequential activation of classic PKC and estrogen receptor α is involved in estradiol 17ß-D-glucuronide-induced cholestasis

Abstract

Estradiol 17ß-D-glucuronide (E17G) induces acute cholestasis in rat with endocytic internalization of the canalicular transporters bile salt export pump (Abcb11) and multidrug resistance-associated protein 2 (Abcc2). Classical protein kinase C (cPKC) and PI3K pathways play complementary roles in E17G cholestasis. Since non-conjugated estradiol is capable of activating these pathways via estrogen receptor alpha (ERα), we assessed the participation of this receptor in the cholestatic manifestations of estradiol glucuronidated-metabolite E17G in perfused rat liver (PRL) and in isolated rat hepatocyte couplets (IRHC). In both models, E17G activated ERα. In PRL, E17G maximally decreased bile flow, and the excretions of dinitrophenyl-glutathione, and taurocholate (Abcc2 and Abcb11 substrates, respectively) by 60% approximately; preadministration of ICI 182,780 (ICI, ERα inhibitor) almost totally prevented these decreases. In IRHC, E17G decreased the canalicular vacuolar accumulation of cholyl-glycylamido-fluorescein (Abcb11 substrate) with an IC50 of 91±1 µM. ICI increased the IC50 to 184±1 µM, and similarly prevented the decrease in the canalicular vacuolar accumulation of the Abcc2 substrate, glutathione-methylfluorescein. ICI also completely prevented E17G-induced delocalization of Abcb11 and Abcc2 from the canalicular membrane, both in PRL and IRHC. The role of ERα in canalicular transporter internalization induced by E17G was confirmed in ERα-knocked-down hepatocytes cultured in collagen sandwich. In IRHC, the protection of ICI was additive to that produced by PI3K inhibitor wortmannin but not with that produced by cPKC inhibitor Gö6976, suggesting that ERα shared the signaling pathway of cPKC but not that of PI3K. Further analysis of ERα and cPKC activations induced by E17G, demonstrated that ICI did not affect cPKC activation whereas Gö6976 prevented that of ERα, indicating that cPKC activation precedes that of ERα.

Conclusion: ERα is involved in the biliary secretory failure induced by E17G and its activation follows that of cPKC.

Figure 1. Estimation of Estrogen Receptor α (ERα) activation by estradiol 17ß-d-glucuronide (E17G).

Isolated rat hepatocytes were incubated with E17G (100 µM) during different time-periods (0 to 20 min). ERα activity was determined by immunoblotting using antibodies against phosphorylated ERα (p-ERα Ser118) and total ERα. Phosphorylation degree of ERα was calculated as the ratio of each p-ERα total ERα band intensity and expressed as percent of this ratio at 0 min of E17G exposure.. Data are expressed as mean ± SEM (n = 3). ^a Significantly different from 0 min (p<0.05). ^b Significantly different from 0 min and 10 min of E17G treatment (p<0.05).

Figure 2. Prevention by ICI 182,780 (ICI) of estradiol 17ß-d-glucuronide (E17G)-induced impairment of canalicular vacuolar accumulation (cVA) of CGamF (upper panels) and GS-MF (lower panels).

IRHC were preincubated with ICI (1 µM) for 15 minutes, and then exposed to E17G (25–800 µM) for an additional 20-min period. cVAs of CGamF and GS-MF were calculated as the percentage of couplets displaying visible fluorescence in their canalicular vacuoles from a total of at least 200 couplets per preparation, referred to control cVA values. cVA control values were: 66±3% for CGamF and 76±3% for GS-MF and cVA of IRHC treated with ICI alone were 65±2% for CGamF and 76±4% for GS-MF. Data are expressed as mean ± SEM (n = 3).

Figure 3. Estrogen receptor inhibitor ICI 182,780 (ICI) prevented estradiol 17ß-d-glucuronide (E17G)-induced endocytic internalization of Abcb11 and Abcc2 in IRHC.

Panel A shows representative confocal images depicting cellular distribution of the canalicular transporters studied under the different treatments. Note that under control or ICI conditions transporter-associated fluorescence is mainly localized at the canalicular membrane. E17G (200 µM) induced a clear internalization of transporter-containing vesicles beyond the limits of the canaliculus, phenomenon significantly prevented by pretreatment with ICI (1 µM, 15 min). Panel B shows the densitometric analysis of the distribution of Abcb11 and Abcc2 fluorescence intensity along an 8-µm line perpendicular to the canalicular vacuole (4 µm to each side of the vacuole center), using the ImageJ 1.44p software (NIH, USA). The canalicular space was identified based on the corresponding DIC image. Each line profile measurement was normalized to the sum of all intensities of the respective measurement. The distribution of Abcb11 or Abcc2 fluorescence, expressed as a percentage of the total, was then calculated for each canaliculus and compared statistically, using the Mann-Whitney test. Statistical analysis of the profiles revealed a significant internalization of Abcb11 and Abcc2 under E17G treatment (p<0.05 vs control), which was completely abolished by ICI (p<0.05 vs E17G). Results are expressed as mean ± SEM. n = 6–8 canalicular vacuoles per preparation, from 3 independent preparations.

Figure 4. Effect of co-incubation with ICI 182,780 (ICI) and classic protein-kinase C (cPKC) or phosphoinositide 3-kinase (PI3K) inhibitors on estradiol 17ß-d-glucuronide (E17G)-induced impairment of canalicular vacuolar accumulation (cVA) of CGamF (panels A and C) and GS-MF (panels B and D).

Left panels: IRHC were incubated with Gö6976 (Gö, cPKC inhibitor, 1 µM) and ICI (1 µM), either alone or together, for 15 min. Right panels: IRHC were incubated with wortmannin (Wm, PI3K inhibitor, 100 nM) and ICI (1 µM), either alone or together, for 15 min. Then, IRHC were exposed to E17G (100 µM) for an additional 20-min period. Finally cVAs of CGamF and GS-MF were calculated as the percentage of couplets displaying visible fluorescence in their canalicular vacuoles from a total of at least 200 couplets per preparation, referred to control cVA values. Control cVA values were:67±3% for CGamF and 75±1% for GS-MF. Data are expressed as mean ± SEM (n = 3). ^a Significantly different from control (p<0.05). ^b Significantly different from E17G and control (p<0.05). ^c Significantly different from E17G, E17G+Wm and E17G+ICI (p<0.05).

Figure 5. Activation of estrogen receptor α (ER α) and classic protein-kinase C α (PKCα) in the presence of the cross inhibitors Gö6976 (Gö) and ICI 182,780 (ICI), respectively.

Panel A. Evaluation by immunoblotting of the effect of ICI on the specific PKCα activation by E17G in primary cultured hepatocytes. Primary cultured hepatocytes were treated with ICI (1 µM) for 15 min, then exposed to E17G (100 µM) for 5, 10 and 15 minutes, and finally the distribution of PKCα between cytosol and membrane was evaluated. The bar graph shows the fold translocation of PKCα isoform. PKCα at time 0 min without ICI (Control cells) were considered to be 1.0-fold activated. Area under the peak of the PKC isoform scanned (both cytosolic and membrane fractions) was determined, and the membrane-to-cytosol ratio was used to calculate fold translocation (or activation). Panel B. Effect of Gö on ERα activation by E17G. Isolated rat hepatocytes were incubated with Gö (1 µM) for 15 min and the exposed to E17G (100 µM) for another 15 min-period. ERα activity was determined by immunoblots using antibodies against phosphorylated ERα (p-ERα, Ser118) and ERα. The ratio of each p- ERα/ERα band density was compared to control bands ratio (100%). Data are expressed as mean ± SEM; n = 3 Western blot analyses, each from different cell culture experiments. ^a Significantly different from control (p<0.05). ^b Significantly different from E17G (p<0.05).

Figure 6. Estrogen receptor α (ERα) knock-down prevents estradiol-17ß-d-glucuronide (E17G)-induced endocytic internalization of Abcc2 in sandwich-cultured rat hepatocytes (SCRH).

Panel A: Representative western blot of ERα in SCRH transfected with four different siRNA. The siRNA1 induced a significant decrease in ERα expression (51±3% of scrambled siRNA-treated SCRH, p<0.05). ^a significantly different from scrambled. Panel B: Representative confocal images showing cellular distribution of Abcc2 (green) in SCRH. Actin network (red) and nuclei (blue) are also shown. E17G induced a clear internalization of Abcc2, visualized as transporter-containing vesicles beyond the canalicular region, reaching the perinuclear zone (white arrowheads). In cells treated with siRNA1 this phenomenon was significantly preventive only in cells effectively transfected. Cells that were not transfected showed the typical pattern of Abcc2 delocalization (pink arrowheads). Scrambled-transfected cells also showed a pattern of Abcc2 delocalization after E17G treatment.

Figure 7. Estrogen Receptor α (ERα) activation by estradiol-17ß-d-glucuronide (E17G) in vivo.

E17G (15 µmol/kg) or solvent was administered through the femoral vein. Immediately after E17G administration, partial hepatectomies were performed at different times (5, 10, 15, 20 min). ERα activity was determined by immunoblots using antibodies against phosphorylated ERα (p-ERα, Ser118) and ERα. The ratio of each p-ERα/ERα band density was compared to bands ratio of time 0 min (100%). Data are expressed as mean ± SEM (n = 3). ^a Significantly different from control sample at the corresponding time (p<0.05).

Figure 8. ICI 182,780 (ICI) protects against estradiol-17ß-d-glucuronide (E17G)-induced impairment of bile flow and biliary secretion of dinitrophenyl-glutathione and taurocholate in the perfused rat liver.

Temporal changes in bile flow (panel A) and in the biliary excretion rate of both total dinitrophenyl-glutathione (DNP-G, panel B) and taurocholate (panel C) throughout the perfusion period. PRLs were treated with a bolus of E17G (3 µmol/liver) or with the E17G vehicle DMSO/BSA 10% in saline (control), in the presence or absence of ICI (0.5 µM). ^a Control significantly different from E17G, ^b Control significantly different from E17G+ICI, ^c E17G significantly different from E17G+ICI. (p<0.05). N = 3–4 animals per group.

Figure 9. Estrogen Receptor inhibition prevents estradiol-17ß-d-glucuronide (E17G)-induced endocytic internalization of Abcb11 and Abcc2 in perfused rat liver.

Panel A: Confocal images of E17G-induced internalization of Abcb11 and Abcc2 and protection by ICI 182,780 (ICI). Representative confocal images of immunostained liver samples displaying a containing of Abcb11 (green) and occludin (red) (upper images), and Abcc2 (red) and occludin (green) (lower images). In control livers, both Abcb11 and Abcc2 were mainly confined to the canalicular space delineated by the tight junction-associated protein occludin. Following E17G (3 µmol/liver), some canaliculi show intracellular fluorescence associated with Abcb11 or Abcc2 at a greater distance from the canalicular membrane, consistent with their delocalization. ICI (0.5 µM, 15 min previous to E17G) prevented the internalization of canalicular transporters, as illustrated by a control-like pattern of Abcb11 and Abcc2 distribution. ICI itself did not induce any changes in transporters localization. Panel B: Densitometric analysis of fluorescence intensity profile of Abcb11, Abcc2 and occluding. Graphs represent the intensity of fluorescence associated with the transporters along an 8-µm line (from −4 µm to +4 µm of the canalicular center) perpendicular to the canaliculus. In control livers, transporter-associated fluorescence was concentrated in the canalicular space. E17G-induced internalization of transporters from the canalicular membrane (P<0.01 versus control) was detected as a decrease in the fluorescence intensity in the canalicular area together with an increased fluorescence at a greater distance from the canaliculus. Distribution profiles of livers treated with E17G+ICI was similar to control and indicated a significantly decreased of Abcb11 and Abcc2 internalization (P<0.01 versus E17G). (n = 20–50 canaliculi per preparation, three independent preparations). Statistical analysis of the distribution profiles of occluding, used to demarcate limits of the canaliculi, showed no changes in the normal distribution by any of the treatments.