Effect of E₂17G on colocalization of Rab11a with Mrp2 or Bsep in IRHCs.

<p>Immunofluorescence confocal images showing staining of Mrp2 or Bsep (green) and Rab11a (red) in IRHCs treated with DMSO (control), the ERK1/2 inhibitor PD98059 (PD; 5 µM), the p38 inhibitor SB203580 (SB; 1 µM), or both inhibitors together. Colocalization of Rab11a with Mrp2 or Bsep in the E₂17G-treated group is indicated by orange-yellow fluorescence in merged images. Insets depict F-actin staining, which was used to demarcate the limits of the canalicular vacuoles.</p

Effect of the inhibition of p38, ERK1/2 and JNK1/2, or the coinhibition of cPKC-ERK1/2, PI3K-p38, or p38-ERK1/2, on E₂17G-induced impairment of the canalicular accumulation of the Bsep and Mrp2 fluorescent substrates in IRHCs.

<p>IRHCs were incubated with E₂17G (200 µM, 20 min) (or DMSO in controls), with or without pretreatment for 15 min with the JNK1/2 inhibitor SP600125 (1 µM), the ERK1/2 inhibitor PD98059 (PD; 5 µM), and/or the p38 inhibitor SB203580 (SB; 1 µM), together or not with the cPKC inhibitor Gö6976 (Gö; 1 µM) or PI3K inhibitor wortmanin (WM; 100 nM). Canalicular accumulation CGamF (Bsep substrate, panel A) and GS-MF (Mrp2 substrate, panel B) was determined as the percentage of couplets displaying visible fluorescence in their canalicular vacuoles from a total of at least 200 couplets per preparation. The results are expressed as percentages of the control group and are shown as mean ± SEM (<i>n</i> = 3–4). *P<0.05 <i>vs.</i> E₂17G, and ^#P<0.05 <i>vs.</i> E₂17G-WM, E₂17G-Gö, E₂17G-PD or E₂17G-SB.</p

Schematic representation of the signalling events involved in E₂17G-induced cholestasis by endocytic internalization and further retention of canalicular transporters relevant to bile formation (Bsep, Mrp2).

<p>p38, acting downstream of cPKC, triggers endocytic internalization of the apical carriers presumably towards apical early endosomes (AEE), the first intracellular endosomal compartment receiving internalized proteins from the apical membrane, in a microtubule-independent manner (solid arrow). These transporters traffic to, and accumulate into, apical recycling endosomes (ARE), from where they can be retargeted to the apical membrane during the recovery of the cholestatic process, in a microtubule-dependent manner (dashed arrows). Activation of the PI3K/Akt/ERK1/2 signalling pathway halts this latter process, thus explaining the increased colocalization of Bsep/Mrp2 with Rab11a, an ARE marker. This prolongs the cholestatic effect of E₂17G by impeding the fast, spontaneous retargeting of intracellular transporters that would lead to a rapid recovery from the cholestatic injury.</p

Effect of inhibition of p38 or ERK1/2, and coinhibition of cPKC-ERK1/2, PI3K-p38, or p38-ERK1/2 on E₂17G-induced retrieval of Bsep and Mrp2 in IRHCs.

<p>The upper panels show representative confocal immunofluorescence images of the localization of Bsep and Mrp2 in DMSO-treated (control) or E₂17G (200 µM)-treated IRHCs, with or without the p38 inhibitor SB203580 (SB; 1 µM) or the ERK1/2 inhibitor PD98059 (PD; 5 µM), in combination or not with the cPKC inhibitor Gö6976 (Gö; 1 µM) or PI3K inhibitor wortmanin (WM; 100 nM). The lower panels show the densitometric analysis of the fluorescence intensity along a line (8 µm) perpendicular to the center of the canalicular vacuole (from +4 to −4 µm). The statistical analysis of the profiles of fluorescence showed a significant change in the E₂17G-treated group (P<0.05; number of analyzed canalicular vacuoles >10), but this reverted to normal in the E₂17G-SB, E₂17G-PD, E₂17G-PD-SB, E₂17G-Gö-PD and E₂17G-WM-SB groups for Bsep and Mrp2.</p

E₂17G activates the MAPK signalling pathway.

<p><i>(A)</i> left panel: representative Western blottings of phospho (p)-p38, p-ERK1/2, p-JNK1/2 and total forms of all these MAPK types were obtained from whole cellular lysates of primary-cultured rat hepatocytes incubated with E₂17G (200 µM) for 10 to 60 min, or with E₂17G (200 µM) for 20 min in cells pretreated with the PI3K inhibitor wortmanin (WM, 100 nM) or with the cPKC inhibitor Gö6976 (Gö, 1 µM) for 15 min. <i>A</i> (right panel), and <i>B</i> and <i>C panels</i> show phosphorylation status of all MAPK types evaluated (calculated as the p-MAPK to total MAPK ratio for each experimental condition). An arbitrary value of 100 was assigned to the band of highest densitometric intensity in every Western blot before the ratio was calculated. The results are shown as mean ± SEM (<i>n</i> = 5). *P<0.05 <i>vs.</i> control (cells treated only with DMSO), and ^#P<0.05 <i>vs.</i> E₂17G (20 min).</p

Sequential Activation of Classic PKC and Estrogen Receptor α Is Involved in Estradiol 17ß-D-Glucuronide-Induced Cholestasis

<div><p>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 <em>via</em> 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.</p> </div

Estrogen Receptor α (ERα) activation by estradiol-17ß-d-glucuronide (E17G) <i>in vivo</i>.

<p>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).</p

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

<p>Isolated rat hepatocytes were incubated with <b>E17G</b> (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).</p

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.

<p>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).</p

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

<p>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.</p