Figure 1.
E217G activates the MAPK signalling pathway.
(A) 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. A (right panel), and B and C panels 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 (n = 5). *P<0.05 vs. control (cells treated only with DMSO), and #P<0.05 vs. E217G (20 min).
Figure 2.
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.
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 (n = 3–4). *P<0.05 vs. E217G, and #P<0.05 vs. E217G-WM, E217G-Gö, E217G-PD or E217G-SB.
Figure 3.
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.
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.
Figure 4.
Effect of E217G on colocalization of Rab11a with Mrp2 or Bsep in IRHCs.
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.
Figure 5.
Effect of inhibition of p38 or ERK1/2 on E217G-induced decrease of bile flow and biliary secretion of the Mrp2 and Bsep substrates DNP-SG and taurocholate, respectively, in the perfused rat liver (IPRL) model.
IPRLs were treated with a portal bolus of E217G (2 µmol/liver), or with the E217G vehicle DMSO (control), in the presence and absence of the ERK1/2 inhibitor PD98059 (PD; 5 µM) or the p38 inhibitor SB203580 (SB; 250 nM). The effect of the treatments on (A) bile flow, (B) DNP-SG excretion, (C) and taurocholate excretion are shown. Results are expressed as the mean ± SEM (n = 4).
Figure 6.
Effect of inhibition of p38 or ERK1/2 on E217G-induced retrieval of Bsep and Mrp2 in perfused rat livers at the end of the perfusion period.
The upper panels show representative confocal images showing co-staining of Mrp2 or Bsep (green) with F-actin (red; used to visualize the bile canaliculus limits), illustrative of the endocytic internalization of Mrp2 and Bsep induced by E217G (2 µmol/liver), and its protection by the ERK1/2 inhibitor PD98059 (PD; 5 µM) or the p38 inhibitor SB203580 (SB; 250 nM). The lower panels show a densitometric analysis of the intensity of fluorescence associated with Bsep and Mrp2 along a 6 µm line perpendicular to the canaliculus (from −3 µm to +3 µm from the canalicular center), corresponding to the confocal images in the upper panels.
Figure 7.
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).
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.