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. 2016 May 12:6:24709.
doi: 10.1038/srep24709.

Rho-kinase/myosin light chain kinase pathway plays a key role in the impairment of bile canaliculi dynamics induced by cholestatic drugs

Ahmad Sharanek  1   2 , Audrey Burban  1   2 , Matthew Burbank  1   2 , Rémy Le Guevel  3 , Ruoya Li  4 , André Guillouzo  1   2 , Christiane Guguen-Guillouzo  1   2   4
Affiliations

Rho-kinase/myosin light chain kinase pathway plays a key role in the impairment of bile canaliculi dynamics induced by cholestatic drugs

Ahmad Sharanek et al. Sci Rep. .

Abstract

Intrahepatic cholestasis represents a frequent manifestation of drug-induced liver injury; however, the mechanisms underlying such injuries are poorly understood. In this study of human HepaRG and primary hepatocytes, we found that bile canaliculi (BC) underwent spontaneous contractions, which are essential for bile acid (BA) efflux and require alternations in myosin light chain (MLC2) phosphorylation/dephosphorylation. Short exposure to 6 cholestatic compounds revealed that BC constriction and dilation were associated with disruptions in the ROCK/MLCK/myosin pathway. At the studied concentrations, cyclosporine A and chlorpromazine induced early ROCK activity, resulting in permanent MLC2 phosphorylation and BC constriction. However, fasudil reduced ROCK activity and caused rapid, substantial and permanent MLC2 dephosphorylation, leading to BC dilation. The remaining compounds (1-naphthyl isothiocyanate, deoxycholic acid and bosentan) caused BC dilation without modulating ROCK activity, although they were associated with a steady decrease in MLC2 phosphorylation via MLCK. These changes were associated with a common loss of BC contractions and failure of BA clearance. These results provide the first demonstration that cholestatic drugs alter BC dynamics by targeting the ROCK/MLCK pathway; in addition, they highlight new insights into the mechanisms underlying bile flow failure and can be used to identify new predictive biomarkers of drug-induced cholestasis.

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Conflict of interest statement

Ruoya Li and Christiane Guguen-Guillouzo are employed by Biopredic International, St Grégoire, France.

Figures

Figure 1
Figure 1. Schematic representation of MLC phosphorylation regulation by Rho-kinase and myosin light chain kinase.
ROCK, Rho-kinase; Ca2+, calcium; CaM, calmodulin; MLCK, myosin light chain kinase; MLCP, myosin light chain phosphatase; MYPT-1, myosin phosphatase target subunit 1; MLC, myosin light chain, ET-1, endothelin-1; ETR, endothelin receptor; IP3, inositol 1,4,5-triphosphate; GPCR, G-protein coupled receptor; PKC, protein kinase C; PLCβ, phospholipase C β; DAG, diacylglycerol; PI3K, phosphatidylinositol 3-kinases ; ERK, extracellular signal-regulated kinases; MAPK, mitogen-activated protein kinases ; GF, growth factor.
Figure 2
Figure 2. Polarity of human HepaRG and primary hepatocytes.
(A) Phase-contrast microscopy examination of the BC in HepaRG hepatocytes, CCHH and SCHH. Rhodamine-phalloidin fluoroprobe staining of pericanalicular F-actin (red); immunolocalization of the junctional ZO-1 protein (green). Merged images showing that the junctional ZO-1 protein (green) co-localizes with the pericanalicular F-actin (red) in HepaRG cells CCHH and SCHH. Immunolocalization of the hepatobiliary transporters MDR1 and MRP2 (green). CDF accumulation in BC (green). Hoechst-labelled nuclei (blue). Fluorescence images were obtained with a Cellomics ArrayScan VTI HCS Reader (bar = 30 μm). (B) Electron microscopy examination of the tight junctions surrounding BC in HepaRG cells (arrows) (bar = 10 μm).
Figure 3
Figure 3. Cytotoxicity evaluation and alteration of the BC morphology by the tested compounds in HepaRG cells.
(A) Cells were incubated for 4 h with different concentrations of CPZ (0–200 μM), CsA (0–200 μM), fasudil (0–200 μM), ANIT (0–200 μM), DCA (0–800 μM) and bosentan (0–400 μM). Cytotoxicity was measured by the MTT colorimetric assay. Data were expressed relative to the untreated cells, which were arbitrarily set at a value of 100%. Data represent the means ± SEM of three independent experiments. (B) Untreated cells and cells treated with 50 μM CPZ, 50 μM CsA, 50 μM fasudil, 50 μM ANIT, 200 μM DCA or 100 μM bosentan. Phase-contrast images were captured after 3 h; BC (arrows); F-actin localized using rhodamine-phalloidin fluoroprobe (red). Immunolabelling of the junctional ZO-1 protein (green) in HepaRG cells treated with the tested compounds compared with that of the control cells. Nuclei stained in blue (Hoechst dye). The images were obtained with a Cellomics ArrayScan VTI HCS Reader (bar = 30 μm).
Figure 4
Figure 4. Alteration of the BC morphology by the tested compounds in CCHH and SCHH.
Untreated cells and cells treated with 50 μM CPZ, 50 μM CsA, 50 μM fasudil, 50 μM ANIT, 200 μM DCA, or 100 μM bosentan. Phase-contrast images were captured after 3 h. F-actin was localized using a rhodamine-phalloidin fluoroprobe (red). Nuclei stained in blue (Hoechst dye). (bar = 30 μm).
Figure 5
Figure 5. Dynamics of BC in human HepaRG and primary hepatocytes.
(A) Time-lapse imaging showing unidirectional rhythmic opening (green arrows) and closing (red arrows) spikes associated with contraction/relaxation of S-BC (white arrows). (B–D) Quantification of S-BC area in arbitrary units (A.U.) and graphic representation of spikes. Imaging was performed using an inverted microscope (bar = 10 μm), and video analysis was performed using a modelling tool (Tracker 4.87).
Figure 6
Figure 6. Disruption of BC rhythmic movements in CPZ- and fasudil-treated HepaRG hepatocytes.
(A,B) Representative time-lapse imaging of HepaRG cells treated with 50 μM CPZ or 50 μM fasudil for 4 h (bar = 10 μm). (C,D) Quantification of the S-BC area and spikes showing the early disappearance of rhythmic spikes (green/red arrows) with both drugs. Occurrence of permanent constriction of S-BC with CPZ and dilation with fasudil (white arrows). (E) Quantification of the BC area based on ZO-1 protein distribution (as in Fig. 3) using Cellomics ArrayScan VTI HCS Reader software as described in the Materials and Methods. The BC were grouped into 3 categories according to their area.
Figure 7
Figure 7. Effects of the tested compounds on BA clearance.
(A) NBD-UDCA and CDF efflux. White arrows indicate fluorescence in the BC (bar = 30 μm). (B) Quantification of CDF accumulation in the BC after fasudil treatment. (C,D) [3H]-TA clearance in the HepaRG cells treated for 2 h with either the tested compounds or different concentrations of unlabelled TA. (E) [3H]-TA clearance in the cells treated with either CPZ or fasudil or co-treated with unlabelled TA. The data were expressed relative to that of the untreated cells arbitrarily set at 100%. The data represent the means ± SEM of 3 independent experiments. *p < 0.05 compared with that of the untreated cells, #p < 0.05 compared with that of TA (50 μM) alone.
Figure 8
Figure 8. Alteration of ROCK activity by the tested compounds.
(A) Untreated and 20 μM Y-27632-treated cells. Phase-contrast images were captured after 3 h. F-actin was localized using a rhodamine-phalloidin fluoroprobe (red). Hoechst-labelled nuclei are shown in blue (bar = 30 μm). Note the BC dilation in the presence of Y-27632 cells. (B) ROCK activity after a 1-h treatment of HepaRG cells using a ROCK activity assay Kit (Millipore, catalogue CSA001). The data were expressed relative to that of the untreated cells and are represented as the means ± SEM of 3 independent experiments. *p < 0.05 compared with that of the untreated cells, #p < 0.05 compared with that of either CPZ (50 μM) or CsA (50 μM) alone.
Figure 9
Figure 9. Alteration of MLC2 phosphorylation/dephosphorylation rhythms by the tested compounds.
(A) Untreated and 20 mM BDM-treated HepaRG cells. Phase-contrast images were captured after 3 h. F-actin was localized using a rhodamine-phalloidin fluoroprobe (red). Hoechst-labelled nuclei are shown in blue (bar = 30 μm). (B) Representative western blots of the p-MLC2/total MLC2 forms at various time points. (C) Graphic representation of MLC2 phosphorylation/dephosphorylation quantified using ImageJ 1.48 software. The data were expressed in arbitrary units (A.U.) and represented as the means ± SEM of 3 independent experiments.
Figure 10
Figure 10. Alteration of the MLCK pathway with the tested compounds.
(A) HepaRG cells exposed to 20 μM ML-9 for 3 h and the controls: phase-contrast microscopy and F-actin localization (bar = 30 μm). Note the BC dilation in the presence of ML-9 cells. (B) Time-lapse imaging of HepaRG cells treated with ANIT, DCA, fasudil or bosentan alone, co-treated with 5 μM CaM; or treated with bosentan + ML-9 or fasudil (bar = 30 μm); BC: green arrows. (C) Quantification of the BC area using ImageJ 1.48 software as described in the Materials and Methods. The data were expressed as the fold change in the BC mean area after 3 h relative to the mean area at T0 (means ± SEM of 3 independent experiments). *p < 0.05 compared with that of the untreated cells, #p < 0.05 compared with that of the cultures treated with either ANIT, DCA or bosentan alone. &p < 0.05 compared with that of the cultures treated with both bosentan and fasudil. (D) [3H]-TA clearance in HepaRG cells treated with bosentan and DCA alone or co-treated with CaM for 2 h. The data were expressed relative to that of the untreated cells arbitrarily set at 100% and are presented as the means ± SEM of 3 independent experiments. *p < 0.05 compared with that of the untreated cells, #p < 0.05 compared with that of the cultures treated with either bosentan or DCA alone.
Figure 11
Figure 11. Schematic representation of the main molecular targets of the tested cholestatic compounds in the ROCK/MLCK pathway.
(1) CsA and CPZ induced the activation of ROCK activity and maintained abnormally high MLC2, thereby leading to BC constriction. However, (2) fasudil and Y-27632 inhibited ROCK activity and caused MLC2 dephosphorylation, thereby leading to BC dilation. (3) DCA, ANIT, bosentan and ML-9 inhibited Ca2+/CaM-dependent MLCK instead of ROCK and led to BC dilation. (4) BDM inhibited myosin II heavy chain ATPase activity and disrupted acto-myosin interaction causing BC dilation. ROCK, Rho-kinase; Ca2+, calcium; CaM, calmodulin; MLCK, myosin light chain kinase; MLCP, myosin light chain phosphatase; MLC, myosin light chain; BC, bile canaliculi.
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References

    1. Padda M. S., Sanchez M., Akhtar A. J. & Boyer J. L. Drug-induced cholestasis. Hepatology 53, 1377–1387 (2011). - PMC - PubMed
    1. Bjornsson E. S. & Jonasson J. G. Drug-induced cholestasis. Clin Liver Dis 17, 191–209 (2013). - PubMed
    1. Morgan R. E. et al. A multifactorial approach to hepatobiliary transporter assessment enables improved therapeutic compound development. Toxicol Sci 136, 216–241 (2013). - PubMed
    1. Stieger B. Role of the bile salt export pump, BSEP, in acquired forms of cholestasis. Drug Metab Rev 42, 437–445 (2010). - PubMed
    1. Welch M. A., Kock K., Urban T. J., Brouwer K. L. & Swaan P. W. Toward predicting drug-induced liver injury: parallel computational approaches to identify multidrug resistance protein 4 and bile salt export pump inhibitors. Drug Metab Dispos 43, 725–734 (2015). - PMC - PubMed

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