Original ContributionOxidative stress and Mrp2 internalization
Introduction
The multidrug resistance-associated protein 2 (Mrp2) is localized on the canalicular membrane of hepatocytes and contributes to bile flow by extensively excreting glutathione (GSH) and a variety of organic anions into bile [1]. Defective expression of MRP2 underlies the jaundice in the human Dubin-Johnson syndrome [1]. In addition to the inherited deficiency of MRP2 expression in these patients, the transporter expression is regulated on a short- and long-term basis in normal subjects, with important implications for the pathogenesis of cholestatic syndromes [1], [2]. Short-term regulation includes rapid retrieval of the transporter from the canalicular membrane of hepatocytes and its translocation into the cytosol in putative vesicles under the influence of hyperosmolarity [3], endotoxin [3], phalloidin [4], bile acids [5], [6], and oxidative stress [7], [8], [9]. On the other hand, long-term regulation includes mrp2 gene expression, which is affected by lipopolysaccharide [3], bile duct ligation [10], aniso-osmolarity [11], dexamethasone [11], or a variety of drugs [12]. As far as the putative regulatory molecules involved in the membrane surface expression of Mrp2 are concerned, several cytosolic proteins including Radixin and PDZ-K1 have been reported [13], [14], [15], [16]. Importantly, impaired steady-state canalicular surface expression of Mrp2 and jaundice have been observed in Radixin knockout mice [14], and so Radixin is now considered as the primary molecule anchoring Mrp2 to the filamentous actin (F-actin). In addition to the static expression of Mrp2 on the canalicular surface, its dynamic insertion and internalization processes are of great importance because the steady-state expression level is directly dependent on these turnover rates.
We have recently reported that Mrp2 is rapidly internalized in rat liver perfused with ethacrynic acid (EA), a highly electrophilic loop diuretic [8]. EA is conjugated with GSH and excreted into bile via Mrp2 and so an excess of EA induces acute oxidative stress by such a futile cycle [8]. As both the GSH itself and the resulting glutathione conjugates, EA-SG, are substrates of Mrp2 [8], [17], rapid down regulation of Mrp2 under GSH-depleted conditions seems a favorable feedback mechanism for hepatocytes to retain their intracellular GSH. Although it is not known whether the down regulation of Mrp2 actually contributes to cyto-protection, constitutive overexpression of human MRP2 in MDCKII cells accelerates 4-hydroxynonenal-induced GSH depletion and necrosis compared with control MDCKII cells [7]. Moreover, a well-regulated molecular mechanism rather than nonspecific cellular damage might be involved in the internalization pathway [8], because the internalization of Mrp2 induced by EA preceded the nonspecific breakdown of tight junction formation and canalicular structure.
Here, we propose the hypothesis that the presence of a specific intracellular signaling pathway produced by EA-induced oxidative stress finally leads to Mrp2 internalization. We used isolated rat hepatocyte couplets (IRHCs) and perfused rat livers to examine this hypothesis in detail. Our results showed that depletion of intracellular GSH is the trigger of this signaling pathway and novel protein kinase C (nPKC) isoforms are translocated to the membranous fraction. Elevation of intracellular Ca2+ and production of nitric oxide (NO) also play important roles in the signaling pathway of nPKC activation. Sequential propagation of these steps finally leads to specific internalization of Mrp2 from the canalicular surface.
Section snippets
Chemicals
EA, Griess reagent, nitric reductase, and rabbit anti-protein kinase Cα, δ, and ε antibodies were obtained from Sigma-Aldrich Chemical (St. Louis, MO). Collagenase S-1, 2-mercaptoethanol, and o-phthalaldehyde were from Wako Pure Chemical Industries (Osaka, Japan). 1-(5-Isoquinolinesulfonyl)-2-methylpiperazine/2HCl (H7) was obtained from Biomol Research Laboratories, Inc. (Plymouth Meeting, PA). PKG 1a inhibitor and PKA inhibitor were from Merck (Darmstadt, Germany). Rabbit anti-Mrp2 antiserum
Effect of EA on the localization of Mrp2 in IRHCs
We have previously demonstrated that Mrp2 is internalized in the liver perfused with EA and that this is possibly related to the intracellular GSH content [8]. To investigate the molecular mechanism of the internalization, an in vitro assay system with qualitative and quantitative characteristics similar to in situ liver conditions is required. IRHCs is an established experimental system especially useful for the study of canalicular ATP-binding cassette (ABC) transporters including Mrp2 and
Discussion
The importance of the intracellular GSH content in regulating Mrp2 internalization was suggested from in situ rat liver perfusion studies using EA [8], tertiary-butyl hydroperoxide (t-BHP) and chlorodinitrobenzene (CDNB) [9]. The signaling process has now been investigated in detail using IRHCs. Severe GSH depletion induced by t-BHP and CDNB (lower than 10% of control) caused Mrp2 internalization in rat liver perfusion experiments as reported previously [9]. In our IRHCs system using EA as a
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2018, Biochimica et Biophysica Acta - Molecular Basis of DiseaseCitation Excerpt :In line with this, a phosphorylation site for PKC, at the C-terminal of MRP2 (Ser1542), has been reported [37]. Interestingly, different PKC isoforms have been shown by our group and others to be involved in the endocytic internalization of MRP2 in different models of cholestasis, including those induced by E17G [7], oxidative stress [38], and taurolithocholate [39]. In addition, our group has also provided evidence for the participation of p38MAPK in the endocytosis of canalicular transporters in E17G-induced cholestasis [9].
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