Ca2+-dependent protein kinase C isoforms are critical to estradiol 17β-D-glucuronide-induced cholestasis in the rat

Abstract

The endogenous estradiol metabolite estradiol 17␤-D-glucuronide (E 2 17G) induces an acute cholestasis in rat liver coincident with retrieval of the canalicular transporters bile salt export pump (Bsep, Abcc11) and multidrug resistance-associated protein 2 (Mrp2, Abcc2) and their associated loss of function. We assessed the participation of Ca 2؉ -dependent protein kinase C isoforms (cPKC) in the cholestatic manifestations of E 2 17G in perfused rat liver (PRL) and in isolated rat hepatocyte couplets (IRHCs). In PRL, E 2 17G (2 mol/liver; intraportal, single injection) maximally decreased bile flow, total glutathione, and [ 3 H] taurocholate excretion by 61%, 62%, and 79%, respectively; incorporation of the specific cPKC inhibitor Gö6976 (500 nM) in the perfusate almost totally prevented these decreases. In dose-response studies using IRHC, E 2 17G (3.75-800 M) decreased the canalicular vacuolar accumulation of the Bsep substrate cholyl-lysylfluorescein with an IC50 of 54.9 ؎ 7.9 M. Gö6976 (1 M) increased the IC50 to 178.4 ؎ 23.1 M, and similarly prevented the decrease in the canalicular vacuolar accumulation of the Mrp2 substrate, glutathione methylfluorescein. Prevention of these changes by Gö6976 coincided with complete protection against E 2 17G-induced retrieval of Bsep and Mrp2 from the canalicular membrane, as detected both in the PRL and IRHC. E 2 17G also increased paracellular permeability in IRHC, which was only partially prevented by Gö6976. The cPKC isoform PKC␣, but not the Ca 2؉ -independent PKC isoform, PKC⑀, translocated to the plasma membrane after E 2 17G administration in primary cultured rat hepatocytes; Gö6976 completely prevented this translocation, thus indicating specific activation of cPKC. This is consistent with increased autophosphorylation of cPKC by E 2 17G, as detected via western blotting. Conclusion: Our findings support a central role for cPKC isoforms in E 2 17G-induced cholestasis, by inducing both transporter retrieval from the canalicular membrane and opening of the paracellular route. (HEPATOLOGY 2008;48:1885-1895 B ile formation represents a key liver function by which xenobiotics and endogenous metabolites such as cholesterol, bilirubin, and hormones are eliminated from the body. 1,2 Efflux of solutes by adenosine triphosphate-dependent transporters at the canalicular membrane of hepatocytes provide the driving force for osmotic bile formation; among these transporters, the bile salt export pump (Bsep, Abcc11) and multidrug re-

Nonalcoholic Steatohepatitis Modulates Membrane Protein Retrieval and Insertion Processes

“…Furthermore, an increase in membrane translocation of PKC« was observed, which suggests activation, although there was no change in phosphorylation. Although these data suggest that MARCKS may not be involved in NASH-related Mrp2 mislocalization, PKC« phosphorylates Mrp2 directly to alter the transport function, and the translocation of PKC« to the membrane may be related to this alternative function (Crocenzi et al, 2008). Furthermore, there may be other substrates for PKC« at the canalicular membrane that mediates Mrp2 retrieval in response to other stimuli.…”

“…Many of these pathways overlap in the use of these mediators, creating a complex regulation of localization, even when looking at just one transporter. For example, activation of PKCa, a calciumdependent protein kinase, has been shown to be involved in both TUDCAstimulated Mrp2 insertion and estradiol-17b-D-glucuronide-induced Mrp2 Altered Membrane Localization in NASH retrieval (Crocenzi et al, 2008;Wimmer et al, 2008). Mrp2 membrane insertion by PKCa is dependent upon the cooperation of PKA; moreover, Mrp2 insertion is also mediated, in a cAMP-dependent manner, by PKCd (Crocenzi et al, 2008;Park et al, 2012).…”

“…For example, activation of PKCa, a calciumdependent protein kinase, has been shown to be involved in both TUDCAstimulated Mrp2 insertion and estradiol-17b-D-glucuronide-induced Mrp2 Altered Membrane Localization in NASH retrieval (Crocenzi et al, 2008;Wimmer et al, 2008). Mrp2 membrane insertion by PKCa is dependent upon the cooperation of PKA; moreover, Mrp2 insertion is also mediated, in a cAMP-dependent manner, by PKCd (Crocenzi et al, 2008;Park et al, 2012). Membrane retrieval of Mrp2 may also be regulated by novel protein kinases, specifically PKC« or PKCd, when stimulated by cholestatic mediators such as TLC to phosphorylate its substrate, MARCKS (Schonhoff et al, 2013).…”

Rapid Modulation of the Organic Anion Transporting Polypeptide 2B1 (OATP2B1, SLCO2B1) Function by Protein Kinase C-mediated Internalization

“…The idea that PKC activation influences transport processes is highlighted by a recent publication of Crocenzi et al (60), which investigated the influence of protein kinase C isoforms on estradiol 17␤-D-glucuronide (E 2 G)-induced acute cholestasis. They demonstrated that E 2 G activates PKC␣ in primary cultured rat hepatocytes and induces retrieval of Mrp2 and Bsep from the apical membrane, which normally provide the driving force for osmotic bile formation.…”

The Role of Canalicular ABC Transporters in Cholestasis

“…Tauroursodeoxycholic acid acts via the same pathway but also via the activation of various protein kinase C isoforms. Protein kinase Ca recruitment by estradiol-17b-D-glucuronoside decreases canalicular ABCB11 expression in rodents, which could partly be responsible for its cholestatic properties (Crocenzi et al, 2008). Inflammation-induced cholestasis finally can lead to a decreased ABCB11 insertion into the canalicular membrane in vitro and in rodents.…”