32 To ascertain that HuH-NTCP cells constitute a valid model, we first determined whether TLC activates PKCϵ and internalizes MRP2 in this cell https://www.selleckchem.com/products/BIBW2992.html line. To determine the effects of PKCϵ and MRP2, cells were treated with TLC for 15 or 25 minutes, respectively. These time points are based on previous studies reporting the effects

of TLC on PKCϵ activation in HuH-NTCP cells34 and biliary excretion of the Mrp2 substrate in perfused rat livers.5 TLC increased PM translocation of PKCϵ and decreased PM-MRP2 in HuH-NTCP cells (Fig. 1). Phorbol myristate acetate (PMA), used as a positive control, also increased PM-PKCϵ. cAMP, used as a negative control, did not affect PM-PKCϵ; cAMP does not activate PKCϵ in rat hepatocytes.31

cAMP also increased PM-MRP2 in HuH-NTCP cells (Fig. 1). Thus, HuH-NTCP cells were considered a valid model for studying the role of PKCϵ in TLC-induced MRP2 internalization. The transfection of HuH-NTCP cells with HA-tagged DN-PKCϵ resulted in the overexpression of total PKCϵ by 2- to 3-fold (Fig. 2). DN-PKCϵ did not affect the basal expression of MRP2 in Ipilimumab clinical trial the PM versus an empty vector. TLC decreased PM expression of MRP2 in cells transfected with an empty vector. However, this effect was reversed in cells transfected with DN-PKCϵ. cAMP, which has been shown to increase PM expression of MRP2 by activating PKCδ,31, 32 was used as a negative control. The ability of cAMP to increase PM-MRP2 was not affected by DN-PKCϵ. These results support the hypothesis that TLC-induced internalization of MRP2 is mediated via PKCϵ and that cAMP-mediated translocation of MRP2 to PM does not involve PKCϵ. Because MARCKS is a substrate for PKC and has been implicated in endocytosis,19 it is possible that TLC-induced MRP2 internalization involves TLC/PKCϵ-mediated phosphorylation of MARCKS. To test this hypothesis, we first determined whether TLC can phosphorylate MARCKS. In these studies, actin instead of MARCKS was used as the loading control because the MARCKS antibody gave inconsistent results on stripped

blots. A time-dependent study showed that TLC increased MARCKS phosphorylation as early as 5 minutes, with significant phosphorylation FAD observed until 25 minutes (Fig. 3). On the other hand, cAMP, which stimulates MRP2 translocation to the PM, did not phosphorylate MARCKS during the same time period. Similar results were obtained in rat hepatocytes (Fig. 3B), and this indicates that this is not an effect specific to transformed cells. Thus, MARCKS phosphorylation may be involved in MRP2 retrieval and not MRP2 translocation to the membrane. One of the consequences of MARCKS phosphorylation is the retrieval of MARCKS from the PM to the cytosol, which results in F-actin disassembly.18 Thus, we determined whether TLC increases cytosolic pMARCKS. TLC increased cytosolic pMARCKS 2.5-fold in comparison with controls (Fig. 4).