Protein kinase Cδ differentially regulates cAMP-dependent translocation of NTCP and MRP2 to the plasma membrane

Abstract

Cyclic AMP stimulates translocation of Na(+)/taurocholate cotransporting polypeptide (NTCP) from the cytosol to the sinusoidal membrane and multidrug resistance-associated protein 2 (MRP2) to the canalicular membrane. A recent study suggested that protein kinase Cδ (PKCδ) may mediate cAMP-induced translocation of Ntcp and Mrp2. In addition, cAMP has been shown to stimulate NTCP translocation in part via Rab4. The aim of this study was to determine whether cAMP-induced translocation of NTCP and MRP2 require kinase activity of PKCδ and to test the hypothesis that cAMP-induced activation of Rab4 is mediated via PKCδ. Studies were conducted in HuH-NTCP cells (HuH-7 cells stably transfected with NTCP). Transfection of cells with wild-type PKCδ increased plasma membrane PKCδ and NTCP and increased Rab4 activity. Paradoxically, overexpression of kinase-dead dominant-negative PKCδ also increased plasma membrane PKCδ and NTCP as well as Rab4 activity. Similar results were obtained in PKCδ knockdown experiments, despite a decrease in total PKCδ. These results raised the possibility that plasma membrane localization rather than kinase activity of PKCδ is necessary for NTCP translocation and Rab4 activity. This hypothesis was supported by results showing that rottlerin, which has previously been shown to inhibit cAMP-induced membrane translocation of PKCδ and NTCP, inhibited cAMP-induced Rab4 activity. In addition, LY294002 (a phosphoinositide-3-kinase inhibitor), which has been shown to inhibit cAMP-induced NTCP translocation, also inhibited cAMP-induced PKCδ translocation. In contrast to the results with NTCP, cAMP-induced MRP2 translocation was inhibited in cells transfected with DN-PKCδ and small interfering RNA PKCδ. Taken together, these results suggest that the plasma membrane localization rather than kinase activity of PKCδ plays an important role in cAMP-induced NTCP translocation and Rab4 activity, whereas the kinase activity of PKCδ is necessary for cAMP-induced MRP2 translocation.

Fig. 1.

Rottlerin inhibits cAMP-induced PKCδ translocation to the plasma membrane. HuH-NTCP cells were treated with or without 5 μM rottlerin for 30 min, followed by treatment with or without 100 μM 8-chlorophenylthio cAMP (CPT-cAMP) for 15 min. PKCδ translocation was measured by a biotinylation method. Top: representative immunoblot of the amount of PKCδ in the plasma membrane (PM). Bottom: densitometric analysis is shown. E-cadherin (E-cad) was used as a loading control. The relative values of PKCδ in the PM are expressed as means ± SE (n = 4). Data were analyzed by paired t-test. *Significantly different (P < 0.05) from control values in the absence of cAMP and rottlerin; #significantly different (P < 0.05) from cAMP treatment.

Fig. 2.

Rottlerin inhibits cAMP-induced activation of Rab4. HuH-NTCP cells were treated with or without 5 μM rottlerin for 30 min, followed by treatment with or without 100 μM CPT-cAMP for 10 min. Rab4 activation was determined by GTP overlay assay as described in materials and methods. A representative immunoblot of Rab4-GTP and total Rab4 is shown at top with the densitometric analysis shown at bottom. Rab4 activity (means ± SE, n = 3) was expressed as a ratio of Rab4-GTP to total Rab4 to correct for variations due to loading and immunoprecipitation. Data were analyzed by 1-way ANOVA. *Significantly different (P < 0.05) from control values in the absence of cAMP and rottlerin; #significantly different (P < 0.05) from cAMP treatment.

Fig. 3.

Rottlerin does not inhibit cAMP-induced Akt phosphorylation. HuH-NTCP cells were treated with or without 5 μM rottlerin for 30 min, followed by treatment with or without 100 μM CPT-cAMP for 10 min. Cell lysates from treated cells were subjected to immunoblot analysis. A representative immunoblot is shown at top with the densitometric analysis shown at bottom. Akt activity was determined from the ratio of phosphorylated Akt to total Akt and is expressed as means ± SE (n = 3). Data were analyzed by 1-way ANOVA. *Significantly different (P < 0.05) from control values in the absence of cAMP and rottlerin.

Fig. 4.

Dominant-negative (DN)- and wild-type (WT)-PKCδ increased Rab4 activity and PM NTCP. HuH-NTCP cells were transfected with empty vector (EV), DN-PKCδ, and WT-PKCδ, followed by treatment with or without 100 μM CPT-cAMP for 15 min. Rab4 activation was determined using a GTP overlay assay (B). NTCP translocation was measured by a biotinylation method (C). A: transfection was confirmed with PKCδ and HA antibodies. B: representative blot of Rab4-GTP and total Rab4 (top) and densitometric analysis (bottom). Rab4 activity (means ± SE, n = 3) was expressed as a ratio of Rab4-GTP to total Rab4. Data were analyzed by paired t-test. C: representative immunoblot of PM NTCP and E-cadherin (top) and densitometric analysis (bottom). Relative values of NTCP in the PM are expressed as means ± SE (n = 3). Data were analyzed by 1-way ANOVA. *Significantly different (P < 0.05) from respective control values in the absence of cAMP in cells transfected with EV.

Fig. 5.

DN- as well as WT-PKCδ increased PM PKCδ. HuH-NTCP cells were transfected with EV, DN-PKCδ, or WT-PKCδ, followed by treatment with or without 100 μM CPT-cAMP for 15 min. A biotinylation method was used to determine PM PKCδ. Representative immunoblots of PM PKCδ (top) and total PKCδ (middle) and the densitometric analysis (bottom). Amount of PKCδ localization in the PM was expressed as a ratio of PM PKCδ to total PKCδ. Relative values of PKCδ in the PM are expressed as means ± SE (n = 3). Data were analyzed by 1-way ANOVA. *Significantly different (P < 0.05) from control values in the absence of cAMP; **significantly different (P < 0.05) from values in the absence of cAMP in cells transfected with WT-PKCδ. Control values in the absence and presence of cAMP are #significantly different (P < 0.05) by paired t-test.

Fig. 6.

Small interfering RNA (siRNA) PKCδ increased PM PKCδ. A: level of PKCδ protein was downregulated up to 80% in cells transfected with siRNA PKCδ for 48 h compared with cells transfected with scrambled siRNA (Con) or nontransfected cells (Non-trans) B: HuH-NTCP cells were transfected with scrambled siRNA and siRNA PKCδ, followed by treatment with or without 100 μM CPT-cAMP for 15 min. A biotinylation method was used to determine PM PKCδ. Representative immunoblots of PM PKCδ (top), total PKCδ (middle), and the densitometric analysis (bottom) are shown. The relative values of PKCδ in the PM are expressed as means ± SE (n = 4). Data were analyzed by paired t-test. *Significantly different (P < 0.05) from control values in the absence of cAMP.

Fig. 7.

siRNA PKCδ increased Rab4 activity and the amount of PM NTCP. HuH-NTCP cells were transfected with scrambled siRNA (control) or siRNA PKCδ, followed by treatment with or without 100 μM CPT-cAMP for 15 min and then determination of Rab4 activity (A). A biotinylation method was used to determine PM NTCP (B). A: representative blot of Rab4-GTP and total Rab4 (top); densitometric analysis (bottom). Rab4 activity (means ± SE, n = 3) was expressed as a ratio of Rab4-GTP to total Rab4. Data were analyzed by 1-way ANOVA. *Significantly (P < 0.05) different from control values in the absence of cAMP in cells transfected with scrambled siRNA. Control values in the absence and presence of cAMP are #significantly different (P < 0.05) by paired t-test. B: representative immunoblot of PM NTCP and E-cadherin (top); densitometric analysis (bottom). Relative values of NTCP in the PM are expressed as means ± SE (n = 3). Data were analyzed by 1-way ANOVA. *Significantly (P < 0.01) different from control values in the absence of cAMP. Control values in the absence and presence of cAMP are #significantly different (P < 0.05) by paired t-test.

Fig. 8.

LY294002 inhibited cAMP-induced PKCδ translocation to the PM. HuH-NTCP cells were treated with or without 20 μM LY294002 for 30 min, followed by treatment with or without 100 μM CPT-cAMP for 15 min. PKCδ translocation was measured by a biotinylation method. Top: representative immunoblot of the amount of PKCδ in the PM. Bottom: densitometric analysis is shown. E-cadherin was used as a loading control. The relative values of PKCδ in the PM are expressed as means ± SE (n = 3). Data were analyzed by 1-way ANOVA. *Significantly (P < 0.05) different from control values in the absence of cAMP and LY294002; #significantly (P < 0.05) different from cAMP treatment.

Fig. 9.

Significant correlation between PM PKCδ and NTCP. Values for PM PKCδ and PM NTCP were from cells transfected with DN-PKCδ or siRNA against PKCδ. Solid line represents best-fit line for PM NTCP = 4.12*PM PKCδ/(2.10+PM PKCδ); R = 0.833, P < 0.0001, n = 18.

Fig. 10.

DN-PKCδ and siRNA PKCδ inhibited cAMP-induced MRP2 translocation. HuH-NTCP cells were transfected with EV and DN-PKCδ (A), or transfected with scrambled siRNA (control) and siRNA PKCδ (B) followed by treatment with or without 100 μM CPT-cAMP for 15 min. A biotinylation method was used to determine PM MRP2. A: representative immunoblot of MRP2 and E-cadherin (top); densitometric analysis (bottom). Relative values of MRP2 in the PM are expressed as means ± SE (n = 3). Data were analyzed by 1-way ANOVA. B: representative blot of PM MRP2 and E-cadherin (top); densitometric analysis (bottom). Relative values of MRP2 in the PM are expressed as means ± SE (n = 3). Data were analyzed by paired t-test. *Significantly different (P < 0.05) from control values in the absence of cAMP; #significantly different (P < 0.05) from control values in the presence of cAMP.