Novel mechanism of impaired function of organic anion-transporting polypeptide 1B3 in human hepatocytes: post-translational regulation of OATP1B3 by protein kinase C activation

Abstract

The organic anion-transporting polypeptide (OATP) 1B3 is a membrane transport protein that mediates hepatic uptake of many drugs and endogenous compounds. Currently, determination of OATP-mediated drug-drug interactions in vitro is focused primarily on direct substrate inhibition. Indirect inhibition of OATP1B3 activity is under-appreciated. OATP1B3 has putative protein kinase C (PKC) phosphorylation sites. Studies were designed to determine the effect of PKC activation on OATP1B3-mediated transport in human hepatocytes using cholecystokinin-8 (CCK-8), a specific OATP1B3 substrate, as the probe. A PKC activator, phorbol-12-myristate-13-acetate (PMA), did not directly inhibit [(3)H]CCK-8 accumulation in human sandwich-cultured hepatocytes (SCH). However, pretreatment with PMA for as little as 10 minutes rapidly decreased [(3)H]CCK-8 accumulation. Treatment with a PKC inhibitor bisindolylmaleimide (BIM) I prior to PMA treatment blocked the inhibitory effect of PMA, indicating PKC activation is essential for downregulating OATP1B3 activity. PMA pretreatment did not affect OATP1B3 mRNA or total protein levels. To determine the mechanism(s) underlying the indirect inhibition of OATP1B3 activity upon PKC activation, adenoviral vectors expressing FLAG-Myc-tagged OATP1B3 (Ad-OATP1B3) were transduced into human hepatocytes; surface expression and phosphorylation of OATP1B3 were determined by biotinylation and by an anti-phosphor-Ser/Thr/Tyr antibody, respectively. PMA pretreatment markedly increased OATP1B3 phosphorylation without affecting surface or total OATP1B3 protein levels. In conclusion, PKC activation rapidly decreases OATP1B3 transport activity by post-translational regulation of OATP1B3. These studies elucidate a novel indirect inhibitory mechanism affecting hepatic uptake mediated by OATP1B3, and provide new insights into predicting OATP-mediated drug interactions between OATP substrates and kinase modulator drugs/endogenous compounds.

Fig. 1.

PKC activation by PMA downregulates [³H]CCK-8 accumulation in human SCH. (A) Time-dependent accumulation of [³H]CCK-8 (1 μM) in human SCH. Data represent the mean ± range of duplicate measurements from a single donor. (B) Effect of PKC activator PMA on [³H]CCK-8 accumulation. [³H]CCK-8 accumulation (1 μM for 3 minutes) was determined in human SCH pretreated with PMA (black bars) or a PMA inactive analog, 4αPDD (gray bars), for 30 minutes at 0.1 or 1 μM. (C) Involvement of PKC activation in PMA-induced downregulation of [³H]CCK-8 accumulation. Human SCH were incubated in medium containing 0.1 or 1 μM PMA for 30 minutes in the presence (white bars) or absence (black bars) of pretreatment (20 minutes) and subsequent coincubation (30 minutes) with the PKC inhibitor BIM I (1 μM). [³H]CCK-8 accumulation (1 μM, 3 minutes) in human SCH was expressed as a percentage of the accumulation measured in vehicle-treated cells. Data represent mean ± S.E.M. from n = 3 donors in triplicate. *P < 0.05 versus control by one-way analysis of variance, followed by Dunnett’s t test. Transport experiments were performed on day 5 or 6 of culture.

Fig. 2.

Indirect inhibition of [³H]CCK-8 accumulation by PMA in human SCH. (A) Time-dependent effect of PMA pretreatment on [³H]CCK-8 accumulation in human SCH. Human SCH were pretreated with PMA (0.1 and 1 μM) for various times (10–60 minutes). [³H]CCK-8 accumulation (1 μM, 3 minutes) was expressed as a percentage of the accumulation measured in vehicle control–treated cells. (B) [³H]CCK-8 accumulation in the presence of vehicle control, PMA, or BIM I at indicated concentrations. [³H]CCK-8 accumulation (1 μM, 3 minutes) in human SCH was expressed as a percentage of the accumulation measured in the absence of PMA or BIM I. Data represent mean ± S.E.M. from n = 3 donors in triplicate. *P < 0.05 versus vehicle control treatment by one-way analysis of variance, followed by Dunnett’s t test. Transport experiments were performed on day 5 or 6 of culture.

Fig. 3.

PMA treatment does not affect OATP1B3 mRNA and total protein levels in human SCH. Human SCH were treated with vehicle control DMSO (CTL) or 0.1 μM PMA for 30 minutes. (A) OATP1B3 mRNA levels were determined by TaqMan real-time RT-PCR with GAPDH as an internal standard; values were expressed as a percentage of CTL. (B) Representative immunoblot (IB) image of OATP1B3 in PMA- and vehicle control–treated SCH (CTL) (n = 3 donors). β-actin was used as a loading control. Densitometry of OATP1B3 was normalized to β-actin expression; results were expressed as fold change versus CTL. Data represent mean ± S.E.M. from n = 3 donors. Statistical analysis was performed versus CTL by Student’s t test. Experiments were performed on day 5 or 6 of culture.

Fig. 4.

PKC activation potently inhibits [³H]CCK-8 accumulation in nontransduced and Ad-OATP1B3-transduced human SCH. Human hepatocytes were transduced with Ad-LacZ or Ad-OATP1B3 at a MOI of 5 and overlaid with Matrigel. Experiments were conducted 48 hours post-transduction on day 2 of culture. (A) Immunoblot (IB) of FLAG in nontransduced (None), Ad-LacZ–, and Ad-OATP1B3–transduced human SCH. β-Actin was used as a loading control. Representative images from n = 3 donors are shown. (B) Potent inhibition of [³H]CCK-8 accumulation in human SCH upon PKC activation. [³H]CCK-8 accumulation (1 μM, 1.5 minutes) in nontransduced (None) and Ad-OATP1B3-transduced (Ad-OATP1B3) human SCH pretreated with vehicle control (CTL) or PMA (0.1 μM) for 30 minutes), or coincubated with BSP (100 μM) without PMA pretreatment. Data represent mean ± S.D. in triplicate from a representative data set from n = 3 and n = 2 donors for PMA and BSP treatment, respectively. *P < 0.05 versus CTL by one-way analysis of variance in each group of nontransduced and Ad-OATP1B3–transduced cells, followed by Dunnett’s t test.

Fig. 5.

PKC activation does not affect total or surface levels of FLAG-Myc-OATP1B3. Human hepatocytes were transduced with Ad-OATP1B3 at a MOI of 5 and cultured without Matrigel overlay. At 48 hours post-transduction on day 2 of culture, cells were treated with PMA (0.1 μM, 30 minutes) or vehicle control (CTL). Surface levels of FLAG-Myc-OATP1B3 were determined by biotinylation followed by immunoblot (IB) with FLAG antibody. GAPDH was used as a cytoplasmic protein marker. Protein levels were determined by densitometry. Fold change of total and surface levels of FLAG-OATP1B3 (PMA versus CTL) were expressed as mean ± S.E.M (n = 3 donors). Representative images from n = 3 donors are shown.

Fig. 6.

PKC activation increases phosphorylation of FLAG-Myc-OATP1B3 in human hepatocytes. Human hepatocytes were transduced with Ad-OATP1B3 at a MOI of 5 and cultured without Matrigel overlay. At 48 hours post-transduction on day 2 of culture, cells were treated with PMA (0.1 μM, 30 minutes) or vehicle control (CTL). (A) FLAG-Myc-OATP1B3 was immunoprecipitated in hepatocytes treated with PMA or CTL. Immunocomplexes were first probed with anti–phospho-Ser/Thr/Tyr antibody (top panel) and then reprobed with OATP1B3 antibody after stripping (middle panel). Immunoprecipitation (IP) with normal mouse IgG served as negative control. (B) Densitometry of the phosphorylation signal was normalized to OATP1B3 levels in IP and was expressed as fold change (PMA versus CTL). Data represent mean ± S.E.M (n = 3 donors). Representative images from n = 3 donors are shown.