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 posttranslational 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.

Protein kinase C-dependent regulation of human hepatic drug transporter expression

“…Previous studies have demonstrated that PKC activation can rapidly modulate drug and bile acid transporter location and activity in hepatocytes, by notably promoting their retrieval from sinusoidal or canalicular membranes [49][50][51] or modulating their phosphorylation status [16]. The present study demonstrates that in vitro PKC activation caused by PMA can also result in marked changes in expression of main hepatic drug transporters.…”

“…PMA, used at a 100 nM concentration previously shown to activate PKCs in various studies [15,16,30], was first demonstrated to not alter cellular viability of HepaRG cells up to 48 h of exposure, as assessed by MTT viability assay (Fig. 1A).…”

“…Indeed, treatment by the PKC-activating phorbol ester 12-myristate 13-acetate (PMA) leads to retrieval of the sodium-taurocholate cotransporting polypeptide (NTCP/SLC10A1) and multidrug resistance-associated protein (MRP) 2 (ABCC2) from hepatocyte sinusoidal and canalicular membranes, respectively [14,15]. It also decreases activity of the sinusoidal organic anion transporting polypeptide (OATP) 1B3 (SLCO1B3) through post-translational regulation [16] and induces cholestasis in rat liver, through notably retrieval of the bile salt export pump (BSEP/ABCB11) from the canalicular membrane [17].…”

Characterization of Long-Lasting Oatp Inhibition by Typical Inhibitor Cyclosporine A and In Vitro–In Vivo Discrepancy in Its Drug Interaction Potential in Rats

“…Activation of protein kinase C inhibited OATP1B3 function without affecting its total and plasma membrane expression levels. 33 In contrast, CsA-activated signaling cascade is induced by protein kinase C in the rat hippocampus. 34 Further investigation is required to clarify the exact mechanism responsible for long-lasting inhibition of Oatps by CsA.…”

“…2), which simply assumes competitive or noncompetitive inhibition by unbound CsA. Powell et al 33 reported posttranslational regulation of OATP1B3 by protein kinase C in human hepatocytes. Activation of protein kinase C inhibited OATP1B3 function without affecting its total and plasma membrane expression levels.…”