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

Drug Metab Dispos. 2014 Nov;42(11):1964-70. doi: 10.1124/dmd.114.056945. Epub 2014 Sep 8.


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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adolescent
  • Adult
  • Aged
  • Base Sequence
  • Cells, Cultured
  • DNA Primers
  • Down-Regulation
  • Enzyme Activation
  • Female
  • Hepatocytes / enzymology
  • Hepatocytes / metabolism*
  • Humans
  • Male
  • Middle Aged
  • Organic Anion Transporters, Sodium-Independent / physiology*
  • Phosphorylation
  • Protein Kinase C / metabolism*
  • Protein Processing, Post-Translational*
  • Real-Time Polymerase Chain Reaction
  • Solute Carrier Organic Anion Transporter Family Member 1B3
  • Tetradecanoylphorbol Acetate / pharmacology
  • Young Adult


  • DNA Primers
  • Organic Anion Transporters, Sodium-Independent
  • SLCO1B3 protein, human
  • Solute Carrier Organic Anion Transporter Family Member 1B3
  • Protein Kinase C
  • Tetradecanoylphorbol Acetate