Protein kinase C affects the internalization and recycling of organic anion transporting polypeptide 1B1

Abstract

Organic anion-transporting polypeptides are members of the solute carrier (SLC) family and key determinants for the transmembrane transport of a wide variety of compounds. OATP1B1 is predominantly expressed at the basolateral membrane of human hepatocytes and play an important role in drug clearance from the body. It has been demonstrated to be responsible for the hepatic uptake of various drugs. Computer-based hydropathy analysis predicted several putative phosphorylation sites at the amino and carboxyl termini and at intracellular loop 3 of OATP family members. Therefore, their transport functions may be regulated by phosphorylation. Previous studies have demonstrated that uptake function of OATP2B1 and OATP1A2 is regulated by protein kinase C (PKC). In the present study, we treated HEK293 cells stably expressing OATP1B1 with different PKC modulators and measured their transport activity for prototypic substrate estrone-3-sulfate. It was found that OATP1B1 uptake function was reduced upon PKC activation. Further studies indicated that PKC may affect OATP1B1 activity through regulation of the cell surface protein level. Moreover, we found out that PKC activator phorbol 12-myristate 13-acetate (PMA) not only affects the internalization of OATP1B1 but its recycling as well. Immunocytochemistry analysis revealed that internalized OATP1B1 co-localized with early and recycling endosomal markers and the co-localization of OATP1B1 with recycling endosome is dependent on PKC activation. Taken together, our present study demonstrated that PKC regulates the function of OATP1B1 by affecting internalization and recycling of the transporter protein.

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.…”

“…Taken together, these data fully establish PKCs as important regulators of Hepatic transporters whose expression is targeted by the PKC activator PMA include major and relevant SLC transporters, such as OATP1B1, OATP2B1 and OCT1, involved in sinusoidal uptake of various marketed drugs such as statins and metformin [2]. These SLC transporters were down-regulated at the mRNA level in both HepaRG cells and primary human hepatocytes exposed to PMA; some of them, i.e., OATP1B1 and OATP2B1, whose membrane location is already known to be rapidly modulated by PKCs [50,52], were concomitantly decreased at the protein level and also targeted by the PKC activator agent ingenol mebutate. Importantly, OATP and OCT1 activity were reduced in parallel, thus indicating that PMA-mediated repression of transporter expression results into functional consequences.…”

Protein Kinases C-Mediated Regulations of Drug Transporter Activity, Localization and Expression

“…The PKC activator agent PMA has thus been shown to stimulate internalization of OATP transporters such as OATP1A2, OATP1B1 and OATP2B1 in cultured cells [66,67,68]. As an example, cell surface expression of OATP2B1 at the sinusoidal pole of human highly-differentiated hepatoma HepaRG cells was markedly decreased in response to a short 1-h treatment with 100 nM of PMA (Figure 2a).…”

“…PKC-triggered OATP1A2 internalization in COS-7 cells transfected with OATP1A2 was blocked by the cPKC inhibitor Gö6976 and was dependent on clathrin-dependent endocytosis, but not on the caveolin-dependent pathway [66]. Similarly, OATP2B1 internalization caused by PMA was related to clathrin-mediated endocytosis, followed by lysosomal degradation in OATP2B1-transfected MDCKII cells [68], whereas internalized OATP1B1 co-localized with early and recycling endosomal markers in OATP1B1-transfected HEK293 cells [67]. Activation of PKCs by PMA also results in altered trafficking of the ENT1 nucleoside transporter, with significant increase in the plasma membrane localization of ENT1 [73].…”

More Power to OATP1B1: An Evaluation of Sample Size in Pharmacogenetic Studies Using a Rosuvastatin PBPK Model for Intestinal, Hepatic, and Renal Transporter‐Mediated Clearances

“…Moreover, recent genetic studies have detected a coordinated expression (colinearity) of OATP1B1 and OATP1B3 in human liver, with respect to mRNA and, to a lesser extent, protein expression . These observations are believed to be linked to posttranscriptional and posttranslational regulation of OATP expression by hepatic nuclear factor 1α (HNF1α) and protein kinase C (PKC), respectively …”