Human organic anion transporter-1 (hOAT1) regulates the absorption, distribution, and excretion of a wide range of clinically important drugs. Our previous work demonstrated that hOAT1 is a dynamic membrane transporter, constitutively internalizing from and recycling back to the cell plasma membrane. Short-term activation (<30 minutes) of protein kinase C (PKC) promotes the attachment of a lysine 48-linked polyubiquitin chain to hOAT1, a process catalyzed by ubiquitin ligase neural precursor cell expressed developmentally down-regulated 4-2 (Nedd4-2). The ubiquitination of hOAT1 then triggers an accelerated endocytosis of the transporter from plasma membrane, which results in reduced hOAT1 expression at the cell surface and decreased hOAT1 transport activity. In the present study, we investigated the long-term effect of PKC on hOAT1. We showed that long-term activation (>2 hours) of PKC significantly enhanced hOAT1 degradation, and such action was partially blocked by ubiquitin mutant Ub-K48R, which has its lysine (K) 48 mutated to arginine (R) and is incapable of forming a K48-linked polyubiquitin chain. The ubiquitin ligase Nedd4-2 was also found to augment hOAT1 degradation. These results suggest that PKC-regulated and Nedd4-2-catalyzed attachment of a lysine 48-linked polyubiquitin chain to hOAT1 is important for hOAT1 stability. We further showed through coimmunoprecipitation experiments that there was a direct association between hOAT1 and Nedd4-2, and such interaction was weakened when the WW3 and WW4 domains of the ligase were mutated. Mutating WW3 and WW4 domains of the ligase also impaired its ability to ubiquitinate hOAT1. Therefore, WW3 and WW4 domains of Nedd4-2 are critical for its association with and modulation of the transporter.

Human organic anion transporter-3 (hOAT3) is richly expressed in the kidney, where it plays critical roles in the secretion of clinically important drugs, including anti-viral therapeutics, anti-cancer drugs, antibiotics, antihypertensives, and anti-inflammatories. In the current study, we examined the role of AG490, a specific inhibitor of the Janus tyrosine kinase 2 (JAK2), in hOAT3 transport activity in the kidney COS-7 cells. AG490 induced a time- and concentration-dependent inhibition of hOAT3-mediated uptake of estrone sulfate, a prototypical substrate for the transporter. The inhibitory effect of AG490 correlated with a reduced expression of hOAT3 at the cell surface. Our lab previously demonstrated that Nedd4-2, a ubiquitin ligase, down regulates OAT expression and transport activity by enhancing OAT ubiquitination, which leads to an internalization of OAT from cell surface to intracellular compartments and subsequent degradation. In the current study, we showed that treatment of hOAT3-expressing cells with AG490 resulted in an enhanced hOAT3 ubiquitination and degradation, which was accompanied by a strengthened association of Nedd4-2 with hOAT3 and a reduction in Nedd4-2 phosphorylation. SiRNA knockdown of endogenous Nedd4-2 abrogated the effects of AG490 on hOAT3. In summary, our study demonstrated that AG490 regulates hOAT3 expression and transport activity through the modulation of Nedd4-2.

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Liver kinase B 1 (LKB1 or STK11) and PTEN (phosphatase and tensin homologue deleted on chromosome 10) are two tumor suppressors that regulate the mTOR signaling pathway. Deletion studies show that loss of either Lkb1 (Lkb+/−) or Pten (PtenloxP/loxP; Alb-Cre+) leads to liver injury and development of hepatocarcinoma. In this study, we investigated the crosstalk of LKB1 and PTEN loss during tumorigenesis and liver development. We show here that haplo-insufficiency of Lkb1 in the liver leads to advanced tumor development in the Pten null mice (PtenloxP/loxP; LkbloxP/+; Alb-Cre+). Our analysis shows that LKB1 and PTEN interacted with each other in their regulation of fatty acid synthase as well as p21 expression. The combined loss of LKB1 and PTEN (PtenloxP/loxP; LkbloxP/loxP; Alb-Cre+) also led to the inability to form zonal structures in the liver. The lack of metabolic zonal structures is consistent with the inability of the livers to store glycogen as well as elevated plasma bilirubin and alanine aminotransferase (ALT), indicative of liver dysfunction. These structural and functional defects are associated with cytoplasm distribution of a canalicular membrane protein MRP2 (multidrug resistant protein 2) which is responsible for clearing bilirubin. This observed regulation of MRP2 by LKB1 likely contributed to the lack of cellular polarity and the early lethality phenotype associated with homozygous loss of Lkb1 alone or in combination with Pten. Finally, Pten deletion does not rescue the precocious ductal plate formation reported for Lkb1 deleted livers. Conclusion: Our study dissected the functional and molecular crosstalk of PTEN and LKB1 and elucidate key molecular targets for such interaction.

Human organic anion transporter-3 (hOAT3) is richly expressed in the kidney, where it plays critical roles in the secretion, from the blood to urine, of clinically important drugs, such as anti-viral therapeutics, anti-cancer drugs, antibiotics, antihypertensives, and anti-inflammatories. In the current study, we examined the role of dexamethasone in hOAT3 transport activity in the kidney HEK293 cells. Cis-inhibition study showed that dexamethasone exhibited a concentration-dependent inhibition of hOAT3-mediated uptake of estrone sulfate, a prototypical substrate for the transporter, with IC value of 49.91 μM. Dixon plot analysis revealed that inhibition by dexamethasone was competitive with a Ki = 47.08 μM. In contrast to the cis-inhibition effect of dexamethasone, prolonged incubation (6 h) of hOAT3-expressing cells with dexamethasone resulted in an upregulation of hOAT3 expression and transport activity, kinetically revealed as an increase in the maximum transport velocity V without meaningful alteration in substrate-binding affinity K. Such upregulation was abrogated by GSK650394, a specific inhibitor for serum- and glucocorticoid-inducible kinases (sgk). Dexamethasone also enhanced sgk1 phosphorylation. Our study demonstrated that dexamethasone exhibits dual effects on hOAT3: it is a competitive inhibitor for hOAT3-mediated transport, and interestingly, when entering the cells, it stimulates hOAT3 expression and transport activity through sgk1.