Protein kinase C regulates organic anion transporter 1 through phosphorylating ubiquitin ligase Nedd4-2.

BACKGROUND: Organic anion transporter 1 (OAT1) is a drug transporter expressed on the basolateral membrane of the proximal tubule cells in kidneys. It plays an essential role in the disposition of numerous clinical therapeutics, impacting their pharmacological and toxicological properties. The activation of protein kinase C (PKC) is shown to facilitate OAT1 internalization from cell surface to intracellular compartments and thereby reducing cell surface expression and transport activity of the transporter. The PKC-regulated OAT1 internalization occurs through ubiquitination, a process catalyzed by a E3 ubiquitin ligase, neural precursor cell expressed developmentally down-regulated 4-2 (Nedd4-2). Nedd4-2 directly interacts with OAT1 and affects ubiquitination, expression and stability of the transporter. However, whether Nedd4-2 is a direct substrate for PKC-induced phosphorylation is unknown. RESULTS: In this study, we investigated the role of Nedd4-2 phosphorylation in the PKC regulation of OAT1. The results showed that PKC activation enhanced the phosphorylation of Nedd4-2 and increased the OAT1 ubiquitination, which was accompanied by a decreased OAT1 cell surface expression and transport function. And the effects of PKC could be reversed by PKC-specific inhibitor staurosporine. We further discovered that the quadruple mutant (T197A/S221A/S354A/S420A) of Nedd4-2 partially blocked the effects of PKC on Nedd4-2 phosphorylation and on OAT1 transport activity. CONCLUSIONS: Our investigation demonstrates that PKC regulates OAT1 likely through direct phosphorylation of Nedd4-2. And four phosphorylation sites (T197, S221, S354, and S420) of Nedd4-2 in combination play an important role in this regulatory process.

Ubiquitin-specific peptidase 8 regulates the trafficking and stability of the human organic anion transporter 1.

Background Organic anion transporter 1 (OAT1) plays a vital role in avoiding the potential toxicity of various anionic drugs through the involvement of kidney elimination. We previously demonstrated that ubiquitin conjugation to OAT1 led to OAT1 internalization from cell surface, followed by degradation. Ubiquitination is a dynamic process, where deubiquitination is catalyzed by a class of ubiquitin-specific peptidases. Methods The role of ubiquitin-specific peptidase 8 (USP8) in hOAT1 function, expression and ubiquitination was assessed by conducting transporter uptake assay, biotinylation assay and ubiquitination assay. Results We demonstrated that USP8 overexpression in hOAT1-expressing cells led to an increased hOAT1 transporter activity and expression, which correlated well with a reduced hOAT1 ubiquitination. Such phenomenon was not observed in inactive USP8 mutant-transfected cells. In addition, the knockdown of endogenous USP8 by USP8-specific siRNA resulted in an increased hOAT1 ubiquitination, which correlated well with a decrease in hOAT1 expression and transport activity. Biotinylation experiments demonstrated that USP8-induced increase in hOAT1 expression and transport activity occurred through a deceleration of the rates of hOAT1 internalization and degradation. Conclusions These results indicated the regulatory role of USP8 in OAT1 function, expression, trafficking, and stability. General significance USP8 could be a new target for modulating OAT1-mediated drug transport.

Interaction of Anticancer Drugs with Human Organic Anion Transporter hOAT4.

Human organic anion transporter 4 (hOAT4) belongs to a family of multispecific organic anion transporters that play critical roles in the disposition of numerous drugs and therefore are the major sites for drug-drug interaction. Drug-drug interactions contribute significantly to the individual variation in drug response. hOAT4 is expressed in the kidney and placenta. In the current study, we examined the interaction of 36 anticancer drugs with hOAT4 in kidney COS-7 cells and placenta BeWo cells. Among the drugs tested, only epirubicin hydrochloride and dabrafenib mesylate exhibited > 50% cis-inhibitory effect, in COS-7 cells, on hOAT4-mediated uptake of estrone sulfate, a prototypical substrate for the transporter. The IC50 values for epirubicin hydrochloride and dabrafenib mesylate were 5.24±0.95 µM and 8.30±3.30 µM, respectively. Dixon plot analysis revealed that inhibition by epirubicin hydrochloride was noncompetitive with a Ki = 3 µM whereas inhibition by dabrafenib mesylate was competitive with a Ki = 4.26 µM. Our results established that epirubicin hydrochloride and dabrafenib mesylate are inhibitors of hOAT4. Furthermore, by comparing our data with clinically relevant exposures of these drugs, we conclude that although the tendency for dabrafenib mesylate to cause drug-drug interaction through hOAT4 is insignificant in the kidney, the propensity for epirubicin hydrochloride to cause drug-drug interaction is high.

AG490, a JAK2-specific inhibitor, downregulates the expression and activity of organic anion transporter-3.

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.

The activity of organic anion transporter-3: Role of dexamethasone.

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 IC50 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 Vmax without meaningful alteration in substrate-binding affinity Km. 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.

Crosstalk of LKB1- and PTEN-regulated signals in liver morphogenesis and tumor development.

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.

PKC/Nedd4-2 Signaling Pathway Regulates the Cell Surface Expression of Drug Transporter hOAT1.

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.

Preparation, characterization and antioxidant activities of polymannuronic acid phosphate, H-phosphonate and sulfate.

Three derivatives of polymannuronic acid (PM) were prepared by chemically polyanionic modification. PM was substituted with phosphate (OPO3(2-)), H-phosphonate (OPO2H(-)) and sulfate (OSO3(-)) groups with the degree of substitution (DS) of 0.41, 1.42 and 1.04, respectively. The structures of all PM derivatives were characterized by FT-IR, (31)P NMR and (13)C NMR spectroscopy. The weight average molecular weight (Mw) was determined by high performance gel permeation chromatography (HPGPC). The antioxidant activities of PM and its derivatives were evaluated in vitro. The results indicated that both phosphate and H-phosphonate groups improved the hydroxyl radical scavenging activity, while sulfate group enhanced the superoxide radical scavenging activity. However, all of the derivatives scavenged DPPH less effectively than PM. The mechanism for how the different anionic substituent groups influenced the antioxidant activities was discussed.