RESUMEN
Human organic anion transporting polypeptides (OATPs) mediate the influx of many important drugs into cells. Casein kinase 2 (CK2) is a critical protein kinase that phosphorylates >300 protein substrates and is dysregulated in a number of disease states. Among the CK2 substrates are several transporters, although whether this includes human OATPs has not been evaluated. The current study was undertaken to evaluate the regulation of human OATP1A2 by CK2. HEK-239T cells in which OATP1A2 was overexpressed were treated with CK2 specific inhibitors or transfected with CK2 specific siRNA, and the activity, expression, and subcellular trafficking of OATP1A2 was evaluated. CK2 inhibition decreased the uptake of the prototypic OATP1A2 substrate estrone-3-sulfate (E3S). Kinetic studies revealed that this was due to a decrease in the maximum velocity (Vmax) of E3S uptake, while the Michaelis constant was unchanged. The cell surface expression, but not the total cellular expression of OATP1A2, was impaired by CK2 inhibition and knockdown of the catalytic α-subunits of CK2. CK2 inhibition decreased the internalization of OATP1A2 via a clathrin-dependent pathway, decreased OATP1A2 recycling, and likely impaired OATP1A2 targeting to the cell surface. Consistent with these findings, CK2 inhibition also disrupted the colocalization of OATP1A2 and Rab GTPase (Rab)4-, Rab8-, and Rab9-positive endosomal and secretory vesicles. Taken together, CK2 has emerged as a novel regulator of the subcellular trafficking and stability of OATP1A2. Because OATP1A2 transports many molecules of physiological and pharmacological importance, the present data may inform drug selection in patients with diseases in which CK2 and OATP1A2 are dysregulated.
Asunto(s)
Quinasa de la Caseína II/metabolismo , Transportadores de Anión Orgánico/metabolismo , Quinasa de la Caseína II/genética , Línea Celular , Silenciador del Gen/fisiología , Humanos , Immunoblotting , Estabilidad Proteica , Transporte de Proteínas/fisiologíaRESUMEN
UNLABELLED: Abstract Context: Solute carrier transporters (SLCs) are membrane proteins responsible for cellular influx of various substances including many pharmaceutical agents; therefore, they largely impact on drug disposition and elimination in body. Punica granatum Linnaeus (Lythraceae), pomegranate, is a fruit with antidiabetic potential. Oleanolic acid (OA), ursolic acid (UA), and gallic acid (GA) are the major bioactive components of pomegranate. Co-administration of these compounds with other drugs could result in altered drug pharmacokinetics, possibly due to competing for transporter proteins. OBJECTIVE: We investigated the interactions of these three compounds with the essential hepatic and renal SLC transporters. MATERIALS AND METHODS: Uptake of radiolabeled transporter model substrates was assessed in HEK293 cells over-expressing SLC transporters including the organic anion transporters (OATs), organic anion transporting polypeptides (OATPs) and organic cation transporters (OCTs), in the presence or absence of 10.0 µM UA, OA, or GA. Their IC50 values on specific SLC transporters were also evaluated using varying concentrations of the particular compound (ranging from 0.10 nM to 80.0 µM). RESULTS: Our results demonstrated UA could significantly inhibit OAT3 and OATP2B1 uptake (IC50: 18.9 ± 8.20 µM and 11.0 ± 5.00 µM, respectively) and GA has a pronounced inhibitory effect on OATP1B3 uptake (IC50: 1.60 ± 0.60 µM). DISCUSSION AND CONCLUSION: Our study reports the interactions of OA, UA, and GA with the essential SLC transporters. This information may contribute to elucidating the drug-drug/herb interactions involved with these three compounds and form the basis of therapeutic optimization when drugs are co-administered.
Asunto(s)
Ácido Gálico/farmacología , Lythraceae/química , Ácido Oleanólico/farmacología , Triterpenos/farmacología , Relación Dosis-Respuesta a Droga , Ácido Gálico/administración & dosificación , Ácido Gálico/aislamiento & purificación , Células HEK293 , Interacciones de Hierba-Droga , Humanos , Concentración 50 Inhibidora , Riñón/efectos de los fármacos , Riñón/metabolismo , Hígado/efectos de los fármacos , Hígado/metabolismo , Ácido Oleanólico/administración & dosificación , Ácido Oleanólico/aislamiento & purificación , Transportadores de Anión Orgánico/antagonistas & inhibidores , Proteínas de Transporte de Catión Orgánico/antagonistas & inhibidores , Triterpenos/administración & dosificación , Triterpenos/aislamiento & purificación , Ácido UrsólicoRESUMEN
The human organic anion transporting polypeptide 1A2 (OATP1A2) is an important membrane protein that mediates the cellular influx of various substances including drugs. Previous studies have shown that PDZ-domain containing proteins, especially PDZK1 and NHERF1, regulate the function of related membrane transporters in other mammalian species. This study investigated the role of PDZK1 and NHERF1 in the regulation of OATP1A2 in an in vitro cell model. Transporter function and protein expression were assessed in OATP1A2-transfected HEK-293 cells that co-expressed PDZK1 or NHERF1. Substrate (estrone-3-sulfate) uptake by OATP1A2 was significantly increased to â¼1.6- (PDZK1) and â¼1.8- (NHERF1) fold of control; this was dependent on the putative PDZ-binding domain within the C-terminus of OATP1A2. The functional increase of OATP1A2 following PDZK1 or NHERF1 over-expression was associated with increased transporter expression at the plasma membrane and in the whole cell, and was reflected by an increase in the apparent maximal velocity of estrone-3-sulfate uptake (V(max): 138.9±4.1 (PDZK1) and 181.4±16.7 (NHERF1) versus 55.5±3.2 pmol*(µg*4 min)⻹ in control; P<0.01). Co-immunoprecipitation analysis indicated that the regulatory actions of PDZK1 and NHERF1 were mediated by direct interaction with OATP1A2 protein. In further experiments PDZK1 and NHERF1 modulated OATP1A2 expression by decreasing its internalization in a clathrin-dependent (but caveolin-independent) manner. Additionally, PDZK1 and NHERF1 enhanced the stability of OATP1A2 protein in HEK-293 cells. The present findings indicated that PDZK1 and NHERF1 regulate the transport function of OATP1A2 by modulating protein internalization via a clathrin-dependent pathway and by enhancing protein stability.
Asunto(s)
Proteínas Portadoras/metabolismo , Transportadores de Anión Orgánico/metabolismo , Fosfoproteínas/metabolismo , Intercambiadores de Sodio-Hidrógeno/metabolismo , Línea Celular , Clatrina/metabolismo , Regulación de la Expresión Génica , Humanos , Espacio Intracelular/metabolismo , Proteínas de la Membrana , Transportadores de Anión Orgánico/genética , Unión Proteica , Estabilidad Proteica , Transporte de Proteínas , Transducción de SeñalRESUMEN
Solute carrier transporters (SLCs), in particular the organic anion transporting polypeptides (OATPs) and organic anion/cation transporters (OATs/OCTs), are responsible for the cellular entry of many clinically important drugs in body. They largely influence drug safety and efficacy. Icariin is a flavonol widely present in many herbal preparations, which is used to improve sexual function and prevent osteogenesis. However, precautions are necessary in therapies containing icariin due to its involvement in drug-drug/herb interactions, possibly mediated through competing drug uptake via membrane-transporter proteins. This study is the first to comprehensively evaluate the interactions between icariin and a range of essential SLCs. Our data demonstrated that icariin can significantly inhibit OATP1B3- and OATP2B1-mediated cellular uptake of specific substrates (IC50 of 3.0 ± 1.3 and 6.4 ± 1.9 µM, respectively). Our study revealed that icariin can potentially compete with coadministrated drugs for particular SLCs, which may impact the therapeutic outcome of regimens.
Asunto(s)
Flavonoides/farmacología , Transportadores de Anión Orgánico/metabolismo , Flavonoides/química , Células HEK293 , Humanos , Riñón/efectos de los fármacos , Riñón/metabolismo , Hígado/efectos de los fármacos , Hígado/metabolismo , Transportadores de Anión Orgánico Sodio-Independiente/metabolismo , Miembro 1B3 de la Familia de los Transportadores de Solutos de Aniones OrgánicosRESUMEN
The human organic cation/ergothioneine transporter 1 (hOCTN1, gene symbol SLC22A4) is responsible for the cellular uptake of substances, such as L-ergothioneine, which is an important antioxidant in mammalian cells. The common-function-altered variant L503F-hOCTN1 has been associated with susceptibility to Crohn's disease in certain populations. Previously, we identified eight novel nonsynonymous single-nucleotide polymorphisms (SNPs) in the SLC22A4 gene in the Chinese and Indian populations of Singapore. The present study evaluated the impact of these novel SNPs on hOCTN1 transport function in HEK-293 cells. Transport uptake assays with L-ergothioneine were used to assess the function of the variant transporters. Cell surface biotinylation and Western blot analysis were used to characterize cellular transporter expression. Comparative modeling was used to locate amino acid substitutions in the topology of hOCTN1 in order to account for altered transport function. Transporter activity was markedly impaired in four of the naturally occurring hOCTN1 variants (R63H, R83P, G482D, and I500N). Multiple glycosylated isoforms of hOCTN1 proteins were identified in the plasma membrane and in the whole cell. Either the total cellular or membrane expression of the functionally deficient transporter variants was lower than that of the wild-type hOCTN1. The underlying mechanism involves both impaired transporter-substrate binding affinity and turnover rate. Considered together, several naturally occurring SNPs in the SLC22A4 gene encode variant hOCTN1 transporters that may impact the cellular uptake of L-ergothioneine and other substrates, with the potential to influence the antioxidant capacity of human cells.