Regulation Of ATP-binding Cassette Transporters Research Articles

Variations in ATP-binding cassette transporter regulation during the progression of human nonalcoholic fatty liver disease.

Transporters located on the sinusoidal and canalicular membranes of hepatocytes regulate the efflux of drugs and metabolites into blood and bile, respectively. Changes in the expression or function of these transporters during liver disease may lead to a greater risk of adverse drug reactions. Nonalcoholic fatty liver disease (NAFLD) is a progressive condition encompassing the relatively benign steatosis and the more severe, inflammatory state of nonalcoholic steatohepatitis (NASH). Here, we present an analysis of the effect of NAFLD progression on the major ATP-binding cassette (ABC) efflux transport proteins ABCC1-6, ABCB1, and ABCG2. Human liver samples diagnosed as normal, steatotic, NASH (fatty), and NASH (not fatty) were analyzed. Increasing trends in mRNA expression of ABCC1, ABCC4-5, ABCB1, and ABCG2 were found with NAFLD progression, whereas protein levels of all transporters exhibited increasing trends with disease progression. Immunohistochemical staining of ABCC3, ABCB1, and ABCG2 revealed no alterations in cellular localization during NAFLD progression. ABCC2 staining revealed an alternative mechanism of regulation in NASH in which the transporter appears to be internalized away from the canalicular membrane. This correlated with a preferential shift in the molecular mass of ABCC2 from 200 to 180 kDa in NASH, which has been shown to be associated with a loss of glycosylation and internalization of the protein. These data demonstrate increased expression of multiple efflux transporters as well as altered cellular localization of ABCC2 in NASH, which may have profound effects on the ability of patients with NASH to eliminate drugs in an appropriate manner.

ABC Transporters in the CNS – An Inventory

In the present review we provide a summary of ATP-binding cassette (ABC) transporters in the central nervous system (CNS). Our review is focused on transporters of the ABC A, B, C, D, and G families that have been detected in the cells of the neurovascular unit/blood-brain barrier including brain capillary endothelial cells, pericytes, astrocytes, and neurons, as well as in other brain cells, such as microglia, oligodendrocytes, and choroid plexus epithelial cells. In this review, we provide an overview, organized by ABC family, of transporter expression, localization, and function. We summarize recent findings on ABC transporter regulation in the CNS and address the role of ABC transporters in CNS diseases including brain cancer, seizures/epilepsy, and Alzheimer's disease. Finally, we discuss new therapeutic strategies focused on ABC transporters in CNS disease.

Long-term regulation of ATP-binding cassette transporters on mRNA and protein level by simvastatin in human rhabdomyosarcoma and neuroblastoma cells

Background Resistance against chemotherapeutics is still a major problem in cancer therapy. One of the mechanisms of multidrug resistance is up-regulation of ATP-binding cassette (ABC) transporters. HMG-CoA reductase inhibitors have been shown to directly inhibit the main representative, ABCB1 (P-glycoprotein). We have previously shown in rhabdomyosarcoma (RD) and neuroblastoma (SH-SY5Y) cells that simvastatin enhanced the intrinsic apoptotic potential of the anthracycline doxorubicin [1,2]. The aim of the study presented here is to investigate the long-term effect of simvastatin on the expression of ABC-transporters in order to evaluate an adjuvant therapeutic potential of statins in chemotherapy.

Regulation of ABC Transporters at the Blood-Brain Barrier: New Targets for CNS Therapy

Worldwide, more than one billion people are affected by CNS disorders. Despite the huge demand for treatments, existing drugs have limited or no efficacy for some neurological diseases, including brain cancer and certain epilepsies. Furthermore, no effective therapies are available at all for some common disorders of the central nervous system (CNS) such as Alzheimer's disease. ATP-binding cassette (ABC) transporters at the blood-brain barrier (BBB) have become increasingly important in the treatment and pathogenesis of CNS disorders. Here we highlight a novel strategy--targeting signaling pathways that control ABC transporters at the BBB--to protect the brain, improve brain drug delivery, and reduce CNS pathology.

Hydrogen sulphide, a novel gasotransmitter involved in guard cell signalling

Hydrogen sulphide (H(2) S) has been proposed as the third gasotransmitter. In animal cells, H(2) S has been implicated in several physiological processes. H(2) S is endogenously synthesized in both animals and plants by enzymes with l-Cys desulphydrase activity in the conversion of l-Cys to H(2) S, pyruvate and ammonia. The participation of H(2) S in both stomatal movement regulation and abscisic acid (ABA)-dependent induction of stomatal closure was studied in epidermal strips of three plant species (Vicia faba, Arabidopsis thaliana and Impatiens walleriana). The effect of H(2) S on stomatal movement was contrasted with leaf relative water content (RWC) measurements of whole plants subjected to water stress. In this work we report that exogenous H(2) S induces stomatal closure and this effect is impaired by the ATP-binding cassette (ABC) transporter inhibitor glibenclamide; scavenging H(2) S or inhibition of the enzyme responsible for endogenous H(2) S synthesis partially blocks ABA-dependent stomatal closure; and H(2) S treatment increases RWC and protects plants against drought stress. Our results indicate that H(2) S induces stomatal closure and participates in ABA-dependent signalling, possibly through the regulation of ABC transporters in guard cells.

Small Molecule Activator of the Human Epithelial Sodium Channel

The epithelial sodium channel (ENaC), a heterotrimeric complex composed of alpha, beta, and gamma subunits, belongs to the ENaC/degenerin family of ion channels and forms the principal route for apical Na(+) entry in many reabsorbing epithelia. Although high affinity ENaC blockers, including amiloride and derivatives, have been described, potent and specific small molecule ENaC activators have not been reported. Here we describe compound S3969 that fully and reversibly activates human ENaC (hENaC) in an amiloride-sensitive and dose-dependent manner in heterologous cells. Mechanistically, S3969 increases hENaC open probability through interactions requiring the extracellular domain of the beta subunit. hENaC activation by S3969 did not require cleavage by the furin protease, indicating that nonproteolyzed channels can be opened. Function of alphabetaG37Sgamma hENaC, a channel defective in gating that leads to the salt-wasting disease pseudohypoaldosteronism type I, was rescued by S3969. Small molecule activation of hENaC may find application in alleviating human disease, including pseudohypoaldosteronism type I, hypotension, and neonatal respiratory distress syndrome, when improved Na(+) flux across epithelial membranes is clinically desirable.

Sonic Hedgehog promotes multiple drug resistance by regulation of drug transport

A major obstacle to successful chemotherapy is intrinsic or acquired multi-drug resistance (MDR). The most common cause of MDR involves increased drug efflux from cancer cells mediated by members of the ATP-binding cassette (ABC) transporter family. The regulation of ABC transporters in the context of cancer is poorly understood, and clinical efforts to inhibit their function have not been fruitful. Constitutive activation of the Hedgehog (Hh) pathway has been shown to contribute to the growth and maintenance of various cancers. Here, we show that inhibition of Hh signaling increases the response of cancer cells to multiple structurally unrelated chemotherapies. We further show that Hh pathway activation induces chemoresistance in part by increasing drug efflux in an ABC transporter-dependent manner. We found that Hh signaling regulates the expression of the ABC transporter proteins multi-drug resistance protein-1 (MDR1, ABCB1, P-glycoprotein) and (BCRP, ABCG2), and that targeted knockdown of MDR1 and BCRP expression by small interfering RNA partially reverses Hh-induced chemoresistance. These results suggest that the Hh pathway may be a target to overcome MDR and increase chemotherapeutic response.

UV irradiation inhibits ABC transporters via generation of ADP-ribose by concerted action of poly(ADP-ribose) polymerase-1 and glycohydrolase.

ATP-binding cassette (ABC) transporters are involved in the transport of multiple substrates across cellular membranes, including metabolites, proteins, and drugs. Employing a functional fluorochrome export assay, we found that UVB irradiation strongly inhibits the activity of ABC transporters. Specific inhibitors of poly(ADP-ribose) polymerase-1 (PARP-1) restored the function of ABC transporters in UVB-irradiated cells, and PARP-1-deficient cells did not undergo UVB-induced membrane transport inhibition. These data suggest that PARP-1 activation is necessary for ABC transporter functional downregulation. The hydrolysis of poly(ADP-ribose) by poly(ADP-ribose) glycohydrolase (PARG) was also required, since specific PARG inhibitors, which limit the production of ADP-ribose molecules, restored the function of ABC transporters. Furthermore, ADP-ribose molecules potently inhibited the activity of the ABC transporter P-glycoprotein. Hence, poly(ADP-ribose) metabolism appears to play a novel role in the regulation of ABC transporters.

Role of nuclear bile acid receptor, FXR, in adaptive ABC transporter regulation by cholic and ursodeoxycholic acid in mouse liver, kidney and intestine

Background/Aims: Adaptive changes in transporter expression in liver and kidney provide alternative excretory pathways for biliary constituents during cholestasis and may thus attenuate liver injury. Whether adaptive changes in ATP-binding cassette (ABC) transporter expression are stimulated by bile acids and their nuclear receptor FXR is unknown. Methods: Hepatic, renal and intestinal ABC transporter expression was compared in cholic acid (CA)- and ursodeoxycholic acid (UDCA)-fed wild-type (FXR +/+) and FXR knock-out mice (FXR −/−). Expression was assessed by reverse transcription–polymerase chain reaction, immunoblotting and immunofluorescence microscopy. Results: CA feeding stimulated hepatic Mrp2, Mrp3, Bsep and renal Mrp2 as well as intestinal Mrp2 and Mrp3 expression. Lack of Bsep induction by CA in FXR −/− was associated with disseminated hepatocyte necrosis which was not prevented by compensatory induction of Mrp2 and Mrp3. With the exception of Bsep, UDCA stimulated expression of hepatic, renal and intestinal ABC transporters independent of FXR without inducing liver toxicity. Conclusions: Toxic CA and non-toxic UDCA induce adaptive ABC transporter expression, independent of FXR with the exception of Bsep. Stimulation of hepatic Mrp3 as well as intestinal and renal Mrp2 by UDCA may contribute to its therapeutic effects by inducing alternative excretory routes for bile acids and other cholephiles.

Rapid quantification of murine ABC mRNAs by real time reverse transcriptase-polymerase chain reaction

Several ATP-binding cassette (ABC) transporters are critically involved in cholesterol and phospholipid efflux, reverse cholesterol transport, and play an important role in the development of atherosclerosis. Quantification of ABC mRNA is important in studying the regulation of cellular cholesterol homeostasis and mechanisms related to the pathogenesis of atherosclerosis. We have developed a one-step real time quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) method for measuring mRNA levels of ABCA1, ABCG1, and ABCA2 in murine tissues using the TaqMan(TM) technology. It has significant methodological benefits when compared to classic Northern blotting or semi-quantitative RT-PCR analysis. Using this method, we found high expression levels of ABCA1 in liver and macrophages, and of ABCG1 in the brain and macrophages. The expression of ABCA1 and ABCG1 were further induced in macrophages loaded with acLDL. In contrast, ABCA2 was expressed exclusively in the brain with low expression levels in the macrophages. This method provides a rapid, highly sensitive, specific, and reproducible quantification of ABC mRNA, and can be performed with nanograms of total RNA sample, thus making it a superior method for studying the regulation of ABC transporters in cholesterol efflux and its role in the pathogenesis of atherosclerosis in murine models.

Trafficking of canalicular ABC transporters in hepatocytes.

ATP-binding cassette (ABC) transporters located in the hepatocyte canalicular membrane of mammalian liver are critical players in bile formation and detoxification. Although ABC transporters have been well characterized functionally, only recently have several canalicular ABC transporters been cloned and their molecular nature revealed. Subsequently, development of specific antibodies has permitted a detailed investigation of ABC transporter intrahepatic distribution under varying physiological conditions. It is now apparent that there is a complex array of ABC transporters in hepatocytes. ABC transporter molecules reside in intrahepatic compartments and are delivered to the canalicular domain following increased physiological demand to secrete bile. Insufficient amounts of ABC transporters in the bile canalicular membrane result in cholestasis (i.e., bile secretory failure). Therefore, elucidation of the intrahepatic pathways and regulation of ABC transporters may help to understand the cause of cholestasis at a molecular level and provide clues for novel therapies.

Inhibition of ATP-Binding Cassette Transporter Downregulates Interleukin-1β-Mediated Autocrine Activation of Human Dermal Fibroblasts

Fibroblasts constitute an important source of cytokines during inflammatory processes in the skin. Interleukin-1 is a potent, pleiotropic cytokine that is induced in activated human dermal fibroblasts. Interleukin-1 further induces many inflammatory mediators, including the chemokine interleukin-8. As fibroblasts express both interleukin-1 and the interleukin-1 receptor complex, the cellular response may be enhanced by autocrine activation. Interleukin-1alpha and interleukin-1beta lack a signal peptide and are translocated at the plasma membrane using an alternative secretory pathway, which may involve ATP-binding cassette transporter proteins. We hypothesize that inhibition of this pathway prevents secretion of interleukin-1, thereby downregulating interleukin-1-dependent autocrine induction of interleukin-8. We used the ATP-binding cassette 1 transporter inhibitor glybenclamide, which has been previously shown to block interleukin-1beta secretion in human monocytes. Using enzyme-linked immunosorbent assay, we assessed the effect of glybenclamide on interleukin-8 production in human dermal fibroblasts. In interleukin-1beta-transfected human dermal fibroblasts, interleukin-8 was induced through an autocrine activity of interleukin-1beta. Glybenclamide disabled this activation loop and significantly reduced interleukin-8. In human dermal fibroblasts that were stimulated with tumor necrosis factor alpha to reach high interleukin-1 expression levels, glybenclamide similarly suppressed interleukin-8. In contrast, glybenclamide did not affect interleukin-8 production in cells stimulated with interleukin-1 only. Glybenclamide did not affect caspase-1 in fibroblasts, which was expressed as an inactive precursor form, irrespective of treatments with tumor necrosis factor alpha and/or glybenclamide. Using overexpressing, interleukin-1-transfected COS-1 cells, inhibition of interleukin-1alpha and interleukin-1beta secretion was directly demonstrated on Western blots. These results are consistent with glybenclamide preventing externalization of interleukin-1 and subsequent autocrine induction of interleukin-8 in human dermal fibroblasts. Acting through such a mechanism, ATP-binding cassette transporter inhibitors may downregulate inflammation locally.