ABSTRACT
PURPOSE: Acneiform rash is frequently observed in patients undergoing cancer treatment with anti-epidermal growth factor receptor (EGFR) antibody drugs and can often necessitate treatment discontinuation. However, the specific changes in skin parameters resulting from anti-EGFR antibody drug administration are poorly understood. Therefore, this study aimed to longitudinally and quantitatively evaluate the changes in skin parameters (transepidermal water loss [TEWL], hydration level, and sebum level) caused by anti-EGFR antibody drugs and investigate their potential as control markers for skin disorders. METHODS: This prospective study included 12 patients with colorectal cancer who received anti-EGFR antibody drugs for the first time. The assessment items included the grade of acneiform rash and skin parameters (TEWL, hydration level, and sebum level), which were observed for up to 6 weeks after administration of the medication. RESULTS: The enrolled patients were classified into two groups based on the grade of acneiform rash caused by anti-EGFR antibody drugs: "Grade 1 and lower," and "Grade 2 and higher." The skin parameters were compared between these groups. The results showed that in the "Grade 2 and higher" group, TEWL in the face (at week 2 of administration), chest (baseline, weeks 2 and 6 of administration), and back (at week 2 of administration) were significantly higher than those in the "Grade 1 and lower" group. However, the two groups showed no significant differences in hydration or sebum levels at any time point. CONCLUSION: TEWL can serve as a marker for acneiform rashes induced by anti-EGFR antibody drugs during cancer treatment.
Subject(s)
Colorectal Neoplasms , Exanthema , Humans , Panitumumab/adverse effects , Prospective Studies , ErbB Receptors , Skin , Exanthema/chemically induced , Colorectal Neoplasms/drug therapy , Cetuximab/adverse effectsABSTRACT
PURPOSE: The aim of this study was to determine the frequency of opioid-induced neurotoxicity (OIN) in cancer patients receiving oral controlled-release oxycodone and to define risk factors for OIN. METHODS: This was a single-center, retrospective study of hospitalized adult cancer patients receiving oral controlled-release oxycodone between April 1, 2013, and April, 30, 2020. The onset of OIN within 30 days after oxycodone initiation in the study patients was investigated. OIN was defined as any of the following: delirium, hallucinations (visual or auditory), seizure, myoclonus, hyperesthesia, and excessive somnolence. Multivariate logistic regression analysis was performed to identify risk factors for OIN in patients receiving oxycodone. RESULTS: In total, 520 patients were included in this study. The number of patients with OIN was 65 (12.5%). The median time until onset of OIN after oxycodone initiation was 7.5 days. Multivariate logistic regression analysis revealed that age ≥ 65 years (OR = 2.74, 95% CI [1.30-5.78], p = 0.008), total bilirubin ≥ 1.3 mg/dL (OR = 4.85, 95% CI [2.13-11.0], p < 0.001), and concomitant use of pregabalin or mirogabalin (OR = 3.11, 95% CI [1.47-6.61], p = 0.003) were significant independent risk factors for OIN. CONCLUSION: Age ≥ 65 years, liver dysfunction, and concomitant use of pregabalin or mirogabalin were independent risk factors for OIN in patients receiving oxycodone. Patients with these risk factors who are receiving oxycodone should be monitored for OIN, especially early in the administration of oxycodone.
Subject(s)
Neoplasms , Neurotoxicity Syndromes , Adult , Humans , Aged , Analgesics, Opioid/therapeutic use , Oxycodone/adverse effects , Delayed-Action Preparations , Retrospective Studies , Pregabalin , Neoplasms/drug therapy , Neurotoxicity Syndromes/etiology , Risk FactorsABSTRACT
BACKGROUND: We previously reported that high body weight was a risk factor affecting the onset of anti-epidermal growth factor receptor (EGFR) antibody drug-induced acneiform rash. The current study investigated the relationship between risk factors for anti-EGFR antibody drug-induced acneiform rash and survival probability in colorectal cancer patients, as well as effects of drug withdrawal, dose reduction, or treatment discontinuation on treatment continuation. METHODS: This retrospective study included 67 patients with unresectable advanced or recurrent colorectal cancer treated with anti-EGFR antibody drugs for the first time. RESULTS: The survival time and acneiform rash grade of patients with high body weight (≥ 67.2 kg) were significantly longer and higher than those of patients with low body weight (< 67.2 kg). Moreover, the treatment continuation time of patients with drug withdrawal or dose reduction was significantly longer than that of patients without drug withdrawal or dose reduction or with/without treatment discontinuation. Meanwhile, the treatment continuation time of patients with treatment discontinuation was significantly shorter than that of patients with drug withdrawal or dose reduction or those without drug withdrawal, dose reduction, or treatment discontinuation. CONCLUSIONS: High body weight is a novel prognostic factor for patients receiving cancer drugs with anti-EGFR antibody drugs. Hence, the results of this study suggest that patients with high body weight should be carefully monitored for the development of acneiform rash when receiving anti-EGFR antibody drugs as cancer drug therapy.
ABSTRACT
Molecular mechanisms underlying the nephrotoxicity associated with bevacizumab are unclear. Endothelin-1 (ET-1) is involved in podocyte injury and proteinuria, and its level increases in most cases of kidney disorders. Forkhead box protein O1 (FoxO1), a transcription factor, is a major determinant of ET-1 promoter activation and is regulated by protein kinase B (Akt) phosphorylation-dependent nuclear exclusion. We evaluated the effect of bevacizumab on ET-1 production in human glomerular microvascular endothelial cells (hGECs). We analyzed the changes in the mRNA and protein levels of ET-1 in hGECs treated with bevacizumab using real-time reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay. Changes in the protein levels and phosphorylation status of Akt and FoxO1 in hGECs treated with bevacizumab were analyzed by western blotting. After cell lysis, FoxO1 protein was isolated from the cytoplasmic and nuclear fractions. We also investigated the effects of AS1842856 (a FoxO1 inhibitor) on bevacizumab-induced ET-1 production. Bevacizumab significantly and dose-dependently increased the mRNA and protein levels of ET-1 in hGECs (p < 0.05). Bevacizumab treatment also led to a decrease in phosphorylated Akt protein levels. Inhibition of Akt activity by LY294002 promoted ET-1 production. Bevacizumab also induced an increase in FoxO1 protein levels in the nucleus. Inhibition of FoxO1 activity by AS1842856 resulted in decreased ET-1 levels in bevacizumab-treated hGECs. ET-1 axis activation, Akt inactivation, and FoxO1 nuclear localization are the molecular mechanisms underlying bevacizumab-induced nephrotoxicity. Therefore, inhibition of renal ET-1 production could be a promising approach to protect against or treat bevacizumab-induced nephrotoxicity.
ABSTRACT
WHAT IS KNOWN AND OBJECTIVE: Cancer drug treatment is often discontinued because of skin disorder aggravation. However, information on risk factors for skin disorders caused by anti-epidermal growth factor receptor (EGFR) antibody drugs is limited. The aim of this study was to analyse the factors associated with skin disorders caused by anti-EGFR antibody drugs and establish a method to minimize such aggravations. METHODS: We retrospectively examined 67 colorectal cancer patients treated with anti-EGFR antibody drugs for the first time. RESULTS AND DISCUSSION: A higher proportion of males than females experienced drug withdrawal, dose reduction or treatment discontinuation. The multiple logistic regression analysis revealed body weight as a risk factor affecting drug withdrawal, dose reduction or treatment discontinuation because of an acneiform rash. An examination of methods to avoid the aggravation of skin disorders revealed the acneiform rash grade in patients who received prophylactic minocycline was significantly lower than that in patients who did not receive prophylactic minocycline. Furthermore, among patients with grade 1 acneiform rash at the initiation of minocycline, the proportion of those who withdrew, required dose reduction or discontinued treatment was lower than that among patients with grade 2 acneiform rash. WHAT IS NEW AND CONCLUSION: High body weight was identified as a novel factor for skin disorder aggravation caused by anti-EGFR antibody drugs. The aggravation of skin disorders during cancer treatment with anti-EGFR antibody drugs can potentially be avoided by carefully observing the onset of acneiform rash in affected patients with high body weight and using minocycline prophylactically or as an early-stage intervention.
Subject(s)
Antineoplastic Agents, Immunological/adverse effects , Antineoplastic Combined Chemotherapy Protocols/adverse effects , Colorectal Neoplasms/drug therapy , ErbB Receptors/antagonists & inhibitors , Skin Diseases/chemically induced , Age Factors , Aged , Antineoplastic Agents, Immunological/therapeutic use , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Body Weight , Female , Humans , Logistic Models , Male , Middle Aged , Minocycline/administration & dosage , Patient Dropouts , Quality of Life , Retrospective Studies , Sex FactorsABSTRACT
Opioids are widely used for the treatment of moderate/severe pain in cancer and noncancer patients. In this study, we searched for safety signals for a wide variety of opioid-related adverse events (AEs) in elderly patients by disproportionality analysis using the Japanese Adverse Drug Event Report (JADER) database. Data from the JADER database from April 2004 to May 2018 were obtained from the Pharmaceuticals and Medical Devices Agency website. Safety signal detection of opioid-related AEs in elderly patients was defined using the relative elderly reporting odds ratio (ROR). Among the analyzed AEs, opioid-induced neurotoxicity (OIN) was assessed based on the time to onset using the Weibull shape parameter. The following safety signals were detected in elderly patients: respiratory depression, somnolence, hallucinations, akathisia and OIN. Fentanyl, tramadol, oxycodone and morphine exhibited a large relative elderly ROR for OIN. The median time to onset of OIN of transdermal fentanyl, oral tramadol, oral oxycodone and oral morphine was 13.5, 6, 9, and 6 d, respectively. These opioids were classified as early failure types using the Weibull distribution. Our results showed that elderly patients who are administered opioids should be closely monitored for AEs, such as respiratory depression, OIN and akathisia.
Subject(s)
Adverse Drug Reaction Reporting Systems/statistics & numerical data , Analgesics, Opioid/adverse effects , Drug-Related Side Effects and Adverse Reactions/epidemiology , Pharmacovigilance , Adult , Age Distribution , Age Factors , Aged , Aged, 80 and over , Causality , Drug-Related Side Effects and Adverse Reactions/etiology , Female , Humans , Japan/epidemiology , Male , Middle Aged , Young AdultABSTRACT
To investigate neuronal activity involved in responses to noxious stimuli in conscious monkeys, the animals were subjected to a task that required them to detect a small change in facial skin temperature or light (second temperature: T2, second light: V2) relative to an initial condition (T1 or V1), and to detect changes in V2 along with a heat task. Recordings were obtained from 57 neurons in the ventral premotor cortex (PMv) during the heat or light detection task. T1 neurons and T2 neurons showed increased activity only during T1 or T2, and T1/T2 neurons were activated by both T1 and T2 stimuli. T1/T2 neurons showed an increase in firing at higher T1 temperatures, whereas T1 neurons did not. About half of the non-light/heat-sensitive T1/T2 neurons showed increased firing at higher T2 temperatures, whereas T2 neurons showed no such increase. The heat responses of heat-sensitive PMv neurons were significantly suppressed when monkeys shifted their attention from heat to light. The present findings suggest that heat-sensitive PMv neurons may be involved in motor responses to noxious heat, whereas light/heat-PMv neurons may be involved in emotional and motivational aspects of pain and inappropriate motor responses to allow escape from noxious stimuli.
Subject(s)
Motor Cortex , Animals , Hot Temperature , Macaca fascicularis , Neurons , NociceptorsABSTRACT
Bright light stimulation of the eye activates trigeminal subnucleus caudalis (Vc) neurons in rats. Sensory information is conveyed to the Vc via the trigeminal ganglion (TG). Thus, it is likely that TG neurons respond to photic stimulation and are involved in photic hypersensitivity. However, the mechanisms underlying this process are unclear. Therefore, the hypothesis in this study is bright light stimulation enhances the excitability of TG neurons involved in photic hypersensitivity. Expressions of calcitonin gene-related peptide (CGRP) and neuronal nitric oxide synthase (nNOS) were significantly higher in TG neurons from 5 min to 12 h after photic stimulation of the eye. Phosphorylation of extracellular signal-regulated kinase1/2 (pERK1/2) was enhanced in TG neurons within 5 min after photic stimulation, while pERK1/2 immunoreactivity in satellite glial cells (SGCs) persisted for more than 12 h after the stimulus. Activation of SGCs was observed from 5 min to 2 h. Expression of CGRP, nNOS, and pERK1/2 was observed in small and medium TG neurons, and activation of SGCs and pERK1/2-immunoreactive SGCs encircling large TG neurons was accelerated after stimulation. These results suggest that upregulation of CGRP, nNOS, and pERK1/2 within the TG is involved in photic hypersensitivity.
Subject(s)
Calcitonin Gene-Related Peptide/metabolism , Eye/radiation effects , Light , MAP Kinase Signaling System , Nitric Oxide Synthase Type I/metabolism , Trigeminal Ganglion/metabolism , Up-Regulation , Animals , Eye/enzymology , Eye/metabolism , Glial Fibrillary Acidic Protein/metabolism , Immunohistochemistry , Male , Neurons/metabolism , Phosphorylation , Rats , Rats, Sprague-Dawley , Trigeminal Ganglion/cytology , Trigeminal Ganglion/enzymologyABSTRACT
Oxytocin (OXT) is a neuropeptide hormone synthesized and secreted by hypothalamic neurons and has been reported to play a significant role in pain modulation. However, the mechanisms underlying OXT's antinociceptive effect on neuropathic pain are not fully understood. In this study, we examined the peripheral effect of OXT on mechanical hypersensitivity induced by partial ligation of the infraorbital nerve (PNL) in rats. Mechanical hypersensitivity in the whisker pad skin after PNL was attenuated by the direct administration of OXT into the trigeminal ganglion (TG). The proportion of vasopressin-1A receptor (V1A-R)-immunoreactive, but not OXT-receptor-immunoreactive, neurons significantly increased among TG neurons innervating the whisker pad skin after PNL. In a patch-clamp recording from TG neurons isolated from PNL rats, the resting membrane potential of OXT-treated neurons was significantly decreased, and the current thresholds of OXT-treated neurons for spike generation (rheobases) were significantly greater than those of vehicle-treated neurons. In addition, OXT increased voltage-gated K channel currents in PNL animals. Furthermore, intra-TG administration of a selective V1A-R antagonist reversed the OXT-induced alleviation of mechanical hypersensitivity, and coapplication of the antagonist opposed OXT's effects on the resting membrane potential, rheobase, and K current. These findings suggest that OXT is effective at suppressing TG neuronal hyperexcitability after nerve injury, likely by modulation of voltage-gated K channels through V1A-R. This signaling mechanism represents a potential therapeutic target for the treatment of orofacial neuropathic pain.
Subject(s)
Facial Pain/complications , Hyperalgesia , Oxytocin/therapeutic use , Receptors, Vasopressin/metabolism , Trigeminal Ganglion/metabolism , Animals , Cells, Cultured , Disease Models, Animal , Facial Pain/drug therapy , Hormone Antagonists/pharmacology , Hyperalgesia/drug therapy , Hyperalgesia/etiology , Hyperalgesia/pathology , Indoles/pharmacology , Male , Membrane Potentials/drug effects , Membrane Potentials/physiology , Neurons/drug effects , Pain Threshold/drug effects , Pyrrolidines/pharmacology , Rats , Rats, Sprague-Dawley , Receptors, Oxytocin/metabolism , Trigeminal Ganglion/pathology , Vibrissae/innervationABSTRACT
Human organic cation transporter 1 (OCT1/SLC22A1) plays important roles in the hepatic uptake of cationic drugs. The functional characteristics of this transporter have been well evaluated, but molecular information regarding transcriptional regulation is limited. In the present study, therefore, we examined the gene regulation of OCT1 gene focusing on basal core expression. An approximately 2.5-kb fragment of the OCT1 promoter region was isolated, and promoter activity was measured by luciferase assay in the human liver cell lines Huh7 and HepG2. Deletion analysis suggested that the region spanning -141/-69 was essential for the basal core transcriptional activity and that this region contained the sequence of a cognate E-box (CACGTG). The E-box is known to be bound by the basal transcription factors, upstream stimulating factors (USFs), and the functional involvements of USF1 and USF2 were confirmed by a transactivation effect, a mutational analysis of the E-box, and an electrophoretic mobility shift assay. The transactivation effect of USFs on the OCT1 promoter was further stimulated by hepatocyte nuclear factor 4alpha, a liver-enriched transcription factor. There were no polymorphisms in the proximal promoter region (about 400 bp) of OCT1 gene (n = 109). These findings indicated that both USF1 and USF2 bind to an E-box sequence located in the OCT1 core promoter region and are required for the basal gene expression of this transporter.
Subject(s)
Octamer Transcription Factor-1/genetics , Promoter Regions, Genetic/genetics , Upstream Stimulatory Factors/physiology , Base Sequence , Cell Line, Tumor , DNA Mutational Analysis , E-Box Elements/genetics , Gene Expression Regulation , Hepatocyte Nuclear Factor 4 , Humans , Molecular Sequence Data , Polymorphism, Single Nucleotide , Transcription Initiation SiteABSTRACT
Organic cation transporters (OCTs) and organic anion transporters (OATs) (SLC22A family) play crucial roles in the renal secretion of various drugs. Messengar ribonucleic acid (mRNA) expression of transporters can be a key factor regulating interindividual differences in drug pharmacokinetics. However, the source of variations in mRNA levels of transporters is unclear. In this study, we focused on single nucleotide polymorphisms (SNP) in the promoter region [regulatory SNPs (rSNPs)] as candidates for the factor regulating mRNA levels of SLC22A. We sequenced the promoter regions of OCT2 and OAT1-4 in 63 patients and investigated the effects of the identified rSNPs on transcriptional activities and mRNA expression. In the OCT2 promoter region, one deletion polymorphism (-578_-576delAAG) was identified; -578_-576delAAG significantly reduced OCT2 promoter activity (p < 0.05), and carriers of -578_-576delAAG tend to have lower OCT2 mRNA levels, but the difference is not significant. There was no rSNP in the OAT1 and OAT2 genes. The five rSNPs of OAT3 and one rSNP of OAT4 were unlikely to influence mRNA expression and promoter activity. This is the first study to investigate the influences of rSNPs on mRNA expression of SLC22A in the kidney and to identify a regulatory polymorphism affecting OCT2 promoter activity.
Subject(s)
Gene Expression Regulation/genetics , Kidney/metabolism , Organic Anion Transporters, Sodium-Independent/genetics , Organic Cation Transport Proteins/genetics , Polymorphism, Single Nucleotide , Animals , Cell Line , Humans , LLC-PK1 Cells , Opossums , Organic Anion Transport Protein 1/genetics , Organic Anion Transporters, Sodium-Independent/metabolism , Organic Cation Transporter 2 , Promoter Regions, Genetic , SwineABSTRACT
A H+/organic cation antiporter (multidrug and toxin extrusion 1: MATE1/SLC47A1) plays important roles in the tubular secretion of various clinically important cationic drugs such as cimetidine. We have recently found that the regulation of this transporter greatly affects the pharmacokinetic properties of cationic drugs in vivo. No information is available about the regulatory mechanisms for the MATE1 gene. In the present study, therefore, we examined the gene regulation of human (h) and rat (r) MATE1, focusing on basal expression. A deletion analysis suggested that the regions spanning -65/-25 and -146/-38 were essential for the basal transcriptional activity of the hMATE1 and rMATE1 promoter, respectively, and that both regions contained putative Sp1-binding sites. Functional involvement of Sp1 was confirmed by Sp1 overexpression, a mutational analysis of Sp1-binding sites, mithramycin A treatment, and an electrophoretic mobility shift assay. Furthermore, we found a single nucleotide polymorphism (SNP) in the promoter region of hMATE1 (G-32A), which belongs to a Sp1-binding site. The allelic frequency of this rSNP was 3.7%, and Sp1-binding and promoter activity were significantly decreased. This is the first study to clarify the transcriptional mechanisms of the MATE1 gene and to identify a SNP affecting the promoter activity of hMATE1.
Subject(s)
Antiporters/genetics , Gene Expression Regulation/physiology , Organic Cation Transport Proteins/genetics , Sp1 Transcription Factor/physiology , Animals , Binding Sites/genetics , Blotting, Western , Cell Line , DNA Mutational Analysis , Electrophoretic Mobility Shift Assay , Gene Frequency , Humans , LLC-PK1 Cells/chemistry , Plicamycin/analogs & derivatives , Plicamycin/pharmacology , Polymorphism, Single Nucleotide , Promoter Regions, Genetic , Rats , Recombinant Proteins/metabolism , Sp1 Transcription Factor/analysis , Swine , Transcription Initiation Site , Transcription, Genetic/physiologyABSTRACT
Multidrug and toxin extrusion 1 (MATE1) has been isolated as an H(+)/organic cation antiporter located at the renal brush-border membranes. Previous studies using rat renal brush-border membrane vesicles indicated that cysteine and histidine residues played critical roles in H(+)/organic cation antiport activity. In the present study, essential histidine and cysteine residues of MATE1 family were elucidated. When 7 histidine and 12 cysteine residues of rat (r)MATE1 conserved among species were mutated, substitution of His-385, Cys-62, and Cys-126 led to a significant loss of tetraethylammonium (TEA) transport activity. Cell surface biotinylation and immunofluorescence analyses with confocal microscopy indicated that rMATE1 mutant proteins were localized at plasma membranes. Mutation of the corresponding residues in human (h)MATE1 and hMATE2-K also diminished the transport activity. The transport of TEA via rMATE1 was inhibited by the sulfhydryl reagent p-chloromercuribenzenesulfonate (PCMBS) and the histidine residue modifier diethyl pyrocarbonate (DEPC) in a concentration-dependent manner. The PCMBS-caused inhibition of the transport via rMATE1 was protected by an excess of various organic cations such as TEA, suggesting that cysteine residues act as substrate-binding sites. In the case of DEPC, no such protective effects were observed. These results suggest that histidine and cysteine residues are required for MATE1 to function and that cysteine residues may serve as substrate-recognition sites.
Subject(s)
Antiporters/metabolism , Cysteine/metabolism , Histidine/metabolism , Organic Cation Transport Proteins/metabolism , 4-Chloromercuribenzenesulfonate/pharmacology , Animals , Antiporters/genetics , Biological Transport/drug effects , Cysteine/genetics , Diethyl Pyrocarbonate/pharmacology , Gene Expression , Histidine/genetics , Humans , Mutagenesis, Site-Directed , Organic Cation Transport Proteins/genetics , Rats , Tetraethylammonium/metabolism , Toxins, Biological/metabolismABSTRACT
Human organic anion transporter 1 (OAT1, SLC22A6), which is localized to the basolateral membranes of renal tubular epithelial cells, plays a critical role in the excretion of anionic compounds. OAT1 is regulated by various pathophysiological conditions, but little is known about the molecular mechanisms regulating the expression of OAT1. In the present study, we investigated the transcriptional regulation of OAT1 and found that hepatocyte nuclear factor (HNF)-4alpha markedly transactivated the OAT1 promoter. A deletion analysis of the OAT1 promoter suggested that the regions spanning -1191 to -700 base pairs (bp) and -140 to -79 bp were essential for the transactivation by HNF-4alpha. These regions contained a direct repeat separated by two nucleotides (DR-2), which is one of the consensus sequences binding to HNF-4alpha, and an inverted repeat separated by eight nucleotides (IR-8), which was recently identified as a novel element for HNF-4alpha, respectively. An electrophoretic mobility shift assay showed that HNF-4alpha bound to DR-2 and IR-8 under the conditions of HNF-4alpha overexpression. Furthermore, under normal conditions, HNF-4alpha bound to IR-8, and a mutation in IR-8 markedly reduced the OAT1 promoter activity, indicating that HNF-4alpha regulates the basal transcription of OAT1 via IR-8. This paper reports the first characterization of the human OAT1 promoter and the first gene in the kidney whose promoter activity is regulated by HNF-4alpha.
Subject(s)
Gene Expression Regulation/physiology , Hepatocyte Nuclear Factor 4/physiology , Kidney/metabolism , Organic Anion Transport Protein 1/genetics , Animals , Cell Line , Cloning, Molecular , DNA Primers , DNA, Complementary/biosynthesis , DNA, Complementary/genetics , Electrophoretic Mobility Shift Assay , Epithelial Cells/metabolism , Gene Expression Regulation/genetics , Humans , Luciferases/genetics , Luciferases/metabolism , Mutagenesis, Site-Directed , Opossums , Promoter Regions, Genetic/genetics , Transcription Factors , TransfectionABSTRACT
Human organic cation transporter 2 (hOCT2; SLC22A2) is abundantly expressed in the kidney, and it plays important roles in the renal tubular secretion of cationic drugs. Although the transport characteristics of hOCT2 have been studied extensively, there is no information available for the transcriptional regulation of hOCT2. The present study was undertaken to identify the cis-element and trans-factor for basal expression of hOCT2. The transcription start site was located 385 nucleotides above the translation start site by using 5'-rapid amplification of cDNA ends. An approximately 4-kilobase fragment of the hOCT2 promoter region was isolated and the promoter activities were measured in the renal epithelial cell line LLC-PK1. A deletion analysis suggested that the region spanning -91 to -58 base pairs was essential for basal transcriptional activity. This region lacked a TATA-box but contained a CCAAT box and an E-box. Electrophoretic mobility shift assays showed that specific DNA/protein complexes were present in the E-box but not in the CCAAT box, and supershift assays revealed that upstream stimulatory factor 1 (USF-1), which belongs to the basic helix-loop-helix-leucine zipper family of transcription factors, bound to the E-box. Mutation of the E-box resulted in a decrease in hOCT2 promoter activity, and overexpression of USF-1 enhanced the hOCT2 promoter activity in a dose-dependent manner. This article reports the first characterization of the hOCT2 promoter and shows that USF-1 functions as a basal transcriptional regulator of the hOCT2 gene via the E-box.
Subject(s)
Organic Cation Transport Proteins/genetics , Promoter Regions, Genetic , Upstream Stimulatory Factors/physiology , 5' Flanking Region , Base Sequence , Electrophoretic Mobility Shift Assay , Humans , Kidney/metabolism , Molecular Sequence Data , Organic Cation Transporter 2 , Polymorphism, Single Nucleotide , Transcription Initiation Site , Transcriptional ActivationABSTRACT
Recently, we have isolated the rat (r) H(+)/organic cation antiporter multidrug and toxin extrusion 1 (MATE1) and reported its tissue distribution and transport characteristics. Functional characterization suggested that an oppositely directed H(+) gradient serves as a driving force for the transport of a prototypical organic cation, tetraethylammonium, by MATE1, but there is no direct evidence to prove this. In the present study, therefore, we elucidated the driving force of tetraethylammonium transport via rMATE1 using plasma membrane vesicles isolated from HEK293 cells stably expressing rMATE1 (HEK-rMATE1 cells). A 70-kDa rMATE1 protein was confirmed to exist in HEK-rMATE1 cells, and the transport of various organic cations including [(14)C]tetraethylammonium was stimulated in intracellular acidified HEK-rMATE1 cells but not mock cells. The transport of [(14)C]tetraethylammonium in membrane vesicles from HEK-rMATE1 cells exhibited the overshoot phenomenon only when there was an outwardly directed H(+) gradient, as observed in rat renal brush-border membrane vesicles. The overshoot phenomenon was not observed in the vesicles from mock cells. The stimulated [(14)C]tetraethylammonium uptake by an H(+) gradient [intravesicular H(+) concentration ([H(+)](in)) > extravesicular H(+) concentration ([H(+)](out))] was significantly reduced in the presence of a protonophore, carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP). [(14)C]tetraethylammonium uptake was not changed in the presence of valinomycin-induced membrane potential. These findings definitively indicate that an oppositely directed H(+) gradient serves as a driving force of tetraethylammonium transport via rMATE1, and this is the first demonstration to identify the driving force of the MATE family. The present experimental strategy is very useful in identifying the driving force of cloned transporters whose driving force has not been evaluated.
Subject(s)
Antiporters/physiology , Hydrogen/physiology , Organic Cation Transport Proteins/physiology , Animals , Cell Line , Cell Membrane/metabolism , Humans , Rats , Tetraethylammonium Compounds/metabolismABSTRACT
Human organic anion transporter (OAT) 3 (SLC22A8) is localized to the basolateral membranes of renal tubular epithelial cells and plays a critical role in the excretion of anionic compounds. We previously reported that interindividual variation in the OAT3 mRNA level corresponded to interindividual differences in the rate of renal excretion of cefazolin. However, there is little information available on the molecular mechanisms regulating the gene expression of OAT3. Therefore, in the present study, we examined the transcriptional regulation of human OAT3. A deletion analysis of the OAT3 promoter suggested that the region spanning -214 to -77 base pairs was essential for basal transcriptional activity. This region contained a perfectly conserved cAMP-response element (CRE), and a mutation here led to a reduction in promoter activity. Electrophoretic mobility shift assays showed that CRE-binding protein (CREB)-1 and activating transcription factor (ATF)-1 bound to CRE. The activity of the OAT3 promoter was increased through the phosphorylation of CREB-1 and ATF-1 by treatment with 8-bromo-cAMP. This paper reports the first characterization of the human OAT3 promoter and shows that CREB-1 and ATF-1 function as constitutive and inducible transcriptional regulators of the human OAT3 gene via CRE.
Subject(s)
Cyclic AMP/physiology , Gene Expression Regulation , Organic Anion Transporters, Sodium-Independent/genetics , Response Elements/physiology , Base Sequence , Cyclic AMP-Dependent Protein Kinases/physiology , Electrophoretic Mobility Shift Assay , Humans , Molecular Sequence Data , Polymorphism, Single Nucleotide , Promoter Regions, Genetic , Transcription Initiation SiteABSTRACT
PURPOSE: Transport characteristics and tissue distribution of the rat H+/organic cation antiporter MATE1 (multidrug and toxin extrusion 1) were examined. METHODS: Rat MATE1 cDNA was isolated by polymerase chain reaction (PCR) cloning. Transport characteristics of rat MATE1 were assessed by HEK293 cells transiently expressing rat MATE1. The mRNA expression of rat MATE1 was examined by Northern blot and real-time PCR analyses. RESULTS: The uptake of a prototypical organic cation tetraethylammonium (TEA) by MATEI-expressing cells was concentration-dependent, and showed the greatest value at pH 8.4 and the lowest at pH 6.0-6.5. Intracellular acidification induced by ammonium chloride resulted in a marked stimulation of TEA uptake. MATE1 transported not only organic cations such as cimetidine and metformin but also the zwitterionic compound cephalexin. MATE1 mRNA was expressed abundantly in the kidney and placenta, slightly in the spleen, but not expressed in the liver. Real-time PCR analysis of microdissected nephron segments showed that MATE1 was primarily expressed in the proximal convoluted and straight tubules. CONCLUSIONS: These findings indicate that MATE1 is expressed in the renal proximal tubules and can mediate the transport of various organic cations and cephalexin using an oppositely directed H+ gradient.
Subject(s)
Antiporters/genetics , Antiporters/metabolism , Organic Cation Transport Proteins/genetics , Organic Cation Transport Proteins/metabolism , Animals , Blotting, Northern , Cells, Cultured , Cloning, Molecular , DNA, Complementary/biosynthesis , DNA, Complementary/genetics , Humans , Nephrons/metabolism , Organic Cation Transporter 1/genetics , Organic Cation Transporter 1/metabolism , Organic Cation Transporter 2 , Rats , Reverse Transcriptase Polymerase Chain Reaction , Tissue DistributionABSTRACT
PURPOSE: Organic cation transporters 1-3 (OCT1-3; Slc22a1-3) mediate the membrane transport of organic cations in the kidney. We previously reported that rat (r)OCT2 expression in the kidney was regulated by testosterone. In this study, we examined the transcriptional mechanisms underlying the testosterone-dependent regulation of rOCT2 expression. METHODS: Approximately 3000-bp fragments of the rOCT1-3 promoter region were isolated, and promoter activities were measured in the renal epithelial cell line LLC-PK1 with the coexpression of rat androgen receptor. RESULTS: Among reporter constructs tested, only rOCT2 promoter activity was stimulated by testosterone. This stimulation was suppressed by nilutamide, an antiandrogen drug. Reporter assays using deletion constructs and mutational constructs of putative androgen response elements (ARE) in the rOCT2 promoter region suggested that two AREs, located at approximately -3000 and -1300, respectively, play an important role in the induction by testosterone. CONCLUSIONS: Testosterone induces the expression of rOCT2, but not of rOCT1 and rOCT3, via the AR-mediated transcriptional pathway. This is the first study to address the transcriptional mechanisms of testosterone-dependent gene regulation of the Slc22 family.