Isoform-Specific Regulation of Mouse Carboxylesterase Expression and Activity by Prototypical Transcriptional Activators
Nuclear receptors and transcription factors regulate the mRNA expression of many drug metabolizing enzymes, including the carboxylesterases (Ces). However, there are few data regarding whether these changes in mRNA expression result in alteration of protein levels or activity. In the present study, we sought to determine the isoform-specific regulation of hepatic Ces mRNA expression and activity following administration of pharmacological activators of the constitutive androstane receptor (CAR), pregnane X receptor (PXR) and nuclear factor-E2-related protein (Nrf2) to mice. The CAR activator 1,4-bis-[2-(3,5-dichloropyridyloxy)] benzene (TCPOBOP) and PXR ligand pregnenolone-16a-carbonitrile (PCN) increased Ces mRNA expression of various Ces2 isoforms, whereas the Nrf2 activator butylated hydroxyanisole (BHA) primarily reduced Ces3a mRNA expression and induced Ces1g mRNA. TCPOBOP and PCN increased Ces2 hydrolytic activity in an isoform-specific manner. Taken together, these data demonstrate that activation of CAR, PXR and Nrf2 regulates not only Ces mRNA expression, but also isoform-specific activity.
Introduction<!>Chemicals<!>Animals<!>Transcriptional Activator Treatment<!>Quantitative Real-time Polymerase Chain Reaction<!>Spectrophotometric Carboxylesterase Activity Assay<!>In-gel Carboxylesterase Activity Assay<!>Statistical Analysis<!>Induction of Target Genes by Transcriptional Activators<!>Basal Expression of Ces Genes and Activity<!>Regulation of Mouse Ces1 mRNA Expression by Transcriptional Activators<!>Regulation of Mouse Ces2 mRNA Expression by Transcriptional Activators<!>Effect of Transcriptional Activators on Ces-Mediated Hydrolysis of p-Nitrophenyl Valerate<!>Effect of Transcriptional Activators on Isoform-Specific Hydrolytic Activity<!>Discussion<!>
<p>Carboxylesterases (Ces) are serine hydrolase enzymes expressed in various tissues including the liver, intestine, kidney, lung, testis, and brain [1,2,3,4]. Ces hydrolyze endogenous lipids and xenobiotics containing ester bonds, to generate free acids and alcohol [4]. Ces form a super family with approximately 20 recognized mouse Ces genes located on mouse chromosome 8 named according to their position in the genome. The 360-kb Ces1 gene cluster includes eight genes (Ces1a-h), similar to the 286-kb Ces2 gene cluster, which also includes eight genes (Ces2a-h). In contrast, smaller (17- or 21-kb) genomic regions contain Ces4a or Ces5a [4,5,6].</p><p>Ces are important enzymes in the clinical efficacy of a variety of drugs, including angiotensin converting enzyme inhibitors and chemotherapeutics [7,8,9]. Additionally, many xenobiotics are extensively metabolized by Ces, including organophosphorus and pyrethroid pesticides [10,11]. Similar to other drug metabolizing enzymes, the activity of Ces is affected by nutritional status, hormonal factors and xenobiotics [5,12]. However, the specific mechanism(s) by which Ces is regulated has only recently begun to be explored.</p><p>Nuclear receptors are a group of ligand activated transcription factors, which are responsible for regulating and modulating expression of genes important for metabolism, developmental functions, and cell differentiation [13]. These receptors play key roles in endogenous physiological processes and regulate mRNA expression of phase I and II drug metabolizing enzymes and transporters [14]. Only recently has attention turned to the potential role of nuclear receptors and transcriptional activators in regulating Ces expression [15,16,17]. Transcriptional activator or nuclear receptor-mediated alterations in the expression and activity for human CES following exposure to xenobiotics have been documented previously in vitro [18,19]. Studies in hepatoma cells found that the nuclear factor-E2-related protein (Nrf2) agonists, sulphorophane and tert-butylhydroquinone, induce CES1 gene expression, protein and activity [19]. Dexamethasone, a glucocorticoid receptor and pregnane × receptor (PXR) agonist and phenobarbital, a constitutive androstane receptor (CAR) agonist, also increased human CES1 and CES2 protein in human hepatocytes [18,20]. In mice, Xu and co-workers observed that mouse Ces2a mRNA expression was induced in liver and intestine following treatment with the PXR and CAR agonists pregnenolone-16a-carbonitrile (PCN) and 1,4-bis-[2-(3,5-dichloropyridyloxy)] benzene (TCPOBOP), respectively [21]. Most recently, Zhang and co-workers reported on the effects of nuclear receptor agonists on the mRNA expression of a large number of Ces isoforms in wild type and nuclear receptor knockout mice [16].</p><p>Based on these studies, it is clear that Ces mRNA expression is affected by transcriptional activation. However, few studies have explored whether these changes in expression translate to effects on Ces activity and protein expression. Because alteration of Ces activity by transcriptional activation may have significant impact on the efficacy of therapeutic drugs or toxicity of xenobiotics, the present study investigated the regulation of isoform-specific Ces mRNA expression catalytic activity in mouse liver following administration of activators of CAR, PXR and Nrf2. Though the current body of literature on Ces regulation by transcriptional activators varies by species, route of exposure, dose and dosing regimen, our study confirms the roles of Nrf2, CAR and PXR by demonstrating induction of Ces at the mRNA level. Our work extends these findings by demonstrating that altered Ces mRNA expression generally corresponds to changes in isoform-specific hydrolytic activity against the model substrate 4-MUBA, with a few exceptions.</p><!><p>1,4-bis-[2-(3,5-dichloropyridyloxy)] benzene (TCPOBOP), pregnenolone-16α-carbonitrile (PCN), butylated hydroxyanisole (BHA), p-nitrophenol valerate, 1-naphthylacetate and 4-methylumbelliferlyacetate (4-MUBA) were all obtained from Sigma-Aldrich (St. Louis, MO).</p><!><p>Male C57BL6J mice (age 8 weeks, 18-20g) were purchased from Jackson Laboratories and were housed in an American Animal Association for Laboratory Animal Care acredited facility. Mice were given access to rodent chow and water ad libitum. Mice were acclimated to the housing facility for 2 weeks before experiments (4 mice per cage, 50% relative humidity, 12-h light/dark cycle). All experiments were conducted in accordance with recommendations from the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Robert Wood Johnson Medical School Institutional Animal Care and Use Committee (Protocol #I08-065).</p><!><p>Mice were administered the nuclear receptor agonists of CAR (TCPOBOP), PXR (PCN) and Nrf2 (BHA) by intraperitoneal injection (5 ml/kg) at doses of 3mg/kg, 200 mg/kg and 350 mg/kg, respectively [22]. Control animals were dosed with corn oil vehicle. Mice were treated for 4 days and the animals sacrificed on day 5. Liver samples for activity assessment were frozen on dry ice and samples for quantitative real time-PCR (qPCR) were frozen in liquid nitrogen.</p><!><p>qPCR analysis was performed as described previously [23]. RNA was isolated using Miniprep kits (Qiagen) per manufacturer's instructions and reverse transcribed using Superscript II and 1 μg RNA. qPCR was performed in duplicate using an ABI 7900HT (Applied Biosystems, Carlsbad, CA) with SYBR Green (Applied Biosystems) detection. To normalize for the amount of mRNA in each sample, actin expression was monitored in parallel samples and data calculated using the ΔΔCt method. Primers were designed using the Primer Blast program (NCBI) and all isoforms were run under identical conditions and exhibited similar amplification efficiencies so that comparison of basal expression levels could be made. Primer sequences are available upon request.</p><!><p>Ces enzyme activity assay was performed as previously described by Ross and Borazjani [24]. Briefly, Ces activity in the liver microsomal fraction was determined by continuous spectrophotometric assay using p-nitrophenol valerate as the substrate in a 96 well plate format. Hydrolysis of p-nitrophenyl valerate was detected via liberation of p-nitrophenol and rate of formation of hydrolysis product was monitored for 5mins at 405nm. Non-Ces mediated hydrolysis was determined in the presence of paraoxon (1 μM) and subtracted from total activity to calculate specific activity (μmol/min/mg protein), as described previously [25].</p><!><p>Liver homogenates were in 100mM potassium phosphate buffer, pH 7.4, and contained 0.2% Lubrol. For electrophoresis, 10 μg of liver homogenate was loaded on a 4-12% Tris-Glycine gel and then stained with 4-MUBA (1mM) for 15 min as previously described by Ross and Borazjani [24]. Staining intensity, which is reflective of enzyme activity, was captured under ultraviolet illumination with an Alpha Innotech Imager and quantified by densitometry using AlphaView Protein simple software.</p><!><p>The effects of agonist treatment on enzyme activity and mRNA expression were analyzed by one-way analysis of variance or Student's t test, where appropriate. For ANOVA, post-hoc analysis was performed using Dunnett's test. Each data point represents a group mean ± SEM. Asterisks represent statistical differences (p ≤ 0.05) between control and treatment groups.</p><!><p>To determine whether treatments activated the appropriate transcription factor pathways, induction of mRNA expression of target genes for the respective CAR, PXR and Nrf2 agonists were evaluated by qPCR (Supplemental Fig. 1). Cyp 2b10 mRNA expression, a marker for CAR activation, was increased by TCPOBOP, and to a lesser extent, BHA and PCN. The PXR target gene Cyp 3a11 was induced by both PCN and TCPOBOP. Nqo1, a marker of Nrf2 activation, was increased by BHA, and to a lesser extent, TCPOBOP.</p><!><p>Ct values for basal hepatic Ces mRNA expression ranged from 19.9 to 25.3, with a general expression pattern of Ces3a ≥ 1c > 1g ≥ 2a ≥ 2c > 2e ≥ 1d ≥ 1e. This is similar to that observed by Jones and co-workers [17].</p><!><p>The effects of nuclear receptor agonist treatment on Ces isoform mRNA expression in the Ces1 family are shown in Figure 1. Ces1d expression was the most significantly impacted in the Ces 1 family, with expression increased 124% and 163% respectively, by TCPOBOP and PCN. Ces 1c was increased by PCN only (52%). Ces1g expression was increased 155% and 151% by BHA and PCN, respectively. Ces1e expression trended toward reduced expression by BHA (−35%) and TCPOBOP (−25%), but did not quite reach statistical significance.</p><!><p>Figure 1 also illustrates the effects of nuclear receptor activation on Ces2 family mRNA expression. Ces2a expression was increased 858% and 252% by TCPOBOP and PCN, respectively. Ces2c expression was also increased by TCPOBOP (223%) and PCN (116%). Ces2e was increased 147% by PCN and 42% by TCPOBOP, although this did not quite reach statistical significance. In contrast, Ces2e was significantly decreased by 32% following BHA treatment. Ces3a was also significantly decreased by 67% and 69% following BHA and TCPOBOP, respectively.</p><!><p>To determine whether alteration in Ces mRNA and protein expression resulted in alteration of function, hydrolytic activity of Ces was determined in mouse liver microsomes using p-nitrophenol valerate, a generic ester substrate hydrolyzed by CES 1 and 2 families [24]. Ces activity was significantly increased 135% and 31% by TCPOBOP and PCN, respectively (Fig. 2). BHA administration did not result in significant changes in activity.</p><!><p>Liver samples were subjected to native gel electrophoresis and stained with 4-methylumbelliferlyacetate (4MUBA) to assess isoform-specific Ces activity. 4-MUBA is a generic ester substrate hydrolyzed by carboxylesterases in the human CES 1 and 2 families [24]. 4-MUBA staining of gels revealed bands for Ces1d, Ces1e, Ces2a, Ces2c, Ces2e and two unidentified Ces1 bands as previously described by Xiao and co-workers [26]. In the BHA treated mouse Ces 1d and Ces 2c hydrolytic activity were significantly decreased by 21% and 12%, respectively (Fig. 3). Ces x, y-1e and Ces 2a-e were not changed in the BHA treated mouse. TCPOBOP treatment increased Ces 2a-e and Ces 2c hydrolytic activity by 55% and 198%, respectively (Fig. 4). Ces-y 1e activity was significantly decreased by 21% following TCPOBOP treatment. PCN treatment resulted in significant increases of Ces 1d and Ces 2a-e hydrolytic activity by 28% and 38% respectively (Fig. 5).</p><!><p>Based on lack of validated isoform-specific mouse antibodies, groups have elected to use molecular approaches to profile the tissue distribution and regulation of Ces mRNA expression [16,17,21]. However, there are few data regarding whether or not these mRNA expression results in changes of Ces activity.</p><p>The transcription factor Nrf2 regulates the transcription of genes encoding antioxidant proteins and phase II enzymes. Zhang and co-workers [16] recently reported the induction of Ces1g and suppression of Ces1e and Ces3a mRNA in mouse liver following BHA treatment, when analyzed by branched DNA assay. Our findings generally confirmed this, with exception of an observed decrease of Ces2e mRNA. This discrepancy is likely the result of differences in detection technology (i.e. branched DNA vs qPCR).</p><p>The nuclear receptor CAR is a xenobiotic sensing transcription factor that regulates the expression and activity of a variety of phase I and II enzymes and some transporters [14]. Previous studies reported Ces mRNA induction by TCPOBOP in mouse liver [16] [17] [21]. Our findings that Ces 2a and Ces 2c mRNA expression was induced by TCPOBOP are in agreement with the aforementioned studies. Previously, Xiao and coworkers demonstrated an apparent increase in Ces2a, Ces2c and Ces2e hydrolytic activity towards 4-MUBA in mice treated with 1.5mg/kg TCPOBOP, although these data were not quantified [29]. Our study confirms these findings and extends them by demonstrating that hepatic Ces hydrolytic activity toward p-nitrophenyl valerate was also increased by TCPOBOP.</p><p>The nuclear receptor PXR is an important xenobiotic sensing transcription factor that regulates expression of Cyp3a, Cyp2b, Cyp2c and phase II GSTs, SULTs and UGTs in liver [14]. Recently PCN (200 mg/kg) was shown to induce Ces2a and Ces2c mRNA expression [16]. In another study, PCN (50 mg/kg) induced Ces2a and suppressed Ces3a mRNA expression in mouse liver [17], while Xu and co-workers [21] showed Ces2a mRNA was induced by PCN (100 mg/kg). Our findings are in general agreement with these studies. We also observed induction of Ces1c, Ces1d and Ces1g mRNA expression. With regard to enzyme activity, Xiao and colleagues demonstrated an apparent increase in Ces1d, Ces2a, Ces2c and Ces2e hydrolytic activity towards 4-MUBA in mice treated with 20 mg/kg PCN [29]. We also observed this with the exception of 2c. Finally, Ces hydrolytic activity toward p-nitrophenol valerate was increased in the PCN treated mouse liver, again supporting the contribution of Ces2 to this activity.</p><p>From a translational standpoint, the data observed here may have relevance to humans, as CES1A mRNA, protein and imidapril hydrolase activity was induced in three human cell lines by Nrf2 activators [19]. However, no effect of Nrf2 agonists on CES2 expression was observed, suggesting there may be dissociation between regulation of human and mouse Ces by Nrf2. The relevance of CAR regulation of CES expression in humans remains to be established, as there are no published data on the ability of CAR to induce CES in human cells. A recent paper where the human PXR ligand-binding domain was knocked-in to the murine PXR reported induction of Ces2a [25], suggesting potential relevance to humans. Taken together, these findings demonstrate the functional relevance of altered Ces mRNA expression by translational activators, which may provide a means to manipulate the efficacy of therapeutics or toxicity of xenobiotics.</p><!><p>Supplemental Figure 1. Effect of transcriptional activator treatment on target gene expression in the liver. Following treatment with the CAR, PXR and Nrf2 activators TCPOBOP, PCN and BHA, mouse liver was harvested and RNA subjected to qPCR to determine the level of target gene mRNA expression (n = 4-6 per group). Messenger RNA was normalized to actin expression and data calculated using the 2−ΔΔCt method. Normalized data are expressed as mean ± SEM. * represents statistically significant differences between control and activator-treated mice.</p>