Dynamic Ir(III) Photosensors for the Major Human Drug-Metabolizing Enzyme Cytochrome P450 3A4.

Probing the activity of cytochrome P450 3A4 (CYP3A4) is critical for monitoring the metabolism of pharmaceuticals and identifying drug-drug interactions. A library of Ir(III) probes that detect occupancy of the CYP3A4 active site were synthesized and characterized. These probes show selectivity for CYP3A4 inhibition, low cellular toxicity, Kd values as low as 9 nM, and are highly emissive with lifetimes up to 3.8 µs in cell growth media under aerobic conditions. These long emission lifetimes allow for time-resolved gating to distinguish probe from background autofluorescence from growth media and live cells. X-ray crystallographic analysis revealed structure-activity relationships and the preference or indifference of CYP3A4 toward resolved stereoisomers. Ir(III)-based probes show emission quenching upon CYP3A4 binding, then emission increases following displacement with CYP3A4 inhibitors or substrates. Importantly, the lead probes inhibit the activity of CYP3A4 at concentrations as low as 300 nM in CYP3A4-overexpressing HepG2 cells that accurately mimic human hepatic drug metabolism. Thus, the Ir(III)-based agents show promise as novel chemical tools for monitoring CYP3A4 active site occupancy in a high-throughput manner to gain insight into drug metabolism and drug-drug interactions.

Ir(III)-Based Agents for Monitoring the Cytochrome P450 3A4 Active Site Occupancy.

Cytochromes P450 (CYPs) are a superfamily of enzymes responsible for biosynthesis and drug metabolism. Monitoring the activity of CYP3A4, the major human drug-metabolizing enzyme, is vital for assessing the metabolism of pharmaceuticals and identifying harmful drug-drug interactions. Existing probes for CYP3A4 are irreversible turn-on substrates that monitor activity at specific time points in end-point assays. To provide a more dynamic approach, we designed, synthesized, and characterized emissive Ir(III) and Ru(II) complexes that allow monitoring of the CYP3A4 active-site occupancy in real time. In the bound state, probe emission is quenched by the active-site heme. Upon displacement from the active site by CYP3A4-specific inhibitors or substrates, these probes show high emission turn-on. Direct probe binding to the CYP3A4 active site was confirmed by X-ray crystallography. The lead Ir(III)-based probe has nanomolar Kd and high selectivity for CYP3A4, efficient cellular uptake, and low toxicity in CYP3A4-overexpressing HepG2 cells.

Negative Regulation of Human Hepatic Constitutive Androstane Receptor by Cholesterol Synthesis Inhibition: Role of Sterol Regulatory Element Binding Proteins.

The squalene synthase inhibitor squalestatin 1 (Squal1) is a potent and efficacious inducer of CYP2B expression in primary cultured rat hepatocytes and rat liver. To determine whether Squal1 is also an inducer of human CYP2B, the effects of Squal1 treatment were evaluated in primary cultured human hepatocytes, differentiated HepaRG cells, and humanized mouse livers. Squal1 treatment did not increase CYP2B6 mRNA levels in human hepatocytes or HepaRG cells and only slightly and inconsistently increased CYP2B6 mRNA content in humanized mouse liver. However, treatment with farnesol, which mediates Squal1's effect on rat CYP2B expression, increased CYP2B6 mRNA levels in HepaRG cells expressing the constitutive androstane receptor (CAR), but not in cells with knocked-down CAR. To determine the impact of cholesterol biosynthesis inhibition on CAR activation, the effects of pravastatin (Prava) were determined on CITCO-mediated gene expression in primary cultured human hepatocytes. Prava treatment abolished CITCO-inducible CYP2B6 expression, but had less effect on rifampicin-mediated CYP3A4 induction, and CITCO treatment did not affect Prava-inducible HMG-CoA reductase (HMGCR) expression. Treatment with inhibitors of different steps of cholesterol biosynthesis attenuated CITCO-mediated CYP2B6 induction in HepaRG cells, and Prava treatment increased HMGCR expression and inhibited CYP2B6 induction with comparable potency. Transfection of HepG2 cells with transcriptionally active sterol regulatory element binding proteins (SREBPs) reduced CAR-mediated transactivation, and inducible expression of transcriptionally active SREBP2 attenuated CITCO-inducible CYP2B6 expression in HepaRG cells. These findings suggest that Squal1 does not induce CYP2B6 in human hepatocytes because Squal1's inhibitory effect on cholesterol biosynthesis interferes with CAR activation. SIGNIFICANCE STATEMENT: The cholesterol biosynthesis inhibitor squalestatin 1 induces rat hepatic CYP2B expression indirectly by causing accumulation of an endogenous isoprenoid that activates the constitutive androstane receptor (CAR). This study demonstrates that squalestatin 1 does not similarly induce CYP2B6 expression in human hepatocytes. Rather, inhibition of cholesterol biosynthesis interferes with CAR activity, likely by activating sterol regulatory element binding proteins. These findings increase our understanding of the endogenous processes that modulate human drug-metabolizing gene expression.

Developmental Expression of SULT1C4 Transcript Variants in Human Liver: Implications for Discordance Between SULT1C4 mRNA and Protein Levels.

The cytosolic sulfotransferases (SULTs) metabolize a variety of xenobiotic and endogenous substrates. Several SULTs are expressed in the fetus, implying that these enzymes have important functions during human development. We recently reported that while SULT1C4 mRNA is abundant in prenatal human liver specimens, SULT1C4 protein is barely detectable. Two coding transcript variants (TVs) of SULT1C4 are indexed in GenBank, TV1 (full-length) and TV2 (lacking exons 3 and 4). The purpose of this study was to evaluate expression of the individual TVs as a clue for understanding the discordance between mRNA and protein levels. Reverse-transcription polymerase chain reaction was initially performed to identify TVs expressed in intestinal and hepatic cell lines. This analysis generated fragments corresponding to TV1, TV2, and a third variant that lacked exon 3 (E3DEL). Using reverse-transcription quantitative polymerase chain reaction assays designed to quantify TV1, TV2, or E3DEL individually, all three TVs were more highly expressed in prenatal than postnatal specimens. TV2 levels were ∼fivefold greater than TV1, while E3DEL levels were minimal. RNA sequencing (RNA-seq) analysis of another set of liver specimens confirmed that TV1 and TV2 levels were highest in prenatal liver, with TV2 higher than TV1. RNA-seq also detected a noncoding RNA, which was also more abundant in prenatal liver. Transfection of HEK293T cells with plasmids expressing individual Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys-tagged SULT1C4 isoforms demonstrated that TV1 produced much more protein than did TV2. These data suggest that the lack of correspondence between SULT1C4 mRNA and protein levels in human liver is likely attributable to the inability of the more abundant TV2 to produce stable protein. SIGNIFICANCE STATEMENT: Cytosolic sulfotransferases (SULTs) metabolize a variety of xenobiotic and endogenous substrates, and several SULTs are highly expressed in the fetus, implying that they have important functions during human development. SULT1C4 is highly expressed in prenatal liver at the mRNA level but not the protein level. This study provides an explanation for this discordance by demonstrating that the predominant SULT1C4 transcript is a variant that produces relatively little protein.

Developmental Expression of the Cytosolic Sulfotransferases in Human Liver.

The liver is the predominant organ of metabolism for many endogenous and foreign chemicals. Cytosolic sulfotransferases (SULTs) catalyze the sulfonation of drugs and other xenobiotics, as well as hormones, neurotransmitters, and sterols, with consequences that include enhanced drug elimination, hormone inactivation, and procarcinogen bioactivation. SULTs are classified into six gene families, but only SULT1 and SULT2 enzymes are expressed in human liver. We characterized the developmental expression patterns of SULT1 and SULT2 mRNAs and proteins in human liver samples using reverse transcription quantitative polymerase chain reaction (RT-qPCR), RNA sequencing, and targeted quantitative proteomics. Using a set of prenatal, infant, and adult liver specimens, RT-qPCR analysis demonstrated that SULT1A1 (transcript variant 1) expression did not vary appreciably during development; SULT1C2, 1C4, and 1E1 mRNA levels were highest in prenatal and/or infant liver, and 1A2, 1B1, and 2A1 mRNA levels were highest in infant and/or adult. Hepatic SULT1A1 (transcript variant 5), 1C3, and 2B1 mRNA levels were low regardless of developmental stage. Results obtained with RNA sequencing of a different set of liver specimens (prenatal and pediatric) were generally comparable results to those of the RT-qPCR analysis, with the additional finding that SULT1A3 expression was highest during gestation. Analysis of SULT protein content in a library of human liver cytosols demonstrated that protein levels generally corresponded to the mRNAs, with the major exception that SULT1C4 protein levels were much lower than expected based on mRNA levels. These findings further support the concept that hepatic SULTs play important metabolic roles throughout the human life course, including early development.

Farnesol induces fatty acid oxidation and decreases triglyceride accumulation in steatotic HepaRG cells.

Non-alcoholic fatty liver disease is manifested by hepatic accumulation of triglycerides (TG) and is commonly associated with metabolic syndrome. The isoprenoid farnesol (FOH) modulates lipid metabolism and reduces hepatic TG content in rodents. This effect involves activation of at least two nuclear receptors, peroxisome proliferator-activated receptor α (PPARα) and farnesoid X receptor. We evaluated the effects of FOH (100 µM) in a cellular model of human hepatic steatosis by loading hepatocyte-like HepaRG cells with oleic acid (OA, 0.66 mM). FOH treatment decreased OA-induced TG accumulation by ~25%. Using PCR arrays, we found that FOH treatment modulated the mRNA levels of several lipid-metabolizing enzymes, both alone and when cells were loaded with OA. While FOH activated PPARα and the constitutive androstane receptor (CAR), most of the FOH-mediated effects on lipid-metabolizing genes could be attributed to activation of PPARα. In OA-loaded HepaRG cells, FOH increased fatty acid oxidation, which was accompanied by up-regulation of PPARα target genes involved in mitochondrial fatty acid oxidation, including hydroxyacyl-CoA dehydrogenase/3-ketoacyl-CoA thiolase/enoyl-CoA hydratase and acetyl-coenzyme A acyltransferase 2. These effects on gene expression were lost when the cells were co-treated with the PPARα antagonist, GW6471. OA treatment alone decreased the mRNA levels of the drug-metabolizing enzymes, cytochrome P450 (CYP)1A2, 2B6, and 3A4, and increased CYP2E1 expression, all of which were attenuated by FOH co-treatment. These findings show that FOH treatment increases fatty acid oxidation and decreases TG accumulation in steatotic HepaRG cells, which is likely attributable to PPARα-mediated induction of mitochondrial fatty acid oxidation.

Regulation of Cytosolic Sulfotransferases in Models of Human Hepatocyte Development.

Cytosolic sulfotransferases (SULTs) are expressed during early life and therefore metabolize endogenous and xenobiotic chemicals during development. Little is currently known about the regulation of individual SULTs in the developing human liver. We characterized SULT expression in primary cultures of human fetal hepatocytes and the HepaRG model of liver cell differentiation. SULT1A1 (transcript variants 1-4), SULT1C2, SULT1C4, SULT1E1, and SULT2A1 were the most abundant transcripts in human fetal hepatocytes. In HepaRG cells, SULT1B1, SULT1C2/3/4, and SULT1E1 mRNA levels increased during the transition from proliferation to confluency and then decreased as the cells underwent further differentiation. By contrast, SULT2A1 mRNA levels increased during differentiation, whereas SULT1A1 and SULT2B1 mRNA levels remained relatively constant. The temporal patterns of SULT1C2, SULT1E1, and SULT2A1 protein content were consistent with those observed at the mRNA level. To identify regulators of SULT expression, cultured fetal hepatocytes and HepaRG cells were treated with a panel of lipid- and xenobiotic-sensing receptor activators. The following effects were observed in both fetal hepatocytes and HepaRG cells: 1) liver X receptor activator treatment increased SULT1A1 transcript variant 5 levels; 2) vitamin D receptor activator treatment increased SULT1C2 and SULT2B1 mRNA levels; and 3) farnesoid X receptor activator treatment decreased SULT2A1 expression. Activators of aryl hydrocarbon receptor, constitutive androstane receptor, pregnane X receptor, and peroxisome proliferator-activated receptors produced additional gene-dependent effects on SULT expression in HepaRG cells. These findings suggest that SULT-regulating chemicals have the potential to modulate physiologic processes and susceptibility to xenobiotic stressors in the developing human liver.

Transcriptional Regulation of Human Cytosolic Sulfotransferase 1C3 by Peroxisome Proliferator-Activated Receptor γ in LS180 Human Colorectal Adenocarcinoma Cells.

Cytosolic sulfotransferase 1C3 (SULT1C3) is the least characterized of the three human SULT1C subfamily members. Originally identified as an orphan SULT by computational analysis of the human genome, we recently reported that SULT1C3 is expressed in human intestine and LS180 colorectal adenocarcinoma cells and is upregulated by agonists of peroxisome proliferator-activated receptor (PPAR) α and γ To determine the mechanism responsible for PPAR-mediated upregulation, we prepared reporter plasmids containing fragments of the SULT1C3 5'-flanking region. During initial attempts to amplify a 2.8-kb fragment from different sources of human genomic DNA, a 1.9-kb fragment was sometimes coamplified with the expected 2.8-kb fragment. Comparison of the 1.9-kb fragment sequence to the published SULT1C3 5'-flanking sequence revealed an 863-nt deletion (nt -146 to -1008 relative to the transcription start site). Transfection analysis in LS180 cells demonstrated that PPARα, δ, and γ agonist treatments induced luciferase expression from a reporter plasmid containing the 2.8-kb but not the 1.9-kb fragment. The PPAR agonists also activated a 1-kb reporter containing the 863-nt deletion region. Computational analysis identified three peroxisome proliferator response elements (PPREs) within the 863-nt region and serial deletions and site-directed mutations indicated that the most distal PPRE (at nt -769) was essential for obtaining PPAR-mediated transcriptional activation. Although agonists of all three PPARs could activate SULT1C3 transcription, RNA interference analysis indicated the predominance of PPARγ These data demonstrate that the PPARγ regulatory network includes SULT1C3 and imply that this enzyme contributes to the control of such PPARγ-regulated intestinal processes as growth, differentiation, and metabolism.

Differential Regulation of Gene Expression by Cholesterol Biosynthesis Inhibitors That Reduce (Pravastatin) or Enhance (Squalestatin 1) Nonsterol Isoprenoid Levels in Primary Cultured Mouse and Rat Hepatocytes.

Squalene synthase inhibitors (SSIs), such as squalestatin 1 (SQ1), reduce cholesterol biosynthesis but cause the accumulation of isoprenoids derived from farnesyl pyrophosphate (FPP), which can modulate the activity of nuclear receptors, including the constitutive androstane receptor (CAR), farnesoid X receptor, and peroxisome proliferator-activated receptors (PPARs). In comparison, 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors (e.g., pravastatin) inhibit production of both cholesterol and nonsterol isoprenoids. To characterize the effects of isoprenoids on hepatocellular physiology, microarrays were used to compare orthologous gene expression from primary cultured mouse and rat hepatocytes that were treated with either SQ1 or pravastatin. Compared with controls, 47 orthologs were affected by both inhibitors, 90 were affected only by SQ1, and 51 were unique to pravastatin treatment (P < 0.05, ≥1.5-fold change). When the effects of SQ1 and pravastatin were compared directly, 162 orthologs were found to be differentially coregulated between the two treatments. Genes involved in cholesterol and unsaturated fatty acid biosynthesis were up-regulated by both inhibitors, consistent with cholesterol depletion; however, the extent of induction was greater in rat than in mouse hepatocytes. SQ1 induced several orthologs associated with microsomal, peroxisomal, and mitochondrial fatty acid oxidation and repressed orthologs involved in cell cycle regulation. By comparison, pravastatin repressed the expression of orthologs involved in retinol and xenobiotic metabolism. Several of the metabolic genes altered by isoprenoids were inducible by a PPARα agonist, whereas cytochrome P450 isoform 2B was inducible by activators of CAR. Our findings indicate that SSIs uniquely influence cellular lipid metabolism and cell cycle regulation, probably due to FPP catabolism through the farnesol pathway.

Transcriptional Regulation of Cytosolic Sulfotransferase 1C2 by Vitamin D Receptor in LS180 Human Colorectal Adenocarcinoma Cells.

The factors that regulate expression of genes in the 1C family of human cytosolic sulfotransferases (SULT1C) are not well understood. In a recent study evaluating the effects of a panel of transcription factor activators on SULT1C family member expression in LS180 human colorectal adenocarcinoma cells, we found that SULT1C2 expression was significantly increased by 1α,25-dihydroxyvitamin D3 (VitD3) treatment. The objective of our current study was to identify the mechanism responsible for VitD3-mediated activation of SULT1C2 transcription. VitD3 treatment of LS180 cells activated transcription of a transfected luciferase reporter plasmid that contained ∼5 kilobase pairs (kbp) of the SULT1C2 gene, which included 402 nucleotides (nt) of the noncoding exon 1, all of intron 1, and 21 nt of exon 2. Although computational analysis of the VitD3-responsive region of the SULT1C2 gene identified a pregnane X receptor (PXR)-binding site within exon 1, the transfected 5 kbp SULT1C2 reporter was not activated by treatment with rifampicin, a prototypical PXR agonist. However, deletion or mutation of the predicted PXR-binding site abolished VitD3-mediated SULT1C2 transcriptional activation, identifying the site as a functional vitamin D response element (VDRE). We further demonstrated that vitamin D receptor (VDR) can interact directly with the SULT1C2 VDRE sequence using an enzyme-linked immunosorbent assay-based transcription factor binding assay. In conclusion, VitD3-inducible SULT1C2 transcription is mediated through a VDRE in exon 1. These results suggest a role for SULT1C2 in VitD3-regulated physiologic processes in human intestine.

Nonsterol Isoprenoids Activate Human Constitutive Androstane Receptor in an Isoform-Selective Manner in Primary Cultured Mouse Hepatocytes.

Our laboratory previously reported that accumulation of nonsterol isoprenoids following treatment with the squalene synthase inhibitor, squalestatin 1 (SQ1) markedly induced cytochrome P450 (CYP)2B1 mRNA and reporter activity in primary cultured rat hepatocytes, which was dependent on activation of the constitutive androstane receptor (CAR). The objective of the current study was to evaluate whether isoprenoids likewise activate murine CAR (mCAR) or one or more isoforms of human CAR (hCAR) produced by alternative splicing (SPTV, hCAR2; APYLT, hCAR3). We found that SQ1 significantly induced Cyp2b10 mRNA (∼3.5-fold) in primary hepatocytes isolated from both CAR-wild-type and humanized CAR transgenic mice, whereas the 3-hydroxy-3-methylglutaryl-CoA reductase inhibitor pravastatin had no effect. In the absence of CAR, basal Cyp2b10 mRNA levels were reduced by 28-fold and the effect of SQ1 on Cyp2b10 induction was attenuated. Cotransfection with an expression plasmid for hCAR1, but not hCAR2 or hCAR3, mediated SQ1-induced CYP2B1 and CYP2B6 reporter activation in hepatocytes isolated from CAR-knockout mice. This effect was also observed following treatment with the isoprenoid trans,trans-farnesol. The direct agonist CITCO increased interaction of hCAR1, hCAR2, and hCAR3 with steroid receptor coactivator-1. However, no significant effect on coactivator recruitment was observed with SQ1, suggesting an indirect activation mechanism. Further results from an in vitro ligand binding assay demonstrated that neither farnesol nor other isoprenoids are direct ligands for hCAR1. Collectively, our findings demonstrate that SQ1 activates CYP2B transcriptional responses through farnesol metabolism in an hCAR1-dependent manner. Further, this effect probably occurs through an indirect mechanism.

Role of Phosphatidic Acid Phosphatase Domain Containing 2 in Squalestatin 1-Mediated Activation of the Constitutive Androstane Receptor in Primary Cultured Rat Hepatocytes.

Farnesyl pyrophosphate (FPP) is a branch-point intermediate in the mevalonate pathway that is normally converted mainly to squalene by squalene synthase in the first committed step of sterol biosynthesis. Treatment with the squalene synthase inhibitor squalestatin 1 (SQ1) causes accumulation of FPP, its dephosphorylated metabolite farnesol, and several oxidized farnesol-derived metabolites. In addition, SQ1 treatment of primary cultured rat hepatocytes increases CYP2B expression through a mechanism that requires FPP synthesis and activation of the constitutive androstane receptor (CAR). Because direct farnesol treatment also increases CYP2B expression, it seems likely that SQ1-mediated CAR activation requires FPP dephosphorylation to farnesol. The lipid phosphatase, phosphatidic acid phosphatase domain containing 2 (PPAPDC2), was recently reported to catalyze FPP dephosphorylation. We therefore determined the effect of overexpressing or knocking down PPAPDC2 on SQ1-mediated CAR activation in primary cultured rat hepatocytes. Cotransfection of rat hepatocytes with a plasmid expressing rat or human PPAPDC2 enhanced SQ1-mediated activation of a CAR-responsive reporter by 1.7- or 2.4-fold over the SQ1-mediated activation that was produced when hepatocytes were cotransfected with empty expression plasmid. Similarly, transduction of rat hepatocytes with a recombinant adenovirus expressing PPAPDC2 enhanced SQ1-mediated CYP2B1 mRNA induction by 1.4-fold over the induction that was seen in hepatocytes transduced with control adenovirus. Cotransfection with a short hairpin RNA targeting PPAPDC2 reduced SQ1-mediated CAR activation by approximately 80% relative to the activation that occurred in hepatocytes transfected with nontargeting short hairpin RNA. These results indicate that PPAPDC2 plays an important role in SQ1-mediated CAR activation, most likely by catalyzing the conversion of FPP to farnesol.

Transcriptional Regulation of Cytosolic Sulfotransferase 1C2 by Intermediates of the Cholesterol Biosynthetic Pathway in Primary Cultured Rat Hepatocytes.

Cytosolic sulfotransferase 1C2 (SULT1C2) is expressed in the kidney, stomach, and liver of rats; however, the mechanisms regulating expression of this enzyme are not known. We evaluated transcriptional regulation of SULT1C2 by mevalonate (MVA)-derived intermediates in primary cultured rat hepatocytes using several cholesterol synthesis inhibitors. Blocking production of mevalonate with the 3-hydroxy-3-methylglutaryl-CoA reductase inhibitor pravastatin (30 µM), reduced SULT1C2 mRNA content by ∼40% whereas the squalene synthase inhibitor squalestatin (SQ1, 0.1 µM), which causes accumulation of nonsterol isoprenoids, increased mRNA content by 4-fold. Treatment with MVA (10 mM) strongly induced SULT1C2 mRNA by 12-fold, and this effect was blocked by inhibiting squalene epoxidase but not by more distal cholesterol inhibitors, indicating the effects of MVA are mediated by postsqualene metabolites. Using rapid amplification of cDNA ends (RACE), we characterized the 5' end of SULT1C2 mRNA and used this information to generate constructs for promoter analysis. SQ1 and MVA increased reporter activity by ∼1.6- and 3-fold, respectively, from a construct beginning 49 base pairs (bp) upstream from the longest 5'-RACE product (-3140:-49). Sequence deletions from this construct revealed a hepatocyte nuclear factor 1 (HNF1) element (-2558), and mutation of this element reduced basal (75%) and MVA-induced (30%) reporter activity and attenuated promoter activation following overexpression of HNF1α or 1ß. However, the effects of SQ1 were localized to a more proximal promoter region (-281:-49). Collectively, our findings demonstrate that cholesterol biosynthetic intermediates influence SULT1C2 expression in rat primary hepatocytes. Further, HNF1 appears to play an important role in mediating basal and MVA-induced SULT1C2 transcription.

Upregulation of UGT2B4 Expression by 3'-Phosphoadenosine-5'-Phosphosulfate Synthase Knockdown: Implications for Coordinated Control of Bile Acid Conjugation.

During cholestasis, the bile acid-conjugating enzymes, SULT2A1 and UGT2B4, work in concert to prevent the accumulation of toxic bile acids. To understand the impact of sulfotransferase deficiency on human hepatic gene expression, we knocked down 3'-phosphoadenosine-5'-phosphosulfate synthases (PAPSS) 1 and 2, which catalyze synthesis of the obligate sulfotransferase cofactor, in HepG2 cells. PAPSS knockdown caused no change in SULT2A1 expression; however, UGT2B4 expression increased markedly (∼41-fold increase in UGT2B4 mRNA content). Knockdown of SULT2A1 in HepG2 cells also increased UGT2B4 expression. To investigate the underlying mechanism, we transfected PAPSS-deficient HepG2 cells with a luciferase reporter plasmid containing ∼2 Kb of the UGT2B4 5'-flanking region, which included a response element for the bile acid-sensing nuclear receptor, farnesoid X receptor (FXR). FXR activation or overexpression increased UGT2B4 promoter activity; however, knocking down FXR or mutating or deleting the FXR response element did not significantly decrease UGT2B4 promoter activity. Further evaluation of the UGT2B4 5'-flanking region indicated the presence of distal regulatory elements between nucleotides -10090 and -10037 that negatively and positively regulated UGT2B4 transcription. Pulse-chase analysis showed that increased UGT2B4 expression in PAPSS-deficient cells was attributable to both increased mRNA synthesis and stability. Transfection analysis demonstrated that the UGT2B4 3'-untranslated region decreased luciferase reporter expression less in PAPSS-deficient cells than in control cells. These data indicate that knocking down PAPSS increases UGT2B4 transcription and mRNA stability as a compensatory response to the loss of SULT2A1 activity, presumably to maintain bile acid-conjugating activity.

Expression of the orphan cytosolic sulfotransferase SULT1C3 in human intestine: characterization of the transcript variant and implications for function.

The cystolic sulfotransferse 1C3 (SULT1C3) gene was identified by computational analysis of the human genome and suggested to contain duplications of its last two exons (7a/b and 8a/b). Although the SULT1C3 isoform containing the more downstream exons 7b and 8b (SULT1C3d) has been expressed in Escherichia coli, crystallized, and characterized for activity, there is currently no evidence that SULT1C3 is expressed in any human tissue. Using reverse-transcription polymerase chain reaction, we detected SULT1C3 mRNA in the colorectal adenocarcinoma cell line (LS180), colon, and small intestine, but the amplified fragment contained the more upstream exons 7a and 8a. 3'-Rapid amplification of cDNA ends (RACE) confirmed that the SULT1C3 transcript expressed in LS180 cells contained exons 7a/8a, whereas 5'-RACE identified a noncoding exon 1. Full-length SULT1C3 transcript containing exons 7a/8a was amplified from LS180 and intestinal RNA, and in vitro transcription-translation of the cloned cDNA indicated that translation primarily began at the first of three in-frame ATG codons. Since SULT1C3 containing exons 7a/8a (SULT1C3a) would differ by 30 amino acids from SULT1C3d containing exons 7b/8b, we considered the functional implications of expressing one or the other isoform by generating structural models based on the reported crystal structure for SULT1C3d. Comparison of the structures indicated that five of the residues forming the substrate-binding pocket differed between the two isoforms, resulting in a change in both electron density and charge distribution along the inner wall of the substrate-binding pocket. These data indicate that SULT1C3 is expressed in human intestine but suggest that the expressed isoform is likely to differ functionally from the isoform that has been previously characterized.

Regulation of human cytosolic sulfotransferases 1C2 and 1C3 by nuclear signaling pathways in LS180 colorectal adenocarcinoma cells.

Cytosolic sulfotransferases (SULTs) catalyze the sulfate conjugation of a myriad of endogenous and xenobiotic substrates. Among the 13 human SULTs, little is known regarding regulation of the SULT1C subfamily. We evaluated the effects of a panel of transcription factor activators on levels of SULT1C mRNA (1C2 and 1C3) and protein (1C2) in LS180 colorectal adenocarcinoma cells. Treatment with 3-[3-[N-(2-chloro-3-trifluoromethylbenzyl)-(2,2-diphenylethyl)amino]propyloxy]phenylacetic acid hydrochloride [GW3965, liver X receptor (LXR) activator], 3-(2,6-dichlorophenyl)-4-(3'-carboxy-2-chlorostilben-4-yl)oxymethyl-5-isopropylisoxazole [GW4064, farnesoid X receptor (FXR)], or rifampicin [pregnane X receptor (PXR)] moderately (≤2-fold) increased both SULT1C2 and SULT1C3 mRNA levels. 1α,25-Dihydroxyvitamin D3 [1,25(OH)2D3, vitamin D receptor (VDR)] selectively upregulated SULT1C2, whereas ciprofibrate [peroxisome proliferator-activated receptor α (PPARα)], rosiglitazone (PPARγ), and 2,3,7,8-tetrachlorodibenzo-p-dioxin [aryl hydrocarbon receptor (AhR)] selectively increased SULT1C3 mRNA levels. SULT1C2 protein content was strongly increased by 1,25(OH)2D3 treatment and moderately increased by GW3965, GW4064, and rifampicin. To evaluate SULT1C2 transcriptional regulation, treatment effects were determined on reporter activity from transfected constructs containing ∼10 kb of the SULT1C2 gene. Treatment with GW3965, GW4064, or 1,25(OH)2D3 increased reporter activity ∼2-, 5-, and 5.5-fold, respectively, from a construct containing mostly intron 1 of the SULT1C2 gene. Expression of AhR, LXRα, LXRß, PPARα, PPARγ, PXR, and VDR was confirmed in LS180 cells using quantitative reverse-transcription polymerase chain reaction; however, FXR expression was negligible, suggesting that GW4064 increased SULT1C expression through an FXR-independent mechanism. Collectively, our findings are the first to characterize the regulation of human SULT1C2 and SULT1C3 expression by several transcription factor activators. Further, we determined that responsive regions for LXR and VDR are likely contained within intron 1 of the SULT1C2 gene.

Expression of the sulfotransferase 1C family: implications for xenobiotic toxicity.

The SULT1C enzymes are a relatively under-studied branch of the cytosolic sulfotransferase (SULT) multigene family. Concrete information about SULT1C tissue-specific expression, substrate preference, role in physiology and regulation is just emerging in the literature. The role of SULT1Cs in normal physiology is uncertain, but SULT1C-catalyzed sulfonation of thyroid hormones may be a mechanism to titrate the pre-receptor levels of biologically active thyroid hormone in target tissues. Both rat and human cytosolic SULT1Cs are most noted for their ability to bioactivate potent procarcinogens such as N-hydroxy-2-acetylaminofluorene. This implicates a possible role for the SULT1Cs as modulators of environmental carcinogen exposure and determinants of neoplastic transformation. In humans, the SULT1Cs are likely to function physiologically in cell proliferation and organogenesis pathways during development, as SULT1Cs appear to be preferentially expressed during fetal life. In recent years, the SULT1C nomenclature as presented in the literature has undergone major changes in response to updated genomic information. The purpose of this review is to summarize the current literature on the SULT1Cs and to clarify perspectives on SULT1C species differences, tissue-specific expression, nomenclature and role in pathophysiology. The ultimate goal is to understand the undiscovered impact of SULT1C expression on hormone homeostasis and xenobiotic toxicity during human development and as a prelude to disease development later in life.

Regulation of murine hepatic hydroxysteroid sulfotransferase expression in hyposulfatemic mice and in a cell model of 3'-phosphoadenosine-5'-phosphosulfate deficiency.

The cytosolic sulfotransferases (SULTs) catalyze the sulfate conjugation of nucleophilic substrates, and the cofactor for sulfonation, 3'-phosphoadenosine-5'-phosphosulfate (PAPS), is biosynthesized from sulfate and ATP. The phenotype of male knockout mice for the NaS1 sodium sulfate cotransporter includes hyposulfatemia and increased hepatic expression of mouse cytoplasmic sulfotransferase Sult2a and Sult3a1. Here we report that in 8-week-old female NaS1-null mice, hepatic Sult2a1 mRNA levels were ∼51-fold higher than they were in a wild-type liver but expression of no other Sult was affected. To address whether hyposulfatemia-inducible Sult2a1 expression might be due to reduced PAPS levels, we stably knocked down PAPS synthases 1 and 2 in HepG2 cells (shPAPSS1/2 cells). When a reporter plasmid containing at least 233 nucleotides (nt) of Sult2a1 5'-flanking sequence was transfected into shPAPSS1/2 cells, reporter activity was significantly increased relative to the activity that was seen for reporters containing 179 or fewer nucleotides. Mutation of an IR0 (inverted repeat of AGGTCA, with 0 intervening bases) nuclear receptor motif at nt -191 to 180 significantly attenuated the PAPSS1/2 knockdown-mediated increase. PAPSS1/2 knockdown significantly activated farnesoid X receptor (FXR), retinoid-related orphan receptor, and pregnane X receptor responsive reporters, and treatment with the FXR agonist GW4064 [3-(2,6-dichlorophenyl)-4-(3'-carboxy-2-chlorostilben-4-yl)oxymethyl-5-isopropylisoxazole] increased Sult2a1 promoter activity when the IR0 was intact. Transfection of shPAPSS1/2 cells with FXR small interfering RNA (siRNA) significantly reduced the Sult2a1 promoter activity. The impact of PAPSS1/2 knockdown on Sult2a1 promoter activity was recapitulated by knocking down endogenous SULT2A1 expression in HepG2 cells. We propose that hyposulfatemia leads to hepatic PAPS depletion, which causes loss of SULT2A1 activity and results in accumulation of nonsulfated bile acids and FXR activation.

Regulation of the cytosolic sulfotransferases by nuclear receptors.

The cytosolic sulfotransferases (SULTs) are a multigene family of enzymes that catalyze the transfer of a sulfonate group from the physiologic sulfate donor, 3'-phosphoadenosine-5'-phosphosulfate, to a nucleophilic substrate to generate a polar product that is more amenable to elimination from the body. As catalysts of both xenobiotic and endogenous metabolism, the SULTs are major points of contact between the external and physiological environments, and modulation of SULT-catalyzed metabolism can not only affect xenobiotic disposition, but it can also alter endogenous metabolic processes. Therefore, it is not surprising that SULT expression is regulated by numerous members of the nuclear receptor (NR) superfamily that function as sensors of xenobiotics as well as endogenous molecules, such as fatty acids, bile acids, and oxysterols. These NRs include the peroxisome proliferator-activated receptors, pregnane X receptor, constitutive androstane receptor, vitamin D receptor, liver X receptors, farnesoid X receptor, retinoid-related orphan receptors, and estrogen-related receptors. This review summarizes current information about NR regulation of SULT expression. Because species differences in SULT subfamily composition and tissue-, sex-, development-, and inducer-dependent regulation are prominent, these differences will be emphasized throughout the review. In addition, because of the central role of the SULTs in cellular physiology, the effect of NR-mediated SULT regulation on physiological and pathophysiological processes will be discussed. Gaps in current knowledge that require further investigation are also highlighted.

p-Anilinoaniline enhancement of dioxin-induced CYP1A1 transcription and aryl hydrocarbon receptor occupancy of CYP1A1 promoter: role of the cell cycle.

The aryl hydrocarbon receptor (AhR) is targeted by ubiquitination for degradation by the proteasome shortly after its activation by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). In silico screening identified p-anilinoaniline (pAA) as a putative inhibitor of an E2 ligase that partners with an E3 ligase implicated in AhR ubiquitination. We investigated whether pAA could modify AhR-dependent activation of its target gene CYP1A1. pAA (1-200 µM) alone did not affect AhR content, or stimulate CYP1A1 mRNA accumulation in human mammary epithelial MCF10A cultures. However, pretreatment with ≥100 µM pAA suppressed TCDD-induced CYP1A1 activation and AhR degradation via its functioning as an AhR antagonist. At a lower concentration (25 µM), pAA cotreatment increased TCDD-induced CYP1A1 mRNA accumulation, without inhibiting AhR turnover or altering CYP1A1 mRNA half-life. Whereas TCDD alone did not affect MCF10A proliferation, 25 µM pAA was cytostatic and induced a G(1) arrest that lasted ∼7 h and induced an S phase arrest that peaked 5 to 8 h later. TCDD neither affected MCF10A cell cycle progression nor did it alter pAA effects on the cell cycle. The magnitude of CYP1A1 activation depended upon the time elapsed between pAA pretreatment and TCDD addition. Maximal AhR occupancy of the CYP1A1 promoter and accumulation of CYP1A1 heterogeneous nuclear RNA and mRNA occurred when pAA-pretreated cultures were exposed to TCDD in late G(1) and early/mid S phase. TCDD-mediated induction of CYP2S1 was also cell cycle-dependent in MCF10A cultures. Similar studies with HepG2 cultures indicated that the cell cycle dependence of CYP1A1 induction is cell context-dependent.