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 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.

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.

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.

Inflammation-associated serum and colon markers as indicators of dietary attenuation of colon carcinogenesis in ob/ob mice.

Although inflammatory cytokines and obesity-associated serum proteins have been reported as biomarkers of colorectal adenoma risk in humans, little is known of biomarkers of response to interventions that attenuate tumorigenesis. Dietary navy beans and their fractions attenuate colon carcinogenesis in carcinogen-induced genetically obese mice. We hypothesized that this attenuation would be associated with changes in inflammatory cytokines and obesity-related serum proteins that may serve as measures of efficacy. ob/ob mice (n = 160) were injected with the carcinogen azoxymethane (AOM) to induce colon cancer and randomly placed on one of four diets (control, whole navy bean, bean residue fraction, or bean extract fraction) for 26 to 28 wk. Serum was analyzed for 14 inflammation- or obesity-related proteins, and colon RNA was analyzed for expression of 84 inflammation-associated genes. Six of 14 serum proteins were increased [i.e., interleukin (IL)-4, IL-5, IL-6, IL-10, IFN gamma, granulocyte macrophage colony-stimulating factor] in hyperplastic/dysplastic stages of colon carcinogenesis. Bean-fed mice had significantly higher monocyte chemoattractant protein-1 and lower IL-6 levels in serum. In colon mucosa, 55 of 84 inflammation-associated genes differed between AOM-induced and noninduced mice. Of the 55 AOM-induced genes, 5 were counteracted by bean diets, including IL-6 whose increase in expression levels was attenuated by bean diets in AOM-induced mice. In summary, IL-6 emerged as a serum protein that was increased in hyperplastic/dysplastic stages of colon carcinogenesis, but attenuated with bean-based diet in serum and colon mucosa. Changes in a subset of inflammation-associated serum proteins and colon gene expression may serve as response indicators of dietary attenuation of colon carcinogenesis.

Dietary cooked navy beans and their fractions attenuate colon carcinogenesis in azoxymethane-induced ob/ob mice.

Based on the protective effects of cooked dry bean consumption in a human intervention study, we evaluated which fraction of cooked dry beans is responsible for its cancer-preventive effects. Cooked navy beans (whole beans), the insoluble fraction (bean residue) or soluble fraction of the 60% (vol:vol) ethanol extract of cooked navy beans (bean extract), or a modified AIN-93G diet (16.6% fat including 12.9% lard) as control diet were fed to 160 male obese ob/ob mice after 2 azoxymethane injections. In comparison to control-fed mice, dysplasia, adenomas, or adenocarcinomas were detected in fewer mice on either bean fraction diet (percent reduction from control: whole beans 54%, P=0.10; bean residue 81%, P=0.003; bean extract 91%, P=0.007), and any type of colon lesions, including focal hyperplasia, were found in fewer mice on each of the 3 bean diets percent reduction from control: whole bean 56%, P=0.04; bean residue 67%, P=0.01; bean extract 87%, P=0.0003. These results suggest that both the soluble and the insoluble fraction of the extract contribute to the cancer-protective effect of cooked navy beans.