Age- and sex-dependent expression of multiple murine hepatic hydroxysteroid sulfotransferase (SULT2A) genes.

Hydroxysteroid sulfotransferase (SULT2A) enzymes play important roles in hepatic steroid and xenobiotic metabolism. Unlike humans, which express one SULT2A, inspection of mouse genome information indicated the presence of seven SULT2A genes within a cluster on chromosome 7. The age- and sex-dependent expressions of the seven murine SULT2A family members were characterized in the livers of C57BL/6 mice using real-time RT-PCR. The transcripts for three of the SULT2A forms (NCBI reference/model sequences XM_001471624, NM_009286 and NM_001111296) were abundant in pre-pubertal male and female mouse liver but were essentially silenced in the livers of adult male mice. The mRNAs of three other SULT2A forms (NM_001101534, XM_894052 and NM_001081325) were also expressed in pre-pubertal male and female mouse liver, but at markedly reduced levels relative to those of the abundant forms. The mRNA levels of these lower-abundance forms were further suppressed in adult animals. A seventh SULT2A mRNA (XM_983034) was expressed in adult male and female mouse liver, but was not detected in pre-pubertal mouse liver of either sex. Full-length amplifications with primers targeting untranslated regions confirmed that all SULT2A forms were expressed. However, while the XM_001471624, NM_001111296, NM_001101534, XM_894052 and NM_001081325 transcripts were detected at their predicted sizes, the NM_009286 and XM_983034 transcripts each lacked two predicted exons. These results demonstrate that seven murine SULT2As display different profiles of age- and sex-dependent hepatic expression.

Positive and negative regulation of human hepatic hydroxysteroid sulfotransferase (SULT2A1) gene transcription by rifampicin: roles of hepatocyte nuclear factor 4alpha and pregnane X receptor.

The effects of rifampicin treatment on SULT2A1 mRNA expression were evaluated in 23 preparations of primary cultured human hepatocytes. In contrast to the consistently occurring induction of CYP3A4, a prototypical pregnane X receptor (PXR) target gene, rifampicin treatment increased SULT2A1 mRNA levels in 12 of the hepatocyte preparations, but it produced little change or even suppression in the others. Transient transfection of HepG2 cells with a series of reporter constructs implicated two SULT2A1 5'-flanking regions as containing rifampicin-responsive information. Each of these regions contained a hepatocyte nuclear factor 4 (HNF4) binding site (at nucleotide [nt] -6160 and -54), as demonstrated by in vitro binding and site-directed mutagenesis. HNF4alpha bound to the HNF4-54 region of the endogenous SULT2A1 gene, as indicated by chromatin immunoprecipitation. Cotransfection of HepG2 cells with pregnane X receptor (PXR) dose-dependently suppressed reporter expression from SULT2A1 constructs containing the HNF4 sites, and rifampicin treatment augmented the suppression. Rifampicin treatment concentration-dependently suppressed SULT2A1 reporter expression at the same concentrations that progressively induced expression from a PXR-responsive CYP3A4 reporter, whereas higher rifampicin concentrations reversed the SULT2A1 suppression. The suppressive effect of rifampicin was diminished, whereas the activating effect was augmented, in HepG2 cells with RNA interference-mediated PXR knockdown. These results suggest that HNF4alpha plays a central role in the control of SULT2A1 transcription and that rifampicin-liganded PXR suppresses SULT2A1 expression by interfering with HNF4alpha activity. By contrast, the rifampicin-inducible SULT2A1 expression that occurs in many human hepatocyte preparations seems to be mediated through a PXR-independent mechanism.

Staphylococcus aureus induces the expression of tumor necrosis factor-alpha in primary human keratinocytes.

BACKGROUND: Staphylococcus aureus induces inflammatory cytokines and causes skin inflammatory diseases, but infection parameters leading to cytokine induction are poorly understood in keratinocytes, the primary skin cells to interface with S. aureus. METHODS: Human primary keratinocytes were infected with S. aureus under various conditions to identify properties of infection that cause the induction of tumor necrosis factor-alpha (TNF-alpha), a proinflammatory cytokine that initiates host inflammatory responses. RESULTS: Staphylococcus aureus induced TNF-alpha mRNA and protein in a dose-dependent manner. Cytochalasin D, an inhibitor of actin polymerization and S. aureus invasion, failed to prevent the induction of TNF-alpha, indicating that invasion was not a requirement. Furthermore, ultraviolet-, heat-, and gentamicin-treated bacteria did not induce TNF-alpha, suggesting that de novo bacterial protein synthesis of viable bacteria was required. Finally, S. aureus infection of primary human keratinocytes also led to an induction of the TNF-alpha receptor, TNFR1 (p55). CONCLUSION: Early (preinvasion) S. aureus-keratinocyte surface interactions that require protein synthesis induce TNF-alpha. Bacterial surface components embedded within the cell wall do not suffice as TNF-alpha mediators, but require active protein synthesis and/or the accompaniment of secreted bacterial products. Furthermore, S. aureus infection leads to the specific induction of the TNF-alpha receptor TNFR1, but not TNFR2.

Cytochrome P450 expression and metabolic activation of cooked food mutagen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in MCF10A breast epithelial cells.

The cytochrome P450 expression profile was determined in the MCF10A human breast epithelial cell line, as was the ability of this cell line to catalyze the bioactivation of the cooked food mutagen, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). Using non-quantitative reverse transcription-polymerase chain reaction (RT-PCR), transcripts for CYP1B1, CYP2J2, CYP2R1, CYP2U1, CYP2W1, CYP4B1, CYP4F, CYP4V2, CYP4X1 and CYP4Z1 were detected in both sub-confluent and confluent MCF10A cells. By contrast, CYP1A2 mRNA was detected only in confluent MCF10A cells, while CYP1A1, CYP2S1 and CYP2F1 were detected predominantly or exclusively in sub-confluent cultures. 2,3,7,8-Tetrachlorodibenzo-p-dioxin treatment of confluent MCF10A cells markedly induced microsomal ethoxyresorufin O-deethylase activity and CYP1A1, CYP1A2 and CYP1B1 mRNA levels, as determined by real-time RT-PCR, while treatment with 10(-4)M PhIP had little effect on these P450 transcript levels. Treatment of confluent MCF10A cells with PhIP (10(-4)M) for 24, 48 or 72 h produced time-dependent increases in the amounts of DNA adducts, as measured by (32)P-post-labeling. These results indicate that multiple P450s, including those known to catalyze PhIP N-oxidation, are expressed in MCF10A cells, and that this non-neoplastic human breast epithelial cell line contains sufficient enzymatic machinery to support PhIP bioactivation and generate DNA damage.

Developmental expression of aryl, estrogen, and hydroxysteroid sulfotransferases in pre- and postnatal human liver.

Aryl- (SULT1A1), estrogen- (SULT1E1), and hydroxysteroid- (SULT2A1) sulfotransferases (SULTs) are active determinants of xenobiotic detoxication and hormone metabolism in the adult human liver. To investigate the role of these conjugating enzymes in the developing human liver, the ontogeny of immunoreactive SULT1A1, SULT1E1, and SULT2A1 expression was characterized in a series of 235 pre- and postnatal human liver cytosols ranging in age from early gestation to a postnatal age of 18 years. Interindividual variability in expression levels was apparent for all three SULTs in pre- and postnatal liver samples. Expression of the three SULTs displayed distinctly different developmental profiles. Semiquantitative Western blot analyses indicated that SULT1A1 and SULT2A1 immunoreactive protein levels were readily detectable in the majority of developmental human liver cytosols throughout the prenatal period. Whereas SULT1A1 expression did not differ significantly among the various developmental stages, SULT2A1 expression increased during the third trimester of gestation and continued to increase during postnatal life. By contrast, SULT1E1, a cardinal estrogen-inactivating enzyme, achieved the highest levels of expression during the earliest periods of gestation in prenatal male livers, indicating a requisite role for estrogen inactivation in the developing male. The present analysis suggests that divergent regulatory mechanisms are responsible for the differential patterns of hepatic SULT1A1, SULT1E1, and SULT2A1 immunoreactive protein levels that occur during pre- and postnatal human development, and implicates a major role for sulfotransferase expression in the developing fetus.

Regulation of glucocorticoid-inducible hydroxysteroid sulfotransferase (SULT2A-40/41) gene transcription in primary cultured rat hepatocytes: role of CCAAT/enhancer-binding protein liver-enriched transcription factors.

The mechanism responsible for glucocorticoid receptor (GR)-mediated induction of rat hepatic hydroxysteroid sulfotransferase (SULT2A-40/41) gene transcription was investigated. We previously reported that the region of the SULT2A-40/41 5'-flanking region delimited by -158 to -77 nucleotides relative to the transcription start site was sufficient to support GR-inducible expression. This region of the SULT2A-40/41 gene does not contain a consensus glucocorticoid receptor-responsive element, but does contain two consensus sites for liver-enriched CCAAT/enhancer-binding protein (C/EBP) transcription factors. In the present study, incubation of primary cultured rat hepatocytes with a GR-activating concentration (10(-7) M) of a potent glucocorticoid, dexamethasone or triamcinolone acetonide (TA), rapidly produced increases in C/EBPalpha and C/EBPbeta nuclear protein contents, as measured by Western blot or in vitro DNA-binding activity analysis, that preceded increases in SULT2A-40/41 mRNA and protein levels. Transient cotransfection of SULT2A-40/41 reporter plasmids with a dominant negative C/EBP expression plasmid completely blocked TA-inducible SULT2A-40/41 reporter gene expression. Linker scanning and site-directed mutagenesis of the proximal SULT2A-40/41 5'-flanking region, complemented by in vitro DNA-binding analyses, indicated that the more distal C/EBP site was important for controlling SULT2A-40/41 promoter activity. These data support a role for GR-inducible C/EBPalpha and C/EBPbeta expression in the transactivation of hepatic SULT2A-40/41 expression.

Transactivation of glucocorticoid-inducible rat aryl sulfotransferase (SULT1A1) gene transcription.

The purpose of the current study was to establish the role of the glucocorticoid receptor (GR) and androgen receptor (AR) transcription factors in the transactivation of rat aryl sulfotransferase (SULT1A1) gene transcription and to identify the functional hormone-responsive element(s) in the SULT1A1 gene. A cis-acting inverted repeat with three intervening bases (IR3) was identified in the 5'-flanking of the SULT1A1 gene that mediates the transactivation of SULT1A1 gene transcription by both the GR and AR. CV-1 cells were cotransfected with SULT1A1-luciferase reporter plasmids and either wild-type or mutant GR or AR expression constructs. In cotransfectants expressing the wild-type GR, treatment with triamcinolone acetonide produced an approximately 4- to 6-fold induction of luciferase activity in IR3-containing SULT1A1 reporter plasmids. IR3-containing SULT1A1 reporter constructs were also activated by treatment with the synthetic androgen R1881 in cells cotransfected with wild-type but not mutant AR. In primary cultured rat hepatocytes, androgen-inducible expression of IR3-containing SULT1A1 reporter plasmids required cotransfection with AR expression plasmid. Targeted disruption of the SULT1A1 IR3 by mutation of a conserved GT sequence in the 3' half-site of the element ablated GR and AR responsiveness. These results indicate that a proximal IR3 element in the 5'-flanking region of the rat SULT1A1 gene is sufficient for the transactivation of SULT1A1 gene transcription by the GR and AR, and that relative to the GR, functional AR activity is reduced in primary cultured rat hepatocytes.

Effects of dexamethasone on aryl (SULT1A1)- and hydroxysteroid (SULT2A1)-sulfotransferase gene expression in primary cultured human hepatocytes.

To determine whether the dexamethasone (DEX)-inducible hepatic sulfotransferase gene expression that has been described in the rat is conserved in humans, the effects of DEX treatment on hydroxysteroid sulfotransferase (SULT2A1) and aryl sulfotransferase (SULT1A1) gene expression were investigated in primary cultured human hepatocytes. Hepatocytes were prepared from nontransplantable human livers by collagenase perfusion of the left hepatic lobe, and cultured in Williams' medium E that was supplemented with 0.25 U/ml insulin. As reported in the rat, DEX treatment produced concentration-dependent increases in SULT2A1 mRNA and protein expression, with maximum increases observed at concentrations of DEX that would be expected to activate the pregnane X receptor (PXR) transcription factor. In contrast to the rat, in which DEX-inducible SULT1A1 expression has been demonstrated, SULT1A1 expression in primary cultured human hepatocytes was not measurably increased by DEX. In transient transfections conducted in primary cultured rat hepatocytes, the PXR ligands DEX and pregnenolone-16 alpha-carbonitrile significantly induced transcription of human and rat SULT2A reporter gene constructs. Cotransfection of either the human or rat SULT2A reporter gene with a PXR dominant negative construct significantly reduced DEX-inducible transcription. These results underscore that while certain features of rat hepatic sulfotransferase gene regulation are conserved in humans, important differences exist across species. The findings also implicate a role for the PXR transcription factor in DEX-inducible rat and human SULT2A gene expression.