Melissa Runge-Morris

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.

24-hydroxycholesterol sulfation by human cytosolic sulfotransferases: formation of monosulfates and disulfates, molecular modeling, sulfatase sensitivity, and inhibition of liver x receptor activation.

24-Hydroxycholesterol (24-OHChol) is a major cholesterol metabolite and the form in which cholesterol is secreted from the brain. 24-OHChol is transported by apolipoprotein E to the liver and converted into bile acids or excreted. In both brain and liver, 24-OHChol is a liver X receptor (LXR) agonist and has an important role in cholesterol homeostasis. 24-OHChol sulfation was examined to understand its role in 24-OHChol metabolism and its effect on LXR activation. 24-OHChol was conjugated by three isoforms of human cytosolic sulfotransferase (SULT). SULT2A1 and SULT1E1 sulfated both the 3- and 24-hydroxyls to form the 24-OHChol-3, 24-disulfate. SULT2B1b formed only 24-OHChol-3-sulfate. The 3-sulfate as a monosulfate or as the disulfate was hydrolyzed by human placental steroid sulfatase, whereas the 24-sulfate was resistant. At physiological 24-OHChol concentrations, SULT2A1 formed the 3-monosulfate and the 3, 24-disulfate as a result of a high affinity for sulfation of the 3-OH in 24-OHChol-24-sulfate. Molecular docking simulations indicate that 24-OHChol-24-sulfate binds in an active configuration in the SULT2A1 substrate binding site with high affinity only when the SULT2A1 homodimer structure was used. 24-OHChol is an LXR activator. In contrast, the 24-OHChol monosulfates were not LXR agonists in a fluorescence resonance energy transfer coactivator recruitment assay. However, both the 24-OHChol-3-sulfate and 24-sulfate were antagonists of LXR activation by N-(2,2,2-trifluoroethyl)-N-[4-[2,2,2-trif-luoro-1-hydroxy-1-(trifluoromethyl)ethyl]phenyl]-benzenesulfonamide (T0901317) with an IC50 of 0.15 and 0.31 μM, respectively. Inhibition of LXR activation by the 24-OHChol monosulfates at low nanomolar concentrations indicates that sulfation has a role in LXR regulation by oxysterols.

Positive and Negative Regulation of Human Hepatic Hydroxysteroid Sulfotransferase (SULT2A1) Gene Transcription by Rifampicin: Roles of Hepatocyte Nuclear Factor 4α 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. HNF4α 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 HNF4α plays a central role in the control of SULT2A1 transcription and that rifampicin-liganded PXR suppresses SULT2A1 expression by interfering with HNF4α activity. By contrast, the rifampicin-inducible SULT2A1 expression that occurs in many human hepatocyte preparations seems to be mediated through a PXR-independent mechanism.

Sulfation of 25-hydroxycholesterol by SULT2B1b decreases cellular lipids via the LXR/SREBP-1c signaling pathway in human aortic endothelial cells

OBJECTIVE 25-Hydroxycholesterol (25HC) and its sulfated metabolite, 25-hydroxycholesterol-3-sulfate (25HC3S), regulate certain aspects of lipid metabolism in opposite ways. Hence, the enzyme for the biosynthesis of 25HC3S, oxysterol sulfotransferase (SULT2B1b), may play a crucial role in regulating lipid metabolism. We evaluate the effect of 25HC sulfation on lipid metabolism by overexpressing the gene encoding SULT2B1b in human aortic endothelial cells (HAECs) in culture. METHODS AND RESULTS The human SULT2B1b gene was successfully overexpressed in HAECs following infection using a recombinant adenovirus. HPLC analysis demonstrated that more than 50% of (3)H-25HC was sulfated in 24h following overexpression of the SULT2B1b gene. In the presence of 25HC, SULT2B1b overexpression significantly decreased mRNA and protein levels of LXR, ABCA1, SREBP-1c, ACC-1, and FAS, which are key regulators of lipid biosynthesis and transport; and subsequently reduced cellular lipid levels. Overexpression of the gene encoding SULT2B1b gave similar results as adding exogenous 25HC3S. However, in the absence of 25HC or in the presence of T0901317, synthetic liver oxysterol receptor (LXR) agonist, SULT2B1b overexpression had no effect on the regulation of key genes involved in lipid metabolism. CONCLUSIONS Our data indicate that sulfation of 25HC by SULT2B1b plays an important role in the maintenance of intracellular lipid homeostasis via the LXR/SREBP-1c signaling pathway in HAECs.

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.

Age and gender-related gene expression of hydroxysteroid sulfotransferase-a in rat liver

Hepatic hydroxysteroid sulfotransferase-a (HST-a) gene expression was examined in young male (age 22-26 days) and female rats (age 22-30 days), and in older male (age 42-45 days) and female (age 49-55 days) rats. Northern and slot blot analyses of poly(A)+RNA revealed that HST-a was differentially expressed with respect to both age and gender with female rats expressing higher levels of HST-a in both age groups. Hepatic HST-a mRNA levels were approximately 4 to 6-fold higher in females compared to males in both age groups examined. HST-a expression increased with age in both male and female rats. HST-a expression was approximately 8 to 10-fold higher in 42-45 day old males relative to 22-26 day old males. HST-a mRNA levels were approximately 3 to 7-fold higher in 49-55 day old females relative to females in the 22-30 day age group. These data suggest that HST-a gene expression is transcriptionally controlled and that HST-a regulation is subject to hormonal and developmental modulation.

Regulation of expression of the rodent cytosolic sulfotransferases.

Understanding the molecular regulation of the sulfotransferases is important because these enzymes are essential to a number of critical biological processes. Sulfotransferase expression clearly plays a role in xenobiotic detoxication, carcinogen activation, prodrug processing, cellular signaling pathways, and the regulation of intratissue active androgen and estrogen levels. Although cytosolic sulfotransferases are present in the gut, adrenal, kidney, lung, skin, brain, and other extrahepatic tissues, the basis for the molecular regulation of this complicated gene family has been best characterized in the rat liver, where sulfotransferase levels are relatively abundant. Advances in genomic cloning and in the molecular characterization of individual sulfotransferase cDNAs have inspired new insights into the mechanisms involved in sulfotransferase gene regulation. In particular, the hypothalamic‐pi‐tuitary‐gonadal‐adrenocortical axis appears to play a significant role in the regulation of individual sulfotransferase genes. The molecular signals that fluctuate with developmental age, gender, and the occurrence of systemic endocrinopathies also influence sulfotransferase gene expression. For example, diabetes, which disrupts glucose and ketone homeo‐ stasis, insulin sensitivity, gonadal and neuroendocrine hormone balance, protein kinase C isoform expression, and P450 metabolism, also disturbs hepatic sulfotransferase gene expression. What role does sulfotransferase expression play in target organ toxicity? Do xenobiotic‐mediated changes in sulfotransferase expression compromise detoxication? Does deregulated sulfotransferase expression during development lead to birth defects by perturbing the delicate balance of active hormone levels in fetal tissues? Do conditions of glucocorticoid excess, such as stress or high‐dose glucocorticoid therapy induce sulfotransferase expression and place toxicant and carcinogen bioactivation systems in overdrive? This review will summarize our current understanding of the molecular and cellular regulation of the major rodent cytosolic sulfotransferases. Only by thoroughly dissecting the regulation of this important multigene family in rodent liver, where sulfotransferase expression is most abundant, can we begin to focus on more pressing questions concerning the role of the sulfotransferases in the genesis of endocrinopathies and cancer in humans.—Runge‐Morris, M. A. Regulation of expression of the rodent cytosolic sulfotransferases. FASEB J. 11, 109‐117 (1997)

Use of dominant negative nuclear receptors to study xenobiotic-inducible gene expression in primary cultured hepatocytes

INTRODUCTION To determine the feasibility of using dominant negative nuclear receptors to dissect the regulation of inducible gene expression in primary cultured hepatocytes, a series of dominant negative nuclear receptor expression plasmids were designed with truncated AF-2 subdomains. METHODS Plasmids expressing dominant negative or wild-type constitutive androstane receptor (CAR), pregnane X receptor (PXR), farnesoid X receptor (FXR), liver X receptor (LXR), or peroxisome proliferator-activated receptor alpha (PPARalpha) were transiently cotransfected into primary cultured rat hepatocytes, together with an appropriate reporter plasmid. RESULTS Treatment with prototypic inducers, 10(-4) M phenobarbital (CAR activator), 10(-5) M pregnenolone 16alpha-carbonitrile (PXR activator), 3x10(-5) M chenodeoxycholate (FXR activator), or 10(-4) M ciprofibrate (PPARalpha activator), significantly activated expression from the corresponding reporter plasmid. Treatment with 22(R)-hydroxycholesterol (LXR activator) only weakly activated the LXR-responsive reporter, while pregnenolone 16alpha-carbonitrile treatment significantly activated this reporter. Cotransfection with wild-type LXRalpha strongly enhanced 22(R)-hydroxycholesterol-inducible expression from the LXR-responsive reporter. Cotransfection of hepatocyte cultures with each of the dominant negative nuclear receptor plasmids significantly inhibited inducible expression of the corresponding reporter while, with one exception (LXRalpha), cotransfection with the wild-type receptor moderately enhanced or had little effect on reporter expression. When each dominant negative nuclear receptor was cross-examined against all inducer-reporter pairs, effects on multiple inducer-reporter pairs were frequently observed. However, in general, only cotransfection with the appropriate dominant negative inhibited inducible reporter expression to a greater extent than did cotransfection with the corresponding wild-type receptor. DISCUSSION We suggest that the application of dominant negative nuclear receptors has utility in transient transfection studies aimed at discerning the regulatory role of individual nuclear receptor transcription factors in inducible hepatic gene expression, provided that appropriate controls are employed.

Expression of estrogenicity genes in a lineage cell culture model of human breast cancer progression

TaqMan Gene Expression assays were used to profile the mRNA expression of estrogen receptor (ERα and ERβ) and estrogen metabolism enzymes including cytosolic sulfotransferases (SULT1E1, SULT1A1, SULT2A1, and SULT2B1), steroid sulfatase (STS), aromatase (CYP19), 17β-hydroxysteroid dehydrogenases (17βHSD1 and 2), CYP1B1, and catechol-O-methyltransferase (COMT) in an MCF10A-derived lineage cell culture model for basal-like human breast cancer progression and in ERα-positive luminal MCF7 breast cancer cells. Low levels of ERα and ERβ mRNA were present in MCF10A-derived cell lines. SULT1E1 mRNA was more abundant in confluent relative to subconfluent MCF10A cells, a non-tumorigenic proliferative breast disease cell line. SULT1E1 was also expressed in preneoplastic MCF10AT1 and MCF10AT1K.cl2 cells, but was markedly repressed in neoplastic MCF10A-derived cell lines as well as in MCF7 cells. Steroid-metabolizing enzymes SULT1A1 and SULT2B1 were only expressed in MCF7 cells. STS and COMT were widely detected across cell lines. Pro-estrogenic 17βHSD1 mRNA was most abundant in neoplastic MCF10CA1a and MCF10DCIS.com cells, while 17βHSD2 mRNA was more prominent in parental MCF10A cells. CYP1B1 mRNA was most abundant in MCF7 cells. Treatment with the histone deacetylase inhibitor trichostatin A (TSA) induced SULT1E1 and CYP19 mRNA but suppressed CYP1B1, STS, COMT, 17βHSD1, and 17βHSD2 mRNA in MCF10A lineage cell lines. In MCF7 cells, TSA treatment suppressed ERα, CYP1B1, STS, COMT, SULT1A1, and SULT2B1 but induced ERβ, CYP19 and SULT2A1 mRNA expression. The results indicate that relative to the MCF7 breast cancer cell line, key determinants of breast estrogen metabolism are differentially regulated in the MCF10A-derived lineage model for breast cancer progression.

Regulation of Sulfotransferase and UDP-Glucuronosyltransferase Gene Expression by the PPARs

During phase II metabolism, a substrate is rendered more hydrophilic through the covalent attachment of an endogenous molecule. The cytosolic sulfotransferase (SULT) and UDP-glucuronosyltransferase (UGT) families of enzymes account for the majority of phase II metabolism in humans and animals. In general, phase II metabolism is considered to be a detoxication process, as sulfate and glucuronide conjugates are more amenable to excretion and elimination than are the parent substrates. However, certain products of phase II metabolism (e.g., unstable sulfate conjugates) are genotoxic. Members of the nuclear receptor superfamily are particularly important regulators of SULT and UGT gene transcription. In metabolically active tissues, increasing evidence supports a major role for lipid-sensing transcription factors, such as peroxisome proliferator-activated receptors (PPARs), in the regulation of rodent and human SULT and UGT gene expression. This review summarizes current information regarding the regulation of these two major classes of phase II metabolizing enzyme by PPARs.