SULT1E1 mRNA Research Articles

Steroidogenesis and androgen/estrogen signaling pathways are altered in in vitro matured testicular tissues of prepubertal mice

Children undergoing cancer treatments are at risk for impaired fertility. Cryopreserved prepubertal testicular biopsies could theoretically be later matured in vitro to produce spermatozoa for assisted reproductive technology. A complete in vitro spermatogenesis has been obtained from mouse prepubertal testicular tissue, although with low efficiency. Steroid hormones are essential for the progression of spermatogenesis, the aim of this study was to investigate steroidogenesis and steroid signaling in organotypic cultures. Histological, RT-qPCR, western blot analyses, and steroid hormone measurements were performed on in vitro cultured mouse prepubertal testicular tissues and age-matched in vivo controls. Despite a conserved density of Leydig cells after 30 days of culture (D30), transcript levels of adult Leydig cells and steroidogenic markers were decreased. Increased amounts of progesterone and estradiol and reduced androstenedione levels were observed at D30, together with decreased transcript levels of steroid metabolizing genes and steroid target genes. hCG was insufficient to facilitate Leydig cell differentiation, restore steroidogenesis, and improve sperm yield. In conclusion, this study reports the failure of adult Leydig cell development and altered steroid production and signaling in tissue cultures. The organotypic culture system will need to be further improved before it can be translated into clinics for childhood cancer survivors.

Testicular steroid sulfatase overexpression is associated with Leydig cell dysfunction in primary spermatogenic failure.

Decreased testosterone (T) to LH ratio and increased 17β-estradiol (E2) serum concentrations represent a common finding among patients with severe spermatogenic failure, suggesting a concurrent Leydig cell steroidogenic dysfunction. Aromatase overexpression has been associated with increased serum and intratesticular E2 in these patients. However, it is unknown whether the sulfatase pathway contributes to the increased availability of active estrogens in patients with primary spermatogenic failure. To assess estrogen sulfotransferase (SULT1E1) and steroid sulfatase (STS) mRNA abundance in testicular tissue of patients with Sertoli cell-only syndrome (SCOS) and normal tissues, its association with serum and intratesticular hormone levels, and to explore the mRNA and protein testicular localization of both enzymes. Testicular tissues of 23 subjects with SCOS (cases) and 22 patients with obstructive azoospermia and normal spermatogenesis (controls) were obtained after biopsy. SULT1E1 and STS transcripts accumulation was quantified by RT-qPCR. For mRNA and protein localization, we performed RT-qPCR in Leydig cell clusters and seminiferous tubules isolated by laser-capture microdissection and immunofluorescence in testicular tissues. Serum and intratesticular hormones were measured by immunoradiometric assays. SULT1E1 mRNA accumulation was similar in both groups. The amount of STS mRNA was higher in cases (p=0.007) and inversely correlated with T/LH ratio (r=-0.402; p=0.02). Also, a near significant correlation was observed with intratesticular E2 (r=0.329, p=0.057), in agreement with higher intratesticular E2 in cases (p<0.001). Strong STS immunoreaction was localized in the wall of small blood vessels but not in Leydig cells. Both SULT1E1 and STS mRNA abundance was similar in Leydig cell clusters and the tubular compartment, except for lower SUTL1E1 mRNA in the seminiferous tubules of SCOS patients (p=0.001). Our results suggest that an unbalance of the STS/SULT1E1 pathway contributes to the testicular hyperestrogenic microenvironment in patients with primary spermatogenic failure and Leydig cell dysfunction.

PXR phosphorylated at Ser350 transduces a glucose signal to repress the estrogen sulfotransferase gene in human liver cells and fasting signal in mouse livers

Hepatic estrogen sulfotransferase (SULT1E1), the enzyme that inactivates estrogen, regulates metabolic estrogen homeostasis. Here, we have demonstrated how nuclear receptor PXR regulated the SULT1E1 gene in response to glucose in human hepatoma-derived cells and in response to fasting in mouse livers. The SULT1E1 gene was activated by a nuclear receptor HNF4α-RORα complex binding on an upstream enhancer of the SULT1E1 promoter in cells cultured in high glucose medium (Hu and Negishi, 2020). The SULT1E1 gene was repressed in cells cultured in low glucose medium, in which PXR was phosphorylated at Ser350 by vaccinia virus-related kinase 1. Phosphorylated PXR interacted with this complex, retaining HNF4α on and dissociating RORα from the enhancer as a phosphorylated PXR complex. Therefore, in response to low glucose, phosphorylated PXR transduced a low glucose signal to repress the SULT1E1 gene in cells. Hepatic Sult1e1 mRNA was induced in PXR wild type (WT) male mice in response to fasting, whereas this induction was synergistically increased in phosphorylation-blocking PXR Ser347Ala (Ser350 in human) KI males over that observed in PXR WT males. As phosphorylated PXR repressed the Sult1e1 gene, it increased its binding to the Sult1e1 promoter in WT males. The absence of phosphorylated PXR resulted in the synergistic activation of the Sult1e1 gene in PXR KI males. Apparently, phosphorylated PXR functioned as a transcriptional repressor to the SULT1E1/Sult1e1 gene in human liver cells and mouse livers.

Nuclear receptor CAR-ER\u03b1 signaling regulates the estrogen sulfotransferase gene in the liver

Estrogen sulfotransferase (SULT1E1) inactivates estrogen and regulates its metabolic homeostats. Whereas SULT1E1 is expressed low in the liver of adult mice, it is induced by phenobarbital (PB) treatment or spontaneously in diabetic livers via nuclear receptors. Utilizing constitutive active/androstane receptor (CAR) KO, estrogen receptor α (ERα KO, phosphorylation-blocked ERα S216A KI mice, it is now demonstrated that, after being activated by PB, CAR binds and recruits ERα onto the Sulte1 promoter for subsequent phosphorylation at Ser216. This phosphorylation tightens CAR interacting with ERα and to activates the promoter. Hepatic SULT1E1 mRNA levels are constitutively up-regulated in type 1 diabetic Akita mice; CAR spontaneously accumulates in the nucleus and activates the Sult1e1 promoter by recruiting phosphorylated ERα in the liver as observed with PB-induced livers. Thus, this CAR-phosphorylated ERα signaling enables these two nuclear receptors to communicate, activating the Sult1e1 gene in response to either PB or diabetes in mice. ERα phosphorylation may integrate CAR into estrogen actions, providing insights into understanding drug-hormone interactions in clinical therapy.

The sulfatase pathway as estrogen supply in endometrial cancer

ObjectiveContradictory results are reported about the level of steroid sulfatase (STS), estrogen sulfotransferase (SULT1E1; together, the sulfatase pathway) and aromatase (CYP19A1) in endometrial cancer (EC). The aim of this study was to explore the levels of these enzymes in a well-characterized cohort of EC patients and postmenopausal controls. Materials and MethodsEndometrial tissues from 31 EC patients (21 grade 1 and 10 grade 2–3) and 19 postmenopausal controls were collected. Levels of mRNA (RT-qPCR) and protein (immunohistochemistry) were determined. STS enzyme activity was measured by HPLC, whereas SULT1E1 enzyme activity was determined using a novel method based on liquid chromatography-mass spectrometry (LC-MS/MS). ResultsNo significant differences in STS, SULT1E1 mRNA or protein levels and STS:SULT1E1 ratio were found. STS enzyme activity and STS:SULT1E1 activity ratio were significantly decreased in ECs compared with controls. CYP19A1 mRNA levels were lower in ECs than in controls. ConclusionA novel highly sensitive and accurate protocol to assess SULT1E1 activity is presented. STS enzyme activity and the STS:SULT1E1 activity ratio seem to be lower in ECs than in controls. STS is an important route for estrogen supply in endometrial cells.

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.

Resveratrol and its methoxy derivatives modulate the expression of estrogen metabolism enzymes in breast epithelial cells by AhR down-regulation.

Our earlier studies have shown that compared to resveratrol, its analogs with ortho-methoxy substituents exert stronger antiproliferative and proapoptotic activity. Since estrogens are considered the major risk factors of breast carcinogenesis, the aim of this study was to evaluate the effect of 3,4,2'-trimethoxy (3MS), 3,4,2',4'-tetramethoxy (4MS), and 3,4,2',4',6'-pentamethoxy (5MS) trans-stilbenes on the constitutive expression of the enzymes involved in estrogen metabolism, as well as receptors: AhR and HER2 in breast epithelial cell line MCF10A. The results showed different effect of resveratrol and its methoxy derivatives on the expression of genes encoding key enzymes of estrogen synthesis and catabolism. Resveratrol at the doses of 1 and 5µmol/L increased the level of CYP19 transcript and protein level, while 5MS reduced mRNA transcript of both CYP19 and STS genes. Resveratrol and all its derivatives reduced also SULT1E1 mRNA transcript level. The reduced expression of AhR, CYP1A1, and 1B1 was also found as a result of treatment with these compounds. The most significant changes were found in the case of AhR. The most potent inhibitor of CYP1A1 and 1B1 genes expression was 5MS, which reduced the levels of mRNA transcript and protein of both CYPs from 31 to 89% of the initial levels. These results indicate that methoxy derivatives of resveratrol might be efficient modulators of estrogen metabolism. Moreover, the number of methoxy groups introduced to stilbene structure may play a certain role in this effect.

Effects of steroid hormone on estrogen sulfotransferase and on steroid sulfatase expression in endometriosis tissue and stromal cells

Endometriosis is an estrogen-dependent disease that afflicts about 10% of women in their reproductive age, causing severe pain and infertility. The potential roles of female steroid hormones in modulating key estrogen-metabolizing enzymes, steroid sulfatase (STS) and estrogen sulfotransferase (SULT1E1), were investigated. The expression of STS and SULT1E1 mRNA in biopsy samples (n=78) of superficial and deep endometriotic lesions, eutopic endometrium of women with endometriosis and endometrium from control patients were compared according to the menstrual cycle phase. Increased STS gene expression was detected in superficial and deep-infiltrating lesions and a reduced SULT1E1 expression was also observed in the eutopic endometrium relative to the superficial lesions. Additionally, a significantly positive correlation was detected between STS and SULT1E1 mRNA expression levels in biopsy specimens collected from the endometriosis patients, and not in control individuals. The actions of female steroid hormones on SULT1E1 and STS expression were evidenced in endometriosis, revealed by increased expression levels in the luteal phase of the cycle. There was an increased STS expression in primary eutopic and ectopic endometrial stromal cells treated with estradiol and progesterone (representative of the luteal phase, n=3). Although an increased STS mRNA expression was observed in hormone-induced endometrial stromal cells in vitro, no difference could be detected between the hormone treatment groups in estradiol formation from estradiol sulfate measured by LC–MS–MS. Interestingly, a greater expression of STS was observed in stromal cells from eutopic endometrium with an agreement in estradiol formation originated from estradiol sulfate. The differential regulation of STS and SULT1E1 could provide insights for novel studies of the therapeutic use of STS inhibitors.

In vitro-assessment of putative antiprogestin activities of phytochemicals and synthetic UV absorbers in human endometrial Ishikawa cells

Critical steps of embryo implantation are controlled by progesterone. These processes can be interrupted by progesterone receptor (PR) antagonists, e.g. drugs used for abortion. Antiprogestin effects induced by natural compounds and environmental chemicals have been rarely addressed. In our in vitro study, we investigated putative antiprogestin activities of the plant compounds apigenin (API) and trans-ferulic acid (t-FA) as well as the UV absorbers octyl methoxycinnamate (OMC) and 4-methylbenzylidene camphor (4-MBC). They were compared with the selective progesterone receptor modulators (SPRMs) mifepristone (RU486) and ulipristal acetate (UPA) as well as the full PR-antagonist ZK137316. Effects of test compounds in combination with progesterone on the progesterone-sensitive target gene estrogen sulfotransferase (SULT1E1) were characterized by sigmoidal concentration–response curves obtained by RT-qPCR. The agonistic effect of progesterone on SULT1E1 mRNA levels was concentration-dependently antagonized by RU486, UPA and ZK137316 as well as, with lower potency, apigenin. t-FA, OMC and 4-MBC had no effect on SULT1E1 mRNA levels. We demonstrated that apigenin, although at higher concentrations, exerts a similar effect as the well-characterized SPRMs RU486 and UPA or the progesterone antagonist ZK137316 in this model. Our endometrium-specific Ishikawa cell assay is a useful complement to artificial transactivation assays for the identification of environmental substances with antiprogestin activities.

Estrogen-specific sulfotransferase (SULT1E1) in bovine placentomes: inverse levels of mRNA and protein in uninucleated trophoblast cells and trophoblast giant cells.

The bovine trophoblast produces significant amounts of estrogens. In maternal and fetal blood, estrogens occur predominantly in sulfonated forms, which are unable to bind to estrogen receptors (ESRs). However, estrogens may act as local factors in ESR-positive trophoblast cells or in the adjacent caruncular epithelium, which in addition to ESR highly expresses steroid sulfatase. Estrogen sulfonation is catalyzed by the cytosolic enzyme SULT1E1. Previous studies clearly indicated the trophoblast as the primary site of estrogen sulfonation. However, investigations into the cellular localization of SULT1E1 yielded conflicting results. In situ hybridization studies detected SULT1E1 mRNA only in trophoblast giant cells (TGCs), whereas in immunohistochemical experiments the SULT1E1 protein was virtually restricted to uninucleated trophoblast cells (UTCs). The aim of this work was to resolve this conflict by analyzing SULT1E1 expression in isolated UTCs and TGCs. Highly enriched pools of UTCs and TGCs were obtained from four bovine placentas (Days 118-130 of gestation) using an optimized fluorescence-activated cell sorting procedure. UTC and TGC pools were analyzed by quantitative RT-PCR and Western blot experiments to measure the amounts of SULT1E1 transcript and protein, respectively. In contrast to previously published results, both SULT1E1 transcript and SULT1E1 protein were clearly present in the UTC and TGC pools. However, some evidence indicated a higher transcript concentration in TGCs and a higher amount of protein in UTCs. Thus, our results resolve the conflicting results on the localization of SULT1E1 from earlier studies and suggest that posttranscriptional mechanisms play an important role in the control of SULT1E1 expression during TGC differentiation.

Downregulation of Sulfotransferase Expression and Activity in Diseased Human Livers

Sulfotransferase (SULT) function has been well studied in healthy human subjects by quantifying mRNA and protein expression and determining enzyme activity with probe substrates. However, it is not well known if sulfotransferase activity changes in metabolic and liver disease, such as diabetes, steatosis, or cirrhosis. Sulfotransferases have significant roles in the regulation of hormones and excretion of xenobiotics. In the present study of normal subjects with nonfatty livers and patients with steatosis, diabetic cirrhosis, and alcoholic cirrhosis, we sought to determine SULT1A1, SULT2A1, SULT1E1, and SULT1A3 activity and mRNA and protein expression in human liver tissue. In general, sulfotransferase activity decreased significantly with severity of liver disease from steatosis to cirrhosis. Specifically, SULT1A1 and SULT1A3 activities were lower in disease states relative to nonfatty tissues. Alcoholic cirrhotic tissues further contained lower SULT1A1 and 1A3 activities than those affected by either of the two other disease states. SULT2A1, on the other hand, was only reduced in alcoholic cirrhotic tissues. SULT1E1 was reduced both in diabetic cirrhosis and in alcoholic cirrhosis tissues, relative to nonfatty liver tissues. In conclusion, the reduced levels of sulfotransferase expression and activity in diseased versus nondiseased liver tissue may alter the metabolism and disposition of xenobiotics and affect homeostasis of endobiotic sulfotransferase substrates.

Effects of PPAR-α agonist and IGF-1 on estrogen sulfotransferase in human vascular endothelial and smooth muscle cells

Estrogen has a protective role in vascular functions and estrogen levels are modulated by estrogen sulfotransferase(SULT1E1). In this study, we investigated the effects of the peroxisome proliferator-activated receptor-α(PPAR-α) agonist WY14643 and insulin-like growth factor-1(IGF-1) on the expression and activity of SULT1E1 in vascular cells. Human umbilical vein endothelial cells (HUVECs) and human umbilical artery smooth muscle cells (HUASMCs) were primarily cultured from fresh umbilical cord. SULT1E1 was highly expressed in HUVECs and HUASMCs according to immunofluorescence microscopy detection. WY14643 decreased, while IGF-1 increased, SULT1E1 mRNA and SULT1E1 protein levels, as demonstrated by RT-qPCR and western blot analysis, respectively, in the HUVECs and HUASMCs. SULT1E1 activity was indicated by counting the transformed 3H-estradiol sulfate from 3H-labeled 17β-estradiol added into the cell culture medium. The activity of SULT1E1 reduced following treatment with WY14643, whereas SULT1E1 activity was enhanced in the presence of IGF-1. The human SULT1E1 promoter-reporter plasmid was constructed. The activity of the SULT1E1 promoter increased 30-fold compared with the pGL3-basic vector. The PPAR-α agonist WY14643 downregulated, while IGF-1 upregulated, the luciferase activity of the SULT1E1 promoter. In conclusion, the PPAR-α agonist WY14643 and IGF-1 may regulate SULT1E1 expression at the transcriptional level and modulate the levels of active estrogens in endothelial cells and smooth muscle cells, thereby affecting the physiology and pathophysiology of vascular walls.

Potential hazards to embryo implantation: A human endometrial in vitro model to identify unwanted antigestagenic actions of chemicals

Embryo implantation is a crucial step in human reproduction and depends on the timely development of a receptive endometrium. The human endometrium is unique among adult tissues due to its dynamic alterations during each menstrual cycle. It hosts the implantation process which is governed by progesterone, whereas 17β-estradiol regulates the preceding proliferation of the endometrium. The receptors for both steroids are targets for drugs and endocrine disrupting chemicals. Chemicals with unwanted antigestagenic actions are potentially hazardous to embryo implantation since many pharmaceutical antiprogestins adversely affect endometrial receptivity. This risk can be addressed by human tissue-specific in vitro assays. As working basis we compiled data on chemicals interacting with the PR. In our experimental work, we developed a flexible in vitro model based on human endometrial Ishikawa cells. Effects of antiprogestin compounds on pre-selected target genes were characterized by sigmoidal concentration–response curves obtained by RT-qPCR. The estrogen sulfotransferase (SULT1E1) was identified as the most responsive target gene by microarray analysis. The agonistic effect of progesterone on SULT1E1 mRNA was concentration-dependently antagonized by RU486 (mifepristone) and ZK137316 and, with lower potency, by 4-nonylphenol, bisphenol A and apigenin. The negative control methyl acetoacetate showed no effect. The effects of progesterone and RU486 were confirmed on the protein level by Western blotting. We demonstrated proof of principle that our Ishikawa model is suitable to study quantitatively effects of antiprogestin-like chemicals on endometrial target genes in comparison to pharmaceutical reference compounds. This test is useful for hazard identification and may contribute to reduce animal studies.

Garlic Extract Diallyl Sulfide (DAS) Activates Nuclear Receptor CAR to Induce the Sult1e1 Gene in Mouse Liver

Constituent chemicals in garlic extract are known to induce phase I and phase II enzymes in rodent livers. Here we have utilized Car +/+ and Car −/− mice to demonstrate that the nuclear xenobiotic receptor CAR regulated the induction of the estrogen sulfotransferase Sult1e1 gene by diallyl sulfide (DAS) treatment in mouse liver. DAS treatment caused CAR accumulation in the nucleus, resulting in a remarkable increase of SULT1E1 mRNA (3,200 fold) and protein in the livers of Car +/+ females but not of Car −/− female mice. DAS also induced other CAR-regulated genes such as Cyp2b10, Cyp3a11 and Gadd45β. Compared with the rapid increase of these mRNA levels, which began as early as 6 hourrs after DAS treatment, the levels of SULT1E1 mRNA began increasing after 24 hours. This slow response to DAS suggested that CAR required an additional factor to activate the Sult1e1 gene or that this activation was indirect. Despite the remarkable induction of SULT1E1, there was no decrease in the serum levels of endogenous E2 or increase of estrone sulfate while the clearance of exogenously administrated E2 was accelerated in DAS treated mice.

Tobacco smoke condensate down‐regulates estrogen sulfotransferase (SULT1E1) expression in MCF10A human breast epithelial cells through an aryl hydrocarbon receptor‐mediated mechanism

Tobacco smoke has been implicated as a risk factor for breast cancer. We evaluated the effect of tobacco smoke condensate (TSC) on expression of the estrogen-inactivating enzyme SULT1E1 in the MCF10A human breast epithelial cell line. Because TSC contains components that are known aryl hydrocarbon receptor (AhR) agonists, effects of TSC treatment were compared to those of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), and effects on SULT1E1 expression were compared to those on CYP1A1. Treatment for 24–72 h with 0.05–10 μg/ml TSC produced concentration-dependent increases in CYP1A1 mRNA content, decreases in SULT1E1 mRNA content, and increases in expression from a transfected AhR-responsive reporter plasmid. Treatment with 10 μg/ml TSC and 30 nM TCDD produced comparable increases in CYP1A1 mRNA levels (~300-fold) and decreases in SULT1E1 mRNA levels (~90%). Treatment with the AhR antagonist 3′-methoxy-4′-nitroflavone (PD168641, at 1 μM) completely inhibited TCDD-inducible CYP1A1 expression and partially reversed TCDD-mediated SULT1E1 suppression. PD168641 also inhibited TSC-mediated CYP1A1 induction and SULT1E1 suppression as indicated by rightward shifts of the TSC concentration-response curves. These findings support a role for the AhR in TSC-mediated regulation of CYP1A1 and SULT1E1 expression in human breast epithelial cells. Supported by NIH Grant (ARRA) R21ES016373 (to M.R.-M.).

Garlic Extract Diallyl Sulfide (DAS) Activates Nuclear Receptor CAR to Induce the Sult1e1 Gene in Mouse Liver

Constituent chemicals in garlic extract are known to induce phase I and phase II enzymes in rodent livers. Here we have utilized Car+/+ and Car−/− mice to demonstrate that the nuclear xenobiotic receptor CAR regulated the induction of the estrogen sulfotransferase Sult1e1 gene by diallyl sulfide (DAS) treatment in mouse liver. DAS treatment caused CAR accumulation in the nucleus, resulting in a remarkable increase of SULT1E1 mRNA (3,000 fold) and protein in the livers of Car+/+ females but not of Car−/− female mice. DAS also induced other CAR‐regulated genes such as Cyp2b10, Cyp3a11 and Gadd45β. Compared with the rapid increase of these mRNA levels which began as early as 6 hrs after DAS treatment, the SULT1E1 mRNA began increasing its levels after 24 hrs. This slow response to DAS suggested that CAR required an additional factor to activate the Sult1e1 gene or that this activation was indirect. Despite the remarkable induction of SULT1E1, there was no decrease in the serum levels of endogenous E2 or increase of estron sulfate while the clearance of exogenously administrated E2 was accelerated in DAS treated mice.

Regulation of estrogen sulfotransferase (SULT1E1) expression by proliferation state in MCF10A human breast epithelial cells: Potential role of microRNAs

The MCF10A cell line represents a model of the normal human breast epithelial cell. Estrogen sulfotransferase (SULT1E1), a major estrogen‐inactivating enzyme, is up‐regulated in confluent relative to preconfluent MCF10A cells. The mechanism responsible for SULT1E1 up‐regulation in confluent MCF10A cells was investigated. Transient or stable tranfection of reporter constructs containing up to 7 kb of the SULT1E1 5′‐flanking region failed to recapitulate confluency‐induced SULT1E1 up‐regulation. The half‐life of SULT1E1 mRNA in preconfluent MCF10A cells was estimated to be ~4h based on analysis of SULT1E1 mRNA levels in MCF10A cells that were replated after reaching confluency. Microarray analysis revealed that 85 microRNAs were significantly up‐regulated and 24 microRNAs were down‐regulated in confluent relative to preconfluent MCF10A cells. Computational analysis of the SULT1E1 3′‐untranslated region identified 4 putative candidate binding sites for confluency‐regulated microRNAs. The confluency‐related decrease in two of these microRNAs (miR‐221 and miR‐100*) corresponded with the confluency‐induced increase in SULT1E1 mRNA observed in MCF10A cells. These results support a role for the post‐transcriptional up‐regulation of SULT1E1 mRNA expression in confluent relative to preconfluent MCF10A cells. Supported by NIH grant (ARRA) ES016373 (MRM) and EHS Center grant ES06636.

Expression and functional activities of selected sulfotransferase isoforms in BeWo cells and primary cytotrophoblast cells

Several cytosolic sulfotransferase enzyme isoforms are functional in placenta but there is limited information available on the utility of cultured trophoblast cells for studying sulfation. The trophoblast cell layer constitutes the rate-determining barrier for trans-placental transfer. The objective of this work was to examine the mRNA expression and enzyme activities of four sulfotransferase isoforms reported to be functional in human placenta (SULT1A1, SULT1A3, SULT1E1, and SULT2A1) in primary cytotrophoblast cells and the trophoblast-like BeWo cell line. Reverse transcription polymerase chain reaction (RT-PCR) was performed to determine mRNA expression. Enzyme activities were assessed using the following substrates: 4-nitrophenol for SULT1A1, dopamine for SULT1A3, 17β-estradiol for SULT1E1, and dehydroepiandrosterone for SULT2A1. For 4-nitrophenol and dopamine sulfation, apparent K m values, response to inhibitors (2,6-dichloro-4-nitrophenol and sodium chloride), and thermal stability profiles indicated that 4-nitrophenol and dopamine sulfation in BeWo cells were being mediated by SULT1A1 and SULT1A3, respectively. SULT1A1 and SULT1A3 were also functional in the cytotrophoblast cells. Both at the protein and at the mRNA levels, SULT1A1 was more abundant in BeWo cells in comparison to the primary cytotrophoblast cells. SULT1E1 and SULT2A1 mRNA were not detected in the cytotrophoblasts. SULT1E1 mRNA was weakly expressed in BeWo but there was negligible functional activity. Although SULT2A1 mRNA was abundantly expressed in BeWo, Western blot and enzyme activities revealed that the protein is not expressed in BeWo cells. The results suggest that the BeWo cells and the cytotrophoblast cells can be used to examine the roles of SULT1A1 and SULT1A3 in placental metabolism.

Expression of Estrogenicity Genes in a Lineage Cell Culture Model of Human Breast Cancer Progression

The mRNA levels of estrogen receptors (ERα and ERβ), cytosolic sulfotransferases (SULT1E1, SULT1A1, SULT2A1 and SULT2B1), steroid sulfatase (STS), aromatase (CYP19), 17β‐hydroxysteroid dehydrogenases (17βHSD1 and 2), CYP1B1 and catechol‐O‐methyltransferase (COMT) were examined in MCF7 and in MCF10A‐derived cell lines representing a lineage cell culture model for breast cancer progression. Relative to MCF7, MCF10A‐derived cells expressed low levels of ERα but comparable levels of ERβ. In benign parental MCF10A breast epithelial cells, SULT1E1 mRNA was induced in confluent relative to subconfluent cell cultures. Proestrogenic 17βHSD1 mRNA was expressed in all of the MCF10A lineage cell lines while 17βHSD2 mRNA was most abundant in MCF10A cells. CYP1B1, SULT1A1 and SULT2B1 were expressed in MCF7 cells, while STS and COMT were widely detected across cell lines. Treatment with the potent histone deacetylase inhibitor trichostatin A (TSA) induced SULT1E1, SULT2B1 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 ER‐positive MCF7 cells, key determinants of breast estrogen metabolism are differentially regulated during neoplastic progression. Supported by ES05823 and ES06636.

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 (ERalpha and ERbeta) and estrogen metabolism enzymes including cytosolic sulfotransferases (SULT1E1, SULT1A1, SULT2A1, and SULT2B1), steroid sulfatase (STS), aromatase (CYP19), 17beta-hydroxysteroid dehydrogenases (17betaHSD1 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 ERalpha-positive luminal MCF7 breast cancer cells. Low levels of ERalpha and ERbeta 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 17betaHSD1 mRNA was most abundant in neoplastic MCF10CA1a and MCF10DCIS.com cells, while 17betaHSD2 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, 17betaHSD1, and 17betaHSD2 mRNA in MCF10A lineage cell lines. In MCF7 cells, TSA treatment suppressed ERalpha, CYP1B1, STS, COMT, SULT1A1, and SULT2B1 but induced ERbeta, 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.