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.

Expression and purification of recombinant extracellular sulfatase HSulf-2 allows deciphering of enzyme sub-domain coordinated role for the binding and 6-O-desulfation of heparan sulfate.

Through their ability to edit 6-O-sulfation pattern of Heparan sulfate (HS) polysaccharides, Sulf extracellular endosulfatases have emerged as critical regulators of many biological processes, including tumor progression. However, study of Sulfs remains extremely intricate and progress in characterizing their functional and structural features has been hampered by limited access to recombinant enzyme. In this study, we unlock this critical bottleneck, by reporting an efficient expression and purification system of recombinant HSulf-2 in mammalian HEK293 cells. This novel source of enzyme enabled us to investigate the way the enzyme domain organization dictates its functional properties. By generating mutants, we confirmed previous studies that HSulf-2 catalytic (CAT) domain was sufficient to elicit arylsulfatase activity and that its hydrophilic (HD) domain was necessary for the enzyme 6-O-endosulfatase activity. However, we demonstrated for the first time that high-affinity binding of HS substrates occurred through the coordinated action of both domains, and we identified and characterized 2 novel HS binding sites within the CAT domain. Altogether, our findings contribute to better understand the molecular mechanism governing HSulf-2 substrate recognition and processing. Furthermore, access to purified recombinant protein opens new perspectives for the resolution of HSulf structure and molecular features, as well as for the development of Sulf-specific inhibitors.

TRF2 positively regulates SULF2 expression increasing VEGF-A release and activity in tumor microenvironment.

The telomeric protein TRF2 is overexpressed in several human malignancies and contributes to tumorigenesis even though the molecular mechanism is not completely understood. By using a high-throughput approach based on the multiplexed Luminex X-MAP technology, we demonstrated that TRF2 dramatically affects VEGF-A level in the secretome of cancer cells, promoting endothelial cell-differentiation and angiogenesis. The pro-angiogenic effect of TRF2 is independent from its role in telomere capping. Instead, TRF2 binding to a distal regulatory element promotes the expression of SULF2, an endoglucosamine-6-sulfatase that impairs the VEGF-A association to the plasma membrane by inducing post-synthetic modification of heparan sulfate proteoglycans (HSPGs). Finally, we addressed the clinical relevance of our findings showing that TRF2/SULF2 expression is a worse prognostic biomarker in colorectal cancer (CRC) patients.

Cytotoxic and partial hepatoprotective activity of sodium ascorbate against hepatocellular carcinoma through inhibition of sulfatase-2 in vivo and in vitro.

Hepatocellular carcinoma (HCC) is characterized by elevation in the activity of sulfatase-2, an extracellular enzyme that catalyzes removal of 6-O-sulfate groups from heparan sulfate. Therefore, we conducted this study to investigate the cytotoxic activity of the strong water-soluble antioxidant, sodium ascorbate, against HCC both in vivo and in vitro. Sodium ascorbate enhanced animal survival in vivo and reduced HepG2 cells survival. The protein levels of heparan sulfate proteoglycans (HSPGs), insulin like growth factor (IGF)-2, sulfatase-2 and glypican-3 were assessed. Inflammation was evaluated by measuring the gene and protein expression of NFκB, TNF-α, IL-1ß, IL-4, IL-6 and IL-10. We found that sodium ascorbate blocked HCC-induced activation of sulfatase-2 leading to restoration of HSPGs receptors associated with reduction in IGF-2 and glypican-3. Sodium ascorbate exerts anti-inflammatory activity by reducing the expression of NFκB, CRP, TNF-α, IL-1ß and IL-6 associated with enhanced expression of the anti-inflammatory cytokines, IL-4 and IL-10. In conclusion, cytotoxic effects of sodium ascorbate against HCC can be partially explained by inhibition of sulfatase-2, restoration of HSPGs receptors and deactivation of the inflammatory pathway.

Whole-cell-dependent biosynthesis of sulfo-conjugate using human sulfotransferase expressing budding yeast.

Cytosolic sulfotransferases (SULTs), one of the predominant phase II drug metabolizing enzymes (DME), play important roles in metabolism of xeno- and endobiotics to generate their sulfo-conjugates. These sulfo-conjugates often have biological activities but are difficult to study, because even though only small amounts are required to evaluate their efficacy and safety, chemical or biological synthesis of sulfo-conjugatesis is often challenging. Previously, we constructed a DME expression system for cytochrome P450 and UGT, using yeast cells, and successfully produced xenobiotic metabolites in a whole-cell-dependent manner. In this study, we developed a yeast expression system for human SULTs, including SULT1A1, 1A3, 1B1, 1C4, 1E1, and 2A1, in Saccharomyces cerevisiae and examined its sulfo-conjugate productivity. The recombinant yeast cells expressing each of the SULTs successfully produced several hundred milligram per liter of xeno- or endobioticsulfo-conjugates within 6 h. This whole-cell-dependent biosynthesis enabled us to produce sulfo-conjugates without the use of 3'-phosphoadenosine-5'-phosphosulfate, an expensive cofactor. Additionally, the production of regiospecific sulfo-conjugates of several polyphenols was possible with this method, making this novel yeast expression system a powerful tool for uncovering the metabolic pathways and biological actions of sulfo-conjugates.

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 5 µmol/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.

Tumour-necrosis factor-α induces heparan sulfate 6-O-endosulfatase 1 (Sulf-1) expression in fibroblasts.

Heparan sulfate (HS) 6-O-endosulfatases (Sulfs) have emerged recently as critical regulators of many physiological and pathological processes. By removing 6-O-sulfates from specific HS sequences, they modulate the activities of a variety of growth factors and morphogens, including fibroblast growth factor (FGF)-1. However, little is known about the functions of Sulfs in inflammation. Tumour-necrosis factor (TNF)-α plays an important role in regulating the behaviour of fibroblasts. In this study, we examined the effect of this inflammatory cytokine on the expression of Sulfs in human MRC-5 fibroblasts. Compositional analysis of HS from TNF-α-treated cells showed a strong reduction in the amount of the trisulfated UA2S-GlcNS6S disaccharide, which suggested a selective reaction of 6-O-desulfation. Real-time PCR analysis revealed that TNF-α increased Sulf-1 expression in a dose- and time-dependent manner, via a mechanism involving NF-ĸB, ERK1/2 and p38 MAPK. In addition, we confirmed that cell stimulation with TNF-α was accompanied by the secretion of an active form of Sulf-1. To study the function of Sulf- 1, we examined the responses induced by FGF-1. We showed that ERK1/2 activation and cell proliferation were markedly reduced in TNF-α-treated MRC-5 cells compared with untreated cells. Silencing the expression of Sulf-1 by RNA interference restored the responses induced by FGF-1, which indicated that TNF-α-mediated induction of the sulfatase indeed resulted in alterations of HS biological properties. Taken together, our results indicate that Sulf-1 is responsive to TNF-α stimulation and may function as an autocrine regulator of fibroblast expansion in the course of an inflammatory response.

Regulation of the Expression of Heparan Sulfate 3-O-Sulfotransferase 3B (HS3ST3B) by Inflammatory Stimuli in Human Monocytes.

Heparan sulfate (HS) is recognized as an important player in a wide range of dynamic steps of inflammatory reactions. Thereby, structural HS remodeling is likely to play an important role in the regulation of inflammatory and immune responses; however, little is known about underlying mechanism. In this study, we analyzed the regulation of expression of HS 3-O-sulfotransferases (HS3STs) in response to inflammatory stimuli. We found that among the seven HS3ST isoenzymes, only the expression of HS3ST3B was markedly up-regulated in human primary monocytes and the related cell line THP1 after exposure to TLR agonists. TNF-α was also efficient, to a lesser extent, to increase HS3ST3B expression, while IL-6, IL-4, and IFN-γ were poor inducers. We then analyzed the molecular mechanisms that regulate the high expression of HS3ST3B in response to LPS. Based on the expression of HS3ST3B transcripts and on the response of a reporter gene containing the HS3ST3B1 promoter, we provide evidence that LPS induces a rapid and strong transcription of HS3ST3B1 gene, which was mainly dependent on the activation of NF-κB and JNK signaling pathways. Additionally, active p38 MAPK and de novo synthesized proteins are involved in post-transcriptional mechanisms to maintain a high level of HS3ST3B mRNA to a steady state. Altogether, our findings indicate that HS3ST3B1 gene behaves as a primary response gene, suggesting that it may play an important role in making 3-O-sulfated HS with specific functions in the regulation of inflammatory and immune responses. J. Cell. Biochem. 117: 1529-1542, 2016. © 2015 Wiley Periodicals, Inc.

Sulfatase 2 promotes breast cancer progression through regulating some tumor-related factors.

In previous studies Sulf2 has been evidenced to play an important role in tumor progression through editing sulfate moieties on heparan sulfate proteoglycans (HSPGs) and modulating heparin binding growth factors. However, the role of Sulf2 in breast cancer progression is still poorly understood. In the present study, we hypothesized that Sulf2 promoted breast cancer progression. Two different breast cancer cell lines, MCF-7 and MDA-MB-231, were chosen for this study because of high and low Sulf2 expression levels. We also altered their Sulf2 expression by establishing Sulf2 knockdown and overexpressing breast cancer cell lines MCF-7 shSulf2 and MDA-MB-231 Sulf2. To evaluate the functions of Sulf2, cell proliferation, apoptosis, cell cycle, invasion, mobility and adhesion of these cell lines were measured in vitro, and xenograft formation, invasion and metastasis ability were examined in vivo. Furthermore, expression of related genes were screened and were certified in these cell lines. We found that Sulf2 increased breast cancer proliferation, invasion, mobility and adhesion both in vitro and in vivo. Sulf2 also decreased cisplatin inducing breast cancer apoptosis without affecting the cell cycle. Sulf2 upregulated c-fos induced growth factor (FIGF) and nuclear receptor subfamily 4 group A member 3 (NR4A3) expression and downregulated the cluster of differentiation 82 (CD82) and platelet-derived growth factor C (PDGFC) expression in breast cancer. Our data confirmed that Sulf2 promoted breast cancer progression and regulated the expression of tumor-related genes in breast cancer.

Prognostic impact of chondroitin-4-sulfotransferase CHST11 in ovarian cancer.

Ovarian cancer (OvCa) accounts for the highest tumor-related mortality among gynecological malignancies, but the underlying mechanisms are poorly understood. Glycosaminoglycans are abundantly present in ovarian tumors, and there is rising evidence that chondroitin sulfate (CS) as well as diverse carbohydrate sulfotransferases (CHSTs), the enzymes involved in the sulfation process of these structures, plays an important role in metastatic spread of tumor cells. mRNA expression levels of CHST3/7/11/12/13/15 were compared between malignant (86 OvCas) and non-malignant tumors (6 borderline tumors and 3 cystadenomas). CHST11 and CHST15 were further chosen for Western blot analysis in a cohort of 216 OvCas. Protein expression levels were correlated with clinicopathologic prognostic parameters and survival data. A significantly higher mRNA expression of CHST11, CHST12, and CHST15 was measured in ovarian cancer samples in comparison to non-malignant ones, and the same trend was observed for CHST13. For CHST3 and CHST7, no significant differences were found between the two groups. At protein level, high CHST11 expression was independently associated with unfavorable progression-free survival (PFS; p = 0.027). A similar trend was observed for CHST15, showing a nearly significant correlation between high expression levels and shorter recurrence-free survival in patients without macroscopic residual tumor after surgery (p = 0.053). We conclude that CHSTs involved in the synthesis of CS-A and CS-E might influence ovarian cancer progression, and we suggest CHST11 as independent unfavorable prognostic factor in this entity.

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.

Sulf1 and Sulf2 expression in the nervous system and its role in limiting neurite outgrowth in vitro.

Sulf1 and Sulf2 are endosulfatases that cleave 6-O-sulphate groups from Heparan Sulphate Proteoglycans (HSPGs). Sulfation levels of HSPGs are critical for their role in modulating the activity of various growth factor receptors. Sulf1 and Sulf2 mRNAs were found to be widely expressed in the rodent nervous system and their full-length proteins were found in many types of neuronal perikarya and axons in the cerebral cortex, cerebellum, spinal cord and dorsal root ganglia (DRG) of adult rats. Sulf1/2 were also strongly expressed by cultured DRG neurons. To determine if blocking Sulf1 or Sulf2 activity affected neurite outgrowth in vitro, cultured DRG neurons were treated with neutralising antibodies to Sulf1 or Sulf2. Blocking Sulf1 and Sulf2 activity did not affect neurite outgrowth from cultured DRG neurons grown on a laminin/polylysine substrate but ameliorated the inhibitory effects of chondroitin sulphate proteoglycans (CSPGs) on neurite outgrowth. Blocking epidermal growth factor receptor (ErbB1) activity also improved neurite outgrowth in the presence of CSPGs, but the effects of ErbB1 antagonists and blocking SULFs were not additive. It is proposed that Sulf1, Sulf2 and ErbB1 are involved in the signalling pathway from CSPGs that leads to inhibition of neurite outgrowth and may regulate structural plasticity and regeneration in the nervous system.

Role for chondroitin sulfate glycosaminoglycan in NEDD9-mediated breast cancer cell growth.

There are lines of evidence demonstrating that NEDD9 (Cas-L, HEF-1) plays a key role in the development, progression, and metastasis of breast cancer cells. We previously reported that NEDD9 plays a critical role for promoting migration and growth of MDA-MB-231. In order to further characterize the mechanisms of NEDD9-mediated cancer migration and growth, stable cells overexpressing NEDD9 were generated using HCC38 as a parental cell line which expresses low level of endogenous NEDD9. Microarray studies demonstrated that core proteins of CD44 and Serglycin were markedly upregulated in HCC38(NEDD9) cells compared to HCC38(Vector) cells, while those of Syndecan-1, Syndecan-2, and Versican were downregulated in HCC38(NEDD9). Importantly, enzymes generating chondroitin sulfate glycosaminoglycans (CS) such as CHST11, CHST15, and CSGALNACT1 were upregulated in HCC38(NEDD9) compared to HCC38(Vector). Immunofluorescence studies using specific antibody, GD3G7, confirmed the enhanced expression of CS-E subunit in HCC38(NEDD9). Immunoprecipitation and western blotting analysis demonstrated that CS-E was attached to CD44 core protein. We demonstrated that removing CS by chondroitinase ABC significantly inhibited anchorage-independent colony formation of HCC38(NEDD9) in methylcellulose. Importantly, the fact that GD3G7 significantly inhibited colony formation of HCC38(NEDD9) cells suggests that CS-E subunit plays a key role in this process. Furthermore, treatment of HCC38(NEDD9) cells with chondroitinase ABC or GD3G7 significantly inhibited mammosphere formation. Exogenous addition of CS-E enhanced colony formation and mammosphere formation of HCC38 parental and HCC38(Vector) cells. These results suggest that NEDD9 regulates the synthesis and expression of tumor associated glycocalyx structures including CS-E, which plays a key role in promoting and regulating breast cancer progression and metastasis and possibly stem cell phenotypes.

Regulation of chondroitin-4-sulfotransferase (CHST11) expression by opposing effects of arylsulfatase B on BMP4 and Wnt9A.

In this report, the gene regulatory mechanism by which decline in arylsulfatase B (ARSB; N-acetylgalactosamine-4-sulfatase) reduces CHST11 (chondroitin-4-sulfotransferase; C4ST) mRNA expression in human colonic epithelial cells and in colonic epithelium of ARSB-deficient mice is presented. ARSB controls the degradation of chondroitin 4-sulfate (C4S) by removing the 4-sulfate group at the non-reducing end of the C4S chain, but has not previously been shown to affect C4S biosynthesis. The decline in CHST11 expression following ARSB reduction is attributable to effects of ARSB on bone morphogenetic protein (BMP)4, since BMP4 expression and secretion declined when ARSB was silenced. Inhibition of BMP4 by neutralizing antibody also reduced CHST11 expression. When C4S was more sulfated due to decline in ARSB, more BMP4 was sequestered by C4S in the cell membrane, and CHST11 expression declined. Exogenous recombinant BMP4, acting through a phospho-Smad3 binding site in the CHST11 promoter, increased the mRNA expression of CHST11. In contrast to the decline in BMP4 that followed decline in ARSB, Wnt9A mRNA expression was previously shown to increase when ARSB was silenced and C4S was more highly sulfated. Galectin-3 bound less to the more highly sulfated C4S, leading to increased nuclear translocation and enhanced galectin-3 interaction with Sp1 in the Wnt9A promoter. Silencing Wnt9A increased the expression of CHST11 in the colonic epithelial cells, and chromatin immunoprecipitation assay demonstrated enhancing effects of Wnt9A siRNA and exogenous BMP4 on the CHST11 promoter through the pSmad3 binding site. These findings suggest that cellular processes mediated by differential effects of Wnt9A and BMP4 can result from opposing effects on CHST11 expression.

MYCN-dependent expression of sulfatase-2 regulates neuroblastoma cell survival.

Heparan sulfate proteoglycans (HSPG) play a critical role in the interaction of tumor cells and their microenvironment. HSPG activity is dictated by sulfation patterns controlled by sulfotransferases, which add sulfate groups, and sulfatases (Sulf), which remove 6-O-sulfates. Here, we report altered expression of these enzymes in human neuroblastoma cells with higher levels of Sulf-2 expression, a specific feature of MYCN-amplified cells (MYCN-A cells) that represent a particularly aggressive subclass. Sulf-2 overexpression in neuroblastoma cells lacking MYCN amplification (MYCN-NA cells) increased their in vitro survival. Mechanistic investigations revealed evidence of a link between Sulf-2 expression and MYCN pathogenicity in vitro and in vivo. Analysis of Sulf-2 protein expression in 65 human neuroblastoma tumors demonstrated a higher level of Sulf-2 expression in MYCN-A tumors than in MYCN-NA tumors. In two different patient cohorts, we confirmed the association in expression patterns of Sulf-2 and MYCN and determined that Sulf-2 overexpression predicted poor outcomes in a nonindependent manner with MYCN. Our findings define Sulf-2 as a novel positive regulator of neuroblastoma pathogenicity that contributes to MYCN oncogenicity. Cancer Res; 74(21); 5999-6009. ©2014 AACR.

Effect on metabolic enzymes and thyroid receptors induced by BDE-47 by activation the pregnane X receptor in HepG2, a human hepatoma cell line.

2,2',4,4'-Tetra-bromodiphenyl ether (BDE-47), an important congener among polybrominated diphenyl ether (PBDE) compounds, has been predominantly in environmental samples and human tissue. Thyroid disruption is the most sensitive endpoint effect among a number of health effects of exposure to BDE-47 in animals and humans. However, the detailed underlying mechanisms in humans are not well understood. In the present study, human pregnane X receptor (hPXR)-overexpressing HepG2 cell model and a dual-luciferase reporter assay system were constructed to investigate the role of hPXR in BDE-47-induced alterations of expression of metabolic enzymes and TR in vitro. The results showed that hPXR was significantly activated by BDE-47, and expression levels of both mRNA and protein of the thyroid receptor (TR) isoforms TRα1 and TRß1 were decreased in hPXR-overexpressing HepG2 cells after BDE-47 treatment. However, the increased expression of hepatic microsomal phase I enzyme CYP3A4 and phase II enzymes, UGT1A3 and SULT2A1 were also found. Taken together, the results indicated that BDE-47 was a strong hPXR activator, activation of hPXR played an important role in BDE-47-induced down-regulation of TR, and up-regulations of CYP3A4, UGT1A3, and SULT2A1 participated in the process, which may provide more toxicological evidence on mechanisms of disruption of thyroid hormone induced by BDE-47.

Fluorescent peptide sensors for tyrosylprotein sulfotransferase activity.

Tyrosine sulfurylation is a post-translational modification important for protein-protein interactions in the extracellular space that are instrumental in cell adhesion, cell signaling, immune responses, and pathogen recognition of host cells. Tyrosine sulfurylation is catalyzed by the tyrosylprotein sulfotransferases (TPSTs), and in humans there are two isoforms: hTPST1 and hTPST2. The study of hTPST function and the development of small molecule probes to examine the role of hTPSTs in cell biology have been delayed by the absence of a continuous direct assay for hTPST activity. We have developed a fluorescent peptide-based assay to directly monitor tyrosine sulfurylation in real time. TPST-mediated tyrosine sulfurylation of the peptides disrupts fluorophore quenching and results in increased fluorescence emission. The assay can be used to study TPST enzymatic activity, and we show that recombinant hTPSTs are active in the absence of divalent metal ions and that optimal activity is at pH 6.0. We further show that the assay can also be used to identify inhibitors of tyrosine sulfurylation. A clear understanding of hTPST function in normal cell biology and in disease states will require the identification of small molecule inhibitors or probes to modulate enzymatic activity, and our results will facilitate that process.

OASIS regulates chondroitin 6-O-sulfotransferase 1 gene transcription in the injured adult mouse cerebral cortex.

Old astrocyte specifically induced substance (OASIS), a basic leucine zipper transcription factor of the cAMP response element binding/Activating transcription factor family, is induced in reactive astrocytes in vivo and has important roles in quality control of protein synthesis at the endoplasmic reticulum. Reactive astrocytes produce a non-permissive environment for regenerating axons by up-regulating chondroitin sulfate proteoglycans (CSPGs). In this study, we focus on the potential role of OASIS in CSPG production in the adult mouse cerebral cortex. CS-C immunoreactivity, which represents chondroitin sulfate moieties, was significantly attenuated in the stab-injured cortices of OASIS knockout mice compared to those of wild-type mice. We next examined expression of the CSPG-synthesizing enzymes and core proteins of CSPGs in the stab-injured cortices of OASIS knockout and wild-type mice. The levels of chondroitin 6-O-sulfotransferase 1 (C6ST1, one of the major enzymes involved in sulfation of CSPGs) mRNA and protein increased after cortical stab injury of wild-type, but not of OASIS knockout, mice. A C-terminal deletion mutant OASIS over-expressed in rat C6 glioma cells increased C6ST1 transcription by interacting with the first intron region. Neurite outgrowth of cultured hippocampal neurons was inhibited on culture dishes coated with membrane fractions of epidermal growth factor-treated astrocytes derived from wild type but not from OASIS knockout mice. These results suggest that OASIS regulates the transcription of C6ST1 and thereby promotes CSPG sulfation in astrocytes. Through these mechanisms, OASIS may modulate axonal regeneration in the injured cerebral cortex. OASIS, an ER stress-responsive CREB/ATF family member, is up-regulated in the reactive astrocytes of the injured brain. We found that the up-regulated OASIS is involved in the transcriptional regulation of C6ST1 gene, which promotes chondroitin sulfate proteoglycan (CSPG) sulfation. We conclude that OASIS functions as an anti-regenerative transcription factor by establishing a non-permissive microenvironment to regenerating axons.

Induction of epoxide hydrolase, glucuronosyl transferase, and sulfotransferase by phenethyl isothiocyanate in male Wistar albino rats.

Phenethyl isothiocyanate (PEITC) is an isothiocyanate found in watercress as the glucosinolate (gluconasturtiin). The isothiocyanate is converted from the glucosinolate by intestinal microflora or when contacted with myrosinase during the chopping and mastication of the vegetable. PEITC manifested protection against chemically-induced cancers in various tissues. A potential mechanism of chemoprevention is by modulating the metabolism of carcinogens so as to promote deactivation. The principal objective of this study was to investigate in rats the effect of PEITC on carcinogen-metabolising enzyme systems such as sulfotransferase (SULT), N-acetyltransferase (NAT), glucuronosyl transferase (UDP), and epoxide hydrolase (EH) following exposure to low doses that simulate human dietary intake. Rats were fed for 2 weeks diets supplemented with PEITC at 0.06 µmol/g (low dose, i.e., dietary intake), 0.6 µmol/g (medium dose), and 6.0 µmol/g (high dose), and the enzymes were monitored in rat liver. At the Low dose, no induction of the SULT, NAT, and EH was noted, whereas UDP level was elevated. At the Medium dose, only SULT level was increased, whereas at the High dose marked increase in EH level was observed. It is concluded that PEITC modulates carcinogen-metabolising enzyme systems at doses reflecting human intake thus elucidating the mechanism of its chemoprevention.

17ß-Estradiol induces sulfotransferase 2A1 expression through estrogen receptor α.

Sulfotransferase (SULT) 2A1 catalyzes sulfonation of drugs and endogenous compounds and plays an important role in xenobiotic metabolism as well as in the maintenance of steroid and lipid homeostasis. A recent study showed that 17ß-estradiol (E2) increases the mRNA levels of SULT2A1 in human hepatocytes. Here we report the underlying molecular mechanisms. E2 enhanced SULT2A1 expression in human hepatocytes and HepG2-ER cells (HepG2 stably expressing ERα). SULT2A1 induction by E2 was abrogated by antiestrogen ICI 182,780, indicating a key role of ERα in the induction. Results from deletion and mutation assays of SULT2A1 promoter revealed three cis-elements located within -257/+140 region of SULT2A1 that are potentially responsible for the induction. Chromatin immunoprecipitation assay verified the recruitment of ERα to the promoter region. Electrophoretic mobility shift assays revealed that AP-1 proteins bind to one of the cis-elements. Interestingly, SULT2A1 promoter assays using ERα mutants revealed that the DNA-binding domain of ERα is indispensable for SULT2A1 induction by E2, suggesting that direct ERα binding to the SULT2A1 promoter is also necessary for the induction. Taken together, our results indicate that E2 enhances SULT2A1 expression by both the classical and nonclassical mechanisms of ERα action.