Targeting glycan sulfation in a CD11c+ myeloid population inhibits early KRAS-mutant lung neoplasia.

Early lung carcinoma development may be modulated by innate host cellular mechanisms that promote tumor growth and invasion. We recently identified how a loss-of-function mutation in the glycan sulfating enzyme N-deacetylase/N-sulfotransferase-1 (Ndst1; involved in heparan sulfate biosynthesis) targeted to antigen presenting cells (APCs) may augment acquired anti-tumor T cell immune mechanisms. Crossing this mutation (Ndst1f/f CD11cCre+) onto a model of inducible spontaneous Kras mutant lung cancer [CCSP-rtTA; (tetO7) CMV-Kras-G12D] allowed us to examine how the APC mutation affects the formation and growth of early lung carcinoma. We examined early bronchocentric adenoma formation in the model, and the frequency of such events was significantly reduced on the mutant background. This was associated with significant reductions in tumor associated FOXP3+ cellular infiltration and CD163+ M2-type macrophage infiltration. The findings evolved prior to effector CD8+ T cell infiltration into tumors. The impact of this unique glycan under-sulfating mutation on inhibiting early Kras G12D mutant bronchocentric adenoma formation along with a cellular phenotype of inhibited tumor infiltration by cells involved in suppressive T-regulatory cell signaling (FOXP3+ cells) or tumor-permissive M2 macrophage functions (CD163+ cells) provides insight on how glycan targeting may modulate innate cellular mechanisms during early lung tumor development. The findings may also impact the future design of host-centered immunologic anti-tumor therapeutic strategies.

Chromosome 2 Fragment Substitutions in Dahl Salt-Sensitive Rats and RNA Sequencing Identified Enpep and Hs2st1 as Vascular Inflammatory Modulators.

Chromosome 2 introgression from normotensive Brown Norway (BN) rats into hypertensive Dahl salt-sensitive (SS) background (SS-chromosome 2BN/Mcwi; consomic S2B) reduced blood pressure and vascular inflammation under a normal-salt diet (NSD). We hypothesized that BN chromosome 2 contains anti-inflammatory genes that could reduce blood pressure and vascular inflammation in rats fed NSD or high-salt diet (HSD). Four- to 6-week old male SS and congenic rats containing the BN chromosome 2 distal portion (SS.BN-[rs13453786-rs66377062]/Aek; S2Ba) and middle segment (SS.BN-[rs106982173-rs65057186]/Aek; S2Bb) were fed NSD or HSD (4% NaCl) up to age 12 to 13 weeks. Systolic blood pressure determined by telemetry was higher in SS rats fed HSD versus NSD. Systolic blood pressure was lower in both congenic rats than in SS under NSD, but similar under HSD versus SS. Reactive oxygen species generation using dihydroethidium staining, expression of vascular cell adhesion molecule-1 and monocyte chemoattractant protein-1, and immune cell infiltration by immunofluorescence demonstrated that S2Ba rats present less inflammation under NSD and more under HSD versus SS rats. RNA sequencing and reverse transcription-quantitative PCR identified 2 differentially expressed genes encoded within BN chromosome 2 distal portion that could act as regulators of vascular inflammation. These were downregulated glutamyl aminopeptidase (Enpep) that was anti-inflammatory under NSD and upregulated heparan sulfate 2-O-sulfotransferase 1 (Hs2st1) that was proinflammatory under HSD. In conclusion, 2 differentially expressed genes encoded within introgressed BN chromosome 2 distal fragment were identified: Enpep associated with reduced vascular inflammation under NSD, and Hs2st1, associated with increased vascular inflammation under HSD.

Dependence between estrogen sulfotransferase (SULT1E1) and nuclear transcription factor Nrf-2 regulations via oxidative stress in breast cancer.

Human estrogen sulfotransferase (SULT1E1) and nuclear factor erythroid 2-related factor 2 (Nrf-2) expression influences each other in advanced human breast carcinogenesis. The difference in the metabolism of estradiol (E2) in pre- and post-menopausal women remains to be connected with post-menopausal breast cancer. A synergism between ROS production and E2 generation has been demonstrated. No definite mechanism for simultaneous functions of Nrf2, oxidative stress E2 regulating enzymes (SULT1E1) has been yet clarified. Our present review demonstrates that ROS dependent regulation of Nrf-2 is one of the most important determinants of E2 regulation by altering SULT1E1 expression. This study also focuses the idea that estrogen receptor cased subtypes of cancer may have different molecular environments which has an impact on the therapeutic efficacy.

Carbohydrate (Chondroitin 4) Sulfotransferase-11-Mediated Induction of Epithelial-Mesenchymal Transition and Generation of Cancer Stem Cells.

INTRODUCTION: We have previously shown that the expression of carbohydrate (chondroitin 4) sulfotransferase-11 (CHST11) is elevated in human breast cancer tissues, and that its expression in human breast cancer cell lines is associated with aggressive behavior of cells. The clinical significance of CHST11 expression is unknown, and its function in breast cancer cells is not fully understood. OBJECTIVE: The current study was performed to define the clinical significance of this gene and address its biological function in promoting the aggressive behavior of breast cancer cells. METHODS: Publicly available datasets were analyzed to determine the correlation of CHST11 expression with breast cancer survival. MCF-7 cells were transfected with the human CHST11 gene, and MCF-7-CHST11 cells with stable expression of the gene were established. Morphology and metastatic capacity of transfected cells were monitored in vitro. E-cadherin and ß-catenin expression was compared by immunofluorescence. The expression of genes involved in epithelial-mesenchymal transition (EMT) and pluripotency was determined using real-time PCR. The Wnt inhibitor, Wnt-C59, was used to examine the involvement of Wnt in CHST11-mediated morphology. RESULTS: The elevated expression of CHST11 in breast tumor specimens was significantly associated with poor survival among patients. MCF-7-CHST11 cells displayed morphological characteristics consistent with EMT, together with a significantly higher proliferation rate, enhanced migratory potential, and more robust anchorage-independent growth. MCF-7-CHST11 cells showed decreased expression of E-cadherin and increased accumulation of ß-catenin, as assessed by immunofluorescence. Consistently, increased expression of CHST11 resulted in upregulation of key EMT and stem cell markers. Morphological transition in MCF-7-CHST11 cells was partially reversed by co-incubation with an inhibitor of the Wnt pathway. CONCLUSIONS: Our findings support a role for CHST11 in induction of EMT and stem cell-like properties. Our data also associate the expression levels of CHST11 in breast tumor specimens with patients' survival. The results have a significant implication for CHST11 expression level as a novel molecular signature for predictive and prognostic purposes in breast cancer. Moreover, with a possible role in driving tumor cell aggressiveness, CHST11 expression might be further considered as a potential therapeutic target for breast cancer.

Analysis of A4gnt Knockout Mice Reveals an Essential Role for Gastric Sulfomucins in Preventing Gastritis Cystica Profunda.

Gastric adenocarcinoma cells secrete sulfomucins, but their role in gastric tumorigenesis remains unclear. To address that question, we generated A4gnt/Chst4 double-knockout (DKO) mice by crossing A4gnt knockout (KO) mice, which spontaneously develop gastric adenocarcinoma, with Chst4 KO mice, which are deficient in the sulfotransferase GlcNAc6ST-2. A4gnt/Chst4 DKO mice lack gastric sulfomucins but developed gastric adenocarcinoma. Unexpectedly, severe gastric erosion occurred in A4gnt/Chst4 DKO mice at as early as 3 weeks of age, and with aging these lesions were accompanied by gastritis cystica profunda (GCP). Cxcl1, Cxcl5, Ccl2, and Cxcr2 transcripts in gastric mucosa of 5-week-old A4gnt/Chst4 DKO mice exhibiting both hyperplasia and severe erosion were significantly upregulated relative to age-matched A4gnt KO mice, which showed hyperplasia alone. However, upregulation of these genes disappeared in 50-week-old A4gnt/Chst4 DKO mice exhibiting high-grade dysplasia/adenocarcinoma and GCP. Moreover, Cxcl1 and Cxcr2 were downregulated in A4gnt/Chst4 DKO mice relative to age-matched A4gnt KO mice exhibiting adenocarcinoma alone. These combined results indicate that the presence of sulfomucins prevents severe gastric erosion followed by GCP in A4gnt KO mice by transiently regulating a set of inflammation-related genes, Cxcl1, Cxcl5, Ccl2, and Cxcr2 at 5 weeks of age, although sulfomucins were not directly associated with gastric cancer development.

Heparan Sulfate Sulfation by Hs2st Restricts Astroglial Precursor Somal Translocation in Developing Mouse Forebrain by a Non-Cell-Autonomous Mechanism.

Heparan sulfate (HS) is a cell surface and extracellular matrix carbohydrate extensively modified by differential sulfation. HS interacts physically with canonical fibroblast growth factor (FGF) proteins that signal through the extracellular signal regulated kinase (ERK)/mitogen activated protein kinase (MAPK) pathway. At the embryonic mouse telencephalic midline, FGF/ERK signaling drives astroglial precursor somal translocation from the ventricular zone of the corticoseptal boundary (CSB) to the induseum griseum (IG), producing a focus of Slit2-expressing astroglial guidepost cells essential for interhemispheric corpus callosum (CC) axon navigation. Here, we investigated the cell and molecular function of a specific form of HS sulfation, 2-O HS sulfation catalyzed by the enzyme Hs2st, in midline astroglial development and in regulating FGF protein levels and interaction with HS. Hs2st-/- embryos of either sex exhibit a grossly enlarged IG due to precocious astroglial translocation and conditional Hs2st mutagenesis and ex vivo culture experiments show that Hs2st is not required cell autonomously by CC axons or by the IG astroglial cell lineage, but rather acts non-cell autonomously to suppress the transmission of translocation signals to astroglial precursors. Rescue of the Hs2st-/- astroglial translocation phenotype by pharmacologically inhibiting FGF signaling shows that the normal role of Hs2st is to suppress FGF-mediated astroglial translocation. We demonstrate a selective action of Hs2st on FGF protein by showing that Hs2st (but not Hs6st1) normally suppresses the levels of Fgf17 protein in the CSB region in vivo and use a biochemical assay to show that Hs2st (but not Hs6st1) facilitates a physical interaction between the Fgf17 protein and HS.SIGNIFICANCE STATEMENT We report a novel non-cell-autonomous mechanism regulating cell signaling in developing brain. Using the developing mouse telencephalic midline as an exemplar, we show that the specific sulfation modification of the cell surface and extracellular carbohydrate heparan sulfate (HS) performed by Hs2st suppresses the supply of translocation signals to astroglial precursors by a non-cell-autonomous mechanism. We further show that Hs2st modification selectively facilitates a physical interaction between Fgf17 and HS and suppresses Fgf17 protein levels in vivo, strongly suggesting that Hs2st acts selectively on Fgf17 signaling. HS interacts with many signaling proteins potentially encoding numerous selective interactions important in development and disease, so this class of mechanism may apply more broadly to other biological systems.

Glial Sulfatides and Neuronal Complex Gangliosides Are Functionally Interdependent in Maintaining Myelinating Axon Integrity.

Sulfatides and gangliosides are raft-associated glycolipids essential for maintaining myelinated nerve integrity. Mice deficient in sulfatide (cerebroside sulfotransferase knock-out, CST-/-) or complex gangliosides (ß-1,4-N-acetylegalactosaminyltransferase1 knock-out, GalNAc-T-/-) display prominent disorganization of proteins at the node of Ranvier (NoR) in early life and age-dependent neurodegeneration. Loss of neuronal rather than glial complex gangliosides underpins the GalNAc-T-/- phenotype, as shown by neuron- or glial-specific rescue, whereas sulfatide is principally expressed and functional in glial membranes. The similarities in NoR phenotype of CST-/-, GalNAc-T-/-, and axo-glial protein-deficient mice suggests that these glycolipids stabilize membrane proteins including neurofascin155 (NF155) and myelin-associated glycoprotein (MAG) at axo-glial junctions. To assess the functional interactions between sulfatide and gangliosides, CST-/- and GalNAc-T-/- genotypes were interbred. CST-/-× GalNAc-T-/- mice develop normally to postnatal day 10 (P10), but all die between P20 and P25, coinciding with peak myelination. Ultrastructural, immunohistological, and biochemical analysis of either sex revealed widespread axonal degeneration and disruption to the axo-glial junction at the NoR. In addition to sulfatide-dependent loss of NF155, CST-/- × GalNAc-T-/- mice exhibited a major reduction in MAG protein levels in CNS myelin compared with WT and single-lipid-deficient mice. The CST-/- × GalNAc-T-/- phenotype was fully restored to that of CST-/- mice by neuron-specific expression of complex gangliosides, but not by their glial-specific expression nor by the global expression of a-series gangliosides. These data indicate that sulfatide and complex b-series gangliosides on the glial and neuronal membranes, respectively, act in concert to promote NF155 and MAG in maintaining the stable axo-glial interactions essential for normal nerve function.SIGNIFICANCE STATEMENT Sulfatides and complex gangliosides are membrane glycolipids with important roles in maintaining nervous system integrity. Node of Ranvier maintenance in particular requires stable compartmentalization of multiple membrane proteins. The axo-glial adhesion molecules neurofascin155 (NF155) and myelin-associated glycoprotein (MAG) require membrane microdomains containing either sulfatides or complex gangliosides to localize and function effectively. The cooperative roles of these microdomains and associated proteins are unknown. Here, we show vital interdependent roles for sulfatides and complex gangliosides because double (but not single) deficiency causes a rapidly lethal phenotype at an early age. These findings suggest that sulfatides and complex gangliosides on opposing axo-glial membranes are responsible for essential tethering of the axo-glial junction proteins NF155 and MAG, which interact to maintain the nodal complex.

Cholesterol sulfate is a DOCK2 inhibitor that mediates tissue-specific immune evasion in the eye.

Although immune responses are essential to protect the body from infection, they can also harm tissues. Certain tissues and organs, including the eye, constitute specialized microenvironments that locally inhibit immune reactivity. Dedicator of cytokinesis protein 2 (DOCK2) is a Rac-specific guanine nucleotide exchange factor (GEF) that is predominantly found in hematopoietic cells. DOCK2 plays a key role in immune surveillance because it is essential for the activation and migration of leukocytes. DOCK2 mutations cause severe immunodeficiency in humans. We found that DOCK2-mediated Rac activation and leukocyte migration were effectively inhibited by cholesterol sulfate (CS), but not by cholesterol or other sulfated steroids. CS bound to the catalytic domain of DOCK2 and suppressed its GEF activity. Mass spectrometric quantification revealed that CS was most abundantly produced in the Harderian gland, which provides the lipids that form the oily layer of the tear film. Sulfation of cholesterol is mediated by the sulfotransferases SULT2B1b and, to a lesser extent, SULT2B1a, which are produced from the same gene through alternative splicing. By genetically inactivating Sult2b1, we showed that the lack of CS in mice augmented ultraviolet- and antigen-induced ocular surface inflammation, which was suppressed by administration of eye drops containing CS. Thus, CS is a naturally occurring DOCK2 inhibitor and contributes to the generation of the immunosuppressive microenvironment in the eye.

The heparan sulfate 3-O-sulfotransferases (HS3ST) 2, 3B and 4 enhance proliferation and survival in breast cancer MDA-MB-231 cells.

Heparan sulfate 3-O-sulfotransferases (HS3STs) catalyze the final maturation step of heparan sulfates. Although seven HS3ST isozymes have been described in human, 3-O-sulfation is a relatively rare modification, and only a few biological processes have been described to be influenced by 3-O-sulfated motifs. A conflicting literature has recently reported that HS3ST2, 3A, 3B and 4 may exhibit either tumor-promoting or anti-oncogenic properties, depending on the model used and cancer cell phenotype. Hence, we decided to compare the consequences of the overexpression of each of these HS3STs in the same cellular model. We demonstrated that, unlike HS3ST3A, the other three isozymes enhanced the proliferation of breast cancer MDA-MB-231 and BT-20 cells. Moreover, the colony forming capacity of MDA-MB-231 cells was markedly increased by the expression of HS3ST2, 3B and 4. No notable difference was observed between the three isozymes, meaning that the modifications catalyzed by each HS3ST had the same functional impact on cell behavior. We then demonstrated that overexpression of HS3ST2, 3B and 4 was accompanied by increased activation of c-Src, Akt and NF-κB and up-regulation of the anti-apoptotic proteins survivin and XIAP. In line with these findings, we showed that HS3ST-transfected cells are more resistant to cell death induction by pro-apoptotic stimuli or NK cells. Altogether, our findings demonstrate that HS3ST2, 3B and 4 share the same pro-tumoral activity and support the idea that these HS3STs could compensate each other for loss of their expression depending on the molecular signature of cancer cells and/or changes in the tumor environment.

Desulfation of Heparan Sulfate by Sulf1 and Sulf2 Is Required for Corticospinal Tract Formation.

Heparan sulfate (HS) has been implicated in a wide range of cell signaling. Here we report a novel mechanism in which extracellular removal of 6-O-sulfate groups from HS by the endosulfatases, Sulf1 and Sulf2, is essential for axon guidance during development. In Sulf1/2 double knockout (DKO) mice, the corticospinal tract (CST) was dorsally displaced on the midbrain surface. In utero electroporation of Sulf1/2 into radial glial cells along the third ventricle, where Sulf1/2 mRNAs are normally expressed, rescued the CST defects in the DKO mice. Proteomic analysis and functional testing identified Slit2 as the key molecule associated with the DKO phenotype. In the DKO brain, 6-O-sulfated HS was increased, leading to abnormal accumulation of Slit2 protein on the pial surface of the cerebral peduncle and hypothalamus, which caused dorsal repulsion of CST axons. Our findings indicate that postbiosynthetic desulfation of HS by Sulfs controls CST axon guidance through fine-tuning of Slit2 presentation.

Sex- and Tissue-Specific Role of Estrogen Sulfotransferase in Energy Homeostasis and Insulin Sensitivity.

Estrogen sulfotransferase catalyzes the sulfoconjugation and deactivation of estrogens. Previously, we showed that loss of Est in male ob/ob mice, but not in female ob/ob mice, exacerbated the diabetic phenotype, but the underlying mechanism was unclear. In this study, we show that transgenic reconstitution of Est in the adipose tissue, but not in the liver, attenuated diabetic phenotype in Est-deficient ob/ob mice (obe mice). Mechanistically, adipose reconstitution of Est in obe mice (oae mice) resulted in reduced local and systemic inflammation, improved insulin sensitivity, and increased energy expenditure. At the molecular level, adipose induction of lipocalin-2 (Lcn2) in oae males may have contributed to the inhibition of inflammation because the level of Lcn2 was negatively associated with tumor necrosis factor (Tnf) α expression, and treatment of differentiated adipocytes with Lcn2 antagonized Tnfα-responsive inhibition of insulin signaling. The metabolic benefit of adipose reconstitution of Est was sex specific, because adipose reconstitution of Est in obe females had little effect. Interestingly, despite their improved metabolic functions, obe male mice with reconstituted Est in their adipose tissue failed to ameliorate the impairment of the structure and function of the pancreatic islets. In summary, our study uncovers a crucial adipose- and male-specific role of Est in maintaining the whole-body energy homeostasis.

Multiple UDP-Glucuronosyltransferase and Sulfotransferase Enzymes are Responsible for the Metabolism of Verproside in Human Liver Preparations.

Verproside, an active iridoid glycoside component of Veronica species, such as Pseudolysimachion rotundum var. subintegrum and Veronica anagallis-aquatica, possesses anti-asthma, anti-inflammatory, anti-nociceptive, antioxidant, and cytostatic activities. Verproside is metabolized into nine metabolites in human hepatocytes: verproside glucuronides (M1, M2) via glucuronidation, verproside sulfate (M3) via sulfation, picroside II (M4) and isovanilloylcatalpol (M5) via O-methylation, M4 glucuronide (M6) and M4 sulfate (M8) via further glucuronidation and sulfation of M4, and M5 glucuronide (M7) and M5 sulfate (M9) via further glucuronidation and sulfation of M5. Drug-metabolizing enzymes responsible for verproside metabolism, including sulfotransferase (SULT) and UDP-glucuronosyltransferase (UGT), were characterized. The formation of verproside glucuronides (M1, M2), isovanilloylcatalpol glucuronide (M7), and picroside II glucuronide (M6) was catalyzed by commonly expressed UGT1A1 and UGT1A9 and gastrointestinal-specific UGT1A7, UGT1A8, and UGT1A10, consistent with the higher intrinsic clearance values for the formation of M1, M2, M6, and M7 in human intestinal microsomes compared with those in liver microsomes. The formation of verproside sulfate (M3) and M5 sulfate (M9) from verproside and isovanilloylcatalpol (M5), respectively, was catalyzed by SULT1A1. Metabolism of picroside II (M4) into M4 sulfate (M8) was catalyzed by SULT1A1, SULT1E1, SULT1A2, SULT1A3, and SULT1C4. Based on these results, the pharmacokinetics of verproside may be affected by the co-administration of relevant UGT and SULT inhibitors or inducers.

Altered expression of chondroitin sulfate structure modifying sulfotransferases in the articular cartilage from adult osteoarthritis and Kashin-Beck disease.

OBJECTIVE: To investigate the expression of enzymes involved in chondroitin sulfate (CS) sulfation in the articular cartilage isolated from adult patients with osteoarthritis (OA) and Kashin-Beck disease (KBD), using normal adults as controls. METHODS: Articular cartilage samples were collected from normal, OA and KBD adults aged 38-60 years old, and divided into three groups with six individual subjects in each group. The morphology and pathology grading of knee joint cartilage was examined by Safranin O staining. The localization and expression of enzymes involved in CS sulfation (CHST-3, CHST-11, CHST-12, CHST-13, carbohydrate (N-acetylgalactosamine 4-sulfate 6-O) sulfotransferase 15 - CHST-15, and uronyl 2-O-sulfotransferase - UST) were examined by immunohistochemical (IHC) staining and semi-quantitative analysis. RESULTS: Positive staining rates for anabolic enzymes CHST-3, CHST-12, CHST-15, and UST were lower in the KBD and OA groups than those in the control group. Meanwhile, reduced levels of CHST-11, and CHST-13 in KBD group were observed, in contrast to those in OA and control groups. The expressions of all six CS sulfation enzymes were less detected in the superficial and deep zones of KBD cartilage compared with control and OA cartilage. CONCLUSION: The reduced expression of the CS structure modifying sulfotransferases in the chondrocytes of both KBD and OA adult patients may provide explanations for their cartilage damages, and therapeutic targets for their treatment.

Sulfatase 2 facilitates lymphangiogenesis in breast cancer by regulating VEGF-D.

In our previous studies, sulfatase 2 (Sulf2) was found to upregulate vascular endothelial growth factor-D (VEGF-D) expression in breast cancer. As VEGF-D plays an important role in lymphangiogenesis, we hypothesized that Sulf2 facilitates lymphangiogenesis in breast cancer by regulating VEGF-D. To evaluate the functions of Sulf2 on lymphangiogenesis in breast cancer, proliferation, apoptosis, cell cycle, cell mobility and tube-formation of lymphatic endothelial cells (LECs) were measured in vitro. Lymphangiogenesis in nude mouse ears and breast cancer xenografts were examined in vivo. Furthermore, the expression levels of related signaling pathway genes were screened and verified in LECs. We found that Sulf2 significantly increased the mobility and tube formation of the LECs, inhibited cisplatin-induced LEC apoptosis, but had no effect on cell proliferation and the cell cycle. Moreover, recombinant Sulf2 (rSulf2) combined with VEGF-D further promoted the proliferation, cell cycle, mobility and tube-like structure formation in the LECs, and at the same time inhibited cisplatin-induced apoptosis especially in the late stage. Sulf2 also significantly increased the density of lymphatic vessels in mouse ears and breast cancer xenografts in vivo. AKT1 was also shown to be upregulated and activated by Sulf2. Our results confirmed that Sulf2 facilitated lymphangiogenesis in breast cancer cells by regulating VEGF-D and that the AKT1­related signaling pathway was involved.

Role of sulfatase 2 in lipoprotein metabolism and angiogenesis.

PURPOSE OF REVIEW: This article summarizes the current evidence to support a role of sulfatase 2 (SULF2) in triglyceride-rich lipoprotein (TRL) metabolism and angiogenesis. RECENT FINDINGS: Heparan sulfate proteoglycans (HSPG) are involved in the hepatic clearance of TRLs in mice and in humans. Different genetically modified mouse models have been instrumental to provide evidence that syndecan1, the core protein of HSPG, but also the degree of sulfation of the heparin sulfate chain, attached to syndecan 1, is important for hepatic TRL metabolism. Studies in humans demonstrate the regulating role of SULF2 in the hepatic uptake of TRL by HSPG and demonstrate the importance of 6-O-sulfation, modulated by SULF2, for HSPG function. The role of SULF2 in angiogenesis is illustrated by increased SULF2 mRNA expression in the stalk cells of angiogenic vascular sprouts that use fatty acids derived from TRL as a source for biomass production. Interestingly, SULF2 also interferes with HSPG-vascular endothelial growth factor binding, which impacts upon the angiogenic properties of stalk cells. SUMMARY: SULF2 is a multifaceted protein involved in TRL homeostasis and angiogenesis. Future investigations should focus on the potential benefits of targeting SULF2 in atherosclerosis and angiogenesis.

[In vitro characterization of glucuronosyl- and sulfotransferases involved in the conjugation of ethanol]. / In vitro Charakterisierung der Glucuronidierung und Sulfatierung von Ethanol.

Ethyl glucuronide (EtG) and ethyl sulfate (EtS) are minor metabolites of ethanol; for some years, both compounds have been used as direct biomarkers of alcohol consumption in forensic and clinical settings as well as in traffic medicine. Drinking experiments showed individual variations of the formation of EtG and EtS. At present, our knowledge on enzymes involved in the conjugation of ethanol is incomplete and partly inconsistent. The purpose of the present study was to characterize those enzymes that are capable of catalyzing glucuronidation and sulfation of ethanol including some potential inhibitors. Following optimization of incubation conditions, the formation rates of EtG and EtS from ethanol via recombinant glucuronosyltransferases (UGTs, hepatic) and sulfotransferases (SULTs, hepatic, intestinal), the kinetics and the inhibitory potential of polyphenols such as quercetin, kaempferol and resveratrol were determined. Analysis was performed following either solid phase extraction due to severe ion suppression of EtG or direct injection of the EtS-containing incubation mixture by high-pressure liquid chromatography/tandem mass spectrometry. Deuterated analogues were used as internal standards. All UGTs were capable of metabolizing ethanol through glucuronidation; UGT1A9 and UGT2B7 exhibited the highest formation rates. All SULTs showed ethanol-sulfating activity with SULT1A1 being most active. Data for all enzymes could best be described by Michaelis-Menten kinetics. All polyphenols inhibited the conjugation of ethanol except UGT2B 15. Inhibition was reversible and competitive for most enzymes; mechanism-based inhibition was evident for UGT2B7 and SULT2A1 with regard to quercetin and for SULT1E1 with regard to kaempferol. These results suggest an influence on the formation rates of EtG and EtS by common food ingredients beside known polymorphisms of UGT and SULT family members. Further studies should be conducted to achieve a better understanding of the extent and significance of this influence.

Sulfation of afimoxifene, endoxifen, raloxifene, and fulvestrant by the human cytosolic sulfotransferases (SULTs): A systematic analysis.

Previous studies demonstrated that sulfate conjugation is involved in the metabolism of three commonly used breast cancer drugs, tamoxifen, raloxifene and fulvestrant. The current study was designed to systematically identify the human cytosolic sulfotransferases (SULTs) that are capable of sulfating raloxifene, fulvestrant, and two active metabolites of tamoxifen, afimoxifene and endoxifen. A systematic analysis using 13 known human SULTs revealed SULT1A1 and SULT1C4 as the major SULTs responsible for the sulfation of afimoxifene, endoxifen, raloxifene and fulvestrant. Kinetic parameters of these two human SULTs in catalyzing the sulfation of these drug compounds were determined. Sulfation of afimoxifene, endoxifen, raloxifene and fulvestrant under metabolic conditions was examined using HepG2 human hepatoma cells and MCF-7 breast cancer cells. Moreover, human intestine, kidney, liver, and lung cytosols were examined to verify the presence of afimoxifene/endoxifen/raloxifene/fulvestrant-sulfating activity.

2-O Heparan Sulfate Sulfation by Hs2st Is Required for Erk/Mapk Signalling Activation at the Mid-Gestational Mouse Telencephalic Midline.

Heparan sulfate (HS) is a linear carbohydrate composed of polymerized uronate-glucosamine disaccharide units that decorates cell surface and secreted glycoproteins in the extracellular matrix. In mammals HS is subjected to differential sulfation by fifteen different heparan sulfotransferase (HST) enzymes of which Hs2st uniquely catalyzes the sulfation of the 2-O position of the uronate in HS. HS sulfation is postulated to be important for regulation of signaling pathways by facilitating the interaction of HS with signaling proteins including those of the Fibroblast Growth Factor (Fgf) family which signal through phosphorylation of extracellular signal-regulated kinases Erk1/2. In the developing mouse telencephalon Fgf2 signaling regulates proliferation and neurogenesis. Loss of Hs2st function phenocopies the thinned cerebral cortex of mutant mice in which Fgf2 or Erk1/2 function are abrogated, suggesting the hypothesis that 2-O-sulfated HS structures play a specific role in Fgf2/Erk signaling pathway in this context in vivo. This study investigated the molecular role of 2-O sulfation in Fgf2/Erk signaling in the developing telencephalic midline midway through mouse embryogenesis at E12.5. We examined the expression of Hs2st, Fgf2, and Erk1/2 activity in wild-type and Hs2st-/- mice. We found that Hs2st is expressed at high levels at the midline correlating with high levels of Erk1/2 activation and Erk1/2 activation was drastically reduced in the Hs2st-/- mutant at the rostral telencephalic midline. We also found that 2-O sulfation is specifically required for the binding of Fgf2 protein to Fgfr1, its major cell-surface receptor at the rostral telencephalic midline. We conclude that 2-O sulfated HS structures generated by Hs2st are needed to form productive signaling complexes between HS, Fgf2 and Fgfr1 that activate Erk1/2 at the midline. Overall, our data suggest the interesting possibility that differential expression of Hs2st targets the rostral telencephalic midline for high levels of Erk signaling by increasing the sensitivity of cells to an Fgf2 signal that is rather more widespread.

Tyrosine sulfation as a protein post-translational modification.

Integration of inorganic sulfate into biological molecules plays an important role in biological systems and is directly involved in the instigation of diseases. Protein tyrosine sulfation (PTS) is a common post-translational modification that was first reported in the literature fifty years ago. However, the significance of PTS under physiological conditions and its link to diseases have just begun to be appreciated in recent years. PTS is catalyzed by tyrosylprotein sulfotransferase (TPST) through transfer of an activated sulfate from 3'-phosphoadenosine-5'-phosphosulfate to tyrosine in a variety of proteins and peptides. Currently, only a small fraction of sulfated proteins is known and the understanding of the biological sulfation mechanisms is still in progress. In this review, we give an introductory and selective brief review of PTS and then summarize the basic biochemical information including the activity and the preparation of TPST, methods for the determination of PTS, and kinetics and reaction mechanism of TPST. This information is fundamental for the further exploration of the function of PTS that induces protein-protein interactions and the subsequent biochemical and physiological reactions.

Activation of the transforming growth factor-ß/SMAD transcriptional pathway underlies a novel tumor-promoting role of sulfatase 1 in hepatocellular carcinoma.

UNLABELLED: In vitro studies have proposed a tumor suppressor role for sulfatase 1 (SULF1) in hepatocellular carcinoma (HCC); however, high expression in human HCC has been associated with poor prognosis. The reason underlying this paradoxical observation remains to be explored. Using a transgenic (Tg) mouse model overexpressing Sulf1 (Sulf1-Tg), we assessed the effects of SULF1 on the diethylnitrosamine model of liver carcinogenesis. Sulf1-Tg mice show a higher incidence of large and multifocal tumors with diethylnitrosamine injection compared to wild-type mice. Lung metastases were found in 75% of Sulf1-Tg mice but not in wild-type mice. Immunohistochemistry, immunoblotting, and reporter assays all show a significant activation of the transforming growth factor-ß (TGF-ß)/SMAD transcriptional pathway by SULF1 both in vitro and in vivo. This effect of SULF1 on the TGF-ß/SMAD pathway is functional; overexpression of SULF1 promotes TGF-ß-induced gene expression and epithelial-mesenchymal transition and enhances cell migration/invasiveness. Mechanistic analyses demonstrate that inactivating mutation of the catalytic site of SULF1 impairs the above actions of SULF1 and diminishes the release of TGF-ß from the cell surface. We also show that SULF1 expression decreases the interaction between TGF-ß1 and its heparan sulfate proteoglycan sequestration receptor, TGFßR3. Finally, using gene expression from human HCCs, we show that patients with high SULF1 expression have poorer recurrence-free survival (hazard ratio 4.1, 95% confidence interval 1.9-8.3; P = 0.002) compared to patients with low SULF1. We also found strong correlations of SULF1 expression with TGF-ß expression and with several TGF-ß-related epithelial-mesenchymal transition genes in human HCC. CONCLUSION: Our study proposes a novel role of SULF1 in HCC tumor progression through augmentation of the TGF-ß pathway, thus defining SULF1 as a potential biomarker for tumor progression and a novel target for drug development for HCC.