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Introduction: The potential role of accumulation of chondroitin sulfates (CSs) in the pathobiology of COVID-19 has not been examined. Accumulation may occur by increased synthesis or by decline in activity of the enzyme arylsulfatase B (ARSB; N-acetylgalactosamine-4-sulfatase) which requires oxygen for activity. Methods: Immunostaining of lung tissue from 28 patients who died due to COVID-19 infection was performed for CS, ARSB, and carbohydrate sulfotransferase (CHST)15. Measurements of mRNA expression of CHST15 and CHST11, sulfotransferase activity, and total sulfated glycosaminoglycans (GAGs) were determined in human vascular smooth muscle cells following angiotensin (Ang) II treatment. Results: CS immunostaining showed increase in intensity and distribution, and immunostaining of ARSB was diminished in COVID-19 compared to normal lung tissue. CHST15 immunostaining was prominent in vascular smooth muscle cells associated with diffuse alveolar damage due to COVID-19 or other causes. Expression of CHST15 and CHST11 which are required for synthesis of CSE and chondroitin 4-sulfate, total sulfated GAGs, and sulfotransferase activity was significantly increased following AngII exposure in vascular smooth muscle cells. Expression of Interleukin-6 (IL-6), a mediator of cytokine storm in COVID-19, was inversely associated with ARSB expression. Discussion/Conclusion: Decline in ARSB and resulting increases in CS may contribute to the pathobiology of COVID-19, as IL-6 does. Increased expression of CHSTs following activation of Ang-converting enzyme 2 may lead to buildup of CSs.

Immunostaining in lungs of patients who died with Covid-19 infection showed increased intensity and distribution of chondroitin sulfate and carbohydrate sulfotransferase (CHST)15 and decline in N-acetylgalactostamine-4-sulfatase (Arylsulfatase B; ARSB). To explain these findings, human small airway epithelial cells were exposed to the SARS-CoV-2 spike protein receptor binding domain (SPRBD) and transcriptional mechanisms investigated. Phospho-p38 MAPK and phospho-Smad3 increased following exposure to the SPRBD, and their inhibition suppressed CHST15 and CHST11 promoter activation. Decline in ARSB was mediated by phospho-38 MAPK-induced N-terminal Rb phosphorylation and the associated decline in E2F1 binding to the ARSB promoter. The increases in chondroitin sulfotransferases were inhibited by the p38-MAPK inhibitor SB203580 and by the Smad3 inhibitor SIS3 and by treatment with the antihistamine desloratadine and the antibiotic monensin. Since accumulation of chondroitin sulfates is associated with fibrotic lung conditions and diffuse alveolar damage, increased attention to p38- MAPK inhibitors may be beneficial in Covid-19 infection.

The enzyme N-acetylgalactosamine-4-sulfatase (Arylsulfatase B; ARSB) was originally identified as a lysosomal enzyme which was deficient in Mucopolysaccharidosis VI (MPS VI; Maroteaux-Lamy Syndrome). The newly directed attention to the impact of ARSB in human pathobiology indicates a broader, more pervasive effect, encompassing roles as a tumor suppressor, transcriptional mediator, redox switch, and regulator of intracellular and extracellular-cell signaling. By controlling the degradation of chondroitin 4-sulfate and dermatan sulfate by removal or failure to remove the 4-sulfate residue at the non-reducing end of the sulfated glycosaminoglycan chain, ARSB modifies the binding or release of critical molecules into the cell milieu. These molecules, such as galectin-3 and SHP-2, in turn, influence crucial cellular processes and events which determine cell fate. Identification of ARSB at the cell membrane and in the nucleus expands perception of the potential impact of decline in ARSB activity. The regulation of availability of sulfate from chondroitin 4-sulfate and dermatan sulfate may also affect sulfate assimilation and production of vital molecules, including glutathione and cysteine. Increased attention to ARSB in mammalian cells may help to integrate and deepen our understanding of diverse biological phenomenon and to approach human diseases with new insights.

BACKGROUND: Carrageenan is a very common food additive in Western diets, but predictably causes inflammation in thousands of cell-based and animal experiments. OBJECTIVE: To assess the impact of carrageenan exposure on the interval to relapse in patients with ulcerative colitis in remission. METHODS: A randomized, double-blind, placebo-controlled, multicenter, clinical trial was conducted to assess if patients with ulcerative colitis in remission would have a longer interval to relapse if they followed a diet with no carrageenan. All participants were instructed in the no-carrageenan diet and were randomized to either placebo capsules or carrageenan-containing capsules. The carrageenan in the capsules was less than the average daily carrageenan intake from the diet. Relapse was defined as an increase of two or more points on the Simple Clinical Colitis Activity Index (SCCAI) and intensification of treatment for ulcerative colitis. Participants were followed by telephone calls every two weeks until relapse or one year of participation. The occurrence of relapse and inflammatory biomarkers were compared between the two groups. RESULTS: Twelve patients completed study questionnaires. Three patients who received carrageenan-containing capsules relapsed, and none of the patients who received placebo-containing capsules relapsed (p = 0.046, log-rank test). Laboratory tests showed increases in Interleukin-6 (p = 0.02, paired t-test, two-tailed) and fecal calprotectin (p = 0.06; paired t-test, two-tailed) between the beginning and the end of study participation in the carrageenan-exposed group, but not in the placebo-group. CONCLUSION: Carrageenan intake contributed to earlier relapse in patients with ulcerative colitis in remission. Restriction of dietary carrageenan may benefit patients with ulcerative colitis.

Background In tissue microarrays, immunostaining of the enzyme arylsulfatase B (ARSB; N-acetylgalactosamine-4-sulfatase) was less in recurrent prostate cancers and in cancers with higher Gleason scores. In cultured prostate stem cells, decline in ARSB increased Wnt signaling through effects on Dickkopf Wnt Signaling Pathway Inhibitor (DKK)3. The effects of androgen exposure on ARSB and the impact of decline in ARSB on Wnt signaling in prostate tissue were unknown. Methods Epithelial and stromal tissues from malignant and normal human prostate were obtained by laser capture microdissection. mRNA expression of ARSB, galactose-6-sulfate-sulfatase (GALNS) and Wnt-signaling targets was determined by QPCR. Non-malignant human epithelial and stromal prostate cells were grown in tissue culture, including two-cell layer cultures. ARSB was silenced by specific siRNA, and epithelial cells were treated with stromal spent media following treatment with IWP-2, an inhibitor of Wnt secretion, and by exogenous recombinant human Wnt3A. Promoter methylation was detected using specific DKK3 and ARSB promoter primers. The effects of DHT and of ARSB overexpression on DKK expression were determined. Cell proliferation was assessed by BrdU incorporation. Results Normal stroma showed higher expression of vimentin, ARSB, and Wnt3A than epithelium. Normal epithelium had higher expression of E-cadherin, galactose 6-sulfate-sulfatase (GALNS), and DKK3 than stroma. In malignant epithelium, expression of ARSB and DKK3 declined, and expression of GALNS and Wnt signaling targets increased. In cultured prostate epithelial cells, Wnt-mediated signaling was greatest when ARSB was silenced and cells were exposed to exogenous Wnt3A. Exposure to 5α-dihydrotestosterone (DHT) increased ARSB and DKK3 promoter rmethylation, and effects of DHT on DKK3 expression were reversed when ARSB was overexpressed. Conclusions Androgen-induced declines in ARSB and DKK3 may contribute to prostate carcinogenesis by sustained activation of Wnt signaling in prostate epithelium in response to stromal Wnt3A.

The precise mechanisms by which Covid-19 infection leads to hypoxia and respiratory failure have not yet been elucidated. Interactions between sulfated glycosaminoglycans (GAGs) and the SARS-CoV-2 spike glycoprotein have been identified as participating in viral adherence and infectivity. The spike glycoprotein binds to respiratory epithelium through the angiotensin converting enzyme 2 (ACE2) receptor, which endogenously interacts with Angiotensin (Ang) II to yield Angiotensin 1-7. Stimulation of human vascular smooth muscle cells by Ang II leads to increased mRNA expression of two chondroitin sulfotransferases (CHST11 and CHST15), which are required for synthesis of chondroitin 4-sulfate (C4S) and chondroitin 4,6-disulfate (CSE), respectively. Total sulfated GAGs, increased sulfotransferase activity, and increased expression of the proteoglycans biglycan, syndecan-1, perlecan, and versican followed treatment by Ang II. Candesartan, an Angiotensin II receptor blocker (Arb), largely, but incompletely, inhibited these increases, and the differences from baseline remained significant. These results suggest that another effect of Ang II also contributes to the increased expression of chondroitin sulfotransferases, total sulfated GAGs, and proteoglycans. We hypothesize that activation of ACE2 may contribute to these increases and suggest that the SARS-CoV-2 spike glycoprotein interaction with ACE2 may also increase chondroitin sulfotransferases, sulfated GAGs, and proteoglycans and thereby contribute to viral adherence to bronchioalveolar cells and to respiratory compromise in SARS-CoV-2 infection.

The spike protein of SARS-CoV-2 binds to respiratory epithelium through the ACE2 receptor, an endogenous receptor for Angiotensin II (AngII). The mechanisms by which this viral infection leads to hypoxia and respiratory failure have not yet been elucidated. Interactions between the sulfated glycosaminoglycans heparin and heparan sulfate and the SARS-CoV-2 spike glycoprotein have been identified as participating in viral adherence and infectivity. In this brief report, we present data indicating that stimulation of vascular smooth muscle cells by AngII leads to increased expression of two chondroitin sulfotransferases (CHST11 and CHST15), which are required for the synthesis of the sulfated glycosaminoglycans chondroitin 4-sulfate (C4S) and chondroitin 4,6-disulfate (CSE). We suggest that increased expression of these chondroitin sulfotransferases and the ensuing production of chondroitin sulfates may contribute to viral adherence to bronchioalveolar cells and to the progression of respiratory disease in Covid-19. The enzyme Arylsulfatase B (ARSB; N-acetylgalactosamine-4-sulfatase), which removes 4-sulfate groups from the non-reducing end of chondroitin 4-sulfate residues, is required for degradation of C4S and CSE. In hypoxic conditions or following treatment with chloroquine, ARSB activity is reduced. Decline in ARSB can contribute to ongoing accumulation and airway obstruction by C4S and CSE. Decline in ARSB leads to increased expression of Interleukin(IL)-6 in human bronchial epithelial cells, and IL-6 is associated with cytokine storm in Covid-19. These findings indicate how chondroitin sulfates, chondroitin sulfotransferases, and chondroitin sulfatases may participate in the progression of hypoxic respiratory insufficiency in Covid-19 disease and suggest new therapeutic targets.

Expression of CHST15 (carbohydrate sulfotransferase 15; chondroitin 4-sulfate-6-sulfotransferase; BRAG), the sulfotransferase enzyme that adds 6-sulfate to chondroitin 4-sulfate (C4S) to make chondroitin 4,6-disulfate (chondroitin sulfate E, CSE), was increased in malignant prostate epithelium obtained by laser capture microdissection and following arylsulfatase B (ARSB; N-acetylgalactosamine-4-sulfatase) silencing in human prostate epithelial cells. Experiments in normal and malignant human prostate epithelial and stromal cells and tissues, in HepG2 cells, and in the ARSB-null mouse were performed to determine the pathway by which CHST15 expression is up-regulated when ARSB expression is reduced. Effects of Wnt-containing prostate stromal cell spent media and selective inhibitors of WNT, JNK, p38, SHP2, β-catenin, Rho, and Rac-1 signaling pathways were determined. Activation of WNT signaling followed declines in ARSB and Dickkopf WNT Signaling Pathway Inhibitor (DKK)3 and was required for increased CHST15 expression. The increase in expression of CHST15 followed activation of non-canonical WNT signaling and involved Wnt3A, Rac-1 GTPase, phospho-p38 MAPK, and nuclear DNA-bound GATA-3. Inhibition of JNK, Sp1, β-catenin nuclear translocation, or Rho kinase had no effect. Consistent with higher expression of CHST15 in prostate epithelium, disaccharide analysis showed higher levels of CSE and chondroitin 6-sulfate (C6S) disaccharides in prostate epithelial cells. In contrast, chondroitin 4-sulfate (C4S) disaccharides were greater in prostate stromal cells. CSE may contribute to increased C4S in malignant epithelium when GALNS (N-aceytylgalactosamine-6-sulfate sulfatase) is increased and ARSB is reduced. These effects increase chondroitin 4-sulfates and reduce chondroitin 6-sulfates, consistent with enhanced stromal characteristics and epithelial-mesenchymal transition.

Epidermal growth factor receptor (EGFR) has a crucial role in cell differentiation and proliferation and cancer, and its expression appears to be up-regulated when arylsulfatase B (ARSB or GalNAc-4-sulfatase) is reduced. ARSB removes 4-sulfate groups from the nonreducing end of dermatan sulfate and chondroitin 4-sulfate (C4S), and its decreased expression has previously been reported to inhibit the activity of the ubiquitous protein-tyrosine phosphatase, nonreceptor type 11 (SHP2 or PTPN11). However, the mechanism by which decline in ARSB leads to decline in SHP2 activity is unclear. Here, we show that SHP2 binds preferentially C4S, rather than chondroitin 6-sulfate, and confirm that SHP2 activity declines when ARSB is silenced. The reduction in ARSB activity, and the resultant increase in C4S, increased the expression of EGFR (Her1/ErbB1) in human prostate stem and epithelial cells. The increased expression of EGFR occurred after 1) the decline in SHP2 activity, 2) enhanced c-Jun N-terminal kinase (JNK) activity, 3) increased nuclear DNA binding by c-Jun and c-Fos, and 4) EGFR promoter activation. In response to exogenous EGF, there was increased bromodeoxyuridine incorporation, consistent with enhanced cell proliferation. These findings indicated that ARSB and chondroitin 4-sulfation affect the activation of an important dual phosphorylation threonine-tyrosine kinase and the mRNA expression of a critical tyrosine kinase receptor in prostate cells. Restoration of ARSB activity with the associated reduction in C4S may provide a new therapeutic approach for managing malignancies in which EGFR-mediated tyrosine kinase signaling pathways are active.

The chondroitin sulfatases N-acetylgalactosamine-4-sulfatase (ARSB) and galactosamine-N-acetyl-6-sulfatase (GALNS) remove either the 4-sulfate group at the non-reducing end of chondroitin 4-sulfate (C4S) and dermatan sulfate, or the 6-sulfate group of chondroitin 6-sulfate, chondroitin 4,6-disulfate (chondroitin sulfate E), or keratan sulfate. In human prostate cancer tissues, the ARSB activity was reduced and the GALNS activity was increased, compared to normal prostate tissue. In human prostate stem cells, when ARSB was reduced by silencing or GALNS was increased by overexpression, activity of SHP2, the ubiquitous non-receptor tyrosine phosphatase, declined, attributable to increased binding of SHP2 with C4S. This led to increases in phospho-ERK1/2, Myc/Max nuclear DNA binding, DNA methyltransferase (DNMT) activity and expression, and methylation of the Dickkopf Wnt signaling pathway inhibitor (DKK)3 promoter and to reduced DKK3 expression. Since DKK3 negatively regulates Wnt/β-catenin signaling, silencing of ARSB or overexpression of GALNS disinhibited (increased) Wnt/β-catenin signaling. These findings indicate that the chondroitin sulfatases can exert profound effects on Wnt-mediated processes, due to epigenetic effects that modulate Wnt signaling.

BACKGROUND: Carrageenan is a very common food additive in Western diets, but predictably causes inflammation in thousands of cell-based and animal experiments. OBJECTIVE: To assess the impact of carrageenan exposure on the interval to relapse in patients with ulcerative colitis in remission. METHODS: A randomized, double-blind, placebo-controlled, multicenter, clinical trial was conducted to assess if patients with ulcerative colitis in remission would have a longer interval to relapse if they followed a diet with no carrageenan. All participants were instructed in the no-carrageenan diet and were randomized to either placebo capsules or carrageenan-containing capsules. The carrageenan in the capsules was less than the average daily carrageenan intake from the diet. Relapse was defined as an increase of two or more points on the Simple Clinical Colitis Activity Index (SCCAI) and intensification of treatment for ulcerative colitis. Participants were followed by telephone calls every two weeks until relapse or one year of participation. The occurrence of relapse and inflammatory biomarkers were compared between the two groups. RESULTS: Twelve patients completed study questionnaires. Three patients who received carrageenan-containing capsules relapsed, and none of the patients who received placebo-containing capsules relapsed (p = 0.046, log-rank test). Laboratory tests showed increases in Interleukin-6 (p = 0.02, paired t-test, two-tailed) and fecal calprotectin (p = 0.06; paired t-test, two-tailed) between the beginning and the end of study participation in the carrageenan-exposed group, but not in the placebo-group. CONCLUSION: Carrageenan intake contributed to earlier relapse in patients with ulcerative colitis in remission. Restriction of dietary carrageenan may benefit patients with ulcerative colitis.

Objectives. Carrageenan is well known to cause inflammation and is used in laboratory experiments to study mediators and treatments of inflammation. However, carrageenan is added to hundreds of processed foods to improve texture. Previous work indicated that low concentrations of carrageenan in drinking water caused marked glucose intolerance and insulin resistance in a mouse model. This exploratory, clinical study tested the impact of the no-carrageenan diet in prediabetes. Research Design and Methods. Participants with prediabetes (n=13), defined as HbA1c of 5.7%-6.4%, enrolled in a 12-week, randomized, parallel-arm, feeding trial. One group (n=8) was provided all meals and snacks with no carrageenan. A second group (n=5) received a similar diet with equivalent content of protein, fat, and carbohydrate, but with carrageenan. Blood samples were collected at baseline and during oral glucose tolerance tests at 6 and 12 weeks. The primary outcome measure was changed in %HbA1c between baseline and 12 weeks. Statistical analysis included paired and unpaired t-tests, correlations, and 2×2 ANOVAs. Results. Subjects on no carrageenan had declines in HbA1c and HOMA-IR (p=0.006, p=0.026; paired t-test, two tailed). They had increases in C-peptide (p=0.029) and Matsuda Index (2.1±0.7 to 4.8±2.3; p=0.052) and declines in serum IL-8, serum galectin-3, and neutrophil phospho-(Ser307/312)-IRS1 (p=0.049, p=0.003, and p=0.006; paired t-tests, two tailed). Subjects on the diet with carrageenan had no significant changes in these parameters. Significant differences between no-carrageenan and carrageenan-containing diet groups for changes from baseline to 12 weeks occurred in C-peptide, phospho-Ser-IRS1, phospho-AKT1, and mononuclear cell arylsulfatase B (p=0.007, p=0.038, p=0.0012, and p=0.0008; 2×2 ANOVA). Significant correlations were evident between several of the variables. Conclusions. Findings indicate improvement in HbA1c and HOMA-IR in participants on no-carrageenan diets, but not in participants on carrageenan-containing diets. Significant differences between groups suggest that removing carrageenan may improve insulin signaling and glucose tolerance. Larger studies are needed to further consider the impact of carrageenan on development of diabetes.

The enzymes arylsulfatase B (ARSB; N-acetylgalactosamine-4-sulfatase) and galactose-6-sulfate sulfatase (GALNS; N-acetylgalactosamine-6-sulfatase) remove either the 4-sulfate group at the non-reducing end of chondroitin 4-sulfate (C4S) and dermatan sulfate, or the 6-sulfate group of chondroitin 6-sulfate, chondroitin 4,6-disulfate (chondroitin sulfate E), or keratan sulfate. In normal and malignant prostate tissues obtained by laser-capture microdissection, ARSB expression is greater in stroma than epithelium and GALNS expression is greater in epithelium than stroma. In malignant epithelium, GALNS expression is greater and ARSB expression is less than in normal epithelium. In cultured, normal human prostate stem and epithelial cells, ARSB silencing and GALNS overexpression exert similar signaling effects, attributable to enhanced binding of C4S with SHP2 (PTPN11), leading to SHP2 inhibition and sustained phosphorylation of phospho-ERK1/2 and other critical signaling molecules. Experiments in developing rat prostate showed that exposure to estrogen significantly reduced ARSB activity, whereas GALNS activity increased at post-natal day 30. To further assess how changes in sulfatases may contribute to prostate development, transcription factor activation profiling plate arrays (Signosis) were performed in cultured human prostate stem cells (CRL-2887, ATCC). Cells were silenced for ARSB (EC 3.1.6.12) or GALNS (EC 3.1.6.4) by specific siRNA (Qiagen) or ARSB was overexpressed [for ARSB (NCBI NM_000046, transcript variant 1; for GALNS (NCBI NM_000512; TrueClone, Origene] by transfection of specific untagged plasmids. Biotin-labeled transcription factor probes based on the known DNA-binding consensus sequences were detected in nuclear extracts of the treated and control stem cell preparations. Nuclear DNA bound transcription factor Gli declined 35% when ARSB was silenced and 63% when GALNS was overexpressed. Inversely, nuclear bound Gli increased 62% when ARSB was overexpressed and 42% when GALNS was silenced. These findings are consistent with the observed inverse signaling effects of ARSB and GALNS in the prostate cells. Also, Gli mRNA expression was 3.50 ± 0.22 times greater in non-malignant epithelium than in malignant epithelium, consistent with decline when GALNS is greater and ARSB is reduced. Nuclear binding of TCF/Lef and c-Myc showed inverse effects in relation to GALNS silencing and overexpression, declining when GALNS was silenced and increasing when GALNS was overexpressed. These effects are consistent with the observed effects on Wnt signaling. These observations indicate that changes in chondroitin sulfates mediated by chondroitin sulfatases may have profound consequences on developmental programming in prostate tissue and suggest that hormonal-mediated effects may be transcribed through changes in sulfatases and chondroitin sulfates. Citation Format: Joanne Kramer Tobacman, Sumit Bhattacharyya, Leo Feferman. Chondroitin sulfatases and transcription factors Gli, Tcf/Lef, and c-Myc in prostate stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2442.

Background The enzyme Arylsulfatase B (ARSB; N-acetylgalactosamine 4-sulfatase), is required for degradation of sulfated glycosaminoglycans (GAGs) which accumulate in cystic fibrosis. ARSB is reduced in cystic fibrosis cells and increases when defective CFTR is repaired by insertion of the normal gene. This study was undertaken to determine if modification of CFTR by small molecule correctors or potentiators could also increase ARSB and reduce the accumulation of chondroitin 4-sulfate (C4S). Methods CF bronchial epithelial cells homozygous for the F508 deletion (ACD#14071) and normal human bronchial epithelial cells (BEC) were grown and differentiated following an established protocol. Cells were treated with either VRT-532, a CFTR potentiator, or VRT-534, a CFTR corrector, or vehicle control. The impact on ARSB activity, protein and mRNA expression, C4S and total sulfated glycosaminoglycan content, Interleukin-8 and Interleukin-6 secretion, and neutrophil chemotaxis was determined by specific assays. Results The CFTR potentiator, but not the corrector, increased ARSB activity and expression to the level in the normal bronchial epithelial cells (BEC). Concomitantly, total sulfated glycosaminoglycans and C4S declined, secreted IL-8 increased, secreted IL-6 declined, and neutrophil chemotaxis to the spent media obtained from the potentiator-treated CF cells increased. Conclusion The CFTR potentiator increased ARSB activity and expression and associated effects. This suggests that a critical interaction between CFTR and ARSB is related to CFTR function in regulation of a ligand-gated anion channel at the cell membrane, rather than to CFTR processing and intracellular trafficking.

Carrageenan has been used in the laboratory for decades because it reliably causes inflammation. Coincident with its use in the laboratory, carrageenan has been used in an ever-increasing list of processed foods, as well as in infant formula, pharmaceuticals, cosmetics, toothpaste, room air deodorizers, and other products. The biological reactivity of carrageenan makes it a useful substance to improve the texture and solubility of ingredients in processed foods. Individuals may consume several grams of carrageenan on a daily basis. However, the potential contribution of carrageenan exposure to chronic human diseases, due to its activation of immune responses and its potent inflammatory properties, must be recognized.

Arylsulfatase B (ARSB; N-acetylgalactosamine-4-sulfatase) is the enzyme that removes 4-sulfate groups from chondroitin 4-sulfate (C4S) and dermatan sulfate. In previous work, we have shown decline in ARSB with increasing aggressiveness of melanoma cell lines. C4S increased as ARSB declined in the cultured melanoma cell lines. Decline in ARSB was associated with increased expression of chondroitin sulfate proteoglycan (CSPG) 4 and matrix metalloproteinase (MMP) 2. The increase in CSPG4 was attributed to reduced binding of galectin-3 with C4S, and the increase of MMP2 was attributed to inhibition of SHP2, the tyrosine-protein phosphatase nonreceptor type 11 (PTPN11), due to increased binding with C4S. The invasiveness of the cultured melanoma cells increased when ARSB was silenced by siRNA. To further address the role of ARSB in melanoma progression, metastatic melanoma tissue was obtained from the biorepository of the University of Illinois at Chicago. ARSB activity and expression were reduced in the metastatic tissues and ARSB promoter methylation was increased. ARSB promoter methylation was also increased in cultured metastatic melanoma cells compared to normal melanocytes and to radial growth phase melanoma cells. In the metastatic tissues from skin and liver, SHP2 activity declined, and mRNA expression of MMP2 and CSPG4 increased. When ARSB was silenced by siRNA in the cultured normal melanocytes, Programmed Death-Ligand (PD-L) 1 increased from 78 ± 7 pg/mg protein to 128 ± 11 pg/mg protein by ELISA (p Citation Format: Joanne K. Tobacman, Sumit Bhattacharyya, Leo Feferman. Arylsulfatase B (ARSB) is reduced in melanoma metastases and decline in ARSB increases programmed death-ligand (PDL) 1 [abstract]. In: Proceedings of the AACR Special Conference on Melanoma: From Biology to Target; 2019 Jan 15-18; Houston, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(19 Suppl):Abstract nr B21.

CHST15 (carbohydrate sulfotransferase 15; chondroitin 4-sulfate-6-sulfotransferase), is the sulfotransferase enzyme which adds 6-sulfate to chondroitin 4-sulfate to make chondroitin 4,6-sulfate, known as CSE. Increased CHST15 expression has been identified in malignant pancreas, renal, colon, ovary, and prostate tissues. We now report a pathway by which expression of CHST15 is increased following decline in the enzyme ARSB (arylsulfatase B; N-acetylgalactosamine-4-sulfatase), which is required for removal of 4-sulfate groups from chondroitin 4-sulfate (C4S). Previous work has shown that decline in ARSB and the resulting increase in C4S leads to decline in activity of SHP2, a ubiquitous non-receptor tyrosine phosphatase, due to enhanced binding of SHP2 with C4S when ARSB is reduced. Decline in SHP2 leads to enhanced ERK1/2 phosphorylation and subsequent downstream effects, including hypermethylation of the DKK (Dickkopf WNT signaling pathway inhibitor)3 promoter and suppressed DKK3 expression. Since DKK3 acts to inhibit Wnt signaling, Wnt signaling is enhanced in prostate epithelium when ARSB is reduced. Experiments were performed to identify the pathway by which CHST15 expression is up-regulated when ARSB is reduced and to assess the role of Wnt. The impact of ARSB silencing on CHST15 expression was determined in normal and malignant human prostate cells, in normal and malignant prostate tissue obtained by laser-capture microdissection, in HepG2 cells, and in prostate tissue of the ARSB-null mouse. Effects of selective inhibitors of Wnt and MAPK signaling pathways on CHST15 expression were evaluated. CHST15 expression was increased in malignant prostate epithelial tissue, in ARSB-null mouse prostate tissue, and following ARSB silencing in human prostate epithelial cells. The activation of Wnt signaling which followed declines in ARSB and DKK3 was required for increased CHST15 expression. Inhibition of Wnt3A blocked the increase in CHST15 expression in prostate epithelial cells co-cultured with prostate stromal cells, whereas DKK3 antibody increased CHST15. Inhibition of Rac-1 GTPase and of phospho-p38 MAPK signaling blocked the observed increase in CHST15 expression, whereas inhibition of JNK, Sp1, or of Rho kinase had no effect. These results indicate that the increase in epithelial CHST15 occurred in the malignant prostate cells and tissue due to effects of non-canonical Wnt signaling leading to enhanced phospho-p38 MAPK. The resultant increase in CHST15 when ARSB activity is reduced may contribute to determination of epithelial vs. stromal characteristics. Citation Format: Joanne Kramer Tobacman, Sumit Bhattacharyya, Leo Feferman. Increased CHST15 expression follows declines in Arylsulfatase B (ARSB) and DKK3 and disinhibition of non-canonical WNT signaling [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4698.

Arylsulfatase B (ARSB; N-acetylgalactosamine-4-sulfatase) is the enzyme that removes 4-sulfate groups from chondroitin 4-sulfate (C4S) and dermatan sulfate. In previous work, we have shown decline in ARSB with increasing aggressiveness of melanoma cell lines and in association with more aggressive prostate cancers. As ARSB decreases, chondroitin 4-sulfate increases, leading to increased binding of SHP2 (PTP11; non-receptor tyrosine phosphatase) and reduced binding of galectin-3. Subsequent effects include increase in phospho-ERK1/2 and transcriptional effects, including hypermethylation of the DKK3 promoter and reduced expression of DKK (Dickkopf WNT pathway signaling inhibitor) 3, leading to activation of Wnt signaling. Other transcriptional events following silencing of ARSB include: increased expression of CSPG4 and MMP-2 in melanoma cells; increased expression of versican in prostate epithelial cells; increased expression of Wnt9A in colonic epithelial cells; and increased MITF-mediated expression of GPNMB in hepatic cells. Invasiveness of cultured melanoma cells increased when ARSB was silenced by siRNA. To further address the role of ARSB, metastatic melanoma tissue was obtained from the biorepository of the University of Illinois at Chicago. Expression of CSPG4 and MMP2 was increased in the metastatic tissues and SHP2 activity was reduced. When ARSB was silenced by siRNA in cultured normal melanocytes (PCS 200-013, ATCC), Programmed Death-Ligand (PD-L)1 increased from 78 ± 7 pg/mg protein to 128 ± 11 pg/mg protein (ELISA assay; p Citation Format: Joanne Kramer Tobacman, Sumit Bhattacharyya, Leo Feferman. Decline in Arylsulfatase B (ARSB) increases PD-L1 expression in melanoma, hepatic, prostate, and mononuclear cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4699.

Exposure to low concentration of the common food additive carrageenan (CGN) leads to impaired glucose tolerance and insulin resistance in C57BL/6J mice, due to CGN’s pro-inflammatory effects and changes in phospho-IRS1 phosphorylation. CGN is a sulfated polysaccharide with α-1,3 and β-1,4 galactosidic bonds. Other studies showed: 1) CGN reduced activity of arylsulfatase B, which removes sulfate groups from N-acetylgalactosamine 4-sulfate residues of chondroitin 4-sulfate (C4S) and dermatan sulfate; and 2) galectin-3 binds less with C4S when ARSB is reduced. Increase in galectin-3, which binds to β-galactosides, was reported to inhibit insulin signaling (Li P et al) by effects on the insulin receptor. This background led to the current assessment of effects of the no-CGN diet on galectin-3 levels in serum and galectin-3 mRNA in circulating mononuclear cells. Blood samples were obtained from adults with prediabetes before and after dietary intervention. Baseline serum galectin-3 values averaged 8.94 ng/ml (range 4.06-12.64). In 7 patients on a no-CGN diet for 12 weeks, the mean level declined to 7.25 ng/ml (p=0.006, paired t-test). In contrast, serum galectin-3 was unchanged in participants on the CGN-containing control diet and increased by over 2 ng/ml in subjects on their regular diet. mRNA expression of galectin-3 (NGALS3) in circulating mononuclear cells of patients on the no-CGN diet declined by 69%, and increased by 36% in the CGN-containing diet group and by 98% in the regular diet group (p=0.0002, 1-way ANOVA). Also, serum galectin-3 levels increased to 1.72 times baseline in C57BL/6J mice given CGN in their water supply for 12 weeks. In HepG2 cells, exogenous galectin-3 (25 µg/ml) inhibited insulin-induced glucose uptake by 39%, and was highly correlated with decline in tyrosine phosphorylation of IRS-1. These findings suggest that CGN contributes to glucose intolerance and insulin resistance, in part, by effects on galectin-3. Disclosure J.K. Tobacman: None. S. Bhattacharyya: None. L. Feferman: None.

Previous animal and cell-based studies indicated that exposure to the common food additive carrageenan impaired glucose tolerance and increased insulin resistance. Carrageenan is widely used in processed foods to improve solubility, but predictably causes inflammation, which contributes to insulin resistance by inhibition of insulin signaling. To test if removal of carrageenan from the diet could reduce insulin resistance, we initiated a double-blinded, controlled feeding study in individuals with prediabetes, defined by Hemoglobin A1c values of 5.7%-6.4%. All procedures were approved by the University of Illinois at Chicago IRB. Participants were provided study diets for 12 weeks, which either contained or did not contain carrageenan, and had similar composition of protein, carbohydrate, and fat. An additional group was comprised of individuals who participated in study tests, but ate their usual diet. Subjects on study diets picked up their food weekly at UIC and had blood tests, including 2h glucose tolerance tests performed at entry, 6, and 12 weeks. Inflammatory parameters, including fecal calprotectin, were measured at entry and conclusion. Preliminary study results from 17 participants indicate that the carrageenan-free diet was associated with significant decline in hemoglobin A1c (p=0.004, unpaired t-test, two-tailed), HOMA-IR (homeostatic model for insulin resistance) score (p=0.0064), insulin resistance (p=0.02), and leukocyte phospho-serine IRS-1 (p=0.003) and NF-kappaB, compared to study participants on the control study diet or their regular diet. Study findings indicate significant decline in insulin resistance by removal of carrageenan from the diet. Additional studies of inflammatory parameters, immune cell subsets, and fecal microbiome are pending. Disclosure J.K. Tobacman: None.

In an established rat model of penetrating ballistic-like brain injury (PBBI), arylsulfatase B (ARSB; N-acetylgalactosamine 4-sulfatase) activity was significantly reduced at the ipsilateral site of injury, but unaffected at the contralateral site or in sham controls. In addition, the ARSB substrate chondroitin 4-sulfate (C4S) and total sulfated glycosaminoglycans increased. The mRNA expression of chondroitin 4-sulfotransferase 1 (C4ST1; CHST11) and the sulfotransferase activity rose at the ipsilateral site of injury (PBBI-I), indicating contributions from both increased production and reduced degradation to the accumulation of C4S. In cultured, fetal rat astrocytes, following scratch injury, the ARSB activity declined and the nuclear hypoxia inducible factor-1α increased significantly. In contrast, sulfotransferase activity and chondroitin 4-sulfotransferase expression increased following astrocyte exposure to TGF-β1, but not following scratch. These different pathways by which C4S increased in the cell preparations were both evident in the response to injury in the PBBI-I model. Hence, findings support effects of injury because of mechanical disruption inhibiting ARSB and to chemical mediation by TGF-β1 increasing CHST11 expression and sulfotransferase activity. The increase in C4S following traumatic brain injury is because of contributions from impaired degradation and enhanced synthesis of C4S which combine in the pathogenesis of the glial scar. This is the first report of how two mechanisms contribute to the increase in chondroitin 4-sulfate (C4S) in TBI. Following penetrating ballistic-like brain injury in a rat model and in the scratch model of injury in fetal rat astrocytes, Arylsulfatase B activity declined, leading to accumulation of C4S. TGF-β1 exposure increased expression of chondroitin 4-sulfotransferase. Hence, the increase in C4S in TBI is attributable to both impaired degradation and enhanced synthesis, combining in the pathogenesis of the glial scar.

Inflammation induced by exposure to the common food additive carrageenan leads to insulin resistance by increase in Ser(P)(307)-insulin receptor substrate 1 (IRS1) and subsequent decline in the insulin-stimulated increase in Ser(P)(473)-AKT. Inhibition of carrageenan-induced inflammation reversed the increase in Ser(P)(307)-IRS1 but did not completely reverse the carrageenan-induced decline in Ser(P)(473)-AKT. To identify the additional mechanism responsible for the decrease in Ser(P)(473)-AKT, studies were performed in human HepG2 cells and in C57BL/6J mice. Following carrageenan, expression of GRB10 (growth factor receptor-bound 10 protein), an adaptor protein that binds to the insulin receptor and inhibits insulin signaling, increased significantly. GRB10 silencing blocked the carrageenan-induced reduction of the insulin-stimulated increase in Tyr(P)-IRS1 and partially reversed the decline in Ser(P)(473)-AKT. The combination of GRB10 silencing with BCL10 silencing and the reactive oxygen species inhibitor Tempol completely reversed the decline in Ser(P)(473)-AKT. After carrageenan, GRB10 promoter activity was enhanced because of activation by GATA2. A direct correlation between Ser(P)(473)-AKT and Ser(P)(401)-GATA2 was evident, and inhibition of AKT phosphorylation by the PI3K inhibitor LY294002 blocked Ser(401)-GATA2 phosphorylation and the increase in GRB10 expression. Studies indicated that carrageenan inhibited insulin signaling by two mechanisms: through the inflammation-mediated increase in Ser(P)(307)-IRS1, a negative regulator of insulin signaling, and through a transcriptional mechanism leading to increase in GRB10 expression and GRB10-inhibition of Tyr(P)-IRS1, a positive regulator of insulin signaling. These mechanisms converge to inhibit the insulin-induced increase in Ser(P)(473)-AKT. They provide internal feedback, mediated by Ser(P)(473)-AKT, Ser(P)(401)-GATA2, and nuclear GATA2, which links the opposing effects of serine and tyrosine phosphorylations of IRS1 and can modulate insulin responsiveness.

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.

BACKGROUND Arylsulfatase B (ARSB) removes the 4-sulfate group from chondroitin 4-sulfate (C4S) and dermatan sulfate and is required for their degradation. Prior work showed that ARSB immunohistochemical scores were lower in malignant prostate tissue, and were associated with higher Gleason scores and recurrence. OBJECTIVE This study aims to confirm that ARSB immunostaining of prostate tissue obtained at the time of radical prostatectomy is prognostic for prostate cancer recurrence. METHODS Intensity and distribution of ARSB immunostaining were digitally analyzed in a large, well-annotated, prostate cancer tissue microarray (TMA). Scores were calculated for stroma and epithelium and compared for 191 cases, including 36 recurrences, defined as PSA > 0.2 ng/ml. RESULTS Epithelial scores were significantly lower in the recurrences (p= 0.010), and among subgroups with age > 60, initial PSA > 6 ng/ml, or Gleason grade = 7. ARSB score did not improve the prediction of recurrence in multifactorial analysis. CONCLUSIONS Study findings validate previous findings and provide further evidence that lower ARSB is associated with prostate cancer recurrence. Additional studies are required to assess if there are specific cutoff values that may help predict recurrence.

GPNMB was increased in a CF gene array and in Arylsulfatase B (ARSB; N-acetylgalactosamine-4-sulfatase)-null mice, consistent with previous reports that ARSB is reduced in cystic fibrosis (CF). Implications of GPNMB increase in CF are unknown.GPNMB levels were determined in serum and circulating leukocytes from CF patients and healthy controls. GPNMB binding with β-1 integrin and measurements of phospho-ERK1/2 and MMP-9 in CFTR-uncorrected, CFTR-corrected, and normal human bronchial epithelial cells (BEC) were determined, following ARSB and GPNMB knockdown, and treatment with RGD peptide, and ERK phosphorylation inhibitor.GPNMB was markedly increased in CF patients compared to controls (p 0.0001, unpaired t-test, two-tailed). Silencing GPNMB, treatment with excess RGD peptide, and treatment with ERK phosphorylation inhibitor blocked ARSB silencing-induced increases in MMP-9 in the normal BEC.Findings suggest that decline in ARSB activity caused by decline in CFTR function leads to increased GPNMB, which may contribute to organ dysfunction in CF by increased MMP-9 expression.

In cultured human colonic epithelial cells and mouse colonic tissue, exposure to the common food additive carrageenan leads to inflammation, activation of Wnt signaling, increased Wnt9A expression, and decline in the activity of the enzyme arylsulfatase B (ARSB; N-acetylgalactosamine-4-sulfatase). In this study, the novel transcriptional mechanism by which carrageenan and decline in ARSB increase Wnt9A expression in NCM460 and HT-29 human colonic epithelial cells and in mouse colon is presented. Increased expression of Wnt9A has been associated with multiple malignancies, including colon carcinoma, and with ectodermal and mesoendodermal morphogenesis. When ARSB activity was reduced by siRNA or by exposure to carrageenan (1 μg/ml for 24 h), degradation of chondroitin 4-sulfate (C4S) was inhibited, leading to accumulation of more highly sulfated C4S, which binds less galectin-3, a β-galactoside-binding protein. Nuclear galectin-3 increased and mediated increased binding of Sp1 to the Sp1 consensus sequence in the Wnt9A promoter, shown by oligonucleotide-binding assay and by chromatin immunoprecipitation assay. When galectin-3 was silenced, the increases in Sp1 binding to the Wnt9A promoter and in Wnt9A expression, which followed carrageenan or ARSB silencing, were inhibited. Mithramycin A, a specific inhibitor of Sp1 oligonucleotide binding, and Sp1 siRNA blocked the carrageenan- and ARSB siRNA-induced increases in Wnt9A expression. These studies reveal how carrageenan exposure can lead to transcriptional events in colonic epithelial cells through decline in arylsulfatase B activity, with subsequent impact on C4S, galectin-3, Sp1, and Wnt9A and can exert significant effects on Wnt-initiated signaling and related vital cell processes.

BACKGROUND The enzyme Arylsulfatase B (ARSB; N-acetylgalactosamine-4-sulfatase) degrades chondroitin-4-sulfate (C4S) and is reduced in malignant colonic and mammary tissues, but has not previously been evaluated in prostate cancer. METHODS ARSB immunostaining was performed on two tissue microarrays (TMA) and analyzed by digital image analysis, generating ARSB H-scores for prevalence and intensity of epithelial, stromal, and combined epithelial and stromal immunostaining. Also, paired malignant and normal prostate tissues were analyzed for ARSB activity, C4S, total sulfated glycosaminoglycans, and versican content. The quantities of C4S and of the epidermal growth factor receptor that co-immunoprecipitated with versican were determined in the normal and malignant paired prostate tissues. RESULTS 44 cases of prostate cancer were paired by age (± 5y), race, Gleason score (in order), and pathologic TNM score. The pairs differed by recurrence vs. non-recurrence of elevated PSA at 4 or more years. When TMA cores were analyzed for ARSB H-score, 18 of the 22 pairs had lower ARSB H-scores in the recurrent member of the pair, whereas higher initial PSA values were associated with recurrence in only 65% of the paired cases. In a second TMA, Gleason scores 6 and 7 were associated with higher ARSB H-scores than Gleason scores 8 and 9 for stroma, epithelium, and stroma and epithelium combined (p=0.052, p=0.015, p

N -acetylgalactosamine-4-sulfatase (Arylsulfatase B; ARSB) is the enzyme that removes sulfate groups from the N-acetylgalactosamine-4-sulfate residue at the non-reducing end of chondroitin-4-sulfate (C4S) and dermatan sulfate (DS). Previous studies demonstrated reduction in cell-bound high molecular weight kininogen in normal rat kidney (NRK) epithelial cells when chondroitin-4-sulfate content was reduced following overexpression of ARSB activity, and chondroitinase ABC produced similar decline in cell-bound kininogen. Reduction in the cell-bound kininogen was associated with increase in secreted bradykinin. In this report, we extend the in vitro findings to in vivo models, and present findings in Dahl salt-sensitive (SS) rats exposed to high (SSH) and low salt (SSL) diets. In the renal tissue of the SSH rats, ARSB activity was significantly less than in the SSL rats, and chondroitin-4-sulfate and total sulfated glycosaminoglycan content were significantly greater. Disaccharide analysis confirmed marked increase in C4S disaccharides in the renal tissue of the SSH rats. In contrast, unsulfated, hyaluronan-derived disaccharides were increased in the rats on the low salt diet. In the SSH rats, with lower ARSB activity and higher C4S levels, cell-bound, high-molecular weight kininogen was greater and urinary bradykinin was lower. ARSB activity in renal tissue and NRK cells declined when exogenous chloride concentration was increased in vitro. The impact of high chloride exposure in vivo on ARSB, chondroitin-4-sulfation, and C4S-kininogen binding provides a mechanism that links dietary salt intake with bradykinin secretion and may be a factor in blood pressure regulation.

The common food additive carrageenan is a known activator of inflammation in mammalian tissues and stimulates both the canonical and noncanonical pathways of NF-κB activation. Exposure to low concentrations of carrageenan (10 μg/mL in the water supply) has produced glucose intolerance, insulin resistance, and impaired insulin signaling in C57BL/6 mice. B-cell leukemia/lymphoma 10 (Bcl10) is a mediator of inflammatory signals from Toll-like receptor (TLR) 4 in myeloid and epithelial cells. Since the TLR4 signaling pathway is activated in diabetes and by carrageenan, we addressed systemic and intestinal inflammatory responses following carrageenan exposure in Bcl10 wild type, heterozygous, and null mice. Fecal calprotectin and circulating keratinocyte chemokine (KC), nuclear RelA and RelB, phospho(Thr559)-NF-κB-inducing kinase (NIK), and phospho(Ser36)-IκBαin the colonic epithelial cells were significantly less (P<0.001) in the carrageenan-treated Bcl10 null mice than in controls. IL-10-deficient mice exposed to carrageenan in a germ-free environment showed an increase in activation of the canonical pathway of NF-κB (RelA) activation, but without increase in RelB or phospho-Bcl10, and exogenous IL-10 inhibited only the canonical pathway of NF-κB activation in cultured colonic cells. These findings demonstrate a Bcl10 requirement for maximum development of carrageenan-induced inflammation and lack of complete suppression by IL-10 of carrageenan-induced inflammation.

Arylsulfatase B (ARSB; N-acetylgalactosamine 4-sulfatase) is reduced in several malignancies, but levels in melanoma have not been investigated previously. Experiments were performed in melanoma cell lines to determine ARSB activity and impact on melanoma invasiveness. ARSB activity was reduced ~50% in melanoma cells compared to normal melanocytes. Silencing ARSB significantly increased the mRNA expression of chondroitin sulfate proteoglycan(CSPG)4 and pro-matrix metalloproteinase(MMP)-2, known mediators of melanoma progression. Also, invasiveness and MMP activity increased when ARSB was reduced, and recombinant ARSB inhibited invasiveness and MMP activity. Since the only known function of ARSB is to remove 4-sulfate groups from the N-acetylgalactosamine 4-sulfate residue at the non-reducing end of chondroitin 4-sulfate (C4S) or dermatan sulfate, experiments were performed to determine the transcriptional mechanisms by which expression of CSPG4 and MMP2 increased. Promoter activation of CSPG4 was mediated by reduced binding of galectin-3 to C4S when ARSB activity declined. In contrast, increased pro-MMP2 expression was mediated by increased binding of the non-receptor tyrosine phosphatase SHP2 to C4S. Increased phospho-ERK1,2 resulted from SHP2 inhibition. Combined effects of increased C4S, CSPG4, and MMP2 increased the invasiveness of the melanoma cells, and therapy with recombinant ARSB may inhibit melanoma progression.

The enzyme Arylsulfatase B (ARSB; N-acetylgalactosamine-4-sulfatase) removes 4-sulfate groups from chondroitin-4-sulfate and dermatan sulfate and is required for the degradation of these sulfated glycosaminoglycans (sGAGs). Since these GAGs accumulate in patients with Cystic Fibrosis (CF), we investigated the activity of ARSB in leukocytes of patients with CF, to consider if reduced activity of ARSB might contribute to the pathophysiology of CF. Previous cell-based experiments had demonstrated that when the deficiency of the cystic fibrosis transmembrane regulator (CFTR) was corrected in bronchial epithelial cells, the ARSB activity increased significantly. De-identified, citrated blood samples were collected from 16 children with CF and 31 control subjects, seen in the Pediatric Clinic at Rush University Medical Center. Polymorphonuclear leukocytes (PMN) and mononuclear cell (MC) populations were separated by density gradient, and blinded determinations of ARSB activity were performed using the exogenous substrate 4-methylumbilliferyl sulfate. Interleukin-6 was measured in the plasma samples by ELISA. ARSB activity was significantly less in the PMN and MC from the CF patients than controls (P

Tumor necrosis factor (TNF)-α, a homotrimeric, pleiotropic cytokine, is secreted in response to inflammatory stimuli in diseases such as rheumatoid arthritis and inflammatory bowel disease. TNF-α mediates both apoptosis and inflammation, stimulating an inflammatory cascade through the non-canonical pathway of NF-κB activation, leading to increased nuclear RelB and p52. In contrast, the common food additive carrageenan (CGN) stimulates inflammation through both the canonical and non-canonical pathways of NF-κB activation and utilizes the adaptor molecule BCL10 (B-cell leukemia/lymphoma 10). In a series of experiments, colonic epithelial cells and mouse embryonic fibroblasts were treated with TNF-α and carrageenan in order to simulate the possible effects of exposure to dietary CGN in the setting of a TNF-α-mediated inflammatory disease process. A marked increase in secretion of IL-8 occurred, attributable to synergistic effects on phosphorylated NF-κB-inducing kinase (NIK) in the non-canonical pathway. TNF-α induced the ubiquitination of TRAF2 (TNF receptor-associated factor 2), which interacts with NIK, and CGN induced phosphorylation of BCL10, leading to increased NIK phosphorylation. These results suggest that TNF-α and CGN in combination act to increase NIK phosphorylation, thereby increasing activation of the non-canonical pathway of NF-κB activation. In contrast, the apoptotic effects of TNF-α, including activation of caspase-8 and PARP-1 (poly(ADP-ribose) polymerase 1) fragmentation, were markedly reduced in the presence of CGN, and CGN caused reduced expression of Fas. These findings demonstrate that exposure to CGN drives TNF-α-stimulated cells toward inflammation rather than toward apoptotic cell death and suggest that CGN exposure may compromise the effectiveness of anti-TNF-α therapy.

Background and aims B-cell lymphoma/leukemia (BCL)-10 and reactive oxygen species mediate two pathways of NF-κB (RelA) activation by lipopolysaccharide (LPS) in human colonic epithelial cells. The pathway for LPS activation of RelB by the non-canonical pathway (RelB) in non-myeloid cells was not yet reported, but important for understanding the range of potential microbial LPS-induced effects in inflammatory bowel disease. Methods Experiments were performed in human colonic epithelial cells and in mouse embryonic fibroblasts deficient in components of the IkappaB kinase (IKK) signalosome, in order to detect mediators of the non-canonical pathway of NF-κB activation, including nuclear RelB and p52 and phospho- and total NF-κB inducing kinase (NIK). BCL10 was silenced by siRNA and effects of mutations of specific phosphorylation sites of BCL10 (Ser138Gly and Ser218Gly) were determined. Results By the non-canonical pathway, LPS exposure increased nuclear RelB and p52, and phospho-NIK, with no change in total NIK. Phosphorylation of BCL10 serine 138 was required for NIK phosphorylation, since mutation of this residue eliminated the increases in phospho-NIK and nuclear RelB and p52. Mutations of either serine 138 or serine 218 reduced RelA, p50, and phospho-IκBα of the canonical pathway. Effects of LPS stimulation and BCL10 silencing on NIK phosphorylation were demonstrated in confocal images. Conclusions LPS induces activation of both canonical and non-canonical pathways of NF-κB in human colonic epithelial cells, and the non-canonical pathway requires phosphorylations of BCL10 (serine 138) and NIK. These findings demonstrate the important role of BCL10 in mediating LPS-induced inflammation in human colonic epithelial cells and may open new avenues for therapeutic interventions.

The common food additive carrageenan (CGN) predictably induces intestinal inflammation in animal models. Mechanisms of CGN-induced nuclear factor κB and interleukin-8 (IL-8) stimulation include an immune-mediated pathway involving toll-like receptor 4 (TLR4) and B-cell lymphoma/leukemia 10 (BCL10) and a reactive oxygen species (ROS)-mediated pathway. To determine how the structure of CGN contributes to its initiation of inflammation through these two distinct mechanisms, we treated CGNs with galactosidases and carrageenases (CGNases) and determined the impact on IL-8 secretion and BCL10 production. Hydrolysis of CGN by the enzyme α-1→(3,6)-galactosidase significantly reduced increases in IL-8 and BCL10, but other galactosidases tested, including α-1→6-galactosidase, β-1→4-galactosidase and β-1→3,6-galactosidase, had no effect. In contrast, specific κ-CGNases or ι-CGNases, which hydrolyze β-1,4-galactosidic bonds, produced increases in IL-8 and BCL10 attributable to increased exposure of the immunogenic α-1→3-galactosidic epitope of CGN to TLR4. These results were consistent with induction of innate immune response by an interaction of TLR4 with the unusual α-d-Gal-(1→3)-d-Gal epitope present in CGN. Activation of the ROS-mediated pathway was unaffected by treatment of κ-CGN with either κ-CGNase (3 mg/L), α-1→(3,6)-galactosidase (20 mU/ml) or these enzymes in combination, indicating that changes in IL-8 production were attributable to the effects of induction of inflammation on the TLR4-BCL10-mediated innate immune pathway. These findings provide new information about the specificity of carbohydrate-protein interaction between CGN and TLR4 and may help to devise treatments that modify the immune reactivity induced by carbohydrate antigens.

The enzyme arylsulfatase B (N-acetylgalactosamine 4-sulfatase; ASB; ARSB), which removes 4-sulfate groups from the nonreducing end of chondroitin-4-sulfate (C4S;CSA) and dermatan sulfate, has cellular effects, beyond those associated with the lysosomal storage disease mucopolysaccharidosis VI. Previously, reduced ASB activity was reported in cystic fibrosis patients and in malignant human mammary epithelial cell lines in tissue culture compared to normal cells. ASB silencing and overexpression were associated with alterations in syndecan-1 and decorin expression in MCF-7 cells and in IL-8 secretion in human bronchial epithelial cells. In this report, we present the role of ASB in the regulation of the kininogen–bradykinin axis owing to its effect on chondroitin-4-sulfation and the interaction of C4S with kininogen. Silencing or overexpression of ASB in normal rat kidney epithelial cells in tissue culture modified the content of total sulfated glycosaminoglycans (sGAGs), C4S, kininogen, and bradykinin in spent media and cell lysates. Treatment of the cultured cells with chondroitinase ABC also increased the secretion of bradykinin into the spent media and reduced the C4S-associated kininogen. When ASB was overexpressed, the cellular kininogen that associated with C4S declined, suggesting a vital role for chondroitin-4-sulfation in regulating the kininogen–C4S interaction. These findings suggest that ASB, owing to its effect on chondroitin-4-sulfation, may impact on the kininogen–bradykinin axis and, thereby, may influence blood pressure.Because ASB activity is influenced by several ions, including chloride and phosphate, ASB activity may provide a link between salt responsiveness and the bradykinin-associated mechanism of blood pressure regulation.

The chemokine IL-8 is critically important in inflammatory processes in human tissues, and IL-8 interactions with sulfated glycosaminoglycans have been implicated in modification of inflammatory responses in bronchial epithelium. To determine the role of chondroitin-4-sulfate (C4S) in mediating effects of IL-8, we silenced the enzyme N-acetylgalactosamine-4-sulfatase (arylsulfatase B [ASB]) that removes the 4-sulfate group from C4S, in the IB3-1 and C38 bronchial epithelial cell lines and in normal primary bronchial epithelial cells. When ASB was silenced and IL-8 production stimulated by exposure to TNF-alpha, ASB activity declined by roughly 75%, cellular C4S content increased by over 7.5 microg/mg protein, cell-bound IL-8 increased by over 530 pg/mg protein, and secreted IL-8 declined by over 520 pg/mg protein in all cell lines (P0.001). When cell lysates were immunoprecipitated with C4S antibody after ASB silencing and TNF-alpha, the IL-8 content of the immunoprecipitate was approximately 500 pg/mg protein, indicating that most of the cell-bound IL-8 was associated with C4S. Cell fractionation demonstrated that the IL-8 content associated with the cell membranes was about twice that of the cytosolic fraction. Also, ASB appeared to localize in the cell membrane, as well as in lysosomes. Neutrophil attraction to the cell lysates increased after ASB silencing, consistent with increased attraction to the cell-bound IL-8. These findings provide evidence for the influential role of ASB and C4S in the regulation of IL-8 secretion, and suggest that changes in ASB activity and C4S content may have a significant impact on IL-8-mediated inflammatory responses.

Dextran sodium sulfate (DSS) is a sulfated polysaccharide that has been very widely used to induce inflammation in experimental models of inflammatory bowel disease in which the effects of pharmacologic and biologic therapies are tested. However, the precise mechanisms by which DSS induces inflammation have not been elucidated.DSS-induced increases in phospho-IkappaBalpha, nuclear NFkappaB (p65), and IL-8 secretion in human colonic epithelial cells in tissue culture are attributable to a reactive oxygen species (ROS)-induced pathway of inflammation, and do not require TLR4, MyD88, or Bcl10, which are associated with the innate immune pathway of NFkappaB-IL-8 activation.DSS-induced increases were inhibited by the ROS scavengers Tempol and Tiron, were associated with decreased phosphorylation of MAPK12 (p38gamma), MAPK 13 (p38delta), and Hsp27, and required the IkappaB kinase (IKK) signalosome component IKKbeta. In ex vivo colonic tissue from TLR4-deficient mice, or following knockdown of MyD88 or Bcl10 or exposure to an IRAK 1/4 inhibitor, DSS effects were not suppressed. Data demonstrated that DSS activates IkappaBalpha, NFkappaB, and IL-8 through an ROS-Hsp27-IKKbeta-mediated pathway, and not through an innate immune cascade.These results suggest that DSS models of inflammation may not be optimal for evaluation of interventions that involve mechanisms of innate immunity.

Arylsulfatase B (ASB; N-acetylgalactosamine-4-sulfatase; 4-sulfatase; ARSB) is the enzyme that removes 4-sulfate groups from N-acetylgalactosamine 4-sulfate, which combines with glucuronate to form the disaccharide unit of chondroitin-4-sulfate (C4S). In this study, we report how variation in expression of ASB affected the migration of human colonic epithelial cells. In the T84 cell line, derived from lung metastasis of malignant colonic epithelial cells, the activity of ASB, as well as steroid sulfatase, arylsulfatase A, and galactose-6-sulfatase, were significantly less than in normal, primary colonic epithelial cells and in the NCM460 cell line which was derived from normal colonocytes. In the T84 cells, matrix metalloproteinase 9 (MMP9), activated RhoA, and cell migration, as well as C4S content, were significantly more than in the NCM460 cells. Silencing and overexpression of ASB had inverse effects on MMP9, activated RhoA, and cell migration, as well as the C4S content, in the NCM460 and T84 cells. When ASB expression was silenced by siRNA in the NCM460 cells, MMP9 secretion increased to over 3 times the basal level, activated RhoA increased * 85%, and cell migration increased * 52%. Following overexpression of ASB, MMP9 declined 51%, activated RhoA declined * 51%, and cell migration decreased * 37%. These findings demonstrate marked effects of ASB expression on the migratory activity of colonic epithelial cells, activated RhoA, and MMP9, and suggest a potential vital role of ASB, due to its impact on chondroitin sulfation, on determination of the invasive phenotype of colonic epithelial cells.