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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.

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.

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.

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.

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.

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.

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.

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.

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.

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 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.

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.

Carrageenans are highly sulfated polysaccharides that are widely used as food additives due to their ability to improve food texture. They are also widely recognized for their ability to induce inflammation in animal models of colitis. Recently, we reported that carrageenan (CGN) activated a pathway of innate immunity in human colonic epithelial cells mediated by Bcl10 (B-cell CLL/lymphoma 10). However, increases in phospho-IkappaBalpha and Interleukin-8 (IL-8) were not completely inhibited by silencing Bcl10, suggesting that CGN also influenced another mechanism, or mechanisms, of inflammation. In this report, we demonstrate that CGN increases production of reactive oxygen species (ROS) in human colonic epithelial cells. The combination of ROS quenching by the free radical scavenger Tempol and of Bcl10 silencing by siRNA completely inhibited the CGN-induced increases in nuclear NFkappaB (p65), phospho-IkappaBalpha, and secretion of IL-8. The CGN-induced increase in ROS was associated with declines in phosphorylation of MAPK 12 (p38gamma), MAPK 13 (p38delta), and heat-shock protein (Hsp) 27. The CGN-induced decline in phospho-Hsp27 was reversed by co-administration of Tempol (100 nM), but unaffected by silencing Bcl10. Since Hsp27 phosphorylation is inversely associated with phosphorylation of the IkappaBalpha kinase (IKK) signalosome, CGN exposure appears to affect the IKK signalosome by both the catalytic component, mediated by ROS-phospho-Hsp27, and the regulatory component, mediated by Bcl10 interaction with IKKgamma (Nemo). Hence, the CGN-activated inflammatory cascades related to innate immunity and to generation of ROS may be integrated at the level of the IKK signalosome.

The sulfatase enzymes, N-acetylgalactosamine-4-sulfatase (arylsulfatase B (ASB)) and galactose-6-sulfatase (GALNS) hydrolyze sulfate groups of CS. Deficiencies of ASB and GALNS are associated with the mucopolysaccharidoses. To determine if expression of ASB and GALNS impacts on glycosaminoglycans (GAGs) and proteoglycans beyond their association with the mucopolysaccharidoses, we modified the expression of ASB and GALNS by overexpression and by silencing with small interference RNA in MCF-7 cells. Content of total sulfated GAG (sGAG), chondroitin 4-sulfate (C4S), and total chondroitin sulfates (CSs) was measured following immunoprecipitation with C4S and CS antibodies and treatment with chondroitinase ABC. Following silencing of ASB or GALNS, total sGAG, C4S, and CS increased significantly. Following overexpression of ASB or GALNS, total sGAG, C4S, and CS declined significantly. Measurements following chondroitinase ABC treatment of the cell lysates demonstrated no change in the content of the other sGAG, including heparin, heparan sulfate, dermatan sulfate, and keratan sulfate. Following overexpression of ASB and immunoprecipitation with C4S antibody, virtually no sGAG was detectable. Total sGAG content increased to 23.39 (+/-1.06) microg/mg of protein from baseline of 12.47 (+/-0.68) microg/mg of protein following ASB silencing. mRNA expression of core proteins of the CS-containing proteoglycans, syndecan-1 and decorin, was significantly up-regulated following overexpression of ASB and GALNS. Soluble syndecan-1 protein increased following increases in ASB and GALNS and reduced following silencing, inversely to changes in CS. These findings demonstrate that modification of expression of the lysosomal sulfatases ASB and GALNS regulates the content of CSs.

Bcl10 (B‐cell CLL/lymphoma 10) is a 233 amino acid CARD (caspase recruitment domain)‐containing cellular protein, increasingly recognized as a mediator of NFκB activation in non‐immune, as well as immune cells. Due to the importance of Bcl10 in diverse cell types, we developed a solid‐phase, enzyme‐linked immunosorbent (ELISA) assay to precisely measure Bcl10 in small volume cell lysates, using recombinant Bcl10 to standardize the assay. Standard curve measures Bcl10 from 0.25 ng/mL to 16 ng/mL, with very low intra‐ and inter‐assay variation. Sample dilution and exogenous Bcl10 recovery experiments, comparisons with Western blot, and linear response to increasing doses of known Bcl10 activators confirm the specificity and precision of the ELISA.

Background The genetic disorder cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, impairing its function as a regulated anion channel involved with fluid secretion across epithelial cells. However, the clinical manifestations of CF are not thoroughly explained by impaired CFTR function. Experimental data have demonstrated oversulfation of glycoconjugates synthesized by CF epithelial cells of lung, pancreas, and other organs, and increases in the glycosaminoglycans dermatan sulfate and chondroitin sulfate in cultured skin fibroblasts from patients with CF. Since the enzyme arylsulfatase B (ASB) catalyzes hydrolysis of the sulfate ester of N-acetylgalactosamine 4-sulfate, a component of dermatan sulfate and chondroitin A sulfate, determination of ASB activity in human airway epithelial cells, corrected and uncorrected for CFTR, was undertaken. Methods Arylsulfatase B (ASB) enzyme activity was measured in three pairs of cells in which the defect in CFTR was corrected or uncorrected. The substrates p-nitrocatechol sulfate and 4-MUS were used to measure activity. Results An increase of 40% in ASB activity occurred in the CF cells when corrected for CFTR deficiency. Conclusions Decline in ASB activity may affect characteristics of secretions in CF, due to impaired metabolism of GAGs containing N-acetylgalactosamine 4-sulfate. ASB activity was markedly reduced when phosphate-buffered saline (PBS) was used as buffer, consistent with inhibition of sulfatase activity by phosphate. Increased attention to sulfatases may help to explain the pathophysiology of CF and lead to new therapies.

Sulfatase enzymes have important roles in metabolism of steroid hormones and of glycosaminoglycans (GAGs). The activity of five sulfatase enzymes, including steroid sulfatase (STS; arylsulfatase C), arylsulfatase A (ASA; cerebroside sulfatase), arylsulfatase B (ASB; N-acetylgalactosamine-4-sulfatase), galactose-6-sulfatase (GALNS), and iduronate-2-sulfatase (IDS), was compared in six different mammary cell lines, including the malignant mammary cell lines MCF7, T47D, and HCC1937, the MCF10A cell line which is associated with fibrocystic disease, and in primary epithelial and myoepithelial cell lines established from reduction mammoplasty. The effects of estrogen hormones, including estrone, estradiol, estrone 3-sulfate, and estradiol sulfate on activity of these sulfatases were determined. The malignant cell lines MCF7 and T47D had markedly less activity of STS, ASB, ASA, and GAL6S, but not IDS. The primary myoepithelial cells had highest activity of STS and ASB, and the normal epithelial cells had highest activity of GALNS and ASA. Greater declines in sulfatase activity occurred in response to estrone and estradiol than sulfated estrogens. The study findings demonstrated marked variation in sulfatase activity and in effects of exogenous estrogens on sulfatase activity among the different mammary cell types.

Purpose : This investigation examined mRNA expression and enzymatic activity of steroid sulfatase (STS) in human mammary myoepithelial cells (MMECs) and MCF-7 cells and assessed the effects of 17-β estradiol on the activity of STS. Methods : The mRNA level of STS in MMECs was determined by RT–PCR analysis using specific primers for STS. STS enzymatic activity prior to and after treatment with 17-β estradiol was determined by measuring 3 H -metabolites formed after exposure to [ 3 H ]estrone 3-sulfate (E1S) and [ 3 H ]dehydroepiandrosterone-sulfate (DHEA-S). Results : Our data demonstrate the presence of STS in the MMECs. Based on RT–PCR analysis, MMECs had slightly lower levels of STS compared to MCF-7 cells. However, sulfatase activity was about 120 times greater in the MMECs than the MCF-7 cells (E1S V max =2640 nmol/(mg DNA h) compared to 20.9 nmol/(mg DNA h)). Exposure to 17-β estradiol was associated with 70% reduction in E1S sulfatase activity in the MCF-7 cells and 9% increase in the MMECs after 6 days. Discussion : Our studies indicate for the first time the presence of STS in MMECs. This is suggestive of a previously undetermined role for MMECs in converting precursor hormones into active steroid hormones within mammary tissue. In addition, differential response of the MMECs and the MCF-7 cells to estrogen demonstrates differences in hormone metabolism between these two cell types, perhaps related to the absence of estrogen receptors in the MMECs and their presence in the MCF-7 cells. The MMECs may have an important role in hormonal regulation within mammary tissue.

Initial experiments in human colonic epithelial cell lines and in the arylsulfatase B (N-acetylgalactosamine-4-sulfatase; ARSB)-deficient mouse showed that reduction in ARSB and the resultant increase in chondroitin-4-sulfate (C4S) were associated with reduction in chondroitin-4-sulfotransferase expression and ~50% reduction in sulfotransferase activity with overall increase in total sulfated glycosaminoglycans and C4S. Our experiments have also shown that decline in ARSB leads to increase in Wnt9A expression in human colonic epithelial cells and mouse intestine. Since modeling of the extracellular matrix by Wnt and BMP4 involves their interactions with sulfated glycosaminoglycans, including chondroitin sulfate, we evaluated the interactions among ARSB, Wnt9A, BMP4, and chondroitin-4-sulfotransferase (CHST11). Following silencing of Wnt9A by siRNA, the CHST11 expression increased to ~3 times baseline and the sulfotransferase activity increased to ~1.6 times the baseline. In contrast, when BMP4 was neutral...

In this article I review the association between exposure to carrageenan and the occurrence of colonic ulcerations and gastrointestinal neoplasms in animal models. Although the International Agency for Research on Cancer in 1982 identified sufficient evidence for the carcinogenicity of degraded carrageenan in animals to regard it as posing a carcinogenic risk to humans, carrageenan is still used widely as a thickener, stabilizer, and texturizer in a variety of processed foods prevalent in the Western diet. I reviewed experimental data pertaining to carrageenan's effects with particular attention to the occurrence of ulcerations and neoplasms in association with exposure to carrageenan. In addition, I reviewed from established sources mechanisms for production of degraded carrageenan from undegraded or native carrageenan and data with regard to carrageenan intake. Review of these data demonstrated that exposure to undegraded as well as to degraded carrageenan was associated with the occurrence of intestinal ulcerations and neoplasms. This association may be attributed to contamination of undegraded carrageenan by components of low molecular weight, spontaneous metabolism of undegraded carrageenan by acid hydrolysis under conditions of normal digestion, or the interactions with intestinal bacteria. Although in 1972, the U.S. Food and Drug Administration considered restricting dietary carrageenan to an average molecular weight > 100,000, this resolution did not prevail, and no subsequent regulation has restricted use. Because of the acknowledged carcinogenic properties of degraded carrageenan in animal models and the cancer-promoting effects of undegraded carrageenan in experimental models, the widespread use of carrageenan in the Western diet should be reconsidered.

Exposure to the common food additive carrageenan was previously associated with increased Wnt9A expression and increased cytoplasmic β-catenin in human colonic epithelial cells. In this report, exposure of human colonic epithelial cells in culture and of mouse colonic epithelium in vivo to low concentrations of carrageenan is shown to activate the Wnt/β-catenin signaling pathway, leading to increases in nuclear β-catenin, T-cell factor/lymphoid enhancer factor activation, and cyclin D1 expression and decline in bone morphogenetic protein-4. These effects are mediated through carrageenan-induced reactive oxygen species (ROS), and inhibited by the ROS scavenger Tempol. Carrageenan exposure and ROS production inhibited thioredoxin reductase activity and increased oxidation of nucleoredoxin, a member of the thioredoxin family of redox proteins. When oxidized, nucleoredoxin co-immunoprecipitation with dishevelled (DVL) declined, enabling DVL to interact with and inhibit the cytoplasmic β-catenin destruction complex, and facilitating nuclear translocation of β-catenin. Both nucleoredoxin silencing and carrageenan exposure produced similar declines in thioredoxin reductase activity. In addition to activation of Wnt signaling, carrageenan exposure also increased Wnt9A mRNA expression in the mouse colonic epithelium and the human colonic epithelial cells, thereby potentially permitting ongoing stimulation of the Wnt/β-catenin pathway. These findings suggest how a common dietary ingredient can contribute to colon carcinogenesis by effects on Wnt signaling and Wnt expression.

Sulfatase enzymes remove sulfate groups from sulfated steroid hormones, including estrone-sulfate and dehydroepiandrosterone-sulfate, and from sulfated glycosaminoglycans (GAGs), including chondroitin sulfates and heparan sulfate. The enzymes N-acetylgalactosamine-4-sulfatase (arylsulfatase B; ARSB) and N-acetylgalactosamine-6-sulfatase (GALNS), which remove sulfate groups from the sulfated GAGs chondroitin 4-sulfate (C4S) and chondroitin 6-sulfate, respectively, have not been studied in prostate development previously. In this report, the endogenous variation and the impact of exogenous estradiol benzoate on the immunohistochemistry and activity of ARSB and GALNS in post-natal (days 1-30) ventral rat prostate are presented, as well as measurements of steroid sulfatase activity (STS), C4S, total sulfated GAGs, and versican, an extracellular matrix proteoglycan with chondroitin sulfate attachments on days 5 and 30. Findings demonstrate distinct and reciprocal localization of ARSB and GALNS, with ARSB predominant in the stroma and GALNS predominant in the epithelium. Control ARSB activity increased significantly between days 5 and 30, but following estrogen exposure (estradiol benzoate 25 μg in 25 μl sesame oil subcutaneously on days 1, 3, and 5), activity was reduced and the observed increase on day 30 was inhibited. However, estrogen treatment did not inhibit the increase in GALNS activity between days 5 and 30, and reduced STS activity by 50% on both days 5 and 30 compared to vehicle control. Sulfated GAGs, C4S, and the extracellular matrix proteoglycan versican declined between days 5 and 30 in the control, but these declines were inhibited following estrogen. Study findings indicate distinct variation in expression and activity of sulfatases, sulfated GAGs, C4S, and versican in the process of normal prostate development, and disruption of these events by exogenous estrogen.

Arylsulfatase B (B-acetylgalactosamine 4-sulfatase; ARSB) is the enzyme that removes 4-sulfate groups from the non-reducing end of the glycosaminoglycans chondroitin 4-sulfate and dermatan sulfate. Decline in ARSB has been shown in malignant prostate, colonic, and mammary cells and tissues, and decline in ARSB leads to transcriptional events mediated by galectin-3 with AP-1 and Sp1. Increased mRNA expression of GPNMB (transmembrane glycoprotein NMB) in HepG2 cells and in hepatic tissue from ARSB-deficient mice followed decline in expression of ARSB and was mediated by the microphthalmia-associated transcription factor (MITF), but was unaffected by silencing galectin-3. Since GPNMB is increased in multiple malignancies, studies were performed to determine how decline in ARSB increased GPNMB expression. The mechanism by which decline in ARSB increased nuclear phospho-MITF was due to reduced activity of SHP2, a protein tyrosine phosphatase with Src homology (SH2) domains that regulates multiple cellular processes. SHP2 activity declined due to increased binding with chondroitin 4-sulfate when ARSB was reduced. When SHP2 activity was inhibited, phosphorylations of p38 mitogen-associated phosphokinase (MAPK) and of MITF increased, leading to GPNMB promoter activation. A dominant negative SHP2 construct, the SHP2 inhibitor PHSP1, and silencing of ARSB increased phospho-p38, nuclear MITF, and GPNMB. In contrast, constitutively active SHP2 and overexpression of ARSB inhibited GPNMB expression. The interaction between chondroitin 4-sulfate and SHP2 is a novel intersection between sulfation and phosphorylation, by which decline in ARSB and increased chondroitin 4-sulfation can inhibit SHP2, thereby regulating downstream tyrosine phosphorylations by sustained phosphorylations with associated activation of signaling and transcriptional events.

This report presents evidence of 1) a role for arylsulfatase B (ARSB; N-acetylgalactosamine-4-sulfatase) in mediating intracellular oxygen signaling; 2) replication between the effects of ARSB silencing and hypoxia on sulfated glycosaminoglycan content, cellular redox status, and expression of hypoxia-associated genes; and 3) a mechanism whereby changes in chondroitin-4-sulfation that follow either hypoxia or ARSB silencing can induce transcriptional changes through galectin-3. ARSB removes 4-sulfate groups from the non-reducing end of chondroitin-4-sulfate and dermatan sulfate and is required for their degradation. For activity, ARSB requires modification of a critical cysteine residue by the formylglycine generating enzyme and by molecular oxygen. When primary human bronchial and human colonic epithelial cells were exposed to 10% O(2) × 1 h, ARSB activity declined by ~41% and ~30% from baseline, as nuclear hypoxia inducible factor (HIF)-1α increased by ~53% and ~37%. When ARSB was silenced, nuclear HIF-1α increased by ~81% and ~61% from baseline, and mRNA expression increased to 3.73 (± 0.34) times baseline. Inversely, ARSB overexpression reduced nuclear HIF-1α by ~37% and ~54% from baseline in the epithelial cells. Hypoxia, like ARSB silencing, significantly increased the total cellular sulfated glycosaminoglycans and chondroitin-4-sulfate (C4S) content. Both hypoxia and ARSB silencing had similar effects on the cellular redox status and on mRNA expression of hypoxia-associated genes. Transcriptional effects of both ARSB silencing and hypoxia may be mediated by reduction in galectin-3 binding to more highly sulfated C4S, since the galectin-3 that co-immunoprecipitated with C4S declined and the nuclear galectin-3 increased following ARSB knockdown and hypoxia.

The commonly used food additive carrageenan, including lambda (λ), kappa (κ) and iota (ι) forms, is composed of galactose disaccharides linked in alpha-1,3 and beta-1,4 glycosidic bonds with up to three sulfate groups per disaccharide residue. Carrageenan closely resembles the endogenous galactose or N-acetylgalactosamine-containing glycosaminoglycans (GAGs), chondroitin sulfate (CS), dermatan sulfate (DS), and keratan sulfate. However, these GAGs have beta-1,3 and beta-1,4 glycosidic bonds, in contrast to the unusual alpha-1,3 glycosidic bond in carrageenan. Since sulfatase activity is inhibited by sulfate, and carrageenan is so highly sulfated, we tested the effect of carrageenan exposure on sulfatase activity in human intestinal and mammary epithelial cell lines and found that carrageenan exposure significantly reduced the activity of sulfatases, including N-acetylgalactosamine-4-sulfatase, galactose-6-sulfatase, iduronate sulfatase, steroid sulfatase, arylsulfatase A, SULF-1,2, and heparan sulfamidase. Consistent with the inhibition of sulfatase activity, following exposure to carrageenan, GAG content increased significantly and showed marked differences in disaccharide composition. Specific changes in CS disaccharides included increases in di-sulfated disaccharide components of CSD (2S6S) and CS-E (4S6S), with declines in CS-A (4S) and CS-C (6S). Specific changes in heparin-heparan sulfate disaccharides included increases in 6S disaccharides, as well as increases in NS and 2S6S disaccharides. Study results suggest that carrageenan inhibition of sulfatase activity leads to re-distribution of the cellular GAG composition with increase in di-sulfated CS and with potential consequences for cell structure and function.

The enzyme arylsulfatase B (N-acetylgalactosamine-4-sulfatase; ARSB; ASB) removes 4-sulfate groups from the sulfated glycosaminoglycans (sGAG) chondroitin-4-sulfate (C4S) and dermatan sulfate (DS). Inborn deficiency of ARSB leads to the lysosomal storage disease mucopolysaccharidosis VI, characterized by accumulation of sGAG in vital organs, disruption of normal physiological processes, severe morbidity, and premature death. Recent published work demonstrated extra-lysosomal localization with nuclear and cell membrane ARSB observed in bronchial and colonic epithelial cells, cerebrovascular cells, and hepatic cells. In this report, the authors present ARSB immunostaining in a colonic microarray and show differences in distribution, intensity, and pattern of ARSB staining among normal colon, adenomas, and adenocarcinomas. Distinctive, intense luminal membrane staining was present in the normal epithelial cells but reduced in the malignancies and less in the grade 3 than in the grade 1 adenocarcinomas. In the normal cores, a distinctive pattern of intense cytoplasmic positivity at the luminal surface was followed by reduced staining deeper in the crypts. ARSB enzymatic activity was significantly greater in normal than in malignant tissue. These study findings affirm extra-lysosomal localization of ARSB and suggest that altered ARSB immunostaining and reduced activity may be useful indicators of malignant transformation in human colonic tissue.

Background The receptors for adhesion of Plasmodium falciparum-infected red blood cells (RBC) in the placenta have been identified as chondroitin-4-sulphate (C4S) proteoglycans, and the more sulphate-rich chondroitin oligosaccharides have been reported to inhibit adhesion. Since the anti-malarial drug chloroquine accumulates in lysosomes and alters normal lysosomal processes, the effects of chloroquine on the lysosomal enzyme arylsulphatase B (ASB, N-acetylgalactosamine-4-sulphatase), which removes 4-sulphate groups from chondroitin-4-sulphate, were addressed. The underlying hypothesis derived from the recognized impairment of attachment of parasite-infected erythrocytes in the placenta, when chondroitin-4-sulphation was increased. If chloroquine reduced ASB activity, leading to increased chondroitin-4-sulphation, it was hypothesized that the anti-malarial mechanism of chloroquine might derive, at least in part, from suppression of ASB. Methods Experimental methods involved cell culture of human placental, bronchial epithelial, and cerebrovascular cells, and the in vitro exposure of the cells to chloroquine at increasing concentrations and durations. Measurements of arylsulphatase B enzymatic activity, total sulphated glycosaminoglycans (sGAG), and chondroitin-4-sulphate (C4S) were performed using in vitro assays, following exposure to chloroquine and in untreated cell preparations. Fluorescent immunostaining of ASB was performed to determine the effect of chloroquine on cellular ASB content and localization. Mass spectrometry and high performance liquid chromatography were performed to document and to quantify the changes in chondroitin disaccharides following chloroquine exposure. Results In the human placental, bronchial epithelial, and cerebrovascular cells, exposure to increasing concentrations of chloroquine was associated with reduced ASB activity and with increased concentrations of sGAG, largely attributable to increased C4S. The study data demonstrated: 1) decline in ASB activity following chloroquine exposure; 2) inverse correlation between ASB activity and C4S content; 3) increased content of chondroitin-4-sulphate disaccharides following chloroquine exposure; and 4) decline in extent of chloroquine-induced ASB reduction with lower baseline ASB activity. Confocal microscopy demonstrated the presence of ASB along the cell periphery, indicating extra-lysosomal localization. Conclusions The study data indicate that the therapeutic mechanism of chloroquine action may be attributable, at least in part, to reduction of ASB activity, leading to increased chondroitin-4-sulphation in human placental, bronchial epithelial, and cerebrovascular cells. In vivo, increased chondroitin-4-sulphation may reduce the attachment of P. falciparum-infected erythrocytes to human cells. Extra-lysosomal localization of ASB and reduced impact of chloroquine when baseline ASB activity is less suggest possible mechanisms of resistance to the effects of chloroquine.

B-cell CLL/lymphoma 10 (BCL10), the caspase recruitment domain (CARD)-containing protein involved in the etiology of the mucosa-associated lymphoid tissue (MALT) lymphomas, has been implicated in inflammatory processes in epithelial cells, as well as in immune cells. Experiments in this report indicate that BCL10 is required for activation of nuclear factor (NF)-kappaB by both canonical and noncanonical pathways, following stimulation by the sulfated polysaccharide carrageenan (CGN). In wild type and IkappaB-kinase (IKK)alpha(-/-) mouse embryonic fibroblasts, increases in phospho-IkappaBalpha, nuclear NF-kappaB p65 (RelA) and p50, and KC, the mouse analog of human interleukin-8, were markedly reduced by silencing BCL10 or by exposure to the free radical scavenger Tempol. In IKKbeta(-/-) cells, BCL10 silencing, but not Tempol, reduced the CGN-induced increases in KC, phospho-NF-kappaB-inducing kinase (NIK), cytoplasmic NF-kappaB p100, and nuclear NF-kappaB p52 and RelB, suggesting a BCL10 requirement for activation of the noncanonical pathway. In NCM460 cells, derived from normal, human colonic epithelium, the CGN-induced increases in NF-kappaB family members, p65, p50, p52, and RelB, were inhibited by BCL10 silencing. Although enzyme-linked immunosorbent assay and confocal images demonstrated no change in total NIK following CGN, increases in phospho-NIK in the wild type, IKKbeta(-/-) and IKKalpha(-/-) cells were inhibited by silencing BCL10. These findings indicate an upstream signaling role for BCL10, in addition to its effects on IKKgamma, the regulatory component of the IKK signalosome, and a requirement for BCL10 in both canonical and noncanonical pathways of NF-kappaB activation. Also, the commonly used food additive carrageenan can be added to the short list of known activators of both pathways.

The sulfated polysaccharide carrageenan (CGN) induces activation of NFκB and interleukin 8 (IL-8) in human colonic epithelial cells through a pathway of innate immunity mediated by Bcl10 (B-cell CLL/lymphoma 10). In this report, we identify Toll-like receptor 4 (TLR4), a member of the family of innate immune receptors, as the surface membrane receptor for CGN in human colonic epithelial cells. Experiments with fluorescence-tagged CGN demonstrated a marked reduction in binding of CGN to human intestinal epithelial cells and to RAW 264.7 mouse macrophages, following exposure to TLR4 blocking antibody (HTA-125). Binding of CGN to 10ScNCr/23 mouse macrophages, which are deficient in the genetic locus for TLR4, was absent. Additional experiments with TLR4 blocking antibody and TLR4 small interfering RNAs showed 80% reductions in CGN-induced increases in Bcl10 and IL-8. Transfection with dominant-negative MyD88 plasmid demonstrated MyD88 dependence of the CGN-TLR4-triggered increases in Bcl10 and IL-8. Therefore, these results indicate that CGN-induced inflammation in human colonocytes proceeds through a pathway of innate immunity, perhaps related to the unusual α-1,3-galactosidic linkage characteristic of CGN, and suggest how dietary CGN intake may contribute to human intestinal inflammation. Because CGN is a commonly used food additive in the Western diet, clarification of its effects and mechanisms of action are vital to issues of food safety.

The Wnt-β-catenin signaling pathway plays a pivotal role in development and disease. It is crucial for basic developmental processes, such as cell-fate specification, progenitor-cell proliferation and the control of asymmetric cell division. Recent studies suggest that self-renewal of stem/progenitor cells by the canonical Wnt cascade is subverted in cancer cells to allow malignant proliferation in intestine. Carrageenans are highly sulfated polysaccharides, which are widely used as food additives to improve the texture of processed foods, and are consumed at levels of about 250 mg/day in the average diet. Reports of many investigators have demonstrated that carrageenan induces inflammation, colonic ulcerations, polyps, and colorectal tumors. We have reported distinct pathways by which carrageenan induces the activation of NF-κB and secretion of IL-8, mediated through TLR4-BCL10 or reactive oxygen species (ROS) in colonic epithelial cells. Our previous work also indicated that carrageenan exposure caused activation of the Wnt-β-catenin pathway in colonic epithelial cells. This investigation was undertaken to elucidate the Wnt signaling mechanisms activated by carrageenan exposure, using NCM460 (INCELL) human colonic epithelial cells exposed to carrageenan (1 μg/ml) x 24 h and colonic tissue from ten-week old C57BL/6J mice exposed to carrageenan (10 - 100 μg/ml) in their water for four weeks. Nuclear β-catenin in NCM460 cells and mouse colonic tissue was determined by Western blot and ELISA and increased to two times the baseline level following carrageenan. This increase in nuclear β-catenin in NCM460 cells was inhibited by the ROS quencher Tempol, but not by TLR4 or BCL10 siRNA. TCF/Lef-1 reporter assay demonstrated increased nuclear binding post carrageenan treatment in the mouse colon (to 4x baseline) and in the NCM460 cells (to 6.3x baseline). Also, mRNA expression of cyclin D1 and wnt9a was increased more than two-fold, and BMP4 expression was decreased. In the NCM460 cells, these responses to carrageenan were inhibited by Tempol, but not BCL10 silencing. Also, carrageenan-induced increases in ROS and lipid peroxidation were inhibited by Tempol. Since Wnt signaling is affected by free dishevelled 2 (dsh2), and dsh2 binding to nucleoredoxin (NRX) is greater when NRX is in its reduced state, we tested whether carrageenan exposure could impact upon the Wnt signaling pathway by modifying the redox status of NRX. The ratio of free NRX to NRX bound to dsh2 increased to six times baseline following carrageenan, and was reversed by Tempol. These profound effects of carrageenan on the Wnt signaling pathway in colonic cells can help to explain how diet can contribute to development of colonic neoplasia. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 163. doi:1538-7445.AM2012-163