Leo Feferman

Increased Expression of Colonic Wnt9A through Sp1-Mediated Transcriptional Effects involving Arylsulfatase B, Chondroitin-4-Sulfate, and Galectin-3

Background: Mechanism by which Wnt expression was increased by carrageenan exposure was unknown. Results: Sp1 activated Wnt9A transcription through changes in arylsulfatase B, chondroitin 4-sulfation, and galectin-3. Conclusion: Decline in arylsulfatase B leads to transcriptional effects mediated by Sp1 and galectin-3. Significance: Extracellular events can regulate transcription through changes in arylsulfatase B and chondroitin 4-sulfation. 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.

Arylsulfatase B (N-acetylgalactosamine-4-sulfatase): potential role as a biomarker in prostate cancer

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 (TMAs) 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 (EGFR) that co-immunoprecipitated with versican were determined in the normal and malignant paired prostate tissues.Results:Forty-four cases of prostate cancer were paired by age (±5 years), race, Gleason score (in order) and pathological TNM (tumor, node, metastasis) score. The pairs differed by recurrence vs non-recurrence of elevated PSA at ⩾4 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<0.0001, respectively) and were inversely correlated (r=−0.98, −0.97 and −0.99, respectively). In other paired normal and malignant prostate tissues, ARSB activity was significantly higher in the normal tissues, and C4S and versican values were lower (P<0.0001). C4S that co-immunoprecipitated with versican was greater in the malignant than in the normal tissue, whereas total EGFR that co-immunoprecipitated with versican was reduced.Conclusions:Study findings suggest that ARSB may be useful as a prognostic biomarker in prostate cancer and that the biological action of ARSB on chondroitin sulfate may impact upon versican’s effects in the tumor microenvironment.

Inhibition of phosphatase activity follows decline in sulfatase activity and leads to transcriptional effects through sustained phosphorylation of transcription factor MITF

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.

Carrageenan Inhibits Insulin Signaling through GRB10-mediated Decrease in Tyr(P)-IRS1 and through Inflammation-induced Increase in Ser(P)307-IRS1.

Background: The common food additive carrageenan inhibits insulin signaling by increasing Ser(P)307-IRS1, leading to reduced Ser(P)473-AKT. Results: Carrageenan also inhibits insulin signaling by increasing expression of GRB10, an inhibitor of Tyr(P)-IRS1. Conclusion: Combined effects of carrageenan on Tyr(P)-IRS1 and Ser(P)307-IRS1 completely block the insulin-induced increase in Ser(P)473-AKT. Significance: Carrageenan impairs glucose tolerance by inflammatory and transcriptional effects that lead to insulin resistance. 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 Ser401-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.

Exposure to Common Food Additive Carrageenan Alone Leads to Fasting Hyperglycemia and in Combination with High Fat Diet Exacerbates Glucose Intolerance and Hyperlipidemia without Effect on Weight

Aims. Major aims were to determine whether exposure to the commonly used food additive carrageenan could induce fasting hyperglycemia and could increase the effects of a high fat diet on glucose intolerance and dyslipidemia. Methods. C57BL/6J mice were exposed to either carrageenan, high fat diet, or the combination of high fat diet and carrageenan, or untreated, for one year. Effects on fasting blood glucose, glucose tolerance, lipid parameters, weight, glycogen stores, and inflammation were compared. Results. Exposure to carrageenan led to glucose intolerance by six days and produced elevated fasting blood glucose by 23 weeks. Effects of carrageenan on glucose tolerance were more severe than from high fat alone. Carrageenan in combination with high fat produced earlier onset of fasting hyperglycemia and higher glucose levels in glucose tolerance tests and exacerbated dyslipidemia. In contrast to high fat, carrageenan did not lead to weight gain. In hyperinsulinemic, euglycemic clamp studies, the carrageenan-exposed mice had higher early glucose levels and lower glucose infusion rate and longer interval to achieve the steady-state. Conclusions. Carrageenan in the Western diet may contribute to the development of diabetes and the effects of high fat consumption. Carrageenan may be useful as a nonobese model of diabetes in the mouse.

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

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

Decline in arylsulfatase B and Increase in chondroitin 4-sulfotransferase combine to increase chondroitin 4-sulfate in traumatic brain injury

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.

A randomized trial of the effects of the no-carrageenan diet on ulcerative colitis disease activity

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.

Decline in arylsulfatase B leads to increased invasiveness of melanoma cells

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.

Restriction of Aerobic Metabolism by Acquired or Innate Arylsulfatase B Deficiency: A New Approach to the Warburg Effect

Aerobic respiration is required for optimal efficiency of metabolism in mammalian cells. Under circumstances when oxygen utilization is impaired, cells survive by anerobic metabolism. The malignant cell has cultivated the use of anerobic metabolism in an aerobic environment, the Warburg effect, but the explanation for this preference is not clear. This paper presents evidence that deficiency of the enzyme arylsulfatase B (ARSB; N-acetylgalactosamine 4-sulfatase), either innate or acquired, helps to explain the Warburg phenomenon. ARSB is the enzyme that removes 4-sulfate groups from the non-reducing end of chondroitin 4-sulfate and dermatan sulfate. Previous reports indicated reduced ARSB activity in malignancy and replication of the effects of hypoxia by decline in ARSB. Hypoxia reduced ARSB activity, since molecular oxygen is needed for post-translational modification of ARSB. In this report, studies were performed in human HepG2 cells and in hepatocytes from ARSB-deficient and normal C57BL/6J control mice. Decline of ARSB, in the presence of oxygen, profoundly reduced the oxygen consumption rate and increased the extracellular acidification rate, indicating preference for aerobic glycolysis. Specific study findings indicate that decline in ARSB activity enhanced aerobic glycolysis and impaired normal redox processes, consistent with a critical role of ARSB and sulfate reduction in mammalian metabolism.