Hypoxia Reduces Arylsulfatase B Activity and Silencing Arylsulfatase B Replicates and Mediates the Effects of Hypoxia

Abstract

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% O2×1 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.

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

“…This finding is consistent with the reported decline in binding of galectin-3, -7, and -9 to more highly sulfated chondroitin, which was observed by frontal affinity chromatography (23). Coincident with reduced binding of galectin-3 to C4S, we have shown increases in nuclear galectin-3, which enabled transcriptional events mediated by AP-1, including increased expression of HIF-1␣ and versican, in human bronchial, colonic, and prostate cells (21,22). Galectin-3 interacts with heterogeneous ribonucleoprotein and may contribute to malignant transformation in various epithelial tissues, including human colonic cells (21, 24 -26).…”

“…Previously, experiments demonstrated that carrageenan exposure reduced ARSB activity (27) and that decline in ARSB led to transcriptional events that were attributable to the reduced association of galectin-3 with the more highly sulfated C4S present when ARSB activity was less (21,22). Wnt9A expression increased to 2.8 times the baseline in NCM460 cells and to 2.4 times the baseline in C57BL/6J mice following exposure to carrageenan, and exposure to the reactive oxygen species scavenger Tempol inhibited the increase in Wnt9A in the NCM460 cells (3).…”

“…In our previous work, we have reported a novel transcriptional mechanism, whereby decline in ARSB can lead to increased transcription of HIF-1␣ and of versican, due to reduced binding of galectin-3 with chondroitin 4-sulfate when chondroitin 4-sulfate (C4S) 2 is more highly sulfated following decline in ARSB activity (21,22). This finding is consistent with the reported decline in binding of galectin-3, -7, and -9 to more highly sulfated chondroitin, which was observed by frontal affinity chromatography (23).…”

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

“…Extracellular and intracellular signals may be integrated through modulation of ARSB activity by oxygen and the associated changes in chondroitin 4-sulfation [15]. Subsequent variation in binding to more or less sulfated C4S can then regulate other cell processes, as shown by effects on galectin-3 leading to increased transcription of versican, HIF-1α, and Wnt9A in human epithelial cells and in the ARSB-deficient mouse [2,15,16]. The studies in this report address the impact of ARSB on BMP4/Wnt mediated CHST11 expression in intestinal epithelium and provide a new perspective on the interaction between degradation and synthesis of CS.…”

Common Food Additive Carrageenan Stimulates Wnt/ β-Catenin Signaling in Colonic Epithelium by Inhibition of Nucleoredoxin Reduction

“…NRX was immunoprecipitated from control and treated samples by the coated beads. Eluted protein was subjected to thiol:disulfide assay, and reduced NRX was detected by applying the methods previously used to detect total cellular thiol and sulfide content (Molecular Probes, Eugene, OR), in which cystamine, papain, and N α -benzoyl-L-arginine p-nitroanilide hydrochloride (L-BAPNA) were used (24)(25)(26). To measure the NRX thiol groups, cystamine was added, and cystamine underwent an exchange reaction with NRX, yielding 2-mercaptoethylamine (cysteamine), which then interacted with the disulfide bond of papain, yielding the active enzyme papain from its inactive S-S form.…”