N-acetylgalactosamine-4-sulfatase (Arylsulfatase B; ARSB) modifies the glycosaminoglycans (GAGs) chondroitin-4-sulfate (C4S) and dermatan sulfate by removing 4-sulfate groups from the non-reducing end and thereby regulating GAG degradation. Previous studies demonstrated that ARSB was reduced in malignant human colonic, mammary, and prostatic tissue and suggested that ARSB acts as a tumor suppressor. In this report, ARSB was silenced by siRNA in the WPMY-1 human prostate stromal myofibroblast cell line (ATCC), and effects on C4S, total sulfated glycosaminoglycans (GAGs), ErbB2, total EGFR, phospho-ERK, versican, and AP-1 were determined. Total sulfated GAGs were measured by Blyscan assay (Biocolor), and C4S was determined by Blyscan assay following immunoprecipitation by C4S antibody. Phospho(T202/Y204 for ERK1 or T185/Y187 for ERK2)-ERK, ErbB2, and total EGFR were measured by ELISA. Versican, an extracellular matrix chondroitin sulfate proteoglycan with EGF-like repeats at its C-terminus, was measured by ELISA and QPCR. ARSB and galectin-3 were silenced by siRNA (Qiagen). ARSB activity declined from 140.4 ± 8.4 to 21.4 ± 0.9 nmol/mg protein/hr following silencing. Total sulfated GAGs increased from 17.9 ± 0.7 μg/mg protein in control cells to 28.6 ± 1.4 μg/mg protein following ARSB silencing, and C4S increased from 7.4 ± 0.6 μg/mg protein to 15.5 ± 0.8 μg/mg protein following ARSB silencing. Total EGFR increased to 2.65 times baseline, and ErbB2 increased to over six-fold times baseline. Phospho-ERK increased to more than three times baseline, from 5.0 ± 0.5 ng/mg protein to 15.2 ± 0.8 ng/mg protein following silencing. Versican protein increased from 122.1 ± 5.3 ng/mg protein in control to 295.2 ± 11.2 ng/mg protein and mRNA increased more than two-fold. ARSB-silencing induced increases in versican mRNA and in activation of c-Jun and c-Fos, and these increases were inhibited when galectin-3 was also silenced. [All findings are highly significant (p<0.001, one-way ANOVA with Tukey-Kramer post-test)]. Previously, we have shown reduced binding of galectin-3 to C4S following ARSB silencing. Since there is an AP-1 binding site in the versican promoter, study data demonstrate that transcriptional effects of ARSB silencing on versican are mediated by galectin-3 and AP-1. The profound effects of ARSB silencing on versican and phospho-ERK in the prostatic myofibroblasts provide further evidence that ARSB acts as a tumor suppressor.
Citation Format: Sumit Bhattacharyya, Leonid Feferman, Joanne Kramer Tobacman. Galectin-3 and AP-1 mediate transcriptional effect of Arylsulfatase B (N-acetylgalactosamine-4-sulfatase) on versican in prostatic cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5205. doi:10.1158/1538-7445.AM2013-5205