Biochemical and Biophysical Research Communications
Histone deacetylase-mediated regulation of chondroitin 4-O-sulfotransferase-1 (Chst11) gene expression by Wnt/β-catenin signaling
Graphical abstract
Introduction
Chondroitin sulfate (CS) is a linear polysaccharide comprising repeating disaccharide units (-4GlcAβ1-3GalNAcβ1-), where GlcA and GalNAc represent glucuronic acid and N-acetylgalactosamine, respectively. The biosynthesis of the CS backbone (i.e., the repeating disaccharide region [(-4GlcAβ1-3GalNAcβ1-)n]) is catalyzed by six homologous glycosyltransferases, and each has been cloned [1], [2], [3], [4], [5], [6], [7], [8], [9], [10], [11]. The CS backbone is further modified by sulfation. Several sulfotransferases responsible for the sulfation of CS chains have been identified [12], [13]. Each sulfotransferase catalyzes the transfer of a sulfate group from 3′-phosphoadenosine 5′-phosphosulfate, the universal donor in sulfation reactions, to the respective sulfation site on GalNAc or GlcA residues in the CS chains. A nonsulfated O unit of GalNAc residues serves as the common acceptor substrate for two types of sulfotransferases: chondroitin 4-O-sulfotransferase (C4ST), which catalyzes 4-O-sulfation, and chondroitin 6-O-sulfotransferase (C6ST), which catalyzes 6-O-sulfation. C4ST and C6ST therefore generate monosulfated A and C units, respectively (Fig. 1A). Subsequent sulfation of the A and C units can also occur via GalNAc 4-sulfate 6-O-sulfotransferase (GalNAc4S-6ST) or uronosyl 2-O-sulfotransferase (UST), forming disulfated disaccharide E and D units, respectively (Fig. 1A). The arrangement of these sulfated units (A, C, D, and E) along the CS chain creates specific sulfation motifs that function as molecular recognition elements for various biological proteins such as growth factors. The expression patterns of each sulfotransferase are, at least partly, responsible for the elaboration of specific sulfation motifs of CS. Therefore, it is important to understand the mechanisms involved in the regulation of the expression of each sulfotransferase.
A recent study shows that the gene expression of enzymes involved in the remodeling of CS is tightly regulated to maintain the specific sulfation motifs of CS [14]. A decrease in the gene expression level of arylsulfatase B (N-acetylgalactosamine-4-sulfatase), involved in the degradation of A units, causes an increase in the amount of A unit [14]. This increase in A unit content in CS chains affects the biosynthesis of A units; the gene expression level of C4ST-1, involved in the synthesis of A units, is suppressed by A-unit-rich CS chains [14]. The mechanism underlying negative feedback between the expression levels of C4ST-1 and the sulfation patterns in CS chains is described below. A-unit-rich CS chains can sequester BMP4 molecules and block BMP4 signaling [14]. Because BMP4 acts through a phospho-Smad3 binding site in the CHST11 promoter, blockage of BMP4 signaling reduces the gene expression of C4ST-1 [14]. There is therefore an unknown mechanism that surveys and controls the sulfation of CS chains within cells; thus, a molecular understanding of how cells control the quality of their sugar chains during homeostasis is required.
We have previously shown that down-regulation of C4ST-1 by Wnt signaling triggers diffusion of Wnt-3a [15]. Wnt-3a is a secreted signaling molecule that regulates cell-cell signaling during morphogenesis of the developing neural tube, during adult tissue homeostasis, and self-renewal of stem cells. Upon secretion from Wnt-3a-producing cells, Wnt-3a molecules traverse the extracellular space through association and dissociation with the surface of the cells and the extracellular matrix, and generate a concentration gradient with positional information that instructs developing cells to pursue particular fates. The binding of Wnt-3a to the surface of the cells and the extracellular matrix is partly mediated by CS chains [15], [16]. Our previous study showed that Wnt-3a binds to CS chains containing E units with high affinity [16] and C4ST-1 is involved in the biosynthesis of E units [15]. Therefore, we have proposed a model in which Wnt molecules down-regulate C4ST-1 gene expression, leading to structural changes in CS chains on the surface of the cells and triggering the release of Wnt molecules from these cells [15]. However, how Wnt-3a down-regulates C4ST-1 gene expression is unclear. Here we report that Wnt signaling controls C4ST-1 gene expression mediated by histone deacetylase.
Section snippets
Cell culture
L-Wnt-3a cells (CRL-2647) were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 100 units/mL penicillin, 100 μg/mL streptomycin sulfate, and 400 μg/mL G418 (Invitrogen), and maintained in an incubator in a 5% CO2 atmosphere at 37 °C. Mouse L fibroblasts kindly provided by Dr. Frank Tufaro (Allera Health Products, Inc.) and human hepatocellular carcinoma HepG2 (HB-8065) cells were cultured in DMEM supplemented with 10% fetal bovine serum, 100 units/mL
Results
We previously indicated that the expression of C4ST-1 was depressed in mouse L cells expressing high amounts of Wnt-3a protein (L-Wnt-3a cells) compared to the parental L cells [15]. Wnt-3a ligands lead to activation of β-catenin, a key transcription factor in the canonical Wnt/β-catenin pathway. In addition, we showed that blockage of Wnt signaling could recover C4ST-1 expression [16]. These findings suggested that Wnt/β-catenin signaling or β-catenin is directly or indirectly involved in
Discussion
During synthesis of the CS backbone, sulfotransferases catalyze various modifications such as sulfation. “Sugar codes” specified by explicit sulfation patterns are thought to define the specificity and affinity of protein-sugar interactions. Thus, we have been studying how the sugar code is regulated. We previously showed a reciprocal relationship between Wnt/β-catenin signaling and specific sulfation patterns of CS chains [15]. We found that the specific sulfation pattern of CS chains
Acknowledgements
We thank Mr. Souta Miyazaki, Mr. Kenji Someya, and Ms. Midori Takami for their technical assistance. This work was supported in part by Grants-in-Aid for Scientific Research (B) (#25293014 and #16H05088, to H.K.) and (C) (#25460080, to S.N.) and by the Supported Program for the Strategic Research Foundation at Private Universities (#S1201040), 2012–2016 (to H.K.) from the Ministry of Education, Culture, Sports, Science & Technology, Japan.
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2018, Journal of Biological ChemistryCitation Excerpt :Based on the substrate preferences of Chn sulfotransferases identified to date, the biosynthetic scheme for CS-type sulfation can be classified into initial “4-O-sulfation” and “6-O-sulfation” pathways. In the initial step, the non-sulfated O-unit [GlcA-GalNAc] serves as a common acceptor substrate for two types of sulfotransferases, chondroitin 4-O-sulfotransferases (C4ST-1 and C4ST-2) (18–20) and chondroitin 6-O-sulfotransferse-1 (C6ST-1), forming monosulfated A-unit (GlcA-GalNAc(4-O-sulfate)) and C-unit (GlcA-GalNAc(6-O-sulfate)), respectively (see Fig. 7A, panel b). Subsequent sulfation of A- and C-units can also occur via GalNAc 4-sulfate 6-O-sulfotransferase (GalNAc4S-6ST) or CS-specific uronyl 2-O-sulfotransferase, resulting in the formation of disulfated disaccharide E-unit (GlcA-GalNAc(4,6-O-disulfate)) and D-unit (GlcA(2-O-sulfate)-GalNAc(6-O-sulfate)), respectively (see Fig. 7A, panel b) (16).
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