Exostosin-like 2 regulates FGF2 signaling by controlling the endocytosis of FGF2

https://doi.org/10.1016/j.bbagen.2018.01.002Get rights and content

Highlights

  • Reduced expression of EXTL2 increased the amounts of heparan sulfate.

  • However, the increased heparan sulfate suppressed FGF2-dependent proliferation.

  • The increased heparan sulfate enhanced the incorporation of FGF2 into the cells.

  • Exostosin glycosyltransferases differentially regulate FGF2 signaling.

Abstract

Background

Heparan sulfate proteoglycans are ubiquitously expressed on cell surfaces and in extracellular matrices, and are engaged in heparin-binding growth factor-related signal transduction. Thus, changes in the amounts, structures, and chain lengths of heparan sulfate have profound effects on aspects of cell growth controlled by heparin-binding growth factors such as FGF2. Exostosin glycosyltransferases (EXT1, EXT2, EXTL1, EXTL2, and EXTL3) control heparan sulfate biosynthesis, and the expression levels of their genes regulate the amounts, chain lengths, and sulfation patterns of heparan sulfate. Unlike EXT1, EXT2, and EXTL3, EXTL2 functions chain termination of heparan sulfate. Here, we examined the importance of EXTL2 in FGF2-dependent signaling.

Methods

We investigated heparan sulfate biosynthesis and FGF2 signaling using four cell lines, EXT1-deficient cells, EXT2-, EXTL2-, or EXTL3-knockdown cells, by HPLC, qRT-PCR, flow cytometry, and western blotting.

Results

Reduced expression of either EXT1, EXT2, or EXTL3 decreased heparan sulfate biosynthesis, and consequently suppressed the FGF2-dependent proliferation of mouse L fibroblasts. In contrast, although knockdown of EXTL2 increased the amounts of heparan sulfate, FGF2-dependent proliferation was significantly inhibited because the increased heparan sulfate enhanced the incorporation of FGF2 into the cells.

Conclusions

EXTL2 controls FGF2 signaling through regulation of heparan sulfate biosynthesis in a manner distinct from that of other exostosins.

General significance

This study provides new insights into the regulatory mechanisms of FGF2 signaling by EXTL2.

Introduction

Heparan sulfate proteoglycans (HS-PGs) are cell-surface and extracellular matrix macromolecules that comprise a core protein to which heparan sulfate (HS) chains are attached. HS-PGs play vital functions in many biological processes. The HS moiety is essential for these functions, and operates as a cofactor for growth factor interaction with their high affinity receptors and thus influences cell adhesion, motility, proliferation, differentiation, and tissue morphogenesis [1]. The sugar structure of HS chains consists of the disaccharide building units, glucuronic acid (GlcA) and N-acetylglucosamine (GlcNAc). These units alternate and have different sulfation patterns, thereby generating diverse sulfation sequences and providing specific binding sites for diverse protein ligands. The interaction of HS binding proteins with HS chains is mediated through positive patches on the protein surface with the negatively charged sulfate groups that decorate HS chains. These decorations do not occur uniformly along the HS chains but rather form organized sub-regions, creating domains with various sulfation patterns that can interact with proteins in distinct manners. Several bioactive proteins have been shown to recognize highly specific HS structures, thereby exerting biological function. Thus, understanding the regulatory mechanisms of HS biosynthesis underlying diverse HS functions is essential.

HS chains are synthesized in the Golgi apparatus in a multi-step process that involves several enzymes. HS biosynthesis is initiated by the transfer of a xylose residue at the HS attachment site(s) of the core protein. Each attachment site contains two to four Ser-Gly sequences. The transfer of xylose is followed by the construction of a tetrasaccharide linkage region, GlcAβ1-3Galβ1-3Galβ1-4Xylβ1-, by multiple glycosyltransferases. Next, the HS chain backbone is synthesized by HS polymerases encoded by EXT1 and EXT2 [2] in the EXT (exostosin) gene family. EXT1 and EXT2 were first identified as causative genes of a genetic bone disorder, hereditary multiple exostoses [3], and were subsequently demonstrated to function as tumor suppressor genes [4]. A connection between HS-synthesizing glycosyltransferases and the hereditary multiple exostoses genes was revealed by two independent studies. In one study, employing an HS-deficient mutant cell line, sog9 and the property of herpes simplex virus to infect cells with HS, cDNA that can rescue HS biosynthesis in that mutant cell line was isolated and identified as EXT1 [5], [6]. Both EXT1 and EXT2 encode bifunctional glycosyltransferases with GlcNAcT-II (N-acetylglucosaminyltransferase II) and GlcAT-II (glucuronyltransferase II) activities that catalyze the polymerization of HS. It has been suggested that the EXT1–EXT2 heterocomplex represents the biologically functional form of HS polymerases [7]. During or after formation of the HS backbone, various modification enzymes are involved in modification processes of HS-PG biosynthesis. N-deacetylase/N-sulfotransferase enzymes, C5 epimerase, and 2-O-, 3-O-, and 6-O-sulfotransferases modify HS chains by catalyzing deacetylation, epimerization, and sulfation at different positions.

The EXT gene family consists of five members: EXT1, EXT2, and three additional members, designated EXTL13 (EXT-like 1–3) on the basis of the amino acid sequence similarity of their gene products to EXT1 and EXT2 proteins [8], [9], [10], [11]. The EXTL genes have not been linked to hereditary multiple exostoses, although the chromosomal loci of the genes imply that they might also encode tumor suppressors. All three EXTL proteins possess glycosyltransferase activities related to HS biosynthesis [6], [12], [13]. Because EXTL2 and EXTL3 possessed activity transferring the first GlcNAc residue to the tetrasaccharide-linkage region (so-called GlcNAcT-I activity), it was speculated that they were implicated in the initiation of HS biosynthesis as a GlcNAcT-I [14]. However, a report that in vitro HS polymerization is induced using tetrasaccharide-linkage analogues as acceptor substrates for the enzyme complex of human EXT1–EXT2, without the aid of EXTL proteins [15], somewhat obscures the biological roles of mammalian EXTLs in HS biosynthesis. Recently, it has been reported that an EXTL2- and EXTL3-mediated mechanism controls the biological function of HS [14], [16], [17], [18], [19], [20]. In addition, it has been suggested that altered EXTL2 and EXTL3 expression is associated with several diseases [21], [22], [23], [24], [25], [26], [27]. Thus, we examined in detail how cellular function is controlled by altered expression of the EXT gene family, and especially EXTL2.

Section snippets

Cell culture and stable transfection

Mouse L fibroblasts (gro2C) and their derivatives were provided by Dr. Frank Tufaro (Allera Health Products, St Petersburg, FL, U.S.A.). To obtain EXT2-, EXTL2-, and EXTL3-knockdown L cells, MISSION™ plasmids for expressing sh (short hairpin) EXT2 and shEXTL2 (Sigma–Aldrich), and pLKO.1-Puro empty vector (Sigma–Aldrich) and Sure Silencing™ shEXTL3 plasmids (SuperArray Bioscience), were transfected into L cells using lipofectamine 2000 transfection reagent (ThermoFisher Scientific) according to

Results

To clarify the roles of the EXT gene family in cellular function, we prepared cells expressing various EXT gene family members: gro2C (EXT1-deficient cells), and EXT2-, EXTL2-, or EXTL3-knocked down cells (shEXT2, shEXTL2, or shEXTL3). As shown in Fig. 1A(a), each cell type expresses HS chains at various levels. Compared to the amount of HS expressed by the parental L cells, loss of EXT1 (gro2C cells) and decreased expression of EXT2 and EXTL3 reduced the amount of HS, whereas decreased

Discussion

HS-PGs are well-known co-receptors for FGF signaling, and thus it is reasonable that FGF signaling is suppressed in gro2C, shEXT2, and shEXTL3 cells, which have a low expression level of HS chains (Fig. 1A(a)). In contrast, although shEXTL2 cells showed a higher expression level of HS chains compared with parental L cells (Fig. 1A(a)), their FGF2 signaling was reduced (Fig. 3B). A previous report demonstrated that HS-PGs are involved in endocytosis of FGF2 [29]. FGF2 bound to HS-PGs enters the

Declaration of interest

The authors declare that they have no conflicts of interest with the contents of this article.

Transparency document

Transparency document.

Acknowledgements

This work was supported in part by Grants-in-Aid for Scientific Research (B) #16H05088 (to H. K.) and (C) 25460080 (to S. N.) from the Ministry of Education, Culture, Sports, Science and Technology, Japan. We are especially grateful to Professor Frank Tufaro (Allera Health Products) for kind provision of the L mutant cell lines.

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