The extostosin family: Proteins with many functions

Heparan sulfates are complex sulfated molecules found in abundance at cell surfaces and in the extracellular matrix. They bind to and influence the activity of a variety of molecules like growth factors, proteases and morphogens and are thus involved in various cell–cell and cell–matrix interactions. The mammalian EXT proteins have glycosyltransferase activities relevant for HS chain polymerization, however their exact role in this process is still confusing. In this review, we summarize current knowledge about the biochemical activities and some proposed functions of the members of the EXT protein family and their roles in human disease.publishedVersio

QSulf1 remodels the 6-O sulfation states of cell surface heparan sulfate proteoglycans to promote Wnt signaling

The 6-O sulfation states of cell surface heparan sulfate proteoglycans (HSPGs) are dynamically regulated to control the growth and specification of embryonic progenitor lineages. However, mechanisms for regulation of HSPG sulfation have been unknown. Here, we report on the biochemical and Wnt signaling activities of QSulf1, a novel cell surface sulfatase. Biochemical studies establish that QSulf1 is a heparan sulfate (HS) 6-O endosulfatase with preference, in particular, toward trisulfated IdoA2S-GlcNS6S disaccharide units within HS chains. In cells, QSulf1 can function cell autonomously to remodel the sulfation of cell surface HS and promote Wnt signaling when localized either on the cell surface or in the Golgi apparatus. QSulf1 6-O desulfation reduces XWnt binding to heparin and HS chains of Glypican1, whereas heparin binds with high affinity to XWnt8 and inhibits Wnt signaling. CHO cells mutant for HS biosynthesis are defective in Wnt-dependent Frizzled receptor activation, establishing that HS is required for Frizzled receptor function. Together, these findings suggest a two-state “catch or present” model for QSulf1 regulation of Wnt signaling in which QSulf1 removes 6-O sulfates from HS chains to promote the formation of low affinity HS–Wnt complexes that can functionally interact with Frizzled receptors to initiate Wnt signal transduction

Fibroblast EXT1-Levels Influence Tumor Cell Proliferation and Migration in Composite Spheroids

Background: Stromal fibroblasts are important determinants of tumor cell behavior. They act to condition the tumor microenvironment, influence tumor growth, support tumor angiogenesis and affect tumor metastasis. Heparan sulfate proteoglycans, present both on tumor and stromal cells, interact with a large number of ligands including growth factors, their receptors, and structural components of the extracellular matrix. Being ubiquitously expressed in the tumor microenvironment heparan sulfate proteoglycans are candidates for playing central roles in tumor-stroma interactions. The objective of this work was to investigate the role of heparan sulfate expressed by stromal fibroblasts in modulating the growth of tumor cells and in controlling the interstitial fluid pressure in a 3-D model. Methodology/Principal Findings: We generated spheroids composed of fibroblasts alone, or composite spheroids, composed of fibroblasts and tumor cells. Here we show that stromal fibroblasts with a mutation in the heparan sulfate elongating enzyme Ext1 and thus a low heparan sulfate content, formed composite fibroblast/tumor cell spheroids with a significant lower interstitial fluid pressure than corresponding wild-type fibroblast/tumor cell composite spheroids. Furthermore, immunohistochemistry of composite spheroids revealed that the cells segregated, so that after 6 days in culture, the wild-type fibroblasts formed an inner core and the tumor cells an outer layer of cells. For composite spheroids containing Ext1-mutated fibroblasts this segregation was less obvious, indicating impaired cell migration. Analysis of tumor cells expressing the firefly luciferase gene revealed that the changes in tumor cell migration in mutant fibroblast/tumor cell composite spheroids coincided with a lower proliferation rate. Conclusions/Significance: This is the first demonstration that stromal Ext1-levels modulate tumor cell proliferation and affect the interstitial fluid pressure in a 3-D spheroid model. Learning how structural changes in stromal heparan sulfate influence tumor cells is essential for our understanding how non-malignant cells of the tumor microenvironment influence tumor cell progression

A549 expression profiling in α11^-/-/A549 and α11^+/+/A549 heterospheroids.

<p>(A) The top 10 up- and down-regulated genes in A549 cells in α11^-/-/A549 spheroids compared with those in α11^+/+/A549 spheroids. Fold changes shown are the average values from 6 individual samples from each type of spheroid. Gene symbols marked in bold are the genes chosen for RT-qPCR verification. (B) Microarray data verification of two selected genes using qRT-PCR. Total RNA was extracted from 6-day-old spheroids prepared in new replicate experiments in conditions identical to those used for Microarray (n = 6). Shown is the fold change of the mRNA expression levels of the genes in α11^-/-/A549 comparing with those in α11^+/+/A549 spheroids (set arbitrarily as 1). Error bars represent standard deviation across 6 independent samples.</p

CXCL5 regulates heterospheroid invasion in 3D collagen gels.

<p>Six-day-old spheroids were embedded in 3D collagen gels (1 mg/ml collagen type I) in the presence of DMSO (control) or different concentrations of the CXCL5 receptor inhibitor SB225002 (upper graph) or recombinant CXCL5 (lower graph). Six spheroids were imbedded into the collagen gels under each condition. Invasion of the cells from the spheroids into the collagen gels was observed under an inverted phase contrast microscope and photographed after 48 h. Size bar  = 100 µm.</p