Heparan sulfate proteoglycans as trastuzumab targets in anoikis-resistant endothelial cells

Anoikis is a form of programmed cell death induced by loss of contact from neighboring cells or from their extracellular matrix (ECM). Many tumorigenic cells are anoikis resistant, facilitating cancer progression and metastasis. Trastuzumab is a monoclonal antibody used for the treatment of breast and gastric cell cancer, but its mechanism of action is not well elucidated and its target molecules not well defined. Heparan sulfate proteoglycans (HSPGs) and glycosaminoglycans (GAGs) play important roles in tumor development and in response of cancer cells to drugs. This study investigates the effect of trastuzumab on the expression of HSPGs and sulfated glycosaminoglycans (SGAGs) in anoikis-resistant endothelial cells. After trastuzumab treatment, endothelial cells resistant to anoikis show an increase in adhesion to fibronectin followed by a decrease in invasion, proliferation, and angiogenic capacity. In addition, a significant increase in the number of cells in the S phase of the cell cycle was also observed. In relation to HSPGs and SGAGs expression, we observed a decrease in syndecan-4 and perlecan expression, as well as in the heparan sulfate biosynthesis in anoikis-resistant endothelial cells after exposure to trastuzumab. Our results suggest that trastuzumab interacts with GAGs and proteoglycans of the cell surface and ECM and through this interaction controls cellular events in anoikis-resistant endothelial cells.     

Heparan sulfate and chondroitin sulfate proteoglycans inhibit E-selectin binding to endothelial cells

E-selectin is a cell adhesion molecule involved in the initial rolling and adhesion of leukocytes to the endothelium during inflammation. In addition, in vitro studies have suggested that an interaction between E-selectin and binding sites such as sialyl Lewis X-containing oligosaccharides on endothelial cells may be important for angiogenesis. In order to investigate the binding of E-selectin to endothelial cells, we developed an ELISA assay using chimeric E-selectin-Ig molecules and endothelial cells fixed on poly-L-lysine coated plates. Our results indicate that E-selectin-Ig binds to both bovine capillary endothelial cells and human dermal microvascular endothelial cells in a calcium-dependent and saturable manner. The binding is inhibited markedly by heparin and by syndecan-1 ectodomain, and moderately by chondroitin sulfate, but not by sialyl Lewis X-containing oligosaccharides. These results suggest that heparan sulfate and chondroitin sulfate proteoglycans on endothelial cells are potential ligands for E-selectin.     

Heparan sulfate proteoglycans and triglyceride-rich lipoprotein metabolism

PURPOSE OF REVIEW: Clearance of triglyceride-rich lipoprotein remnants by the liver is a key step in preventing hypertriglyceridemia, an independent risk factor for cardiovascular disease. We review recent genetic evidence that heparan sulfate proteoglycans work in concert with the LDL receptor in the liver to facilitate binding and clearance of both triglyceride and cholesterol-rich lipoproteins from the circulation. RECENT FINDINGS: Partial reduction of sulfation of liver heparan sulfate using the Cre-loxP system caused accumulation of hepatic and dietary triglyceride-rich lipoprotein particles due to delayed clearance. Compounding the mutation with LDL receptor deficiency caused enhanced accumulation of both cholesterol and triglyceride-rich particles compared with mice lacking only LDL receptors. These findings provide the first genetic evidence that hepatic heparan sulfate proteoglycans play a central role in the clearance of lipoproteins by the liver and work independently of LDL receptors. SUMMARY: A role for hepatocyte heparan sulfate in lipoprotein metabolism has now been genetically established in mice. Given this finding, mild, but clinically relevant, hyperlipidemias in human patients may be a result of alterations in heparan sulfate structure or possible genetic polymorphisms in the relevant biosynthetic genes.     

Heparan sulfate proteoglycans regulate autophagy in Drosophila

Heparan sulfate-modified proteoglycans (HSPGs) are important regulators of signaling and molecular recognition at the cell surface and in the extracellular space. Disruption of HSPG core proteins, HS-synthesis, or HS-degradation can have profound effects on growth, patterning, and cell survival. The Drosophila neuromuscular junction provides a tractable model for understanding the activities of HSPGs at a synapse that displays developmental and activity-dependent plasticity. Muscle cell-specific knockdown of HS biosynthesis disrupted the organization of a specialized postsynaptic membrane, the subsynaptic reticulum (SSR), and affected the number and morphology of mitochondria. We provide evidence that these changes result from a dysregulation of macroautophagy (hereafter referred to as autophagy). Cellular and molecular markers of autophagy are all consistent with an increase in the levels of autophagy in the absence of normal HS-chain biosynthesis and modification. HS production is also required for normal levels of autophagy in the fat body, the central energy storage and nutritional sensing organ in Drosophila. Genetic mosaic analysis indicates that HS-dependent regulation of autophagy occurs non-cell autonomously, consistent with HSPGs influencing this cellular process via signaling in the extracellular space. These findings demonstrate that HS biosynthesis has important regulatory effects on autophagy and that autophagy is critical for normal assembly of postsynaptic membrane specializations.     

Heparan sulfate proteoglycans in invasion and metastasis

Because heparan sulfate proteoglycans mediate cell adhesion and control the activities of numerous growth and motility factors, they play a critical role in regulating the metastatic behavior of tumor cells. Due to their utilitarian nature, heparan sulfate proteoglycans may at times act as inhibitors of cell invasion and at other times as promoters of cell invasion, with their function being determined by their location (cell surface or extracellular matrix), the heparin-binding molecules they associate with, the presence of modifying enzymes (proteases, heparanases) and the precise structural characteristics of the proteoglycan. Also, the tissue type and pathophysiological state of the tumor influence proteogylcan function. This review summarizes our current knowledge of the role heparan sulfate proteoglycans play in regulating tumor cell metastasis, proposes mechanisms of how these molecules function and examines the potential for discovery of new therapeutic approaches designed to block metastatic cancer.     

Heparan sulfate proteoglycans in Drosophila neuromuscular development

Heparan sulfate proteoglycans (HSPGs) are glycoconjugates bearing heparan sulfate (HS) chains covalently attached to core proteins, which are ubiquitously distributed on the cell surface and in the extracellular matrix. HSPGs interact with a number of molecules mainly through HS chains, which play critical roles in diverse physiological and disease processes. Among these, recent vertebrate studies showed that HSPGs are closely involved in synapse development and function. However, the detailed molecular mechanisms remain elusive. Genetic studies from fruit flies, Drosophila melanogaster, have begun to reveal the molecular mechanisms by which HSPGs regulate synapse formation at neuromuscular junctions (NMJs). In this review, we introduce Drosophila studies showing how HSPGs regulate various signaling pathways in developing NMJs. This article is part of a Special Issue entitled Neuro-glycoscience, edited by Kenji Kadomatsu and Hiroshi Kitagawa.     

Heparan sulfate proteoglycans and the emergence of neuronal connectivity

With the identification of the molecular determinants of neuronal connectivity, our understanding of the extracellular information that controls axon guidance and synapse formation has evolved from single factors towards the complexity that neurons face in a living organism. As we move in this direction - ready to see the forest for the trees - attention is returning to one of the most ancient regulators of cell-cell interaction: the extracellular matrix. Among many matrix components that influence neuronal connectivity, recent studies of the heparan sulfate proteoglycans suggest that these ancient molecules function as versatile extracellular scaffolds that both sculpt the landscape of extracellular cues and modulate the way that neurons perceive the world around them.     

Regulation of hematopoietic stem cells in the niche

Hematopoiesis provides a suitable model for understanding adult stem cells and their niche. Hematopoietic stem cells(HSCs) continuously produce blood cells through orchestrated proliferation, self-renewal, and differentiation in the bone marrow(BM). Within the BM exists a highly organized microenvironment termed "niche" where stem cells reside and are maintained. HSC niche is the first evidence that a microenvironment contributes to protecting stem cell integrity and functionality in mammals. Although multiple models exist, recent progress has principally elucidated the cellular complexity of the HSC niche that maintains and regulates HSCs in BM. Here we introduce the development and summarize the achievements of HSC niche studies.     

Heparan sulfate proteoglycans as regulators of fibroblast growth factor-2 signaling in brain endothelial cells. Specific role for glypican-1 in glioma angiogenesis

Fibroblast growth factor-2 (FGF2) is a potent angiogenic factor in gliomas. Heparan sulfate promotes ligand binding to receptor tyrosine kinase and regulates signaling. The goal of this study was to examine the contribution of heparan sulfate proteoglycans (HSPGs) to glioma angiogenesis. Here we show that all brain endothelial cell HSPGs carry heparan sulfate chains similarly capable of forming a ternary complex with FGF2 and fibroblast growth factor receptor-1c and of promoting a mitogenic signal. Immunohistochemical analysis revealed that glypican-1 was overexpressed in glioma vessel endothelial cells, whereas this cell-surface HSPG was consistently undetectable in normal brain vessels. To determine the effect of increased glypican-1 expression on FGF2 signaling, we transfected normal brain endothelial cells, which express low base-line levels of glypican-1, with this proteoglycan. Glypican-1 expression enhanced growth of brain endothelial cells and sensitized them to FGF2-induced mitogenesis despite the fact that glypican-1 remained a minor proteoglycan. In contrast, overexpression of syndecan-1 had no effect on growth or FGF2 sensitivity. We conclude that the glypican-1 core protein has a specific role in FGF2 signaling. Glypican-1 overexpression may contribute to angiogenesis and the radiation resistance characteristic of this malignancy.     

Heparan sulfate proteoglycans serve as alternative receptors for low affinity LCMV variants

Members of the Old World Arenaviruses primarily utilize α-dystroglycan (α-DAG1) as a cellular receptor for infection. Mutations within the glycoprotein (GP) of lymphocytic choriomeningitis virus (LCMV) reduce or abrogate the binding affinity to α-DAG1 and thus influence viral persistence, kinetics, and cell tropism. The observation that α-DAG1 deficient cells are still highly susceptible to low affinity variants, suggests the use of an alternative receptor(s). In this study, we used a genome-wide CRISPR Cas9 knockout screen in DAG1 deficient 293T cells to identify host factors involved in α-DAG1-independent LCMV infection. By challenging cells with vesicular stomatitis virus (VSV), pseudotyped with the GP of LCMV WE HPI (VSV-GP), we identified the heparan sulfate (HS) biosynthesis pathway as an important host factor for low affinity LCMV infection. These results were confirmed by a genetic approach targeting EXTL3, a key factor in the HS biosynthesis pathway, as well as by enzymatic and chemical methods. Interestingly, a single point mutation within GP1 (S153F or Y155H) of WE HPI is sufficient for the switch from DAG1 to HS binding. Furthermore, we established a simple and reliable virus-binding assay, using directly labelled VSV-GP by intramolecular fusion of VSV-P and mWasabi, demonstrating the importance of HS for virus attachment but not entry in Burkitt lymphoma cells after reconstitution of HS expression. Collectively, our study highlights the essential role of HS for low affinity LCMV infection in contrast to their high affinity counterparts. Residual LCMV infection in double knockouts indicate the use of (a) still unknown entry receptor(s).     

Heparan sulfate proteoglycans and heparin regulate melanoma cell functions

Background

The solid melanoma tumor consists of transformed melanoma cells, and the associated stromal cells including fibroblasts, endothelial cells, immune cells, as well as, soluble macro- and micro-molecules of the extracellular matrix (ECM) forming the complex network of the tumor microenvironment. Heparan sulfate proteoglycans (HSPGs) are an important component of the melanoma tumor ECM. Importantly, there appears to be both a quantitative and a qualitative shift in the content of HSPGs, in parallel to the nevi–radial growth phase–vertical growth phase melanoma progression. Moreover, these changes in HSPG expression are correlated to modulations of key melanoma cell functions.

Scope of review

This review will critically discuss the roles of HSPGs/heparin in melanoma development and progression.

Major conclusions

We have correlated HSPGs' expression and distribution with melanoma cell signaling and functions as well as angiogenesis.

General significance

The current knowledge of HSPGs/heparin biology in melanoma provides a foundation we can utilize in the ongoing search for new approaches in designing anti-tumor therapy. This article is part of a Special Issue entitled Matrix-mediated cell behaviour and properties.     

Heparan sulfate proteoglycans mediate the invasion of cardiomyocytes by Trypanosoma cruzi

Cytoadherence is an important step for the invasion of a mammalian host cell by Trypanosoma cruzi. Cell surface macromolecules are implicated in the T. cruzi-cardiomyocyte recognition process. Therefore, we investigated the role of cell surface proteoglycans during this invasion process and analyzed their expression after the parasite infected the target cells. Treatment of trypomastigote forms of T. cruzi with soluble heparan sulfate resulted in a significant inhibition in successful invasion, while chondroitin sulfate had no effect. Removal of sulfated glycoconjugates from the cardiomyocyte surface using glycosaminoglycan (GAG) lyases demonstrated the specific binding of the parasites to heparan sulfate proteoglycans. Infection levels were reduced by 42% whenthe host cells were previously treated with heparitinase II. No changes were detected in the expression of GAGs infected cardiomyocytes even after 96 h of infection. Our data demonstrate that heparan sulfate proteoglycans, but not chondroitin sulfate, mediate both attachment and invasion of cardiomyocytes by T. cruzi.     

Heparan sulfate regulates self-renewal and pluripotency of embryonic stem cells

Embryonic stem (ES) cell self-renewal and pluripotency are maintained by several signaling cascades and by expression of intrinsic factors, such as Oct3/4 and Nanog. The signaling cascades are activated by extrinsic factors, such as leukemia inhibitory factor, bone morphogenic protein, and Wnt. However, the mechanism that regulates extrinsic signaling in ES cells is unknown. Heparan sulfate (HS) chains are ubiquitously present as the cell surface proteoglycans and are known to play crucial roles in regulating several signaling pathways. Here we investigated whether HS chains on ES cells are involved in regulating signaling pathways that are important for the maintenance of ES cells. RNA interference-mediated knockdown of HS chain elongation inhibited mouse ES cell self-renewal and induced spontaneous differentiation of the cells into extraembryonic endoderm. Furthermore, autocrine/paracrine Wnt/beta-catenin signaling through HS chains was found to be required for the regulation of Nanog expression. We propose that HS chains are important for the extrinsic signaling required for mouse ES cell self-renewal and pluripotency.     

Metabolic regulation of hematopoietic stem cells in the hypoxic niche

Tissue homeostasis over the life of an organism relies on both self-renewal and multipotent differentiation of stem cells. Hematopoietic stem cells (HSCs) reside in a hypoxic bone marrow environment, and their metabolic status is distinct from that of their differentiated progeny. HSCs generate energy mainly via anaerobic metabolism by maintaining a high rate of glycolysis. This metabolic balance promotes HSC maintenance by limiting the production of reactive oxygen species, but leaves HSCs susceptible to changes in redox status. In this review, we discuss the importance of oxygen homeostasis and energy metabolism for maintenance of HSC function and long-term self-renewal.     

Heparan sulfate proteoglycans from mouse mammary epithelial cells. Cell surface proteoglycan as a receptor for interstitial collagens

A heparan sulfate-rich proteoglycan is on the surface of NMuMG mouse mammary epithelial cells apparently intercalated into their plasma membranes. Mild treatment of the cells with trypsin releases the GAG-bearing region (ectodomain) of this molecule as a discrete proteoglycan which is readily purified. At physiological pH and ionic strength, the ectodomain binds collagen types I, III, and V but not types II, IV, or denatured type I. The proteoglycan binds to a single class of high affinity saturable sites on type I collagen fibrils, sites which are selective for heparin-like glycosaminoglycans. The binding of NMuMG cells to type I collagen duplicates that of their cell surface proteoglycan; cells bind to native but not denatured collagen, and binding is inhibited by heparin but not by other glycosaminoglycans. These binding properties suggest that cell surface heparan sulfate proteoglycans could act as receptors for interstitial collagens and mediate changes in cell behavior induced by collagenous matrices.     

Heparan sulfate proteoglycans as extracellular docking molecules for matrilysin (matrix metalloproteinase 7)

Many matrix metalloproteinases (MMPs) are tightly bound to tissues; matrilysin (MMP-7), although the smallest of the MMPs, is one of the most tightly bound. The most likely docking molecules for MMP-7 are heparan sulfate proteoglycans on or around epithelial cells and in the underlying basement membrane. This is established by extraction experiments and confocal microscopy. The enzyme is extracted from homogenates of postpartum rat uterus by heparin/heparan sulfate and by heparinase III treatment. The enzyme is colocalized with heparan sulfate in the apical region of uterine glandular epithelial cells and can be released by heparinase digestion. Heparan sulfate and MMP-7 are expressed at similar stages of the rat estrous cycle. The strength of heparin binding by recombinant rat proMMP-7 was examined by affinity chromatography, affinity coelectrophoresis, and homogeneous enzyme-based binding assay; the K(D) is 5-10 nM. Zymographic measurement of MMP-7 activity is greatly enhanced by heparin. Two putative heparin-binding peptides have been identified near the C- and N-terminal regions of proMMP-7; however, molecular modeling suggests a more extensive binding track or cradle crossing multiple peptide strands. Evidence is also found for the binding of MMP-2, -9, and -13. Binding of MMP-7 and other MMPs to heparan sulfate in the extracellular space could prevent loss of secreted enzyme, provide a reservoir of latent enzyme, and facilitate cellular sensing and regulation of enzyme levels. Binding to the cell surface could position the enzyme for directed proteolytic attack, for activation of or by other MMPs and for regulation of other cell surface proteins. Dislodging MMPs by treatment with compounds such as heparin might be beneficial in attenuating excessive tissue breakdown such as occurs in cancer metastasis, arthritis, and angiogenesis.     

Noncanonical Wnt signaling maintains hematopoietic stem cells in the niche

Wnt signaling is involved in self-renewal and maintenance of hematopoietic stem cells (HSCs); however, the particular role of noncanonical Wnt signaling in regulating HSCs in vivo is largely unknown. Here, we show Flamingo (Fmi) and Frizzled (Fz) 8, members of noncanonical Wnt signaling, both express in and functionally maintain quiescent long-term HSCs. Fmi regulates Fz8 distribution at the interface between HSCs and N-cadherin(+) osteoblasts (N-cad(+)OBs that enrich osteoprogenitors) in the niche. We further found that N-cad(+)OBs predominantly express noncanonical Wnt ligands and inhibitors of canonical Wnt signaling under homeostasis. Under stress, noncanonical Wnt signaling is attenuated and canonical Wnt signaling is enhanced in activation of HSCs. Mechanistically, noncanonical Wnt signaling mediated by Fz8 suppresses the Ca(2+)-NFAT- IFNγ pathway, directly or indirectly through the CDC42-CK1α complex and also antagonizes canonical Wnt signaling in HSCs. Taken together, our findings demonstrate that noncanonical Wnt signaling maintains quiescent long-term HSCs through Fmi and Fz8 interaction in the niche.