Elsevier

Experimental Cell Research

Volume 315, Issue 10, 10 June 2009, Pages 1715-1723
Experimental Cell Research

Research Article
Galectin-8 interacts with podoplanin and modulates lymphatic endothelial cell functions

https://doi.org/10.1016/j.yexcr.2009.02.021Get rights and content

Abstract

Podoplanin is a small, mucin-like membrane glycoprotein highly expressed by lymphatic but not by blood vascular endothelial cells. Although it was shown to be indispensable for the correct formation and function of the lymphatic vasculature, its precise molecular function has remained unknown. In the present study, we identified the mammalian lectin galectin-8 as a novel, glycosylation-dependent interaction partner of podoplanin. Galectin-8 is a tandem-repeat type galectin, which interacts with cell surface glycoproteins, including certain integrins, as well as with extracellular matrix molecules such as fibronectin. Here we show that, similar to podoplanin, galectin-8 is more highly expressed by lymphatic than by blood vascular endothelial cells, and that it promotes lymphatic endothelial cell adhesion as well as haptotactic migration when immobilized onto a surface, while inhibiting the formation of tube-like structures by lymphatic endothelial cells in a collagen matrix when incorporated into the matrix. Importantly, functions of blood vascular endothelial cells, which lack podoplanin expression, are not affected by galectin-8. These data suggest a role for galectin-8 and podoplanin in supporting the connection of the lymphatic endothelium to the surrounding extracellular matrix, most likely in cooperation with other glycoproteins on the surface of lymphatic endothelial cells.

Introduction

The lymphatic vascular system has become the subject of intense research during the last two decades, since diverse molecular markers for lymphatic vessels were discovered and a number of in vitro and in vivo models became available to study various aspects of lymphatic biology.

Lymphatic vessels are found in nearly all organs and play important physiological roles, including the drainage of tissue fluid, the transport of immune cells to lymph nodes and the uptake of dietary fat in the intestine. Moreover, the lymphatic system is crucially involved in a number of pathological conditions, such as cancer metastasis and certain inflammatory diseases. Despite the growing interest in lymphatic biology, however, the molecular events underlying the development and the function of the lymphatic system are still much less well understood than those taking place in the blood vascular system.

A long-standing open question in the field of lymphatic biology concerns the presumable molecular function of podoplanin, a sialomucin-like membrane glycoprotein, which is highly expressed on the surface of lymphatic endothelial cells (LECs) but not blood vascular endothelial cells (BECs) in vitro as well as in vivo [1], [2]. Human podoplanin is a 162 amino acid type I transmembrane protein, comprising a short cytoplasmic tail, a single membrane-spanning domain and an extensively O-glycosylated, sialylated extracellular portion [3]. Although studies in podoplanin knockout mice demonstrated that this protein is essential for the correct formation and function of the lymphatic vascular system [2], and it is one of the most commonly exploited markers for lymphatic vessels [4], the exact molecular function of podoplanin on the lymphatic endothelium remains unclear. Several reports suggested a role for podoplanin in endothelial cell cytoskeletal organization and migration. Podoplanin overexpression induces the formation of filopodia-like plasma membrane extensions in a variety of cell types [2], [3], [5], [6], and a conserved cluster of three basic amino acids in its cytoplasmic domain mediates its interaction with the membrane cytoskeleton linkers ezrin and moesin [7]. Additionally, two binding partners of the extracellular domain of podoplanin have been identified so far. The podoplanin ectodomain and the lymphatic-specific chemokine CCL21 form a complex, which presumably contributes to the recruitment of CCR7-positive immune cells towards the lymphatic vessels after being shed from the lymphatic endothelium into the perivascular stroma [8]. The interaction of podoplanin with the C-type lectin-like receptor 2 (CLEC-2) on platelets is responsible for the platelet-aggregating activity of podoplanin [9], which is of relevance mainly for podoplanin-expressing cancer cells, but might also be of importance in the lymphatic vasculature, to prevent mixing of blood and lymphatic vessels. The interactions with CCL21 and with CLEC-2 both require the glycosylation of the podoplanin ectodomain [8], [9].

Galectins are a subgroup of animal lectins – i.e. non-enzymatic, sugar-binding proteins – which are defined by their binding specificity for β-galactosides and the presence of at least one structurally conserved carbohydrate-recognition domain (CRD) [10]. Based on their domain organization, galectins can be classified into prototypes, chimera types and tandem-repeat types [11]. They are found throughout all animal kingdoms and exert extraordinarily diverse functions both inside and outside the cell, through protein–carbohydrate as well as protein–protein interactions (reviewed in [12]). Fifteen mammalian galectins have been identified up to date, eleven of which are also expressed in humans. One of those is galectin-8, which is found in many tissues including lung, liver, kidney, spleen and others. Galectin-8 is a soluble, 35 kDa protein belonging to the subclass of tandem-repeat type galectins, since it has two homologous CRDs, connected by a short, unconserved linker peptide. Alternative mRNA splicing gives rise to at least six different potential galectin-8 protein isoforms, including proteins with only one CRD which have, however, never been isolated so far [13]. Despite having features of a cytosolic protein, galectin-8 – similar to other galectins – may be externalized under certain conditions through a non-classical secretion pathway, without the requirement for a signal peptide [14], [15]. Upon secretion, it is retained at the cell surface due to its interaction with certain integrins, including α3β1 and α6β1 and to a very limited extent also α4 and β3 [15]. The binding of galectin-8 to integrins is sugar-dependent (in contrast to the protein–protein interactions of integrins with their ligands) [15] and triggers integrin-mediated signaling cascades and cytoskeletal re-organization [16]. Additionally, galectin-8 also associates with extracellular matrix (ECM) glycoproteins, such as fibronectin [17].

When immobilized onto a surface, galectin-8 promotes cell adhesion and spreading as potently as fibronectin [17]. Soluble galectin-8, however, was shown to inhibit cell adhesion to ECM molecules such as fibronectin and laminin, possibly by masking the ligand binding sites of integrin receptors and thereby preventing cell–matrix interactions. Its ability to both positively and negatively regulate cell adhesion qualifies galectin-8 as a matricellular protein [17], i.e. an adhesion-modulating protein similar to SPARC, thrombospondin, tenascin and others [18].

In the present study, we identified galectin-8 as a novel, glycosylation-dependent interaction partner of podoplanin. Moreover, we found that galectin-8 is more highly expressed by LECs than by BECs and that it promotes LEC adhesion and haptotactic migration while inhibiting the formation of tube-like structures by LECs. Our results suggest a role for galectin-8 and podoplanin in supporting the connection of the lymphatic endothelium to the surrounding ECM, most likely in cooperation with other glycoproteins on the surface of LECs.

Section snippets

Cell culture

Primary human lymphatic (LECs) and blood vascular endothelial cells (BECs), isolated from neonatal foreskins as previously described [1], as well as human umbilical vein endothelial cells (HUVECs; ScienCell, Carlsbad, CA) were kept in type I collagen-coated (50 μg/ml in PBS; Inamed, Fremont, CA) culture dishes in endothelial cell basal medium (EBM; Lonza, Walkersville, MD) supplemented with 20% fetal bovine serum (FBS), 100 U/ml penicillin, 100 μg/ml streptomycin, 2 mM l-glutamine (all from

Galectin-8 interacts with podoplanin in a glycosylation-dependent manner

In order to clarify the yet unknown molecular function of podoplanin on the lymphatic endothelium, we sought to identify novel interaction partners of this membrane glycoprotein. To this aim, we probed a human protein microarray with a recombinant soluble fusion protein consisting of the extracellular domain of podoplanin linked to the Fc domain of human IgG1 (podoplanin-Fc), or the human IgG1 Fc domain alone as a control. Podoplanin-Fc used for probing the array was produced in Chinese hamster

Discussion

In the present study we identified galectin-8 as a novel interaction partner of podoplanin, an extensively O-glycosylated membrane protein which is within the vasculature specifically expressed on the lymphatic endothelium. As expected for a lectin, the interaction of galectin-8 with podoplanin required sugar-moieties, which as such are naturally present in the ectodomain of podoplanin, as shown by the interaction of endogenous podoplanin from primary human lymphatic endothelial cells (LECs)

Acknowledgments

We thank Dr. Yehiel Zick and Denise Ronen for providing us with their anti-galectin-8 antibody. This work was supported by the National Institutes of Health grant CA69184, Swiss National Fund grant 3100A0-108207, Austrian Science Foundation grant S9408-B11, Cancer League Zurich, Oncosuisse and Commission of the European Communities grant LSHC-CT-2005-518178 (M.D.).

References (27)

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