RESEARCH ARTICLE VEGF-A stimulates podosome-mediated collagen-IV proteolysis in microvascular endothelial cells Thomas Daubon 1, * ,§ , Pirjo Spuul 1,2, ‡,§ , Florian Alonso 1,2 , Isabelle Fremaux 1,2 and Elisabeth Ge ́ not 1,2,¶ ABSTRACT Podosomes are dynamic cell–matrix contact structures that combine several key abilities, including adhesion, matrix degradation and mechanosensing. These actin-based cytoskeletal structures have been mostly studied in monocytic cells, but much less is known about those formed in other lineages. In this study, we characterise podosomes in capillary-derived microvascular endothelial cells. We identify two types of podosomes: constitutive podosomes that form in the absence of specific stimulation and induced podosomes that arise in response to the angiogenic factor VEGF-A. Constitutive and VEGF- A-induced podosomes share similar components but exhibit marked differences in terms of gelatinolytic activity. We also show that the extracellular matrix proteins laminin and collagen-IV are key determinants of the VEGF-A response, but neither collagen-I nor fibronectin are conducive for podosome induction. Moreover, only collagen-IV elicits the formation of proteolytically active podosomes through a mechanism involving increased Src phosphorylation, p190RhoGAP-B (also known as ARHGAP5) relocalisation and MT1-MMP (also known as MMP14) cell surface exposure at podosome sites. We hypothesise that by promoting podosome formation, VEGF-A enables endothelial cells to overcome the basement membrane barrier to allow sprouting outwards from the existing vasculature. KEY WORDS: Podosomes, Endothelial cells, VEGF-A, Collagen-IV, Basement membrane INTRODUCTION Vascular endothelial cells line the entire circulatory system from the heart to the smallest capillaries and play a key role in the maintenance and remodelling of the vascular system. The physiology of these cells depends heavily on cytoskeleton rearrangements that are controlled by soluble factors, matrix proteins, cell–cell interactions and mechanical forces. Actin-based cytoskeletal structures called podosomes have been described in endothelial cells (Billottet et al., 2008) and the elucidation of their precise role in endothelial biology thus represents a major challenge. So far, most of our knowledge on podosomes comes from studies performed in macrophages, osteoclasts or immature dendritic cells where they are most commonly found. They are described as dynamic cell–matrix contact structures that combine several key abilities, including adhesion, matrix degradation and mechanosensing (Linder and Kopp, 2005; Linder and Wiesner, 2015). Although the triggering signal for podosome initiation is not known yet, Src family kinases have a pivotal role in their formation (Murphy and Courtneidge, 2011). Structurally, podosomes comprise a core of F-actin associated with actin regulatory proteins such as Arp2/3, WASp or N-WASp (also known as WAS and WASL, respectively), cortactin, dynamin, gelsolin and cofilin, which contribute to their dynamic behaviour, and the scaffolding protein and Src substrate Tks5 (also known as SH3PXD2A), which constitutes a reliable podosome marker. Around the central core, integrins, adaptors and signalling proteins, including FAK (also known as PTK2), vinculin, talin and paxillin, form an adhesive ring domain. Recent studies have shown that acto-myosin filaments radiating from the dense F-actin cores interconnect podosomes and enable their collective behaviour (Proag et al., 2015; van den Dries et al., 2013). In contrast to cells of the myelomonocytic lineage, where podosomes develop evenly in the first steps of integrin-mediated cell spreading, podosome formation is an inducible phenomenon for endothelial cells. Most studies have been performed with human umbilical vein endothelial cells (HUVECs) (Osiak et al., 2005) exposed to phorbol esters (Guegan et al., 2008; Seano et al., 2014; Tatin et al., 2006) or with aortic endothelial cells exposed to TGFβ (Curado et al., 2014; Varon et al., 2006). In these situations, podosomes arise in clusters or ring-shaped superstructures that are often called rosettes. However, recent studies have shown that matrix proteins are also involved in podosome induction, architecture and arrangement. In the absence of any specific cytokine (or pharmacological) stimulation, a collagen-I (Col-I) substrate elicits the formation of atypical linear podosomes aligning along collagen fibrils (Juin et al., 2013), whereas in a confined environment, fibronectin induces the formation of either isolated entities or podosome rosettes (Spuul et al., 2015). Such diversity points to a multiplicity of regulatory mechanisms for podosome formation in endothelial cells and raises the question of whether these podosomes are functionally equivalent. Podosome-forming cells share the common feature of traveling across tissues, suggesting that podosome lytic enzymes overcome extracellular matrix (ECM) barriers. Among them, the transmembrane matrix metalloprotease MT1-MMP (also known as MMP14) is found at podosomes in all types of cells forming these structures and plays a prominent role (Linder, 2007). However, in vitro, many studies are limited to the use of the traditional fluorescent gelatin invasion assay that highlights the matrix-degrading capabilities of podosomes, showing only the gelatinolytic activity on the denatured Col-I matrix. Thus, it remains unclear whether podosomes are used by cells to travel across all types of ECM encountered during their transtissular migration or if there are cell type and matrix protein specificities for their induction and function. Received 21 January 2016; Accepted 19 May 2016 1 Université de Bordeaux, 33 000 Bordeaux, France. 2 INSERM U1045, 33 000 Bordeaux, France. *Present address: Department of Biomedecine, University of Bergen, Postboks 7804, Bergen N-5009, Norway. ‡ Present address: Department of Gene Technology, Tallinn University of Technology, Akadeemia Rd 15, Tallinn 12618, Estonia. § These authors contributed equally to this work ¶ Author for correspondence (elisabeth.genot@inserm.fr) T.D., 0000-0003-0622-7946; P.S., 0000-0001-9410-721X; F.A., 0000-0002- 6794-1513; I.F., 0000-0002-3632-6016 2586 © 2016. Published by The Company of Biologists Ltd | Journal of Cell Science (2016) 129, 2586-2598 doi:10.1242/jcs.186585 Journal of Cell Science