Research Article
Regulation of fibronectin matrix assembly and capillary morphogenesis in endothelial cells by Rho family GTPases
Introduction
Both genetic and pharmacological studies have provided strong evidence pointing to a crucial role for fibronectin (FN) and its receptor α5β1 integrin in neovascularization [1], [2], [3]. Assembly of a provisional FN matrix by activated endothelial cells occurs during angiogenic blood vessel remodeling [4], thus signaling components that control this event are interesting targets for anti-angiogenic therapies [5]. Beyond its role as an autocrine matrix component, recent evidence suggests that the FN matrix assembled by endothelial cells in the tumor microenvironment may be crucial for endothelial-tumor cell interactions and for the recruitment of infiltrating immune cells and bone marrow-derived progenitor cells [6].
In vitro studies have provided insights into the molecular mechanisms that underlie remodeling of the extracellular FN matrix, otherwise known as FN fibrillogenesis (reviewed in [7], [8]. In cultured cells, this occurs at specific integrin-based adhesive structures, called fibrillar adhesions, rich in α5β1 integrin and tensin [9]. FN fibrillogenesis is regulated by extracellular factors, such as the physical properties of the pre-existing matrix [10] and the conformational state of α5β1 integrin [11], but also by cytoskeletal generated acto-myosin contractility (see [7]. Accordingly, the Rho/Rho-associated kinase (ROCK) pathway has been shown to play a crucial role in FN matrix assembly in various cultured cells, including fibroblasts, osteoscarcoma cells and platelets [12], [13], [14], [15].
We previously reported that integrin-linked kinase (ILK), a cytoplasmic adaptor of β1 integrin plays a pivotal role in extracellular FN matrix assembly and capillary morphogenesis in cultured bovine aortic endothelial cells (BAEC). ILK depletion in these cells abolished the formation of fibrillar, matrix-forming adhesions without affecting α5β1 integrin binding to FN, or FN expression [16]. Although the precise mechanism by which ILK controls fibrillogenesis remains to be deciphered, Rho family GTPases have been placed downstream of ILK-mediated integrin signaling cascades. ILK depletion markedly attenuates the activation of Rac1 and Cdc42 [17], [18], [19]. RhoA activation following adhesion is only modestly affected in ILK-deficient cells [16], yet ILK has been reported to stimulate cytoskeletal contractility by promoting phosphorylation of myosin light chain (MLC) at the myosin light chain kinase (MLCK) sites in platelets [20], and by inducing inhibitory phosphorylation of the myosin phosphatase regulatory subunit, MYPT1 [21] (see scheme, Fig. 1). In vascular smooth muscle cells, ILK was proposed to be the kinase responsible for diphosphorylation of myosin [22].
In addition to the Rho/ROCK pathway, other Rho GTPases such as Cdc42 and Rac1 are activated in response to angiogenic agents and participate in cytoskeletal reorganization and capillary morphogenesis of endothelial cells [23], [24], [25], [26], [27]. In light of the established link between the actin cytoskeleton and remodeling of the FN matrix we set out to define the respective contribution of RhoA, Rac1 and Cdc42 in this process in endothelial cells. Our findings reveal a previously unappreciated influence of these GTPases on the topology of the fibrillar FN network and they document the presence of a low tension RhoA-independent mode of matrix-forming adhesion assembly and fibril growth.
Section snippets
Materials
Tissue-culture plasticware was from Nunc (Roskilde, Denmark) and Falcon (BD Biosciences San Diego, CA), for time lapse analysis. The inhibitors 1-[5-Iodonaphtalene-1-sulfonyl]-1H-hexahydro-1,4-diazepine hydrochloride (ML-7) and (R)-(+)-trans-4-(1-Aminoethyl)-N-(4-Pyridyl)cyclohexanecarboxamide dihydrochloride (Y-27632) were purchased from Sigma-Aldrich (St. Louis, MO). ML-7 was reconstituted in a mixed solution of distilled water and 10% ethanol. Y-27632 was reconstituted in distilled water.
Cell culture
The Rho/ROCK pathway and FN fibrillogenesis in BAEC
First we examined the effect of pharmacologic inhibition of Rho-associated kinase (p160 ROCK, ROCK-1, ROCK-2, referred to as ROCK), MLCK, and acto-myosin contractility on FN fibrillogenesis in BAEC. As shown in Fig. 2A, BAEC accumulate an extensive network of extracellular FN fibrils during an overnight incubation on glass coverslips. Treatment of cells with the ROCK inhibitor, Y-27632, or the MLCK inhibitor, ML-7, resulted in a significant inhibition of FN matrix assembly. The dose dependency
Functional studies of Rho family proteins by RNA interference
The subendothelial matrix plays both a structurally and functionally important role in maintaining vascular homeostasis and regulating the outgrowth of new vessels during tumor angiogenesis (reviewed in [4], [40]). Although absent from the extracellular matrix of blood vessels in normal tissues, FN is a major constituent of the matrix that sheaths tumor-associated vessels [5]. Therefore, understanding the cellular signaling events that regulate FN production and assembly is of utmost importance
Acknowledgments
We kindly acknowledge Sébastien Schaub and the PRISM Platform (UMR6543 CNRS/UNS/IBDC) for assistance with cell imaging, Dr Pierre Roux for providing the plasmids encoding GFP-tagged Rac1 and Cdc42 mutants and Dr Elisabeth Genot for helpful discussions. This study was funded by the Association for International Cancer Research. We are grateful to the Association pour la Recherche contre le Cancer the Centre National de la Recherche Scientifique, the University of Nice (UMR6543) and the Emerald
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Current address: INSERM, UMR 911-CRO2, Aix Marseille Université, Faculté de Médecine Secteur Nord, IFR Jean Roche, Marseille, F-13000, France.