Epithelial cells utilize cortical actin/myosin to activate latent TGF-β through integrin α_vβ₆-dependent physical force

Abstract

Transforming Growth Factor Beta (TGF-β) is involved in regulating many biological processes and disease states. Cells secrete cytokine as a latent complex that must be activated for it to exert its biological functions. We previously discovered that the epithelial-restricted integrin α_vβ₆ activates TGF-β and that this process is important in a number of in vivo models of disease. Here, we show that agonists of G-protein coupled receptors (Sphingosine-1-Phosphate and Lysophosphatidic Acid) which are ligated under conditions of epithelial injury directly stimulate primary airway epithelial cells to activate latent TGF-β through a pathway that involves Rho Kinase, non-muscle myosin, the α_vβ₆ integrin, and the generation of mechanical tension. Interestingly, lung epithelial cells appear to exert force on latent TGF-β using sub-cortical actin/myosin rather than the stress fibers utilized by fibroblasts and other traditionally “contractile” cells. These findings extend recent evidence suggesting TGF-β can be activated by integrin-mediated mechanical force and suggest that this mechanism is important for an integrin (α_vβ₆) and a cell type (epithelial cells) that have important roles in biologically relevant TGF-β activation in vivo.

Introduction

Transforming Growth Factor Beta (TGF-β) is a central mediator in multiple in vivo disease models, including fibrosis in the lungs, kidney, and biliary tract [1], [2], [3], [4], [5], acute lung injury [6], [7], and pulmonary emphysema [8]. Cells secrete the pleiotropic cytokine as a large latent complex, which must be activated to exert its biological functions. There are many ways to activate latent TGF-β in vitro, for example, via heat, acidic pH, and matrix-metalloproteinases. However, the relevance of these processes in vivo is poorly understood [9]. Several studies have demonstrated that a subset of integrins that interact with an arginine–glycine–aspartic acid (RGD)-binding site motif also directly bind and activate latent TGF-β [1], [10].

Integrins are a widely expressed family of cell surface receptors that mediate cell adhesion and bidirectional signaling to regulate cellular processes. The importance of integrins in TGF-β activation in vivo is highlighted by transgenic mice that harbor a knock-in mutation of Tgfb1, so that the RGD binding site is mutated to RGE. These mice exhibit multi-organ inflammation and autoimmunity, a phenotype that completely phenocopies that of Tgfb1^−/− mice, presumably due to a lack of binding and activation of TGF-β by RGD-binding integrins [11]. However, the only RGD integrins that have been definitively shown to regulate TGF-β activation in vivo are α_vβ₆ and α_vβ₈ [1], [12]. In particular, Itgb8^−/− mice treated with blocking antibodies against α_vβ₆ from birth demonstrate all of the developmental phenotypes of mice lacking both TGF-β isoforms 1 and 3 [13].

We previously demonstrated that the epithelial-restricted integrin α_vβ₆ activates TGF-β and that Itgb6^−/− mice exhibit a phenotype mildly similar to Tgfb1^−/− mice [1], [14]. These mice are also dramatically protected in models of diseases that are mediated by TGF-β, supporting a role for α_vβ₆ in regulating TGF-β bioactivity in vivo [1], [4], [5], [9], [14]. Because of the importance of α_vβ₆-mediated TGF-β activation in the pathogenesis of various disease states, we wanted to determine the molecular signals and the mechanisms that regulate activation of this pathway.

Binding of α_vβ₆ to latent TGF-β1 and 3 in vitro is insufficient for activation of the cytokine, and cytoplasmic interactions between the integrin and the actin cytoskeleton are required for this process [1]. However, the mechanism and significance of these interactions in modulating α_vβ₆-mediated TGF-β activation are unknown. Furthermore, tethering of the latent complex to the extracellular matrix (ECM) by latent TGF-β binding protein-1 (LTBP-1) is required [15]. These findings suggest that TGF-β activation by α_vβ₆ potentially involves a mechanical mechanism. In vitro evidence supporting a role for mechanical force in integrin-dependent TGF-β activation has been provided by studies of α_vβ₃ and α_vβ₅-mediated TGF-β activation in fibroblasts [16], but thus far, there are no convincing data demonstrating roles for either of these integrins in activating TGF-β in vivo. The recently solved crystal structure of the small latent complex of TGF-β1 provides a model for how an RGD-binding integrin could exert mechanical force on a tethered latent complex and activate it. Electron microscopic analysis of purified integrin α_vβ₆ and the small latent complex provided further evidence that this integrin binds to the latent complex in an appropriate fashion to exert activating mechanical force [17]. However, the relevance of mechanical force in TGF-β activation in vivo remains unknown.

The purpose of this study was to identify signals that regulate α_vβ₆-mediated TGF-β activation and to determine what role, if any, mechanical force plays in the activation process. Here, we describe a novel activator, Sphingosine 1-Phosphate (S1P) that regulates α_vβ₆-mediated TGF-β activation in primary lung epithelial cells. We also demonstrate that both S1P and Lysophosphatidic Acid (LPA) induced α_vβ₆-mediated TGF-β activation require cell contraction and the generation of cellular tension. Finally, we show that cell tension generated by these primary epithelial cells is associated with organization of sub-cortical actin rings without evidence of actin stress fibers. Our findings highlight the potential for the development of additional therapeutic strategies for diseases that involve aberrant α_vβ₆-mediated TGF-β activation signaling and demonstrate a role for mechanical force in mediating TGF-β activation in a cell type and by an integrin that have been clearly shown to be relevant in vivo.