cAMP-induced Epac-Rap activation inhibits epithelial cell migration by modulating focal adhesion and leading edge dynamics
Introduction
Epithelial cell migration is a complex process induced by specific growth factors that takes place during certain stages of embryonic development, organogenesis and wound healing. In response to oncogenic signals, epithelial cell migration also mediates tumor invasion and metastasis [1]. Epithelial cell migration requires the disruption of cell–cell adhesion [1], the modification of the integrin–extracellular matrix (ECM) interactions [2] and engagement of the actomyosin-based migration machinery that induces polarized membrane protrusion [3]. Beneath this leading edge protrusion of a migrating cell, integrin-mediated focal contacts are initiated and subsequently reinforced by tension generated in the actomyosin cytoskeleton [4]. As a consequence, they grow larger and alter their composition to become focal adhesions (FAs) [5]. Contraction of the actomyosin cytoskeleton attached to leading edge FAs pulls the cell body forward and is used to disassemble rear-end FAs [6], [7]. The efficiency of migration in two-dimensional culture also depends on the balance between ECM concentration and the extent of integrin-activation [8].
Several growth factors implicated in tumor metastasis can induce the processes described above in cultured cells, resulting in the scattering of initially clustered epithelial cells. The most well-known inducers of epithelial cell scattering are transforming growth factor-β (TGFβ) and hepatocyte growth factor (HGF) [9]. TGFβ induces scattering in many different cell lines, invariantly accompanied by silencing of the E-cadherin gene through Smad signaling [10]. The most prominent induction of scattering by HGF occurs in MDCK cells [11] and does not involve down regulation of E-cadherin protein levels or adhesive capacity, but correlates with increased integrin-mediated adhesion and depends on actomyosin based tension [12]. Given the lethal consequences of tumor metastasis, we aim to understand the cellular machinery that governs epithelial cell migration.
cAMP is a pivotal second messenger that regulates a wide range of cellular processes. Signaling through cAMP and protein kinase A (PKA) has been implicated in cytoskeletal regulation and cell migration [13]. The effects of PKA on cell migration can be both stimulatory and inhibitory, depending on the cell type and matrix used [13], [14], [15], [16]. cAMP also activates the guanine nucleotide exchange factor (GEF) Epac that can subsequently activate the small GTPase Rap [17]. Rap is an important regulator of both integrin- and cadherin-mediated adhesion (reviewed in [18], [19], [20]). Although it is not yet completely understood how Rap regulates these two processes, several proteins that interact with its GTP-bound form have been identified that may serve as effector proteins [18]. In the case of integrin-mediated adhesion, Rap regulates both integrin affinity and integrin avidity, or clustering, depending on the type of integrin and the cell type [21], [22], [23], [24]. Two effectors of Rap1, Riam and RapL, have been shown to be important in the regulation of integrin affinity [25], [26], although they induce integrin activation via distinct mechanisms [27], [28]. In the regulation of (V)E-cadherin-mediated adhesion, Rap effectors likely recruit junctional proteins to sites of developing cell–cell contacts to stabilize the connection between the actin cytoskeleton and the junctional complex [20], [29], [30]. Because of its established function as a regulator of integrin-mediated cell adhesion, a role for Rap in cell migration has been suggested. Direct evidence comes from studies in leukocytes, where chemokine-induced integrin activation by Rap1 leads to an increase in adhesion to the endothelium and subsequent endothelial transmigration [31], [32]. Clearly, in addition to PKA, also Epac/Rap signaling may be involved in regulation of cell migration via cAMP.
Previously, we reported that Rap is involved in cell surface expression of E-cadherin and the stabilization of cell–cell junctions and that cAMP-induced activation of Rap through Epac1 inhibits HGF-induced cell scattering. These observations suggested that scattering is inhibited by stabilization of adherens junctions [33]. However, a deficiency in C3G, another RapGEF, results in increased migration velocity [34], indicating that Rap might also have a restraining effect on cell migration itself. Moreover, Zhang et al. recently described that TGFβ-induced transformation of cells is inhibited by cAMP, independently of PKA [35]. As TGFβ, in contrast to HGF, down regulates the E-cadherin expression levels, cAMP-Epac-Rap signaling may regulate cell migration directly rather than through a stabilizing effect on cell–cell junctions.
Here, we show that activation of Rap through Epac1 inhibits epithelial cell migration in a number of different model systems in response to both HGF and TGFβ, irrespective of the presence of cell–cell junctions. Interestingly, forced integrin activation, by the integrin-activating antibody TS2/16, does not inhibit migration, even though it induces adhesion to the ECM to the same extent as Rap activation does. Apparently, the effects of Rap on cadherin-mediated adhesion and integrin activation are not sufficient to inhibit epithelial cell migration, indicating that inhibition of the basal cell migration machinery is the critical step downstream of Rap that mediates its effects on scattering. To further understand the mechanism of Rap-induced inhibition of cell migration, we studied the migration machinery in more detail and observed that Rap activation impairs the dynamics of focal adhesions and blocks protrusive activity at the leading edge in migrating cells. These effects are also not mimicked by integrin activating antibodies. Together, these data show that Rap regulates focal adhesion and leading edge dynamics, independently of integrin activation, to restrain epithelial cell migration.
Section snippets
cAMP-induced Rap activation inhibits HGF-stimulated epithelial cell migration
To investigate the effect of Rap activation on epithelial cell migration we used MDCK cells, which do not express endogenous Epac, and MDCK cells stably expressing GFP-tagged human Epac1 (MDCK-GFP-Epac cells). Cells were plated in a 48-well plate coated with collagen (10 µg/mL), simultaneously filmed by phase-contrast microscopy for 2 h, stimulated with HGF, and filmed for an additional 18 h. Parental MDCK cells exhibited a typical response to HGF; the cells initially spread, disrupted
Discussion
For cells to migrate efficiently, both cell–cell junctions and cell-ECM interactions need to be regulated and tension within the actomyosin cytoskeleton needs to be induced [1], [2], [3]. Polarized membrane protrusion and efficient turnover of focal adhesions are also required for efficient cell migration [6]. As the small GTPase Rap is a known regulator of cell junctions and integrin-mediated adhesion [18] and has been suggested to be involved in cell migration [34], we investigated how Rap
Conclusion
We conclude that activation of endogenous Rap leads to an inhibition of growth factor-induced epithelial cell migration by targeting the basal migration machinery. This effect is independent of E-cadherin stabilization and cannot be explained solely by affinity modulation of β1-integrins. Rap inhibits epithelial cell migration through the stabilization of focal adhesions and the inhibition of membrane protrusion, possibly by stabilizing the connection between the actin cytoskeleton and
Cell lines and culture
Stable MDCK-GFP-Epac cells were created by transfection of MDCK with pEGFP-C1-Epac1 followed by selection with G418. Polyclonal MDCK cells stably expressing moderate levels of GFP-Epac were isolated by fluorescence activated cell sorting (FACS) from this cell line. MDCK-Epac1 cells were described previously [33]. Stable Epac1-expressing A549 cells were created by infecting A549 cells with Epac1 ecotrophic virus. The Epac1 gene was linked via an IRES sequence to a zeocin resistance gene.
Acknowledgements
We thank members of our labs for comments and discussion, Livio Kleij for assistance with live cell imaging experiments, Dr Jeroen Bakkers for providing the GFP-CAAX construct and Dr Mark Ginsberg for pEGFP-paxillin. We thank Dr Jan Willem Akkerman for sparing precious LIBS6 antibody, which originated from Dr Mark Ginsberg, whom we thank for it as well. We thank Arnoud Sonnenberg for support, suggestions and critical reading of the manuscript. This work was supported by a Long-Term Fellowship
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- 1
Current address: Wittmann Laboratory, Department of Cell and Tissue Biology, University of California-San Francisco, 513 Parnassus Avenue, HSW-618, San Francisco, CA, United States.
- 2
These authors contributed equally to this paper.