Research Article: Migfilin and Filamin as Regulators of Integrin Activation in Endothelial Cells and Neutrophils

Date Published: October 17, 2011

Publisher: Public Library of Science

Author(s): Mitali Das, Sujay Subbayya Ithychanda, Jun Qin, Edward F. Plow, Maddy Parsons.

Abstract: Cell adhesion and migration depend on engagement of extracellular matrix ligands by integrins. Integrin activation is dynamically regulated by interactions of various cytoplasmic proteins, such as filamin and integrin activators, talin and kindlin, with the cytoplasmic tail of the integrin β subunit. Although filamin has been suggested to be an inhibitor of integrin activation, direct functional evidence for the inhibitory role of filamin is limited. Migfilin, a filamin-binding protein enriched at cell-cell and cell-extracellular matrix contact sites, can displace filamin from β1 and β3 integrins and promote integrin activation. However, its role in activation and functions of different β integrins in human vascular cells is unknown. In this study, using flow cytometry, we demonstrate that filamin inhibits β1 and αIIbβ3 integrin activation, and migfilin can overcome its inhibitory effect. Migfilin protein is widely expressed in different adherent and circulating blood cells and can regulate integrin activation in naturally-occurring vascular cells, endothelial cells and neutrophils. Migfilin can activate β1, β2 and β3 integrins and promote integrin mediated responses while migfilin depletion impairs the spreading and migration of endothelial cells. Thus, filamin can act broadly as an inhibitor and migfilin is a promoter of integrin activation.

Partial Text: During homeostatic processes, such as hemostasis, angiogenesis and inflammation, cells must respond with immediacy and precision to different physiological and pathological cues. The orchestration of cellular responses often depends upon remodeling of the actin cytoskeleton which is tightly regulated by a protein circuitry that connects the intracellular milieu to the extracellular environment. Integrin heterodimeric adhesion receptors play important roles in this bidirectional communication between cells and their environment by engaging extracellular ligands via their extracellular regions and interacting with different cytoskeletal proteins via their cytoplasmic tails (CTs). Mechanistically, the ability of integrins to transit between high and low affinity states for extracellular ligands, inside-out signaling, provides a means to dynamically regulate cellular responses. Such regulation of integrin activation is initiated by their interactions with different intracellular adaptor proteins.

Regulation of integrin activation influences many cellular processes including adhesion, spreading and migration that occur in response to different physiological and pathological agonists. FLN was shown to inhibit integrin mediated CHO cell migration, and talin, which is essential for integrin activation [40], associated with integrin only when the FLN-integrin association was diminished [6]. This competition between FLN and talin is likely to regulate integrin activation [1] but also could influence talin- and FLN-dependent outside-in signaling [8]. If FLN is a negative regulator of integrin activation, some mechanism can be anticipated to relieve the suppressive effect of FLN by influencing its release from the β CT. Migfilin seems to be at least one of the good candidates to perform this task. In our earlier work, we demonstrated that migfilin binds to FLN repeat 21 at an identical site as β integrin CT [4]. The higher affinity of migfilin for FLN compared to the β integrin CT thereby facilitates integrin activation. However, this competition will not only be governed by affinity but also by the relative local concentrations of the competitors and other factors within the microenvironment of the cell. Accordingly, in the present work, we investigated the role of FLN in inside-out integrin activation, examined the migfilin levels in various cell types and determined how the migfilin-FLN-integrin axis influences integrin function in these cells. The major findings of our study are: (1) Migfilin is expressed at variable levels in a wide variety of physiologically relevant cell types. (2) Migfilin influences activation of three major subfamilies of integrins – β1, β2 and β3. (3) Migfilin can play a functional role in controlling integrin-mediated responses in intact cells as migfilin knockdown suppresses spreading and impairs migration of endothelial cells, and over-expression enhances adhesion of differentiated HL60 cells to an integrin-dependent ligand, iC3b. (4) FLN inhibits activation of β1 and β3 integrins. (5) The inhibitory effect of FLN on integrin activation can be overcome by co-expression with migfilin. (6) Mechanistically, the Nterminal region of migfilin plays a dominant role in the integrin activation response.