Migfilin and filamin as regulators of integrin activation in endothelial cells and neutrophils

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.

Figure 1. Migfilin expression in different human vascular cells.

(A) Immunoblots showing migfilin in whole cell lysates of different circulating and non-circulating cell types. Migfilin immunoblots reflect signals after 5 min of exposure while the actin bands were exposed for ∼2 seconds on the X-ray film. (B) The intensities of the 50 kDa migfilin band were quantified by densitometry and normalized to actin band intensities in the same gels.

Figure 2. Migfilin activates β1, β2 & β3 integrins in bovine aortic endothelial (BAE) and model granulocytic cells.

Compared to vector control, migfilin induces 2–5 fold increase in β1 and β3 integrin activation in BAEC as monitored by HUTS-4 binding (A) and Alexa Fluor 647-labeled fibrinogen binding (B), respectively. (C–F) HL60 cells were differentiated for 6 days with DMSO and processed for flow cytometry as described in Methods. Transfection of the undifferentiated HL60 cells (C, E) did not significantly affect activation of the β2 integrins compared to the vector, as monitored by the β2 activation-specific antibodies, CBRM1/5 or mAb24. In contrast, transfection of the dHL60 cells with migfilin caused a >2.5 fold increase (p<0.05) in binding of these antibodies compared to vector control (D, F). Data are representative of 3 independent experiments. Values are means± S.E. ** denotes p<0.01 and * denotes p<0.05. In all experiments, Mn²⁺ was used as a positive control for integrin activation assay.

Figure 3. Integrin activation of β1 and β2 integrins with MT and WT migfilin peptides.

(A) HUVECS or (B) human neutrophils were treated with 50 µM wild type (WT) and mutant (MT) migfilin peptides for 5 min at 37°C. Using flow cytometry, HUTS-4 binding or CBRM1/5 binding was then measured to evaluate the extent of β1 or β2 integrin activation, respectively. Data are representative of 3 independent experiments. Values are means± S.E * denotes p<0.05. As a positive control Mn²⁺ was again used for β1 integrin activation assay.

Figure 4. Effect of migfilin depletion on integrin-dependent functions in HUVECs.

(A) Migfilin siRNA causes a >70% reduction in migfilin protein levels in HUVECs 48 hours after transfection with a migfilin targeting siRNA compared to a control siRNA. Western blot of migfilin is shown along with actin as a loading control. (B, C) Knockdown of migfilin impairs cell spreading on fibronectin- (15 µg/ml) or fibrinogen- (20 µg/ml) coated coverslips. Area of at least 100 cells was measured for each ligand and each time-point. (D) Migfilin knockdown impairs VEGF-induced directed migration of HUVEC across fibronectin and fibrinogen. The upper wells were seeded with HUVECs, resuspended in non-COMPLETE HUVEC media with 0.1% BSA, and underside of the transwell filters were coated with fibronectin and fibrinogen as in the cell spreading assay. The lower chamber contained above media and 15 µg/ml (for fibronectin coated filters) or 20 µg/ml (fibrinogen coated filters) of VEGF. Cells were allowed to migrate for 8 hrs at 37°C. Data are representative of 3 independent experiments. Values are means± S.E. ** denotes p<0.01 and * denotes p<0.05.

Figure 5. Role of migfilin in β2 integrin dependent function.

Empty vector or migfilin were nucleofected into HL60 or dHL60 cells; and, after 24 hours, adhesion of the transfected cells to immobilized iC3b, a ligand for β2 integrins, was assessed as described in Materials and methods. No difference was observed between vector control and migfilin in HL60 cells (A), but with the dHL60 cells, migfilin transfection resulted in a higher (p<0.05) adhesion compared to vector alone (B). Data (means ±S.E.) are representative of 3 independent experiments. * denotes p<0.05. The positive control for this assay was Mn²⁺ as in earlier experiments.

Figure 6. FLNA inhibits integrin activation.

(A) Full length FLNA or FLNA repeat 21 inhibit β1 integrin activation as monitored by HUTS-4 binding in CHO cells. (B) PAC1 binding is inhibited by full length FLNA in M2 cells transiently over-expressing αIIbβ3 integrin (p<0.05). Data are optimized for surface expression of αIIbβ3 integrin as measured by 2G12 antibody. In both cases, talin head and migfilin activated the respective integrins. Results are representative of 3 independent experiments for HUTS-4 and PAC-1 binding. Values are means± S.E. ** denotes p<0.01 and * denotes p<0.05.

Figure 7. Migfilin can rescue FLNA-induced inhibition of integrin activation & its N-terminal mediates integrin activation.

(A) For analyzing the role of single proteins on integrin activation, EGFP-migfilin was transfected with empty DsRed vector (the FLN vector) and DsRed-FLNA16-24 with empty EGFP vector (the migfilin vector) into M2 cells. Empty EGFP and DsRed vectors were co-transfected and used as controls. HUTS-4 binding of EGFP and DsRed positive cells was used to monitor the activation of the endogenous β1 integrins of the cells by FACS, correcting for changes in protein expression. While migfilin activates, FLNA16-24 inhibits β1 integrins. Migfilin enhances the activation of the integrin when co-expressed with FLNA16-24 (p<0.05). (B) The N-terminal (residues 1–85) FLN binding region of migfilin (Nter) or its triple mutant (NterMT) with reduced FLN binding activity was transfected into A7 cells, and β1 integrin activation monitored by HUTS-4 binding. Both migfilin full length and the Nter segment of migfilin activated β1 integrins, but the NterMT was significantly less activating than Nter alone (p<0.05). Data are representative of multiple independent experiments. Values are means± S.E. * denotes p<0.05.