Akt1 signaling regulates integrin activation, matrix recognition, and fibronectin assembly

Abstract

Akt, a serine-threonine kinase, regulates multiple cellular processes in vascular cells. We have previously documented that Akt activates integrins and Akt1 deficiency results in matrix abnormalities in skin and blood vessels in vivo. Based on these observations, we hypothesized that Akt1 is necessary for integrin activation and matrix assembly by fibroblasts. In this study, using various cell systems, we show that Akt1 is essential for the inside-out activation of integrins in endothelial cells and fibroblasts, which in turn, mediates matrix assembly. Fibronectin is a major extracellular matrix component of the skin and the vascular basement membrane, which possesses binding sites for many integrins and extracellular matrix proteins. Akt1(-/-) fibroblasts and NIH fibroblasts expressing dominant negative Akt1 (K179M-Akt1) showed impaired fibronectin assembly compared with control fibroblasts. In contrast, expression of constitutively active Akt1 (myrAkt1) resulted in enhanced fibronectin assembly. Although increased fibronectin assembly by myrAkt1-expressing human foreskin fibroblasts was abolished by treatment with anti-integrin beta(1) blocking antibodies, treatment with beta(1)-stimulating antibodies rescued the impaired fibronectin assembly that was due to lack of Akt activity. Finally, expression of myrAkt1 corrected the phenotype of Akt1(-/-) fibroblasts thus showing that Akt1 regulates fibronectin assembly through activation of integrin alpha(5)beta(1).

FIGURE 1. Akt1^−/− ECs exhibit impaired adhesion and migration to ECM proteins

A, wild-type and Akt1^−/− mouse lung ECs (1 × 10⁵/well) were introduced into 12-well plates coated with laminin, fibronectin, vitronectin, or fibrinogen (1 µg/well) and were allowed to attach by incubating for 45 min at 37 °C in the presence and absence of 20 ng/ml VEGF. Adhered cells were fixed in 70% methanol and stained using toluidine blue and quantified using a phase-contrast microscope. B, wild-type and Akt1^−/− lung ECs were plated at about 90% confluence in 6-well culture plates coated with 1 µg/ml each of laminin, fibronectin, vitronectin, or fibrinogen and were allowed to form a monolayer. The endothelial monolayers were wounded across the diameter of the well with a 2.5-mm wide cell scraper. The media and dislodged cells were aspirated, and the cells were incubated in fresh 2% serum-containing medium with 20 ng/ml VEGF. 12h after wounding, plates were processed and analyzed as described under “Experimental Procedures.” C, a time course effect of the abovementioned scratch wound assay on 2% gelatin, laminin (10 µg/ml), and fibronectin (1 µg/ml) at 6 and 12 h.D, a short-term Boyden chamber assay for migration of WT and Akt1^−/− mouse lung ECs on 1 µg/ml each of fibronectin, vitronectin, and fibrinogen.

FIGURE 2. Akt1 mediates activation of integrins

A, confluent monolayers of HUVECs were serum-starved overnight and treated with 50 ng/ml insulin-like growth factor-I or 20 ng/ml bFGF. Lysates prepared at different time points (0, 5, and 15 min) after treatment with insulin-like growth factor-I and bFGF were subjected to Western analysis for pS473 Akt and total Akt. B, confluent monolayers were treated with 20 ng/ml VEGF for 0, 5, 15, and 30 min, and then lysates were prepared and subjected to Western analysis for pS473/pT308 Akt and total Akt. C, HUVECs were either kept in suspension or plated on fibronectin or fibrinogen and were treated with either control (0.01%BSA in PBS) or 20 ng/ml VEGF for 15 min. Lysates were prepared and subjected for Western analysis for pS473 Akt and total Akt. Serum-starved HUVECs treated with PI3K inhibitors LY294002 (10 µm) or wortmannin (100 nm) 30 min prior to VEGF treatment followed by Western analysis of the lysates with pS473 Akt and total Akt. D, HUVECs were infected with adenovirus encoding control GFP or myrAkt1 and were then subjected to FACS analysis for WOW-1 Fab binding. E and F, WT and Akt1^−/− mouse embryonic fibroblasts as well as NIH 3T3 fibroblasts expressing WT-Akt1, myrAkt1, and DN-Akt1 were subjected to Alexa 633-labeled fibrinogen binding in the presence of Mn²⁺ and were subsequently analyzed by FACS.

FIGURE 3. Akt1^−/− fibroblasts exhibit impaired adhesion and migration to fibronectin

A, wild-type and Akt1^−/− MEFs (1 × 10⁵ cells/well) were introduced into 12-well plates coated with BSA, fibronectin, or vitronectin (1 µg/ml) and were allowed to attach by incubating for 45 min at 37 °C in the presence of 20 ng/ml bFGF. Adhered cells were fixed in 70% methanol and stained using toluidine blue and visualized in a phase-contrast microscope. B, wild-type and Akt1^−/− MEFs were plated at ~90% confluency in 6-well culture plates coated with BSA, fibronectin, or vitronectin and allowed to form a monolayer. Monolayers were wounded across the diameter of the well with a 2.5-mm-wide cell scraper. The media and dislodged cells were aspirated, and the cells were incubated in fresh 2% serum-containing medium with 20 ng/ml bFGF. 12 h after wounding, plates were fixed with 70% methanol, stained with toluidine blue, and analyzed microscopically at 12 h (5× magnification). C, a time course effect of scratch wound assay on fibronectin (1 µg/ml) at 3, 6, 9, and 12 h. D, monolayers of WT MEFs were serum-starved overnight and were then treated with VEGF (20 ng/ml), bFGF (20 ng/ml), 1 µm VEGFR2 in hibitor (SU1498), and/or 10 µm LY294002, and the lysates were subjected to Western analysis. E, Western blots of lysates from the wild-type and Akt1^−/− MEFs, probed with antibodies to Akt1, Akt2, Akt3, panAkt, and β-actin. F, Western analysis of lysates from the wild-type and Akt1^−/− MEFs after treatment with bFGF (20 ng/ml) at time intervals of 0, 5, 10, 15, 30, and 60 min probed for pS473 Akt and panAkt. G, histogram showing band densitometry analysis of the above data analyzed using Kodak molecular imaging software (version 4.0).

FIGURE 4. Deficiency of Akt1 results in impaired fibronectin assembly by fibroblasts

A, equal numbers of WT and Akt1^−/− MEFs (1 × 10⁵/well) were plated in 12-well plates onto glass coverslips and allowed to form a monolayer. The monolayer was fixed using 1% paraformaldehyde and stained using anti-fibronectin antibodies. Fluorescent images of fibronectin meshwork created by WT and Akt1^−/− MEFs were visualized using a fluorescence microscope. Bar = 20 µm. B, these results were quantified using Image ProPlus software for the percentage of the total area positive for fibronectin and were normalized to the number of nuclei counted per field (three different fields taken from four sets of samples). C, NIH 3T3 cells were transfected with control vector (pBabe-Puromycin), WT-Akt1, myrAkt1, and DN-Akt1. 36 h after transfection, lysates were prepared and subjected to Western analysis for pS473Akt and Akt1. D, NIH 3T3 cells were transfected with control vector, WT-Akt1, myrAkt1, and DN-Akt1 and were used to study the role of Akt1 in assembly of fibronectin. Monolayers were fixed in 1% paraformaldehyde and were stained with anti-fibronectin antibody to study matrix assembly. Bar = 20 µm. E, histogram showing quantification of the above data as analyzed by Image ProPlus.

FIGURE 5. Akt1 is necessary for the assembly of exogenously supplied fibronectin

A, NIH 3T3 cells (1 × 10⁵/well) were plated onto coverslips in 12-well plates and were allowed to adhere and spread for 1 h. To all the wells (except control), 10 µg of fibronectin was added exogenously in the presence or absence of PI3K or Akt inhibitors (10 µm LY294002 and 1 µm SH5, respectively). The effects of PI3K and Akt inhibitors on assembly of exogenous fibronectin were studied after fixing the wells with 2% paraformaldehyde 4 h after the addition of fibronectin by staining with anti-fibronectin antibodies. Assembly of fibronectin was analyzed by fluorescence microscopy. Bar = 20 µm. B, effect of PI3K and Akt inhibitors on the assembly of exogenous fibronectin was quantified by Image ProPlus. C and D, WT and Akt1^−/− MEFs (1 × 10⁵/well) subjected to fibronectin assembly assay and image analysis as mentioned above, showing the importance of Akt1 in assembly of exogenous fibronectin. Bar = 20 µm. E and F, effect of expression of WT-Akt1, myrAkt1, and DN-Akt1 in NIH 3T3 fibroblasts on assembly of exogenous fibronectin. Bar = 20 µm.

FIGURE 6. Akt1 is essential for the activation of β₁ integrins in fibroblasts

A, HFFs were infected with adenovirus encoding control GFP or myrAkt1 and were then subjected to FACS analysis for HUTS-4 antibody binding, which specifically binds to the active form of integrin β₁. B, HFFs were treated with SH-5 (1 µm) and/or integrin β₁-stimulating antibodies (TS2/16, 1 µg/ml) for 30 min in suspension and were subjected to adhesion assay on fibronectin coated wells. Bar = 50 µm. C, quantification of HFFs adhered to fibronectin-coated wells after treatment in suspension with SH-5 (1 µm) and/or integrin β₁-stimulating antibodies (TS2/16, 1 µg/ml) for 30 min.

FIGURE 7. Integrin β₁-stimulating antibodies rescue the defect in fibronectin assembly due to the lack of Akt activity

A, HFFs, transfected with control vector (pBabe-puromycin), myrAkt1, and DN-Akt1 by retroviral infection, were selected by puromycin. Lysates were prepared and subjected to Western analysis for pS473Akt and Akt1. B, human fibroblasts were fixed in 1% para-formaldehyde, and intact cells were stained with β₁-blocking antibodies. Bar = 20 µm. C and D, HFFs, transfected and selected as above, were plated on laminin-coated coverslips in 12-well plates and allowed to spread. Wells were treated with 1 µg/ml integrin β₁-blocking antibodies four times with intervals of 3 h (total 12 h). The effect of β₁-blocking antibodies on Akt-mediated fibronectin assembly was studied by fluorescence microscopy. Bar = 20 µm. E and F, HFFs (1 × 10⁵/well) treated in suspension with SH-5 and/or TS2/16 for 30 min were plated onto coverslips in 12-well plates and were allowed to adhere and spread. To all the wells (except control), 10 µg of fibronectin was added exogenously. Cells were fixed with 1% para-formaldehyde 4 h after the addition of fibronectin and stained with anti-fibronectin antibodies. Assembly of fibronectin was analyzed by fluorescence microscopy. Bar = 20 µm.

FIGURE 8. Reconstitution of Akt1 in Akt1^−/− ECs and fibroblasts rescues the impaired migration on fibronectin and impaired fibronectin assembly, respectively

A, WT and Akt1^−/− ECs were infected with adenovirus expressing GFP and myrAkt1, respectively, and were subjected to 12 h scratch wound assay on fibronectin. B and C, WT and Akt1^−/− MEFs were infected with adenovirus expressing GFP or myrAkt1 and subjected to assembly of exogenously supplied fibronectin. Cells were fixed, stained for fibronectin, and analyzed using microscopy and Image ProPlus analysis.