Integrin-linked kinase functions as a downstream signal of platelet-derived growth factor to regulate actin polymerization and vascular smooth muscle cell migration

Abstract

Background: Vascular smooth muscle cell migration and accumulation in response to growth factors extensively contribute to the development of intimal thickening within the vessel wall. Cumulative evidence has shown that actin cytoskeleton polymerization and rearrangement are critical steps during cellular spreading and migration. Integrin-linked kinase, an intracellular serine/threonine kinase, is a cytoplasmic interactor of integrin beta-1 and beta-3 receptors regulating cell-cell and/or cell-extracellular matrix interaction, cell contraction, extracellular matrix modification, and cell spreading and migration in response to various stimuli. However, the regulatory role of ILK during vascular smooth muscle cell migration and the importance of integrin signaling in occlusive vascular diseases are not yet fully elucidated.

Results: In the present study, we report that integrin-linked kinase controls mouse aortic smooth muscle cell migration in response to platelet-derived growth factor. We have also identified p38 mitogen activated protein kinase as a downstream signaling pathway of the integrin-linked kinase that regulates platelet-derived growth factor-induced actin polymerization and smooth muscle cell migration.

Conclusion: This study will provide new insights into the potential therapeutic value of modulating integrin signaling in an attempt to block or delay smooth muscle cell migration and the progression of vascular diseases.

Figure 1

PDGF treatment increases integrin-linked kinase activity in mouse aortic smooth muscle cell culture. A) Primary mouse aortic SMCs were serum-starved overnight and then treated with 25 ng/ml of PDGF-BB. ILK kinase activity was measured at various timepoints following treatment. PDGF-BB increased ILK kinase activity (two fold increase). SMC culture transfected with ILK siRNA was used as negative control in the assay. As shown in first lane of the blot, ILK expression was completely abolished in negative control group that led to a significant decrease in ILK kinase activity in this group. B) Transfection of mouse aortic SMCs with ILK siRNA for 96 hours completely blocked ILK protein expression. Data presents three independent experiments and is presented as mean ± STDEV (n = 3), where P < 0.05 was considered significant.

Figure 2

PDGF induces SMC migration through anILK-dependent pathway. A) Inhibition of ILK protein expression with 20 nM ILK siRNA markedly decreased SMC migration in response to PDGF-BB. Data represents three independent experiments (n = 3 culture plates in each replicate) and the value is presented as mean ± STDEV where P < 0.05 was considered significant. B) ILK inhibition decreased the number of migrated SMCs into the site of injury in response to PDGF-BB treatment as compared to the control group (contorl siRNA). Original magnification, ×40. Data present one of three independent experiments.

Figure 3

ILK inhibition abolishes PDGF-induced p38 MAPK activation in mouse aortic SMCs. A) Mouse aortic SMCs were treated with specific MAPKs inhibitors at specified concentrations for 2 hours prior to seeding onto Transwell Boyden Chambers and PDGF-BB treatment (25 ng/ml) and during the incubation period (12 hours), and cell migration was measured at 12 hours post culture. As shown, inhibition of all three members of MAPKs family significantly reduced SMC migration in response to PDGF-BB. Data is the representative of three independent experiments (n = 3 cell culture replicates for each experiments). B) Mouse aortic SMCs were transfected with 20 nM of specific ILK siRNA and then treated with 25 ng/ml of PDGF-BB for specified timepoints. Inhibition of ILK protein expression markedly reduced PDGF-induced p38 MAPK phosphorylation in mouse aortic SMCs. The blot presents one of three independent experiments. The value in the graph is presented as the mean ± STDEV of three independent experiments. C) ILK inhibition by specific siRNA had no detectable effect on Erk1/2 and JNK phosphorylation in response to PDGF-BB treatment in mouse aortic SMC culture. Data presents one of three independent experiments (n = 3 cell cultures for each independent experiments).

Figure 4

Dominant negative form of p38 MAPK significantly decreases SMC migration in response to PDGF. A) Photomicrograph of mouse aortic SMCs showing the morphology and GFP expression at 36 hours following transfection. Sub-confluent SMCs were infected with adenoviral vector at the multiplicity of infection of 100. Note the high level of GFP expression as well as the normal phenotype of Ad-GFP-transfected SMCs. Original magnification, ×200. B) Over-expression of a dominant negative form of p38 MAPK markedly increased total p38 MAPK expression while blocking p38 MAPK phosphorylation in response to serum treatment in SMCs (serum is used as a strong activator for p38 MAPK). Also, note the considerable increase in ILK expression level in SMCs transfected with adenoviral ILK constructs. C) For migration assay, transfected SMCs were cultured in Transwell Boyden Chambers in the presence or absence of 25 ng/ml of PDGF-BB and cell migration was measured 12 hours post culture. Dominant negative p38 MAPK significantly decreases SMC migration in response to PDGF-BB treatment while expression of a wild type form of p38 MAPK increases SMC migration in response to PDGF-BB. In addition, over-expression of a wild type mutant of p38 MAPK subverted the inhibitory effect of p38 inhibition on SMC migration. Data represents three independent experiments (n = 3 cell cultures for each independent experiments).

Figure 5

ILK regulates PDGF-induced SMC migration through a p38 MAPK-dependent pathway. A) Kinase deficient mutant of ILK significantly reduced PDGF-induced SMC migration in a Traswell Boyden Chamber migration assay. As shown, expression of a wild type form of ILK rescued SMC migratory response indicating the specificity of ILK inhibition by the adenoviral construct. B) Over-expression of an active form of MKK3/MKK6 (specific upstream activators of p38 MAPK), but not a wild type form of p38 MAPK, rescued SMC migration in response to PDGF-BB indicating that an active form of ILK is required for the initiation of a p38 MAPK-dependent migratory response to PDGF-BB in mouse aortic SMCs. (n = 3 cell cultures for each independent experiments).

Figure 6

ILK and p38 MAPK regulate PDGF-induced actin cytoskeleton polymerization in vascular SMCs. A) Mouse SMCs were treated with either vehicle (DMSO) or with 10 μM of p38 MAPK specific inhibitor SB202190 for 1 hour prior to PDGF treatment. Following treatment, cells were washed and fixed and then stained for actin filaments. Inhibition of p38 MAPK markedly blocks PDGF-induced actin polymerization and reorganization (original magnification ×400). B) Mouse aortic SMCs were transfected with 20 nM of specific ILK siRNA and then treated with 25 ng/ml of PDGF-BB for 20 minutes. Cells were then fixed and stained for actin filaments. Inhibition of ILK expression in SMCs significantly reduced PDGF-induced actin polymerization and reorganization in mouse aortic SMCs. Images are representative image of three independent experiments (original magnification ×400).