The association of cortactin with profilin-1 is critical for smooth muscle contraction

Abstract

Profilin-1 (Pfn-1) is an actin-regulatory protein that has a role in modulating smooth muscle contraction. However, the mechanisms that regulate Pfn-1 in smooth muscle are not fully understood. Here, stimulation with acetylcholine induced an increase in the association of the adapter protein cortactin with Pfn-1 in smooth muscle cells/tissues. Furthermore, disruption of the protein/protein interaction by a cell-permeable peptide (CTTN-I peptide) attenuated actin polymerization and smooth muscle contraction without affecting myosin light chain phosphorylation at Ser-19. Knockdown of cortactin by lentivirus-mediated RNAi also diminished actin polymerization and smooth muscle force development. However, cortactin knockdown did not affect myosin activation. In addition, cortactin phosphorylation has been implicated in nonmuscle cell migration. In this study, acetylcholine stimulation induced cortactin phosphorylation at Tyr-421 in smooth muscle cells. Phenylalanine substitution at this position impaired cortactin/Pfn-1 interaction in response to contractile activation. c-Abl is a tyrosine kinase that is necessary for actin dynamics and contraction in smooth muscle. Here, c-Abl silencing inhibited the agonist-induced cortactin phosphorylation and the association of cortactin with Pfn-1. Finally, treatment with CTTN-I peptide reduced airway resistance and smooth muscle hyperreactivity in a murine model of asthma. These results suggest that the interaction of cortactin with Pfn-1 plays a pivotal role in regulating actin dynamics, smooth muscle contraction, and airway hyperresponsiveness in asthma. The association of cortactin with Pfn-1 is regulated by c-Abl-mediated cortactin phosphorylation.

Keywords: Actin; Adapter Protein; Contraction; Cytoskeleton; Excitation-Contraction Coupling; Protein Phosphorylation; Smooth Muscle.

FIGURE 1.

Contractile activation increases the association of cortactin with Pfn-1 in smooth muscle cells/tissues. A, cortactin immunoprecipitates of HASM cells that had been treated with ACh (100 μm; 5 min) or left unstimulated (US) were separated by SDS-PAGE and blotted with antibodies against cortactin and Pfn-1. Cortactin antibody, but not IgG, immunoprecipitates cortactin and Pfn-1. The ratios of Pfn-1 over cortactin are significantly higher in stimulated cells than in unstimulated cells (*, p < 0.05). Data are mean values of four independent experiments. Error bars indicate S.E. B, blots of Pfn-1 immunoprecipitates from HASM cells treated with or without ACh (100 μm; 5 min) were blotted with antibodies against cortactin and Pfn-1. Pfn-1 antibody, but not IgG, immunoprecipitates cortactin and Pfn-1. The ratios of cortactin/Pfn-1 are significantly higher in stimulated cells than in unstimulated cells (*, p < 0.05). Data are mean values of four independent experiments. Error bars indicate S.E. C, extracts of human bronchial rings treated with or without ACh were precipitated with cortactin antibody and blotted with antibodies against cortactin and Pfn-1. The Pfn-1/cortactin ratios are significantly higher in stimulated tissues than in unstimulated tissues (*, p < 0.05; n = 3). D, pretreatment with atropine (1 μm; 10 min) inhibits the association of cortactin with Pfn-1 induced by ACh (100 μm; 5 min) (*, p < 0.05; n = 4). E, pretreatment with atropine (1 μm; 10 min) inhibits contraction in human bronchial tissues induced by ACh (100 μm; 5 min) (*, p < 0.05; n = 4). F, top panel, time dependence of F/G-actin ratios in HASM cells in response to ACh stimulation. F/G-actin ratios were determined by the fractionation assay (n = 3). Bottom panel, force development in human bronchial rings during ACh stimulation was determined using a muscle research system (n = 3). Force is expressed as the percentage of maximal response to 100 μm ACh. IP, immunoprecipitation; IB, immunoblot.

FIGURE 2.

Cortactin and Pfn-1 undergo spatial redistribution in smooth muscle cells upon contractile activation. A, HASM cells were stimulated with ACh (100 μm; 5 min) or left unstimulated. The cellular localization of cortactin and Pfn-1 was evaluated by immunofluorescence microscopy. Representative images illustrate the spatial redistribution of cortactin and Pfn-1 in response to ACh activation. Arrows indicate a single line scan to analyze the fluorescence signal for each cell. Cort, cortactin. The line scan graphs show co-localization and relative fluorescence intensity of the two proteins. B, the percentage of cells with colocalization was calculated as follows: (Number of cells with colocalization/Number of total cells observed) × 100. *, significantly higher cell numbers after stimulation with ACh compared with unstimulated cells (p < 0.05). Data are mean values of three independent experiments. Error bars indicate S.E. C, representative gel images illustrating the binding of Pfn-1 to cortactin beads but not to control beads. Binding of Pfn-1 to cortactin is concentration-dependent (n = 3). a.u., arbitrary units; FL, fluorescence.

FIGURE 3.

Treatment with CTTN-I peptide inhibits cortactin/Pfn-1 coupling but not cortactin phosphorylation stimulated with ACh. A, HASM cells were pretreated with control or CTTN-I peptide (2.5 μg/ml) for 20 min. Cells were then stimulated with ACh (100 μm; 5 min) or left unstimulated. The protein/protein interaction was evaluated by co-immunoprecipitation. Data are mean values of four independent experiments. Error bars indicate S.E. (*, p < 0.05). B, HASM cells pretreated with control or CTTN-I peptide were stimulated with ACh (100 μm; 5 min). The spatial localization of cortactin and Pfn-1 in the cells was assessed by immunostaining. Arrows indicate a single line scan to analyze the fluorescence signal for each cell. The line scan graphs show co-localization and relative fluorescence intensity of the two proteins. Cort, cortactin. C, the percentage of cells with colocalization was calculated as follows: (Number of cells with colocalization/Number of total cells observed) × 100. *, significantly different between cells treated with CTTN-I and cells treated with control peptide (p < 0.05; n = 3). D, HASM cells that had been pretreated with peptides were stimulated with ACh (100 μm; 5 min) or left unstimulated. Cortactin phosphorylation at Tyr-421 in these cells was evaluated. Cortactin phosphorylation is normalized to unstimulated cells treated with control peptide (p > 0.05; n = 6). a.u., arbitrary units; IP, immunoprecipitation; p-Cortactin, phosphocortactin.

FIGURE 4.

Treatment with CTTN-I peptide attenuates increases in F/G-actin ratios and force development without affecting myosin phosphorylation. A, HASM cells that had been pretreated with control or CTTN-I peptide were stimulated with ACh (100 μm; 5 min) or left unstimulated. F/G-actin ratios in cells were evaluated using the assay described under “Experimental Procedures.” Data are mean values of six independent experiments. Error bars indicate S.E. (*, p < 0.05). B, contractile response of human bronchial rings to ACh was determined, after which they were pretreated with peptides (2.5 μg/ml) for 20 min. They were then stimulated with ACh. Contractile force is compared before and after the treatment with peptides (*, p < 0.05; n = 3). C, mouse tracheal segments were treated with peptides for 20 min. ACh dose response was then determined. *, significantly lower contractile force in CTTN-I peptide-treated segments than in control peptide-treated tissues at corresponding concentrations (p < 0.01; n = 14). D, myosin light chain phosphorylation in HASM cells pretreated with peptides was assessed by immunoblot analysis. Myosin phosphorylation was similar in cells pretreated with control peptide and in cells pretreated with CTTN-I peptide (p > 0.05; n = 6).

FIGURE 5.

Cortactin is required for airway smooth muscle contraction. A, human bronchial rings were transduced with lentiviruses encoding control shRNA or cortactin shRNA. These tissues were then incubated in the serum-free medium for 3 days. Immunoblot analysis was used to assess the expression of cortactin in tissues. UI, uninfected; C, control shRNA; Cort, cortactin shRNA. *, significantly lower protein ratios of cortactin/GAPDH in tissues transduced with virus encoding cortactin shRNA than in uninfected tissues and tissues expressing control shRNA (p < 0.05). Data are mean values of three independent experiments. Error bars indicate S.E. B, contraction of human bronchial rings was evaluated, after which they were transduced with lentiviruses as described above. Contractile responses of tissues expressing cortactin shRNA were normalized to tissues expressing control shRNA. *, significantly lower contractile force in bronchial rings treated with cortactin shRNA as compared with tissues infected with virus encoding control shRNA (p < 0.05; n = 3). C, cells expressing control shRNA or cortactin shRNA were stimulated with ACh (100 μm; 5 min) or left unstimulated. F/G-actin ratios in cells were evaluated using the fractionation assay. Data are mean values of six independent experiments. Error bars indicate S.E. (*, p < 0.05). D, myosin light chain phosphorylation in HASM cells transduced with lentivirus encoding control or cortactin shRNA was assessed by immunoblot analysis. Myosin phosphorylation was similar in cells expressing control shRNA and cells expressing cortactin shRNA (p > 0.05; n = 6).

FIGURE 6.

Cortactin undergoes phosphorylation at Tyr-421 in cells in response to stimulation with ACh. A, HASM cells were stimulated with 100 μm ACh for different time periods or left unstimulated. Cortactin phosphorylation in these cells was evaluated by immunoblot analysis. Cortactin phosphorylation upon ACh activation is time-dependent. Data are mean values of seven independent experiments. Error bars indicate S.E. B, cells were treated with different concentrations of ACh for 5 min. Cortactin phosphorylation was then assessed. ACh-induced cortactin phosphorylation at Tyr-421 is dose-dependent. Cortactin phosphorylation in cells induced by ACh is normalized to the levels in unstimulated cells (n = 8). p-Cortactin, phosphocortactin.

FIGURE 7.

c-Abl regulates cortactin phosphorylation and cortactin/Pfn-1 coupling in response to ACh stimulation. A, silencing of c-Abl in HASM cells by lentivirus-mediated RNAi. Blots of extracts from uninfected (UI) cells and cells transduced with lentivirus encoding control (Con) shRNA or c-Abl shRNA were probed with antibodies against c-Abl and GAPDH. Protein ratios of c-Abl/GAPDH in cells expressing control or c-Abl shRNA are normalized to uninfected cells (n = 6). *, significantly lower c-Abl/GAPDH ratios in c-Abl KD cells than in uninfected cells and cells expressing control shRNA (p < 0.05). B, cells stably expressing control shRNA or c-Abl shRNA were stimulated with ACh (100 μm; 5 min) or left unstimulated. Cortactin phosphorylation of these cells was then evaluated. Cortactin phosphorylation is normalized to unstimulated cells expressing control shRNA. Data are mean values of five independent experiments. Error bars indicate S.E. (*, p < 0.05). C, cortactin phosphorylation by c-Abl in vitro. c-Abl-mediated cortactin phosphorylation was determined by in vitro kinase assay. Cortactin phosphorylation after c-Abl treatment is normalized to the phosphorylation level in the absence of c-Abl (*, p < 0.05; n = 4). D, control cells and c-Abl KD cells were stimulated with ACh (100 μm; 5 min), or they were unstimulated. Cortactin/Pfn-1 coupling was evaluated by co-immunoprecipitation (n = 5; *, p < 0.05). E, tracheal rings from c-Abl-flox mice or c-Abl-KO mice were treated with 100 μm ACh for 5 min or left untreated. Cortactin/Pfn-1 coupling was evaluated by co-immunoprecipitation analysis (n = 4; *, p < 0.05). F, cells expressing WT or mutant (Y421F) cortactin were stimulated with 100 μm ACh for 5 min or left unstimulated. Cortactin/Pfn-1 coupling was evaluated by co-immunoprecipitation analysis. Data are mean values of six independent experiments. Error bars indicate S.E. (*, p < 0.05). IP, immunoprecipitation; p-Cortactin, phosphocortactin; Mut, mutant.

FIGURE 8.

Knockdown of Abi1 does not affect the association of cortactin with Pfn-1 upon ACh stimulation. A, knockdown of Abi1 in cells by lentivirus-mediated RNAi. Blots of extracts from uninfected (UI) cells and cells transduced with lentivirus encoding control (Ctrl) shRNA or Abi1 shRNA were probed with antibodies against Abi1 and GAPDH. Abi1/GAPDH ratios in cells expressing control or Abi1 shRNA are normalized to uninfected cells (n = 4; *, p < 0.05). B, control cells and Abi1 KD cells were stimulated with ACh (100 μm; 5 min) or left unstimulated. Cortactin/Pfn-1 association was evaluated by co-immunoprecipitation. CTTN, cortactin. Data are mean values of four independent experiments. Error bars indicate S.E. (n = 4; p > 0.05). C, control cells and Abi1 KD cells were stimulated with ACh (100 μm; 5 min) or left unstimulated. F/G-actin ratios in cells were evaluated using the assay described under “Experimental Procedures” (n = 5; *, p < 0.05). IP, immunoprecipitation.

FIGURE 9.

Treatment with CTTN-I peptide diminishes airway resistance and contractile response of tracheal rings from OVA-sensitized and -challenged mice. A, sensitization, challenge, and treatment protocol for the chronic asthma animal model. I.P., intraperitoneal injection. I.N., intranasal instillation. B, BALB/c mice were sensitized and challenged with OVA in the presence of control (Con) or CTTN-I peptide. Airway resistance (R_aw) in these mice was then measured. Intranasal instillation of CTTN-I peptide inhibits airway resistance in mice sensitized and challenged by OVA. Data are mean values of six independent experiments. Error bars indicate S.E. (*, p < 0.05). C, treatment with CTTN-I peptide attenuates OVA-sensitized tracheal contraction ex vivo. Contractile force is normalized to maximal force of rings from OVA- and vehicle-treated mice. Data are mean values of six independent experiments. Error bars indicate S.E. (*, p < 0.05). MCh, methacholine.

FIGURE 10.

Proposed mechanism for smooth muscle contraction. In addition to myosin activation, contractile agonists may promote the association of cortactin (CTTN) with Pfn-1, which induces actin polymerization and smooth muscle contraction. The interaction of cortactin with Pfn-1 is regulated by cortactin phosphorylation and c-Abl tyrosine kinase.