Muscarinic m2 receptor-mediated actin polymerization via PI3 kinase γ and integrin-linked kinase in gastric smooth muscle

Abstract

Background: Actin polymerization plays an important role in smooth muscle contraction. Integrin-linked kinase (ILK) was shown to mediate actin polymerization in airway smooth muscle. The role of ILK in actin polymerization in response to m2 receptor activation was not in gastric smooth muscle.

Methods: Phosphorylation of paxillin, neuronal Wiskott-Aldrich syndrome protein (N-WASp), and association of paxillin with GEF proteins (Cool2/αPix [Cool2/PAK-interacting exchange factor alpha], Cool1/βPix [Cool1/PAK-interacting exchange factor beta], and DOCK 180 [Dedicator of cytokinesis]) and N-WASp with Arp2/3 complex were measured by western blot. Activation of Cdc42 was determined using an antibody for activated Cdc42. Actin polymerization was measured as an increase in F-actin/G-actin ratio.

Results: Phosphorylation of paxillin, an association of paxillin with GEF proteins, Cdc42 activity, and actin polymerization were increased in response to m2 receptor activation in gastric smooth muscle cells. The increases in paxillin phosphorylation, Cdc42 activity, and actin polymerization were inhibited by a PI3Kγ inhibitor (AS-605240), ILK siRNA, and ILK dominant negative mutant (ILK [R211]). Increase in actin polymerization was also inhibited by Cdc42 dominant negative mutant (Cdc42 [T17N]). Increases in the association of paxillin with GEF proteins, phosphorylation of N-WASp and its association with Arp2/3 complex were inhibited by ILK (R211).

Conclusion: In gastric smooth muscle cells, activation of PI3Kγ by muscarinic m2 receptors causes ILK-dependent phosphorylation of paxillin, an association of paxillin with Cdc42 GEF proteins and activation of Cdc42, which, in turn, causes phosphorylation of N-WASp and its association with Arp2/3 complex leading to actin polymerization.

Keywords: ILK; PI3 Kinase; actin polymerization; m2 receptors; smooth muscle.

Figure 1.. PI3Kγ dependent phosphorylation of paxillin by m2 receptor activation.

Cultured smooth muscle cells were treated for 5 min with ACh (1 µM) plus m3 receptor antagonist (4-DAMP, 1 µM) in the presence or absence of PI3Kγ inhibitor (AS605240, 25 nM). 4-DAMP and AS-605240 were added 10 min prior to the addition of ACh. Phosphorylation of paxillin (p-Paxillin) was measured by immunocytochemistry (A) and western blot (B). Values are means ± SE of 3–4 experiments. **P<0.001

Figure 2.. ILK dependent phosphorylation of paxillin by m2 receptor activation.

Cultured smooth muscle cells transfected with vector alone or dominant negative mutant ILK(R211A) (A) or siRNA for ILK (B) were treated for 5 min with ACh (1 µM) plus 4-DAMP (1 µM). Phosphorylation of paxillin (p-Paxillin) was measured by western blot. Values are means ± SE of 3 experiments. **P<0.001

Figure 3.. Association of p-paxillin with Cdc42 GEFs by m2 receptor activation.

Cultured smooth muscle cells transfected with vector alone or dominant negative mutant ILK(R211A) (A) or siRNA for ILK (B) were treated for 5 min with ACh (1 µM) plus 4-DAMP (1 µM). Association of paxillin with Cdc42 GEFs by m2 receptor activation. Values are means ± SE of 3 experiments. **P<0.001

Figure 4.. PI3Kγ- and ILK-dependent Cdc42 activity by m2 receptor activation.

Cultured smooth muscle cells were treated for 5 min with ACh (1 µM) plus m3 receptor antagonist (4-DAMP, 1 µM) in the presence or absence of PI3Kγ inhibitor (AS605240, 25 nM). 4-DAMP and AS-605240 were added 10 min prior to the addition of ACh (A). Cultured smooth muscle cells transfected with vector alone or dominant negative mutant ILK(R211A) (B) or transfected with siRNA for ILK (C) were treated for 5 min with ACh (1 µM) plus 4-DAMP (1 µM). Cdc42 activity was determined by using pull-down assay for activated Cdc42. Activated GTP-bound Cdc42 was affinity-precipitated by glutathione beads and quantitated by western blot. Values are means ± SE of 3–4 experiments. **P<0.001

Figure 5.. ILK-dependent phosphorylation of N-WASp and association of N-WASp with Arp2/3 by m2 receptor activation.

(A) Cultured smooth muscle cells transfected with vector alone, dominant negative mutant ILK(R211A) were treated for 5 min with ACh (1 µM) plus 4-DAMP (1 µM). Phosphorylation of N-WASp (pNWASp) was measured by western blot. Values are means ± SE of 3–4 experiments. (B) Cultured smooth muscle cells transfected with vector alone or dominant negative mutant ILK(R211A) were treated for 5 min with ACh (1 µM) plus 4-DAMP (1 µM). Association of N-WASp with Arp2/3 was measured by immunoprecipitation followed by western blot. Values are means ± SE of 3 experiments. **P<0.001

Figure 6.. PI3Kγ-dependent actin polymerization by m2 receptor activation.

Cultured smooth muscle cells were treated for 5 min with ACh (1 µM) plus m3 receptor antagonist (4-DAMP, 1 µM) in the presence or absence of PI3Kγ inhibitor (AS605240, 25 nM) or Arp2/3 inhibitor (CK-666, 200 µM. 4-DAMP and AS-605240 were added 10 min and CK-666 was added 1h prior to the addition of ACh. Actin polymerization was measured by western blot. Values are means ± SE of 3 experiments. . **P<0.001

Figure 7.. ILK-dependent actin polymerization by m2 receptor activation.

Cultured smooth muscle cells transfected with vector alone or dominant negative mutant ILK(R211A) (A) or transfected with siRNA for ILK (B) were treated for 5 min with ACh (1 µM) plus 4-DAMP (1 µM). Actin polymerization was measured by western blot. Values are means ± SE of 5–6 experiments. **P<0.001

Figure 8.. Cdc42-dependent actin polymerization by m2 receptor activation.

Cultured smooth muscle cells transfected with vector alone or dominant negative Cdc42 [T17N] were treated for 5 min with ACh (1 µM) plus 4-DAMP (1 µM). Actin polymerization was measured by western blot. Values are means ± SE of 3 experiments. **P<0.001

Figure 9.. Signaling mechanism of actin polymerization by m2 receptor activation.

Activation of muscarinic m2 receptors causes sequential activation of PI3Kγ via Gβγ_i3 and recruitment of ILK/paxillin complex to PIP3. This results in an increase in phosphorylation of paxillin and its association with Cdc42 GEF proteins and Cdc42 activity, which in turn causes an increase in phosphorylation of N-WASp and its association with Arp2/3 complex to mediate actin polymerization.