IQGAP1-dependent scaffold suppresses RhoA and inhibits airway smooth muscle contraction

Abstract

The intracellular scaffold protein IQGAP1 supports protein complexes in conjunction with numerous binding partners involved in multiple cellular processes. Here, we determined that IQGAP1 modulates airway smooth muscle contractility. Compared with WT controls, at baseline as well as after immune sensitization and challenge, Iqgap1-/- mice had higher airway responsiveness. Tracheal rings from Iqgap1-/- mice generated greater agonist-induced contractile force, even after removal of the epithelium. RhoA, a regulator of airway smooth muscle contractility, was activated in airway smooth muscle lysates from Iqgap1-/- mice. Likewise, knockdown of IQGAP1 in primary human airway smooth muscle cells increased RhoA activity. Immunoprecipitation studies indicated that IQGAP1 binds to both RhoA and p190A-RhoGAP, a GTPase-activating protein that normally inhibits RhoA activation. Proximity ligation assays in primary airway human smooth muscle cells and mouse tracheal sections revealed colocalization of p190A-RhoGAP and RhoA; however, these proteins did not colocalize in IQGAP1 knockdown cells or in Iqgap1-/- trachea. Compared with healthy controls, human subjects with asthma had decreased IQGAP1 expression in airway biopsies. Together, these data demonstrate that IQGAP1 acts as a scaffold that colocalizes p190A-RhoGAP and RhoA, inactivating RhoA and suppressing airway smooth muscle contraction. Furthermore, our results suggest that IQGAP1 has the potential to modulate airway contraction severity in acute asthma.

Figure 3. IQGAP1 acts as a scaffold that colocalizes p190A-RhoGAP and RhoA.

(A) Co-immunoprecipitation of IQGAP1 and p190A-RhoGAP in lysates of human airway smooth muscle. (B) Co-immunoprecipitation of IQGAP1 and FLAG in lysates of human airway smooth muscle transfected with Rho-FLAG or GFP control. (C) Association of IQGAP1 with both RhoA and p190A-RhoGAP was also demonstrated by PLA in cultured human airway smooth muscle cells. In the control condition, PLA probes were applied without primary antibody. Nuclei were stained with DAPI. Original magnification, ×63. (D) Spatial association of p190A-RhoGAP and RhoA, tested by PLA in cultured human airway smooth muscle cells with and without IQGAP1 knockdown and seeded at equal density. Nuclei were stained with DAPI. Number of PLA foci per high-power field (hpf) was calculated for 10 images per condition. **P ≤ 0.001. Original magnification, ×63. (E) Spatial association of p190A-RhoGAP and RhoA, tested by PLA in 10-μm mouse tracheal sections. Smooth muscle was stained with anti–α-SMA antibody (red). Number of PLA foci per unit area of smooth muscle is also shown (n = 4 per group). *P ≤ 0.05. Original magnification, ×63. (F) Immunoblots for IQGAP1, α-SMA, and GAPDH were performed for lysates of airway biopsies from separate healthy control and asthmatic human subjects. Bands were measured by densitometry, and IQGAP1 was quantified normalized to α-SMA (n = 4 per group). *P ≤ 0.05.

Figure 2. IQGAP1 suppresses airway smooth muscle contraction.

(A) Force of contraction for Iqgap1^–/– and WT tracheal rings in response to methacholine (Mch; n = 6 per group). *P ≤ 0.001 vs. WT. (B) Tracheal immunofluorescence for IQGAP1 and α-SMA. IQGAP1 staining was seen in both the epithelium (arrow) and the smooth muscle (arrowheads). Original magnification, ×10. (C) Force of contraction in response to methacholine for Iqgap1^–/– and WT tracheal rings with and without epithelium removed (n = 6 per group). *P ≤ 0.001 vs. respective WT. (D) Western blots with lysates of posterior trachea from Iqgap1^–/– and WT mice. p-MLC, phosphorylated MLC; p-MYPT1, phosphorylated MLC phosphatase-1. (E) GST-rhotekin bead pulldown of RhoA-GTP (RhoA active form) with lysates of posterior trachea from Iqgap1^–/– and WT mice. Results in D and E are representative of at least 3 individual experiments. (F) GST-rhotekin bead pulldown of RhoA-GTP with lysates of human airway smooth muscle cells with and without knockdown (KD) of IQGAP1 or p190A-RhoGAP. EV, empty vector.

Figure 1. Airway responsiveness and inflammation in Iqgap1^–/– mice.

(A) Pulmonary resistance in anesthetized, mechanically ventilated Iqgap1^–/– and WT mice treated with increasing doses of i.v. acetylcholine (n = 7–8 per group). *P ≤ 0.01, **P ≤ 0.001 vs. WT. (B) Pulmonary resistance in OVA-sensitized Iqgap1^–/– and WT mice (n = 10–11 per group). *P ≤ 0.01, **P ≤ 0.001 vs. WT. (C) Bronchoalveolar lavage cell count and differential in OVA-sensitized Iqgap1^–/– and WT mice (n = 3–6 per group). (D) Inflammation in Iqgap1^–/– and WT lung sections, quantified by scoring of periodic acid–Schiff (PAS) and H&E stain (n = 4–6 per group). (E) Serum OVA-specific IgE, measured by ELISA (n = 4–6 per group).