DOI: 10.5061/dryad.jh9w0vtnj
title: Data from: IL-13 and IL-17A activate β1 integrin through an NF-kB/Rho kinase/PIP5K1γ pathway to enhance force transmission in airway smooth muscle

description: Integrin activation resulting in enhanced adhesion to the extracellular
 matrix plays a key role in fundamental cellular processes. Although
 integrin activation has been extensively studied in circulating cells such
 as leukocytes and platelets, much less is known about the regulation and
 functional impact of integrin activation in adherent cells such as smooth
 muscle. Here, we show that two different asthmagenic cytokines, IL-13 and
 IL-17A, activate type I and IL-17 cytokine receptor families,
 respectively, to enhance adhesion of airway smooth muscle. These cytokines
 also induce activation of β1 integrins detected by the
 conformation-specific antibody HUTS-4. Moreover, HUTS-4 binding is
 increased in the smooth muscle of patients with asthma compared to
 nonsmokers without lung disease, suggesting a disease-relevant role for
 integrin activation in smooth muscle. Indeed, integrin activation induced
 by the β1-activating antibody TS2/16, the divalent cation manganese, or
 the synthetic peptide β1-CHAMP that forces an extended-open integrin
 conformation dramatically enhances force transmission in smooth muscle
 cells and airway rings even in the absence of cytokines. We demonstrate
 that cytokine-induced activation of β1 integrins is regulated by a common
 pathway of NF-κB-mediated induction of RhoA and its effector Rho kinase,
 which in turn stimulates PIP5K1γ-mediated synthesis of PIP 2 at focal
 adhesions, resulting in β1 integrin activation. Taken together, these data
 identify a pathway by which type I and IL-17 cytokine receptor family
 stimulation induces functionally relevant β1 integrin activation in
 adherent smooth muscle and help to explain the exaggerated force
 transmission that characterizes chronic airway diseases such as asthma.
description: # Data from: IL-13 and IL-17A activate β1 integrin through an NF-kB/Rho
 kinase/PIP5K1γ pathway to enhance force transmission in airway smooth
 muscle
 ([https://doi.org/10.1073/pnas.2401251121](https://doi.org/10.1073/pnas.2401251121)) ## Description of data files ### Figure 1 folder #### File: Figure 1A and 1B.xlsx **Description:** Data for adhesion assay as measured by absorbance of crystal violet at 595 nm of Human Airway Smooth Muscle (HASM) cells to various concentrations of collagen and fibronectin after treatment with vehicle, IL-13 (100 ng/mL), or IL-17A (100 ng/mL) for 12 h. #### Folder:Figure 1C **Description:** Contains flow cytometry data (file type:fcs) comparing the expression of active beta-1 integrin in HASM cells treated with vehicle, IL-13 (100 ng/mL), IL-17A (100 ng/mL) for 12 h, or Mn2+ (1 mM) for 20 min followed by labeling with antibody specific for active beta-1 integrin (HUTS-4) and secondary (2°) conjugated to allophycocyanin (APC). #### Folder: Figure 1D **Description:** Contains flow cytometry data (file type:fcs) comparing the expression of total beta-1 integrin in HASM cells treated with vehicle, IL-13 (100 ng/mL), IL-17A (100 ng/mL) for 12 h, or Mn2+ (1 mM) for 20 min followed by labeling with antibody specific for total beta-1 integrin (P5D2) and secondary (2°) conjugated to allophycocyanin (APC). ### Figure 2 folder #### File: Figure 2A and 2B.xlsx **Description:** Median fluorescence intensity (MFI) data of HUTS-4 in human bronchial rings (A) or endobronchial airway biopsy specimens from de-identified asthma and healthy control (nonsmokers without lung disease) (B). MFI of HUTS-4 was measured in five sections per sample (n = 3 donors per group). #### Folder: Figure 2A **Description:** Contains representative immunofluorescence (file type: TIFF) from de-identified human bronchial rings treated with vehicle, IL-13 (100 ng/mL) for 12 h, or Mn2+ (1 mM) for 2 h, then sectioned, and stained with antibodies specific for active β1 integrin (HUTS-4, red), anti-α-smooth muscle actin (α-sma, green), and DAPI (blue). #### Folder: Figure 2B **Description:** Contains representative immunofluorescence from endobronchial airway biopsy specimens from de-identified asthma and control stained with antibodies specific for active β1 integrin (HUTS-4, red), anti-α-smooth muscle actin (green), and DAPI (blue). ### Figure 3 folder #### File: Figure 3.xlsx **Description:** Quantification data of percentage change in area after histamine (100 μM) relative to baseline after HASM embedded in collagen gels are exposed to (A) IgG control, β1-activating antibody (TS2/16, 10 μM) for 12 h, or (B) vehicle, Mn2+ (1 mM) for 20 min, followed by the α2 integrin inhibitor c15 (20 μM) for 1 h. (C) Contractile force measured in mouse tracheal rings after incubation with vehicle or Mn2+ (1 mM) for 2 h, followed by the α2 integrin inhibitor c15 (20 μM) for 1 h. (D) Contractile force measured in mouse tracheal rings after incubation with vehicle or IL-13 (100 ng/mL) for 12 h, followed by Mn2+ (1 mM) for 2 h.(E) Contractile force measured in human bronchial rings after incubation with vehicle or Mn2+ (1 mM) for 2 h. ### Figure 4 folder #### Folder: Figure 4A **Description:** Contains flow cytometry data (file type:fcs) comparing the expression of active beta-1 integrin in HASM cells treated with lipid carrier 1 in the presence or absence of diC16-PIP2 (10 μM) for 1 h, followed by labeling with antibody specific for active beta-1 integrin (HUTS-4) and secondary (2°) conjugated to allophycocyanin (APC). #### File: Figure 4B\_E **Description:** (B) Contractile force measured in mouse tracheal rings after incubation with lipid carrier 1 in the presence or absence of diC16-PIP2 (10 μM) for 1 h. (C) Quantification of PIP5K1γ activity normalized to vehicle as measured by transfer of [γ-32P] to PI4P from [γ-32P] ATP in HASM after treatment with vehicle or IL-13 (100 ng/mL) for 12 h followed by lysis and IP with anti-PIP5K1γ antibody. (D) Contractile force measured in mouse tracheal rings after incubation with vehicle or IL-13 (100 ng/mL) for 12 h, followed by UNC3230 (200 nM) for 1 h. (E) PIP5K1γ activity normalized to vehicle in HASM after treatment with vehicle or IL-13 (100 ng/mL) in the presence of vehicle or Y-27632 (100 μM) for 12 h, followed by lysis and IP with anti-PIP5K1γ antibody. #### Folder: Figure 4F **Description:** Contains flow cytometry data (file type:fcs) comparing the expression of active beta-1 integrin in HASM cells treated with treated with vehicle, Mn2+ (1 mM) for 20 min, IL-13 (100 ng/mL), or IL-17A (100 ng/mL) in the presence of vehicle or Y-27632 (100 μM) for 12 h followed by labeling with antibody specific for active beta-1 integrin (HUTS-4) and secondary (2°) conjugated to allophycocyanin (APC). #### Folder: Figure 4G **Description:** Contains Western Blot picture of immunoprecipitation from lysates of mouse trachea after pulldown with rabbit IgG or PIP5K1γ antibody followed by immunoblot (IB) for Rho kinase. ### Figure 5 folder #### Folder: Figure 5A **Description:** Contains flow cytometry data (file type:fcs) comparing the expression of active beta-1 integrin in HASM cells treated with treated with vehicle, Mn2+ (1 mM) for 20 min, IL-13 (100 ng/mL), or IL-17A (100 ng/mL) for 1 h followed by labeling with antibody specific for active beta-1 integrin (HUTS-4) and secondary (2°) conjugated to allophycocyanin (APC). #### Folder: Figure 5B **Description:** Contains flow cytometry data (file type:fcs) comparing the expression of active beta-1 integrin in HASM cells treated with treated with vehicle, Mn2+ (1 mM) for 20 min, IL-13 (100 ng/mL), or IL-17A (100 ng/mL) in the presence of vehicle or cycloheximide (CHX, 15 ug/mL) for 12 h followed by labeling with antibody specific for active beta-1 integrin (HUTS-4) and secondary (2°) conjugated to allophycocyanin (APC). #### Folder: Figure 5C **Description:** Contains Western Blot pictures of phospho- and total-IκBα and GAPDH. #### Figure 5C.xlsx **Description:** Quantification data of densitometry for HASM treated with vehicle or IL-13 (100 ng/mL) for 45 min or 12 h followed by histamine (100 μM) for 15 min followed by lysis, separation by SDS-PAGE, and transfer to membrane probed with antibodies to phospho- and total-IκBα and GAPDH. #### Folder: Figure 5D **Description:** Contains flow cytometry data (file type:fcs) comparing the expression of active beta-1 integrin in HASM cells treated with treated with vehicle, Mn2+ (1 mM) for 20 min, IL-13 (100 ng/mL), or IL-17A (100 ng/mL) in the presence of vehicle or BAY 11-7082 (2 μM) for 12 h followed by labeling with antibody specific for active beta-1 integrin (HUTS-4) and secondary (2°) conjugated to allophycocyanin (APC).