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
Abstract
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.
(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).