Integrin α7 expression is increased in asthmatic patients and its inhibition reduces Kras protein abundance in airway smooth muscle cells

Abstract

Airway smooth muscle (ASM) cells exhibit plastic phenotypic behavior marked by reversible modulation and maturation between contractile and proliferative phenotypic states. Integrins are a class of transmembrane proteins that have been implicated as novel therapeutic targets for asthma treatment. We previously showed that integrin α7 is a novel marker of the contractile ASM phenotype suggesting that targeting this protein may offer new avenues to counter the increase in ASM cell mass that underlies airways hyperresponsiveness (AHR) in asthma. We now determine whether inhibition of integrin α7 expression would revert ASM cells back to a proliferative phenotype to cause an increase in ASM cell mass. This would be detrimental to asthmatic patients who already exhibit increased ASM mass in their airways. Using immunohistochemical analysis of the Melbourne Epidemiological Study of Childhood Asthma (MESCA) cohort, we show for the first time that integrin α7 expression in patients with severe asthma is increased, supporting a clinically relevant role for this protein in asthma pathophysiology. Moreover, inhibition of the laminin-integrin α7 signaling axis results in a reduction in smooth muscle-alpha actin abundance and does not revert ASM cells back to a proliferative phenotype. We determined that integrin α7-induced Kras isoform of p21 Ras acts as a point of convergence between contractile and proliferative ASM phenotypic states. Our study provides further support for targeting integrin α7 for the development of novel anti-asthma therapies.

Figure 1

Integrin α7 expression is increased with asthma severity. (a) Representative matched immunohistochemical images of human lung biopsies taken from the MESCA (Melbourne Epidemiological Study on Childhood Asthma) cohort stained for smooth muscle (sm)-α-actin and integrin α7. Brown staining represents positive staining for either sm-α-actin or integrin α7, while blue staining represents nuclear staining; ASM = airway smooth muscle, E = epithelium. Bar = 50μm. (b) Quantitation of integrin α7 staining intensity in biopsies from subjects with increasing asthma severity. Individual and median values from non-asthma (n = 9), mild asthma (n = 13), moderate asthma (n = 8) and severe asthma (n = 8) subjects are shown. *P < 0.05, Kruskal–Wallis test.

Figure 2

Inhibition of laminin-binding or laminin receptor expression (integrin α7β1) maintains the ASM cells in a low proliferative state. (a) Time course and representative histograms showing a typical distribution of cells in G0/G1, S, and G2/M phases with increasing days of serum deprivation. Grouped (b) and representative histograms (c) showing the effect of the laminin-selective competing peptide (YIGSR, 10 μM) on ASM cell cycle S-phase following day 1 serum deprivation. Grouped (d) and representative histograms (e) showing the effect of integrin α7 siRNA (1 μM) on ASM cell cycle S-phase following day 1 serum deprivation. Grouped (f) and representative histograms (g) showing the effect of the laminin-selective competing peptide (YIGSR, 10 μM) on ASM cell cycle S-phase following day 7 serum deprivation. Grouped (h) and representative histograms (i) showing the effect of integrin α7 siRNA (1 μM) on ASM cell cycle S-phase following day 7 serum deprivation. Transfection agent (TA) served as vehicle control, green fluorescence protein (GFP) siRNA served as negative control. Results are representative of 3 independent experiments. *P < 0.05 compared with Day 0. Portions of this study have been deposited in scholarbank.nus.edu.sg.

Figure 3

Laminin and integrin α7β1 are not required for ERK, cyclin D1, p38 MAPK, PKC, p70s6K, or Rac1 protein abundance. Effect of laminin-selective competing peptide (YIGSR, 10 μM, (a) or integrin α7 siRNA (1 μM, (b) treatment on ERK and cyclin D1 protein abundance. Effect of laminin-selective competing peptide (YIGSR, 10 μM, (c) or integrin α7 siRNA (1 μM, (d) on p38 MAPK, PKC, p70s6K, and Rac1 protein abundance.  Transfection agent (TA) served as vehicle control, green fluorescence protein (GFP) siRNA served as negative control. Data are expressed as fold increment over basal (Day 0) relative to β-actin protein abundance. Results are representative of 3 independent experiments. *P < 0.05 compared with Day 0; ^†P < 0.05 compared with Day 7 response without YIGSR or integrin α7 siRNA.

Figure 4

(a) Validation of integrin α7 siRNA on integrin α7 protein abundance. NT = non-targeting siRNA. Effect of integrin α7 siRNA on bFGF or FBS-induced increases in (b) cyclin D1 protein abundance following 20 hr mitogen stimulation, and (c) cell number following 48 hr mitogen stimulation. Data are expressed as fold increment over control (Day 0). Results are representative of 3–5 independent experiments. *P < 0.05 compared with control; ns = not significant compared with respective mitogen.

Figure 5

Laminin-binding (a) and integrin α7β1 (b) expression is required for p21 Ras protein abundance. Transfection agent (TA) served as vehicle control, green fluorescence protein (GFP) siRNA served as negative control. Data are expressed as fold increment over basal (Day 0) relative to β-actin protein abundance. Results are representative of 3 independent experiments. *P < 0.05 compared with Day 0; ^†P < 0.05 compared with Day 7 response without YIGSR or integrin α7 siRNA.

Figure 6

Grouped (a) and representative western blots (b) of time course experiments showing the typical distribution of p21 Ras protein isoforms with increasing days of serum deprivation. (c) Laminin-binding and (d) integrin α7β1 expression are required for Kras protein abundance. (e) Effect of α2-chain laminin siRNA (50 nM) on Kras protein abundance. Transfection agent (TA) served as vehicle control, green fluorescence protein (GFP) and non-targeting (NT) siRNAs served as negative control, LN, Laminin. Results are representative of 3 independent experiments. Data are expressed as fold increment over basal (Day 0) relative to β-actin protein abundance. *P < 0.05 compared with Day 0; ^†P < 0.05 compared with Day 7 response without YIGSR, integrin α7 siRNA or α2-chain laminin siRNA.

Figure 7

Silencing of Kras protein markedly reduces sm-α-actin expression with no change in cyclin D1 protein abundance. Effect of Kras siRNA (50 nM) on (a) p21 Ras isoforms, sm-α-actin, and (b) cyclin D1 protein abundance. Transfection agent (TA) served as vehicle control, non-targeting (NT) siRNA served as negative control. Results are representative of 4–6 independent experiments. Data are expressed as fold increment over basal (Day 0) relative to β-actin protein abundance. *P < 0.05 compared with Day 0; ^†P < 0.05 compared with Day 7 response without Kras siRNA.