Laminin-binding integrin alpha7 is required for contractile phenotype expression by human airway myocytes

Abstract

Contractile airway smooth muscle (ASM) cells retain the ability for phenotype plasticity in response to multiple stimuli, which equips them with capacity to direct modeling and remodeling during development, and in disease states such as asthma. We have shown that endogenously expressed laminin is required for maturation of human ASM cells to a contractile phenotype, as occurs during ASM thickening in asthma. In this study, we profiled the expression of laminin-binding integrins alpha3beta1, alpha6beta1, and alpha7beta1, and tested whether they are required for laminin-induced myocyte maturation. Immunoblotting revealed that myocyte maturation induced by prolonged serum withdrawal, which was marked by the accumulation of contractile phenotype marker protein desmin, was also associated with the accumulation of alpha3A, alpha6A, and alpha7B. Flow cytometry revealed that alpha7B expression was a distinct feature of individual myocytes that acquired a contractile phenotype. siRNA knockdown of alpha7, but not alpha3 or alpha6, suppressed myocyte maturation. Thus, alpha7B is a novel marker of the contractile phenotype, and alpha7 expression is essential for human ASM cell maturation, which is a laminin-dependent process. These observations provide new insight into mechanisms that likely underpin normal development and remodeling associated with airways disease.

Figure 1.

(A) Western blot and (B) real-time PCR analysis showing the protein and mRNA expression, respectively, of selective variants of laminin-binding integrins before (Day 0, open bars) and after 7-day serum deprivation (Day 7, solid bars). Grouped data, which are expressed as relative units to Day 0, represent results obtained from at least four different cultures. *P < 0.05, compared with Day 0, relative to β-actin. (C) PCR analysis showing expression of laminin-binding integrins and their respective A and B cytoplasmic splice variants in airway smooth muscle (ASM) cells dissected from human bronchial tissue.

Figure 2.

Flow cytometry analysis of surface expression of laminin-binding integrins in human ASM cells serum deprived for 7 days. Each histogram contains data obtained from 10,000 gated cells. Representative frequency histograms showing the distribution of myocytes stained for integrin (A) α3A, (B) α3B, (C) α6A, (D) α6B, (E) α7A, (F) α7B, (G) α5, or (H) β1. The gray lines in each panel show the distribution of corresponding negative controls that included myocytes incubated in diluted mouse IgG1 (A–D), mouse IgG2a (G), or rabbit (E and F).

Figure 3.

Dual fluorescence analysis of integrin subunit and desmin expression in 7-day serum-deprived human ASM cells. (A) Representative two-parameter histogram dot plot showing the isotype controls for Zenon Alexa-Fluor 488 (AF-488) and Zenon R-PE (R-PE). Representative two-parameter histogram dot plots showing distribution of fluorescence staining of myocytes with AF-488 conjugated primary antibodies for integrins α3A (B), α6A (D), or α7B (F) and R-PE–conjugated primary antibody for desmin are shown. Histograms showing the distribution of positive (+) and negative (−) staining for desmin in myocytes gated for (+) or (−) labeling of integrins α3A (C), α6A (E), or α7B (G). Grouped data from three replicate two-parameter histogram dot plots are shown.

Figure 4.

Matched immunofluorescence images of primary human ASM cells and intact human bronchial tissue double-labeled for integrin α7B and desmin or smMHC. High magnification fields showing typical co-expression of integrin α7B (A) with desmin (B), and α7B (C) with smMHC (D), after prolonged serum withdrawal of primary cultured human ASM cells. Low-power images show coincident expression of the contractile phenotype marker proteins desmin (E) and smMHC (F). Representative immunofluorescence imaging of integrin α7B in smooth muscle layer from 3rd generation human mainstem bronchus is shown in G. Labeling of bronchial tissue with isotype-matched control antibody and fluorescein isothiocyanate–conjugated secondary antibody is shown in H. Arrows indicate matching cells in A and B, C and D, and F and G, respectively. Abbreviations: EPI, epithelium; SM, airway smooth muscle. Bar = 50 μm.

Figure 5.

Analysis of the effect of siRNA-induced suppression of integrin α7 mRNA and protein over 6 days of serum-free culture. (A) PCR analysis; α7 PCR product size = 599 base pairs (bp). (B) Western blotting for integrin α7B. Also shown is Western blot analysis of the effects of α7 siRNA on desmin and smooth muscle α-actin accumulation after 6 days of serum deprivation. Grouped data, which are expressed as relative units to Day 0, represent results obtained from at least four different cultures. *P < 0.05, compared with Day 0, relative to β-actin; ^†P < 0.05, compared with control cultures (6-day serum-free media exposed to transfection reagent without siRNA).

Figure 6.

Western blot analysis showing the effect of siRNA silencing of integrin α7 on the accumulation of integrins α3A (A), α6A (B), α5 (C), and β1 (D) over 6 days of serum deprivation. Grouped data, which are expressed as relative units to Day 0, represent results obtained from at least four different cultures. *P < 0.05, compared with Day 0, relative to β-actin; ^†P < 0.05; ns = not significant compared with control cultures (6-day serum free media exposed to transfection reagent without siRNA). Panels E–H show phase contrast images of human ASM cells in the absence (transfection reagent alone; E) or presence of siRNA directed at integrin α3 (F), α6 (G), or α7 (H). Bar = 50 μm.

Figure 7.

Western blot analysis showing confirmation of effect of siRNA for integrin α3 (A) or α6 (B) on protein abundance of integrin α3A and α6A. Grouped data, which are expressed as relative units to Day 0, represent results from at least four different cultures. *P < 0.05, compared with Day 0, relative to β-actin; ^†P < 0.05; ns = not significant compared with control cultures (6 days of serum-free media exposed to transfection reagent without siRNA).

Figure 8.

Western blot analysis showing the effect of siRNA silencing of integrin α3 (A) or integrin α6 (B) on the accumulation of desmin and smooth muscle α-actin over 6 days of serum free culture. Grouped data, which are expressed as relative units to Day 0, represent results obtained from at least four different cultures. *P < 0.05, compared with Day 0, relative to β-actin; ns = not significant compared with control cultures (6 days of serum-free media exposed to transfection reagent without siRNA).