Unique mechanisms of connective tissue growth factor regulation in airway smooth muscle in asthma: Relationship with airway remodelling

Abstract

Neovascularization, increased basal membrane thickness and increased airway smooth muscle (ASM) bulk are hallmarks of airway remodelling in asthma. In this study, we examined connective tissue growth factor (CTGF) dysregulation in human lung tissue and animal models of allergic airway disease. Immunohistochemistry revealed that ASM cells from patients with severe asthma (A) exhibited high expression of CTGF, compared to mild and non-asthmatic (NA) tissues. This finding was replicated in a sheep model of allergic airways disease. In vitro, transforming growth factor (TGF)-β increased CTGF expression both in NA- and A-ASM cells but the expression was higher in A-ASM at both the mRNA and protein level as assessed by PCR and Western blot. Transfection of CTGF promoter-luciferase reporter constructs into NA- and A-ASM cells indicated that no region of the CTGF promoter (-1500 to +200 bp) displayed enhanced activity in the presence of TGF-β. However, in silico analysis of the CTGF promoter suggested that distant transcription factor binding sites may influence CTGF promoter activation by TGF-β in ASM cells. The discord between promoter activity and mRNA expression was also explained, in part, by differential post-transcriptional regulation in A-ASM cells due to enhanced mRNA stability for CTGF. In patients, higher CTGF gene expression in bronchial biopsies was correlated with increased basement membrane thickness indicating that the enhanced CTGF expression in A-ASM may contribute to airway remodelling in asthma.

Keywords: airway remodelling; airway smooth muscle; asthma; connective tissue growth factor.

Figure 1

Connective tissue growth factor (CTGF) expression is increased in house dust mite (HDM)‐induced allergic airway disease in sheep lungs. CTGF expression in a model of allergic airway disease was assessed by immunohistochemistry in HDM‐ and saline‐exposed (sham control) lung segments from the same sheep30 (n = 5). Isotype‐matched negative control antibody on serial sections shown for comparison. Representative images shown for each group

Figure 2

Asthmatic airway smooth muscle (ASM) cells have different kinetics of connective tissue growth factor (CTGF) induction. ASM cells from A‐ and NA‐donors were stimulated with transforming growth factor (TGF)‐β (1 ng/mL) for up to 72 h and CTGF transcript (A; NA‐ASM [n = 7] and A‐ASM [n = 5]) and protein (B; NA‐ASM [n = 4] and A‐ASM [n = 7]) levels examined by Q‐PCR and Western blot, respectively. Representative images of Western blots are shown. Changes in CTGF expression by Western blot were quantified using image J software (C). *P < .05, **P < .01 and ***P < .001 denotes significance between bovine serum albumin and TGF‐β. #P < .05, ##P < .01 and ####P < .0001, # indicates significant difference between NA‐ and A‐ASM cells

Figure 3

Basal regulation of the connective tissue growth factor (CTGF) promoter is the same in NA‐ and A‐ASM cells. A. Schematic of the 5′ deleted CTGF promoter constructs used to examine regulation in NA‐ and A‐ASM cells. Different lengths of the human CTGF promoter (−1500 to +200; ) were placed upstream of a Luciferase reporter construct (). Secretion of alkaline phosphatase (SEAP) expression was driven by CMV promoter in the same construct and was used as a control for transfection efficiency. Luciferase activity in conditioned media was detected after stimulation of transfected ASM ± TGF‐β (1 ng/mL; NA‐ [B, n = 5] and A‐ASM [D, n = 5]). CTGF mRNA in NA‐ (C) and A‐ASM (E) was measured by Q‐PCR in the same cells used for luciferase assays to determine the effectiveness of induction for the endogenous gene. “&” Denotes significance between promoter‐less (0) and luciferase reporter (&P < .05, &&P < .01, &&&P < .001). *Indicates significant difference of CTGF mRNA expression between TGF‐β and BSA, *P < .05, **P < .01, ****P < .0001. ASM, asthmatic airway smooth muscle; BSA; bovine serum albumin

Figure 4

Differential usage of promoter elements endows cell‐type specific regulation of connective tissue growth factor (CTGF) in asthmatic airway smooth muscle. A, H3K27ac profiling from human lung fibroblasts and HUVECs surrounding the CTGF gene by ChIP‐Seq. B, Plasmid CTGF promotor construct used in this project. C, Validated SMAD and transforming growth factor (TGF)‐β transcription factor binding sites across difference species. HUVECs, human umbilical vein endothelial cells; SMAD, similar to mothers against decapentaplegic. Analysis was performed using the ENCODE database.

Figure 5

Connective tissue growth factor (CTGF) mRNA stability is enhanced in A‐ASM cells. NA‐ (n = 4) and A‐ASM cells (n = 5) were treated with TGF‐β (1 ng/mL) with actinomycin D (10 μg/mL) added after 8 h for up to 16 h. CTGF mRNA expression was measured by Q‐PCR to assess the rate of turnover. *Means significant difference in CTGF mRNA expression to time 0, *P < .05, ***P < .001, ****P < .0001. #P < .05 indicates a significant difference between NA‐ and A‐ASM. ASM, asthmatic airway smooth muscle; TGF, transforming growth factor

Figure 6

Connective tissue growth factor (CTGF) expression and correlations with clinical indices in asthmatic patients. A, CTGF expression was assessed by immunohistochemistry in human lung tissue (n = 5 for healthy control, mild asthma and severe asthma). Representative images shown for each group. B‐E, CTGF mRNA expression fragments per kilobase million (FPKM) was detected in bronchial biopsies from healthy controls and mild asthmatic patients (B). A linear model comparing the association between CTGF expression in asthmatic bronchial biopsies and BM thickness (μmol/L) (C), FEV1% predicted (D), % of sputum eosinophils (E) and PC20 mg/mL (F) was conducted correcting for age, gender and smoking status. β, correlation co‐efficient; P, significance value of the correlation. BM basement membrane, FEV1% predicted forced expiratory volume in 1 s percentage predicted, PC20 the concentration of methacholine needed to produce a 20% fall in FEV(1) from baseline. See Ref. 32 (Table 1) for lung function on this cohort