Comprehensive gene expression profiles reveal pathways related to the pathogenesis of chronic obstructive pulmonary disease

Abstract

To better understand the molecular basis of chronic obstructive pulmonary disease (COPD), we used serial analysis of gene expression (SAGE) and microarray analysis to compare the gene expression patterns of lung tissues from COPD and control smokers. A total of 59,343 tags corresponding to 26,502 transcripts were sequenced in SAGE analyses. A total of 327 genes were differentially expressed (1.5-fold up- or down-regulated). Microarray analysis using the same RNA source detected 261 transcripts that were differentially expressed to a significant degree between GOLD-2 and GOLD-0 smokers. We confirmed the altered expression of a select number of genes by using real-time quantitative RT-PCR. These genes encode for transcription factors (EGR1 and FOS), growth factors or related proteins (CTGF, CYR61, CX3CL1, TGFB1, and PDGFRA), and extracellular matrix protein (COL1A1). Immunofluorescence studies on the same lung specimens localized the expression of Egr-1, CTGF, and Cyr61 to alveolar epithelial cells, airway epithelial cells, and stromal and inflammatory cells of GOLD-2 smokers. Cigarette smoke extract induced Egr-1 protein expression and increased Egr-1 DNA-binding activity in human lung fibroblast cells. Cytomix (tumor necrosis factor alpha, IL-1beta, and IFN-gamma) treatment showed that the activity of matrix metalloproteinase-2 (MMP-2) was increased in lung fibroblasts from EGR1 control (+/+) mice but not detected in that of EGR1 null (-/-) mice, whereas MMP-9 was regulated by EGR1 in a reverse manner. Our study represents the first comprehensive analysis of gene expression on GOLD-2 versus GOLD-0 smokers and reveals previously unreported candidate genes that may serve as potential molecular targets in COPD.

Fig. 1.

Localization of Egr-1, Cyr61, and CTGF in the lung tissues of GOLD-2 and GOLD-0 smokers. Egr-1, Cyr61 and CTGF expression, identified by SAGE and microarray analysis and confirmed by QRT-PCR, were examined for protein expression by immunofluoresence staining. (A-D) Egr-1 expression (red stain) was located in small airways (arrow) and alveolar epithelia (arrowhead) of GOLD-0 and GOLD-2 smokers. (E-H) Cyr61 expression (red stain) was located in small airways (arrow), alveolar epithelia (arrowhead), and inflammatory cells (star) of GOLD-0 and GOLD-2 smokers. (I-L) CTGF expression (green stain) was located in small airways (arrow), alveolar epithelia (arrowhead), small vessel endothelial cells (diamond), stromal cells (pentagon), and inflammatory cells (star) of GOLD-0 and GOLD-2 smokers. (Insets) Egr-1, Cyr61, or CTGF staining at higher magnification. Green (Egr-1 and Cyr61) and yellow (CTGF) stains are the autofluorescence. (Scale bars inside the box indicate 20 μm; scale bars outside the box indicate 100 μm.)

Fig. 2.

The mean relative expression level of candidate genes in human lung fibroblasts as determined by QRT-PCR. QRT-PCR was performed on total RNA isolated from human lung fibroblasts. Open bars, normal lung (n = 2); filled bars, emphysema lung (n = 6). Significant difference between emphysema and normal lung fibroblasts is shown (^*, P < 0.05; ^**, P < 0.01).

Fig. 3.

Dose-response of Egr-1 protein expression in human lung fibroblast cells after exposure to CSE. (A) CSE induces Egr-1 expression in a dose-dependent manner in non-COPD smoker fibroblast cells (NL9). (B) CSE induces Egr-1 expression in a dose-dependent manner in normal fibroblast cells (MRC-5). Fibroblasts were incubated in the absence (CTL) or in the presence of CSE at varying concentrations for 1 h, then whole cell lysates were subjected to Western blot analysis. The same membrane was probed with an anti-β-actin antibody to assess equal loading of the gel.

Fig. 4.

Time course induction of Egr1 protein expression and Egr-1 DNA-binding activity in human lung fibroblast cells after exposure to CSE. (A) CSE induces Egr-1 expression in a time-dependent manner in NL9 cells. NL9 cells were incubated in the absence (CTL) or in the presence of CSE (50%) and whole-cell lysates were subjected to Western blot analysis. The same membrane was probed with an anti-β-actin antibody to assess equal loading of the gel. (B) Nuclear extracts were assayed for the activity of transcription factor Egr-1 by an ELISA-based TransFactor kit. Significant difference between 1 and 4 h CSE (50%) treatment and CTL is shown (^*, P < 0.05).

Fig. 5.

Gelatin zymography. Gelatin zymography was performed to identify matrix MMP-2 (A) and MMP-9 (B) in the condition media of mouse lung EGR1 null (-/-) and +/+ fibroblasts. Media were harvested after 1-4 days of culturing in the presence of cytomix. KO, knockout.