Gene expression analysis reveals matrilysin as a key regulator of pulmonary fibrosis in mice and humans

Abstract

Pulmonary fibrosis is a progressive and largely untreatable group of disorders that affects up to 100,000 people on any given day in the United States. To elucidate the molecular mechanisms that lead to end-stage human pulmonary fibrosis we analyzed samples from patients with histologically proven pulmonary fibrosis (usual interstitial pneumonia) by using oligonucleotide microarrays. Gene expression patterns clearly distinguished normal from fibrotic lungs. Many of the genes that were significantly increased in fibrotic lungs encoded proteins associated with extracellular matrix formation and degradation and proteins expressed in smooth muscle. Using a combined set of scoring systems we determined that matrilysin (matrix metalloproteinase 7), a metalloprotease not previously associated with pulmonary fibrosis, was the most informative increased gene in our data set. Immunohistochemisry demonstrated increased expression of matrilysin protein in fibrotic lungs. Furthermore, matrilysin knockout mice were dramatically protected from pulmonary fibrosis in response to intratracheal bleomycin. Our results identify matrilysin as a mediator of pulmonary fibrosis and a potential therapeutic target. They also illustrate the power of global gene expression analysis of human tissue samples to identify molecular pathways involved in clinical disease.

Figure 1

Gene expression infogram for all 7,129 genes (A) and for the 164 most informative genes (TNoM = 0) (B). To eliminate outlier effect, genes were normalized to a range of (0,1), meaning that the maximum value for every gene was set to be 1, the minimum value to be 0, and the rest of the values were linearly fitted to this range. Yellow is maximal expression and blue is minimal.

Figure 2

MMP-7 gene and protein expression levels are significantly increased in fibrotic lungs. The Gaussian distribution for MMP-7 reveals minimal overlap (score of 0.00625) between normal lungs (green line, A) and fibrotic lungs (red line, A). All actual expression levels of MMP-7 in fibrotic lungs (red X marks, A) were higher than in normal lungs (green X marks, A) and no samples were misclassified (TNoM and Info = 0). The y axis in the Gaussian figure (A) is the estimated density of the expression level. This density is computed based on the normality assumption of expression level (for each class). MMP-7 staining verified the gene expression predictions and demonstrated both cell-associated and matrix MMP-7 in distal airspaces (B) and proximal airway (C) from fibrotic lungs. No MMP-7 immunoreactivity was seen in normal lungs (D). (Magnification: B–D, ×40).

Figure 3

MMP7^−/− mice are protected against bleomycin-induced pulmonary fibrosis. Increase in lung hydroxyproline content in lungs of wild-type (MMP-7^+/+) mice 14 days after bleomycin injection is significantly higher than in MMP-7^−/− mice on both C57BL6 and 129SvJ background. Increase values were calculated by subtracting mean hydroxyproline content (μg hydroxyproline/lung) of lungs of saline-injected animals from the hydroxyproline content of lungs 14 days after bleomycin (A). Comparison of semiquantitative morphometric analysis of lung histology 14 days after bleomycin (B) and representative histology of MMP-7^+/+ (C) and MMP-7^−/− mice (D). Data (means ± SE) are representative of at least two comparable experiments with six mice per group; *, P < 0.01 relative to bleomycin-treated MMP^+/+ mice. (Magnification: B and C, ×10).