Individual matrix metalloproteinases control distinct transcriptional responses in airway epithelial cells infected with Pseudomonas aeruginosa

Abstract

Airway epithelium is the initial point of host-pathogen interaction in Pseudomonas aeruginosa infection, an important pathogen in cystic fibrosis and nosocomial pneumonia. We used global gene expression analysis to determine airway epithelial transcriptional responses dependent on matrilysin (matrix metalloproteinase 7 [MMP-7]) and stromelysin-2 (MMP-10), two MMPs induced by acute P. aeruginosa pulmonary infection. Extraction of differential gene expression (EDGE) analysis of gene expression changes in P. aeruginosa-infected organotypic tracheal epithelial cell cultures from wild-type, Mmp7-/-, and Mmp10-/- mice identified 2,091 matrilysin-dependent and 1,628 stromelysin-2-dependent genes that were differentially expressed. Key node network analysis showed that these MMPs controlled distinct gene expression programs involved in proliferation, cell death, immune responses, and signal transduction, among other host defense processes. Our results demonstrate discrete roles for these MMPs in regulating epithelial responses to Pseudomonas infection and show that a global genomics strategy can be used to assess MMP function.

FIG. 1.

MMP-7 and MMP-10 mRNA expression is induced in lung and ALI epithelium after Pseudomonas aeruginosa infection. Mice and mouse tracheal epithelial cells cultured at an ALI were infected with P. aeruginosa, and total RNA was isolated at 0 to 24 h after infection. qRT-PCR was performed for Mmp7 and Mmp10, and expression levels are shown as change (fold) compared to expression level at time zero. Lung data are pooled from three animals per time point and run in duplicate. ALI data are pooled from three experiments with two to four samples per time point run in duplicate. Values are means ± standard error. ALI Mmp7 and Mmp10 changes are significant over the time course. †, P < 0.0001 by analysis of variance; *, P < 0.01 by Dunnett's multiple-comparison posttest for individual time points as compared to t = 0 h.

FIG. 2.

Differential response between Mmp7^−/− and Mmp10^−/− mice to bacterial infection. Mouse lung tissues were isolated at 4 and 24 h after infection with P. aeruginosa and formalin fixed, and sections were stained with hematoxylin and eosin. Bars, 200 μm. Samples are representative of 8 to 10 mice of each genotype at each time point.

FIG. 3.

PCA of microarrays. PCA was performed in the R environment. Each microarray chip is represented individually by shape and color: circles, wild type (WT); triangles, Mmp7^−/−; squares, Mmp10^−/−; blue, 0 h; green, 1 h; red, 24 h.

FIG. 4.

Matrilysin and stromelysin-2 control different transcriptional responses. The Venn diagram represents significantly changed genes identified by EDGE analysis (false discovery rate, <0.05), from a twofold-change filtered data set, of wild-type (WT) versus specific MMP gene-deleted mice. Unique probe sets are presented in row-normalized heat maps. All numbers are totals of unique ENTREZ gene IDs represented by the Affymetrix probe sets.

FIG. 5.

Key nodes reveal differences in GO biological processes influenced by matrilysin and stromelysin-2. The results of Ingenuity Pathway Analysis of MMP-dependent data sets were filtered to identify genes that had 10 or more network interactions, and these interactions were drawn based on the connectivity matrix of its members using Pajek's visualization software. Unique matrilysin key nodes are cyan, unique stromelysin-2 key nodes are magenta, and key nodes in both data sets are yellow. The table indicates the number of genes in each biological process from key node and all significant gene data sets identified by DAVID analysis as significantly overrepresented GO biological processes (Benjamini corrected P value of <0.001). NS, not significant.

FIG. 6.

Key nodes reflect changes in entire MMP-dependent data sets. Shown are heat maps for key nodes and all significantly changing genes in the matrilysin-dependent cell proliferation module and stromelysin-2-dependent immune response GO biological process modules.

FIG. 7.

Matrilysin and stromelysin-2 differentially control apoptotic transcriptional response. Shown are the results of Ingenuity Pathway Analysis of significantly changed genes from the MMP-7 and MMP-10 data sets in the apoptosis pathway. Unique matrilysin-dependent genes are cyan, unique stromelysin-2-dependent genes are magenta, and genes present in both data sets are yellow. Gray lines indicate network connections with the connections for the Bcl2 key node indicated in black to demonstrate distinct Bcl2 interactions between data sets. EC, extracellular; MEM, membrane; CYT, cytoplasmic; NUC, nuclear.