Matrix metalloproteinase-7 coordinates airway epithelial injury response and differentiation of ciliated cells

Abstract

Matrix metalloproteinase-7 (MMP7) expression is quickly up-regulated after injury, and functions to regulate wound repair and various mucosal immune processes. We evaluated the global transcriptional response of airway epithelial cells from wild-type and Mmp7-null mice cultured at an air-liquid interface. The analysis of differentially expressed genes between genotypes after injury revealed an enrichment of functional categories associated with inflammation, cilia, and differentiation. Because these analyses suggested that MMP7 regulated ciliated cell formation, we evaluated the recovery of the airway epithelium in wild-type and Mmp7-null mice in vivo after naphthalene injury, which revealed augmented ciliated cell formation in the absence of MMP7. Moreover, in vitro studies evaluating cell differentiation in air-liquid interface cultures also showed faster ciliated cell production under Mmp7-null conditions compared with wild-type conditions. These studies identified a new role for MMP7 in attenuating ciliated cell differentiation during wound repair.

Figure 1.

(A) Correspondence analysis of wild-type (WT) and Mmp7-null (Mmp7^−/−) airway epithelial transcriptome at baseline and in response to scratch injury at 24 hours. This analysis showed that the variability in gene expression was relatively small among biological replicates under each experimental condition, and that global changes occurred in gene expression, attributable to genotype (WT or Mmp7^−/−) at baseline and after injury. (B) Differentially expressed genes between Mmp7^−/−and WT cells at baseline (before injury) were functionally grouped according to their gene ontology (GO) annotation. The tree-like structure of GO is depicted for the statistically overrepresented functional categories (false discovery rate, < 0.01). Modules are colored according to whether they are comprised of up-regulated genes under Mmp7^−/− (magenta) or WT (cyan) conditions. MHC, major histocompatibility complex; MMP7, matrix metalloproteinase–7.

Figure 2.

Baseline-adjusted, genotype-specific differential gene expression in response to injury. Several expression patterns are demarcated by the yellow lines: (1) genes that were primarily up-regulated in WT cells after injury, (2) genes that were differentially expressed at baseline between Mmp7^−/− and WT cells, but lost this difference after injury, and (3) genes that were primarily up-regulated in Mmp7^−/− cells in response to injury. Group 3 genes mapped to distinct functional groups, including processes involved in ciliary structure and function, as shown at lower right.

Figure 3.

Altered recovery of ciliated cells in airway epithelia of Mmp7-null mice after naphthalene-induced lung injury. (A) Lung sections were immunostained for acetylated tubulin (green; Clone 6-11B-1; Sigma-Aldrich, St. Louis, MO) to mark ciliated cells, club cell–specific protein (CCSP) (red; Biovendor, Candler, NC) to identify club cells, and 4′,6-diamidino-2-phenylindole (DAPI) (blue) as a nuclear stain. Epifluorescence images were captured using an Olympus BX-51 microscope (Olympus America, Center Valley, PA) with a ×20/0.70 air objective. Scale bar = 200 μm. Acetylated tubulin⁺ cells (B) and CCSP⁺ cells (C) were counted per millimeter of airway length. Only airways between 200 and 350 μm in diameter were evaluated. A minimum of five airways was randomly evaluated in various lung lobes for each sample. Four mice per genotype were evaluated at each time point. *P < 0.05. (D) Scanning electron micrographs of naphthalene-injured lungs. Dome-shaped club cells (arrowhead) are seen interspersed with ciliated cells (arrows) under uninjured (corn oil) conditions. Scale bar = 20 μm.

Figure 4.

Ciliated cells appear more rapidly in airway epithelial cells differentiated at an air–liquid interface (ALI). (A) ALI cultures were immunostained for acetylated tubulin (green; Sigma-Aldrich) to mark ciliated cells, CCSP (red; Biovendor) to identify club cells, and DAPI (blue) as a nuclear stain. Epifluorescence images were captured using an Olympus BX-51 fluorescence/DIC microscope with a Plan Apo ×60/1.4 oil objective. Scale bar = 10 μm. Ciliated cells (B) and club cells (C) were quantified under both conditions. Four independent cultures were evaluated per genotype at each time point. *P < 0.05.