Signaling through the EGF receptor controls lung morphogenesis in part by regulating MT1-MMP-mediated activation of gelatinase A/MMP2

Abstract

Epithelial-mesenchymal interactions during lung development require extracellular signaling factors that facilitate branching morphogenesis. We show here that matrix metalloproteinases (MMPs) originating in the mesenchyme are necessary for epithelial branching and alveolization. We found that the delayed lung maturation characterized by abnormal branching and poor alveolization seen in mice deficient in epidermal growth factor receptor (Egfr(-/-)) is accompanied by aberrant expression of MMPs. By in situ zymography, the lungs from newborn Egfr(-/-) mice had low gelatinolytic activity compared with wildtype. Inhibition of MMPs in developing lungs in vivo or in vitro severely retarded morphogenesis. Egfr(-/-) mice had low expression of MT1-MMP/MMP14, which is a potent activator of gelatinase A/MMP2, in their lungs. Egf ligand increased MT1-MMP mRNA by tenfold in lung fibroblasts from wild type, but not from Egfr(-/-) mice. Extracts from lungs of Egfr(-/-) mice showed a tenfold reduction in active MMP-2, but only a slight decrease in proMMP-2 by zymography. At birth, MMP-2(-/-) mice had a lung phenotype characterized by abnormal lung alveolization which phenocopied that of Egfr(-/-) mice, albeit somewhat less severe. We conclude that proteolysis mediates epithelial/mesenchymal interactions during lung morphogenesis. From the phenotypes of the Egfr(-/-) mice, we identify MT1-MMP as a major downstream target of Egfr signaling in lung in vivo and in vitro. MT1-MMP is, in turn, necessary for activation of MMP-2, a mesenchymal enzyme that is required for normal lung morphogenesis.

Fig. 1

Analysis of activity of protease in lungs of Egfr^−/− mice by in situ zymography. Cryostat sections of lungs from newborn wildtype (a,c,d) and Egfr^−/− (b) after 3 hours of incubation with DQ gelatin. (a) Note gelatinolytic activity throughout the parenchyma and around the airway of wild-type lung (arrowhead), (d) which was inhibited in the presence of 1,10-phenanthroline. (c) Corresponding phase-contrast image from a.

Fig. 2

Inhibition of MMP-2 activation in Egfr^−/− mice. Whole lung homogenates from newborn wildtype and Egfr^−/− mice were analyzed by gelatin zymography. Quantification of zymograms (n=4) is shown in panel a, and the negative images of a representative zymogram in panel b. *P≤0.05 (Student’s t-test).

Fig. 3

MMP function is required for normal branching morphogenesis. Timed-pregnant mice were treated with 100 mg/kg of GM6001 (b,d) or vehicle control (PBS) (a,c) starting at E10.5 for three days. (a,b) Equivalent sections from left lung of embryos treated by sham inhibition (a) or with MMP inhibitor (b), then stained with H and E. Dark boxes indicate the region of epithelial mesenchymal contact presented at higher magnification in c and d. The arrow in c indicates a newly forming branch. Arrowheads in b and d point to loose or poorly organized mesenchyme. Bars, 200 μm (a,b); 50 μm (c,d). Li, liver; Lu, lung; H, heart.

Fig. 4

(A) MMPs regulate lung bud branching in lung organ cultures. Numbers above the panels represent the number of days in culture. E11.5 lung buds were isolated from timed-pregnancies and placed in serum-free media in the presence or absence of MMP inhibitor (GM6001). Following four days growth in vitro, lung buds show retardation of branching in the presence of MMP inhibitor as compared to control. (B) Analysis of branching inhibition. Quantification by counting the number of newly formed secondary and tertiary branches in E10.5 (n=4) and E11.5 (n=3) whole lungs following four days of culture. *P≤0.05 (Student’s t-test).

Fig. 5

Constitutive expression of MMP-2, MT1-MMP and TIMP-2 mRNA during lung development. Lung sections from normal fetuses were taken at E12.5. For ISH analysis the following antisense probes were used: (a) MMP-2, (b) MT1-MMP, (c) TIMP-2, (d) MMP-9, (e) MMP-3, (f) TIMP-1. Note the presence of message in (a,b,c) and its absence in (d,e,f). Arrows point to the presence and arrowheads to the absence of message. Bar, 200 μm (a–f).

Fig. 6

Localization of MMP-2 mRNA in the mesenchyme. Embryos from normal fetuses were taken at E18.5. For ISH analysis the following antisense probes were used: (a) TIMP-2, (c) MT1-MMP, (e) MMP-2. (b,d,f) H and E counterstain of darkfield images are shown. Note localization of TIMP-2 and MT1-MMP to the epithelia and mesenchyme (arrows), and absence of MMP-2 in the epithelia (arrowheads). Inset in each panel is 60× magnification of bronchial region from the corresponding image. aAo, ascending aorta; Ao, aorta; Br, bronchus; L, lung; Pa, pulmonary artery. Bar, 200 μm (a–f).

Fig. 7

Decreased expression of MT1-MMP, but not MMP-2, mRNA in Egfr^−/− mice. Lung from 18.5 dpc (E18.5) wildtype (a,b) or Egfr^−/−(c,d) used to detect MMP-2 (a,c) and MT1-MMP (b,d) mRNA by in situ hybridization. Note the abundant presence of MMP-2 and MT1-MMP mRNA in a,b,c, and absence of MT1-MMP in d. Bar, 100 μm (a–d).

Fig. 8

Quantification of MT1-MMP and MMP-2 mRNA expression by RPA. (a) There was little to no detectable protected MT1-MMP mRNA in the 1- or 9-day-old Egfr^−/− lungs, while protected mRNA fragments were detected in the control littermate. (b) By contrast, MMP-2 mRNA was protected from degradation using the MMP-2 RNA probe with RNA from wild-type and Egfr^−/− mice. (c) Egf regulates MT1-MMP expression in lung fibroblasts. Fibroblasts were isolated from E12 lungs produced by mating of Egfr^+/− mice. Egfr^−/− and wild-type lung fibroblasts were deprived of serum factors overnight, followed by stimulation with 100 ng of murine Egf or Fgf-2 for 16 hours. Total RNA was then isolated and used for quantification of MT1-MMP mRNA by RPA. Major protected bands were quantified and the amount of increase over baseline are as shown.

Fig. 9

Lungs from newborn MMP-2^−/− mice phenocopy branching defects in Egfr^−/− mice. HandE stained sections from lungs of newborn (a) MMP-2^−/−, (c) Egfr^−/−, (b,d) their wild type littermate controls, respectively. Note that the distal airway phenotype of MMP-2^−/− mice resembles that of the Egfr^−/− mice. Bar, 200 μm (a–d).