Fig. 7.

MMP14-MMP2 proteolytic cascade regulates AMYF differentiation during alveolar formation. (A) Representative FACS dot plots of lung cell suspensions from WT C57/BL6 mice treated with DMSO (Upper) or MMPi (Lower), an inhibitor of MMP2 and MMP14. The x axis and y axis represent Cy3-α-SMA and APC-PDGFRα fluorescent intensity, respectively. The colored boxes show AMYF fractions. (B) Percentages of each quadrant of three independent mice per treatment are summarized. The fractions of AMYF precursor, AMYF, and SMC+RMYF contain PDGFRα⁺/α-SMA^– cells, PDGFRα⁺/α-SMA⁺ cells, and PDGFRα^–/α-SMA⁺ cells, respectively. (C) Sections were obtained from the lungs of P14 mice continuously treated with DMSO (Upper) or MMPi (Lower) from P1 to P14 and then stained with H&E. (Scale bar, 100 μm.) (D and E) The lung volumes (D) and alveolar numbers (E) per total lungs in P14 mice with (gray bars) or without (pink bars) MMPi were examined as described in Materials and Methods. The values represent means ± SD. In B, D, and E the analyses were performed with three to five independent mice per genotype. **P < 0.01, *P < 0.05; NS, P > 0.05. (F) A model illustrating the non–cell-autonomous role of DA-Raf in AMYF differentiation during alveolar formation. Ablation of DA-Raf in mice leads to augmented phosphorylation of MEK1/2 in AEC2s and non–cell-autonomously prevents AMYF differentiation. TIMP4 produced by DA-Raf–deficient AEC2s leads to inactivation of MMP14–MMP2 signaling pathway, which is required for AMYF differentiation and the secondary septation in developing lungs.