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. 2009 Oct;136(19):3347-56.
doi: 10.1242/dev.032979. Epub 2009 Aug 26.

Mig-6 is required for appropriate lung development and to ensure normal adult lung homeostasis

Nili Jin  1 , Sung-Nam Cho, M Gabriela Raso, Ignacio Wistuba, Yvonne Smith, Yanan Yang, Jonathan M Kurie, Rudolph Yen, Christopher M Evans, Thomas Ludwig, Jae-Wook Jeong, Francesco J DeMayo
Affiliations

Mig-6 is required for appropriate lung development and to ensure normal adult lung homeostasis

Nili Jin et al. Development. 2009 Oct.

Abstract

Mitogen-inducible gene 6 [Mig-6; Errfi1 (ErbB receptor feedback inhibitor 1); RALT (receptor-associated late transducer); gene 33] is a ubiquitously expressed adaptor protein containing CRIB, SH3 and 14-3-3 interacting domains and has been shown to negatively regulate EGF signaling. Ablation of Mig-6 results in a partial lethal phenotype in which surviving mice acquire degenerative joint diseases and tumors in multiple organs. We have determined that the early lethality in Mig-6(-/-) mice occurs in the perinatal period, with mice displaying abnormal lung development. Histological examination of Mig-6(-/-) lungs (E15.5-P3) revealed reduced septation, airway over-branching, alveolar type II cell hyperplasia, and disturbed vascular formation. In neonatal Mig-6(-/-) lungs, cell proliferation increased in the airway epithelium but apoptosis increased in the blood vessels. Adult Mig-6(-/-) mice developed features of chronic obstructive pulmonary disease (COPD); however, when Mig-6 was inducibly ablated in adult mice (Mig-6(d/d)), the lungs were normal. Knockdown of MIG-6 in H441 human bronchiolar epithelial cells increased phospho-EGFR and phospho-AKT levels as well as cell proliferation, whereas knockdown of MIG-6 in human lung microvascular endothelial (HMVEC-L) cells promoted their apoptosis. These results demonstrate that Mig-6 is required for prenatal and perinatal lung development, in part through the regulation of EGF signaling, as well as for maintaining proper pulmonary vascularization.

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Figures

Fig. 1.
Fig. 1.
Loss of Mig-6 expression results in high neonatal mortality. (A) Mig-6+/+ (left) and Mig-6-/- (right) newborn mice. Arrow indicates stomach (left, with milk). (B) Relative birth weight of Mig-6+/+, Mig-6+/- and Mig-6-/- pups (*P<0.05). (C) The floating lung assay for Mig-6+/+ and Mig-6-/- newborns indicating that Mig-6-/- lungs inflate well with air at birth.
Fig. 2.
Fig. 2.
Differential expression of Mig-6 during lung development. (A) Quantitative real-time RT-PCR of Mig-6 mRNA levels (normalized to 18S rRNA) in wild-type lungs from E13.5 through to P10 and in adult (3 months old, AD). The first lane shows the absence of Mig-6 mRNA in adult Mig-6-/- lungs. *P<0.05 versus E18.5; #P<0.05 versus Mig-6-/-. (B-J) Dual immunofluorescence staining of MIG-6 and CCSP in E18.5 (B-D), P3.5 (E-G) and 2-month-old (H-J) mouse lungs. Clara cells are indicated by arrows. (K-S) Dual immunofluorescence staining of MIG-6 and SP-C in E18.5 (K-M), P3.5 (N-P) and 2-month-old (Q-S) mouse lungs. Type II cells are indicated by arrows. Scale bars: 50 μm in B-J; 100 μm in K-S.
Fig. 3.
Fig. 3.
Perinatal Mig-6-/- mice exhibit altered lung architecture. (A-H) H&E-stained lung sections from E18.5 (A-D) and P3 (E-H) Mig-6+/+ and Mig-6-/- mice. The boxed areas in A,B are shown at high magnification in C,D, respectively. Scale bars: 100 μm in A-D; 500 μm in E,F; 200 μm in G,H. Primitive alveoli are indicated by arrows in A-D; alveolar septa are indicated by arrows in G,H. (I) Quantification of primitive alveoli in terminal airways in E18.5 Mig-6+/+ and Mig-6-/- mouse lungs. *P<0.05. (J) Quantification of secondary septa in alveolar sacs in Mig-6+/+ and Mig-6-/- mouse lungs. ***P<0.001.
Fig. 4.
Fig. 4.
Mig-6-/- neonates exhibit over-branching and airway epithelial and alveolar type II cell hyperplasia. (A-D) CCSP-immunostaining of P3 Mig-6+/+ proximal (A) and distal (C) airways, and of Mig-6-/- proximal (B) and distal (D) airway epithelium. (E,F) Pro-SP-C immunostaining of P3 Mig-6+/+ (E) and Mig-6-/- (F) lungs. Boxed areas are shown at high magnification in the insets. Scale bars: 100 μm.
Fig. 5.
Fig. 5.
Abnormal vascular formation in prenatal and neonatal Mig-6-/- mouse lungs. (A,B) Whole lungs from E15.5 Mig-6+/+ (A) and Mig-6-/- (B) mice. Arrows indicate blood vessels. (C,D) PECAM1 immunostaining (red) of P3 Mig-6+/+ (C) and Mig-6-/- (D) lungs. Nuclei are stained with DAPI (blue). (E,F) α-SMA immunostaining of P3 Mig-6+/+ (E) and Mig-6-/- (F) lungs. Blood vessels are indicated by arrows. Scale bars: 2 mm in A,B; 50 μm in C-F.
Fig. 6.
Fig. 6.
Abnormal cell proliferation and apoptosis in neonatal Mig-6-/- mouse lungs. (A-D) Phospho-histone H3 immunostaining of P3 Mig-6+/+ (A,C) and Mig-6-/- (B,D) mouse lungs. The boxed areas in A,B are shown at high magnification in C,D, respectively. Proliferating cells are indicated by arrows in A-D. (E) Percentage of proliferating cells in different lung compartments. *P<0.05. (F-K) Cleaved caspase 3 immunostaining of P3 Mig-6+/+ (F,H,J) and Mig-6-/- (G,I,K) mouse lungs in the upper airways (F,G), blood vessels (H,I) and alveoli (J,K; arrows indicate alveolar septa). Scale bars: 50 μm.
Fig. 7.
Fig. 7.
Altered pulmonary morphology in adult Mig-6-/- mice. (A) Alveolar morphology of 3-month-old Mig-6+/+ and Mig-6-/- lungs by H&E staining. (B) Mean alveolar chord length (μm) of 3-month-old Mig-6+/+ (n=13) and Mig-6-/- (n=12) lungs. *P<0.05. (C,D) H&E-stained lungs from 1-month-old Mig-6+/+ (C) and Mig-6-/- (D) lungs. (E-H) Masson's trichrome staining of proximal airways (E,F) and alveoli (G,H) of 3-month-old Mig-6+/+ (E,G) and Mig-6-/- (F,H) lungs. (I,J) MAC3 (LAMP2) immunostaining of 3-month-old Mig-6+/+ (I) and Mig-6-/- (J) lungs. Arrow in J, crystal secreted by macrophage (K,L) PAS staining of 3-month-old Mig-6+/+ (K) and Mig-6-/- (L) lungs. Br, bronchiol; Vs, blood vessels; Is, interstitium; Pl, plural membrane. Scale bars: 100 μm in A,E-L; 50 μm in C,D.
Fig. 8.
Fig. 8.
Normal pulnomary morphology in adult Mig-6d/d mice. (A,B) Immunofluorescent staining of MIG-6 (green) in Mig-6f/f and Mig-6d/d lungs. Nuclei were stained with DAPI (blue). (C,D) H&E staining of Mig-6f/f and Mig-6d/d lungs. (E,F) PAS staining of Mig-6f/f and Mig-6d/d lungs. (G) Mig-6 mRNA levels in Mig-6+/+, Mig-6-/-, Mig-6f/f and Mig-6d/d lungs as determined by quantitative real-time PCR. **P<0.01 and ***P<0.001 versus Mig-6+/+ lungs. (H) Mean alveolar chord length (μm) of Mig-6f/f and Mig-6d/d lungs. Scale bars: 50 μm.
Fig. 9.
Fig. 9.
Increased EGF signaling but decreased angiogenetic gene expression in neonatal Mig-6-/- mouse lungs. (A-D) Immunofluorescent staining of EGFR (A,B, green) and phospho-EGFR (C,D, green) in P3 Mig-6+/+ and Mig-6-/- lungs. Nuclei were stained with DAPI (blue). (E-L) Immunohistochemistry of AKT (E,F), phospho-AKT (G,H), mTOR (I,J) and phospho-mTOR (K,L) in P3 Mig-6+/+ and Mig-6-/- lungs. (M) Western blot of AKT, phospho-AKT, mTOR, phospho-mTOR and α-tubulin in P3 Mig-6+/+ and Mig-6-/- lungs. Asterisk indicates mTOR bands. (N) The mRNA expression levels of EGF, AREG, HbEGF, VEGF-A, VEGF-C, VEGF-D, angiopoietin 1 (ANG1) and angiopoietin 2 (ANG2) in P3 Mig-6-/- relative to Mig-6+/+ lungs as determined by quantitative real-time PCR. *P<0.05, **P<0.01 and ***P<0.001 versus Mig-6+/+ littermates. Scale bars: 50 μm.
Fig. 10.
Fig. 10.
Knockdown of Mig-6 expression leads to increased epithelial cell proliferation and endothelial cell apoptosis. (A) Western blot of MIG-6, EGFR, phospho-EGFR, AKT, phospho-AKT and α-tubulin in human H441 cells with no siRNA (1), control siRNA (2) and MIG-6 siRNA (3). (B-D) MTT assay in H441 cells (B) and HMVEC-L cells (D), and relative MIG-6 mRNA levels in HMVEC-L cells as determined by quantitative real-time PCR (C) with no siRNA, control siRNA and MIG-6 siRNA. ***P<0.001 versus control siRNA. (E) Immunofluorescent staining of cleaved caspase 3 on HMVEC-L cells with no siRNA, control siRNA and MIG-6 siRNA. Scale bars: 50 μm.
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