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Mouse Embryonic Lung Culture, A System to Evaluate the Molecular Mechanisms of Branching
Published on: July 1, 2010
MEK-1/2 inhibition reduces branching morphogenesis and causes mesenchymal cell apoptosis in fetal rat lungs
David E Kling1, Hans K Lorenzo, Alexander M Trbovich
1Pediatric Surgical Research Laboratories, Pediatric Surgical Services, and the Department of Surgery, Harvard Medical School, Boston, Massachusetts 02114, USA.
Abstract:
The roles of the mitogen-activated protein (MAP) kinases extracellular signal-regulated kinases-1 and -2 (ERK-1/2) in fetal lung development have not been extensively characterized. To determine if ERK-1/2 signaling plays a role in fetal lung branching morphogenesis, U-0126, an inhibitor of the upstream kinase MAP ERK kinase (MEK), was added to fetal lung explants in vitro. Morphometry as measured by branching, area, perimeter, and complexity were significantly reduced in U-0126-treated lungs. At the same time, U-0126 treatment reduced ERK-1/2, slightly increased p38 kinase, but did not change c-Jun NH(2)-terminal kinase activities, indicating that U-0126 specifically inhibited the ERK-1/2 enzymes. These changes were associated with increased apoptosis as measured by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling and immunofluorescent labeling of anti-active caspase-3 in the mesenchyme of explants after U-0126 treatment compared with the control. Mitosis characterized by immunolocalization of proliferating cell nuclear antigen was found predominantly in the epithelium and was reduced in U-0126-treated explants. Thus U-0126 causes specific inhibition of ERK-1/2 signaling, diminished branching morphogenesis, characterized by increased mesenchymal apoptosis, and decreased epithelial proliferation in fetal lung explants.
Insights
Mitogen-activated protein (MAP) kinases, specifically extracellular signal-regulated kinases-1 and -2 (ERK-1/2), are crucial for fetal lung development. Inhibiting ERK-1/2 signaling via MEK inhibitor U-0126 impaired lung branching morphogenesis.
Area of Science:
- Developmental Biology
- Cell Signaling
Background:
- The role of mitogen-activated protein (MAP) kinases, particularly extracellular signal-regulated kinases-1 and -2 (ERK-1/2), in fetal lung development remains incompletely understood.
- Understanding these signaling pathways is critical for elucidating mechanisms of normal lung formation.
Purpose of the Study:
- To investigate the involvement of ERK-1/2 signaling in fetal lung branching morphogenesis.
- To determine the effects of inhibiting ERK-1/2 on key developmental processes in fetal lung explants.
Main Methods:
- Fetal lung explants were treated in vitro with U-0126, a specific inhibitor of MAP ERK kinase (MEK), which upstream of ERK-1/2.
- Morphometric analysis (branching, area, perimeter, complexity) was performed.
- Apoptosis was assessed using terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) and anti-active caspase-3 immunofluorescence.
- Proliferation was evaluated by immunolocalization of proliferating cell nuclear antigen (PCNA).
- Kinase activities (ERK-1/2, p38, c-Jun NH(2)-terminal kinase) were measured to confirm inhibitor specificity.
Main Results:
- U-0126 treatment significantly reduced fetal lung branching, area, perimeter, and complexity.
- Inhibition of ERK-1/2 signaling was confirmed, with minimal effects on p38 and c-Jun NH(2)-terminal kinase activities.
- Mesenchymal apoptosis significantly increased in U-0126-treated explants.
- Epithelial proliferation, indicated by PCNA staining, was reduced following U-0126 treatment.
Conclusions:
- Specific inhibition of ERK-1/2 signaling by U-0126 disrupts fetal lung branching morphogenesis.
- The observed defects are associated with increased mesenchymal apoptosis and decreased epithelial proliferation.
- ERK-1/2 signaling is essential for normal fetal lung development, regulating both cell proliferation and survival.
