Reduced viability of mice with lung epithelial-specific knockout of glucocorticoid receptor

Abstract

Glucocorticoid (GC)-responsive epithelial-mesenchymal interactions regulate lung development. The GC receptor (GR) mediates GC signaling. Mice lacking GR in all tissues die at birth of respiratory failure. To determine the specific need for epithelial GR in lung development, we bred triple transgenic mice that carry SPC/rtTA, tet-O-Cre, and floxed, but not wild-type, GR genes. When exposed to doxycycline in utero, triple transgenic (GRepi⁻) mice exhibit a Cre-mediated recombination event that inactivates the floxed GR gene in airway epithelial cells. Immunofluorescence confirmed the elimination of GR in Cre-positive airway epithelial cells of late gestation GRepi⁻ mice. Embryonic Day 18.5 pups had a relatively immature appearance with increased lung cellularity and increased pools of glycogen in the epithelium. Postnatal Day 0.5 pups had decreased viability. We used quantitative RT-PCR to demonstrate that specific elimination of epithelial immunoreactive GR in GRepi⁻ mice is associated with reduced mRNA expression for surfactant proteins (SPs) A, B, C, and D; β- and γ-ENaC; T1α; the 10-kD Clara cell protein (CCSP); and aquaporin 5 (AQP5). Western blots confirmed reduced levels of AQP5 protein. No reduction in the levels of the GR transport protein importin (IPO)-13 was observed. Our findings demonstrate a requirement for lung epithelial cell GR in normal lung development. We speculate that impaired epithelial differentiation, leading to decreased SPs, transepithelial Na, and liquid absorption at birth, may contribute to the reduced survival of newborn mice with suppressed lung epithelial GR.

Figure 1.

Epithelial-specific suppression of glucocorticoid receptor (GR) in lungs of triple transgenic (GRepi⁻) mice. Immunofluorescence for Cre (A and D; green), GR (B and E; red) and both (C and F). All lungs from E18.5 fetuses of dox-treated females. (A–C) show tissue sections of lungs from fetuses that express wild-type (WT) GR. (D–F) show tissue sections of lungs from fetuses that express floxed, but not WT, GR. All express Cre exclusively in alveolar epithelial cells (AECs). Cre and GR colocalize (yellow) in C (control), but there is no colocalization in F (GRepi⁻), reflecting widespread suppression of GR expression in AECs.

Figure 2.

GR mRNA levels are reduced in lungs of Embryonic Day (E)18.5 GRepi⁻ fetuses. Values normalized relative to 18S rRNA. Mean ± SEM; n = 10 in each group. *P < 0.05, t test.

Figure 3.

Epithelial GR suppression is associated with a less mature histologic appearance than controls. Lung cellularity and septal thickness were increased in GRepi⁻ mice (B) versus Control (A). Tissue to airspace ratio (a measure of tissue cellularity inversely related to histological maturity) is higher in GRepi⁻ than in Control mice (C). *P < 0.05, n = 4.

Figure 4.

Surfactant protein mRNA levels are reduced and metabolism of glycogen stores is delayed in GRepi⁻ mice. Lungs of E18.5 GRepi⁻ mice, compared with littermate controls, have (A) reduced mRNA levels of surfactant proteins (SPs)-A, -B, -C, and -D, but not (B) ABCA3; and (C) increased histochemical staining for Periodic Acid Schiff (glycogen, dark purple). All mRNA levels normalized to18S rRNA levels, in arbitrary units. Mean ± SEM, n = 10 in each group. *P < 0.05, Student's t test or Mann-Whitney U-test as appropriate.

Figure 5.

Electrolyte clearance: epithelial Na channel (ENaC) subunits are reduced in GRepi⁻ mice. Lungs of E18.5 GRepi⁻ mice, compared with controls, had (A) reduced levels of mRNA encoding ENaC β and γ, but not α, subunits; (B) no differences in mRNA for Na⁺, K+ ATPase α and β subunits. All mRNA levels normalized to 18S rRNA levels, in arbitrary units. Mean ± SEM, n = 10 in each group. *P < 0.05, Student's t test or Mann-Whitney U-test as appropriate.

Figure 6.

Epithelial cell markers are reduced in GRepi⁻ mice. Lungs of E18.5 GRepi⁻ mice had reduced mRNA levels for the epithelial type I cell markers (A) T1 α and (B) the water channel aquaporin (AQP) 5. (C) AQP5 protein by Western blot was also reduced. All mRNA levels normalized to18S rRNA levels, in arbitrary units. Mean ± SEM, n = 10 in each group. *P < 0.05, Student's t test or Mann-Whitney U-test as appropriate.

Figure 7.

The levels of products of three GC-responsive genes that are not epithelial-specific are unaltered in whole lungs of GRepi⁻ mice. (A) Levels of protein (IPO13) by Western blot or mRNA ([B] late gestation lung 1 (Lgl1) and [C] midkine) were not altered in newborn GRepi⁻ mice whole lung. P not significant, n = 9.