The p66Shc adapter protein regulates the morphogenesis and epithelial maturation of fetal mouse lungs

Abstract

Many signaling pathways are mediated by Shc adapter proteins that, in turn, are expressed as three isoforms with distinct functions. The p66Shc isoform antagonizes proliferation, regulates oxidative stress, and mediates apoptosis. It is highly expressed in the canalicular but not the later stages of mouse lung development, and its expression persists in bronchopulmonary dysplasia, a chronic disease associated with premature birth. These observations suggest that p66Shc has a developmental function. However, constitutive p66Shc deletion yields no morphological phenotype, and the structure of the Shc gene precludes its inducible deletion. To elucidate its function in lung development, we transfected p66Shc or nonsilencing small-interfering RNA (siRNA) into the epithelia of embryonic day 11 mouse lungs that were then cultured for 3 days and analyzed morphometrically. To assess cellular proliferation and epithelial differentiation, lung explants were immunostained and immunoblotted for p66Shc, proliferating cell nuclear antigen (PCNA), the proximal airway differentiation antigens Clara cell 10-kDa protein (CC10) and thyroid transcription factor (TTF)-1, and the alveolar surfactant proteins (SP)-A, -B, and -C. Explants transfected with nonsilencing siRNA demonstrated specific epithelial uptake and normal morphological development relative to uninjected controls. In contrast, transfection with p66Shc siRNA significantly increased lumenal cross-sectional areas, decreased branching, and increased epithelial proliferation ( P < 0.05 for all). Relative to controls, the expression of SP-B, SP-C, CC10, and TTF-1 was decreased by p66Shc knockdown. SP-A was not expressed in either control or treated lungs. These data suggest that p66Shc attenuates epithelial proliferation while promoting both distal and proximal epithelial maturation.