Lee MK, Smith SM, Banerjee MM, Li C, Minoo P, Volpe MV, Nielsen HC. The p66 adapter protein regulates the morphogenesis and epithelial maturation of fetal mouse lungs. Am J Physiol Lung Cell Mol Physiol 306: L316–L325, 2014. First published December 27, 2013; doi:10.1152/ajplung.00062.2013.—Many signaling pathways are mediated by Shc adapter proteins that, in turn, are expressed as three isoforms with distinct functions. The p66 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 p66 has a developmental function. However, constitutive p66 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 p66 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 p66, 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 p66 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 p66 knockdown. SP-A was not expressed in either control or treated lungs. These data suggest that p66 attenuates epithelial proliferation while promoting both distal and proximal epithelial maturation.
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