Concomitant deletion of HRAS and NRAS leads to pulmonary immaturity, respiratory failure and neonatal death in mice.

We reported previously that adult (HRAS^-/-; NRAS^-/-) double knockout (DKO) mice showed no obvious external phenotype although lower-than-expected numbers of weaned DKO animals were consistently tallied after crossing NRAS-KO and HRAS-KO mice kept on mixed genetic backgrounds. Using mouse strains kept on pure C57Bl/6 background, here we performed an extensive analysis of the offspring from crosses between HRAS-KO and NRAS-KO mice and uncovered the occurrence of very high rates of perinatal mortality of the resulting DKO littermates due to respiratory failure during the first postnatal 24-48 h. The lungs of newborn DKO mice showed normal organ structure and branching but displayed marked defects of maturation including much-reduced alveolar space with thick separating septa and significant alterations of differentiation of alveolar (AT1, AT2 pneumocytes) and bronchiolar (ciliated, Clara cells) cell lineages. We also observed the retention of significantly increased numbers of undifferentiated progenitor precursor cells in distal lung epithelia and the presence of substantial accumulations of periodic acid-Schiff-positive (PAS+) material and ceramide in the lung airways of newborn DKO mice. Interestingly, antenatal dexamethasone treatment partially mitigated the defective lung maturation phenotypes and extended the lifespan of the DKO animals up to 6 days, but was not sufficient to abrogate lethality in these mice. RNA microarray hybridization analyses of the lungs of dexamethasone-treated and untreated mice uncovered transcriptional changes pointing to functional and metabolic alterations that may be mechanistically relevant for the defective lung phenotypes observed in DKO mice. Our data suggest that delayed alveolar differentiation, altered sphingolipid metabolism and ceramide accumulation are primary contributors to the respiratory stress and neonatal lethality shown by DKO mice and uncover specific, critical roles of HRAS and NRAS for correct lung differentiation that are essential for neonatal survival and cannot be substituted by the remaining KRAS function in this organ.

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