HOIL-1L and PKCζ are required for Na,K-ATPase stabilization during hypoxia

Rationale: The ability of organisms to adapt to hypoxia is critical for survival. Cells downregulate energy consuming enzymes such as Na,K-ATPase in order to decrease O2 consumption. The Na,K-ATPase activity is crucial for alveolar epithelium function and cell homeostasis. In alveolar epithelial cells (AEC) exposed to short term hypoxia, Na,K-ATPase abundance at the plasma membrane is regulated by protein kinase C zeta (PKCζ) through phosphorylation of its α1 subunit at Ser-18 triggering its endocytosis and decreasing the enzyme’s activity. We have previously described that during prolonged hypoxia, HIF up regulates HOIL-1L, which acts as the E3-ubiquitin ligase for PKCζ targeting it for proteasomal degradation. Here, we evaluated the alveolar epithelial function in response to continuous deprived oxygen supply studying whether HOIL-1L interaction with PKCζ prevents further Na,K-ATPase down-regulation as a mechanism of adaptation to hypoxia. Methods and results: C57BL/6 WT mice or SPC-Cre-HOIL-1Lfl/fl mice, which have a lung epithelial specific deletion in HOIL-1L, were kept in room-air or exposed to hypoxia (7% O2) for 7 days. Protein expression in alveolar type 2 (ATII) cells were analyzed by Western blot. We found that absence of HOIL-1L prevents hypoxia-induced PKCζ degradation, along with increased plasma membrane Na,K-ATPase depletion compared to WT mice. The integrity of the alveolo-capillary barrier was evaluated by permeability to small solutes, using FITC-labeled dextran (4KDa) and cell count in broncho-alveolar lavage fluid. Interestingly, we were able to rescue the increased epithelial damage observed in SPC-Cre-HOIL-1Lfl/fl mice exposed to low O2, by infection with PKCζ phosphorylation resistant S18A-α1 Na,K-ATPase. Moreover, histological analysis showed an increased thickening in the alveolar septa and apoptosis in SPC-Cre-HOIL-1Lfl/fl mice after hypoxia. To further explore the mechanism behind PKCζ degradation, PKCζ interaction with HOIL-1L was studied in vitro in AEC exposed to 1.5% O2, using gain and loss of function mutations in the kinase activation loop. Our results suggest that hypoxia-induced phosphorylation of PKCζ T410 is required for translocation to the plasma membrane and interaction with HOIL-1L, which triggers PKCζ ubiquitination and degradation. Conclusions: Collectively, these data show that interaction of HOIL-1L with PKCζ is required to avoid an excessive depletion of Na,K-ATPase safeguarding the alveolar epithelial function as a critically necessary step to ensure cell viability during hypoxic conditions.