HOIL‐1L and PKCζ are Required for Na,K‐ATPase Stabilization at the Plasma Membrane During Hypoxia
Authors: Magnani Natalia D. , Queisser Markus A. , Dada Laura A. , Welch Lynn C. , Brazee Patricia , Misharin Alexander , Budinger Scott G.R. , Sznajder Jacob I. 
Published: November 2023

The ability of organisms to adapt to hypoxia is critical for survival. Cells down regulate energy consuming enzymes such as Na,K‐ATPase, in order to decrease O2 (oxygen) consumption. The Na,K‐ATPase activity is crucial for the alveolar epithelium function and cell homeostasis. In alveolar epithelial type 2 (ATII) cells exposed to short term hypoxia, Na,K‐ATPase abundance at the plasma membrane is tightly regulated by protein kinase C zeta (PKCζ) through phosphorylation of its α1 catalytic subunit at Ser‐18 which triggers its endocytosis decreasing the enzyme's activity. We have previously described that during prolonged hypoxia, HIF promotes transcription of HOIL‐1L, which acts as the E3‐ubiquitin ligase for PKCζ targeting it for proteasomal degradation. In the present study, we focused on elucidating whether PKCζ degradation by HOIL‐1L prevents further Na,K‐ATPase down‐regulation as a mechanism of adaptation to hypoxia. C57BL/6 WT mice or SPC‐Cre‐HOIL‐1Lfl/fl mice, which have a lung epithelial specific deletion of HOIL‐1L, were exposed to room‐air or hypoxia (7% O2) for up to 14 days. Protein expression in ATII cells was analyzed by Western blot. We found that hypoxia‐induced PKCζ degradation was prevented in SPC‐Cre‐HOIL‐1Lfl/fl mice. Moreover, the absence of HOIL‐1L failed to prevent plasma membrane Na,K‐ATPase depletion as compared to hypoxia‐exposed WT mice. The alveolo‐capillary barrier permeability to small solutes, measured by FITC‐labeled dextran (4KDa), cell count and protein content in broncho‐alveolar lavage fluid were increased in SPC‐Cre‐HOIL‐1Lfl/fl mice. Also, histological analysis showed an increased thickening in the alveolar septa and apoptosis in SPC‐Cre‐HOIL‐1Lfl/fl mice after 7 days of hypoxia, suggesting that HOIL‐1L is crucial for adaptation to hypoxia. Interestingly, the increased epithelial barrier damage observed due to the lack of HOIL‐1L was independent of the inflammatory response. In AEC exposed to 1.5% O2 in vitro, the silencing of HOIL‐1L led to Na,K‐ATPase downregulation and cell death as compared to cells transfected with control siRNA, and was rescued by transfection with PKCζ phosphorylation resistant S18A‐α1 Na,K‐ATPase. To further explore the mechanism leading to PKCζ degradation, PKCζ interaction with HOIL‐1L was studied 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 the hypoxia‐induced translocation to the plasma membrane and interaction with HOIL‐1L, which triggers PKCζ ubiquitination and degradation. Collectively, these data provide evidence for an adaptive mechanism to hypoxia where the coordinated action of HOIL‐1L and PKCζ regulates Na,K‐ATPase protein abundance at the plasma membrane, and is therefore a critically necessary step in the cellular adaptation and survival in hypoxic conditions.Support or Funding InformationSupported in part by HL‐71643 and HL‐48129, AG‐49665