HIF and HOIL-1L–mediated PKCζ degradation stabilizes plasma membrane Na,K-ATPase to protect against hypoxia-induced lung injury

Abstract

Organisms have evolved adaptive mechanisms in response to stress for cellular survival. During acute hypoxic stress, cells down-regulate energy-consuming enzymes such as Na,K-ATPase. Within minutes of alveolar epithelial cell (AEC) exposure to hypoxia, protein kinase C zeta (PKCζ) phosphorylates the α-Na,K-ATPase subunit and triggers it for endocytosis, independently of the hypoxia-inducible factor (HIF). However, the Na,K-ATPase activity is essential for cell homeostasis. HIF induces the heme-oxidized IRP2 ubiquitin ligase 1L (HOIL-1L), which leads to PKCζ degradation. Here we report a mechanism of prosurvival adaptation of AECs to prolonged hypoxia where PKCζ degradation allows plasma membrane Na,K-ATPase stabilization at ∼50% of normoxic levels, preventing its excessive down-regulation and cell death. Mice lacking HOIL-1L in lung epithelial cells ( ) were sensitized to hypoxia because they express higher levels of PKCζ and, consequently, lower plasma membrane Na,K-ATPase levels, which increased cell death and worsened lung injury. In AECs, expression of an α-Na,K-ATPase construct bearing an S18A (α-S18A) mutation, which precludes PKCζ phosphorylation, stabilized the Na,K-ATPase at the plasma membrane and prevented hypoxia-induced cell death even in the absence of HOIL-1L. Adenoviral overexpression of the α-S18A mutant Na,K-ATPase in vivo rescued the enhanced sensitivity of mice to hypoxic lung injury. These data suggest that stabilization of Na,K-ATPase during severe hypoxia is a HIF-dependent process involving PKCζ degradation. Accordingly, we provide evidence of an important adaptive mechanism to severe hypoxia, whereby halting the exaggerated down-regulation of plasma membrane Na,K-ATPase prevents cell death and lung injury.

Linear ubiquitin assembly complex regulates lung epithelial–driven responses during influenza infection

“…It has been reported that IFN-α and IFN-γ can regulate the expression of LUBAC components in SHARPIN-deficient mice (43). In our mod-HOIL-1L has several LUBAC-independent functions, such as acting as an E3 ubiquitin ligase to target proteins such as PKCζ for degradation as seen in adaptation to hypoxia and cancer (23,31,39). Nondenaturing native gel electrophoresis of lysates from WT AT2 cells or A549 cells exposed to WSN in vitro revealed that HOIL-1L runs consistent with the 600 kDa molecular weight of LUBAC (25) ( Figure 4C and Supplemental Figure 5K).…”

“…by flow cytometry using defined lineage-specific cell surface markers and gated as described in Methods and but instead express a 30-kDa truncated variant (21) that results in reduced levels of 600-kDa LUBAC (32) as well as impaired NF-κB signaling (21). Immunoblots of mouse alveolar type 2 (AT2) cell lysates using a specific antibody against either the C-terminus (21,31) or the N-terminus of HOIL-1L (33) showed a band at approximately 55 kDa in WT AT2 cells, corresponding to the molecular weight of full-length HOIL-1L, that was absent in SPC Cre /HOIL-1L fl/fl AT2 cell lysates (Supplemental Figure 2, A and B). Additionally, using the HOIL-1L N-terminus antibody, we observed a low-abundance band at approximately 30 kDa in SPC Cre /HOIL-1L fl/fl AT2 cells (Supplemental Figure 2A and ref.…”

“…Linear ubiquitin assembly complex regulates lung epithelial-driven responses during influenza infection relative to total IκBα and IRF3, respectively), NEMO ubiquitination, and cytokine production ( Loss of HOIL-1L from the lung epithelium reduces lung injury and improves survival in mice infected with IAV. To evaluate whether LUBAC activity in the lung epithelium regulates IAV-induced lung injury, we generated mice with a tissue-specific deletion of full-length HOIL-1L (SPC Cre /HOIL-1L fl/fl ) (31). These mice were generated using the same construct described for the well-defined HOIL-1L -/mice (21, 31), which do not express full-length HOIL-1L alveolar epithelial LUBAC in regulating NF-κB-dependent inflammation downstream of RIG-I during IAV infection is unknown.…”

Plumbagin-induced oxidative stress leads to inhibition of Na+/K+-ATPase (NKA) in canine cancer cells

“…Hypoxia results in oxidative stress and is known to damage the NKA complex through at least two mechanisms. The oxidized NKA complex is proteosomally degraded resulting in decrease in the expression of this ion pump on the cell membrane [14][15][16][17]. As a result, there is membrane depolarization and hence, cell death.…”

“…As a result, there is membrane depolarization and hence, cell death. Oxidative stress also is known to activate protein kinase C (PKC) which in turn phosphorylates NKA [14][15][16][17][18][19]. The phosphorylated form of NKA is internalized from the cell membrane, again resulting in membrane depolarization and cell death (6)(7)(8)(9).…”