We provide evidence that hypoxia, via mitochondrial ROS generated at complex III, leads to calcium influx through CRAC channels, which is necessary and sufficient to phosphorylate AMPK via CaMKKβ, leading to Na,K-ATPase endocytosis in alveolar epithelial cells.
We have previously reported that the effects of hypoxia on Na,K-ATPase involved mitochondrial ROS-mediated activation of AMPK, which phosphorylates PKCζ, triggering Na,K-ATPase endocytosis (11
). It has been described that the specificity of the AMPK isoform activated is dependent on the stimulus that regulates the upstream kinases (49
). For example, the increase in AMPK activity via the catalytic α2 isoform has been associated with metabolic homeostasis due to increases in the AMP/ATP ratio and LKB1-dependent phosphorylation, whereas α1-containing complexes can be activated either by LKB1 or by CaMKKβ (40
). We found that during hypoxia AMPK activation is mediated by calcium signals and CaMKKβ in the absence of an increased AMP/ATP ratio, which is in agreement with previous reports that short-term hypoxia does not decrease the ATP levels (11
) and activates the AMPK-α1 isoform (14
). Moreover, it has been described that oxygen becomes rate limiting and inhibits the mitochondrial respiratory chain in alveolar epithelial cells only when its concentration is lower than 0.5% (43
). In ATII cells, inhibition of CaMKKβ by STO-609 prevented the AMPK activation and the hypoxia-induced Na,K-ATPase endocytosis. STO-609 has been described to inhibit other kinases in vitro
). To confirm the effects of STO-609, we used an siRNA approach in A549 cells reconstituted with LKB1. The A549 cell line bears some of the characteristics of ATII cells, including the regulation of Na,K-ATPase (4
). Transfection of siRNA against CaMKKβ abolished hypoxia-induced AMPK activation in A549+LKB1 cells and the endocytosis of the Na,K-ATPase. Both CaMKK isoforms were expressed in A549 cells, but the transfection with siRNA affected only the expression of CaMKKβ, suggesting a role for CaMKKβ in hypoxia-induced AMPK activation. The hypoxia-induced AMPK activation is transient (14
), which coincides with previous studies where CaMKKβ-mediated responses returned to baseline levels when the intracellular Ca2+
levels declined (18
), while AMPK activation via LKB1 was sustained (54
). Taken together, these results suggest that CaMKKβ is the main kinase mediating the hypoxia-induced AMPK activation in alveolar epithelial cells.
Low oxygen concentrations have been shown to modulate the intracellular calcium concentrations in excitable and nonexcitable cells (1
). In alveolar epithelial cells, we found that hypoxia increased [Ca2+
and that it occurs in a fashion consistent with store-operated calcium entry through CRAC channels. Several pieces of evidence support this conclusion. First, removal of extracellular calcium during hypoxia resulted in ER Ca2+
store depletion, followed by Ca2+
influx when cells were perfused with 2 mM Ca2+
. Second, preventing calcium influx with La3+
during hypoxia had no influence on Ca2+
release from stores, yet it was able to prevent the extracellular calcium influx, which was sufficient to inhibit AMPK activation and Na,K-ATPase endocytosis. Finally, hypoxia caused STIM1 to redistribute into discrete puncta at the cell periphery, and silencing of STIM1 and Orai1 in alveolar epithelial cells prevented the hypoxia-induced Ca2+
influx. STIM1 appears to be the limiting step of this process leading to Ca2+
entry by interacting with Orai1, the pore-forming component of CRAC channels (7
). We found that STIM1 depletion prevented AMPK and PKCζ activation and Na,K-ATPase endocytosis during hypoxia, while overexpression of STIM1 mutants with impaired Ca2+
binding was sufficient to enhance AMPK and PKCζ phosphorylation and mimicked the effect of hypoxia and ROS on Na,K-ATPase endocytosis. These data suggest that AMPK activation is dependent on the rise of the Ca2+
level following Ca2+
entry via CRAC channels.
An important finding of the present study is that mitochondrial ROS generated during hypoxia are required and sufficient for Ca2+
release from ER stores and activation of CRAC channels. It has been proposed that mitochondria act as the oxygen sensor (5
), and recent reports indicate that mitochondrial ROS are required during hypoxia-induced AMPK activation (12
). Here we report that t-H2
-induced AMPK activation was prevented in alveolar epithelial cells pretreated with BAPTA-AM or STO-609. Similar to hypoxia, H2
secondary to Ca2+
release from ER stores, which led to CRAC channel activity. Pretreatment of ATII cells with the antioxidant Eukarion-134 prevented the hypoxia-induced redistribution of STIM1 near the plasma membrane. Furthermore, hypoxia failed to induce release of ER Ca2+
influx via CRAC channels in ρ0
-A549 cells, which lack functional mitochondrial electron transfer complexes III and IV (3
). We also transfected A549 cells with shRNA against Rieske Fe-S (a component of complex III), and these cells displayed a significant decrease in Fe-S protein levels, and, importantly, had no increase in cytosolic Ca2+
or AMPK activation in response to hypoxia. Importantly, these effects were rescued by treatment H2
. Moreover, in ρ0
-A549 cells STIM1 D76A expression resulted in the Na,K-ATPase endocytosis, further supporting the role of mitochondrial ROS in the hypoxia induced CRAC channels' activity. Collectively, our data suggest that the mitochondrial ROS generated at complex III are required to release Ca2+
from the ER, and that the Ca2+
influx through CRAC channels is sufficient to activate CaMKKβ and AMPK.
Alveolar epithelial cells through the concerted actions of the Na,K-ATPase and ENaC clear fluid from airspaces to maintain normal gas exchange. Hypoxia impairs this important function of the alveolar epithelium (26
). The data showing the role of CRAC channels in Na,K-ATPase downregulation during hypoxia in isolated alveolar epithelial cells were confirmed in animals, where rats were instilled in vivo
with an shRNA against STIM1. The absence of STIM1 protected the rats from the hypoxia-induced impairment in fluid reabsorption, suggesting a role for Ca2+
influx in the regulation of Na,K-ATPase activity and alveolar epithelial function.
In summary, we propose that the influx of Ca2+ through CRAC channels is a key link between mitochondrial ROS and CaMKKβ/AMPK activation leading to Na,K-ATPase endocytosis and alveolar epithelial dysfunction during hypoxia.