No abstract

Hypercapnia (elevated CO 2 levels) occurs as a consequence of poor alveolar ventilation and impairs alveolar fluid reabsorption (AFR) by promoting Na,K-ATPase endocytosis. We studied the mechanisms regulating CO 2 -induced Na,K-ATPase endocytosis in alveolar epithelial cells (AECs) and alveolar epithelial dysfunction in rats. Elevated CO 2 levels caused a rapid activation of AMP-activated protein kinase (AMPK) in AECs, a key regulator of metabolic homeostasis. Activation of AMPK was mediated by a CO 2 -triggered increase in intracellular Ca 2+ concentration and Ca 2+ /calmodulin-dependent kinase kinase-β (CaMKK-β). Chelating intracellular Ca 2+ or abrogating CaMKK-β function by gene silencing or chemical inhibition prevented the CO 2 -induced AMPK activation in AECs. Activation of AMPK or overexpression of constitutively active AMPK was sufficient to activate PKC-ζ and promote Na,K-ATPase endocytosis. Inhibition or downregulation of AMPK via adenoviral delivery of dominant-negative AMPK-α 1 prevented CO 2 -induced Na,K-ATPase endocytosis. The hypercapnia effects were independent of intracellular ROS. Exposure of rats to hypercapnia for up to 7 days caused a sustained decrease in AFR. Pretreatment with a β-adrenergic agonist, isoproterenol, or a cAMP analog ameliorated the hypercapnia-induced impairment of AFR. Accordingly, we provide evidence that elevated CO 2 levels are sensed by AECs and that AMPK mediates CO 2 -induced Na,K-ATPase endocytosis and alveolar epithelial dysfunction, which can be prevented with β-adrenergic agonists and cAMP.

-Patients with acute lung injury develop hypoxia, which may lead to lung dysfunction and aberrant tissue repair. Recent studies have suggested that epithelial-mesenchymal transition (EMT) contributes to pulmonary fibrosis. We sought to determine whether hypoxia induces EMT in alveolar epithelial cells (AEC). We found that hypoxia induced the expression of ␣-smooth muscle actin (␣-SMA) and vimentin and decreased the expression of E-cadherin in transformed and primary human, rat, and mouse AEC, suggesting that hypoxia induces EMT in AEC. Both severe hypoxia and moderate hypoxia induced EMT. The reactive oxygen species (ROS) scavenger Euk-134 prevented hypoxia-induced EMT. Moreover, hypoxia-induced expression of ␣-SMA and vimentin was prevented in mitochondria-deficient 0 cells, which are incapable of ROS production during hypoxia. CoCl2 and dimethyloxaloylglycine, two compounds that stabilize hypoxiainducible factor (HIF)-␣ under normoxia, failed to induce ␣-SMA expression in AEC. Furthermore, overexpression of constitutively active HIF-1␣ did not induce ␣-SMA. However, loss of HIF-1␣ or HIF-2␣ abolished induction of ␣-SMA mRNA during hypoxia. Hypoxia increased the levels of transforming growth factor (TGF)-␤1, and preincubation of AEC with SB431542, an inhibitor of the TGF-␤1 type I receptor kinase, prevented the hypoxia-induced EMT, suggesting that the process was TGF-␤1 dependent. Furthermore, both ROS and HIF-␣ were necessary for hypoxia-induced TGF-␤1 upregulation. Accordingly, we have provided evidence that hypoxia induces EMT of AEC through mitochondrial ROS, HIF, and endogenous TGF-␤1 signaling. alveolar epithelial cells; pulmonary fibrosis; transforming growth factor-␤1 EPITHELIAL-MESENCHYMAL TRANSITION (EMT) is a cellular process during which epithelial cells acquire mesenchymal properties while losing cell-cell interactions and apicobasal polarity (33,44). EMT is characterized by changes in cell morphology and acquisition of mesenchymal markers such as ␣-smooth muscle actin (␣-SMA) and vimentin as well as loss of epithelial makers, including E-cadherin (53). Transforming growth factor (TGF)-␤1 is considered to be the prototypical cytokine for the induction of EMT (53). Active TGF-␤1 binds to the transmembrane serine-threonine kinase receptor II and receptor I and activates Smad-mediated transcription of target genes, including ␣-SMA and vimentin, which leads to EMT (33,53,54). TGF-␤1 is reported to induce EMT in renal proximal tubular epithelial cells, lens epithelial cells, and, most recently, alveolar epithelial cells (AEC) (19,23,40,48,55).AEC perform many tasks necessary for normal alveolus functioning, including surfactant protein production and fluid and ion transport (17, 57). Recent evidence suggests that AEC may undergo EMT, contributing to the pathogenesis of pulmonary fibrosis (26,49).AEC are exposed to hypoxia in human lung diseases, including acute lung injury and pulmonary fibrosis (20, 41, 57). It has been described that during hypoxia, mitochondria increase the production of reactive oxy...

2؉concentration while a STIM1 mutant rescued the AMPK activation, suggesting that ROS act upstream of Ca 2؉ signaling. Furthermore, inhibition of CRAC channel function in rat lungs prevented the impairment of alveolar fluid reabsorption caused by hypoxia. These data suggest that during hypoxia, calcium entry via CRAC channels leads to AMPK activation, Na,K-ATPase downregulation, and alveolar epithelial dysfunction.

Background: CO 2 retention and skeletal muscle atrophy occur in patients with lung diseases and are associated with poor clinical outcomes. Results: Hypercapnia leads to AMPK/FoxO3a/MuRF1-dependent muscle fiber size reduction. Conclusion: Hypercapnia activates a signaling pathway leading to skeletal muscle atrophy. Significance: High CO 2 levels directly activate a proteolytic program of skeletal muscle atrophy which is of relevance to patients with lung diseases.