-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...

Protein modification with ubiquitin chains is an essential signaling event catalyzed by E3 ubiquitin ligases. Most human E3s contain a signature RING domain that recruits a ubiquitin-charged E2 and a separate domain for substrate recognition. How RING-E3s can build polymeric ubiquitin chains while binding substrates and E2s at defined interfaces remains poorly understood. Here, we show that the RING-E3 APC/C catalyzes chain elongation by strongly increasing the affinity of its E2 for the distal acceptor ubiquitin in a growing conjugate. This function of the APC/C requires its coactivator as well as conserved residues of the E2 and ubiquitin. APC/C's ability to track the tip of an emerging conjugate is required for APC/C-substrate degradation and accurate cell division. Our results suggest that RING-E3s tether the distal ubiquitin of a growing chain in proximity to the active site of their E2s, allowing them to assemble polymeric conjugates without altering their binding to substrate or E2.

Hypoxia promotes Na,K-ATPase endocytosis via protein kinase C (PKC)-mediated phosphorylation of the Na,K-ATPase ␣ subunit. Here, we report that hypoxia leads to the phosphorylation of 5-AMPactivated protein kinase (AMPK) at Thr172 in rat alveolar epithelial cells. The overexpression of a dominant-negative AMPK ␣ subunit (AMPK-DN) construct prevented the hypoxia-induced endocytosis of Na,K-ATPase. The overexpression of the reactive oxygen species (ROS) scavenger catalase prevented hypoxia-induced AMPK activation. Moreover, hypoxia failed to activate AMPK in mitochondrion-deficient 0 -A549 cells, suggesting that mitochondrial ROS play an essential role in hypoxia-induced AMPK activation. Hypoxia-induced PKC translocation to the plasma membrane and phosphorylation at Thr410 were prevented by the pharmacological inhibition of AMPK or by the overexpression of the AMPK-DN construct. We found that AMPK ␣ phosphorylates PKC on residue Thr410 within the PKC activation loop. Importantly, the activation of AMPK ␣ was necessary for hypoxia-induced AMPK-PKC binding in alveolar epithelial cells. The overexpression of T410A mutant PKC prevented hypoxia-induced Na,KATPase endocytosis, confirming that PKC Thr410 phosphorylation is essential for this process. PKC activation by AMPK is isoform specific, as small interfering RNA targeting the ␣1 but not the ␣2 catalytic subunit prevented PKC activation. Accordingly, we provide the first evidence that hypoxia-generated mitochondrial ROS lead to the activation of the AMPK ␣1 isoform, which binds and directly phosphorylates PKC at Thr410, thereby promoting Na,K-ATPase endocytosis.When exposed to low oxygen levels (hypoxia), cells develop adaptative strategies to maintain adequate levels of ATP (21). These strategies include increasing the efficiency of energyproducing pathways, mostly through anaerobic glycolysis, while decreasing energy-consuming processes such as Na,K-ATPase activity (30). Alveolar hypoxia occurs in many respiratory disorders, and it has been shown to decrease epithelial active Na ϩ transport, leading to impaired fluid reabsorption (37,41,42). Active Na ϩ transport and, thus, alveolar fluid reabsortion are effected mostly via apical sodium channels and the basolateral Na,K-ATPase (32, 38, 42). We have reported previously that hypoxia inhibits Na,K-ATPase activity by promoting its endocytosis from the plasma membrane by a mechanism that requires the generation of mitochondrial reactive oxygen species (ROS) and the phosphorylation of the Na,K-ATPase ␣ subunit at Ser18 by protein kinase C (PKC) (8, 9).The 5Ј-AMP-activated protein kinase (AMPK) is a heterotrimeric Ser/Thr kinase composed of a catalytic ␣ subunit and regulatory ␤ and ␥ subunits. Both isoforms of the AMPK catalytic subunit (␣1 and ␣2) form complexes with noncatalytic subunits. The ␣1 subunit is ubiquitously expressed, whereas the ␣2 subunit isoform is expressed predominantly in tissues like the liver, heart, and skeletal muscle (36). The ␣1 and ␣2 subunit isoforms have ϳ90% homology in their N-terminal ca...

Modification of proteins with ubiquitin chains is an essential regulatory event in cell cycle control. Differences in the connectivity of ubiquitin chains are believed to result in distinct functional consequences for the modified proteins. Among eight possible homogenous chain types, canonical Lys48-linked ubiquitin chains have long been recognized to drive the proteasomal degradation of cell cycle regulators, and Lys48 is the only essential lysine residue of ubiquitin in yeast. It thus came as a surprise that in higher eukaryotes atypical K11-linked ubiquitin chains regulate the substrates of the anaphase-promoting complex and control progression through mitosis. Here, we discuss recent findings that shed light on the assembly and function of K11-linked chains during cell division.

Elevated CO2 levels (hypercapnia) occur in patients with respiratory diseases and impair alveolar epithelial integrity, in part, by inhibiting Na,K-ATPase function. Here, we examined the role of c-Jun N-terminal kinase (JNK) in CO2 signaling in mammalian alveolar epithelial cells as well as in diptera, nematodes and rodent lungs. In alveolar epithelial cells, elevated CO2 levels rapidly induced activation of JNK leading to downregulation of Na,K-ATPase and alveolar epithelial dysfunction. Hypercapnia-induced activation of JNK required AMP-activated protein kinase (AMPK) and protein kinase C-ζ leading to subsequent phosphorylation of JNK at Ser-129. Importantly, elevated CO2 levels also caused a rapid and prominent activation of JNK in Drosophila S2 cells and in C. elegans. Paralleling the results with mammalian epithelial cells, RNAi against Drosophila JNK fully prevented CO2-induced downregulation of Na,K-ATPase in Drosophila S2 cells. The importance and specificity of JNK CO2 signaling was additionally demonstrated by the ability of mutations in the C. elegans JNK homologs, jnk-1 and kgb-2 to partially rescue the hypercapnia-induced fertility defects but not the pharyngeal pumping defects. Together, these data provide evidence that deleterious effects of hypercapnia are mediated by JNK which plays an evolutionary conserved, specific role in CO2 signaling in mammals, diptera and nematodes.