Humberto E. Trejo

Hypoxia-induced alveolar epithelial-mesenchymal transition requires mitochondrial ROS and hypoxia-inducible factor 1.

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 alpha-smooth muscle actin (alpha-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 alpha-SMA and vimentin was prevented in mitochondria-deficient rho(0) cells, which are incapable of ROS production during hypoxia. CoCl(2) and dimethyloxaloylglycine, two compounds that stabilize hypoxia-inducible factor (HIF)-alpha under normoxia, failed to induce alpha-SMA expression in AEC. Furthermore, overexpression of constitutively active HIF-1alpha did not induce alpha-SMA. However, loss of HIF-1alpha or HIF-2alpha abolished induction of alpha-SMA mRNA during hypoxia. Hypoxia increased the levels of transforming growth factor (TGF)-beta1, and preincubation of AEC with SB431542, an inhibitor of the TGF-beta1 type I receptor kinase, prevented the hypoxia-induced EMT, suggesting that the process was TGF-beta1 dependent. Furthermore, both ROS and HIF-alpha were necessary for hypoxia-induced TGF-beta1 upregulation. Accordingly, we have provided evidence that hypoxia induces EMT of AEC through mitochondrial ROS, HIF, and endogenous TGF-beta1 signaling.

AMP-activated protein kinase regulates CO2-induced alveolar epithelial dysfunction in rats and human cells by promoting Na,K-ATPase endocytosis

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-beta (CaMKK-beta). Chelating intracellular Ca(2+) or abrogating CaMKK-beta 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-zeta and promote Na,K-ATPase endocytosis. Inhibition or downregulation of AMPK via adenoviral delivery of dominant-negative AMPK-alpha(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 beta-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 beta-adrenergic agonists and cAMP.

Recruitment of vimentin to the cell surface by β3 integrin and plectin mediates adhesion strength

Much effort has been expended on analyzing how microfilament and microtubule cytoskeletons dictate the interaction of cells with matrix at adhesive sites called focal adhesions (FAs). However, vimentin intermediate filaments (IFs) also associate with the cell surface at FAs in endothelial cells. Here, we show that IF recruitment to FAs in endothelial cells requires β3 integrin, plectin and the microtubule cytoskeleton, and is dependent on microtubule motors. In CHO cells, which lack β3 integrin but contain vimentin, IFs appear to be collapsed around the nucleus, whereas in CHO cells expressing β3 integrin (CHOwtβ3), vimentin IFs extend to FAs at the cell periphery. This recruitment is regulated by tyrosine residues in the β3 integrin cytoplasmic tail. Moreover, CHOwtβ3 cells exhibit significantly greater adhesive strength than CHO or CHO cells expressing mutated β3 integrin proteins. These differences require an intact vimentin network. Therefore, vimentin IF recruitment to the cell surface is tightly regulated and modulates the strength of adhesion of cells to their substrate.

High CO2 levels impair alveolar epithelial function independently of pH.

Background In patients with acute respiratory failure, gas exchange is impaired due to the accumulation of fluid in the lung airspaces. This life-threatening syndrome is treated with mechanical ventilation, which is adjusted to maintain gas exchange, but can be associated with the accumulation of carbon dioxide in the lung. Carbon dioxide (CO2) is a by-product of cellular energy utilization and its elimination is affected via alveolar epithelial cells. Signaling pathways sensitive to changes in CO2 levels were described in plants and neuronal mammalian cells. However, it has not been fully elucidated whether non-neuronal cells sense and respond to CO2. The Na,K-ATPase consumes ∼40% of the cellular metabolism to maintain cell homeostasis. Our study examines the effects of increased pCO2 on the epithelial Na,K-ATPase a major contributor to alveolar fluid reabsorption which is a marker of alveolar epithelial function. Principal Findings We found that short-term increases in pCO2 impaired alveolar fluid reabsorption in rats. Also, we provide evidence that non-excitable, alveolar epithelial cells sense and respond to high levels of CO2, independently of extracellular and intracellular pH, by inhibiting Na,K-ATPase function, via activation of PKCζ which phosphorylates the Na,K-ATPase, causing it to endocytose from the plasma membrane into intracellular pools. Conclusions Our data suggest that alveolar epithelial cells, through which CO2 is eliminated in mammals, are highly sensitive to hypercapnia. Elevated CO2 levels impair alveolar epithelial function, independently of pH, which is relevant in patients with lung diseases and altered alveolar gas exchange.

Hypoxia Leads to Na,K-ATPase Downregulation via Ca2+ Release-Activated Ca2+ Channels and AMPK Activation

ABSTRACT To maintain cellular ATP levels, hypoxia leads to Na,K-ATPase inhibition in a process dependent on reactive oxygen species (ROS) and the activation of AMP-activated kinase α1 (AMPK-α1). We report here that during hypoxia AMPK activation does not require the liver kinase B1 (LKB1) but requires the release of Ca2+ from the endoplasmic reticulum (ER) and redistribution of STIM1 to ER-plasma membrane junctions, leading to calcium entry via Ca2+ release-activated Ca2+ (CRAC) channels. This increase in intracellular Ca2+ induces Ca2+/calmodulin-dependent kinase kinase β (CaMKKβ)-mediated AMPK activation and Na,K-ATPase downregulation. Also, in cells unable to generate mitochondrial ROS, hypoxia failed to increase intracellular Ca2+ concentration while a STIM1 mutant rescued the AMPK activation, suggesting that ROS act upstream of Ca2+ 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.

Elevated CO2 selectively inhibits interleukin-6 and tumor necrosis factor expression and decreases phagocytosis in the macrophage

Elevated blood and tissue CO2, or hypercapnia, is common in severe lung disease. Patients with hypercapnia often develop lung infections and have an increased risk of death following pneumonia. To explore whether hypercapnia interferes with host defense, we studied the effects of elevated PCO2 on macrophage innate immune responses. In differentiated human THP‐1 macrophages and human and mouse alveolar macrophages stimulated with lipopolysaccharide (LPS) and other Toll‐like receptor ligands, hypercapnia inhibited expression of tumor necrosis factor and interleukin (IL)‐6, nuclear factor (NF)‐KB‐dependent cytokines critical for antimicrobial host defense. Inhibition of IL‐6 expression by hypercapnia was concentration dependent, rapid, reversible, and independent of extracellular and intracellular acidosis. In contrast, hypercapnia did not down‐regulate IL‐10 or interferon‐ß, which do not require NF‐κB. Notably, hypercapnia did not affect LPS‐induced degradation of IκBα, nuclear translocation of RelA/p65, or activation of mitogen‐activated protein kinases, but it did block IL‐6 promoter‐driven luciferase activity in mouse RAW 264.7 macrophages. Elevated PCO2 also decreased phagocytosis of opsonized polystyrene beads and heat‐killed bacteria in THP‐1 and human alveolar macrophages. By interfering with essential innate immune functions in the macrophage, hypercapnia may cause a previously unrecognized defect in resistance to pulmonary infection in patients with advanced lung disease.—Wang, N., Gates, K. L., Trejo, H., Favoreto, Jr., S., Schleimer, R P., Sznajder, J. I., Beitel, G. J., Sporn, P. H. S. Elevated CO2 selectively inhibits interleukin‐6 and tumor necrosis factor expression and decreases phagocytosis in the macrophage. FASEB J. 24, 2178–2190 (2010). www.fasebj.org

Extracellular signal-regulated kinase (ERK) participates in the hypercapnia-induced Na,K-ATPase downregulation.

Hypercapnia has been shown to impair alveolar fluid reabsorption (AFR) by decreasing Na,K‐ATPase activity. Extracellular signal‐regulated kinase pathway (ERK) is activated under conditions of cellular stress and has been known to regulate the Na,K‐ATPase. Here, we show that hypercapnia leads to ERK activation in a time‐dependent manner in alveolar epithelial cells (AEC). Inhibition of ERK by U0126 or siRNA prevented both the hypercapnia‐induced Na,K‐ATPase endocytosis and impairment of AFR. Moreover, ERK inhibition prevented AMPK activation, a known modulator of hypercapnia‐induced Na,K‐ATPase endocytosis. Accordingly, these data suggest that hypercapnia‐induced Na,K‐ATPase endocytosis is dependent on ERK activation in AEC and that ERK plays an important role in hypercapnia‐induced impairment of AFR in rat lungs.

Insulin regulates alveolar epithelial function by inducing Na+/K+-ATPase translocation to the plasma membrane in a process mediated by the action of Akt

Stimulation of Na+/K+-ATPase translocation to the cell surface increases active Na+ transport, which is the driving force of alveolar fluid reabsorption, a process necessary to keep the lungs free of edema and to allow normal gas exchange. Here, we provide evidence that insulin increases alveolar fluid reabsorption and Na+/K+-ATPase activity by increasing its translocation to the plasma membrane in alveolar epithelial cells. Insulin-induced Akt activation is necessary and sufficient to promote Na+/K+-ATPase translocation to the plasma membrane. Phosphorylation of AS160 by Akt is also required in this process, whereas inactivation of the Rab GTPase-activating protein domain of AS160 promotes partial Na+/K+-ATPase translocation in the absence of insulin. We found that Rab10 functions as a downstream target of AS160 in insulin-induced Na+/K+-ATPase translocation. Collectively, these results suggest that Akt plays a major role in Na+/K+-ATPase intracellular translocation and thus in alveolar fluid reabsorption.

Endothelin-1 Impairs Alveolar Epithelial Function via Endothelial ETB Receptor

RATIONALE Endothelin-1 (ET-1) is increased in patients with high-altitude pulmonary edema and acute respiratory distress syndrome, and these patients have decreased alveolar fluid reabsorption (AFR). OBJECTIVES To determine whether ET-1 impairs AFR via activation of endothelial cells and nitric oxide (NO) generation. METHODS Isolated perfused rat lung, transgenic rats deficient in ETB receptors, coincubation of lung human microvascular endothelial cells (HMVEC-L) with rat alveolar epithelial type II cells or A549 cells, ouabain-sensitive 86Rb+ uptake. MEASUREMENTS AND MAIN RESULTS The ET-1-induced decrease in AFR was prevented by blocking the endothelin receptor ETB, but not ETA. Endothelial-epithelial cell interaction is required, as direct exposure of alveolar epithelial cells (AECs) to ET-1 did not affect Na,K-ATPase function or protein abundance at the plasma membrane, whereas coincubation of HMVEC-L and AECs with ET-1 decreased Na,K-ATPase activity and protein abundance at the plasma membrane. Exposing transgenic rats deficient in ETB receptors in the pulmonary vasculature (ET-B(-/-)) to ET-1 did not decrease AFR or Na,K-ATPase protein abundance at the plasma membrane of AECs. Exposing HMVEC-L to ET-1 led to increased NO, and the ET-1-induced down-regulation of Na,K-ATPase was prevented by the NO synthase inhibitor l-NAME, but not by a guanylate cyclase inhibitor. CONCLUSIONS We provide the first evidence that ET-1, via an endothelial-epithelial interaction, leads to decreased AFR by a mechanism involving activation of endothelial ETB receptors and NO generation leading to alveolar epithelial Na,K-ATPase down-regulation in a cGMP-independent manner.

Asbestos-induced alveolar epithelial cell apoptosis: The role of endoplasmic reticulum stress response

Asbestos exposure results in pulmonary fibrosis (asbestosis) and malignancies (bronchogenic lung cancer and mesothelioma) by mechanisms that are not fully understood. Alveolar epithelial cell (AEC) apoptosis is important in the development of pulmonary fibrosis after exposure to an array of toxins, including asbestos. An endoplasmic reticulum (ER) stress response and mitochondria-regulated (intrinsic) apoptosis occur in AECs of patients with idiopathic pulmonary fibrosis, a disease with similarities to asbestosis. Asbestos induces AEC intrinsic apoptosis, but the role of the ER is unclear. The objective of this study was to determine whether asbestos causes an AEC ER stress response that promotes apoptosis. Using human A549 and rat primary isolated alveolar type II cells, amosite asbestos fibers increased AEC mRNA and protein expression of ER stress proteins involved in the unfolded protein response, such as inositol-requiring kinase (IRE) 1 and X-box-binding protein-1, as well as ER Ca²(2+) release ,as assessed by a FURA-2 assay. Eukarion-134, a superoxide dismutase/catalase mimetic, as well as overexpression of Bcl-XL in A549 cells each attenuate asbestos-induced AEC ER stress (IRE-1 and X-box-binding protein-1 protein expression; ER Ca²(2+) release) and apoptosis. Thapsigargin, a known ER stress inducer, augments AEC apoptosis, and eukarion-134 or Bcl-XL overexpression are protective. Finally, 4-phenylbutyric acid, a chemical chaperone that attenuates ER stress, blocks asbestos- and thapsigargin-induced AEC IRE-1 protein expression, but does not reduce ER Ca²(2+) release or apoptosis. These results show that asbestos triggers an AEC ER stress response and subsequent intrinsic apoptosis that is mediated in part by ER Ca²(2+) release.