Haiying Sun

Elevated CO2 Levels Cause Mitochondrial Dysfunction and Impair Cell Proliferation

Background: Cells are exposed to elevated levels of CO2 (hypercapnia) in many diseases. Results: Hypercapnia decreased cell proliferation, which was prevented with α-ketoglutarate, IDH2 overexpression, and microRNA-183 inhibition. Conclusion: Hypercapnia causes mitochondrial dysfunction by up-regulation of microRNA-183, which decreases the levels of IDH2. Significance: Hypercapnia causes mitochondrial dysfunction, which is relevant for patients with lung diseases. Elevated CO2 concentrations (hypercapnia) occur in patients with severe lung diseases. Here, we provide evidence that high CO2 levels decrease O2 consumption and ATP production and impair cell proliferation independently of acidosis and hypoxia in fibroblasts (N12) and alveolar epithelial cells (A549). Cells exposed to elevated CO2 died in galactose medium as well as when glucose-6-phosphate isomerase was knocked down, suggesting mitochondrial dysfunction. High CO2 levels led to increased levels of microRNA-183 (miR-183), which in turn decreased expression of IDH2 (isocitrate dehydrogenase 2). The high CO2-induced decrease in cell proliferation was rescued by α-ketoglutarate and overexpression of IDH2, whereas proliferation decreased in normocapnic cells transfected with siRNA for IDH2. Also, overexpression of miR-183 decreased IDH2 (mRNA and protein) as well as cell proliferation under normocapnic conditions, whereas inhibition of miR-183 rescued the normal proliferation phenotype in cells exposed to elevated levels of CO2. Accordingly, we provide evidence that high CO2 induces miR-183, which down-regulates IDH2, thus impairing mitochondrial function and cell proliferation. These results are of relevance to patients with hypercapnia such as those with chronic obstructive pulmonary disease, asthma, cystic fibrosis, bronchopulmonary dysplasia, and muscular dystrophies.

Role of the small GTPase RhoA in the hypoxia-induced decrease of plasma membrane Na,K-ATPase in A549 cells

Hypoxia impairs alveolar fluid reabsorption by promoting Na,K-ATPase endocytosis, from the plasma membrane of alveolar epithelial cells. The present study was designed to determine whether hypoxia induces Na,K-ATPase endocytosis via reactive oxygen species (ROS)-mediated RhoA activation. In A549 cells, RhoA activation occurred within 15 minutes of cells exposure to hypoxia. This activation was inhibited in cells infected with adenovirus coding for gluthatione peroxidase (an H2O2 scavenger), in mitochondria depleted (ρ0) cells or cells expressing decreased levels of the Rieske iron-sulfur protein (inhibitor of mitochondrial complex III), which suggests a role for mitochondrial ROS. Moreover, exogenous H2O2 treatment during normoxia mimicked the effects of hypoxia on RhoA, further supporting a role for ROS. Cells expressing dominant negative RhoA failed to endocytose the Na,K-ATPase during hypoxia or after H2O2 treatment. Na,K-ATPase endocytosis was also prevented in cells treated with Y-27632, a Rho-associated kinase (ROCK) inhibitor, and in cells expressing dominant negative ROCK. In summary, we provide evidence that in human alveolar epithelial cells exposed to hypoxia, RhoA/ROCK activation is necessary for Na,K-ATPase endocytosis via a mechanism that requires mitochondrial ROS.

Na,K-ATPase α1-subunit dephosphorylation by protein phosphatase 2A is necessary for its recruitment to the plasma membrane

In alveolar epithelial cells, G‐protein coupled‐receptors agonists (GPCR) induce the recruitment of the Na,K‐ATPase to the plasma membrane. Here we report that for the recruitment of the Na,K‐ ATPase to occur, dephosphorylation of its α1‐subunit at serine 18 is necessary, as demonstrated by in vitro phosphorylation, mutation of the serine 18 to alanine, and use of a specific phospho‐antibody. Several approaches strongly suggest dephosphorylation to be mediated by protein phosphatase 2A (PP2A): 1) Na,K‐ ATPase dephosphorylation and recruitment were prevented by okadaic acid (OA); 2) the Na,K‐ATPase α1‐subunit is an in vitro substrate for PP2A; and 3) glutathione S‐transferase (GST)‐fusion proteins binding assays demonstrate a direct interaction between the catalytic subunit of PP2A and the first 90 amino acids of the Na,K‐ATPase α1‐subunit. Finally, GPCR agonists induced a rapid translocation of PP2A from the cytosol to the membrane fraction, which corresponded with increased coimmunoprecipitation and colocalization of PP2A and the Na,K‐ATPase. Accordingly, we provide evidence that GPCR agonists promote PP2A translocation to the membrane fraction, leading to the dephosphorylation of the Na,K‐ATPase α1‐subunit at the serine 18 residue and its recruitment to the cell plasma membrane, which is of biological and physiological importance.—Lecuona, E., Dada, L. A., Sun, H., Butti, M. L., Zhou, G., Chew, T.‐L., Sznajder, J. I. Na,K‐ ATPase α1‐subunit dephosphorylation by protein phosphatase 2A is necessary for its recruitment to the plasma membrane. FASEB J. 20, E2146–E2155 (2006)

Myosin-Va restrains the trafficking of Na+/K+-ATPase-containing vesicles in alveolar epithelial cells

Stimulation of Na+/K+-ATPase activity in alveolar epithelial cells by cAMP involves its recruitment from intracellular compartments to the plasma membrane. Here, we studied the role of the actin molecular motor myosin-V in this process. We provide evidence that, in alveolar epithelial cells, cAMP promotes Na+/K+-ATPase recruitment to the plasma membrane by increasing the average speed of Na+/K+-ATPase-containing vesicles moving to the cell periphery. We found that three isoforms of myosin-V are expressed in alveolar epithelial cells; however, only myosin-Va and Vc colocalized with the Na+/K+-ATPase in intracellular membrane fractions. Overexpression of dominant-negative myosin-Va or knockdown with specific shRNA increased the average speed and distance traveled by the Na+/K+-ATPase-containing vesicles, as well as the Na+/K+-ATPase activity and protein abundance at the plasma membrane to similar levels as those observed with cAMP stimulation. These data show that myosin-Va has a role in restraining Na+/K+-ATPase-containing vesicles within intracellular pools and that this restrain is released after stimulation by cAMP allowing the recruitment of the Na+/K+-ATPase to the plasma membrane and thus increased activity.

Protein Kinase A-Iα Regulates Na,K-ATPase Endocytosis in Alveolar Epithelial Cells Exposed to High CO2 Concentrations

Elevated concentrations of CO2 (hypercapnia) lead to alveolar epithelial dysfunction by promoting Na,K-ATPase endocytosis. In the present report, we investigated whether the CO2/HCO3(-) activated soluble adenylyl cyclase (sAC) regulates this process. We found that hypercapnia increased the production of cyclic adenosine monophosphate (cAMP) and stimulated protein kinase A (PKA) activity via sAC, which was necessary for Na,K-ATPase endocytosis. During hypercapnia, cAMP was mainly produced in specific microdomains in the proximity of the plasma membrane, leading to PKA Type Iα activation. In alveolar epithelial cells exposed to high CO2 concentrations, PKA Type Iα regulated the time-dependent phosphorylation of the actin cytoskeleton component α-adducin at serine 726. Cells expressing small hairpin RNA for PKAc, dominant-negative PKA Type Iα, small interfering RNA for α-adducin, and α-adducin with serine 726 mutated to alanine prevented Na,K-ATPase endocytosis. In conclusion, we provide evidence for a new mechanism by which hypercapnia via sAC, cAMP, PKA Type Iα, and α-adducin regulates Na,K-ATPase endocytosis in alveolar epithelial cells.

Ubiquitination Participates in the Lysosomal Degradation of Na,K-ATPase in Steady-State Conditions

The alveolar epithelial cell (AEC) Na,K-ATPase contributes to vectorial Na(+) transport and plays an important role in keeping the lungs free of edema. We determined, by cell surface labeling with biotin and immunofluorescence, that approximately 30% of total Na,K-ATPase is at the plasma membrane of AEC in steady-state conditions. The half-life of the plasma membrane Na,K-ATPase was about 4 hours, and the incorporation of new Na,K-ATPase to the plasma membrane was Brefeldin A sensitive. Both protein kinase C (PKC) inhibition with bisindolylmaleimide (10 microM) and infection with an adenovirus expressing dominant-negative PKCzeta prevented Na,K-ATPase degradation. In cells expressing the Na,K-ATPase alpha1-subunit lacking the PKC phosphorylation sites, the plasma membrane Na,K-ATPase had a moderate increase in half-life. We also found that the Na,K-ATPase was ubiquitinated in steady-state conditions and that proteasomal inhibitors prevented its degradation. Interestingly, mutation of the four lysines described to be necessary for ubiquitination and endocytosis of the Na,K-ATPase in injurious conditions did not have an effect on its half-life in steady-state conditions. Lysosomal inhibitors prevented Na,K-ATPase degradation, and co-localization of Na,K-ATPase and lysosomes was found after labeling and chasing the plasma membrane Na,K-ATPase for 4 hours. Accordingly, we provide evidence suggesting that phosphorylation and ubiquitination are necessary for the steady-state degradation of the plasma membrane Na,K-ATPase in the lysosomes in alveolar epithelial cells.

Identification of the amino acid region involved in the intercellular interaction between the β1 subunits of Na+/K+ -ATPase.

Epithelial junctions depend on intercellular interactions between β1 subunits of the Na+/K+-ATPase molecules of neighboring cells. The interaction between dog and rat subunits is less effective than the interaction between two dog β1 subunits, indicating the importance of species-specific regions for β1–β1 binding. To identify these regions, the species-specific amino acid residues were mapped on a high-resolution structure of the Na+/K+-ATPase β1 subunit to select those exposed towards the β1 subunit of the neighboring cell. These exposed residues were mutated in both dog and rat YFP-linked β1 subunits (YFP–β1) and also in the secreted extracellular domain of the dog β1 subunit. Five rat-like mutations in the amino acid region spanning residues 198–207 of the dog YFP–β1 expressed in Madin–Darby canine kidney (MDCK) cells decreased co-precipitation of the endogenous dog β1 subunit with YFP–β1 to the level observed between dog β1 and rat YFP–β1. In parallel, these mutations impaired the recognition of YFP–β1 by the dog-specific antibody that inhibits cell adhesion between MDCK cells. Accordingly, dog-like mutations in rat YFP–β1 increased both the (YFP–β1)–β1 interaction in MDCK cells and recognition by the antibody. Conversely, rat-like mutations in the secreted extracellular domain of the dog β1 subunit increased its interaction with rat YFP–β1 in vitro. In addition, these mutations resulted in a reduction of intercellular adhesion between rat lung epithelial cells following addition of the secreted extracellular domain of the dog β1 subunit to a cell suspension. Therefore, the amino acid region 198–207 is crucial for both trans-dimerization of the Na+/K+-ATPase β1 subunits and cell–cell adhesion.

Donor pulmonary intravascular nonclassical monocytes recruit recipient neutrophils and mediate primary lung allograft dysfunction

Donor nonclassical monocytes mediate primary lung allograft dysfunction by recruiting neutrophils via MyD88-dependent production of CXCL2. Nonclassical monocytes prompt primary graft dysfunction Despite concerted efforts, primary graft dysfunction is a major cause of graft failure after organ transplantation. In lung transplantation, primary graft dysfunction is known to be mediated by early neutrophil infiltration. Zheng et al. used syngeneic and allogeneic mouse models of lung transplantation to show that nonclassical monocytes were the key cell population recruiting these destructive neutrophils. These intravascular cells were donor-derived and were also detectable in human lung grafts being used for transplant. Because depletion of nonclassical monocytes prevented primary graft dysfunction in the mouse models, targeting this cell population during human transplant could lead to improved rates of graft success. Primary graft dysfunction is the predominant driver of mortality and graft loss after lung transplantation. Recruitment of neutrophils as a result of ischemia-reperfusion injury is thought to cause primary graft dysfunction; however, the mechanisms that regulate neutrophil influx into the injured lung are incompletely understood. We found that donor-derived intravascular nonclassical monocytes (NCMs) are retained in human and murine donor lungs used in transplantation and can be visualized at sites of endothelial injury after reperfusion. When NCMs in the donor lungs were depleted, either pharmacologically or genetically, neutrophil influx and lung graft injury were attenuated in both allogeneic and syngeneic models. Similar protection was observed when the patrolling function of donor NCMs was impaired by deletion of the fractalkine receptor CX3CR1. Unbiased transcriptomic profiling revealed up-regulation of MyD88 pathway genes and a key neutrophil chemoattractant, CXCL2, in donor-derived NCMs after reperfusion. Reconstitution of NCM-depleted donor lungs with wild-type but not MyD88-deficient NCMs rescued neutrophil migration. Donor NCMs, through MyD88 signaling, were responsible for CXCL2 production in the allograft and neutralization of CXCL2 attenuated neutrophil influx. These findings suggest that therapies to deplete or inhibit NCMs in donor lung might ameliorate primary graft dysfunction with minimal toxicity to the recipient.

Decreased CXCL12 is associated with impaired alveolar epithelial cell migration and poor lung healing after lung resection.

BACKGROUND Prolonged air leak (PAL) is an important cause of morbidity and mortality after lung resection, but its pathogenesis has not been elucidated. Migration of alveolar type II epithelial cells is essential for lung wound repair. Here we determined the role of C-X-C motif chemokine 12 (CXCL12) on alveolar epithelial cell migration and lung wound healing. METHODS CXCL12 in the pleural fluid of patients was analyzed using enzyme-linked immunosorbent assay. Human A549 and murine MLE12 alveolar epithelial cell lines were used for wound closure, cell migration, and proliferation assays. Western blot was used to analyze Rac1 and cofilin. RESULTS Pleural CXCL12 was decreased in patients with PAL (1,389 ± 192 vs 3,270 ± 247 pg/mL; P < .0001). CXCL12 enhanced scratch wound closure in both A549 (77.9 ± 0.7% vs 71.5 ± 0.4%; P = .0016) and MLE12 (92.9 ± 4.9% vs 66.0 ± 4.8%; P = .017). CXCL12 enhanced migration by 57% in A549 (P = .0008) and by 86% in MLE12 (P < .0001). AMD3100, a selective CXCR4 antagonist, prevented the effects of CXCL12. CXCL12 increased Rac1 and cofilin activation but did not change bromodeoxyuridine incorporation or cell counts. CONCLUSION Reduced pleural CXCL12 is associated with PAL. CXCL12 promotes alveolar epithelial cell migration by binding to its receptor CXCR4 and may have a role in lung healing. CXCL12-mediated alveolar epithelial cell migration is associated with Rac1 and cofilin activation.

Lung Injury Combined with Loss of Regulatory T Cells Leads to De Novo Lung-Restricted Autoimmunity

More than one third of patients with chronic lung disease undergoing lung transplantation have pre-existing Abs against lung-restricted self-Ags, collagen type V (ColV), and k-α1 tubulin (KAT). These Abs can also develop de novo after lung transplantation and mediate allograft rejection. However, the mechanisms leading to lung-restricted autoimmunity remain unknown. Because these self-Ags are normally sequestered, tissue injury is required to expose them to the immune system. We previously showed that respiratory viruses can induce apoptosis in CD4+CD25+Foxp3+ regulatory T cells (Tregs), the key mediators of self-tolerance. Therefore, we hypothesized that lung-tissue injury can lead to lung-restricted immunity if it occurs in a setting when Tregs are impaired. We found that human lung recipients who suffer respiratory viral infections experienced a decrease in peripheral Tregs. Pre-existing lung allograft injury from donor-directed Abs or gastroesophageal reflux led to new ColV and KAT Abs post respiratory viral infection. Similarly, murine parainfluenza (Sendai) respiratory viral infection caused a decrease in Tregs. Intratracheal instillation of anti-MHC class I Abs, but not isotype control, followed by murine Sendai virus infection led to development of Abs against ColV and KAT, but not collagen type II (ColII), a cartilaginous protein. This was associated with expansion of IFN-γ–producing CD4+ T cells specific to ColV and KAT, but not ColII. Intratracheal anti-MHC class I Abs or hydrochloric acid in Foxp3-DTR mice induced ColV and KAT, but not ColII, immunity, only if Tregs were depleted using diphtheria toxin. We conclude that tissue injury combined with loss of Tregs can lead to lung-tissue–restricted immunity.