TRAF2 Is a Novel Ubiquitin E3 Ligase for the Na,K-ATPase ß-Subunit That Drives Alveolar Epithelial Dysfunction in Hypercapnia.

Several acute and chronic lung diseases are associated with alveolar hypoventilation leading to accumulation of CO2 (hypercapnia). The ß-subunit of the Na,K-ATPase plays a pivotal role in maintaining epithelial integrity by functioning as a cell adhesion molecule and regulating cell surface stability of the catalytic α-subunit of the transporter, thereby, maintaining optimal alveolar fluid balance. Here, we identified the E3 ubiquitin ligase for the Na,K-ATPase ß-subunit, which promoted polyubiquitination, subsequent endocytosis and proteasomal degradation of the protein upon exposure of alveolar epithelial cells to elevated CO2 levels, thus impairing alveolar integrity. Ubiquitination of the Na,K-ATPase ß-subunit required lysine 5 and 7 and mutating these residues (but not other lysines) prevented trafficking of Na,K-ATPase from the plasma membrane and stabilized the protein upon hypercapnia. Furthermore, ubiquitination of the Na,K-ATPase ß-subunit was dependent on prior phosphorylation at serine 11 by protein kinase C (PKC)-ζ. Using a protein microarray, we identified the tumor necrosis factor receptor-associated factor 2 (TRAF2) as the E3 ligase driving ubiquitination of the Na,K-ATPase ß-subunit upon hypercapnia. Of note, prevention of Na,K-ATPase ß-subunit ubiquitination was necessary and sufficient to restore the formation of cell-cell junctions under hypercapnic conditions. These results suggest that a hypercapnic environment in the lung may lead to persistent epithelial dysfunction in affected patients. As such, the identification of the E3 ligase for the Na,K-ATPase may provide a novel therapeutic target, to be employed in patients with acute or chronic hypercapnic respiratory failure, aiming to restore alveolar epithelial integrity.

The O-glycosylated ectodomain of FXYD5 impairs adhesion by disrupting cell-cell trans-dimerization of Na,K-ATPase ß1 subunits.

FXYD5 (also known as dysadherin), a regulatory subunit of the Na,K-ATPase, impairs intercellular adhesion by a poorly understood mechanism. Here, we determined whether FXYD5 disrupts the trans-dimerization of Na,K-ATPase molecules located in neighboring cells. Mutagenesis of the Na,K-ATPase ß1 subunit identified four conserved residues, including Y199, that are crucial for the intercellular Na,K-ATPase trans-dimerization and adhesion. Modulation of expression of FXYD5 or of the ß1 subunit with intact or mutated ß1-ß1 binding sites demonstrated that the anti-adhesive effect of FXYD5 depends on the presence of Y199 in the ß1 subunit. Immunodetection of the plasma membrane FXYD5 was prevented by the presence of O-glycans. Partial FXYD5 deglycosylation enabled antibody binding and showed that the protein level and the degree of O-glycosylation were greater in cancer than in normal cells. FXYD5-induced impairment of adhesion was abolished by both genetic and pharmacological inhibition of FXYD5 O-glycosylation. Therefore, the extracellular O-glycosylated domain of FXYD5 impairs adhesion by interfering with intercellular ß1-ß1 interactions, suggesting that the ratio between FXYD5 and α1-ß1 heterodimer determines whether the Na,K-ATPase acts as a positive or negative regulator of intercellular adhesion.

Novel concepts of acute lung injury and alveolar-capillary barrier dysfunction.

In this review we summarize recent major advances in our understanding on the molecular mechanisms, mediators, and biomarkers of acute lung injury (ALI) and alveolar-capillary barrier dysfunction, highlighting the role of immune cells, inflammatory and noninflammatory signaling events, mechanical noxae, and the affected cellular and molecular entities and functions. Furthermore, we address novel aspects of resolution and repair of ALI, as well as putative candidates for treatment of ALI, including pharmacological and cellular therapeutic means.

Megalin mediates transepithelial albumin clearance from the alveolar space of intact rabbit lungs.

The alveolo-capillary barrier is effectively impermeable to large solutes such as proteins. A hallmark of acute lung injury/acute respiratory distress syndrome is the accumulation of protein-rich oedema fluid in the distal airspaces. Excess protein must be cleared from the alveolar space for recovery; however, the mechanisms of protein clearance remain incompletely understood. In intact rabbit lungs 29.8 ± 2.2% of the radio-labelled alveolar albumin was transported to the vascular compartment at 37°C within 120 min, as assessed by real-time measurement of 125I-albumin clearance from the alveolar space. At 4°C or 22°C significantly lower albumin clearance (3.7 ± 0.4 or 16.2 ± 1.1%, respectively) was observed. Deposition of a 1000-fold molar excess of unlabelled albumin into the alveolar space or inhibition of cytoskeletal rearrangement or clathrin-dependent endocytosis largely inhibited the transport of 125I-albumin to the vasculature, while administration of unlabelled albumin to the vascular space had no effect on albumin clearance. Furthermore, albumin uptake capacity was measured as about 0.37 mg ml−1 in cultured rat lung epithelial monolayers, further highlighting the (patho)physiological relevance of active alveolar epithelial protein transport. Moreover, gene silencing and pharmacological inhibition of the multi-ligand receptor megalin resulted in significantly decreased albumin binding and uptake in monolayers of primary alveolar type II and type I-like and cultured lung epithelial cells. Our data indicate that clearance of albumin from the distal air spaces is facilitated by an active, high-capacity, megalin-mediated transport process across the alveolar epithelium. Further understanding of this mechanism is of clinical importance, since an inability to clear excess protein from the alveolar space is associated with poor outcome in patients with acute lung injury/acute respiratory distress syndrome.