Neuronal Wiskott‐Aldrich syndrome protein regulates TGF‐β1‐mediated lung vascular permeability

Abstract

TGF-b1 induces an increase in paracellular permeability and actin stress fiber formation in lung microvascular endothelial and alveolar epithelial cells via small Rho GTPase. The molecular mechanism involved is not fully understood. Neuronal Wiskott-Aldrich syndrome protein (N-WASP) has an essential role in actin structure dynamics. We hypothesized that N-WASP plays a critical role in these TGF-b1-induced responses. In these cell monolayers, we demonstrated that N-WASP down-regulation by short hairpin RNA prevented TGF-b1-mediated disruption of the cortical actin structure, actin stress filament formation, and increased permeability. Furthermore, N-WASP down-regulation blocked TGF-b1 activation mediated by IL-1b in alveolar epithelial cells, which requires actin stress fiber formation. Control short hairpin RNA had no effect on these TGF-b1-induced responses. TGF-b1-induced phosphorylation of Y256 of N-WASP via activation of small Rho GTPase and focal adhesion kinase mediates TGF-b1-induced paracellular permeability and actin cytoskeleton dynamics. In vivo, compared with controls, N-WASP down-regulation increases survival and prevents lung edema in mice induced by bleomycin exposure-a lung injury model in which TGF-b1 plays a critical role. Our data indicate that N-WASP plays a crucial role in the development of TGF-b1-mediated acute lung injury by promoting pulmonary edema via regulation of actin cytoskeleton

Neuronal Wiskott‐Aldrich syndrome protein regulatesPseudomonas aeruginosa‐induced lung vascular permeability through the modulation of actin cytoskeletal dynamics

“…We have previously shown that N‐WASP is an important modulator of lung microvascular endothelial permeability To study whether N‐WASP modulates P aeruginosa‐ induced endothelial permeability, we downregulated N‐WASP expression in rat microvascular endothelial cells (RMVECs) and evaluated changes in endothelial paracellular permeability after P aeruginosa infection. The downregulation of N‐WASP expression was mediated by lentiviral shRNA as previously reported and confirmed at protein and mRNA levels . P aeruginosa infection decreased electrical resistance in RMVECs by 81% at 24 hours (Figure A,B, red).…”

“…Measurement of active TGF‐β1 was described previously . In brief, cells were challenged with P aeruginosa and the culture medium was collected after 6 hours.…”

“…In patients with ALI, transforming growth factor beta‐1 (TGF‐β1) expression and activation are elevated in bronchoalveolar lavage fluid . We have previously reported that N‐WASP mediates lung vascular permeability in response to TGF‐β1 …”

“…28 We have previously reported that N-WASP mediates lung vascular permeability in response to TGF-β1. 29 N-WASP has three independent domains at the C terminus, collectively termed the verprolin homology, central, and acidic regions (VCA) domain. 29,30 The middle region of N-WASP contains a GTPase-binding domain (GBD) and Rac interactive binding domain, which binds to small Rho GTPases and activates N-WASP.…”

Tristetraprolin Down-Regulation Contributes to Persistent TNF-Alpha Expression Induced by Cigarette Smoke Extract through a Post-Transcriptional Mechanism

“…Primary type II alveolar epithelial cells were derived from mice and cultured by the procedures described previously [33]. The rat alveolar epithelial cells (L2) were purchased from ATCC and were propagated and maintained in F-12K medium supplemented with 10% FBS and 100 units/ml penicillin/streptomycin as per ATCC instructions and as described [34]. …”