Care Med

Care Med. 155, 1187C1205 [PubMed] [Google Scholar] 14. Q. Neuronal WiskottCAldrich syndrome protein regulates TGF-1Cmediated lung vascular permeability. a RhoA-v6 integrinCdependent mechanism, and inhibition of active TGF-1 prevented the development of pulmonary edema induced by IL-1 in mice (11). Finally, adenoviral-mediated transfer of active TGF-1 to rat lungs induced perivascular and peribronchial edema (15). Taken together, these findings indicate that TGF-1 is a critical mediator in the development of lung edema associated with ALI. We and others have previously reported that activation of small Rho GTPase is responsible for actin stress fiber formation and increased paracellular permeability in lung endothelial cell monolayers induced by TGF-1 or other proinflammatory mediators (11, 16C21). Neuronal WiskottCAldrich syndrome protein (N-WASP) is a downstream effector of small Rho GTPases (22C26). The middle region Cipargamin of N-WASP contains the GTPase-binding domain (GBD)/Cdc42 and Rac interactive binding domain, which binds to small Rho GTPases and activates N-WASP (22C26). N-WASP has 3 independent small domains at the Cipargamin C terminus, collectively termed the verprolin homology, central, and acidic regions (VCA) domain. The VCA domain of N-WASP is necessary to activate actin polymerization an actin-related protein (Arp) 2/3-dependent mechanism (22C26). N-WASP regulates cytoskeletal dynamics and transmits upstream signals to the cellular machinery that is directly involved in modulation of actin filament structures, such Cipargamin as induction of new actin polymerization and actin structures (22, 23, 27C30); however, the role of N-WASP in actin stress fiber formation and increased paracellular permeability induced by a TGF-1 challenge is still unknown. In this scholarly study, we hypothesized that N-WASP would mediate TGF-1Cinduced changes in actin cytoskeleton dynamics in both lung microvascular endothelial Cipargamin cells and alveolar epithelial cells. We determined that changes in actin dynamics were associated with increased paracellular permeability across these cell monolayers. We demonstrate that IL-1Cdependent activation of TGF-1 RhoA-v6 integrin is N-WASP dependent also. Furthermore, TGF-1 induces phosphorylation of Y256 of N-WASP, and this phosphorylation mediates GREM1 increases in paracellular actin and permeability stress fiber formation. Activation of small Rho GTPase and focal adhesion kinase (FAK) is upstream of N-WASP in the signaling pathway activated by TGF-1 and, thus, is required for N-WASP activation. Finally, we show that N-WASP deficiency increases survival and protects mice against development of bleomycin-induced lung edema. In summary, we demonstrate that N-WASP is a critical mediator of TGF-1Cmediated lung permeability that is dependent on both small Rho GTPase and FAK. MATERIALS AND METHODS Reagents TGF-1 and IL-1 were obtained from R&D Systems (Minneapolis, MN, USA). The following purified antibodies were purchased: antiCN-WASP, anti-FAK, antiCphospho-Y256 of N-WASP (Millipore, Billerica, MA, USA); antiCphospho-Y397 of FAK (Cell Signaling Technology, Danvers, MA, USA); antiCglyceraldehyde 3-phosphate dehydrogenase (GAPDH) and anti-myc (Santa Cruz Biotechnology, Santa Cruz, CA, USA); and Alexa Fluor 488 phalloidin (Thermo Fisher Scientific Life Sciences, Waltham, MA, USA). Rho inhibitor I was purchased from Cytoskeleton (Denver, CO, USA) and PF573228 {6-[4-(3-methanesulfonyl-benzylamino)-5-trifluoromethyl-pyrimidin-2-ylamino]-3,4-dihydro-1bleomycin-induced ALI model All animal interventions were approved by the Institutional Animal Care and Use Committee at the University of Alabama at Birmingham. The administration of bleomycin and of adenoviral vectors were previously described (41). In brief, animals (8C12 wk old) were challenged with bleomycin (4 U/kg body weight) or saline by using an intratracheal catheter. Saline with or without recombinant adenoviral vectors [50 l, 108 plaque-forming units (pfu)] were instilled intratracheally 1 d before bleomycin (or saline) challenge as previously described (41). Measurement of lung edema Lung wet-to-dry ratios were determined as described (7 previously, 42). In brief, whole lungs were excised and dissected away from the thymus and heart. Lungs were weighed to obtain wet weight immediately, and were then dried and placed in an oven at 75C for 8 d to obtain dry weight. Survival Wild-type or N-WASPflox/flox mice.