http://hdl.handle.net/10027/7420 Sun, 26 May 2013 07:25:15 GMT 2013-05-26T07:25:15Z http://hdl.handle.net/10027/8458 An Angiotensin I-Converting Enzyme Mutation (Y465D) Causes a Dramatic Increase in Blood ACE via Accelerated ACE Shedding Danilov, Sergei M.; Gordon, Kerry; Nesterovitch, Andrew B.; Lu¨ nsdorf, Heinrich; Chen, Zhenlong; Castellon, Maricela; Popova, Isolda A.; Kalinin, Sergey; Mendonca, Emma; Petukhov, Pavel A.; Schwartz, David E.; Minshall, Richard D.; Sturrock, Edward D. Background: Angiotensin I-converting enzyme (ACE) metabolizes a range of peptidic substrates and plays a key role in blood pressure regulation and vascular remodeling. Thus, elevated ACE levels may be associated with an increased risk for different cardiovascular or respiratory diseases. Previously, a striking familial elevation in blood ACE was explained by mutations in the ACE juxtamembrane region that enhanced the cleavage-secretion process. Recently, we found a family whose affected members had a 6-fold increase in blood ACE and a Tyr465Asp (Y465D) substitution, distal to the stalk region, in the N domain of ACE. Methodology/Principal Findings: HEK and CHO cells expressing mutant (Tyr465Asp) ACE demonstrate a 3- and 8-fold increase, respectively, in the rate of ACE shedding compared to wild-type ACE. Conformational fingerprinting of mutant ACE demonstrated dramatic changes in ACE conformation in several different epitopes of ACE. Cell ELISA carried out on CHOACE cells also demonstrated significant changes in local ACE conformation, particularly proximal to the stalk region. However, the cleavage site of the mutant ACE - between Arg1203 and Ser1204 - was the same as that of WT ACE. The Y465D substitution is localized in the interface of the N-domain dimer (from the crystal structure) and abolishes a hydrogen bond between Tyr465 in one monomer and Asp462 in another. Conclusions/Significance: The Y465D substitution results in dramatic increase in the rate of ACE shedding and is associated with significant local conformational changes in ACE. These changes could result in increased ACE dimerization and accessibility of the stalk region or the entire sACE, thus increasing the rate of cleavage by the putative ACE secretase (sheddase). Copyright © 2011 Danilov et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.DOI: 10.1371/journal.pone.0025952 Sat, 01 Oct 2011 05:00:00 GMT http://hdl.handle.net/10027/8458 2011-10-01T05:00:00Z http://hdl.handle.net/10027/8439 Src Phosphorylation of Endothelial Cell Surface ICAM-1 Mediates Neutrophil Adhesion and Contributes to the Mechanism of Lung Inflammation Liu, Guoquan; Vogel, Stephen M.; Gao, Xiaopei; Javaid, Kamran; Hu, Guochang; Danilov, Sergei M.; Malik, Asrar B.; Minshall, Richard D. Objective: To determine whether TNFα-induced Src activation and ICAM-1 phosphorylation rapidly increases endothelial cell adhesivity and PMN sequestration independent of de novo ICAM-1 synthesis. Methods and Results: TNFα exposure of mouse lungs for 5 min produced a 3-fold increase in 125I-anti-ICAM-1 mAb binding and 111In oxine-labeled PMN sequestration as well as Src activation, ICAM-1 Tyr518 phosphorylation, and pTyr518-ICAM-1 co-immunoprecipitation with actin. The response was absent in Nox2-/- lungs or following Src inhibition. In COS-7 cells transfected with wild-type (WT), phospho-defective (Y518F), or phospho-mimicking (Y518D) mouse ICAM-1 cDNA constructs, TNFα increased the Bmax of YN1/1.7.4 anti-ICAM-1 mAb binding to WT-ICAM-1 but not to Y518F-ICAM-1 indicating increased binding avidity secondary to ICAM-1 phosphorylation. This effect was mimicked by expression of the Y518DICAM- 1 mutant. TNFα also increased the staining intensity and cell surface clustering of YN1/1.7.4 mAb-labeled WT-ICAM-1 that co-localized with F-actin which was not observed with Y518F-ICAM-1 but was recapitulated with Y518D-ICAM-1. Finally, overexpression of ICAM-1 in mouse lungs significantly increased LPS-induced transvascular albumin leakage and bronchoalveolar lavage PMN counts at 2 and 24 hrs after LPS inhalation compared to lungs expressing Y518F ICAM-1 mutant. Conclusions: Src-dependent phosphorylation of endothelial cell ICAM-1 Tyr518 induces PMN adhesion by promoting ICAM-1 clustering which we propose mediates rapid-phase lung vascular accumulation of PMNs during inflammation. Post print version of article may differ from published version. The definitive version is available through American Heart Association at DOI: 10.1161/ATVBAHA.110.222208 Wed, 01 Jun 2011 05:00:00 GMT http://hdl.handle.net/10027/8439 2011-06-01T05:00:00Z http://hdl.handle.net/10027/8365 Nitric oxide–dependent Src activation and resultant caveolin-1 phosphorylation promote eNOS/caveolin-1 binding and eNOS inhibition Chen, Zhenlong; Bakhshi, Farnaz R.; Shajahan, Ayesha N.; Sharma, Tiffany; Mao, Mao; Trane, Andy; Bernatchez, Pascal; van Nieuw Amerongen, Geerten P.; Bonini, Marcelo G.; Skidgel, Randal A.; Malik, Asrar B.; Minshall, Richard D. Endothelial nitric oxide synthase (eNOS)–mediated NO production plays a critical role in the regulation of vascular function and pathophysiology. Caveolin-1 (Cav-1) binding to eNOS holds eNOS in an inactive conformation; however, the mechanism of Cav-1–mediated inhibition of activated eNOS is unclear. Here the role of Src-dependent Cav-1 phosphorylation in eNOS negative feedback regulation is investigated. Using fluorescence resonance energy transfer (FRET) and coimmunoprecipitation analyses, we observed increased interaction between eNOS and Cav-1 following stimulation of endothelial cells with thrombin, vascular endothelial growth factor, and Ca2+ ionophore A23187, which is corroborated in isolated perfused mouse lung. The eNOS/Cav-1 interaction is blocked by eNOS inhibitor l-NGnitroarginine methyl ester (hydrochloride) and Src kinase inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl) pyrazolo [3, 4-d] pyrimidine. We also observe increased binding of phosphomimicking Y14D-Cav-1 mutant transduced in human embryonic kidney cells overexpressing eNOS and reduced Ca2+-induced NO production compared to cells expressing the phosphodefective Y14F-Cav-1 mutant. Finally, Src FRET biosensor, eNOS small interfering RNA, and NO donor studies demonstrate NO-induced Src activation and Cav-1 phosphorylation at Tyr-14, resulting in increased eNOS/Cav-1 interaction and inhibition of eNOS activity. Taken together, these data suggest that activation of eNOS promotes Src-dependent Cav-1–Tyr-14 phosphorylation and eNOS/Cav-1 binding, that is, eNOS feedback inhibition. © 2012 Chen et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0). “ASCB®,“ “The American Society for Cell Biology®,” and “Molecular Biology of the Cell®” are registered trademarks of The American Society of Cell Biology. Sun, 01 Jan 2012 06:00:00 GMT http://hdl.handle.net/10027/8365 2012-01-01T06:00:00Z http://hdl.handle.net/10027/7743 The mTOR kinase inhibitor rapamycin decreases iNOS mRNA stability in astrocytes Lisi, Lucia; Navarra, Pierluigi; Feinstein, Douglas L.; Russo, Cinzia D. Background: Reactive astrocytes are capable of producing a variety of pro-inflammatory mediators and potentially neurotoxic compounds, including nitric oxide (NO). High amounts of NO are synthesized following up-regulation of inducible NO synthase (iNOS). The expression of iNOS is tightly regulated by complex molecular mechanisms, involving both transcriptional and post-transcriptional processes. The mammalian target of rapamycin (mTOR) kinase modulates the activity of some proteins directly involved in post-transcriptional processes of mRNA degradation. mTOR is a serine-threonine kinase that plays an evolutionarily conserved role in the regulation of cell growth, proliferation, survival, and metabolism. It is also a key regulator of intracellular processes in glial cells. However, with respect to iNOS expression, both stimulatory and inhibitory actions involving the mTOR pathway have been described. In this study the effects of mTOR inhibition on iNOS regulation were evaluated in astrocytes. Methods: Primary cultures of rat cortical astrocytes were activated with different proinflammatory stimuli, namely a mixture of cytokines (TNF alpha, IFN gamma, and IL-1 beta) or by LPS plus IFN gamma. Rapamycin was used at nM concentrations to block mTOR activity and under these conditions we measured its effects on the iNOS promoter, mRNA and protein levels. Functional experiments to evaluate iNOS activity were also included. Results: In this experimental paradigm mTOR activation did not significantly affect astrocyte iNOS activity, but mTOR pathway was involved in the regulation of iNOS expression. Rapamycin did not display any significant effects under basal conditions, on either iNOS activity or its expression. However, the drug significantly increased iNOS mRNA levels after 4 h incubation in presence of pro-inflammatory stimuli. This stimulatory effect was transient, since no differences in either iNOS mRNA or protein levels were detected after 24 h. Interestingly, reduced levels of iNOS mRNA were detected after 48 hours, suggesting that rapamycin can modify iNOS mRNA stability. In this regard, we found that rapamycin significantly reduced the half-life of iNOS mRNA, from 4 h to 50 min when cells were co-incubated with cytokine mixture and 10 nM rapamycin. Similarly, rapamycin induced a significant up-regulation of tristetraprolin (TTP), a protein involved in the regulation of iNOS mRNA stability. Conclusion: The present findings show that mTOR controls the rate of iNOS mRNA degradation in astrocytes. Together with the marked anti-inflammatory effects that we previously observed in microglial cells, these data suggest possible beneficial effects of mTOR inhibitors in the treatment of inflammatory-based CNS pathologies. © 2011 Lisi et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The original source is available through BioMed Central at DOI: 10.1186/1742-2094-8-1 Tue, 05 Jan 2010 06:00:00 GMT http://hdl.handle.net/10027/7743 2010-01-05T06:00:00Z