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  • 1
    Online Resource
    Online Resource
    Mimicking transient activation of protein kinases in living cells
    Proceedings of the National Academy of Sciences ; 2016
    In:  Proceedings of the National Academy of Sciences Vol. 113, No. 52 ( 2016-12-27), p. 14976-14981
    Details
    In: Proceedings of the National Academy of Sciences, Proceedings of the National Academy of Sciences, Vol. 113, No. 52 ( 2016-12-27), p. 14976-14981
    Abstract: Physiological stimuli activate protein kinases for finite periods of time, which is critical for specific biological outcomes. Mimicking this transient biological activity of kinases is challenging due to the limitations of existing methods. Here, we report a strategy enabling transient kinase activation in living cells. Using two protein-engineering approaches, we achieve independent control of kinase activation and inactivation. We show successful regulation of tyrosine kinase c-Src (Src) and Ser/Thr kinase p38α (p38), demonstrating broad applicability of the method. By activating Src for finite periods of time, we reveal how the duration of kinase activation affects secondary morphological changes that follow transient Src activation. This approach highlights distinct roles for sequential Src-Rac1– and Src-PI3K–signaling pathways at different stages during transient Src activation. Finally, we demonstrate that this method enables transient activation of Src and p38 in a specific signaling complex, providing a tool for targeted regulation of individual signaling pathways.
    Type of Medium: Online Resource
    ISSN: 0027-8424 , 1091-6490
    URL: Article
    DOI: 10.1073/pnas.1609675114
    RVK:
    TA 1000
    RVK:
    WA 15000
    Language: English
    Publisher: Proceedings of the National Academy of Sciences
    Publication Date: 2016
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  • 2
    Online Resource
    Online Resource
    TNFα-stimulated gene-6 (TSG6) activates macrophage phenotype transition to prevent inflammatory lung injury
    Proceedings of the National Academy of Sciences ; 2016
    In:  Proceedings of the National Academy of Sciences Vol. 113, No. 50 ( 2016-12-13)
    Details
    In: Proceedings of the National Academy of Sciences, Proceedings of the National Academy of Sciences, Vol. 113, No. 50 ( 2016-12-13)
    Abstract: TNFα-stimulated gene-6 (TSG6), a 30-kDa protein generated by activated macrophages, modulates inflammation; however, its mechanism of action and role in the activation of macrophages are not fully understood. Here we observed markedly augmented LPS-induced inflammatory lung injury and mortality in TSG6 −/− mice compared with WT ( TSG6 +/+ ) mice. Treatment of mice with intratracheal instillation of TSG6 prevented LPS-induced lung injury and neutrophil sequestration, and increased survival in mice. We found that TSG6 inhibited the association of TLR4 with MyD88, thereby suppressing NF-κB activation. TSG6 also prevented the expression of proinflammatory proteins (iNOS, IL-6, TNFα, IL-1β, and CXCL1) while increasing the expression of anti-inflammatory proteins (CD206, Chi3l3, IL-4, and IL-10) in macrophages. This shift was associated with suppressed activation of proinflammatory transcription factors STAT1 and STAT3. In addition, we observed that LPS itself up-regulated the expression of TSG6 in TSG6 +/+ mice, suggesting an autocrine role for TSG6 in transitioning macrophages. Thus, TSG6 functions by converting macrophages from a proinflammatory to an anti-inflammatory phenotype secondary to suppression of TLR4/NF-κB signaling and STAT1 and STAT3 activation.
    Type of Medium: Online Resource
    ISSN: 0027-8424 , 1091-6490
    URL: Article
    DOI: 10.1073/pnas.1614935113
    RVK:
    TA 1000
    RVK:
    WA 15000
    Language: English
    Publisher: Proceedings of the National Academy of Sciences
    Publication Date: 2016
    detail.hit.zdb_id: 209104-5
    detail.hit.zdb_id: 1461794-8
    SSG: 11
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  • 3
    Online Resource
    Online Resource
    Transport of nitrated albumin across continuous vascular endothelium
    Proceedings of the National Academy of Sciences ; 2002
    In:  Proceedings of the National Academy of Sciences Vol. 99, No. 21 ( 2002-10-15), p. 13932-13937
    Details
    In: Proceedings of the National Academy of Sciences, Proceedings of the National Academy of Sciences, Vol. 99, No. 21 ( 2002-10-15), p. 13932-13937
    Abstract: Because modification of plasma albumin on tyrosine residues generates nitrated albumin (NOA) that may function as a mechanism of nitrogen monoxide clearance from microcirculation, we investigated biochemicaly and morphologically the cell surface binding and the transendothelial transport of NOA. An electron microscopic study was carried out with mouse lungs and hearts perfused in situ with NOA and NOA-Au complexes. The results indicate that NOA-Au can bind to the endothelial cell surface, and its binding can be blocked by albumin plus nitrotyrosine (NO-tyrosine) or abolished by excess NOA. We detected NOA-Au into perivascular spaces as early as 30 sec after the beginning of its perfusion. NOA, unlike native albumin, leaves the vascular lumina via both endothelial caveolae and open junctions. By cross-linking and ligand blotting analysis, we showed that NOA interacted with the same albumin binding proteins of 16–18, 30–32, 60, and 74 kDa as native albumin. ELISA performed on tissue homogenates obtained from the same specimens showed that NOA transport was 2- to 4-fold greater than native albumin. The augmented transendothelial transport of NOA reflects its transcytosis as well as its exit from the microcirculation via open junctions. The increased transport of NOA may serve as an important mechanism that protects a vascular bed against the damaging effects of nitrogen monoxide and peroxynitrite.
    Type of Medium: Online Resource
    ISSN: 0027-8424 , 1091-6490
    URL: Article
    DOI: 10.1073/pnas.212253499
    RVK:
    TA 1000
    RVK:
    WA 15000
    Language: English
    Publisher: Proceedings of the National Academy of Sciences
    Publication Date: 2002
    detail.hit.zdb_id: 209104-5
    detail.hit.zdb_id: 1461794-8
    SSG: 11
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  • 4
    Online Resource
    Online Resource
    Nanoparticle targeting of de novo profibrotic macrophages mitigates lung fibrosis
    Proceedings of the National Academy of Sciences ; 2022
    In:  Proceedings of the National Academy of Sciences Vol. 119, No. 15 ( 2022-04-12)
    Details
    In: Proceedings of the National Academy of Sciences, Proceedings of the National Academy of Sciences, Vol. 119, No. 15 ( 2022-04-12)
    Abstract: The pathogenesis of lung fibrosis involves hyperactivation of innate and adaptive immune pathways that release inflammatory cytokines and growth factors such as tumor growth factor (TGF)β1 and induce aberrant extracellular matrix protein production. During the genesis of pulmonary fibrosis, resident alveolar macrophages are replaced by a population of newly arrived monocyte-derived interstitial macrophages that subsequently transition into alveolar macrophages (Mo-AMs). These transitioning cells initiate fibrosis by releasing profibrotic cytokines and remodeling the matrix. Here, we describe a strategy for leveraging the up-regulation of the mannose receptor CD206 in interstitial macrophages and Mo-AM to treat lung fibrosis. We engineered mannosylated albumin nanoparticles, which were found to be internalized by fibrogenic CD206 + monocyte derived macrophages (Mo-Macs). Mannosylated albumin nanoparticles incorporating TGFβ1 small-interfering RNA (siRNA) targeted the profibrotic subpopulation of CD206 + macrophages and prevented lung fibrosis. The findings point to the potential utility of mannosylated albumin nanoparticles in delivering TGFβ-siRNA into CD206 + profibrotic macrophages as an antilung fibrosis strategy.
    Type of Medium: Online Resource
    ISSN: 0027-8424 , 1091-6490
    URL: Article
    DOI: 10.1073/pnas.2121098119
    RVK:
    TA 1000
    RVK:
    WA 15000
    Language: English
    Publisher: Proceedings of the National Academy of Sciences
    Publication Date: 2022
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    detail.hit.zdb_id: 1461794-8
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  • 5
    Online Resource
    Online Resource
    STAT6 induces expression of Gas6 in macrophages to clear apoptotic neutrophils and resolve inflammation
    Proceedings of the National Academy of Sciences ; 2019
    In:  Proceedings of the National Academy of Sciences Vol. 116, No. 33 ( 2019-08-13), p. 16513-16518
    Details
    In: Proceedings of the National Academy of Sciences, Proceedings of the National Academy of Sciences, Vol. 116, No. 33 ( 2019-08-13), p. 16513-16518
    Abstract: Efferocytosis of apoptotic neutrophils (PMNs) by alveolar macrophages (AMФs) is vital for resolution of inflammation and tissue injury. Here, we investigated the role of AMФ polarization and expression of the efferocytic ligand Gas6 in restoring homeostasis. In the murine model of lipopolysaccharide (LPS)-induced acute lung injury (ALI), we observed augmented temporal generation of cytokines IL-4 and TSG6 in bronchoalveolar fluid (BALF). Interestingly, we also observed increased expression of antiinflammatory markers consistent with a phenotype shift in AMФs. In particular, AMФs expressed the efferocytic ligand Gas6. In vitro priming of bone marrow–derived macrophages (BMMФs) with IL-4 or TSG6 also induced MФ transition and expression of Gas6. TSG6- or IL-4–primed BMMФs induced efferocytosis of apoptotic PMNs compared with control BMMФs. Adoptive transfer of TSG6- or IL-4–primed BMMФs i.t. into LPS-challenged mice more rapidly and effectively cleared PMNs in lungs compared with control BMMФs. We demonstrated that expression of Gas6 during AMФ transition was due to activation of the transcription factor signal transducer and activator of transcription-6 (STAT6) downstream of IL-4 or TSG6 signaling. Adoptive transfer of Gas6-depleted BMMФs failed to clear PMNs in lungs following LPS challenge and mice showed severely defective resolution of lung injury. Thus, activation of STAT6-mediated Gas6 expression during macrophage phenotype transition resulting in efferocytosis of PMNs plays a crucial role in the resolution of inflammatory lung injury.
    Type of Medium: Online Resource
    ISSN: 0027-8424 , 1091-6490
    URL: Article
    DOI: 10.1073/pnas.1821601116
    RVK:
    TA 1000
    RVK:
    WA 15000
    Language: English
    Publisher: Proceedings of the National Academy of Sciences
    Publication Date: 2019
    detail.hit.zdb_id: 209104-5
    detail.hit.zdb_id: 1461794-8
    SSG: 11
    SSG: 12
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  • 6
    Online Resource
    Online Resource
    Endothelial cell Piezo1 mediates pressure-induced lung vascular hyperpermeability via disruption of adherens junctions
    Proceedings of the National Academy of Sciences ; 2019
    In:  Proceedings of the National Academy of Sciences Vol. 116, No. 26 ( 2019-06-25), p. 12980-12985
    Details
    In: Proceedings of the National Academy of Sciences, Proceedings of the National Academy of Sciences, Vol. 116, No. 26 ( 2019-06-25), p. 12980-12985
    Abstract: Increased pulmonary microvessel pressure experienced in left heart failure, head trauma, or high altitude can lead to endothelial barrier disruption referred to as capillary “stress failure” that causes leakage of protein-rich plasma and pulmonary edema. However, little is known about vascular endothelial sensing and transduction of mechanical stimuli inducing endothelial barrier disruption. Piezo1, a mechanosensing ion channel expressed in endothelial cells (ECs), is activated by elevated pressure and other mechanical stimuli. Here, we demonstrate the involvement of Piezo1 in sensing increased lung microvessel pressure and mediating endothelial barrier disruption. Studies were made in mice in which Piezo1 was deleted conditionally in ECs ( Piezo1 iΔEC ), and lung microvessel pressure was increased either by raising left atrial pressure or by aortic constriction. We observed that lung endothelial barrier leakiness and edema induced by raising pulmonary microvessel pressure were abrogated in Piezo1 iΔEC mice. Piezo1 signaled lung vascular hyperpermeability by promoting the internalization and degradation of the endothelial adherens junction (AJ) protein VE-cadherin. Breakdown of AJs was the result of activation of the calcium-dependent protease calpain and degradation of the AJ proteins VE-cadherin, β-catenin, and p120-catenin. Deletion of Piezo1 in ECs or inhibition of calpain similarly prevented reduction in the AJ proteins. Thus, Piezo1 activation in ECs induced by elevated lung microvessel pressure mediates capillary stress failure and edema formation secondary to calpain-induced disruption of VE-cadherin adhesion. Inhibiting Piezo1 signaling may be a useful strategy to limit lung capillary stress failure injury in response to elevated vascular pressures.
    Type of Medium: Online Resource
    ISSN: 0027-8424 , 1091-6490
    URL: Article
    DOI: 10.1073/pnas.1902165116
    RVK:
    TA 1000
    RVK:
    WA 15000
    Language: English
    Publisher: Proceedings of the National Academy of Sciences
    Publication Date: 2019
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    SSG: 11
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  • 7
    Online Resource
    Online Resource
    Albumin mediates the transcytosis of myeloperoxidase by means of caveolae in endothelial cells
    Proceedings of the National Academy of Sciences ; 2004
    In:  Proceedings of the National Academy of Sciences Vol. 101, No. 20 ( 2004-05-18), p. 7699-7704
    Details
    In: Proceedings of the National Academy of Sciences, Proceedings of the National Academy of Sciences, Vol. 101, No. 20 ( 2004-05-18), p. 7699-7704
    Abstract: Myeloperoxidase (MPO), the phagocyte hemoprotein involved in neutrophil host defense and consuming nitric oxide ( • NO), induces the nitration of extracellular matrix proteins and tissue remodeling subsequent to its transcytosis across the endothelial barrier. We addressed the role of an interaction of MPO with albumin as a requirement for MPO transport across the endothelium. Matrix-assisted laser desorption/ionization MS analysis of 80- and 60-kDa proteins purified from human lung tissue [with a human serum albumin (HSA)-affinity column] identified these albumin-binding proteins as MPO and MPO-heavy chain. A peptide corresponding to the MPO-heavy chain residues 425–454 demonstrated high-affinity binding to HSA. Replacement of the positively charged residues, R and K with G, prevented the binding of HSA to the peptide. We observed that albumin increased the binding of 125 I-MPO to lung microvascular endothelial cells by 2-fold and the rate of transendothelial flux of 125 I-MPO in cultured monolayers and intact vessels. Disruption of caveolae with cyclodextrin prevented the albumin-induced increase in transendothelial flux of 125 I-MPO. We also observed by confocal imaging that albumin induced the rapid internalization of MPO and its colocalization with albumin-labeled vesicles. MPO colocalized with the caveolae markers cholera toxin subunit B and caveolin 1 in the endocytosed vesicles. Thus, transcytosis of MPO by caveolae induced by its charge-dependent interaction with albumin is an important means of delivering MPO to the subendothelial space. Albumin-mediated transport of MPO may thereby regulate NO bioavailability and formation of NO-derived oxidants in the vessel wall.
    Type of Medium: Online Resource
    ISSN: 0027-8424 , 1091-6490
    URL: Article
    DOI: 10.1073/pnas.0401712101
    RVK:
    TA 1000
    RVK:
    WA 15000
    Language: English
    Publisher: Proceedings of the National Academy of Sciences
    Publication Date: 2004
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    detail.hit.zdb_id: 1461794-8
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  • 8
    Online Resource
    Online Resource
    G protein-coupled receptor kinase-5 regulates thrombin-activated signaling in endothelial cells
    Proceedings of the National Academy of Sciences ; 2000
    In:  Proceedings of the National Academy of Sciences Vol. 97, No. 13 ( 2000-06-20), p. 7440-7445
    Details
    In: Proceedings of the National Academy of Sciences, Proceedings of the National Academy of Sciences, Vol. 97, No. 13 ( 2000-06-20), p. 7440-7445
    Abstract: We studied the function of G protein-coupled receptor kinases (GRKs) in the regulation of thrombin-activated signaling in endothelial cells. GRK2, GRK5, and GRK6 isoforms were expressed predominantly in endothelial cells. The function of these isoforms was studied by expressing wild-type and dominant negative (dn) mutants in endothelial cells. We determined the responses to thrombin, which activates intracellular signaling in endothelial cells by cleaving the NH 2 terminus of the G protein-coupled proteinase-activated receptor-1 (PAR-1). We measured changes in phosphoinositide hydrolysis and intracellular Ca 2+ concentration ([Ca 2+ ] i ) in response to thrombin as well as the state of endothelial activation. In the latter studies, the transendothelial monolayer electrical resistance, a measure of the loss of endothelial barrier function, was measured in real time. Of the three isoforms, GRK5 overexpression was selective in markedly reducing the thrombin-activated phosphoinositide hydrolysis and increased [Ca 2+ ] i . GRK5 overexpression also inhibited the thrombin-induced decrease in endothelial monolayer resistance by 75%. These effects of GRK5 overexpression occurred in association with the specific increase in the thrombin-induced phosphorylation of PAR-1. In contrast to the effects of GRK5 overexpression, the expression of the dn-GRK5 mutant produced a long-lived increase in [Ca 2+ ] i in response to thrombin, whereas dn-GRK2 had no effect. These results indicate the crucial role of the GRK5 isoform in the mechanism of thrombin-induced desensitization of PAR-1 in endothelial cells.
    Type of Medium: Online Resource
    ISSN: 0027-8424 , 1091-6490
    URL: Article
    DOI: 10.1073/pnas.97.13.7440
    RVK:
    TA 1000
    RVK:
    WA 15000
    Language: English
    Publisher: Proceedings of the National Academy of Sciences
    Publication Date: 2000
    detail.hit.zdb_id: 209104-5
    detail.hit.zdb_id: 1461794-8
    SSG: 11
    SSG: 12
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  • 9
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    Online Resource
    Protease-activated receptor-3 (PAR3) regulates PAR1 signaling by receptor dimerization
    Proceedings of the National Academy of Sciences ; 2007
    In:  Proceedings of the National Academy of Sciences Vol. 104, No. 13 ( 2007-03-27), p. 5662-5667
    Details
    In: Proceedings of the National Academy of Sciences, Proceedings of the National Academy of Sciences, Vol. 104, No. 13 ( 2007-03-27), p. 5662-5667
    Abstract: Thrombin activates endothelial cell signaling by cleaving the protease-activated receptor-1 (PAR1). However, the function of the apparently nonsignaling receptor PAR3 also expressed in endothelial cells is unknown. We demonstrate here the crucial role of PAR3 in potentiating the responsiveness of PAR1 to thrombin. We tested the hypothesis that PAR1/PAR3 heterodimerization and its effect in modifying G protein selectivity was responsible for PAR3 regulation of PAR1 sensitivity. Using bioluminescent resonance energy transfer-2, we showed that PAR1 had comparable dimerization affinity for PAR3 as for itself. We observed increased Gα 13 coupling between the PAR1/3 heterodimer compared with the PAR1/1 homodimer. Moreover, knockdown of PAR3 moderated the PAR1-activated increase in endothelial permeability. These results demonstrate a role of PAR3 in allosterically regulating PAR1 signaling governing increased endothelial permeability. Because PAR3 is a critical determinant of PAR1 function, targeting of PAR3 may mitigate the effects of PAR1 in activating endothelial responses such as vascular inflammation.
    Type of Medium: Online Resource
    ISSN: 0027-8424 , 1091-6490
    URL: Article
    DOI: 10.1073/pnas.0700763104
    RVK:
    TA 1000
    RVK:
    WA 15000
    Language: English
    Publisher: Proceedings of the National Academy of Sciences
    Publication Date: 2007
    detail.hit.zdb_id: 209104-5
    detail.hit.zdb_id: 1461794-8
    SSG: 11
    SSG: 12
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