Lipopolysaccharide Induces Actin Reorganization and Tyrosine Phosphorylation of Pyk2 and Paxillin in Monocytes and Macrophages

Williams, Lynn M.; Ridley, Anne J.

doi:10.4049/jimmunol.164.4.2028

Cited by 149 publications

(146 citation statements)

References 72 publications

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“…RAFTK/pyk2 activity and/or paxillin phosphorylation depends upon Src (35,41) because Src kinase inhibitors prevent increased phosphorylation of RAFTK/pyk2 as well as paxillin (42). Our results are consistent with RAFTK/pyk2-mediated loss of paxillin promoting apoptotic signaling by promotion of Src kinase activity.…”

Section: Discussionsupporting

confidence: 80%

Cardiomyocyte Apoptosis Triggered by RAFTK/pyk2 via Src Kinase Is Antagonized by Paxillin

Meléndez

Turner

Avraham

et al. 2004

Journal of Biological Chemistry

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Altered cellular adhesion and apoptotic signaling in cardiac remodeling requires coordinated regulation of multiple constituent proteins that comprise cytoskeletal focal adhesions. One such protein activated by cardiac remodeling is related adhesion focal tyrosine kinase (RAFTK, also known as pyk2). Adenoviral-mediated expression of RAFTK in neonatal rat cardiomyocytes involves concurrent increases in phosphorylation of Src, c-Jun N-terminal kinase, and p38 leading to characteristic apoptotic changes including cleavage of poly(ADP-ribose) polymerase, caspase-3 activation, and increased DNA laddering. DNA laddering was decreased by mutation of the Tyr 402 Src-binding site in RAFTK, suggesting a central role for Src activity in apoptotic cell death that was confirmed by adenoviralmediated Src expression. Multiple apoptotic signaling cascades are recruited by RAFTK as demonstrated by prevention of apoptosis using caspase-3 inhibitor IV (caspase-3 specific inhibitor), PP2 (Src-specific kinase inhibitor), or Csk (cellular negative regulator for Src), as well as dominant negative constructs for p38␤ or MKP-1. These RAFTK-mediated phenotypic characteristics are prevented by concurrent expression of wildtype or a phosphorylation-deficient paxillin mutated at Tyr 31 and Tyr 118 . Wild-type or mutant paxillin protein accumulation in the cytoplasm has no overt effect upon cell structure, but paxillin accumulation prevents losses of myofibril organization as well as focal adhesion kinase, vinculin, and paxillin protein levels mediated by RAFTK. Apoptotic signaling cascade inhibition by paxillin indicates interruption of signaling proximal to but downstream of RAFTK activity. Chronic RAFTK activation in cardiac remodeling may represent a maladaptive reactive response that can be modulated by paxillin, opening up novel possibilities for inhibition of cardiomyocyte apoptosis and structural degeneration in heart failure.Heart failure is characterized by cellular remodeling and apoptosis of cardiomyocytes (1-3). The precipitating stimulus of chronically impaired calcium handling promotes maladaptive remodeling via activation of calcium-dependent signaling cascades (4, 5) with chronic elevation of calcium influx leading to hypertrophy and heart failure in transgenic mice (6). Earlier work from our group demonstrated activation of related adhesion focal tyrosine kinase (RAFTK, also known as pyk2) signaling in cultured cardiomyocytes treated with ionomycin (7) as well as a murine model of dilated cardiomyopathy exhibiting chronic elevation of intracellular calcium levels (8, 9). Concurrent with RAFTK/pyk2 activation, heart samples from cardiomyopathic mice showed enhanced paxillin phosphorylation (7). Thus, paxillin was implicated in the RAFTK/pyk2 signaling cascade leading to heart failure as a target substrate of RAFTK/pyk2-mediated phosphorylation.RAFTK/pyk2 signaling has been extensively characterized in nonmuscle cells where postulated effects include coordinate regulation of cytoskeletal protein phosphorylation in combination...

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Section: Discussionsupporting

confidence: 80%

Cardiomyocyte Apoptosis Triggered by RAFTK/pyk2 via Src Kinase Is Antagonized by Paxillin

Meléndez

Turner

Avraham

et al. 2004

Journal of Biological Chemistry

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“…These differences between AM and NM are consistent with previous literature reporting macrophage polarization upon stimulation with LPS. 47 …”

Section: Culture Of Chondrocytes and Activation Of Macrophagesmentioning

confidence: 99%

A Three-Dimensional Chondrocyte-Macrophage Coculture System to Probe Inflammation in Experimental Osteoarthritis

Samavedi

Díaz‐Rodríguez

Erndt-Marino

et al. 2017

Tissue Engineering Part A

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The goal of the present study was to develop a fully three-dimensional (3D) coculture system that would allow for systematic evaluation of the interplay between activated macrophages (AMs) and chondrocytes in osteoarthritic disease progression and treatment. Toward this end, our coculture system was first validated against existing in vitro osteoarthritis models, which have generally cultured healthy normal chondrocytes (NCs)-in two-dimensional (2D) or 3D-with proinflammatory AMs in 2D. In this work, NCs and AMs were both encapsulated within poly(ethylene glycol) diacrylate hydrogels to mimic the native 3D environments of both cell types within the osteoarthritic joint. As with previous studies, increases in matrix metalloproteinases (MMPs) and proinflammatory cytokines associated with the early stages of osteoarthritis were observed during NC-AM coculture, as were decreases in protein-level Aggrecan and collagen II. Thereafter, the coculture system was extended to osteoarthritic chondrocytes (OACs) and AMs to evaluate the potential effects of AMs on pre-existing osteoarthritic phenotypes. OACs in coculture with AMs expressed significantly higher levels of MMP-1, MMP-3, MMP-9, MMP-13, IL-1b, TNF-a, IL-6, IL-8, and IFN-g compared to OACs in mono-culture, indicating that proinflammatory macrophages may intensify the abnormal matrix degradation and cytokine secretion already associated with OACs. Likewise, AMs cocultured with OACs expressed significantly more IL-1b and VEGF-A compared to AM mono-culture controls, suggesting that OACs may intensify abnormal macrophage activation. Finally, OACs cultured in the presence of nonactivated macrophages produced lower levels of MMP-9 and proinflammatory cytokines IL-1b, TNF-a, and IFN-g compared to OACs in the OAC-AM system, results that are consistent with anti-inflammatory agents temporarily reducing certain OA symptoms. In summary, the 3D coculture system developed herein captures several key features of inflammatory OA and may prove useful in future screening of therapeutic agents and/or assessment of disease progression mechanisms.

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“…Various external stimuli have been shown to alter the morphologic phenotype of macrophages which in turn governs the functions of the cells (22,23). Accordingly, J774 macrophages were examined by videomicroscopy after TNF-␣ or oxidant addition.…”

Section: Tnf-␣ and H 2 O 2 Induced Morphologic Changes In J774 Macropmentioning

confidence: 99%

TNF-α Inhibits Macrophage Clearance of Apoptotic Cells via Cytosolic Phospholipase A2 and Oxidant-Dependent Mechanisms

McPhillips

Janssen

Ghosh

et al. 2007

The Journal of Immunology

100

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Removal of apoptotic cells from inflammatory sites is an important step in the resolution of inflammation. Both murine and human macrophages stimulated with TNF-α or directly administered arachidonic acid showed an impaired ability to ingest apoptotic cells (efferocytosis). The inhibition was shown to be due to generation of reactive oxygen species, was blocked with a superoxide dismutase mimetic, MnTBAP, and was mimicked by direct addition of H2O2. To determine the mechanism of TNF-α-stimulated oxidant production, bone marrow-derived macrophages from gp91phox-deficient mice were examined but shown to still produce oxidants and exhibit defective apoptotic cell uptake. In contrast, a specific cytosolic phospholipase A2 inhibitor blocked the oxidant production and reversed the inhibited uptake. The suppressive effect of endogenous or exogenous oxidants on efferocytosis was mediated through activation of the GTPase, Rho. It was reversed in macrophages pretreated with C3 transferase to inactivate Rho or with an inhibitor of Rho kinase. During maturation of human monocyte-derived macrophages, only mature cells exhibited TNF-α-induced suppression of apoptotic cell clearance. The resistance of immature macrophages to such inhibition was shown to result not from defective generation of oxidants, but rather, from lack of response of these cells to the oxidants. Overall, the data suggest that macrophages in a TNF-α- and oxidant-rich inflammatory environment are less able to remove apoptotic cells and, thereby, may contribute to the local intensity of the inflammatory response.

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Lipopolysaccharide Induces Actin Reorganization and Tyrosine Phosphorylation of Pyk2 and Paxillin in Monocytes and Macrophages

Cited by 149 publications

References 72 publications

Cardiomyocyte Apoptosis Triggered by RAFTK/pyk2 via Src Kinase Is Antagonized by Paxillin

Cardiomyocyte Apoptosis Triggered by RAFTK/pyk2 via Src Kinase Is Antagonized by Paxillin

A Three-Dimensional Chondrocyte-Macrophage Coculture System to Probe Inflammation in Experimental Osteoarthritis

TNF-α Inhibits Macrophage Clearance of Apoptotic Cells via Cytosolic Phospholipase A2 and Oxidant-Dependent Mechanisms

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