TNF induces caspase-dependent inflammation in renal endothelial cells through a Rho- and myosin light chain kinase-dependent mechanism

Wu, Xiaoyan; Guo, Rongqing; Chen, Peili; Wang, Quan; Cunningham, Patrick N.

doi:10.1152/ajprenal.00089.2009

Cited by 83 publications

(73 citation statements)

References 72 publications

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“…Few studies have proposed that cytokines might induce endothelial barrier dysfunction via apoptosis (17,(48)(49)(50)(51). However, one report suggests that primary cultures of human BECs are resistant to cell death mediated by TNF-a (18), although this resistance to apoptosis is not observed in primary cultures of BECs of other species (13,48).…”

Section: Discussionmentioning

confidence: 99%

Role of Caspases in Cytokine-Induced Barrier Breakdown in Human Brain Endothelial Cells

Lopez-Ramirez

Fischer

Torres-Badillo

et al. 2012

The Journal of Immunology

114

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During neuroinflammation, cytokines such as TNF-α and IFN-γ secreted by activated leukocytes and/or CNS resident cells have been shown to alter the phenotype and function of brain endothelial cells (BECs) leading to blood–brain barrier breakdown. In this study, we show that the human BEC line hCMEC/D3 expresses the receptors for TNF-α, TNF receptor 1 and TNF receptor 2, and for IFN-γ. BEC activation with TNF-α alone or in combination with IFN-γ induced endothelial leakage of paracellular tracers. At high cytokine concentrations (10 and 100 ng/ml), this effect was associated with caspase-3/7 activation and apoptotic cell death as evidenced by annexin V staining and DNA fragmentation (TUNEL) assays. In addition, inhibition of JNK and protein kinase C activation at these doses partially prevented activation of caspase-3/7, although only JNK inhibition was partially able to prevent the increase in BEC paracellular permeability induced by cytokines. By contrast, lower cytokine concentrations (1 ng/ml) also led to effector caspase activation, increased paracellular flux, and redistribution of zonula occludens-1 and VE-cadherin but failed to induce apoptosis. Under these conditions, specific caspase-3 and caspase-9, but not caspase-8, inhibitors partially blocked cytokine-induced disruption of tight and adherens junctions and BEC paracellular permeability. Our results suggest that the concentration of cytokines in the CNS endothelial microenvironment determines the extent of caspase-mediated barrier permeability changes, which may be generalized as a result of apoptosis or more subtle as a result of alterations in the organization of junctional complex molecules.

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Section: Discussionmentioning

confidence: 99%

Role of Caspases in Cytokine-Induced Barrier Breakdown in Human Brain Endothelial Cells

Lopez-Ramirez

Fischer

Torres-Badillo

et al. 2012

The Journal of Immunology

114

View full text Add to dashboard Cite

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“…Inflammatory stimuli such as thrombin and tumor necrosis factor-␣ induced cell adhesion molecule expression in endothelial cells via Rho kinase and NF-B (22,40). However, there have been no reports to date, outlining the contributions of ROCK isoforms to NF-B-mediated expression of cell adhesion molecules.…”

Section: Discussionmentioning

confidence: 99%

Rho Kinase-2 Activation in Human Endothelial Cells Drives Lysophosphatidic Acid-mediated Expression of Cell Adhesion Molecules via NF-κB p65

Shimada

Rajagopalan

2010

Journal of Biological Chemistry

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The trans-endothelial migration of activated leukocytes into inflamed tissue is an important mechanism in the pathogenesis of inflammatory diseases. Endothelial cells play a key role in this step by up-regulating the synthesis and secretion of chemokines and the cell surface expression of adhesion molecules (CAMs) 2 (1). The adhesion molecules ICAM-1 and VCAM-1 bind leukocytes expressing the major integrins lymphocyte function associated antigen-1 (LFA-1) and the VLA-4 (very late activation antigen-4), respectively, and are induced on endothelial cells by inflammatory stimuli (2, 3). Both are required for the attachment of circulating leukocytes to the endothelium and mediate not only transcellular migration but also the induction of intracellular signals resulting in transient junctional disruption (1) and the paracellular passage of leukocytes across the endothelial monolayer.Lysophosphatidic acid (LPA), a pleiotropic proinflammatory lipid mediator, is elevated in multiple disease states. High concentrations of the lipid have been detected in the bronchoalveolar lavage fluid of preclinical animal models of allergic asthma (4). LPA plays a role in regulating the secretion of pro-and anti-inflammatory mediators by airway epithelial cells and in modulating airway epithelial barrier integrity (5). Additionally, synovial fluid obtained from rheumatoid arthritis patients contain significant amounts of LPA and the LPA-synthesizing enzyme autotaxin driving cytokine secretion by and migration of resident synoviocytes (6, 7). The lipid mediator has also been implicated in the initiation of neuropathic pain (8). Blood platelets also store LPA and are an important source of the lipid. Serum and plasma contain nanomolar levels of LPA and de novo generation or release from platelet stores during inflammation may result in higher LPA levels at disease loci. LPA engages a family of at least five closely related G protein-coupled receptors LPA1-5 (9). It has been reported that HUVECs express LPA1 and LPA3 receptors and are responsive to LPA with induced expression of chemokines and cell adhesion molecules (CAMs) (10 -12). In vitro and in vivo studies employing Rho kinase inhibitors, isoform-specific LPA receptor inhibitors, and targeted receptor deletions link LPA acting through the LPA1 receptor, to the downstream activation of Rho kinase (8,13,14).Rho kinase, a serine/threonine kinase, was initially characterized as a mediator of the formation of RhoA-induced stress fibers and focal adhesions (15). Activation of the Rho kinase pathway leads to the phosphorylation of downstream substrates including the 20-kDa myosin light chain (MLC) (16), the myosin phosphatase subunit (MYPT-1) (17), and CPI-17A (18), which have been postulated to control a variety of fundamental cellular functions such as proliferation, survival, contraction, migration, and the transcriptional regulation of gene expression. However, abnormal activation of the pathway has been * This work was supported by Pfizer, Inc.

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“…In previous studies, conventional cell lysis methods, which do not involve TCA fixation and collagenase treatment, were often used for WB (Engler et al 2006;Tsutsumi et al 2009;Wu et al 2009). In these studies, the cells were cultured on polyacrylamide gels, plastic plates, or dishes.…”

Section: Resultsmentioning

confidence: 99%

“…Thus, by means of the collagenase treatment, we were able to dissolve the collagen gel and remove it from cell lysates. In previous studies, researchers often lysed the cells without TCA and collagenase treatment for WB (Wang et al 2003;Engler et al 2006;Tsutsumi et al 2009;Wu et al 2009). We refer to this approach as ''the conventional method'' in this study.…”

Section: Introductionmentioning

confidence: 99%

An improved method for western blotting when extracting proteins from mammalian cells cultured on a collagen gel under serum-free conditions

et al. 2014

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Western blotting is a widely used method for detection and quantification of specific proteins extracted from mammalian cells. In the conventional method of protein extraction, we found that collagencontaining gels interfered with detection of the p65 protein (one of the subunits in the NF-jB family of proteins) in human lung adenocarcinoma A549 cells cultured on a collagen gel containing serum. In contrast, the collagen gels did not affect detection of the GAPDH protein. Then, we established an improved method for preparation of protein extracts (using trichloroacetic acid fixation and collagenase treatment) from the cells cultured on the collagen gel. Using the improved method, we were able to detect p65 proteins without loss in A549 cells cultured on a collagen gel under serum-free conditions, but we could not detect the proteins if serum was present in cell culture. Thus, using western blotting and serumfree culture conditions, we succeeded in comparing the p65 expression between the cells grown in a plastic dish and cells grown on a collagen gel.

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TNF induces caspase-dependent inflammation in renal endothelial cells through a Rho- and myosin light chain kinase-dependent mechanism

Abstract: Wu X, Guo R, Chen P, Wang Q, Cunningham PN. TNF induces caspase-dependent inflammation in renal endothelial cells through a Rho-and myosin light chain kinase-dependent mechanism.

Cited by 83 publications

References 72 publications

Role of Caspases in Cytokine-Induced Barrier Breakdown in Human Brain Endothelial Cells

Role of Caspases in Cytokine-Induced Barrier Breakdown in Human Brain Endothelial Cells

Rho Kinase-2 Activation in Human Endothelial Cells Drives Lysophosphatidic Acid-mediated Expression of Cell Adhesion Molecules via NF-κB p65

An improved method for western blotting when extracting proteins from mammalian cells cultured on a collagen gel under serum-free conditions

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