Subcellular proteomics of cell differentiation: Quantitative analysis of the plasma membrane proteome of Caco‐2 cells

Pshezhetsky, Alexey V.; Fedjaev, Michael; Ashmarina, L.I.; Mazur, Alexander; Budman, Lorne; Sinnett, Daniel; Labuda, Damian; Beaulieu, Jean‐François; Ménard, Daniel; Nifant’ev, Ilya E.; Lévy, Émile

doi:10.1002/pmic.200600956

Cited by 54 publications

(55 citation statements)

References 52 publications

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“…Two complementary proteomic approaches were adopted to characterize the fibrogenic proteins secreted by apoptotic HUVECs downstream of caspase activation: SDS-PAGE-liquid chromatography (LC)-tandem mass spectrometry (MS/MS) and two-dimensional (2D)-LC-MS/MS. 23,24 To be considered of potential significance in this fibrogenic loop, the identified proteins had to meet two screening criteria: (1) human proteins identified at a relative abundance ratio of 2.5 and above in SSC-DMSO as compared with SSC-ZVAD (intensity of peptides identified in SSC-DMSO/intensity of peptides identified in SSC-ZVAD) and (2) human proteins with a known fibrogenic activity. CTGF was the only protein that met these criteria (Figure 5a).…”

Section: Resultsmentioning

confidence: 99%

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Caspase-3-mediated secretion of connective tissue growth factor by apoptotic endothelial cells promotes fibrosis

et al. 2009

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Apoptosis of endothelial cells (ECs) is an early pathogenic event in various fibrotic diseases. In this study, we evaluated whether paracrine mediators produced by apoptotic ECs play direct roles in fibrogenesis. C3H mice injected subcutaneously with serumfree medium conditioned by apoptotic ECs (SSC) showed increased skin thickness and heightened protein levels of a-smoothmuscle actin (aSMA), vimentin and collagen I as compared with mice injected with medium conditioned by non-apoptotic ECs. Fibroblasts exposed to SSC in vitro showed cardinal features of myofibroblast differentiation with increased stress fiber formation and expression of aSMA. Caspase-3 silencing in ECs prevented the release of mediators favoring myofibroblast differentiation. To identify the fibrogenic factor(s) released by ECs, the protein contents of media conditioned by either apoptotic or non-apoptotic ECs were compared using SDS-PAGE-liquid chromatography (LC)-tandem mass spectrometry (MS/MS) and two-dimensional LC-MS/MS. Connective tissue growth factor (CTGF) was the only fibrogenic protein found increased in SSC. Pan-caspase inhibition with ZVAD-FMK or caspase-3 silencing in ECs confirmed that CTGF was released downstream of caspase-3 activation. The fibrogenic signaling signatures of SSC and CTGF on fibroblasts in vitro were similarly Pyk2-, Src-family kinases-and PI3K dependent, but TGF-b-independent. CTGF-immunodepleted SSC failed to induce myofibroblast differentiation in vitro and skin fibrosis in vivo. These results identify caspase-3 activation in ECs as a novel inducer of CTGF release and fibrogenesis.

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Section: Resultsmentioning

confidence: 99%

“…The tryptic peptides were analyzed by LC-MS/MS as previously described. 24 SDS-PAGE-LC-MS/MS analysis was performed essentially as described. 24 In all, 125-mg weight of total protein from each sample was resolved by SDS-PAGE on a 15% acrylamide gel.…”

Section: Methodsmentioning

confidence: 99%

Caspase-3-mediated secretion of connective tissue growth factor by apoptotic endothelial cells promotes fibrosis

et al. 2009

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“…A total of 30 ml from each fraction were analyzed by LC-MS/MS. SDS-PAGE LC-MS/MS analysis was performed essentially as outlined elsewhere 14 . Proteins (125 mg) were solubilized in 4 Â Laemmli buffer (62.5 mM Tris-HCl, pH 6.8, 20% glycerol, 2% SDS, 0.5% (w/v) bromophenol blue), reduced in the presence of 4 mM DTT for 1 h at room temperature and then alkylated with 10 mM phenylmaleimide for 1 h at room temperature.…”

Section: Methodsmentioning

confidence: 99%

Caspase-3-dependent export of TCTP: a novel pathway for antiapoptotic intercellular communication

et al. 2010

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The apoptotic program incorporates a paracrine component of importance in fostering tissue repair at sites of apoptotic cell deletion. As this paracrine pathway likely bears special importance in maladaptive intercellular communication leading to vascular remodeling, we aimed at further defining the mediators produced by apoptotic endothelial cells (EC), using comparative and functional proteomics. Apoptotic EC were found to release nanovesicles displaying ultrastructural characteristics, protein markers and functional activity that differed from apoptotic blebs. Tumor susceptibility gene 101 and translationally controlled tumor protein (TCTP) were identified in nanovesicle fractions purified from medium conditioned by apoptotic EC and absent from purified apoptotic blebs. Immunogold labeling identified TCTP on the surface of nanovesicles purified from medium conditioned by apoptotic EC and within multivesicular blebs in apoptotic EC. These nanovesicles induced an extracellular signal-regulated kinases 1/2 (ERK 1/2)-dependent antiapoptotic phenotype in vascular smooth muscle cells (VSMC), whereas apoptotic blebs did not display antiapoptotic activity on VSMC. Caspase-3 biochemical inhibition and caspase-3 RNA interference in EC submitted to a proapoptotic stimulus inhibited the release of nanovesicles. Also, TCTP siRNAs in EC attenuated the antiapoptotic activity of purified nanovesicles on VSMC. Collectively, these results identify TCTP-bearing nanovesicles as a novel component of the paracrine apoptotic program of potential importance in vascular repair.

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“…Characterization of the Proteases Produced by Apoptotic and Non-apoptotic EC-To characterize the complete set of proteases secreted by apoptotic EC downstream of caspase activation, we tested two comparative proteomic approaches: SDS-PAGE-LC-MS/MS and two-dimensional LC-MS/MS (27). SSC-apo and SSC-no-apo were centrifuged to eliminate cell debris and apoptotic bodies (28).…”

Section: Methodsmentioning

confidence: 99%

Caspase-3 Activation Triggers Extracellular Cathepsin L Release and Endorepellin Proteolysis

Cailhier

Sirois

Laplante

et al. 2008

Journal of Biological Chemistry

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121

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Proteolysis of extracellular matrix components and the production of cryptic bioactive factors play key roles in vascular remodeling. We showed previously that extracellular matrix proteolysis is triggered by the apoptosis of endothelial cells (EC), resulting in the release of an anti-apoptotic C-terminal fragment of endorepellin (LG3). Here, we characterize the endorepellin-cleaving proteases released by apoptotic EC using a multifaceted proteomics strategy. Cathepsin L (CathL), a cysteine protease known to be associated with cardiovascular disease progression in animal models and humans, was isolated from medium conditioned by apoptotic EC. CathL cleaved recombinant endorepellin in vitro, leading to LG3 release. Inhibition of CathL activity in EC exposed to pro-apoptotic stimuli prevented LG3 release without modulating the development of apoptosis in EC. Inhibition of caspase-3 activation in EC with the biochemical inhibitor DEVD-fluoromethyl ketone or small interfering RNAs concomitantly prevented CathL release by EC, LG3 production, and the development of paracrine anti-apoptotic activity. These data demonstrate that caspase-3 activation is a novel pathway of importance for triggering extracellular CathL release and the cleavage of extracellular matrix components. Apoptosis of endothelial cells (EC)4 is increasingly recognized as an important component of the "response to injury" process, as most clinical risk factors of atherosclerosis (such as hypertension (1, 2), hyperglycemia (3, 4), oxidized low density lipoproteins (LDLs) (5, 6) and oxidative stress (7)) induce EC apoptosis. Interventions aimed at preventing EC apoptosis in animal models of transplant vasculopathy, an immune-mediated form of atherosclerosis, prevent neointima formation, indicating that EC apoptosis is an important pro-atherosclerotic trigger (8 -13). During vascular remodeling, EC injury and apoptosis are followed by migration of ␣-smooth muscle actinpositive cells (smooth muscle cells (SMC) and myofibroblasts) that accumulate within the intima through a state of resistance to apoptosis largely dependent on Bcl-xl overexpression (14 -16).Recent evidence from our group and others suggests that apoptotic EC favor neointima formation through the release of paracrine mediators, which in turn, increase Bcl-xl expression and inhibit the apoptosis of vascular SMC and fibroblasts (17)(18)(19)(20). The production of biologically active mediators by apoptotic EC is at least partially dependent on pericellular proteolysis, leading to basement membrane and extracellular matrix (ECM) degradation with the release of cryptic anti-apoptotic factors (18 -20). A C-terminal fragment of endorepellin (perlecan domain V) released in association with EC apoptosis was found to heighten Bcl-xl expression in SMC and fibroblasts (18 -20). Perlecan is a basement membrane modular proteoglycan composed of five structural domains (21). The C-terminal domain, also called endorepellin, comprises three laminin-like globular (LG1-LG3) modules interspaced by four epiderm...

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Subcellular proteomics of cell differentiation: Quantitative analysis of the plasma membrane proteome of Caco‐2 cells

Cited by 54 publications

References 52 publications

Caspase-3-mediated secretion of connective tissue growth factor by apoptotic endothelial cells promotes fibrosis

Caspase-3-mediated secretion of connective tissue growth factor by apoptotic endothelial cells promotes fibrosis

Caspase-3-dependent export of TCTP: a novel pathway for antiapoptotic intercellular communication

Caspase-3 Activation Triggers Extracellular Cathepsin L Release and Endorepellin Proteolysis

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