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

Abstract

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.

Figure 1

Serum starvation induces a pure apoptotic response in EC. (a) Percentage of cells with increased chromatin condensation and cell membrane permeabilization (as evaluated with HO and PI staining) in EC exposed to normal medium (N) or serum starvation (SS) for 1–4 h. ^*P≤0.02 versus N, n=4. (b) LDH activity in media conditioned by normal EC and EC serum starved for 4 h or heated at 65 °C for 30 min (positive control for cell membrane permeabilization). ^*P<1 × 10⁻⁶ versus heat, n=3. (c) JC-1 staining for evaluation of mitochondrial permeabilization in EC treated as described in (a) or preincubated with the pan-caspase inhibitor (ZVAD-FMK) 100 μM or vehicle (DMSO) and serum starved for 4 h. ^*P≤0.02 versus normal, n=6. (d) Immunoblots for active forms of caspase-9 (p25) and caspase-3 (p17–p19) in EC treated as in (c). Representative of five experiments. (e) Immunoblots for uncleaved and cleaved PARP in EC treated as in (c–d). Representative of two experiments. Ponceau red staining is shown as loading control. (f) Immunoblots for active forms of caspase-9 and caspase-3 in EC exposed for 4 h to N or preincubated with vehicle (DMSO), 100 μM of the caspase-9 inhibitor (LEHD) or the caspase-3 inhibitor (DEVD) as in (c–d). Representative of five experiments. (g) Percentage of cells with increased chromatin condensation and cell membrane permeabilization (as evaluated with HO and PI staining) in EC as treated in (c). ^*P<0.05 versus serum starvation DMSO, n≥12. (h) Immunoblots for p53 in EC as treated in (c). Representative of four experiments. Ponceau red staining is shown as loading control for (e) and (h). The color reproduction of this figure is available on the html full text version of the manuscript

Figure 2

Characterization of the secretome of apoptotic EC. (a) Schematic representation of the experimental strategy for generating serum-free media (conditioned by equal EC numbers in equal volumes of serum-free media) by apoptotic (SSC-Apo) and non-apoptotic EC (SSC-No-Apo). (b) Flow cytometry analysis providing evidence of depletion of apoptotic blebs before MS/MS analysis. (c) In-solution fractionation (2D-LC-MS/MS) of SSC-Apo and SSC-No-Apo generated as described in (a). Tryptic protein digests (500 μg) from conditioned media (SSC-Apo and SSC-No-Apo) were fractionated with high-performance liquid chromatography into 25 fractions as represented by the chromatograms (O.D. 280 nm in function of elution time). Tryptic digests from fractions 3–22 of both samples were submitted individually to peptide sequencing by LC-MS/MS. (d) In-gel fractionation of SSC-Apo and SSC-No-Apo generated as described in (a). Serum-free conditioned media were concentrated on a 30-kDa membrane by ultrafiltration. 125 μg of proteins were separated on 15% SDS-PAGE gel and stained with Coomassie blue. The gel was cut into 20 pairs of bands, followed by in-gel trypsinization and peptide sequencing by LC-MS/MS. ^* represents bands corresponding to TCTP. The color reproduction of this figure is available on the html full text version of the manuscript

Figure 3

Caspase(s) activation in apoptotic EC fosters the release of antiapoptotic exosome-like nanovesicles. (a) Immunoblots for TCTP, TSG 101, Grp96 and tubulin in cytosolic (25 μg) and total protein extracts from equal volumes (1 ml) of total serum-free media (Total SSC) conditioned by equal numbers of EC serum starved for 1–4 h. Ponceau red staining is representative of total protein content. Representative of three experiments. (b) Immunoblots for TCTP in proteins precipitated from equal volumes of serum-free media conditioned by equal numbers of apoptotic (Apo) and non-apoptotic EC (No-Apo): 1 ml of unfractionated serum-free conditioned media (Total SSC), exosome-like nanovesicle fraction (Exo) purified from 12 ml of total serum-free conditioned media and nanovesicle-free supernatant (SN) corresponding to the soluble fraction of ultracentrifuged serum-free conditioned media (12 ml). Representative of three experiments. (c) Immunoblots for TCTP. Left panel: Proteins isolated from 1 ml of SSC-Apo compared with 1 ml of SSC-Apo supplemented with TCEP 10% (SSC-Apo + TCEP) before electrophoresis. TCEP decreased the intensity of high-molecular-weight TCTP and increased the intensity of the non-oligomeric form (monomer 23 kDa). Middle panel: Quantification by densitometry of TCTP oligomers and monomer from three immunoblots. ^*P≤0.005 versus SSC-Apo. Right panel: A total of 0.1 μg of GST-tagged recombinant TCTP (37 kDa) showing formation of oligomers. (d) Electron micrographs. Exosome-like nanovesicles purified from SSC-Apo (cleared of cell debris and apoptotic blebs). Left panel: Immunogold labeling of nanovesicle extracts revealed TCTP antigenic sites on the surface of a nanovesicle. Bar: 25 nm. Uranyl oxalate and methyl cellulose-uranyl acetate negative staining. Right panel: Electron micrograph presenting a population of exosome-like nanovesicles positive for TCTP immunogold labeling. Bar: 100 nm. (e) Percentage of apoptotic cells as defined by increased chromatin condensation without cell membrane permeabilization (as evaluated with HO and PI staining) in VSMC exposed for 24 h to 0.5 ml of: normal medium (N), serum-free medium (SS), unfractionated serum-free media (Total) conditioned either by apoptotic EC (SSC-Apo) or non-apoptotic EC (SSC-No-Apo), as described in Figure 2a, exosome-like fraction (Exo) purified from 12 ml of total serum-free conditioned media and resuspended in 12 ml of RPMI (SS) and the corresponding nanovesicle-free supernatant (SN). ^*P≤2.0 × 10⁻⁵ versus SSC-Apo, n≥6. (f) Percentage of apoptotic cells as defined by increased chromatin condensation without cell membrane permeabilization (as evaluated with HO and PI staining) in VSMC exposed for 24 h to 0.5 ml of: normal medium (N), serum starvation (SS), total serum-free medium conditioned by apoptotic EC (total), supernatant (SSC WO blebs) after removal of apoptotic blebs by centrifugation at 50 000 × g (12 ml) and apoptotic blebs (SS + blebs) purified from 12 ml of total conditioned medium by centrifugation and resuspended in 12 ml of SS. ^*P≤1 × 10⁻¹¹ versus SS, n=8. (g) Immunoblots for TCTP and TSG 101 in apoptotic blebs (Blebs) and exosome-like nanovesicle fraction (Exo) isolated from 35 ml of serum-free media conditioned by apoptotic serum-starved EC. The immunoblot for TCTP corresponds to two parts of the same gel. Representative of three experiments. The color reproduction of this figure is available on the html full text version of the manuscript

Figure 4

TCTP is a novel antiapoptotic component of the apoptotic secretome. (a) Equal numbers of EC were transfected with control siRNA (siRNA Ctrl) or TCTP siRNA (siRNA TCTP) and serum starved for 4 h in equal volumes of serum-free media. Immunoblots for TCTP in: 25 μg of cytosolic extracts (Cytosolic), proteins precipitated from 1 ml of unfractionated serum-free conditioned media (Total) and exosome-like nanovesicle fraction (Exo) purified from 25 ml of total conditioned media. Tubulin is shown as a loading control for cytosolic extracts. Ponceau red staining indicates total protein content. Representative of three experiments. (b) Percentage of apoptotic cells as defined by increased chromatin condensation without cell membrane permeabilization (as evaluated with HO and PI staining) in EC pretreated with either control siRNA or TCTP siRNA exposed to normal medium or SS for 4 h. ^*P≤0.05 versus N, n=6. (c) Percentage of apoptotic cells as defined by increased chromatin condensation without cell membrane permeabilization (as evaluated with HO and PI staining) in VSMC exposed for 24 h to 0.5 ml of: normal medium (N), serum starvation (SS), total unfractionated serum-free media (Total) conditioned by apoptotic EC transfected with either control siRNA (SSC siRNA Ctrl) or TCTP siRNA (SSC siRNA TCTP), exosome-like nanovesicle fraction (Exo) purified from 12 ml of total unfractionated serum-free conditioned media and resuspended in 12 ml of RPMI (SS) or the corresponding nanovesicle-free supernatant (SN). *P<1 × 10⁻⁵ versus siRNA control, n≥7. Lower panel: Immunoblots for phosphorylated and total ERK 1/2 in cytosolic extracts (25 μg) of VSMC exposed for 30 min to normal medium (N), serum starvation (SS), Total, Exo and SN from SSC siRNA control and SSC siRNA TCTP. Immunoblots are representative of three experiments. (d) Percentage of apoptotic cells as defined by increased chromatin condensation without cell membrane permeabilization (as evaluated with HO and PI staining) in VSMC exposed for 24 h to: normal medium (N), serum starvation in RPMI (SS) and recombinant TCTP (0.03 pM to 26.7 nM) resuspended in RPMI (SS). ^*P≤0.009 versus SS, n≥4. (e) Upper panel: Immunoblots for phosphorylated and total ERK 1/2 in cytosolic extracts (25 μg) of VSMC exposed for 30 min to 0.03 pM and 2.67 nM of recombinant TCTP resuspended in RPMI (SS). Representative of three experiments. Lower panel: Quantification by densitometry for the percentage of ERK 1/2 phosphorylation relative to SS treatment in VSMC. ^*P≤0.02 versus SS, n=3. (f) Percentage of apoptotic cells as defined by increased chromatin condensation without cell membrane permeabilization (as evaluated with HO and PI staining) in VSMC exposed for 24 h to: normal medium (N), serum starvation in RPMI (SS), recombinant TCTP 26.7 nM resuspended in RPMI (SS) and vehicle (DMSO) or recombinant TCTP 26.7 nM resuspended in RPMI (SS) + PD98059 50 μM. ^*P≤0.0008 versus SS or PD 98059; n≥8. The color reproduction of this figure is available on the html full text version of the manuscript

Figure 5

TCTP export is specific of apoptotic cell death. (a) Quantification of apoptotic and autophagic cells by fluorescence microscopy in HO-PI and acridine orange-stained EC exposed to: MMC 0.01 mg/ml in normal medium or vehicle in normal medium for 24 h and rapamycin 0.1 μg/ml in normal medium or vehicle in normal medium for 4 h. Apoptosis refers to the percentage of cells with increased chromatin condensation without cell membrane permeabilization (as evaluated with HO and PI staining). Autophagy referred to the percentage of cells with increased formation of cytoplasmic vacuolization as assessed by acridine orange staining. ^*P=1 × 10⁻⁶ versus vehicle (24 h), n=3. ^**P=1 × 10⁻⁶ versus vehicle (4 h), n=6. (b) Immunoblots for PARP in cytosolic extracts of EC treated with rapamycin or MMC as described in (a). (c) Left upper panel: Immunoblots showing conversion of LC3-I to LC3-II in EC treated with rapamycin 0.1 μg/ml or vehicle in normal medium for 4 h. Electron microscopy for morphological characterization of autophagosomes. Right upper panel: Quantification of volume density of autophagosomes in EC treated with rapamycin 0.1 μg/ml or vehicle as described in (a). A total of 25 cytoplasmic fields were evaluated for each condition. ^*P=0.017. Lower panel: Three different autophagosomes in rapamycine-treated EC (arrows) used for quantification. Bar: 0.5 μm. (d) Immunoblots for TCTP in 1 ml of total serum-free media (cleared of cell debris and apoptotic blebs) conditioned in equal volumes and by equal numbers of EC exposed to MMC and rapamycin, as described in (a). Representative of three experiments. Ponceau red is shown as loading control for b and c. The color reproduction of this figure is available on the html full text version of the manuscript

Figure 6

Activated caspase-3 is a novel regulator of TCTP export. (a) Immunoblots for TCTP in exosome-like nanovesicle extracts (Exo) purified from 25 ml of total unfractionated serum-free media conditioned by equal numbers of serum-starved EC in RPMI (SS) for 1–4 h. Representative of three experiments. (b) Percentage of apoptotic cells as defined by increased chromatin condensation without cell membrane permeabilization (as evaluated with HO and PI staining) in VSMC exposed for 24 h to 0.5 ml of normal medium (N), serum starvation with 0.5 ml of RPMI (SS), total serum-free media conditioned by equal numbers of apoptotic EC (SSC-DMSO) or non-apoptotic EC preincubated with either the caspase-3 inhibitor DEVD-FMK (SSC-DEVD) or the pan-caspase inhibitor ZVAD-FMK (SSC-ZVAD), as described in Figure 1e–g. ^*P<1 × 10⁻¹⁰ versus SS, n≥3. (c) Immunoblots for TCTP in exosome-like nanovesicle extracts (Exo) purified from 12 ml of total unfractionated serum-free media conditioned by equal numbers of EC preincubated as in (b) either with vehicle (SSC-DMSO) or the caspase-3 inhibitor DEVD-FMK (SSC-DEVD) and serum starved for 4 h. Representative of three experiments. (d) Percentage of apoptotic cells as defined by increased chromatin condensation without cell membrane permeabilization (as evaluated with HO and PI staining) in EC exposed for 4 h to 0.5 ml of normal medium (N) or serum starvation with RPMI (SS); or EC transfected with either siRNA control (SS siRNA Ctrl) or siRNA caspase-3 (SS siRNA C3) before serum starvation (0.5 ml of SS) for 4 h. ^*P<0.001 versus SS siRNA control, n=3. (e) Immunoblots for the proform of caspase-3 (p37) in cytosolic extracts (50 μg) of EC transfected with caspase-3 siRNA (C3) or control siRNA (Ctrl) and exposed to normal medium for 4 h. Representative of four experiments. Tubulin is shown as loading control. (f) Percentage of apoptotic cells as defined by increased chromatin condensation without cell membrane permeabilization (as evaluated with HO and PI staining) in VSMC exposed for 24 h to: 0.5 ml of normal medium (N), serum starvation with RPMI (SS) and total unfractionated serum-free media conditioned by EC transfected with either siRNA control (SSC siRNA Ctrl) or siRNA C3 (SSC siRNA C3), as described above. ^*P≤0.004 versus SS, n≥3. (g) Immunoblots for TCTP in protein extracts precipitated from equal volumes of serum-free media conditioned by equal EC numbers and transfected with siRNA control (Ctrl) or caspase-3 siRNA (C3) and serum starved for 4 h; 1 ml of total unfractionated serum-free media (Total); exosome-like nanovesicle fraction (Exo) purified from 25 ml of conditioned media. Representative of three experiments. (h) Immunoblots for PARP and TCTP exposed to recombinant activated caspase-3 in vitro. A total of 300 ng (2.6 pmol) of recombinant PARP (left panel) and 270 ng (7.3 pmol) of recombinant TCTP (right panel) were incubated at 37 °C for 1 h with 50 and 250 units of activated caspase-3. PARP is a known cleavable substrate of activated caspase-3 and was used as a positive control. Representative of two experiments