TCR-induced, PKC-θ-mediated NF-κB activation is regulated by a caspase-8-caspase-9-caspase-3 cascade - PubMed (original) (raw)

TCR-induced, PKC-θ-mediated NF-κB activation is regulated by a caspase-8-caspase-9-caspase-3 cascade

Yixia Zhao et al. Biochem Biophys Res Commun. 2014.

Abstract

It has been documented that caspase-8, a central player in apoptosis, is also crucial for TCR-mediated NF-κB activation. However, whether other caspases are also involved this process is unknown. In this report, we showed that in addition to caspase-8, caspase-9 is required for TCR-mediated NF-κB activation. Caspase-9 induces activation of PKC-θ, phosphorylation of Bcl10 and NF-κB activation in a caspase-3-dependent manner, but it appears that Bcl10 phosphorylation is uncoupled from NF-κB activation. Furthermore, caspase-8 lies upstream of caspase-9 during T cell activation. Therefore, TCR ligation elicits a caspase cascade involving caspase-8, caspase-9 and caspase-3 which initiates PKC-θ-dependent pathway leading to NF-κB activation and PKC-θ-independent Bcl10 phosphorylation which limits NF-kB activity.

Keywords: Caspase-9; NF-κB; T cell activation.

Copyright © 2014 Elsevier Inc. All rights reserved.

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Figures

Figure 1

Figure 1. Caspase-9 is required for NF-κB mediated T cell activation

(A) Splenic T cells from C57BL/6 mice were pretreated for 15 min with zLEHD-FMK or zVAD-FMK at various concentrations, and stimulated for 72 h at 37°C with plate-bound anti-CD3 (2 g/ml). T cell proliferation was determined by [3H]-thymidine incorporation. (B) C57BL/6 T cells were stimulated for 1, 5, 15, 30, 60, and 120 min with plate-bound anti-CD3, and lysed at each time-point. The cell lysates were blotted with anti-caspase-9 Ab, pro-caspase-9 (pro-casp-9) and its p20 fragment. Equal loading was confirmed by anti-actin. Alternatively, cytosolic extracts from time-point of stimulation were prepared by Dounce homogenization in hypotonic extraction buffer (25 mM HEPES, pH 7.5, 5 mM MgCl2, 1 mM EGTA, 1 mM Pefablock, and 1 mg/ml each pepstatin, leupeptin, and aprotinin) and subsequently centrifuged. The protein concentration of supernatant was adjusted to 1 mg/ml with extraction buffer. An equal volume of reagents and 10 mg/ml cytosolic protein were added to a white-walled 96-well plate and incubated at room temperature for 1 h. The luminescence of each sample was measured in a plate-reading luminometer. (C) Jurkat T cells were transfected with GeneSuppressor-Caspase-9 synthetic siRNA. The cells were harvested 48 h post-transfection. The caspase-9 protein expression was significantly reduced in cells transfected with IMG-8003 containing caspase-9 siRNA. The cells were then stimulated with anti-CD3 or anti-CD3 and anti-CD28 for 12 h, and IL-2 production in the culture supernatants was measured by ELISA. (D) C57BL/6 T cells were pretreated with zLEHD-FMK at 1, 5, 10, 25, and 50 M for 15 min, stimulated with anti-CD3, and lysed. The cell lysates were blotted with anti-phospho-Abs against I B, ERK, p38 MAPK, and JNK. The membrane was stripped and reprobed with anti-actin. (E) The extracts of C57BL/6 T cells were treated with recombinant active caspase-9 or caspase-8 at 3.4, 17, and 34 U/ml for 5 or 15 min, and blotted with anti-phospho-IKK / and anti-phospho-I B, respectively. (F) Caspase-9 was knocked down in Jurkat T cells. NF-κB-luciferase reporter gene (pNF-B-luc) was transfected into WT Jurkat (casp-9 WT) or casp-9 KD Jurkat T cells together with pRLTK-Luc (Renilla). The cells were stimulated with anti-CD3 (2 g/ml) plus anti-CD28 (2 g/ml), PMA (50 ng/ml), or unstimulated, and after 24 hours dual luciferase assays (Promega) were performed on an AutoLuman Model LB953 instrument (EG&G Berthold). To obtain relative fluorescence units from each sample, firefly luciferase fluorescence units were normalized to Renilla luciferase fluorescence units. Fold-induction was calculated as relative fluorescence units of stimulated samples divided by relative fluorescence units of unstimulated samples. (G) Casp-9 WT and casp-9 KD cells were stimulated with anti-CD3 and anti-CD28, and lysed. The cell lysates were blotted with phospho-Abs against IκBα ERK, and JNK, respectively and reprobed with anti-actin as loading controls.

Figure 2

Figure 2. Caspase-9 induces phosphorylation of Bcl10 and IκBα in a caspase-3-dependent manner

(A) T cell extracts from C57BL/6 mice were incubated with caspase-9 or caspase-8 for 5 and 15 min, blotted with anti-CARMA1, anti-Bcl10, and anti-MALT1, respectively, and reprobed with anti-actin. (B) T cell extracts were incubated with different doses of caspase-6, and blotted with anti-Bcl10. Addition of caspase-9 was served as a positive control. (C) T cell extracts were incubated with caspase-9 in the presence or absence of λ-phosphatase, and blotted with anti-Bcl10 and anti-actin. (D) C57BL/6 T cells were stimulated with anti-CD3 with or without anti-CD28 for 15 min, and lysed. The cell extracts were blotted with anti-Bcl10. Incubation of the cell extracts with caspase-9 for 15 min was used as a positive control.

Figure 3

Figure 3. Caspase-9-mediated Bcl10 phosphorylation requires caspase-3 which activate PKC-θ whereas PKC-θ is not required for Bcl10 phosphorylation

(A) T cell extracts from WT and caspase-3−/− T cells were incubated with caspase-9, and blotted with anti-Bcl10, anti-phospho-I B, and anti-actin, respectively. (B) C57BL/6 T cell extracts were treated with different doses of caspase-3 for 15 min and blotted with anti-Bcl10 and anti-phospho-I B and reprobed with anti-actin. (C) T cells from caspase-3+/+ and caspase-3−/− mice were stimulated with anti-CD3 and anti-CD28 for 5 and 15 min, and lysed. The cell lysates were blotted with anti-phospho-I B and actin, respectively. (D) T cells isolated from C57BL/6 mice were stimulated with PMA or anti-CD3 and anti-CD28 for 5, 15, and 30 min, and lysed. The cell lysates were blotted with anti-caspase-3 p17 and anti-actin, respectively. (E and F) T cells from caspase-3+/+ and caspase-3−/− mice were stimulated with anti-CD3 and anti-CD28 (E) or caspase-9 (F) for 5 and 15 min, immunoprecipitated with anti-PKC-α and in vitro kinase activity associated with PKC-immunoprecipitates was measured using MBP as a substrate. The cell lysates from the same samples were blotted with anti-PKC-θ (A) and anti-actin. (G) T cell extracts from caspase-3+/+ and caspase-3−/− mice were incubated with caspase-9 at 37°C for 15 min, and immunoprecipitated with anti-PKC-θ and blotted with anti-PKC-θ. (H) T cell extracts from BALB/c mice were incubated with caspase-9 in the presence or absence of Rotterin for 15 min at 37°C. The cell extracts were blotted with anti-Bcl10 and anti-actin. (I) WT and PKC-θ−/− T cell extracts were incubated with caspase-9 for 15 min, blotted with anti-Bcl10 and anti-phospho-I B and anti-actin.

Figure 4

Figure 4. Impaired T cell proliferation and cytokine production is observed in caspase-3−/− mice

(A) Splenic T cells from caspase-3−/− mice and their WT littermates (3 wks of age) were labeled with CFSE, and stimulated for 72 h with plate-bound anti-CD3 (2 g/ml) and anti-CD28 (1 g/ml) and T cell proliferation was determined by flow cytometry. An aliquot of these samples was stained with FITC-conjugated Annexin V, and apoptotic cells were determined by flow cytometry. (B) T cells from caspase-3+/+ and caspase-3−/− mice (3 wks of age) were stimulated with anti-CD3 and anti-CD28 for 72 h, and the expression of CD44 and CD25 was determined by flow cytometry. (C) T cells from caspase-3+/+ and caspase-3−/− mice were stimulated with anti-CD3 or anti-CD3 plus anti-CD28 for 72 h, and the supernatants were collected, and the production of IL-2, IL-4, and IFN-α was measured by ELISA. Sequence: (1) medium only (no cells), (2) cells without stimulation, (3) anti-CD3 (2 g/ml), (4) anti-CD3 (2 g/ml) + anti-CD28 (0.5 g/ml), and (5) anti-CD3 + anti-CD28 (2 g/ml).

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