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Ro52/SSA sensitizes cells to death receptor-induced apoptosis by down-regulating c-FLIP(L)
Jing Zhang, Lei Fang, Xuguo Zhu, Yiting Qiao, Mei Yu, Lu Wang, Yuan Chen, Wu Yin and Zi‑Chun Hua1
The State Key Laboratory of Pharmaceutical Biotechnology, College of Life Sciences, Nanjing University, Nanjing 210093, Peoples Republic of China and Changzhou HighTech Research Institute of Nanjing University, Changzhou 213164, Peoples Republic of China
Ro52/SSA is an autoantigen that presents in patients with SS (Sjögren's syndrome) and SLE (systemic lupus erythematosus). It increases cell death and redistributes itself to apoptotic blebs, but its pro-apoptotic function has not been completely identified. Overexpression of Ro52/SSA promoted cell apoptosis induced by DR (death receptor) in caspase-8-dependent manner. Ro52/SSA expression down-regulated c-FLIP(L) [cellular (Fas-associated death domain)-like interleukin 1β-converting enzyme-inhibitory protein long form] expression, and Ro52/SSA siRNAs (small interfering RNAs) increased c-FLIP(L) production, indicating that Ro52/SSA plays a role in c-FLIP(L) regulation. Ro52/SSA negatively regulated c-FLIP(L) transcriptional level probably by suppressing NF-κB (nuclear factor κB) signalling. The data suggest that Ro52/SSA is involved in DR-mediated apoptosis by regulating c-FLIP(L).
Key words: caspase-8, cellular (Fas-associated death domain)-like interleukin 1β-converting enzyme-inhibitory protein (c-FLIP), nuclear factor κB (NF-κB), Ro52/SSA, tumour-necrosis-factor-related apoptosis-inducing ligand (TRAIL)
Abbreviations: c-FLIP(L), cellular (Fas-associated death domain)-like interleukin 1β-converting enzyme-inhibitory protein long form, DR, death receptor, FADD, Fas-associated death domain, GAPDH, glyceraldehyde-3-phosphate dehydrogenase, IFN, interferon, IL, interleukin, IRF, IFN regulatory factor, NF-κB, nuclear factor κB, PI, propidium iodide, QPCR, quantitative PCR, RT–PCR, reverse transcription–PCR, RING, really interesting new gene, siRNA, small interfering RNA, SLE, systemic lupus erythematosus, SS, Sjögren's syndrome, TRAIL, tumour-necrosis-factor-related apoptosis-inducing ligand
1To whom correspondence should be addressed (email firstname.lastname@example.org).
Ro52/SSA is an autoantigen recognized by antibodies in sera of patients with SS (Sjögren's syndrome) and SLE (systemic lupus erythematosus); systemic autoimmune diseases of unknown cause (Nakken et al., 2001). Its transcript levels increase in PBMCs (peripheral blood mononuclear cells) of patients with SLE and SS, suggesting a role for Ro52/SSA in autoimmune diseases (Espinosa et al., 2006).
Ro52/SSA, also known as TRIM21 (tripartite motif 21), is widely expressed in many cells. It has a RING (really interesting new gene) finger, B-box and coiled-coil domain in the N-terminal region, and a PRY/SPRY domain in the C terminus (Rhodes et al., 2002). As a newly characterized RING-finger-type E3 ubiquitin ligase, Ro52/SSA conjugates ubiquitin molecules with itself and other substrate proteins, such as IRF-8 [IFN (interferon) regulatory factor-8] and IRF-3 (Kong et al., 2007; Higgs et al., 2008), indicating that Ro52/SSA plays important roles in antiviral defences. Ro52/SSA seems to monoubiquitinate IKKβ (inhibitor of nuclear factor κB kinase) and down-regulate NF-κB (nuclear factor κB) signalling. Ro52/SSA-deficient mice develop uncontrolled inflammation, implying another role for Ro52/SSA in the negative regulation of inflammation and systemic autoimmunity (Wada et al., 2009; Yoshimi et al., 2009). Ro52/SSA also plays a role in T-cell responses, since IL-2 (interleukin-2) production increases with exogenous expression of Ro52/SSA in Jurkat T-cells stimulated by anti-CD28 Ab (antibody) (Ishii et al., 2003).
Despite increasing understanding of Ro52/SSA, how the intracellular autoantigen Ro52/SSA presented to the immune system remains obscure. Several studies show that Ro52/SSA redistributed itself to apoptotic blebs in cardiac monocytes, epithelial cells, salivary gland cells and keratinocytes after exposure to pro-apoptotic agents (Miranda et al., 1998; McArthur et al., 2002; Ohlsson et al., 2002). Moreover, Ro52/SSA may be involved in increased apoptotic cell death seen in pSS and SLE (Espinosa et al., 2006). We have extended these findings and show that Ro52/SSA overexpression promotes cell apoptosis following activation of the caspase-8-mediated pathway. The expression of c-FLIP(L) [cellular (Fas-associated death domain)-like IL-1β-converting enzyme-inhibitory protein long form] is negatively regulated by Ro52/SSA in DR (death receptor)-induced apoptosis, confirming Ro52/SSA is a pro-apoptotic factor.
2. Materials and methods
2.1. Cell lines and antibodies
B16F10 cells and A549 cells were cultured in DMEM (Dulbecco's modified Eagle's medium) supplemented with 10% fetal bovine serum (GIBCO). Antibodies were obtained from the following sources: anti-HA (F-7), anti-tubulin (DM1A), anti-Ro52 (D-12) and anti-ubiquitin (P4D1) from Santa Cruz Biotechnology. Anti-FLAG M2 from Stratagene, anti-Fas from Pharmingen, anti-FLIP (NF-6) from Axelis, anti-caspase-8 (Ab-3) from Calbiochem, anti-caspase-3 (8G10) from Cell Signaling Technology, GAPDH (glyceraldehyde-3-phosphate dehydrogenase; KC-5G5) from KangChen. Recombinant TRAIL (tumour-necrosis-factor-related apoptosis-inducing ligand) was prepared in-house (Cao et al., 2008).
2.2. Plasmid construction
pRK5-HA-tagged c-FLIP(L) was gifted by Dr Zhen Xing (University of California at Berkeley). Plasmids of FLAG-Ro52 and FLAG-Ro52ΔRING were provided by Dr Shigetsugu Hatakeyama (Hokkaido University, Japan). Transfection with GenEscort™ reagent (Wisegen Biotechnology Co.) is previously described (Dong et al., 2006).
2.3 RNAi (RNA interference) of Ro52
Ro52 siRNAs (small interfering RNAs) were synthesized by GenePharma Co. The target sequences correspond to nucleotide sequences 113–133 downstream of the start codon of Ro52 mRNA. siRNA duplexes were transfected into cells with Lipofectamine™ 2000 (Invitrogen). After 48 h, cells were prepared for Western blotting.
2.4. Apoptosis assay
After transfection for 24 h, B16F10 cells were cultured for a further 24 h in 1 μg/ml anti-Fas antibody. Cell death was determined with PI (propidium iodide) staining by flow cytometry. For apoptosis, A549 cells were transfected with indicated plasmid construct or siRNAs. After 36 h, cells were treated with TRAIL 100 ng/ml for 18 h. Cells were collected and apoptosis monitored by the Annexin V-FITC/PI double staining method.
2.5. Luciferase assay
Cells seeded in 24-well plates were transiently co-transfected with NF-κB luciferase reporter plasmids together with the Renilla Luciferase reporter vector pRL-null, and expression vector Ro52 or Ro52ΔRING. After 24 h transfection, luciferase assays were done with a Dual Luciferase Kit (Promega).
2.6. QPCR (quantitative PCR)
Total cellular RNA was isolated with TRIzol® Reagent (Invitrogen). cDNA was prepared and amplified by PCR with the following primers: GAPDH: 5-CACCATCTTCCAGGAGCGAG/5-GCAGGAGGCATTGCTGAT; c-FLIP(L) 5-AATTCAAGGCTCAGAAGCGA/5-GGCAGAAACTCTGCTGTTCC.
Amplification was done in an ABI PRISM 7000 Real-Time PCR engine. Relative gene expression of c-FLIP(L) to GAPDH was calculated by the formula: 2−ΔΔCT (Livak method). Reactions were performed in triplicate for statistical evaluation.
3.1. Ro52/SSA promotes DR-induced apoptosis in a caspase-8-dependent manner
DRs regulate important homoeostatic aspects of the immune system and provide important nodes of coordination in immune signalling networks. To examine the functional relevance of Ro52/SSA expression on DR-induced apoptosis, B16F10 cells were transfected with FLAG-Ro52 and induced with anti-Fas antibody. Apoptosis increased in a dose-dependent manner (Figure 1B), and in relation to the amount of Ro52/SSA expression (Figure 1A). Increased apoptosis mediated by Ro52/SSA expression was dramatically inhibited by the caspase-8 inhibitor, FMK007, indicating that apoptosis induced by Ro52/SSA is caspase-8 dependent (Figure 1C). Analogous with Fas-induced apoptosis, TRAIL also signals apoptosis through FADD (Fas-associated death domain) and caspase-8. The increased apoptosis mediated by Ro52 expression also occurred in A549 cells with TRAIL treatment and was clearly suppressed by caspase-8 inhibitor FMK007 (Figure 1D), which implies that Ro52/SSA-mediated apoptosis relates to caspase-8 activation. Immunoblots following apoptosis induced by TRAIL were examined. Overexpression of Ro52/SSA hastened activation of caspase-8 and caspase-3 (Figure 1E) further indicating that Ro52/SSA promotes DR-induced apoptosis in a caspase-8-dependent manner.
3.2. Sensitization to DR-induced apoptosis by Ro52/SSA is associated with c-FLIP(L)
The FLICE-like inhibitory protein, c-FLIP, is an inactive caspase-8 homologue that functions as an apoptotic modulator. Down-regulation of c-FLIP(L) is an important mechanism in sensitizing cells to DR-mediated apoptosis (Safa et al., 2008). To analyse whether Ro52/SSA is associated with down-regulation of the anti-apoptotic action of c-FLIP(L), its expression level of c-FLIP was assessed in cells transfected with increasing amounts of the Ro52/SSA expression vector. The protein level of c-FLIP(L) was reduced by Ro52/SSA overexpression in a concentration-dependent manner (Figure 2A). To address the role of endogenous Ro52/SSA in regulating c-FLIP(L), siRNA was used to reduce endogenous Ro52/SSA levels. Endogenous Ro52/SSA was effectively knocked down with Ro52-specific siRNAs (Figure 2B). As a result, more c-FLIP(L) was produced, indicating that Ro52/SSA expression affects the c-FLIP(L) level. We hypothesized that c-FLIP is a critical factor for the sensitivity of apoptosis mediated by Ro52/SSA.
When cells were transfected with Ro52-specific siRNAs, they proved insensitive to TRAIL-induced apoptosis compared with control siRNAs (P<0.05; Figure 2C). By Western blot assay, repression of Ro52/SSA induced an increased expression of c-FLIP, and then repressed cleavage of caspase-8 and caspase-3 (Figure 2D), which suggests that Ro52/SSA sensitizing is tightly associated with c-FLIP(L) level.
3.3. Ro52/SSA down-regulates c-FLIP(L) via NF-κB signalling
Considering c-FLIP(L) is actively regulated by ubiquitin-proteasome mechanism (Kim et al., 2002) and Ro52/SSA functions as an E3 ligase (Wada and Kamitani, 2006), we investigated whether Ro52/SSA takes a role in the ubiquitination and degradation of c-FLIP(L). After knocking down endogenous Ro52/SSA with an siRNA duplex, cells were treated with the proteasome inhibitor, MG132, for 6 h before being harvested for immunoprecipitation. Polyubiquitination of HA-tagged c-FLIP(L) was not effected when Ro52/SSA was obviously decreased (Figure 3A), indicating that Ro52/SSA is not crucial for c-FLIP(L) degradation. To characterize the transcriptional level of c-FLIP(L) may be regulated by Ro52/SSA, quantitative RT–PCR (reverse transcription–PCR) was used to determine c-FLIP mRNA expression. Expression of c-FLIP mRNA was measured in cells with Ro52/SSA overexpression compared with control transfection. TRAIL treatment induced a modest (∼2-fold) increase in c-FLIP expression, whereas this expression returned to baseline levels with Ro52/SSA overexpression (Figure 3B). A similar pattern of down-regulation also occurred in the absence of TRAIL. These results indicated that down-regulation of c-FLIP mRNA expression by Ro52/SSA plays a key role in enhancing sensitivity of cells to apoptosis.
c-FLIP is one of important downstream anti-apoptotic genes of NF-κB (Micheau et al., 2001). We hypothesized that NF-κB activation is necessary to c-FLIP(L) induction by TRAIL treatment in A549 cells. In the presence of TRAIL, significant accumulation of c-FLIP(L) mRNA was detected, but c-FLIP(L) mRNA was suppressed when cells were treated with TRAIL plus PDTC, a potent inhibitor of NF-κB activation (Figure 4A), suggesting that NF-κB activation is crucial for c-FLIP(L) mRNA production under TRAIL stimulation. We also evaluated the effect of Ro52/SSA on NF-κB-mediated transcription using reporter assays. TRAIL treatment elevated reporter activity, which was repressed by Ro52/SSA overexpression (Figure 4B). The specificity of NF-κB activity repression was confirmed by using Ro52ΔRING, a Ro52-mutant lacking the RING domain (Figure 4B), since the RING finger domain of Ro52/SSA plays an important role in suppressing NF-κB activation (Wada et al., 2009). From these observations, we concluded that Ro52/SSA inhibited NF-κB activation required for c-FLIP production. To examine why Ro52/SSA down-regulates c-FLIP mRNA via the NF-κB pathway, we knocked down Ro52/SSA and inhibited NF-κB activation at the same time to compare the c-FLIP level with that when only Ro52/SSA is knocked down. c-FLIP mRNA was up-regulated by Ro52 siRNA not by control siRNA (Figure 4C), similar results were observed in the c-FLIP protein level (Figure 2B). Moreover, enrichment of c-FLIP mRNA by Ro52 siRNAs was completely suppressed by the NF-κB inhibitor, PDTC, suggesting Ro52 regulates c-FLIP(L) through a NF-κB-dependent pathway.
To see whether Ro52/SSA sensitized cells to apoptosis was associated with the NF-κB pathway, its mutant Ro52ΔRING was transfected into A549 cells and its sensitivity to TRAIL tested. Compared with control vector, Ro52ΔRING overexpression did not promote apoptosis; only Ro52 overexpression effectively sensitized cells to apoptosis (Figures 4D and 4E), suggesting the importance of the RING domain of Ro52/SSA in regulating sensitivity to apoptosis as well as to NF-κB activation.
Although Ro52/SSA autoantibodies have been used clinically in the diagnosis as disease marker for decades, the function of Ro52/SSA is not completely clear. We found that Ro52/SSA is a new pro-apoptotic factor involved in classical caspase-8-mediated apoptosis in DR signalling.
The clustering of Ro52/SSA in small surface blebs of apoptotic cells may be important in the induction of autoantigenic responses (Miranda et al., 1998; McArthur et al., 2002; Ohlsson et al., 2002; Blake et al., 2008). Espinosa et al. (2006) found that Ro52/SSA expression increased in both patients with SS and SLE, and overexpression of Ro52/SSA led to increased apoptosis in tumour B cells after activation with anti-CD40 mAb (monoclonal antibody), but the potential mechanism by which Ro52/SSA promotes activation-induced cell death has not been well clarified. We found that ectopic expression of Ro52/SSA dose-dependently enhanced DR-mediated apoptosis, which was inhibited by caspase-8 inhibitor FMK007 (Figure 1), and increased activation of caspase-8 and caspase-3 were easily detected in cells transfected with Ro52/SSA (Figure 2). This suggested that apoptosis induced by Ro52/SSA is caspase-8-dependent. Some studies demonstrated that steady-state levels of Ro52/SSA mRNA could be rapidly induced by IFN-γ treatment and IFN-induced death was mostly prevented by inhibitors of caspase-8 or caspase-3 or by dominant negative mutants of FADD (Balachandran et al., 2000; Rhodes et al., 2002). These findings strengthen the idea that Ro52/SSA is involved in classical caspase-8-mediated apoptosis.
c-FLIP(L) plays an important function in the DR- or caspase-8-induced apoptotic pathway. In the present study, overexpression of Ro52/SSA reduced c-FLIP(L) expression and knockdown of Ro52/SSA resulted in accumulation of c-FLIP(L), indicating a role for Ro52/SSA as a negative regulator. Although Ro52/SSA is an E3 ligase that catalyses ubiquitination of several proteins, Ro52/SSA actually had no impact on c-FLIP(L) polyubiquitination for its degradation. In fact, Ro52/SSA down-regulates c-FLIP(L) at the transcriptional level. We present evidence that Ro52/SSA regulates c-FLIP(L) indirectly through an NF-κB-dependent pathway, and have confirmed that NF-κB activation is required for c-FLIP production (Figure 4A). Ro52/SSA plays a repressor role on TRAIL-induced activation of NF-κB signalling (Figure 3B). Down-regulation of NF-κB sensitizes tumours to TRAIL (Aydin et al., 2010). We have also demonstrated that Ro52ΔRING mutant did not promote cell apoptosis, for it lacks the RING domain important for suppressing NF-κB activation. In conclusion, Ro52/SSA has a pro-apoptotic role and primes cells to DR-mediated apoptosis by repressing c-FLIP(L) through NF-κB-dependent pathway.
Increased expression of Ro52/SSA in patients may be directly responsible for the increased apoptosis leading to autoantigenic exposure and the generation of Ro52/SSA autoantibodies. Ro52/SSA is not only a biomarker of these autoimmune diseases but also a pro-apoptotic factor regulating cell death by reducing levels of c-FLIP(L). Further work on Ro52/SSA may lead to the development of new therapeutic targets for these diseases.
Jing Zhang designed and performed experiments and wrote the paper. Lei Fang was responsible for the apoptosis assay. Xuguo Zhu and Lu Wang contributed to real-time PCR. Yiting Qiao was a co-worker for most experiments. Mei Yu and Yuan Chen were responsible for cell culture. Wu Yin provided guidance for paper revision. Zi-Chun Hua was involved with scientific supervision, project design and discussion of the paper.
This work was supported by the
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Received 11 June 2011/8 December 2011; accepted 30 January 2012
Published as Cell Biology International Immediate Publication 30 January 2012, doi:10.1042/CBI20110322
© The Author(s) Journal compilation © 2012 International Federation for Cell Biology
ISSN Print: 1065-6995
ISSN Electronic: 1095-8355
Published by Portland Press Limited on behalf of the International Federation for Cell Biology (IFCB)