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J. Biol. Chem., Vol. 280, Issue 45, 37536-37546, November 11, 2005
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-mediated Suppression of Apoptosis and Antiapoptotic c-IAP2 Gene Expression in Human Monocytic Cells*
112
3¶||4
From the
Departments of ¶Pathology || and Laboratory Medicine and Biochemistry, Microbiology, and Immunology, University of Ottawa, Ottawa, Ontario K1H 8M5, Infectious Disease and Vaccine Research Centre, Research Institute, Children's Hospital of Eastern Ontario, Ottawa, Ontario K1H 8L1, and Aegera Therapeutics Inc., Ottawa, Ontario K1H 8L1, Canada
Received for publication, May 5, 2005 , and in revised form, September 8, 2005.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(TNF-), enhance monocytic cell survival through the induction of the antiapoptotic c-IAP2 gene in a human promonocytic THP-1 cell line. We also investigated the role of upstream signaling molecules including the mitogen-activated protein kinases, phosphatidylinositol 3-kinase, and the calcium signaling pathways in the regulation of c-IAP2 expression and eventual survival of monocytic cells. Our results suggest that LPS and TNF--induced c-IAP2 expression was regulated by calmodulin (CaM) through the activation of calmodulin-dependent protein kinase-II (CaMKII). In addition, CaM and CaMKII regulated c-IAP2 expression in LPSand TNF--stimulated cells through NF-
B activation. Moreover, the CaM/CaMKII pathway also regulated LPS- and TNF--mediated inhibition of apoptosis in these cells. Taken together, these results suggest that LPS- and TNF--induced c-IAP2 expression and its associated antiapoptotic survival signals in THP-1 cells are regulated selectively by CaM/CaMKII through NF-B activation. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Mononuclear phagocytes play a central role in both innate and acquired immunity and are a major source of inflammatory/growth cytokines following exposure to bacterial endotoxins/lipopolysaccharides (LPS). Activation of macrophages by cytokines or a mild bacterial infection is shown to confer antiapoptotic survival signals (12, 13); however, in the absence of appropriate stimulation, monocytes are programmed to undergo apoptosis (14). Macrophage survival may thus influence inflammatory and immune responses and susceptibility to microbial pathogens. The molecular mechanism by which LPS confers antiapoptotic survival signals in monocytic cells is poorly understood. Recently, LPS was shown to induce the expression of the antiapoptotic gene, c-IAP2, in PMA-differentiated human monoblastic U-937 cells; however, its role in suppressing apoptosis in monocytic cells is not clear (15).
LPS may rescue monocytic cells from apoptosis directly through the activation of the CD14 ·Toll-like receptor complex or indirectly via the induction of cytokines such as TNF- in an autocrine manner (13, 14), suggesting a key role for TNF- in monocytic cell survival. TNF- generates two opposing signals: one that triggers apoptosis and another that inhibits apoptosis (16). The outcome of TNF--mediated effects is determined by the balance between these two signals. It has been suggested that TNF--induced cell death is mediated by TNF receptor 1 (TNF-RI), whereas both TNF-RI and TNF-RII are required to transduce signals for antiapoptotic activity primarily through NF-B activation (17, 18). The protective role of NF-B against apoptosis is believed to be mediated by the induction of antiapoptotic genes including c-IAP2 (19–22). It has been suggested that TNF- stimulation of Jurkat T cells induced the expression of the c-IAP2 gene through the activation of NF-B, and conversely, c-IAP2 activated NF-B via an IB-targeting mechanism to further enhance c-IAP2 expression (21). c-IAP2 was shown to enhance NF-B activity following its recruitment to the TNF-R via its association with the adaptor proteins, TNF-R-associated factors: TRAF-1 and TRAF-2 (4). Moreover, c-IAP1 and c-IAP2 were originally identified as TRAF-1 and TRAF-2 binding partners (4). c-IAP2 exerts its antiapoptotic activity by directly binding and inhibiting the downstream protease caspase-3, -7, and -9 (7). Interestingly, TRAF-1, TRAF-2, c-IAP1, and c-IAP2, the molecules involved in antiapoptotic activity, are all components of the TNF-RI and -RII complex, and TRAF-1 and c-IAP2 are also transcriptionally regulated by NF-B (23). Overall, these observations suggest that NF-B plays a central role in the induction of a group of genes that function cooperatively to suppress apoptosis by inhibiting the activity of caspases.
Expression of c-IAP2 has been suggested to be regulated by multiple regulatory elements in its promoter region. In addition to NF-B (19, 21, 24), c-IAP2 induction was recently shown to be regulated through a putative glucocorticoid response element in A549 human lung cancer cells in response to stimulation with dexamethasone (25). In another study, the cAMP-responsive element was shown to be vital for c-IAP2 induction in T84 colon cancer cells (26). However, the upstream signaling molecules involved in the activation of these transcription factors regulating c-IAP2 expression are not well understood. Recently, c-IAP2 expression was shown to be regulated by the extracellular signal-regulated kinases (ERKs), p38 mitogen-activated protein kinases (MAPK), and protein kinase C-
in human colon cancer cells (27, 28). In addition, phosphatidylinositol 3-kinase (PI3K) and ERK MAPKs were implicated in endoplasmic reticulum (ER) stress-induced cell death (29), whereas the JAK2-STAT-3 pathway was suggested to regulate granulocyte colony-stimulating factor-stimulated c-IAP2 expression in human neutrophils (30).
Herein, we investigated the molecular mechanism by which LPS and the proinflammatory cytokine TNF- induce antiapoptotic survival signals in human monocytic cells by employing promonocytic THP-1 cells as a model system. We demonstrate for the first time that both LPS and TNF- induce survival of human monocytic cells through the induction of the c-IAP2 gene. Furthermore, LPS induced c-IAP2 expression, at least in part, through the endogenous production of TNF- in an autocrine manner. The molecular mechanism involved in the up-regulation of c-IAP2 following stimulation of monocytic cells either with LPS or TNF- is not known. We investigated the role of upstream signaling molecules including the members of the MAPK, PI3K, and calcium signaling pathways involved in the regulation of c-IAP2 expression and consequent survival of monocytic cells. Our results suggest that LPS- and TNF--induced c-IAP2 expression in THP-1 cells and their survival are regulated selectively by calmodulin-dependent protein kinase-II (CaMKII) through the activation of NF-B.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(BIOSOURCE, Montreal, Canada), and anti-TNF--R1 antibody (R&D Systems, Minneapolis, MN) capable of neutralizing TNF- activity were also purchased. The following calcium signaling inhibitors were employed: EGTA (Sigma), a calcium-chelating agent; SKF-96365 hydrochloride (Calbiochem), specifically inhibiting receptor-mediated Ca2+ entry (32); 2-APB (Calbiochem), inhibiting inositol 1,4,5-trisphosphate-induced Ca 2+ release from the ER (33); W-7 hydrochloride (W-7; Calbiochem), a calmodulin antagonist; KN-93 (Calbiochem), a specific cellpermeable inhibitor of CaMKII; FK506 (AG Scientific Inc., San Diego, CA), interacting with FK506-binding protein, forming a FK506-FK506-binding protein complex, which binds to and blocks calcineurin; and cyclosporine A (CysA; Sigma), binding to cyclophilin and inhibiting the Ca2+-dependent phosphatases. Caffeic acid phenethyl ester (CAPE) has been shown to act as a potent inhibitor of NF-B activation (Calbiochem). The dominant negative (DN) mutant for the human CaMKII 
isoform in pSR vector (pCaMK-IIB) was kindly provided by Drs. Alain Lilienbaum and Alain Isreal from the Institute Pasteur, Paris (34). The control vector pSR was generated from pCaMK-IIB by digesting with EcoRI. Endotoxin-free preparations of pCaMK-IIB and control vectors were used to transfect cells. Real Time PCR—c-IAP2 mRNA levels were measured using real time quantitative reverse transcription-PCR as per the Taqman method. Total RNA was isolated using RNeasy minispin columns combined with DNase I treatment (Qiagen, Mississauga, Canada). The reverse transcribed RNA was amplified by PCR using the Taqman EZ reverse transcription-PCR kit (PerkinElmer Life Sciences). All of the reverse transcription-PCR steps were performed on an ABI Prism 7700 sequence detector and quantified using the cycle threshold method and normalized to glyceraldehyde-3-phosphate dehydrogenase mRNA using PE-ABI supplied primers (600 nM) and probe (200 nM, JOE-labeled). The thermal cycling conditions for the reverse transcription step were as follows: 50 °C for 2 min, 60 °C for 30 min, and 95 °C for 5 min followed by 45 PCR cycles at 94 °C for 20 s and 60 °C for 1 min/cycle.
Ca2 + Influx—THP-1 cells were washed with Ca2+-free phosphatebuffered saline for 5 min at room temperature and resuspended in Buffer A (RPMI 1640 containing 20 mM HEPES, pH 7.4). The cells were washed again and resuspended in Buffer A containing 1 mM Fluo-3/AM (Molecular Probes, Inc., Eugene, OR) in 1 mM Me2SO and 3.75% pluronic F-127 solution (Sigma) followed by incubation in dark for 45 min in a 37 °C shaking water bath. The reaction was stopped by adding an equal volume of Buffer B (Buffer A containing 5% fetal bovine serum, pH 7.4), followed by incubation for 15 min in a 37 °C water bath. The cells were washed and resuspended in Buffer B at a final concentration of 0.5 x 106 cells/ml. The cells were washed again, aliquoted, and analyzed for Ca2+ levels by the FACScan flow cytometer (BD Biosciences) equipped with CellQuest software, version 3.2.1fl. Cell samples were maintained at 37 °C during data acquisition. Intracellular Ca2+ levels at base line and following stimulation with LPS/TNF- were measured. Ca2+ ionophore A23187
[GenBank]
(20 mM)and5mM EGTA (Sigma) were used as positive and negative controls, respectively.
Cell Stimulation and Western Blot Analysis—Cells (1 x 106 cells/ml) were treated with the indicated concentration of inhibitors for 2 h followed by stimulation with either LPS (1 µg/ml) or TNF- (10 ng/ml) for 15–60 min for detection of kinase activation and for 24 h to determine c-IAP2 expression by Western blot analysis as described earlier (31, 35). Briefly, total proteins were subjected to SDS-PAGE, followed by transfer onto a polyvinylidene difluoride membrane (Bio-Rad). The membranes were probed with anti-c-IAP2 (RIAP-1) polyclonal antibody (36), followed by donkey anti-rabbit polyclonal antibodies conjugated to horseradish peroxidase (Amersham Biosciences). To determine NFAT4 phosphorylation, Western blot analysis was performed by employing anti-phospho-NFAT4 antibodies (Cell Signaling). To control for total protein loading, the membranes were stripped and reprobed with mouse monoclonal antibodies specific for 
-actin (Sigma). All immunoblots were visualized by enhanced chemiluminescence (Santa Cruz Biotechnology, Inc., Santa Cruz, CA).
Measurement of CaMKII Activity—The CaMKII assay was performed using a CaMKII kit (Upstate Biotechnology, Inc., Missisauga, Canada) as per the manufacturer's instructions. Cells were pretreated with inhibitors for 2 h followed by stimulation of cells with either LPS or TNF- for 30 min. Cell pellets were lysed with lysis buffer (50 mM HEPES (pH 7.5), 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1.5 mM MgCl2, 100 mM NaF, 100 mM sodium orthovanadate, 1 mM EGTA (pH 7.7), 10 µg/ml leupeptin, 10 µg/ml aprotinin, 10 µg/ml pepstatin A, and 10 µg/ml phenylmethylsulfonyl fluoride) followed by centrifugation for 20 min at 14,000 x g, 4 °C. CaMKII activity was assayed from total cellular proteins utilizing a peptide substrate (KKALRRQETVDAL) specific for CaMKII. Total proteins (200 µg) were added to 10 µl of CaMK substrate, 0.4 µM each of peptide inhibitors for protein kinase A and protein kinase C, and 100 µCi of MgCl2-[-32P]ATP in ADB II buffer (20 mM MOPS, pH 7.2, 2.5 mM -glycerol phosphate, 1 mM sodium orthovanadate, 1 mM dithiothreitol, and 1 mM CaCl2). The reaction was incubated at 30 °C for 10 min. The phosphorylated substrate was separated from the residual [-32P]ATP using P81 phosphocellulose paper. The papers were washed twice in 0.75% H3PO4 and once in acetone for 2 min and were placed in 24-well Wallac plates (Turko, Finland) in scintillation fluid. Radioactivity was measured by scintillation counting using a Microbeta counter (Wallac, Turko, Finland). Blanks to correct for nonspecific binding of [-32P]ATP and its breakdown products to the phosphocellulose paper and controls for phosphorylation of endogenous proteins in the sample were performed. CaMKII activity was expressed as counts/min/µg of protein.
Analysis of Cellular Apoptosis—Cells were incubated with staurosporine (2 µM) for 4 h, and then apoptotic cells with DNA fragmentation were analyzed by flow cytometric analysis with propidium iodide (PI) staining in permeabilized cells. Briefly, cells (1 x 106) were washed twice with phosphate-buffered saline containing 1% fetal calf serum, fixed with methanol for 15 min at 4 °C, and treated with 1 µg/ml of RNase A, followed by staining with 50 µg/ml of PI at 4 °C for 1 h. The DNA content was then analyzed by FACScan, and data were analyzed using Win-MDI version 2.8 software (J. Trotter, Scripps Institute, San Diego, CA).
Plasmid Construction and Mutagenesis—The full-length c-IAP2 promoter (3.5 kb) was amplified by PCR (with Pfu turboTM enzyme) from a previously characterized bacterial artificial chromosome containing the genomic region encompassing both c-IAP1 and c-IAP2 genes (GenBankTM accession number AF070674
[GenBank]
) (24, 37). The primers used were as follows: sense, 5'-GAT GGT ACC ACT AGT ACT AGA ATA ATG C-3'; antisense, 5'-GCT GAA TTC GCA TGC ACC AGC AAG GAC-3'. The underlined bases indicate restriction sites for cloning that together with the preceding bases do not correspond to sequences in the promoter. The amplified promoter fragment was cloned into pCR2.1 TOPO, sequenced, and then subcloned into the pGL3B vector. Since two NF-B sites (sites 1 and 3) are critical in induction of c-IAP2, sitespecific mutagenesis of these two sites was performed with the QuikChangeTM multisite-directed mutagenesis kits (Stratagene, La Jolla, CA) as per the manufacturer's protocol with 5'-phosphorylated primers: site 1, 5'-CTT TTG GGT CAT GGA AAT AGC CGA GTG GGT TTG CCA G-3'; site 3, 5'-GGT TAT TAC CGC TGG AGT TAA CCT AAG TCC TAA AAG G-3'. The mutagenized bases are indicated (underlined and boldface type) that created convenient EcoRI restriction site for analysis. The primers were used together in the mutagenesis reactions to create the double mutant. Successful mutants were identified by EcoRI digests and sequencing.
Transient Transfection and Luciferase Assay—Cells were transiently transfected with plasmids containing the c-IAP2 promoter by Lipofectamine 2000 (Invitrogen) as described previously (31, 35). Briefly, 5 µg of the test plasmid and 3 µg of pSV--galactosidase vector (Promega) were incubated for 30 min at room temperature with 16 µl of Lipofectamine reagent in 200 µl of OPTI-MEM I medium to allow formation of DNA-liposome complexes. These complexes were then added to the cell suspension (2 x 106 cells/ml) for 24 h followed by stimulation either with LPS or TNF- in the presence or the absence of the indicated inhibitors. The cells were harvested and assayed for luciferase and -galactosidase activity by using luciferase assay and -galactosidase assay kits (both from Promega) in a Bio Orbit 1250 Luminometer (Fisher) and spectrophotometer, respectively. THP-1 cells were also transfected with either 2–5 µg of antisense oligonucleotides for c-IAP2 (5'-GAU GTT TTG GTT CTT CUU C-3') or control oligonucleotides (5'-CUU CTT CTT GGT TTT GUA G-3').
Electrophoretic Mobility Shift Assays—Electrophoretic mobility shift assays were performed as described earlier (31, 35). Briefly, cells were stimulated either with LPS or TNF- for 45–60 min in the presence or the absence of the indicated inhibitors. The nuclear proteins (5 µg) were mixed with 32P-labeled NF-B oligonucleotide probes for 20 min, and the resulting complexes were separated on a 5% nondenaturing gel. The oligonucleotide probes contained sequences corresponding to the NF-B sites 1 and 3 in the c-IAP2 promoter as follows: site 1, sense (5'-ATG GAA ATC CCC GA-3') and antisense (5'-TCG GGG ATT TCC AT-3'); site 3, sense (5'-GCT GGA GTT CCC CT-3') and antisense (5'-AGG GGA ACT CCA GC-3'). To determine specificity of NF-B probes, parallel electrophoretic mobility shift assay reactions were incubated with 50–200-fold excess of unlabeled specific and nonspecific oligonucleotide probes (Egr-1) for 20 min prior to the addition of labeled probe. Supershift experiments were also performed by using mouse anti-NF-B p50 and p65 monoclonal antibodies (Santa Cruz Biotechnology).
Statistical Analysis—Means were compared by the two-tailed Student's t test. The results are expressed as mean ± S.D.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-induced Suppression of Apoptosis in THP-1 Cells Is Mediated by c-IAP2 Induction—To determine the role of c-IAP2 in LPS-induced suppression of apoptosis, we first demonstrated that LPS induced the expression of c-IAP2 in THP-1 cells as determined by Western blot (Fig. 1A) and real time reverse transcription-PCR analysis (Fig. 1B). c-IAP2 protein expression was detectable as early as 4 h, and maximum induction to the extent of 20-fold was observed 24 h following LPS stimulation compared with the unstimulated cells (Fig. 1A). Since TNF- is produced in response to LPS stimulation in THP-1 cells (38), we investigated whether LPS-induced c-IAP2 expression is mediated by endogenously produced TNF- by employing neutralizing antibodies specific for TNF--R1 as described previously (38). The results show that anti-TNF--R1 antibodies at concentrations of 20 µg/ml inhibited LPS-induced c-IAP2 expression to almost undetectable levels (Fig. 1C). In addition, TNF- induced c-IAP2 expression in THP-1 cells as determined by both Western blot and real time PCR analysis (Fig. 1, A and B). Similar to the results obtained with LPS, c-IAP2 protein expression in response to TNF- was detectable as early as 4 h, and maximum induction to the extent of 15-fold was observed at 24 h compared with the unstimulated cells (Fig. 1A). It may be noted that cells expressed c-IAP1 constitutively that was not inducible by either LPS or TNF-.
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-induced inhibition of apoptosis, we determined whether LPS and TNF- stimulation could prevent staurosporine-induced apoptosis in THP-1 cells. LPS- and TNF--stimulated cells were treated with staurosporine for 4 h followed by determination of apoptosis by PI staining. Treatment of unstimulated THP-1 cells with staurosporine resulted in 
30% cell death. Stimulation of cells with either LPS or TNF- resulted in significant inhibition of staurosporine-induced apoptosis from 30 to 10% (Fig. 1E).
To determine whether c-IAP2 is involved in LPS- or TNF--induced antiapoptotic cell survival, cells were transfected with c-IAP2 antisense or control oligonucleotides prior to stimulation with either LPS or TNF- for 24 h followed by determination of c-IAP2 expression and staurosporine-induced apoptosis. Antisense c-IAP2 oligonucleotides significantly decreased by 4-fold the LPSas well as the TNF--induced expression of c-IAP2 as compared with the cells treated with control oligonucleotides (Fig. 1D). Furthermore, treatment of LPS- and TNF-stimulated cells with antisense c-IAP2 oligonucleotides enhanced staurosporine-induced apoptosis compared with the cells treated with control oligonucleotides (Fig. 1E). However, antisense c-IAP2 oligonucleotides did not affect staurosporine-induced apoptosis in unstimulated cells compared with the cells treated with control oligonucleotides (data not shown). It may be noted that abrogation of c-IAP2 expression by antisense oligonucleotides may not be possible because of low transfection efficiency in monocytic cells. These results suggest that stimulation with either LPS or TNF- enhanced monocytic cell survival that was mediated by c-IAP2 induction.
LPS- and TNF--induced c-IAP2 Expression Is Selectively Regulated by the Ca2+ Signaling Pathway in Monocytic Cells—To elucidate the upstream signaling pathways involved in the regulation of c-IAP2 expression, we first investigated the role of MAPKs and PI3K in LPS and TNF--stimulated THP-1 cells by employing their specific inhibitors. The results show that c-IAP2 expression was not affected by any of the inhibitors specific for p38, ERK, and c-Jun N-terminal kinase MAPKs (SB202190, PD98059, and SP600125) or PI3K (wortmannin and LY294002) at any concentration (supplemental text and supplemental Figs. 1, 2, 3). Subsequently, we investigated the role of the signalcalcium ing pathway, since changes intracellular Ca2+ in concentrations play a major role in the regulation of transcription, protein synthesis, and apoptosis (40). We first determined whether LPS and TNF- activated calcium signaling by examining calcium influx by flow cytometry using Fluo-3 as a Ca2+ binding dye. Both LPS and TNF- enhanced calcium influx at 12 and 8 min poststimulation, respectively. To determine the involvement of calcium, cells were treated with EGTA after stimulation with either LPS or TNF-. EGTA inhibited LPS- and TNF--induced Ca2+ influx to the basal level (Fig. 2A). To determine the role of Ca2+ in the regulation of c-IAP2 expression, we analyzed c-IAP2 expression in THP-1 cells treated with EGTA for 2 h prior to stimulation with either LPS or TNF- for 24 h. LPS-induced as well as TNF--induced c-IAP2 expression was inhibited in a dose-dependent manner. EGTA at concentrations of 10 mM decreased the expression of c-IAP2 to undetectable levels following LPS stimulation and by 6-fold following TNF- stimulation (Fig. 2B), suggesting the involvement of the Ca2+ signaling pathway in LPS- and TNF--induced expression c-IAP2.
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-induced c-IAP2 expression (Fig. 2C). Subsequently, we investigated the role of receptor-mediated entry of extracellular Ca2+ by employing SKF-96365, a specific inhibitor for receptor-mediated Ca2+ entry (32). SKF-96365 treatment at concentration of 100 µM significantly reduced both LPS- and TNF--induced c-IAP2 expression by 11- and 7-fold, respectively, and in a dose-dependent manner (Fig. 2D). SKF-96365 and 2-APB were biologically active, since both of these agents inhibited TNF--induced CD44 expression (data not shown). These results suggest that receptor-mediated Ca2+ entry rather than the Ca2+ release from the ER may be involved in LPS- and TNF-induced c-IAP2 expression.
Calmodulin (CaM) and CaMKII Regulate LPS- and TNF--induced c-IAP2 Expression—CaM, a major Ca2+ receptor, is present in both cytoplasmic and nuclear compartments. The complex of Ca2+-CaM regulates several downstream targets, including many protein kinases and protein phosphatases (42). To understand the role of CaM, we employed its specific antagonist, W7-hydrochloride (43), which inhibited LPS- and TNF--induced c-IAP2 expression in a dose-dependent manner. W7 at concentrations of 50 µM decreased the expression of c-IAP2 to undetectable levels following LPS stimulation and by 4-fold following TNF--stimulation (Fig. 3B). To gain further insight into the role of the CaM pathway, we examined the involvement of CaMKII, which is activated subsequent to the binding of Ca2+ to CaM (44), by employing its specific inhibitor KN-93 (42, 43). KN-93 inhibited both LPS- and TNF--induced c-IAP2 expression by 7- and 11-fold respectively and in a dose-dependent manner (Fig. 3B). We also demonstrated that stimulation of cells with either LPS or TNF- for 30 min induced CaMKII activity that was inhibited by both W7 and KN-93 inhibitors in a dose-dependent manner (Fig. 3A).
To confirm the involvement of CaMKII in LPS and TNF--induced c-IAP2 expression, cells were transfected with a DN CaMKII plasmid or a control vector. c-IAP2 expression was significantly inhibited in cells transfected with the DN CaMKII plasmid following LPS as well as TNF- stimulation by 4-fold compared with the cells transfected with the control vector (Fig. 3C). In addition, LPS- and TNF--induced CaMKII activity was inhibited by transfecting cells with DN CaMKII plasmid. Following transfection with the DN CaMKII plasmid, CaMKII activity was observed as 3 ± 1pM following stimulation with either LPS or TNF- compared with the activity of 6.5 ± 1pM in LPS-stimulated and 5.5 ± 1pM in TNF--stimulated cells transfected with the control vector.
Calcineurin is also activated by the binding of Ca2+ to CaM, which dissociates the two components and allows the catalytic site of calcineurin to become accessible (43, 45). To determine the role of calcineurin, cells were treated with CysA or FK506, the inhibitors of calcineurin, prior to stimulation with either LPS or TNF-. Neither CysA nor FK506 inhibited LPS- or TNF--induced c-IAP2 expression at any concentration (Fig. 3D). The biological activity of CysA and FK506 was determined by analysis of NFAT4 expression in Jurkat T cells as described (46, 47). PMA and ionomycin are potent activators of calcineurin and cause dephosphorylation of NFAT proteins. CysA and FK506 are potent inhibitors of calcineurin phosphatase activity and restore PMA- and ionomycin-induced NFAT dephosphorylation and, thus, NFAT-mediated gene induction (46, 47). Our results show that NFAT4 phosphorylation was significantly reduced after stimulation of Jurkat T cells with PMA and ionomycin. Pretreatment of cells with either CysA or FK506 prior to stimulation with PMA and ionomycin restored NFAT4 phosphorylation (Fig. 3E). These results suggest that LPS and TNF--induced c-IAP2 expression is regulated by calmodulin through the activation of CaMKII. It should be noted that none of these inhibitors were found to be apoptotic at the concentrations used as determined by PI staining (data not shown).
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B Binding Sites within the c-IAP2 Promoter in the Regulation of LPS and TNF--induced c-IAP2 Expression—To determine whether NF-B regulates LPS- and TNF--induced c-IAP2 expression, THP-1 cells were transfected with the human c-IAP2 promoter (–606 to +121 bp) linked to the luciferase reporter construct (pc-IAP2Pr) followed by stimulation with either LPS or TNF-. The results show a significant 6–10-fold increase in luciferase activity at 24 h following stimulation with either LPS or TNF- compared with the unstimulated cells (Fig. 4A). Since two NF-B sites (sites 1 and 3) (supplemental Fig. 4) have been shown to regulate c-IAP2 gene transcription (24), these NF-B sequences were mutated by site-directed mutagenesis and cloned into the pGL3B vector (pc-IAP2Pr-mNF-B). Stimulation of THP-1 cells transfected with pc-IAP2Pr-mNF-B with either LPS or TNF- resulted in a significant decrease in luciferase activity compared with cells transfected with the wild type pc-IAP2Pr (Fig. 4B). To confirm the involvement of NF-B, cells were cotransfected with either IB superrepressor or control vector along with the WT pc-IAP2Pr followed by stimulation with either LPS or TNF-. LPS or TNF- stimulation of cells cotransfected with IB superrepressor plasmids resulted in a significantly decreased luciferase activity compared with the cells cotransfected with control IB vector and the WT pc-IAP2Pr (Fig. 4B). In addition, THP-1 cells transfected with the WT pc-IAP2Pr were treated with CAPE, a broad spectrum NF-B inhibitor, for 2 h prior to stimulation with either LPS or TNF- followed by determination of c-IAP2 expression by luciferase activity. Treatment of cells with CAPE inhibited LPS- and TNF--induced c-IAP2 luciferase activity in a dosedependent manner (Fig. 4C). Treatment of THP-1 cells with CAPE prior to stimulation with either LPS or TNF- also resulted in downregulation of c-IAP2 expression as determined by Western blot analysis. CAPE at concentrations of 100 µM decreased the expression of c-IAP2 to undetectable levels following LPS stimulation and by 5-fold following TNF- stimulation (Fig. 4D). These results suggest that NF-B activation plays a key role in LPS, and TNF--induced c-IAP2 expression in THP-1 cells.
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-stimulated THP-1 Cells through NF-B Activation—It has been previously demonstrated that CaMKII acts as a mediator of IB kinase activation in response to T cell receptor/CD3 and PMA stimulation (48, 49). To determine whether CaM/CaMKII regulated LPS- and TNF--induced c-IAP2 expression through NF-B activation, cells transfected with WT pc-IAP2Pr were treated for 2 h with various pharmacological inhibitors of the calcium signaling pathway (EGTA, W7, 2-APB, SKF-96365, KN-93, FK506, and CysA) prior to stimulation with either LPS or TNF- followed by measurement of luciferase activity. EGTA, W7, SKF-96365, and KN-93 significantly decreased the LPS or TNF--induced luciferase activity in a dose-dependent manner (Fig. 5). In contrast, pretreatment of cells with either 2-APB, FK506, or CysA did not have any effect on luciferase activity as compared with LPS- or TNF--stimulated cells (data not shown).
CaM and CaMKII Regulate Binding of NF-B to Its Binding Sites in the c-IAP2 Promoter in LPS- and TNF--stimulated Cells—To confirm the role of CaM and CaMKII in NF-B-dependent c-IAP2 gene transcription, we investigated whether LPS and TNF--stimulation of THP-1 cells induced the binding of NF-B to its binding site 1 present in the c-IAP2 promoter. The nuclear extracts harvested from cells stimulated with either LPS or TNF- for 0–4 h were analyzed by electrophoretic mobility shift assay for binding of NF-B to NF-B oligonucleotide probes corresponding to their sites in the c-IAP2 promoter. The results show a significant binding of NF-B to site 1 following stimulation of cells with either LPS or TNF-. The specificity of NF-B binding was demonstrated by competition with specific and nonspecific oligonucleotides and by supershift analysis with mouse anti-NF-B p50 and p65 antibodies (Fig. 6A). To determine whether binding of NF-B transcription factor to the NF-B binding site 1 in the c-IAP2 promoter was regulated by CaM/CaMKII, cells were treated with inhibitors of the calcium signaling pathway for 2 h before stimulation with either LPS or TNF- followed by analysis of NF-B binding. As before, EGTA, W7, SKF-96365, and KN-93 treatment inhibited binding of NF-B to its probe in LPS- and TNF--stimulated cells (Fig. 6B). Similar results were obtained by analysis of NF-B binding to the oligonucleotide probe containing NF-B binding site 3 on the c-IAP-2 promoter (data not shown). These results suggest that LPS- and TNF--induced c-IAP2 gene transcription may be selectively regulated by CaM and CaMKII through NF-B activation.
CaM and CaMKII Regulate LPS- and TNF--mediated Inhibition of Apoptosis—In view of the above results suggesting the involvement of CaM and CaMKII in c-IAP2 expression, we determined whether CaM and CaMKII also regulated LPS- and TNF--mediated inhibition of apoptosis in THP-1 cells. Cells were treated with various inhibitors of the calcium signaling pathway for 2 h before stimulation with either LPS or TNF-, followed by analysis of staurosporine-induced apoptosis by PI staining. As expected, stimulation with either LPS or TNF- inhibited staurosporine-induced apoptosis. Significantly, prior treatment of cells with EGTA, SKF-96365, W7, and KN-93 reversed the LPS- and TNF--mediated inhibition of staurosporine-induced apoptosis, whereas inhibitors of MAPK, PI3K, and calcineurin did not have any effect (Fig. 7A).
To further confirm the involvement of CaMKII in LPS- and TNF--mediated inhibition of apoptosis, THP-1 cells were transfected with a DN CaMKII mutant construct. After 24 h, transfected cells were stimulated with either LPS or TNF- followed by analysis of staurosporineinduced apoptosis. Stimulation with either LPS or TNF- of cells transfected with control vector inhibited staurosporine-induced apoptosis. In contrast, transfection of cells with DN CaMKII mutant construct reversed the LPS- and TNF--mediated inhibition of staurosporineinduced apoptosis (Fig. 7B).
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DISCUSSION |
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have been shown to induce the expression of various inhibitory proteins associated with apoptosis, including members of the Bcl2 and IAP families in various cell types such as Jurkat T cells, primary endothelial cells, and human monocytic cells (15, 19, 21, 24–26, 50). However, the molecular mechanism by which LPS and TNF- induce antiapoptotic activity in monocytic cells remains unknown. In this study, we have demonstrated that LPS and TNF- suppress apoptosis through the selective induction of c-IAP2 in THP-1 monocytic cells. Subsequently, we investigated the signaling pathways underlying c-IAP2 induction and its antiapoptotic activity. Our results suggest that LPS- and TNF--mediated c-IAP2 induction and its associated antiapoptotic activity may be regulated by CaM and CaMKII through the activation of NF-B in THP-1 cells.
It is interesting to observe that c-IAP1 and c-IAP2 are differentially induced following stimulation with LPS and TNF- in THP-1 cells, suggesting a cell type-specific role for these molecules. Specific inhibition of c-IAP2 by antisense oligonucleotides reversed LPS- and TNF--mediated protection from staurosporine-induced apoptosis, suggesting a key role for c-IAP2 in this antiapoptotic effect. Furthermore, LPS-induced c-IAP2 expression was regulated by the endogenous production of TNF-. The IAP family proteins have been shown to suppress apoptosis induced by a variety of stimuli including TNF-, Fas, and growth factor withdrawal by inhibiting activation of procaspase-9 and active caspase-3 and caspase-7 (7, 51). In addition, c-IAP2 appears to be a part of the signaling complex recruited to the cytoplasmic domain of TNF-R by binding to the TRAF-1/TRAF-2 heterocomplexes and functions to suppress caspase-8 activation (4, 7, 52). These observations suggest that LPS- and TNF--induced c-IAP2 may inhibit apoptosis by acting on caspases in THP-1 cells.
Apoptosis has been shown to be regulated by a number of upstream signaling molecules, such as MAPK and PI3K in epithelial and leukemic cell systems (29, 53–56). Recently, c-Jun N-terminal kinase was shown to mediate the antiapoptotic activity of XIAP (53), whereas p38 and ERK MAPKs were shown to regulate c-IAP2 expression in colon cancer cell lines (27). Although both LPS and TNF- induced the activation of p38, ERK, and c-Jun N-terminal kinase MAPK and PI3K in this study, none of these kinases were involved in LPS- or TNF--induced c-IAP2 expression in monocytic cells. Because of the lack of involvement of these major signaling pathways, we investigated the role of upstream Ca2+ signaling proteins, which are important intracellular messengers in many biological processes, including apoptosis (40, 41). Influx of Ca2+ through ligand and voltage-gated calcium channels in the plasma membrane, together with Ca2+ release from ER stores, results in complex calcium signaling cascades (40, 41). Several mechanisms may control Ca2+ entry in response to external stimuli, including membrane depolarization, activation of intracellular messengers, and depletion of intracellular calcium storage (42). The release of Ca2+ from internal stores (ER) is controlled by Ca2+ itself or by an expanding group of messengers. For example, the inositol 1,4,5-trisphosphate, produced in response to a signal from the membrane lipid phosphatidyl inositol, triggers Ca2+ release from the ER after binding to the inositol 1,4,5-trisphosphate receptor (42).
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-induced regulation of several genes (57–63). Both LPS and TNF- have been shown to induce calcium flux (57–59, 64–67). LPS has also been shown to increase phosphatidylinositol 1,4,5-triphosphate (64, 65). Furthermore, the lipid A component of LPS was suggested to markedly enhance free intracellular calcium (64). There is also evidence to suggest that the sources of increased cytosolic calcium in LPS-stimulated cells are extracellular calcium as well as stored calcium from the endoplasmic reticulum (68). Our results also suggest that both LPS and TNF- induced calcium influx into the cytosol, although TNF- acted more quickly than LPS. This is consistent with the induction of TNF- by LPS playing a role in c-IAP2 expression and antiapoptotic activity. Although LPS has been found to increase intracellular calcium, and the lipid A component has been specifically found to trigger calcium flux (64, 69), the precise mechanism by which LPS and TNF- impact calcium signaling is still not well understood.
Calmodulin, a key signaling protein responsible for integrating the Ca2+ signal with transcription factor activation, is known to regulate cell cycle and related cytoskeletal functions and ion channel activity (42, 70, 71). Following binding to Ca2+, CaM undergoes a conformational change that renders it active and able to recognize and bind target proteins with high affinity (42, 71). Among the possible downstream targets of CaM are calcineurin and CaMKII (72–74). As with other kinases, CaMKII undergoes autophosphorylation on a threonine residue contained in a phosphopeptide common to its and subunits and converts it into a Ca2+/CaM-independent enzyme (24). The results obtained by employing specific inhibitors suggested that CaMKII may act as a key link in LPS- and TNF--induced CaM activation and c-IAP2 expression.
Intracellular mobilization of Ca2+ triggered by various stimuli is known to act as a key second messenger necessary for the induction of NF-B activity (43, 72, 74). It has been shown that two NF-B elements are required for c-IAP2 promoter activity, and they function cooperatively in inducing c-IAP2 expression (24). The results of this study suggest that both NF-B binding sites are involved in the up-regulation of LPS- and TNF--induced c-IAP2 expression in monocytic cells. Furthermore, receptor-mediated Ca2+ entry into the cells rather than Ca2+ release from the ER may regulate LPS- and TNF--induced NF-B activity in the c-IAP2 promoter and c-IAP2 transcription. This identified a calcium-triggered signaling cascade, which may stimulate p50/p65 NF-B-transactivating potential, eventually leading to c-IAP2 gene expression. Analysis of c-IAP2 promoter and NF-B activities in the presence of specific inhibitors of NF-B and the calcium signaling pathway revealed that LPS- and TNF--induced c-IAP2 expression may be regulated by NF-B through intracellular calcium mobilization and subsequent activation of CaM/CaMKII. CaMKII has also been previously shown to act as a mediator of IB kinase activation specifically in response to T cell receptor/CD3 and PMA stimulation (48, 49). Overall, these observations suggest that CaM and CaMKII establish a link between receptor-mediated intracellular Ca2+ mobilization on one hand and activation of downstream p50/p65 NF-B on the other to regulate c-IAP2 expression.
At present, the role of c-IAP1 in LPS- and TNF--mediated antiapoptotic effect is not clear. Both c-IAP1 and c-IAP2 have been suggested to contribute toward apoptotic resistance of different cancers (23). The genes encoding c-IAP1 and c-IAP2 share 75% homology at the level of nucleotide and amino acid sequence and are thought to have arisen from a gene duplication event (37). The molecular mechanism involved in the regulation of c-IAP1 expression is not clear at present. Herein, we show that c-IAP1 is expressed constitutively and is not induced by either LPS or TNF-. Furthermore, constitutive expression of c-IAP1 may be regulated selectively by the calcium pathway, since its expression levels were down-regulated by EGTA, SKF, and W-7. However, c-IAP1 does not seem to be regulated by the CaMKII pathway as its inhibitor KN93 inhibited LPS/TNF--induced expression of c-IAP2 alone. Since expression levels of c-IAP1 are not affected by antisense c-IAP2 oligonucleotides, LPS- and TNF--induced antiapoptotic activity may not be regulated by c-IAP1. It is likely that c-IAP1 may have a role in spontaneous survival of THP-1 cells. Furthermore, a role of other inhibitory proteins including Bcl2 and other members of the IAP family in LPS- and TNF--induced antiapoptotic activity cannot be ruled out and requires further investigation.
In summary, this is the first study that demonstrates the involvement of c-IAP2 in LPS and TNF--induced antiapoptotic survival of human monocytic cells. Our results clearly suggest that LPS- and TNF--induced c-IAP2 expression and its associated antiapoptotic activity are regulated distinctly by calmodulin/CaMKII through the activation of the NF-B pathway. Since c-IAP2 is one of the antiapoptotic genes, the results suggest that strategies based on suppression of c-IAP2 induction by agents known to inhibit the calcium/NF-B signaling pathways may be helpful in controlling infection with pathogenic organisms and inflammatory responses.
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FOOTNOTES |
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The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. 1, 2, 3, 4.
1 Supported by scholarships from the OGSST and OGS.
2 Supported by fellowships from the Ontario HIV Treatment Network (OHTN) and Strategic Areas of Development, University of Ottawa.
3 Supported by a Natural Sciences and Engineering Research Council-Industrial Research Fellowship.
4 Recipient of the Career Scientist Award from the OHTN. To whom correspondence should be addressed: Division of Virology, Research Institute, Children's Hospital of Eastern Ontario, University of Ottawa, 401 Smyth Rd., Ottawa, Ontario K1H 8L1, Canada. Tel.: 613-737-7600 (ext. 3920); Fax: 613-738-4825; E-mail: akumar{at}uottawa.ca.
5 The abbreviations used are: IAP, inhibitor of apoptosis protein; LPS, lipopolysaccharide; MAPK, mitogen-activated protein kinase; CaM, calmodulin; CaMK, calmodulin-dependent protein kinase; CaMKII, calmodulin-dependent protein kinase-II; ER, endoplasmic reticulum; TRAF, TNF receptor-associated factor; PMA, phorbol 12-myristate 13-acetate; TNF-, tumor necrosis factor; TNF-R, TNF receptor; ERK, extracellular signal-regulated kinase; PI3K, phosphatidylinositol 3-kinase; CysA, cyclosporine A; CAPE, caffeic acid phenethyl ester; DN, dominant negative; MOPS, 4-morpholinepropanesulfonic acid; PI, propidium iodide; 2APB, (2-aminoethoxy) diphenylborate.
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ACKNOWLEDGMENTS |
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REFERENCES |
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