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J. Biol. Chem., Vol. 281, Issue 51, 39051-39061, December 22, 2006
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B
Protein
1
From the
Department of Pharmacology and the Cell Death Disease Center of Medical Research Center (MRC), College of Medicine, Catholic University of Korea, Seoul 137-701, Korea and the ¶Department of Vascular Biology, Institute of Development, Aging, and Cancer, Tohoku University, Sendai 980-8575, Japan
Received for publication, May 15, 2006 , and in revised form, October 23, 2006.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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B transactivation using glioblastoma cells stably transfected with DSCR1.4 or its truncation mutants (DSCR1.4-(1–133) and DSCR1.4-(134–197)). Overexpression of DSCR1.4 significantly attenuated the induction of cyclooxygenase-2 (COX-2) expression by phorbol 12-myristate 13-acetate (PMA) via a calcineurin-independent mechanism. Experiments using inhibitors of the signaling molecules for NF-B activation showed that NF-B is responsible for the induction of COX-2. Full-length and truncated DSCR1.4 decreased the steady-state activity of NF-B as well as PMA-induced activation of NF-B, which correlated with attenuation of COX-2 induction. DSCR1.4 did not affect the PMA-stimulated phosphorylation or degradation kinetics of IB
; however, DSCR1.4 significantly decreased the basal turnover rate of IB and consequently up-regulated its steady-state level. In the same context, knockdown of endogenous DSCR1.4 increased the turnover rate of IB as well as COX-2 induction. These results suggest that DSCR1 attenuates NF-B-mediated transcriptional activation by stabilizing its inhibitory protein, IB. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Elucidation of the functional roles of DSCR1 in neuronal tissue is important because elevated levels of this protein have been implicated in the pathogenesis of neurodegenerative diseases such as Alzheimer disease and Down syndrome (4, 7, 8). Recent studies in which the DSCR1 gene was overexpressed in endothelial cells showed that DSCR1 down-regulates transcription of several pro-inflammatory factors and enzymes (9–12). In addition, the expression of DSCR1 is up-regulated by mitogens (9–11, 13), inflammatory cytokines (10, 12, 14), infection with Candida albicans (15), depolarization (10, 16), and oxidative stress (17), all of which are frequently implicated in the initiation and maintenance of the inflammatory process. Therefore, DSCR1 is likely to play a significant role as a negative regulatory element in the inflammatory reaction.
Nuclear factor B (NF-B) is a transcription factor involved in the regulation of many cellular target genes that play a central role in immune and inflammatory response (18). In resting cells, most NF-B dimers are sequestered in the cytoplasm through association with inhibitory proteins known as inhibitors of B (IB), of which IB is the best characterized. NF-B-activating signals generated by stimulation with mitogens or cytokines result in rapid degradation of IB, allowing NF-B to localize to the nucleus, where it acts on its target genes. This stimulation-induced IB degradation is initiated by phosphorylation of IB at serines 32 and 36 by a large IB kinase (IKK) complex (IKK·IKK
·IKK
·NEMO), which labels IB for degradation by the ubiquitin/proteasome pathway (18–22). On the other hand, phosphorylation of the C-terminal region of IB, which is rich in proline (P), glutamic acid (E), serine (S), and threonine (T) residues (called the PEST domain), has been implicated as the proteolytic signal that determines the basal turnover rate of IB in resting cells (23, 24). Phosphorylation of the PEST domain is mediated by the constitutively active protein kinase casein kinase 2 (CK2) (25, 26). Down-regulation of CK2 expression by transforming growth factor 1 increases the half-life of IB, which is followed by a decrease in constitutive NF-B activity in murine hepatocyte cells (27). In contrast, ultraviolet radiation increases CK2-mediated degradation of IB, resulting in activation of NF-Bin HeLa cells (28). These studies show that regulation of the basal turnover of IB is another potential means of controlling NF-B activity.
Here, we investigated the molecular basis of the mechanism by which DSCR1 inhibits the expression of genes associated with inflammation. This study shows for the first time that DSCR1.4 decreases the basal turnover rate of IB and consequently attenuates the steady-state and stimulus-induced transcriptional activity of NF-B in human U87MG glioblastoma cells. In addition, we demonstrate that this inhibition of the NF-B pathway by DSCR1.4 is functionally significant by showing that overexpression of DSCR1.4 reduced induction of the NF-B target genes cyclooxygenase-2 (COX-2) and interleukin-1 (IL-1), which were induced by phorbol 12-myristate 13-acetate (PMA) or pro-inflammatory cytokines.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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, PD 98059, SB 203580, Ro-31-8220, calmodulin kinase IINtide, hypoestoxide, and tumor necrosis factor- (TNF-) were purchased from Calbiochem. PMA, A23187
[GenBank]
, and cycloheximide were purchased from Sigma. An antibody specific for COX-2 was purchased from Cayman Chemical (Ann Arbor, MI). Antibodies for NF-B (p65), protein kinase C (PKC), IB (clone C-20), and IL-1 were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Antibodies for IB, phospho-IB Ser-32, phospho-IB Thr-291, and phospho-ERK1/2 Thr-202/Tyr-204 were purchased from Cell Signaling Biotechnology (Beverly, MA).
Cell Culture and Treatments—Human U87MG glioblastoma cells were obtained from American Type Culture Collection (Manassas, VA). The cells were maintained in minimal essential medium supplemented with 0.1 mM nonessential amino acids, 1.0 mM sodium pyruvate, and 10% fetal bovine serum under an atmosphere of 5% CO2 at 37 °C. For experiments, cells (5 x 105/well) were plated in a gelatin-coated 6-well culture dish and incubated for 20 h in complete medium and then for 4 h under serum-reduced conditions (minimal essential medium with 0.1% fetal bovine serum) prior to treatment. For COX-2 induction experiments, cultures were stimulated with PMA (25 ng/ml) plus A23187
[GenBank]
(1 µM), PMA (25 ng/ml) alone, TNF- (10 ng/ml), or IL-1 (5 ng/ml) for the times indicated in the figure legends. In some experiments, cells were pretreated with inhibitors of signaling molecules for 15 min before PMA stimulation.
For IB turnover studies, quiescent cells were incubated for the indicated times in the presence of cycloheximide (20 µg/ml). At the ends of the experiments, whole cell extracts were prepared and subjected to immunoblot analysis as described below.
Plasmid Construction—To construct plasmids encoding recombinant DSCR1.4 protein containing an N-terminal (ntDSCR1) or C-terminal (ctDSCR1) green fluorescent protein (GFP) tag, human wild-type DSCR1.4 cDNA was amplified by PCR and cloned in-frame into the pcDNA3.1/NT-GFP-TOPO or pcDNA3.1/CT-GFP-TOPO vector (Invitrogen), respectively. Primer sets for PCR were as follows: for ntDSCR1, 5'-GGATCCAAATGCATTTTAGAAACTTTAAC-3' (forward) and 5'-CTCGAGTCAGCTGAGGTGGATCGGCGTGTA-3' (reverse); and for ctDSCR1, 5'-GAAAGCAAGATGCATTTTAGAAACTTT-3' (forward) and 5'-CAGTCCAGCTGAGGTGGATCGGCGTGT-3' (reverse).
To construct plasmids for expression of proteins without GFP fusion tags, full-length or truncated DSCR1.4 cDNA fragments were subcloned into the BamHI and EcoRI sites of the pIRES-hrGFP-2 vector (Stratagene, La Jolla, CA). Primer sets for PCR were as follows: for full-length DSCR1.4, 5'-CGGATCCAGCAAGATGCATTTTAG-3' (forward) and 5'-GAATTCTCAGCTGAGGTGGATCGG-3' (reverse); for the DSCR1.4 mutant with a C-terminal deletion up to amino acid 133 (DSCR1
C), 5'-CGGATCCAGCAAGATGCATTTTAG-3' (forward) and 5'-GAATTCTCAATATAAGAGATCATA-3' (reverse); and for the DSCR1.4 mutant with an N-terminal deletion up to amino acid 134 (DSCR1N), 5'-GGATCCAATATGGCCATCTCCAAG-3' (forward) and 5'-GAATTCTCAGCTGAGGTGGATCGG-3' (reverse).
The derivatives of IB containing alanine replacements at Ser-32 and Ser-36 (IB-mutN) or at Ser-283 and Thr-291 (IB-mutC) were generated by overlap extension PCR (29). The PCR fragments were ligated in-frame into the pcDNA3.1D/V5-HisTOPO vector (Invitrogen) to generate vectors expressing V5-His-tagged mutant IB proteins. The overlapping primers used for the generation of IB-mutN and IB-mutC were 5'-CATGGCGTCCAGGCCGGCGTC-3' and 5'-GGCGTCATAGCTCTCCTCATCCTCGGC-3', respectively (with the mutation sites underlined). The N-terminal forward and C-terminal reverse primers for ligation into the expression vector were 5'-CACCCGCGCCATGTTCCAG-3' and 5'-TCTAGATAACGTCAGACGCTGGCC-3', respectively.
To generate an expression construct for the glutathione S-transferase (GST)-tagged PEST domain of IB (GST-PEST), a truncated IB cDNA was PCR-amplified using primers 5'-GGATCCCAGCTGACACTAGAAAAC-3' (forward) and 5'-CTCGAGCCATAACGTCAGACGCTG-3' (reverse). The fragments were digested and then ligated into the BamHI and XhoI sites of pGEX4T-1 (Amersham Biosciences). The insert sequences of all constructs were confirmed by sequencing.
Creation of Stable Cell Lines—Parallel cultures of U87MG cells were transfected with vectors containing each of the DSCR1.4 constructs or with an empty vector using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. After selection against 400 µg/ml G418 (Roche Applied Science, Mannheim, Germany) for 3 weeks, GFP-positive populations were purified using a FACSCalibur machine (BD Biosciences). The selected cells were maintained in complete medium supplemented with 100 µg/ml G418. Transcripts of DSCR1.4 constructs in these cells were confirmed by reverse transcription-PCR amplification (data not shown).
RNA Interference—The DSCR1.4 small interfering RNA (siRNA) used was 5'-CUGUGUGGCAAACAGUGAUdTdT-3'. The CK2 and negative control siRNAs were purchased from Santa Cruz Biotechnology, Inc. In experiments for COX-2 induction studies, cells (3 x 105/well in a 6-well culture dish) were transfected with the siRNA (100 pmol/well) using Lipofectamine 2000 according to the manufacturer's instructions. At 48 h after transfection, the cells were stimulated with PMA for 6 h and then harvested for immunoblot analysis. In experiments for mutant IB turnover studies, the cells were cotransfected with 100 pmol of siRNA and 1.5 µg of IB-mutN or IB-mutC. At 48 h after transfection, the cells were treated with cycloheximide as described for IB turnover experiments.
Luciferase Assay—An NF-B promoter-luciferase reporter plasmid (pNF-B-Luc) was obtained from Stratagene, and a herpes simplex virus thymidine kinase-Renilla luciferase reporter plasmid (pTK-Luc) from Promega Corp. (Madison, WI). Cells (1 x 105/well) were plated in a 24-well culture dish and transfected with 0.5 µg of pNF-B-Luc and 0.2 µg of pTK-Luc using Lipofectamine 2000. At 24 h after transfection, cells were stimulated with the indicated reagents for 6 h, after which luciferase activity was measured with a MiniLumat luminometer (Berthold Technologies GmbH, Bad Wildbad, Germany) using a Dual-Luciferase reporter assay system (Promega Corp.) according to the manufacturer's instructions. Luciferase activities were normalized on the basis of Renilla luciferase luminescence signals.
Preparation of GST-PEST Protein and In Vitro Phosphorylation—Escherichia coli BL21 cells (Invitrogen) were transformed with the GST-PEST construct. The transformants were grown to early log phase, and then protein expression was induced for 3 h by the addition of 1 mM isopropyl -D-thiogalactopyranoside. Cells were harvested and disrupted in B-PER reagent (Pierce) containing protease inhibitor mixture (Roche Applied Science). After insoluble material had been removed by centrifugation, the proteins were recovered from the supernatant by incubation with glutathione-Sepharose 4B (Amersham Biosciences).
For in vitro phosphorylation of the PEST domain of IB by CK2, whole cell extracts (0.5 mg/ml protein) were prepared in kinase buffer (40 mM HEPES (pH 7.5), 130 mM KCl, 10 mM MgCl2, 5 mM -glycerophosphate, 1 mM sodium orthovanadate, and 1 mM dithiothreitol) containing protease inhibitor mixture. The assay was conducted in a reaction mixture containing 100 µM ATP with 1 µg of GST-PEST fusion protein as a substrate in a total volume of 30 µl of kinase buffer. The reactions were started by the addition of 10 µl of cell lysate and were incubated for 15 min at 30 °C. The reaction was stopped by the addition of 2x Laemmli sample buffer, and the samples were subjected to immunoblot analysis.
Immunoblot Analysis—Immediately after the treatments were completed, cultures were lysed in an appropriate volume of lysis buffer (50 mM Tris (pH 7.5), 150 mM NaCl, 1% Triton X-100, 5 mM sodium fluoride, 1 mM sodium orthovanadate, and protease inhibitor mixture). After insoluble material had been removed by centrifugation, the supernatants were mixed with 3x Laemmli sample buffer and denatured for 5 min at 90 °C. The proteins were separated by SDS-PAGE (7 or 12%) and transferred to nitrocellulose membranes. The membranes were blocked for 1 h at room temperature in 1% (w/v) Hammerstengrade casein in phosphate-buffered saline containing 0.05% Tween 20. Immunoblotting was done with appropriate antibodies in 0.5% casein in phosphate-buffered saline. The blots were incubated with horseradish peroxidase-conjugated anti-IgG secondary antibody (Sigma), washed three times with phosphate-buffered saline containing 0.05% Tween 20, and then visualized using the SuperSignal West Dura chemiluminescence substrate (Pierce). The band intensity was analyzed using an LAS-3000 image analyzer (Fujifilm, Tokyo, Japan).
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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To confirm these results, U87MG cells were stably transfected with an expression vector coding for DSCR1.4 without a fusion tag and then tested for stimulus-induced COX-2 expression. As shown in Fig. 1B, overexpression of DSCR1.4 attenuated both PMA/A23187- and PMA-induced COX-2 expression, whereas calcineurin inhibitors reduced only PMA/A23187-induced COX-2 expression.
Knockdown of DSCR1.4 Promotes PMA-induced COX-2 Expression—Next, we conducted knockdown experiments with endogenous DSCR1.4. Cells were transfected with synthetic siRNA oligonucleotides targeted against sequences in a region of exon 4 (DSCR1.4 siRNA). We found a 75–80% reduction in DSCR1.4 mRNA levels using real-time PCR analysis (data not shown). We applied this condition to investigate the effects of decreased endogenous DSCR1.4 levels on COX-2 induction by PMA. As shown Fig. 2, knockdown of DSCR1.4 enhanced COX-2 induction, resulting in a 2–4-fold increase in its expression in cells transfected with DSCR1.4 siRNA compared with negative control siRNA.
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B Transactivation Is Attenuated by DSCR1.4—On the basis of the site of cleavage of DSCR1.4 by calpain (30), we generated plasmids encoding truncated forms of DSCR1.4 lacking a C-terminal region (amino acids 134–197) or an N-terminal region (amino acids 1–133), termed DSCR1C and DSCR1N, respectively. After stable transfection with DSCR1C, DSCR1N, or a vector coding for full-length DSCR1.4, U87MG cells were stimulated with PMA. As shown in Fig. 3A, full-length DSCR1.4, DSCR1C, and DSCR1N reduced COX-2 expression by 20, 77, and 80%, respectively.
Overexpression of DSCR1 inhibits the expression of genes associated with cell adhesion or inflammation such as COX-2, IL-8, monocyte chemoattractant protein 1 (MCP-1), tissue factor 1, E-selectin, intercellular adhesion molecule 1 (ICAM-1), and vascular cell adhesion molecule 1 (VCAM-1) (9–11). It is worth noting that transcription of these genes is regulated by NF-B (18). Therefore, we carried out a promoter assay to evaluate whether DSCR1.4 inhibits the transcriptional activity of NF-B. Control and DSCR1-modified cell lines were transfected with an NF-B-dependent luciferase reporter plasmid (Fig. 3B). Stimulation with PMA increased luciferase activity to a similar extent in the control and DSCR1-modified cell lines (
2.5-fold that of their respective non-stimulated controls). However, the luciferase activities of the DSCR1-modified cells were markedly compared with the control both at steady state and after stimulation with PMA.
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N strongly down-regulated NF-B activity and because overexpression of full-length DSCR1 or DSCR1C caused aggregate formation,3 we used DSCR1N in subsequent experiments. Moreover, DSCR1N encodes a region that is common to all DSCR1 isoforms. Therefore, this fragment may be of use for elucidating the functions of other isoforms.
Inhibitors of Molecules Upstream of NF-B Activation Decrease COX-2 Induction—Next, we determined whether reduction of NF-B activity by DSCR1.4 is responsible for the observed attenuation of COX-2 induction. We first investigated signal transduction pathways that could lead to induction of COX-2 expression after stimulation with PMA. Among the inhibitors of signal molecules reportedly involved in PMA-mediated COX-2 expression (31–34), Ro-31-8220, PD 98059, and hypoestoxide inhibited the induction of COX-2 (Fig. 4A), indicating involvement of PKC, MEK1, and NF-B signaling. We then determined whether PKC and MEK1 are upstream signal transduction molecules of NF-B activation. The final obligatory step for activation of NF-B is the degradation of IB. Among the different IB proteins, IB plays a major role in PMA-induced activation of NF-B (35, 36). Therefore, we tested the effects of inhibitors of PKC and MEK1 on the degradation of IB after stimulation with PMA. Ro-31-8220 and PD 98059 significantly inhibited IB degradation (Fig. 4, B and C), indicating that PKC and ERK are upstream activators of NF-B.
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N. PMA-stimulated PKC degradation (Fig. 4A, middle panel, third lane) or phosphorylation of ERK (Fig. 4D) was not decreased in DSCR1N-transfected cells. In addition, PMA-stimulated phosphorylation at Ser-32 of IB was not inhibited by DSCR1N, suggesting that DSCR1 affects a step downstream of IB phosphorylation in NF-B-activating signaling.
DSCR1 Decreases the Basal Degradation and Increases the Steady-state Level of IB—Next, we investigated whether PMA-stimulated degradation of IB is inhibited by DSCR1N. For this, the time course of IB degradation in DSCR1N-transfected cells was compared with that in control cells following stimulation with PMA (Fig. 5, A and B). PMA stimulation resulted in a similar rapid reduction of IB in both control and DSCR1N-transfected cells. However, the level of IB in DSCR1N-transfected cells was higher than that in control cells throughout the time course. The steady-state level of IB was elevated by 30–40% in DSCR1N-transfected cells compared with control cells.
To test whether the discrepancy in IB levels between the two cell lines is due to a difference in the stimulation-induced degradation rate of IB, we calculated the half-life of the initial "rapid degradation" phase of IB (0–30 min). As shown in Fig. 5 (C and D), the estimated half-lives of IB in DSCR1N-transfected (19.8 min) and control (22.6 min) cells were not significantly different.
Next, we examined the basal turnover rate of IB in the same two cell lines. After incubation under serum-reduced conditions for 20 h, the cells were treated with cycloheximide. The protein level of IB was then observed every 2 h for 6 h. As shown in Fig. 6 (A and B), the IB half-life in control cells was 129.6 min, whereas that in DSCR1N-transfected cells was 280.8 min, indicating a decrease in basal IB degradation in these cells.
Knockdown of DSCR1.4 Increases IB Degradation by an IKK- and CK2-independent Mechanism—We wished to determine whether endogenous DSCR1.4 has a role in the regulation of IB degradation. In addition, we also tested whether IKK-dependent phosphorylation of IB is involved in the mechanism by which DSCR1.4 affects IB degradation. For these experiments, cells were cotransfected with DSCR1.4 siRNA and an expression vector encoding IB-mutN, which has a double mutation at Ser-32 and Ser-36. The degradation of endogenous wild-type IB and IB-mutN was observed following treatments with cycloheximide. As expected, knockdown of DSCR1.4 increased the rate of wild-type IB degradation (Fig. 7A and B), suggesting that endogenous DSCR1.4 plays an important role in IB protein stability. Similarly, the half-life of IB-mutN was also shortened from 122.3 to 81.6 min (Fig. 7, A and C), suggesting that this function of DSCR1.4 does not involve phosphorylation of IB at Ser-32 and Ser-36.
Next, we tested whether CK2-dependent phosphorylation plays a role in the DSCR1.4-mediated stabilization of the IB protein. For this, we cotransfected cells with DSCR1.4 siRNA and IB-mutC, which has a double mutation at Ser-283 and Thr-291; these residues are the most critical phosphorylation sites for CK2 (23, 24). As expected, in the presence of cycloheximide, the double mutation increased the half-life of IB by 2.7-fold compared with that of endogenous wild-type IB. However, the half-life of IB-mutC was shortened from 327 to 234 min by cotransfection with DSCR1.4 siRNA (Fig. 8, A and B).
Taken together, these results show that knockdown of DSCR1.4 decreases the stability of the IB protein by a mechanism that is independent of IKK- and CK2-mediated phosphorylation of IB. Notably, cotransfection with DSCR1.4 siRNA decreased the steady-state expression levels of endogenous wild-type IB (Fig. 2, middle panel), IB-mutN (Fig. 7A), and IB-mutC (Fig. 8A) by 30, 22, and 37%, respectively. These results suggest that endogenous DSCR1.4 plays a significant role in maintaining the level of IB.
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B Thr-291—Next, we tested whether DSCR1.4 inhibits CK2-mediated phosphorylation of IB. For this, we conducted in vitro kination assays using GST-PEST as a substrate in the presence of whole cell extracts from cells stably transfected with full-length DSCR1.4, DSCR1C, DSCR1N, and control vector. After incubation with cell extracts, the substrates were analyzed by immunoblotting with anti-phospho-IB Thr-291 antibody. As shown in Fig. 9 (A and B), overexpression of full-length DSCR1.4 and its truncation mutants increased the phosphorylation of GST-PEST. The specificity of CK2-mediated phosphorylation was evidenced by complete inhibition of phosphorylation with heparin (Fig. 9A, fifth lane).
Next, we performed in vitro kination assays using a cell extract from DSCR1.4 siRNA-transfected cells to test the effects of knockdown of DSCR1.4 on CK2-mediated phosphorylation of IB. As shown Fig. 9C, transfection of DSCR1.4 siRNA decreased GST-PEST phosphorylation.
DSCR1.4 Attenuates TNF-- and IL-1-induced NF-B Activation—The multiple signaling pathways that activate NF-B converge at one critical step in which IB proteins are degraded by a proteasome system. Thus, the elevated steady-state level of IB in the DSCR1N-transfected cell line would be predicted to attenuate NF-B activation by diverse upstream signals and inhibit the expression of NF-B target genes. To test this hypothesis, we assessed the effect of DSCR1.4 and DSCR1N on the expression of COX-2 and IL-1 after stimulation with IL-1 and TNF-, respectively. As shown in Fig. 10A, DSCR1.4 reduced IL-1- and TNF--induced expression of their target genes. In addition, DSCR1.4 attenuated the basal level of IL-1 expression (Fig. 10A, lower panel, second lane). As expected, its truncated version, DSCR1N, also inhibited induction of COX-2 expression by IL-1 or TNF- (Fig. 10B). Consistent with these results, DSCR1N significantly attenuated NF-B-driven luciferase activity that was stimulated with IL-1 or TNF- (Fig. 10C).
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DISCUSSION |
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B and thereby reduces the induction of COX-2. We suggest that different regions of DSCR1.4 may be involved in these two mechanisms on the basis of two observations. First, tagging the C terminus of DSCR1.4 with GFP abolished the inhibitory effect of DSCR1.4 on PMA-mediated COX-2 induction, whereas inhibition of PMA/A23187-mediated COX-2 induction was preserved. Second, the region encoded by exon 7 of DSCR1 is necessary for binding to and inhibition of calcineurin (30, 38). However, the C-terminally truncated protein (DSCR1C) lacks this region and yet more potently inhibited the activity of NF-B and COX-2 induction compared with full-length DSCR1.4.
An important question is how DSCR1 decreases activation of NF-B. Cytoplasmic retention of NF-B by IB is the major mechanism that controls NF-B activity. In this study, we found that both the half-life of IB in the presence of cycloheximide and the steady-state level of IB were increased in cells stably transfected with DSCR1N. In the same context, knockdown of endogenous DSCR1.4 decreased both values. IB degradation is regulated mainly through phosphorylation. Several phosphorylation sites have been identified on IB, including the signal-induced IKK phosphorylation sites located at Ser-32 and Ser-36 (20) and the constitutive CK2 phosphorylation sites located in the PEST region (39). However, the IKK-dependent pathway does not appear to be inhibited in DSCR1N-transfected cells because PMA-induced phosphorylation at Ser-32 and degradation of IB were not decreased. Moreover, the increase in IB degradation, induced by knockdown of DSCR1.4, was not blocked by the double mutation of Ser-32 and Ser-36.
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B (39). However, CK2 phosphorylation also did not appear to be affected by DSCR1.4: the double mutation of Ser-283 and Thr-291 did not block the decreased steady-state level of IB and the elevated turnover rate of IB that was induced by the knockdown of endogenous DSCR1.4. In addition, we have shown that overexpression of DSCR1.4 or its truncation mutants unexpectedly increased, rather than decreased, CK2-dependent phosphorylation of IB at Thr-291 using an in vitro kinase assay. Furthermore, a similar increase in CK2 activity due to DSCR1.4 overexpression was observed in a kinase assay using the synthetic peptide substrate RRRADDSDDDDD (data not shown). On the basis of these results, we suggest that DSCR1.4 modulates the degradation of IB by an IKK- and CK2-independent pathway.
Previous studies have demonstrated the calcineurin dependence of NF-B activation. Calcineurin has been reported to synergize with other signaling molecules such as Raf (40) and PKC (41, 42) to activate the NF-B pathway. Additionally, full activation of NF-B requires simultaneous input through Ca2+- and PMA-activated signaling pathways (43). Finally, many genes are under the dual regulation of NFAT and NF-B (12). Taken together, these data suggest that inhibition of calcineurin would attenuate PMA/A23187 induction of some NF-B target genes. Here, we have shown that DSCR1.4 inhibited NF-B pathways as well as calcineurin in separate mechanisms. This explains how expression of DSCR1.4 can inhibit gene expression by PMA/A23187 more potently compared with pharmacological calcineurin inhibitors, as observed by Hesser et al. (9). In addition, DSCR1.4 is expected to be a broad-acting endogenous suppressor of NF-B target gene expression because it elevates IB levels, affecting activation of NF-B via the classical pathway. Consistent with this hypothesis, this study has shown that DSCR1.4 attenuated induction of NF-B target genes by IL-1 and TNF-, which utilize different adaptor molecules to activate IKK and subsequently degrade IB (44, 45).
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B and induction of its target genes, such as COX-2 and IL-1, in a glial cell line suggests its significant role in preventing inflammatory reactions. This is supported by previous studies demonstrating that NF-B expression is increased and can be activated by amyloid -peptides in the brains of Alzheimer disease patients and that pharmacological prevention of this activation results in reduction of the inflammatory responses (46, 47). In Down syndrome cases, the density of activated microglia overexpressing IL-1 is as much as 30-fold higher than in age-matched control samples (48). During cerebral ischemia, NF-B is involved in acute phase and inflammatory responses, which potentiate ischemic injury (49–51). In transgenic mice that express mutant IB with inhibited degradation, cerebral ischemia causes reduced NF-B target gene expression and reduced infarct size (52). These finding suggest that DSCR1 could be a potential therapeutic target for treatment of these neurodegenerative diseases.
In conclusion, this study has shown that DSCR1.4 inhibits IB metabolism and may have physiologically relevant anti-inflammatory properties. The mechanism by which DSCR1.4 inhibits the basal turnover rate of IB and its pathophysiological relevance in neuroinflammatory diseases remain to be addressed in future studies.
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FOOTNOTES |
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1 To whom correspondence should be addressed: Dept. of Pharmacology, College of Medicine, Catholic University of Korea, Banpo-dong 505, Seo-cho-gu, Seoul 137-701, Korea. Tel.: 82-2-590-1202; Fax: 82-2-536-2485; E-mail: haechee{at}catholic.ac.kr.
2 The abbreviations used are: DSCR1, Down syndrome candidate region 1; NF-B, nuclear factor B; IB, inhibitors of B; IKK, IB kinase; CK2, casein kinase 2; COX-2, cyclooxygenase-2; IL-1, interleukin-1; PMA, phorbol 12-myristate 13-acetate; TNF-, tumor necrosis factor-; PKC, protein kinase C; ERK1/2, extracellular signal-related kinase 1/2; GFP, green fluorescent protein; GST, glutathione S-transferase; siRNA, small interfering RNA; MEK1, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase 1.
3 K.-O. Cho, Y. S. Kim, H. J. Lee, S.-K. Kim, S. Y. Kim, and Y.-J. Cho, unpublished data.
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) of I
