Department of Biomolecular Science and Orthopedics, Fukushima Medical University School of Medicine, Fukushima, Japan
Submitted 6 October 2004 ; accepted in final form 23 February 2005
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ABSTRACT |
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nuclear factor-B; rheumatoid arthritis; joint lining cell; hyperproliferation
NF-B is abundant in the rheumatoid synovium, and immunohistochemical analysis has demonstrated p65 and p50 NF-
B proteins in the nuclei of synovial lining cells (10). Nuclear translocation and activation of NF-
B occurs rapidly after stimulation with interleukin (IL)-1 or tumor necrosis factor-
(TNF-
) and induces a series of cytokines and growth-associated gene products such as ErbB2. Recent studies have demonstrated that poly(ADP-ribose) polymerase 1 (PARP-1) is required for the expression of NF-
B-dependent genes (11, 14, 21, 24). In the present study, we have examined the involvement of PARP-1 in the transcription of the ERBB2 gene and have shown that RA synovial cells express the PARP-1 protein at high levels. PARP-1 binds to NF-
B and upregulates the promoter activity of the ERBB2 gene. Treatment of RA synovial cells with PARP-1 small interfering RNA (siRNA) attenuates the expression of ErbB2. These results suggest that PARP-1 may play an important role in the overexpression of ErbB2 in RA synovial cells.
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MATERIALS AND METHODS |
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Plasmid constructions.
The mammalian expression plasmid for human PARP-1 was generated using pcDNA3 (Invitrogen, Carlsbad, CA). The luciferase reporter plasmid containing the human ERBB2 promoter sequence from 443 to 144 relative to the start site of transcription (443LUC) was generated using pGL3-Basic (Promega, Madison, WI). To obtain the 5' deletion construct (404LUC), the promoter sequence was amplified by performing PCR using primers with synthetic SacI and BamHI sites at the 5' and 3' ends, respectively. The amplified fragment was digested and subcloned into the SacI and BglII sites of pGL3-Basic. Similarly, 368LUC and 348LUC were generated by performing PCR using primers with synthetic NheI and BamHI sites at the 5' and 3' ends, respectively. 404LUC(7cg) and 404LUC(0cg) were generated by performing PCR using primers with a scrambled sequence of the region 393 to 369. 404LUCd(368), a deletion construct lacking an NF-B-binding site 368 to 348, was also produced by performing PCR using primers with corresponding sequences. Expression vectors for human PARP-1, PARP-1/pCMV, and PARP-1/pcDNA3 were made by ligating the cDNA encoding the wild-type human PARP-1 (2), which was cloned into pCMV-Tag2 (Stratagene, La Jolla, CA) and pcDNA3 (Invitrogen, Carlsbad, CA), respectively. The expression plasmid for a DNA-binding mutant of PARP-1, PARP-1(C21G/C125G), was generated by performing site-directed mutagenesis as described previously (11).
Transfection. PARP-1 siRNA (Santa Cruz Biotechnology, Santa Cruz, CA) was introduced into RA synovial cells using Polyfect transfection reagent (Qiagen, Hilden, Germany). Briefly, RA synovial cells (2 x 105 cells) plated on 10-cm dishes were treated for 72 h with various amounts of PARP-1 siRNA embedded in Polyfect reagent according to the manufacturers instructions. A luciferase assay was performed using MDCK cells as a model system. MDCK cells (2 x 104) plated on 24-well plates were transfected using Effectene reagent (Qiagen) with 0.2 µg of each reporter construct, 5 ng of pRL-TK (Promega), and the indicated amounts of a PARP-1 expression plasmid. After incubation for 48 h, the relative luciferase activities were determined using a dual luciferase assay system (Promega) in a Turner TD-20/20 luminometer (Turner Designs, Sunnyvale, CA). The effect of 3-aminobenzamide (3-AB; Sigma, St. Louis, MO) was evaluated by incubating transfected or RA synovial cells with various concentrations of 3-AB for 48 h (8, 11).
Western blot analysis.
Cell lysate was prepared from confluent cultures of synovial cells. Cells were washed with cold phosphate-buffered saline, and cytosolic and membrane proteins were extracted in a lysis buffer consisting of 20 mM Tris·HCl (pH 7.5), 1% Nonidet P-40 (NP-40), 0.5 mM dithiothreitol, 10 µg/ml leupeptin, 10 µg/ml aprotinin, and 100 µM phenylmethylsulfonyl fluoride. After centrifugation for 5 min, the supernatant was used for the detection of ErbB2 and an internal control GAPDH. Nuclear proteins were extracted further by incubating the pellet in a buffer consisting of 20 mM Tris·HCl (pH 7.5), 6 M urea, 0.5 mM dithiothreitol, 10 µg/ml leupeptin, 10 µg/ml aprotinin, and 100 µM phenylmethylsulfonyl fluoride. Cell debris was removed by performing centrifugation for 20 min, and the supernatant fraction was used for the detection of PARP-1 and NF-B. Cell lysate (50 µg of protein) or nuclear extract (20 µg of protein) was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and examined using Western blot analysis with polyclonal anti-ErbB2 antibody (Calbiochem, La Jolla, CA), anti-PARP-1 antibody (Santa Cruz Biotechnology), anti-NF-
B p65 antibody (Santa Cruz Biotechnology), and anti-GAPDH antibody (Chemicon International, Temecula, CA). A horseradish peroxidase-conjugated antibody was used for the secondary antibody (Promega). Positive bands were visualized using an enhanced chemiluminescence system (Amersham Biosciences, Piscataway, NJ).
Southwestern blot analysis. Nuclear protein extracts prepared from RA synovial cells were used in the DNA-binding reactions. The extract was treated with the sample buffer for 3 min at room temperature and subjected to SDS-PAGE. Transfer of nuclear proteins to nitrocellulose filters was performed for 1 h at room temperature with buffer containing 25 mM Tris, 190 mM glycine, and 10% methanol. After pretreatment with hybridization buffer containing 50 mM HEPES (pH 7.8), 5 mM MgCl2, 50 mM NaCl, 1 mM DTT, 1 mM EDTA, and 0.05% Tween 20 for 1 h, the blots were incubated overnight at 4°C with the 32P-labeled, double-stranded probe and 10 µg/ml poly(dI-dC) in the presence or absence of a 20-fold molar excess of the unlabeled probe. The blots were washed three times in hybridization buffer, dried, and subjected to autoradiography.
Chromatin immunoprecipitation. Chromatin immunoprecipitation (ChIP) assays were performed using a ChIP assay kit (Upstate Biotechnology, Lake Placid, NY). Synovial cells were fixed in 1% formaldehyde for 10 min. Soluble chromatin was prepared and immunoprecipitated with anti-PARP-1 antibody or a control immunoglobulin (IgG). The final DNA preparations were amplified by performing PCR using a set of primers for the ERBB2 promoter sequences 5'-ACCTGAGACTTAAAAGGGTGTTAAGAGTGG-3' and 5'-CAACTGCATTCCAACGAAGTCTGGG-3'.
DNA affinity purification and mass spectrometry. The oligonucleotide sequence used for the preparation of the DNA affinity resins was 5'-GAACGGCTGCAGGCAACCCAGGCGTCCCGGCGCTAGGA-GGGACGC-3'. Double-stranded DNA was obtained by annealing two complementary oligonucleotides and immobilizing the product on N-hydroxysuccinimide-activated Sepharose beads (Amersham Biosciences). Nuclear extracts from RA synovial cells were preincubated with heparin-agarose beads equilibrated with a buffer containing 25 mM HEPES (pH 7.8), 12.5 mM MgCl2, 0.2 mM EDTA, 1 mM DTT, 1 mM phenylmethylsulfonyl fluoride, and 20% glycerol. After being washed extensively, the bound proteins were eluted with a linear gradient of KCl. An aliquot (2 µl) of each fraction was assayed for DNA-binding activity using EMSA. The pooled sample containing the DNA-binding activity was precleared with glycine-coupled Sepharose beads and incubated with the DNA affinity resin equilibrated with a buffer containing 25 mM HEPES (pH 7.8), 12.5 mM MgCl2, 1 mM DTT, 0.1% NP-40, 0.1 M KCl, 20% glycerol, and 60 µg/ml poly(dI-dC). After being washed extensively, the bound proteins were eluted with a solution containing 8 M urea, 2% 3-([3-cholamidopropyl]dimethylammonio)-2-hydroxy-1-propanesulfonate, and 0.1 mM DTT. Eluates were subjected to SDS-PAGE and stained with Coomassie brilliant blue. To obtain sequence information, protein bands were excised, digested with trypsin, and subjected to QSTAR quadrupolar time-of-flight mass spectrometry (Applied Biosystems, Foster City, CA).
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RESULTS |
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Association of PARP-1 with NF-B in RA synovial cells.
The presence of a direct interaction between PARP-1 and NF-
B was investigated using RA synovial cells. When crude nuclear extracts were immunoprecipitated with an anti-PARP-1 antibody, p65 was recovered in the immune complex. Similar results were obtained when an anti-p65 antibody was used for immunoprecipitation (Fig. 4A). On the other hand, when the anti-PARP-1 antibody was used for chromatin immunoprecipitation, fragments of the ERBB2 gene were recovered in the precipitates. A band of the same size was obtained in the immunocomplex with the anti-p65 antibody, while none of the ERBB2 gene was present in the complex obtained with control IgG (Fig. 4B). No specific band was observed when the immunoprecipitates were assayed for different regions (
2 kb downstream) of the ERBB2 gene. These results suggest that PARP-1 binds to NF-
B and interacts with a proximal region of the NF-
B binding site, which enhances the transcription of the ERBB2 gene.
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Requirement of PARP-1 for ErbB2 transcription in RA cells. We examined the effect of 3-AB, a potent inhibitor of the poly(ADP-ribose) polymerase activity of PARP-1 (8, 11), on its enhancer activity for the expression of ERBB2. MDCK cells were transfected with reporter vectors (443LUC and pRL-TK), together with the expression plasmid PARP-1/pcDNA3. The level of transcription was not significantly affected when the transfected cells were maintained in the presence of 2 mM 3-AB (Fig. 6A). Similar results were obtained using RA synovial cells; no effect of 3-AB on the expression level of ErbB2 was observed (Fig. 6B).
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DISCUSSION |
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The mechanism underlying the high level of PARP-1 expression in RA synovial cells is not clearly understood. PARP-1 is expressed in all tissues to varying degrees (14, 18). A decrease in the amount of PARP-1 transcript is associated with cellular differentiation and cell senescence (17), whereas an increase is observed upon the activation of lymphocytes (18) and often in hyperproliferative cells, including cancer cells (5, 12). The PARP-1 gene promoter possesses a structure similar to that of housekeeping genes, which lack a functional consensus TATA box, are GC-rich, and contain a consensus initiator sequence surrounding the transcription site (16). This region contains binding sites for transcription factors Sp1 (15), activator protein-2 (33), YY1 (22), and Ets (30). Therefore, hyperproliferative states of RA synovial cells might be associated with the high level of PARP-1 expression.
We have demonstrated that PARP-1 binds to NF-B and activates the transcription of the ERBB2 gene. DNA binding of PARP-1 is not required for the transcriptional enhancer function. Recent studies have shown that NF-
B-dependent transcriptional activation is severely affected in immortalized PARP-1/ cells, whereas translocation of NF-
B to the nuclei is normally observed (24). Thus it is conceivable that NF-
B is indispensable for the transcription of ERBB2 and that PARP-1 enhances the activity of NF-
B, probably by stabilizing the transcription complex. In addition, Hassa et al. (11) reported that the DNA binding activity of PARP-1 is not required for the NF-
B coactivator function. PARP-1/ cells overexpressing a PARP-1 mutant lacking the DNA binding region can normally interact with NF-
B and augment
B-dependent transcription (11). These findings are consistent with our present observations (Fig. 5). Therefore, an interaction between PARP-1 and NF-
B may be enough to activate ERBB2 transcription in RA synovial cells.
On the other hand, it would also be interesting to know whether PARP-1 recognizes and binds to a specific sequence. Our Southwestern blot analysis results (Fig. 3C) show that PARP-1 binds to a region adjacent to the NF-B-binding sites in the promoter of ERBB2 gene, likely in a sequence-specific manner. To clarify this issue, we performed casting purification to identify a consensus PARP-1-binding sequence (28, 32). Although a number of different oligonucleotides were cloned and sequenced, no consensus sequence was obtained from these clones. This discrepancy was consistent with previous reports in which various sequences were identified as the consensus binding sequence of PARP-1 (1, 3, 6, 11, 21, 26). Therefore, further studies are required to clarify the underlying mechanisms by which PARP-1 recognizes and binds to DNA regions.
Recent studies have clearly demonstrated the role of PARP-1 activation in various types of local inflammation induced by typical stimuli and have shown that inhibitors of poly(ADP-ribose) polymerase activity, including 3-AB, reduces the infiltration of neutrophils (31), joint swelling, and paw edema (7). In contrast to the successful application of PARP-1 inhibitors to immune cells and inflammation, we have shown in the present study that a PARP-1 inhibitor has no effect on the PARP-1-induced transactivation of ERBB2. ErbB2 plays an important role in the hyperproliferation of RA synovial cells, and these inhibitors are unlikely to reduce their growth properties. Rather, novel drugs that interfere with the interaction between PARP-1 and NF-B might be effective in preventing the abnormal growth of RA synovial cells.
In conclusion, we demonstrated in the present study that RA synovial cells express the PARP-1 protein at high levels. PARP-1 binds to NF-B and upregulates promoter activity of the ERBB2 gene. Treatment of RA synovial cells with PARP-1 siRNA attenuates the expression of ErbB2. These results suggest that PARP-1 is involved in the expression of ErbB2 in concert with NF-
B, which might be associated with the proliferation of RA synovial cells.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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