Involvement of poly(ADP-ribose) polymerase 1 in ERBB2 expression in rheumatoid synovial cells

Takuya Kitamura, Masayuki Sekimata, Shin-ichi Kikuchi, and Yoshimi Homma

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


    ABSTRACT
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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Hyperplasia of synovial lining cells is one of the main features of rheumatoid arthritis (RA). We previously reported that ERBB2 is highly expressed in RA synovial cells and that it plays an important role in their hyperproliferative growth. Recent findings have suggested that poly(ADP-ribose) polymerase-1 (PARP-1) is involved in the transactivation of NF-{kappa}B-dependent genes such as ERBB2. In the present study, we investigated the role of PARP-1 in ERBB2 transcription in RA synovial cells. The expression level of PARP-1 was significantly high in synovial cells derived from three patients with RA, compared with three patients with osteoarthritis (OA). Luciferase assays revealed that PARP-1 augments the transcription of the ERBB2 gene and that a region between –404 and –368 is responsible for this activation. A protein with an apparent molecular mass of 115 kDa was isolated mainly from nuclear extracts of RA synovial cells with an affinity matrix harboring a DNA fragment identical to the above region. Mass spectrometric analysis demonstrated this protein to be PARP-1. Southwestern blot analysis showed that PARP-1 binds to this region, but not to adjacent regions. PARP-1 associates directly with NF-{kappa}B, and a chromatin immunoprecipitation assay indicated that these proteins interact with this enhancer region in the ERBB2 gene. Treatment of RA synovial cells with PARP-1 small interfering RNA attenuated their ERBB2 expression, while an inhibitor of the polymerase activity of PARP-1 had no effect. PARP-1 DNA binding is not required for transcriptional activation. These findings suggest that PARP-1 is involved in the expression of ERBB2 in concert with NF-{kappa}B, which might be associated with the proliferation of RA synovial cells.

nuclear factor-{kappa}B; rheumatoid arthritis; joint lining cell; hyperproliferation


RHEUMATOID ARTHRITIS (RA) is characterized by hyperplasia of synovial lining cells, excessive infiltration of mononuclear cells, and extensive destruction of the articular cartilage (9). The proliferation of synovial fibroblasts and their invasive growth are due to an impairment in the regulation of the cell cycle. There is growing evidence that the cellular and metabolic alterations of synoviocytes are caused by the upregulated transcription of members of a diverse group of genes, including protooncogenes (20). ERBB2, which encodes a 185-kDa transmembrane tyrosine kinase (ErbB2), was identified on the basis of its close similarity to the epidermal growth factor receptor (EGFR) genes. The human ERBB2 gene is frequently amplified and overexpressed in adenocarcinomas, especially in those arising in the breast (29) and ovary (4). We previously reported that ErbB2 is expressed in rheumatoid synovium and synovial cells in primary cultures from RA patients. We also demonstrated that the growth of synovial cells is inhibited by genistein, a tyrosine kinase inhibitor, and herceptin, an anti-ErbB2 monoclonal antibody (27). These results suggested that ErbB2 plays an important role in RA synovial hyperproliferation. Although ErbB2 overexpression is generally associated with amplification of the ERBB2 gene, we have never observed amplification of this gene in any surgical or fibroblast samples from RA patients.

NF-{kappa}B is abundant in the rheumatoid synovium, and immunohistochemical analysis has demonstrated p65 and p50 NF-{kappa}B proteins in the nuclei of synovial lining cells (10). Nuclear translocation and activation of NF-{kappa}B occurs rapidly after stimulation with interleukin (IL)-1 or tumor necrosis factor-{alpha} (TNF-{alpha}) 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-{kappa}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-{kappa}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.


    MATERIALS AND METHODS
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 MATERIALS AND METHODS
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Cell culture. Synovial tissue samples were obtained at the time of surgery in three patients with RA and three patients with osteoarthritis (OA) with approval from Fukushima Medical University Review Board. Synovial cells were isolated from synovial tissue samples as previously described (27), and they were maintained in RPMI 1640 medium with 20% fetal calf serum (FCS). When the cells were grown to subconfluence, they were passaged at a ratio of 1:2–1:4. The medium was changed every 3–4 days, and the cells from passages 410 were used in the experiments. Normal lung fibroblasts TIG-7 and Madin-Darby canine kidney (MDCK) cells were maintained in Dulbecco’s modified Eagle’s medium with 10% FCS.

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-{kappa}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 manufacturer’s 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-{kappa}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-{kappa}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).


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Enhanced expression of PARP-1 in RA synovial cells. To evaluate the involvement of the PARP-1 protein in the activation of ERBB2 in synovial cells, we first examined the expression levels of PARP-1 in synovial cells derived from patients with RA or OA. When nuclear proteins of primary cells from three individual RA and OA patients were analyzed using Western blot analysis, the expression of PARP-1 in the three RA samples was obviously high compared with that in synovial cells derived from OA patients (Fig. 1). The level in RA patients was two times higher than the level in OA patients. Because the level in OA samples was high compared with normal TIG-7 fibroblasts (data not shown), it seems apparent that the level of PARP-1 expression in RA is abnormally high.



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Fig. 1. Expression levels of poly(ADP-ribose) polymerase-1 (PARP-1) in synovial cells. The nuclear extracts (20 µg of protein) derived from three patients with rheumatoid arthritis (RA) and three patients with osteoarthritis (OA) were examined using Western blot analysis. The examination was repeated four times, and representative results are shown. The intensity of each band was quantified using NIH Image software, and the statistical significance of differences between RA and OA samples was calculated using Student’s t-test. *P < 0.01.

 
Identification of a PARP-1-dependent enhancer region in the ERBB2 gene. Recent studies have suggested that PARP-1 enhances gene expression, including the NF-{kappa}B-dependent pathway. Because NF-{kappa}B functions as a critical activator for the transcription of ERBB2, we examined the effect of PARP-1 on the promoter activity of the ERBB2 gene using luciferase reporter genes harboring different deletions in the 5' enhancer region (Fig. 2A). When MDCK cells were cotransfected with PARP-1 and the reporter vectors, the transcription level was significantly enhanced by PARP-1 in the case of –404LUC and –443LUC, but not in the case of –348LUC and –368LUC (Fig. 2B). This finding suggests that the PARP-1-dependent enhancer region might be present between –404 and –368, which is adjacent to the NF-{kappa}B-binding sites (13).



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Fig. 2. Deletional analysis of human ErbB2 promoter activity. A: luciferase (LUC) reporter constructs used in this study. The translation initiation site is designated as nucleotide +1. B: effect of PARP-1 on the promoter activity of ERBB2. Madin-Darby canine kidney (MDCK) cells (2 x 104) were transfected with one of the ERBB2-LUC reporter genes (1 µg) and the pRL-TK vector (2.5 pg). Luciferase activity was measured 48 h after transfection and normalized to Renilla luciferase activity. PARP-1 significantly potentiated the promoter activity in cells transfected with –443LUC and –404LUC, but not with –368LUC and –348LUC. The statistical significance of the differences between vehicle and PARP-1 overexpression groups was calculated using Student’s t-test. *P < 0.01.

 
To explore the above results in RA synovial cells, we prepared an affinity matrix on which a double-stranded DNA fragment corresponding to the region between –403 and –359 was immobilized (Fig. 3A), and we purified the binding proteins from nuclear extracts of RA cells. The eluate from the matrix was fractionated using SDS-PAGE and examine using Western blot analysis with anti-PARP-1 and anti-NF-{kappa}B antibodies. The protein bands, visualized using Coomassie brilliant blue staining, showed a 115-kDa band to be considerably enriched by this purification, and the band appeared to be PARP-1. Next, we analyzed the amino acid sequences of peptide fragments derived from the 115-kDa band using mass spectrometry and confirmed the 115-kDa protein to be PARP-1. Immunoblot analysis also revealed that the eluate from the DNA matrix contained NF-{kappa}B together with PARP-1 (Fig. 3B).



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Fig. 3. Examination of sequence-specific proteins. A: fragment of double-stranded DNA identical to the region from –443 to –368 was prepared as an affinity resin. X, amino linker. B: sequence-specific proteins were purified from nuclear extracts of synovial cells derived from an RA patient (RA1 in Fig. 1). An aliquot of eluate (~0.1 µg of protein) was separated by performing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the proteins were visualized by staining with Coomassie brilliant blue (left). An aliquot of eluate was also examined using Western blot analysis with anti-PARP-1 (middle) and anti-p65 NF-{kappa}B (right) antibodies. C: to confirm the PARP-1-binding region, Southwestern blot analysis was performed using three different fragments as probes: –404 to –380 (lane 1), –393 to –378 (lane 2), and –383 to –368 (lane 3). Positive bands were visualized using autoradiography. The same sample was blotted with anti-PARP-1 (lane 4).

 
To confirm that the PARP-1 protein present in RA synovial cells actually binds to a region between –404 and –368 of the ERBB2 gene, we performed Southwestern blot analysis using crude nuclear extracts derived from RA synovial cells. Among three fragments identical to regions –404 to –380, –393 to –378, or –383 to –368 used as probes, a positive band with an apparent molecular mass of 115 kDa appeared only when the fragment –393 to –378 was used as a probe (Fig. 3C, lane 2). This fragment from –393 to –378 did not bind to recombinant p65 NF-{kappa}B, and the band was attenuated by excess amounts of cold probe (data not shown). Thus interaction of PARP-1 with the region from –393 to –378 might take place in a sequence-specific manner.

Association of PARP-1 with NF-{kappa}B in RA synovial cells. The presence of a direct interaction between PARP-1 and NF-{kappa}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-{kappa}B and interacts with a proximal region of the NF-{kappa}B binding site, which enhances the transcription of the ERBB2 gene.



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Fig. 4. Direct interaction of PARP-1 with NF-{kappa}B and a promoter region of the ERBB2 gene. A: nuclear extracts (20 µg of protein) were incubated with anti-PARP-1 or anti-NF-{kappa}B p65, the resultant precipitates were resolved by performing SDS-PAGE, and the proteins were detected using Western blot analysis (WB) with anti-PARP-1 or anti-p65. B: chromatin immunoprecipitation was performed with RA synovial cells (2 x 105) using anti-PARP-1 (lane 2), anti-p65 (lane 3), or control IgG (lane 4) in addition to an aliquot of input (lane 1). DNA was isolated and analyzed after PCR with a primer pair from the ERBB2 promoter locus and a region 2 kb downstream from the promoter. The PCR product was resolved in a 1.5% agarose gel and visualized using ethidium bromide staining.

 
DNA binding activity of PARP-1 is not required for enhancer function. To assess whether DNA binding activity of PARP-1 is required for the transactivation of ERBB2 gene, we constructed reporter plasmids –404LUC(7cg) and –404LUC(0cg) harboring scrambled DNA sequences for the region from –393 to –369. When MDCK cells were transfected with one of these reporters and an expression plasmid of PARP-1, significant transcriptional enhancement was observed (Fig. 5B). We confirmed that these scrambled sequences did not bind to PARP-1. Alternatively, a DNA-binding mutant of PARP-1(C21G/C125G) also showed significant enhancement in cells possessing a reporter vector, –443LUC or –404LUC (Fig. 5C). These results suggest that the DNA-binding activity of PARP-1 might not be necessary for the enhancer function of PARP-1.



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Fig. 5. Assessment of DNA-binding activity of PARP-1. A: luciferase reporter constructs derived from –404LUC are shown. –404LUC(7cg) and –404LUC(0cg) contain scrambled sequences of the –393 to –369 region. –404LUCd(–368) is a deletion construct lacking a NF-{kappa}B-binding site (–368 to –348). B: effect of PARP-1 on the promoter activity of the ERBB2 gene. MDCK cells (2 x 104) were transfected with one of the ERBB2-LUC reporter genes (1 µg) and the pRL-TK vector (2.5 pg). Luciferase activity was measured 48 h after transfection with PARP-1/pCMV or vehicle and normalized to the Renilla luciferase activity. PARP-1 significantly enhanced the promoter activity in cells transfected with –404LUC, –404LUC(0cg), and –404LUC(7cg), but not in cells transfected with –404LUCd(–368). C: effect of PARP-1 and a DNA binding mutant PARP-1(C21G/C125G) on the promoter activity of ERBB2. MDCK cells (2 x 104) were transfected with –443LUC or –404LUC reporter gene (1 µg) and the pRL-TK vector (2.5 pg). Luciferase activity was measured 48 h after transfection with PARP-1, PARP-1(C21G/C125G), or vehicle and normalized to the Renilla luciferase activity. Statistical significance of the differences between vehicle and PARP-1-overexpression groups was calculated using Student’s t-test. *P < 0.01.

 
We also tested the effect of deletion of NF-{kappa}B binding site (the –368 to –348 region) on the enhancer activity of PARP-1 using a reporter plasmid, –404LUCd(–368). As shown in Fig. 5B, no significant enhancement was induced by the expression of PARP-1, revealing that the NF-{kappa}B site is indispensable for the enhancer activity of PARP-1.

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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Fig. 6. Effect of 3-aminobenzamide (3-AB) and PARP-1 small interfering RNA (siRNA) on PARP-1-induced ErbB2 transactivation. A: MDCK cells (2 x 104) were transfected with reporter vectors (–443LUC and pRL-TK), along with an expression plasmid for PARP-1 (PARP-1/pcDNA3) (lanes 3 and 4) or control pcDNA3 (lanes 1 and 2; 1 µg each). Assays were performed in the absence (lanes 1 and 3) or presence of 3-AB (2 mM), a potent inhibitor of polymerase activity of PARP-1 (lanes 2 and 4). Cells were harvested 48 h after transfection, luciferase activity was measured, and the activity was measured and normalized to Renilla luciferase activity. B: RA synovial cells were treated with the indicated doses of 3-AB for 3 days, and the level of ErbB2 was determined using Western blot analysis. The intensity of each band was quantified using NIH Image software. C: RA synovial cells were treated with the indicated doses of PARP-1 siRNA for 3 days, and the level of cellular proteins was determined using Western blot analysis. Aliquots of cell lysate (50 µg of protein) or nuclear extract (20 µg of protein) were used for the detection of ErbB2 and GAPDH or PARP-1 and NF-{kappa}B, respectively. The examination was repeated three times, and representative results are shown. D: intensity of each band was quantified using NIH Image software. Statistical significance of the differences among samples was calculated using Student’s t-test. *P < 0.01.

 
On the other hand, we examined the effect of siRNA on ErbB2 expression in these cells. When RA synovial cells were treated with different doses of siRNA, the expression level of PARP-1 was attenuated in a dose-dependent manner. A significant loss in PARP-1 expression was observed in cells treated with 2 nM siRNA (Fig. 6, C and D). Expression of ErbB2 was also reduced by the treatment with 2 nM siRNA, while that of p65 NF-{kappa}B was not affected. These results confirm an enhancer function of PARP-1 on ErbB2 expression in RA synovial cells that is not related to its poly(ADP-ribose) polymerase activity.


    DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
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 DISCUSSION
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Although PARP-1 was originally identified as a DNA damage sensor and signaling molecule that binds to both single- and double-stranded DNA breaks, its biological role as reported to date is complex and includes various important functions. In immunological and inflammatory spheres, it is interesting that PARP-1 regulates the expression of a number of cellular proteins at the transcription level (24, 25, 34). Of importance is the enhancement of the production of inflammatory mediators such as the inducible nitric oxide synthase and intercellular adhesion molecule-1. NF-{kappa}B is a key transcription factor in the regulation of this set of proteins, and PARP-1 has been shown to act as a coactivator in NF-{kappa}B-mediated transcription (11, 14, 21, 24). We previously demonstrated that ErbB2 plays an important role in the proliferation of RA synovial cells (27). Because ErbB2 is one of the NF-{kappa}B-mediated transcripts, we investigated the expression of PARP-1 in RA synovial cells in the present study. The results of study demonstrate that the level of PARP-1 expression is significantly high in RA cells and that this protein is actually involved in the transactivation of ERBB2 in concert with NF-{kappa}B. Namely, PARP-1 binds directly to NF-{kappa}B and also to DNA. Suppression of PARP-1 by siRNA reduces ErbB2 expression in RA synovial cells. These findings suggest that PARP-1 is involved in the pathogenesis of RA.

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-{kappa}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-{kappa}B-dependent transcriptional activation is severely affected in immortalized PARP-1–/– cells, whereas translocation of NF-{kappa}B to the nuclei is normally observed (24). Thus it is conceivable that NF-{kappa}B is indispensable for the transcription of ERBB2 and that PARP-1 enhances the activity of NF-{kappa}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-{kappa}B coactivator function. PARP-1–/– cells overexpressing a PARP-1 mutant lacking the DNA binding region can normally interact with NF-{kappa}B and augment {kappa}B-dependent transcription (11). These findings are consistent with our present observations (Fig. 5). Therefore, an interaction between PARP-1 and NF-{kappa}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-{kappa}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-{kappa}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-{kappa}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-{kappa}B, which might be associated with the proliferation of RA synovial cells.


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 ABSTRACT
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This study was supported by grants from the Fukushima Society for the Promotion of Medicine.


    ACKNOWLEDGMENTS
 
We thank Dr. Margaret Dooly Ohto for comments on the manuscript.


    FOOTNOTES
 

Address for reprint requests and other correspondence: Y. Homma, Dept. of Biomolecular Science, Fukushima Medical Univ. School of Medicine, Fukushima 960-1295, Japan (e-mail: yoshihom{at}fmu.ac.jp)

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.


    REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
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