Abstract
Background
Interleukin (IL)-1β is a key cytokine in the pathogenesis of periodontitis, and it induces inflammatory mediators in periodontal diseases. We developed immortalized human gingival fibroblasts (HGFs), investigated the effects of IL-1β on the gene expression using expression arrays containing ~40,000 genes, and tested the role of nuclear factor-kappa B (NF-κB) in maintaining an activated HGF population.
Methods
Total RNA was isolated from IL-1β–induced and mock-induced control cells. Gene expression analyses were performed using expression arrays and confirmed by quantitative real-time polymerase chain reaction. Western blot analysis to show inhibitor of kappa B-alpha (IκBα) phosphorylation and immunostaining of cells for NF-κB nuclear translocation were performed. Apoptosis was confirmed by assay of poly ADP-ribose polymerase (PARP) cleavage.
Results
A total of 382 probe sets corresponding to 254 genes were differentially expressed in IL-1β–induced cells (P <0.001). A total of 215 genes were upregulated, and 39 genes were downregulated. Most notable NF-κB pathway members (NFκB1, NFκB2, IκBα, IκBε, IκBζ, REL, RELB, and TA-NFKBH) were upregulated. IκBα was phosphorylated, and NF-κB accumulated in the nucleus. An IL-1β–induced set of 27 genes was downregulated by an NF-κB inhibitor, leading to a decreased number of viable cells and suggesting an antiapoptotic role for NF-κB.
Conclusions
IL-1β leads to a large number of significant expression changes consistent with a pathologic role in periodontitis, including enhancement of inflammatory cytokines, chemokines, transcription factors, matrix metalloproteinases, adhesion molecules, and especially NF-κB–dependent antiapoptotic genes. NF-κB activation blocks apoptosis, thereby stabilizing the HGF population in inflammation.
Keywords: Fibroblasts, gene expression, interleukin-1 beta, microarrays, nuclear factor-kappa B, real-time polymerase chain reaction
Periodontal diseases are chronic inflammatory conditions that result in the degradation of supporting connective tissue structures surrounding teeth. Periodontal inflammation is driven by the overexpression of proinflammatory cytokines, mainly interleukin (IL)-1β and tumor necrosis factor-alpha (TNF-α). IL-1 is one of the major cytokines produced at inflamed sites and is involved in the initiation and progression of connective tissue degradation.1,2 IL-1 production is mainly upregulated by microbial products, such as lipopoly-saccharide (LPS).1 Gingival and periodontal ligament fibroblasts secrete high levels of cytokines, chemokines, matrix metalloproteinases, and adhesion molecules during the progression of periodontal inflammation.2 The synthesis, secretion, and biologic activity of IL-1 cytokines have been identified as therapeutic targets for the treatment of periodontitis.3
LPS induces macrophages and fibroblasts to produce inflammatory cytokines, including IL-1β and −6 and TNF-α, which, in turn, promote the destruction of periodontal tissues, aggravate inflammation, and initiate alveolar bone resorption.4 IL-1β is prominent in the periodontal tissue and gingival crevicular fluid of patients with periodontitis.5 Numerous genes downstream of IL-1β have been identified in gingival fibroblasts and implicated in periodontitis. However, there are only limited data showing the important target genes of nuclear factor-kappa B (NF-κB) in gingival inflammation because these studies6,7 focused on only a single gene or a few genes. Thus, the full spectrum of expression changes induced by IL-1β has not been fully characterized. Targeting IL-1–signaling pathways is the subject of intense research, and a number of promising compounds have emerged that may modulate IL-1 signaling for therapeutic advantage. Understanding the immunobiology of IL-1β in human gingival fibroblasts (HGFs) by gene expression arrays will provide broader insight into the pathogenesis of periodontal diseases and may help to identify novel therapeutic targets.
Microarray techniques have been developed recently that may facilitate comprehensive expression analyses of large numbers of defined genes. Microarray analysis is an efficient method for exploring the functions of uncharacterized genes and can be used to find differentially expressed genes that may have diagnostic and therapeutic target potential.8 We previously developed and characterized an immortalized line of HGFs that provide a stable model for systematic studies (unpublished observations). In the present study, we identified gene-expression patterns of IL-1β–stimulated HGFs by using pangenomic oligonucleotide microarrays and identified many important inflammatory intermediates responsive to IL-1β that might be important targets for novel therapeutic strategies in the treatment of periodontal diseases. IL-1β seems to play a central role in the regulation of inflammatory genes, such as interleukins, chemokines, and adhesion molecules. Further, we identified the NF-κB transcription factor family and downstream targets as potential intermediates. Functional studies, using an inhibitor of NF-κB prior to IL-1β induction, support a role for NF-κB in blocking apoptosis and stabilizing activated HGFs during inflammation. Inhibition of NF-κB may be an important treatment strategy in periodontal inflammation.
MATERIALS AND METHODS
Cell Culture
Primary HGFs‡ were immortalized by human telomerase reverse transcriptase (hTERT) infection and selected with G418.§9 Cells were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, 4 mM L-glutamine, and 100 µg/ml penicillin-streptomycin. Cultures were kept at 37°C in a humidified incubator in an atmosphere of 95% air and 5% CO2. Cells were subcultured at a ratio of 1:4. Cells with a low passage number (<25) after immortalization were used for all experiments. We stimulated cells with 5 ng/ml recombinant human IL-1 β∥ for 2 hours; this duration allowed us to identify early and intermediate genes and minimize the number of secondary gene inductions.
Gene Expression Analysis
All microarray hybridization analyses were performed in replicates using expression arrays¶ including 54,675 probe sets to analyze >47,000 transcripts, including 38,500 well-characterized human genes. Isolated total RNA samples were processed as recommended by the manufacturer. In brief, total RNA was initially isolated using reagent# and passed through a spin column** for further purification. Eluted total RNAs were quantified with a portion of the recovered total RNA adjusted to a final concentration of 1.25 µg/µl. All total RNA samples were assessed for quality prior to beginning target preparation/processing steps by the application of a small amount of each sample (typically 25 to 250 ng/well) onto an RNA device†† that was evaluated on a bioanalyzer.‡‡ Single-stranded and then double-stranded (ds) complement (c) DNA was synthesized from the poly(A)+ mRNA present in the isolated total RNA (typically 10 mg total RNA starting material each sample reaction) using a kit§§ and poly (T)-nucleotide primers that contained a sequence recognized by T7 RNA polymerase. A portion of the resulting ds-cDNA was used as a template to generate biotin-tagged complement cRNA from an in vitro transcription reaction, using a kit.∥∥ Fifteen micrograms of the resulting biotin-tagged cRNA was fragmented to an average strand length of 100 bases (range, 35 to 200 bases) following prescribed protocols.¶¶ Subsequently, 10 mg this fragmented target cRNA was hybridized at 45°C with rotation for 16 hours## to probe sets present on expression arrays.*** The arrays were washed and then stained (streptavidin-phycoerythrin),††† followed by scanning on a scanner. ‡‡‡ The results were quantified and analyzed using software§§§ set at default values (scaling, target signal intensity = 500; normalization, all probe sets; and parameters, all set at default values). All arrays analyzed here exhibited >60% present call intensities. Data were analyzed with a statistical software program.∥∥∥ The analysis module computes regularized t tests using a Bayesian estimate of the variance among the gene measurements to infer significant gene changes. P<0.001 genes were accepted as differentially expressed.
Quantitative Real-Time Polymerase Chain Reaction (PCR)
Thirty-one genes (29 were upregulated and two showed no change), including NFκB1, NFκB2, NFκBIA, REL, TRAF1, IRAK2, CSF1, CSF2, ICAM1, VCAM1, IRF1, IL-6, IL-8, PTGS2, CCL2, CCL5, CCL20, CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL10, CXCL11, BDKRB1, BDKRB2, CD44, ETS1, STAT5A, TNFRSF6, and TNFAIP2 were analyzed by quantitative real-time PCR. Total RNA was isolated using reagent¶¶¶ and passed through an RNeasy spin column### for further purification. cDNA was synthesized from 2 mg total RNA using a kit.**** mRNA from cDNA samples was amplified with specific primers. The sequence detection system†††† was used for quantitative assessment of gene expression. This was done using green dye. Reaction mixtures contained cDNA reverse-transcribed from 2 µg total-RNA from each sample, 20 µM each primer, and 12.5 µl 2× SYBR green PCR master mix.‡‡‡‡ Thermal cycling conditions were 50°C for 2 minutes (for uracil-DNA N-glycosylase enzyme activity), 95°C for 10 minutes, and 40 cycles at 95°C for 15 seconds followed by 60°C for 1 minute. Experiments were performed in triplicate for each sample in the same reaction and repeated when a coefficient of variation >5% was observed. The GAPDH gene was used as an internal control. The relative quantification was given by the cycle threshold (Ct) values determined for triplicate reactions for test and reference samples for each target and for the internal control gene (GAPDH). The relative expression level was determined as 2-ΔΔCt. Evaluation of 2-ΔΔCt indicates the fold change in gene expression relative to the reference sample.10,11 Values are expressed as n-fold change in target gene expression.
Western Blot Analyses
Immunoblotting was performed by resolving whole-cell lysates on 10% Tris-acetate gel§§§§ and blotting onto membranes.∥∥∥∥ The membranes were blocked for 1 hour in 5% skim milk, rinsed, and incubated with primary antibodies for phospho-IκBα, phospho-c-Jun N terminal kinase (JNK), phospho-c-Jun, and poly (ADP-ribose) polymerase (PARP)-1¶¶¶¶ in phosphate buffered saline Tween-20 overnight at 4°C. After washings, the membrane was incubated with 0.1 mg/ml peroxidase-labeled secondary antibodies for 45 minutes. Antibody–antigen complexes were detected with a kit.####
Immunostaining of Cells in Culture
A major role of NF-κB in mediating the response to IL-1β predicts that cytoplasmic NF-κB is activated, leading to localization in the nucleus, chromatin binding, and gene activation.12 Therefore, we tested whether inhibitor of kappa B-alpha (IκBα) is inactivated by phosphorylation, leading to the accumulation of NF-κB in the nucleus after IL-1β stimulation of HGFs. Cells treated with IL-1β and untreated cells were fixed with 4% formaldehyde for 20 minutes, per-meabilized with 0.1% Triton X-100, and incubated with 3% hydrogen peroxide to block endogenous peroxidase activity. Cells were incubated with 5% goat serum for 1 hour to block non-specific binding. Cells were rinsed with phosphate buffered saline and incubated with rabbit anti-NF-κB p65 primary antibody***** (1:250) overnight at 4°C, secondary biotin-conjugated goat anti-rabbit antibody (1:200) for 20 minutes, and tertiary antibody (peroxidase-conjugated streptavidin)††††† (1:1,000) for 45 minutes. Staining was visualized with a peroxidase substrate kit‡‡‡‡‡ using diaminobenzidine as chromogen.
Cell Proliferation Assays
NF-κB is believed to mediate apoptotic responses in many settings;12,13 therefore, we tested whether IL-1β is involved in cell proliferation. The cell proliferation rate was analyzed by WST-1 (4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate)-based colorimetric assay.§§§§§ Briefly, cells were seeded at a concentration of 1,000 cells/well in a 96-well plate in 100 µl culture medium and treated with IL-1 β and/or NF-κB inhibitor (10 µM).∥∥∥∥∥ After the incubation of cells for 24,48, and 72 hours, 10 µl WST-1 reagent was added to the cultured cells (1:10 final dilution), and they were shaken for 5 minutes on a shaker. Cells were incubated for 2 hours in the cell culture incubator. Absorbances of samples were measured against the background control using a microplate (enzyme-linked immunosor-bent assay) reader at 450 nm wavelength.
Apoptosis Assays
We performed an apoptosis assay using a PARP cleavage detection kit¶¶¶¶¶ to test whether the inhibition of cell growth by the NF-κB inhibitor in this study was due to apoptosis. The kit is designed to detect PARP cleavage by Western blot. PARP is an enzyme implicated in DNA damage-and-repair mechanisms. During apoptosis, PARP is cleaved by the protease caspase-3, an important downstream apoptotic caspase. Cleavage of PARP from the native 116kDato 85kDa is a hall-mark of apoptosis. Cell lysate preparation and the detection of 85-kDa cleaved PARP was described above in the section on Western blot analysis.
RESULTS
In the present study, we stimulated cells with IL-1β for 2 hours, which would allow us to identify genes of early and intermediate expression change, thereby minimizing the number of secondary gene-regulating events. Our preliminary studies indicated that genes of the NF-κB and mitogen-activated protein kinase pathways and known IL-1β–responsive genes, including IL-6 and −8 and cyclooxygenase-2, exhibited expression changes within 1 hour after IL-1β stimulation (data not shown).
Gene Expression Analyses
A total of 382 probe sets corresponding to 254 genes were differentially expressed in IL-1β–induced cells and compared to control cells. Of 254 genes, 215 genes were upregulated (Table 1) and 39 genes were downregulated (Table 2) by IL-1β. For these microarray gene expression data, a stringent cutoff P value of 0.001 was chosen. Thus, the false positive rate (FPR) was 54,675 × 0.001 = 54.7 probe sets or ~40,000 × 0.001 = 40 genes. The FPR corresponds to 54.7/ 382 = 14.3% for probe sets and 40/254 = 15.7% for genes, indicating that 214 of 254 genes are real differentially expressed genes in this experiment. In our quantitative real-time PCR validation analyses, we validated 29 of 31 genes (93.5%), indicating that significantly differentially expressed genes identified here accurately characterize mRNA changes.
Table 1.
Genes Upregulated by IL-1β (P <0.001)
| ID | Gene Title | Gene Symbol | Fold Change |
|---|---|---|---|
| 209239_at |
Nuclear factor of kappa light polypeptide gene enhancer in B-cells 1 (p105) |
NFKB1 | 4.86E+00 |
| 207535_s_at |
Nuclear factor of kappa light polypeptide gene enhancer in B-cells 2 (p49/p100) |
NFKB2 | 6.67E+00 |
| 201502_s_at |
Nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha (IκB-α) |
NFKBIA | 1.49E+01 |
| 203927_at |
Nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, epsilon (IκB-ε) |
NFKBIE | 8.61E+00 |
| 223218_s_at |
Nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, zeta (IκB-zeta) |
NFKBIZ | 2.67E+01 |
| 206036_s_at | V-rel reticuloendotheliosis viral oncogene homolog (avian) | REL | 5.95E+00 |
| 205205 at |
V-rel reticuloendotheliosis viral oncogene homolog B, nuclear factor of kappa light polypeptide gene enhancer in B-cells 3 (avian) |
RELB | 9.58E+00 |
| 230052_s_at | T-cell activation NFKB-like protein | TA-NFKBH | 1.83E+01 |
| 202672_s_at | Activating transcription factor 3 | ATF3 | 4.51E+00 |
| 204420_at | FOS-like antigen 1 | FOSL1 | 2.90E+00 |
| 201464_x_at | V-jun sarcoma virus 17 oncogene homolog (avian) | JUN | 2.46E+00 |
| 201473_at | Jun B proto-oncogene | JUNB | 3.89E+00 |
| 206115 at | Early growth response 3 | EGR3 | 6.81E+00 |
| 224833_ at | V-ets erythroblastosis virus E26 oncogene homolog 1 (avian) | ETS1 | 3.09E+00 |
| 203010_ at | Signal transducer and activator of transcription 5A | STAT5A | 4.09E+00 |
| 202531_at | Interferon regulatory factor 1 | IRF1 | 2.23E+01 |
| 210162_s_at |
Nuclear factor of activated t-cells, cytoplasmic, calcineurin-dependent 1 |
NFATC1 | 7.02E+00 |
| 203973_s_at | CCAAT/enhancer binding protein (c/ebp), delta | CEBPD | 4.09E+00 |
| 221249_s_at | C/EBP-induced protein /// C/EBP-induced protein | LOC81558 | 4.79E+00 |
| 209706_at | NK3 transcription factor related, locus 1 (Drosophila) | NKX3-1 | 1.51E+01 |
| 203554_x_at | Pituitary tumor-transforming 1 | PTTG1 | 3.33E+00 |
| 236982_at | Pituitary tumor-transforming 1 interacting protein | PTTG1IP | 6.91E+00 |
| 205193_at | V-maf musculoaponeurotic fibrosarcoma oncogene homolog f (avian) | MAFF | 3.75E+00 |
| 216598_s_at | Chemokine (C-C motif) ligand 2 | CCL2 | 4.61E+02 |
| 205476_at | Chemokine (C-C motif) ligand 20 (MIP-3) | CCL20 | 3.79E+04 |
| 1405_i_at | Chemokine (C-C motif) ligand 5 | CCL5 | 1.65E+01 |
| 204470_at | Chemokine (C-X-C motif) ligand 1 | CXCL1 | 1.92E+03 |
| 204533_at | Chemokine (C-X-C motif) ligand 10 | CXCL10 | 8.23E+00 |
| 1569203_at | Chemokine (C-X-C motif) ligand 2 | CXCL2 | 4.40E+01 |
| 207850_at | Chemokine (C-X-C motif) ligand 3 | CXCL3 | 1.13E+03 |
| 206336_at | Chemokine (C-X-C motif) ligand 6 (granulocyte chemotactic protein 2) | CXCL6 | 1.55E+02 |
| 1555464_at | Interferon induced with helicase C domain 1 | IFIH1 | 5.48E+00 |
| 226757_at | Interferon-induced protein with tetratricopeptide repeats 2 | IFIT2 | 5.00E+00 |
| 229450_at | Interferon-induced protein with tetratricopeptide repeats 3 | IFIT3 | 5.20E+00 |
| 227125_at | Interferon (alpha, β and omega) receptor 2 | IFNAR2 | 1.18E+01 |
| 201642 at | Interferon gamma receptor 2 (interferon gamma transducer 1) | IFNGR2 | 3.06E+00 |
| 219684_at | 28 kDa interferon responsive protein | IFRG28 | 8.80E+00 |
| 202270_at |
Guanylate binding protein 1, interferon-inducible, 67 kDa /// guanylate binding protein 1, interferon-inducible, 67kDa |
GBP1 | 3.56E+00 |
| 242907_at | Guanylate binding protein 2, interferon-inducible | GBP2 | 9.60E+00 |
| 211506_s_at | Interleukin-8 | IL-8 | 1.52E+04 |
| 222974_at | Interleukin-22 | IL-22 | 2.00E+02 |
| 205207_at | Interleukin-6 (interferon, β 2) | IL-6 | 3.07E+01 |
| 1553740_a_at | Interleukin-1 receptor-associated kinase 2 | IRAK2 | 1.62E+01 |
| 227697_at | Suppressor of cytokine signaling 3 | SOCS3 | 4.98E+00 |
| 206157_at | Pentraxin-related gene, rapidly induced by IL-1 β | PTX3 | 2.08E+00 |
| 202638_s_at |
Intercellular adhesion molecule 1 (CD54), human rhinovirus receptor |
ICAM1 | 5.24E+02 |
| 203868_s_at | Vascular cell adhesion molecule 1 | VCAM1 | 1.14E+02 |
| 217523_at | CD44 antigen (homing function and Indian blood group system) | CD44 | 4.99E+00 |
| 205173_x_at | CD58 antigen, (lymphocyte function-associated antigen 3) | CD58 | 3.03E+00 |
| 204440_at |
CD83 antigen (activated B lymphocytes, immunoglobulin superfamily) |
CD83 | 1.48E+01 |
| 204170_s_at | CDC28 protein kinase regulatory subunit 2 | CKS2 | 2.91E+00 |
| 227458_at | CD274 antigen | PDCD1LG1 | 1.21E+01 |
| 215078_at | Superoxide dismutase 2, mitochondrial | SOD2 | 3.15E+04 |
| 205027_s_at | Mitogen-activated protein kinase kinase kinase 8 | MAP3K8 | 4.02E+01 |
| 201631_s_at | Immediate early response 3 | IER3 | 1.18E+01 |
| 224341_x_at | Toll-like receptor 4 | TLR4 | 3.13E+02 |
| 242982_x_at | Integrin, β 8 | ITGB8 | 4.73E+00 |
| 204580_at | Matrix metallopeptidase 12 (macrophage elastase) | MMP12 | 5.07E+01 |
| 205828 at | Matrix metallopeptidase 3 (stromelysin 1, progelatinase) | MMP3 | 7.29E+00 |
| 209716_at | Colony stimulating factor 1 (macrophage) | CSF1 | 8.41E+00 |
| 210229_s_at | Colony stimulating factor 2 (granulocyte-macrophage) | CSF2 | 4.88E+01 |
| 206432_ at | Hyaluronan synthase 2 | HAS2 | 5.85E+00 |
| 223541_at | Hyaluronan synthase 3 | HAS3 | 6.01E+00 |
| 232458_at | Collagen, type III, alpha 1 (Ehlers-Danlos syndrome type IV, autosomal dominant) | COL3A1 | 3.77E+00 |
| 205479_s_at | Plasminogen activator, urokinase | PLAU | 3.37E+00 |
| 201939_at | Polo-like kinase 2 (Drosophila) | PLK2 | 2.30E+00 |
| 213506_at | Coagulation factor II (thrombin) receptor-like 1 | F2RL1 | 4.80E+00 |
| 204363_at | Coagulation factor III (thromboplastin, tissue factor) | F3 | 5.73E+00 |
| 229830_at | Platelet-derived growth factor alpha polypeptide | PDGFA | 2.45E+00 |
| 231382_at | Fibroblast growth factor 18 | FGF18 | 3.37E+00 |
| 210310_s_at | Fibroblast growth factor 5 | FGF5 | 2.42E+00 |
| 38037_at | Heparin-binding EGF-like growth factor | HBEGF | 5.94E+00 |
| 223689_at | IGF-II mRNA-binding protein 1 | IMP-1 | 1.98E+02 |
| 207113_s_at | Tumor necrosis factor (TNF superfamily, member 2) | TNF | 4.22E+01 |
| 202510_s_at | Tumor necrosis factor, alpha-induced protein 2 | TNFAIP2 | 6.19E+01 |
| 202644_s_at | Tumor necrosis factor, alpha-induced protein 3 (A20) | TNFAIP3 | 7.06E+01 |
| 206025_s_at | Tumor necrosis factor, alpha-induced protein 6 | TNFAIP6 | 1.96E+01 |
| 208296_x_at | Tumor necrosis factor, alpha-induced protein 8 | TNFAIP8 | 6.34E+00 |
| 221085 at | Tumor necrosis factor (ligand) superfamily, member 15 | TNFSF15 | 5.70E+00 |
| 215719_x_at | Fas (TNF receptor superfamily, member 6) | FAS | 2.53E+00 |
| 207196_s_at | TNFAIP3 interacting protein 1 | TNIP1 | 2.72E+00 |
| 205599_at | TNF receptor-associated factor 1 | TRAF1 | 2.06E+01 |
| 202870_s_at | TNF receptor-associated factor 4 | TRAF4 | 3.50E+00 |
| 235971_at | TRAF-interacting protein with a forkhead-associated domain | TIFA | 8.40E+00 |
| 220975_s_at |
C1q and tumor necrosis factor related protein 1 /// C1q and tumor necrosis factor related protein 1 |
C1QTNF1 | 6.13E+00 |
| 205681_at | BCL2-related protein A1 | BCL2A1 | 1.19E+01 |
| 204908_s_at | B-cell CLL/lymphoma 3 | BCL3 | 6.27E+00 |
| 223566_s_at | BCL6 co-repressor | BCOR | 5.16E+00 |
| 202076_at | Baculoviral IAP repeat-containing 2 | BIRC2 | 1.89E+00 |
| 210538_s_at | Baculoviral IAP repeat-containing 3 | BIRC3 | 9.25E+01 |
| 202095_s_at | Baculoviral IAP repeat-containing 5 (survivin) | BIRC5 | 3.18E+00 |
| 221436_s_at | Cell division cycle associated 3 /// cell division cycle associated 3 | CDCA3 | 3.61E+00 |
| 209714_s_at |
Cyclin-dependent kinase inhibitor 3 (CDK2-associated dual specificity phosphatase) |
CDKN3 | 3.31E+00 |
| 210563_x_at |
CASP8 and FADD-like apoptosis regulator (anti-apoptosis protein) |
CFLAR | 2.82E+00 |
| 214710_s_at | Cyclin B1 | CCNB1 | 3.74E+00 |
| 202705_at | Cyclin B2 | CCNB2 | 2.94E+00 |
| 203725_at | Growth arrest and DNA-damage-inducible, alpha | GADD45A | 2.12E+00 |
| 204641_at | NIMA (never in mitosis gene a)-related kinase 2 | NEK2 | 3.57E+00 |
| 202954_at | Ubiquitin-conjugating enzyme E2C | UBE2C | 3.32E+00 |
| 202779_s_at | Ubiquitin-conjugating enzyme E2S | UBE2S | 1.95E+00 |
| 227489_at | SMAD specific E3 ubiquitin protein ligase 2 | SMURF2 | 2.08E+00 |
| 207510_at | Bradykinin receptor B1 | BDKRB1 | 2.75E+01 |
| 205870_at | Bradykinin receptor B2 | BDKRB2 | 4.92E+00 |
| 229055_at | G protein-coupled receptor 68 | GPR68 | 3.76E+00 |
| 223767_at | G protein-coupled receptor 84 | GPR84 | 5.64E+00 |
| 209324_s_at | Regulator of G-protein signaling 16 | RGS16 | 1.27E+01 |
| 202388 at | Regulator of G-protein signaling 2, 24kDa | RGS2 | 3.93E+00 |
| 203823 at | Regulator of G-protein signaling 3 | RGS3 | 5.70E+00 |
| 228176_at |
Endothelial differentiation, sphingolipid G-protein-coupled receptor, 3 |
EDG3 | 3.08E+00 |
| 208394_x_at | Endothelial cell-specific molecule 1 | ESM1 | 1.51E+01 |
| 232017_at | Tight junction protein 2 (zona occludens 2) | TJP2 | 5.72E+00 |
| 218113_at | Transmembrane protein 2 | TMEM2 | 4.03E+00 |
| 216005_at | Tenascin C (hexabrachion) | TNC | 1.52E+01 |
| 210190_at | Syntaxin 11 | STX11 | 6.24E+00 |
| 214520 at | Forkhead box C2 (MFH-1, mesenchyme forkhead 1) | FOXC2 | 7.35E+00 |
| 205659_at | Histone deacetylase 9 | HDAC9 | 4.82E+00 |
| 218755_at | Kinesin family member 20A | KIF20A | 3.21E+00 |
| 209212_s_at | Kruppel-like factor 5 (intestinal) | KLF5 | 8.60E+00 |
| 230636_s_at | Kruppel-like factor 9 | KLF9 | 6.45E+00 |
| 214470_at |
Killer cell lectin-like receptor subfamily B, member 1 /// killer cell lectin-like receptor subfamily B, member 1 |
KLRB1 | 5.23E+00 |
| 220091_at | Solute carrier family 2 (facilitated glucose transporter), member 6 | SLC2A6 | 3.63E+00 |
| 212110_at | Solute carrier family 39 (zinc transporter), member 14 | SLC39A14 | 3.21E+00 |
| 202357_s_at | B-factor, properdin | BF | 7.89E+00 |
| 205013_s_at | Adenosine A2a receptor | ADORA2A | 2.55E+01 |
| 201272_at | Aldo-keto reductase family 1, member B1 (aldose reductase) | AKR1B1 | 1.66E+00 |
| 207992_s_at | Adenosine monophosphate deaminase (isoform E) | AMPD3 | 1.02E+01 |
| 1552619_a_at | Anillin, actin binding protein (scraps homolog, Drosophila) | ANLN | 3.45E+00 |
| 203586_s_at | ADP-ribosylation factor 4-like | ARF4L | 2.93E+00 |
| 233620 at | Rho guanine nucleotide exchange factor (GEF) 12 | ARHGEF12 | 7.35E+02 |
| 221485_at | UDP-Gal:βGlcNAc β 1,4- galactosyltransferase, polypeptide 5 | B4GALT5 | 2.73E+00 |
| 221530_s_at | Basic helix-loop-helix domain containing, class B, 3 | BHLHB3 | 8.14E+00 |
| 229437_at | BIC transcript | BIC | 1.16E+02 |
| 227143_s_at | BH3 interacting domain death agonist | BID | 3.41E+00 |
| 220987_s_at | Chromosome 11 open reading frame 17 /// chromosome 11 open reading frame 17 /// NUAK family, SNF1-like kinase, 2 /// NUAK family, SNF1-like kinase, 2 |
C11orf17 /// NUAK2 | 1.17E+01 |
| 221182_at | Chromosome 1 open reading frame 129 | C1orf129 | 4.14E+02 |
| 226936_at | Chromosome 6 open reading frame 173 | C6orf173 | 3.02E+00 |
| 228695_at | Chromosome 8 open reading frame 46 | C8orf46 | 7.70E+00 |
| 203921_at | Carbohydrate (N-acetylglucosamine-6-O) sulfotransferase 2 | CHST2 | 3.42E+00 |
| 1554862_at | Cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMP-N-acetylneuraminate monooxygenase) |
CMAH | 1.37E+01 |
| 221903_s_at | Cylindromatosis (turban tumor syndrome) | CYLD | 3.48E+00 |
| 228915_at | Dachshund homolog 1 (Drosophila) | DACH1 | 6.77E+00 |
| 235545_at | DEP domain containing 1 | DEPDC1 | 3.57E+00 |
| 203764_at | Discs, large homolog 7 (Drosophila) | DLG7 | 3.56E+00 |
| 204135_at | Downregulated in ovarian cancer 1 | DOC1 | 6.07E+00 |
| 209039_x_at | EH-domain containing 1 | EHD1 | 2.45E+00 |
| 227180_at | ELOVL family member 7, elongation of long chain fatty acids (yeast) | ELOVL7 | 5.63E+02 |
| 201341_at | Ectodermal-neural cortex (with BTB-like domain) | ENC1 | 4.12E+00 |
| 227844_at | Formin-like 3 | FMNL3 | 4.86E+00 |
| 234830_at | Similar to FSHD region Gene 2 protein | FRG2 | 5.09E+02 |
| 204224_s_at | GTP cyclohydrolase 1 (dopa-responsive dystonia) | GCH1 | 6.02E+03 |
| 204472_at | GTP binding protein overexpressed in skeletal muscle | GEM | 7.53E+00 |
| 232080_at | HECT, C2 and WW domain containing E3 ubiquitin protein ligase 2 | HECW2 | 2.15E+00 |
| 212642_s_at | Human immunodeficiency virus type I enhancer binding protein 2 | HIVEP2 | 5.95E+00 |
| 221458_at | 5-hydroxytryptamine (serotonin) receptor 1F | HTR1F | 6.41E+00 |
| 230640_at | Huntingtin interacting protein C | HYPC | 5.49E+00 |
| 207826_s_at | Inhibitor of DNA binding 3, dominant negative helix-loop-helix protein | ID3 | 3.16E+00 |
| 202503_s_at | KIAA0101 | KIAA0101 | 2.14E+00 |
| 228325_at | KIAA0146 protein | KIAA0146 | 5.92E+00 |
| 202859_x_at | KIAA0476 | KIAA0476 | 4.66E+05 |
| 219218_at | KIAA1447 protein | KIAA1447 | 4.62E+00 |
| 211762_s_at |
Karyopherin alpha 2 (RAG cohort 1, importin alpha 1) /// karyopherin alpha 2 (RAG cohort 1, importin alpha 1) |
KPNA2 | 2.04E+00 |
| 205266_at | Leukemia inhibitory factor (cholinergic differentiation factor) | LIF | 2.04E+01 |
| 211596_s_at | Leucine-rich repeats and immunoglobulin-like domains 1 /// leucine-rich repeats and immunoglobulin-like domains 1 |
LRIG1 | 1.52E+01 |
| 205381_at | Leucine rich repeat containing 17 | LRRC17 | 4.02E+00 |
| 209841_s_at | Leucine rich repeat neuronal 3 | LRRN3 | 5.28E+00 |
| 228885_at | MAM domain containing 2 | MAMDC2 | 2.99E+00 |
| 237849_at | Mannosidase, alpha, class 1A, member 1 | MAN1A1 | 5.12E+00 |
| 225316_at | Major facilitator superfamily domain containing 2 | MFSD2 | 9.63E+00 |
| 212859_x_at | Metallothionein 1E (functional) | MT1E | 2.32E+00 |
| 217165_x_at | Metallothionein 1F (functional) | MT1F | 2.69E+00 |
| 206461_x_at | Metallothionein 1H | MT1H | 2.14E+00 |
| 216336_x_at | Metallothionein 1M | MT1K | 2.33E+00 |
| 204326_x_at | Metallothionein 1X | MT1X | 2.15E+00 |
| 212185_x_at | Metallothionein 2A | MT2A | 1.75E+00 |
| 213906_at | V-myb myeloblastosis viral oncogene homolog (avian)-like 1 | MYBL1 | 3.63E+00 |
| 201976_s_at | Myosin X | MYO10 | 2.46E+00 |
| 203045_at | Ninjurin 1 | NINJ1 | 7.92E+00 |
| 223484_at | Normal mucosa of esophagus specific 1 | NMES1 | 1.49E+01 |
| 222877_at | Neuropilin 2 | NRP2 | 6.63E+00 |
| 218706_s_at | HCV NS3-transactivated protein 2 | NS3TP2 | 2.03E+00 |
| 232666_at | 2′-5′-oligoadenylate synthetase 3, 100kDa | OAS3 | 4.84E+00 |
| 1565111_x_at | Orofacial cleft 1 candidate 1 | OFCC1 | 4.90E+01 |
| 219582_at | Opioid growth factor receptor-like 1 | OGFRL1 | 3.51E+00 |
| 243905_at | Organic solute transporter alpha | OSTalpha | 2.19E+01 |
| 202759_s_at | PALM2-AKAP2 protein | PALM2-AKAP2 | 2.03E+00 |
| 219148_at | PDZ binding kinase | PBK | 3.31E+00 |
| 218691_s_at | PDZ and LIM domain 4 | PDLIM4 | 5.06E+00 |
| 212094_at | Paternally expressed 10 | PEG10 | 3.45E+00 |
| 217997_at | Pleckstrin homology-like domain, family A, member 1 | PHLDA1 | 2.31E+00 |
| 209193_at | Pim-1 oncogene | PIM1 | 4.37E+00 |
| 230673_at | Polycystic kidney and hepatic disease 1 (autosomal recessive)-like 1 | PKHD1L1 | 1.17E+02 |
| 228343 at | POU domain, class 2, transcription factor 2 | POU2F2 | 6.78E+00 |
| 201490_s_at | Peptidylprolyl isomerase F (cyclophilin F) | PPIF | 3.07E+00 |
| 239488_at | Protein phosphatase 1 (formerly 2C)-like | PPM1L | 1.84E+01 |
| 218009_s_at | Protein regulator of cytokinesis 1 | PRC1 | 3.04E+00 |
| 201762_s_at | Proteasome (prosome, macropain) activator subunit 2 (PA28 β) | PSME2 | 2.15E+00 |
| 221872_at | Retinoic acid receptor responder (tazarotene induced) 1 | RARRES1 | 5.80E+00 |
| 1554834_a_at | Ras association (RalGDS/AF-6) domain family 5 | RASSF5 | 1.43E+01 |
| 209545_s_at | Receptor-interacting serine-threonine kinase 2 | RIPK2 | 4.65E+00 |
| 211456_x_at | Similar to 60S ribosomal protein L35 | RPL35 | 1.97E+00 |
| 204803_s_at | Ras-related associated with diabetes | RRAD | 7.41E+00 |
| 206027_at | S100 calcium binding protein A3 | S100A3 | 5.70E+00 |
| 1556472_s_at | Sex comb on midleg-like 4 (Drosophila) | SCML4 | 1.61E+01 |
| 236599_at |
Serpin peptidase inhibitor, clade E (nexin, plasminogen activator inhibitor type 1), member 2 |
SERPINE2 | 3.29E+00 |
| 211767_at | SLD5 homolog /// SLD5 homolog | SLD5 | 3.51E+00 |
| 210357_s_at | Spermine oxidase | SMOX | 3.92E+00 |
| 1562287_at | Syntrophin, gamma 1 | SNTG1 | 4.18E+02 |
| 226075_at | SplA/ryanodine receptor domain and SOCS box containing 1 | SPSB1 | 4.61E+00 |
| 1565579_at | TatD DNase domain containing 2 | TATDN2 | 6.26E+00 |
| 225868 at | Tripartite motif-containing 47 | TRIM47 | 4.44E+00 |
| 202589_at | Thymidylate synthetase | TYMS | 2.22E+00 |
| 205480_s_at | UDP-glucose pyrophosphorylase 2 | UGP2 | 1.58E+00 |
| 203137_at | Wilms tumor 1 associated protein | WTAP | 3.95E+00 |
| 218810_at | Zinc finger CCCH-type containing 12A | ZC3H12A | 3.78E+01 |
Bold indicates genes that are related to each other in the literature.
Table 2.
Genes Downregulated by IL-1β (P<0.001)
| ID | Gene Title | Gene Symbol | Fold Change |
|---|---|---|---|
| 202912_at | Adrenomedullin | ADM | −2.33E+00 |
| 1564699_at | Chromosome 5 open reading frame 4 | C5orf4 | −3.77E+00 |
| 229218_at | Collagen, type I, alpha 2 | COL1A2 | −2.42E+00 |
| 209101_at | Connective tissue growth factor | CTGF | −2.09E+00 |
| 219179_at | Dapper, antagonist of β-catenin, homolog 1 (Xenopus laevis) | DACT1 | −4.73E+00 |
| 209383_at | DNA-damage-inducible transcript 3 | DDIT3 | −4.01E+00 |
| 208215_x_at | Dopamine receptor D4 | DRD4 | −7.43E+02 |
| 225275_at | EGF-like repeats and discoidin I-like domains 3 | EDIL3 | −1.66E+00 |
| 205782_at | Fibroblast growth factor 7 (keratinocyte growth factor) | FGF7 | −3.07E+00 |
| 206377_at | Forkhead box F2 | FOXF2 | −2.01E+00 |
| 218665_at | Frizzled homolog 4 (Drosophila) | FZD4 | −2.34E+00 |
| 204602 at | Dickkopf homolog 1 (Xenopus laevis) | DKK1 | −3.98E+00 |
| 204457_s_at | Growth arrest-specific 1 | GAS1 | −2.74E+00 |
| 208937_s_at |
Inhibitor of DNA binding 1, dominant negative helix-loop-helix protein |
ID1 | −4.29E+00 |
| 201565_s_at |
Inhibitor of DNA binding 2, dominant negative helix-loop-helix protein |
ID2 | −3.39E+00 |
| 209184_s_at | Insulin receptor substrate 2 | IRS2 | −4.92E+00 |
| 205175_s_at | Ketohexokinase (fructokinase) | KHK | −4.36E+02 |
| 205051_s_at |
V-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog |
KIT | −3.75E+00 |
| 234880_x_at | Keratin associated protein 1–3 | KRTAP1–3 | −5.99E+00 |
| 233533_at | Keratin associated protein 1–5 | KRTAP1–5 | −4.74E+00 |
| 206163_at | Mab-21-like 1 (C. elegans) | MAB21L1 | −4.20E+00 |
| 213568 at | Odd-skipped related 2 (Drosophila) | OSR2 | −5.23E+00 |
| 225975_at | Protocadherin 18 | PCDH18 | −2.39E+00 |
| 1559341_at | Protocadherin 9 | PCDH9 | −2.14E+01 |
| 225816_at | PHD finger protein 17 | PHF17 | −4.40E+00 |
| 228481_at | Periostin, osteoblast specific factor | POSTN | −5.52E+00 |
| 203083 at | Thrombospondin 2 | THBS2 | −1.71E+00 |
| 204284_at | Protein phosphatase 1, regulatory (inhibitor) subunit 3C | PPP1R3C | −4.53E+00 |
| 226069_at | Prickle-like 1 (Drosophila) | PRICKLE1 | −3.50E+00 |
| 237719_x_at | R7 binding protein | R7BP | −1.91E+01 |
| 226492_at | Sema domain, transmembrane domain (TM), and cytoplasmic domain, (semaphorin) 6D |
SEMA6D | −5.30E+00 |
| 222258_s_at | SH3-domain binding protein 4 | SH3BP4 | −2.53E+00 |
| 231145_at | Solute carrier family 25 (mitochondrial oxodicarboxylate carrier), member 21 |
SLC25A21 | −3.31E+00 |
| 220924_s_at | Solute carrier family 38, member 2 | SLC38A2 | −2.09E+00 |
| 232481_s_at | SLIT and NTRK-like family, member 6 | SLITRK6 | −5.05E+00 |
| 201416_at | SRY (sex determining region Y)-box 4 | SOX4 | −5.17E+00 |
| 234989_at | Trophoblast-derived noncoding RNA | TncRNA | −2.64E+00 |
| 201010_s_at | Thioredoxin interacting protein | TXNIP | −2.87E+00 |
| 210599_at | Zinc finger protein 614 | ZNF614 | −1.17E+03 |
Bold indicates genes that are related to each other in the literature.
A large number of transcription factors were significantly altered by IL-1β stimulation. Potentially more cytokine-regulated genes would be identified at later time points. The literature-supported pathway-analysis program##### showed that, of the 254 significant genes, 126 genes were related to each other in the literature(Tables 1 and 2; bold genes). This means that when we put the differentially expressed genes into a pathway-analysis program that draws the pathways of these genes according to the current literature, 126 genes seemed to be related to each other in one of the pathways. No connectivity in the literature is apparent for 128 genes, suggesting that up to 128 IL-1β–induced genes may represent new regulatory relationships of IL-1β, one of the most valuable findings of the present study (Tables 1 and 2). In particular, we noted that one of the largest functionally related gene groups included NFκB and NFκB pathway–related genes (e.g., NFκB1, NFκB2, IkBα, IκB∈, IκBζ, REL, and RELB). This observation suggests that the NF-κB pathway may play an important role in the IL-1β response in HGFs during inflammation.
Experimental Confirmation and Role of NF-κB
Thirty-one genes were analyzed by quantitative realtime PCR, and 29 genes were validated (93.5%). Of the 29 upregulated genes, 27 genes, including NF-κB family members (NFκB1, NFκB2, NFκBIA, and REL), TRAF1, IRAK2, CSF1, CSF2, ICAM1, VCAM1, IRF1, IL-6, IL-8, PTGS2, several chemokines (CCL2, CCL5, CCL20, CXCL1, CXCL2, CXCL3, CXCL6, and CXCL10), BDKRB1, BDKRB2, CD44, ETS1, and STAT5A, were downregulated by NFκB inhibitor plus IL1β, whereas the addition of the inhibitor alone hadno regular effect (Table 3). These results indicate that NF-κB is involved in mediating the IL-1β– induced expression changes of this cohort (Table 3).
Table 3.
Gene Expression Changes (fold change) of HGFs After Treatment With IL-1β and/or NF--κB Inhibitor Relative to Untreated Control Cells Quantified by Quantitative Real-Time PCR
| Genes (fold change) |
|||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| Treatment | NFKB1 | NFKB2 | NFKBIA | REL | TRAF1 | CSF1 | CSF2 | ICAM1 | IRF1 | BDKRB1 | |
| No treatment | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | |
| NF-κB-I | 0.92 | 0.99 | 0.88 | 1.03 | 1.03 | 1.32 | 0.45 | 1.06 | 1.18 | 1.07 | |
| IL-1β | 4.4 | 8.95 | 26.2 | 6.92 | 9.33 | 5.83 | 482.8 | 739.7 | 33.9 | 10.45 | |
| NF-κB-I + IL-1β | 0.97 | 1.0991 | 1.90678 | 2.08 | 0.5 | 1.07 | 1.86 | 5.27 | 1.76 | 1.7 | |
| Genes (fold change) | |||||||||||
| Treatment | CCL2 | CCL5 | CCL20 | CXCL1 | CXCL2 | CXCL3 | CXCL5 | CXCL6 | CXCL10 | CXCL11 | PTGS2 |
| No treatment | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 |
| NF-κB-I | 1.03 | 0.46 | 0.76 | 1.88 | 0.08 | 0.23 | 0.3 | 6.12 | 0.39 | 0.3 | 2.53 |
| IL-1β | 397.6 | 66.4 | 173.3 | 17.77 | 566.9 | 67.3 | 0.6 | 38.5 | 302 | 0.6 | 21.23 |
| NF-κB-I + IL-1β | 0.6 | 2.15 | 1.12 | 0.01 | 26.8 | 1.6 | 0.2 | 1.12 | 13.8 | 0.1 | 13.8 |
| Genes (fold change) |
|||||||||||
| Treatment | IL-6 | IL-8 | CD44 | ETS1 | IRAK2 | BDKRB2 | VCAM1 | STAT5A | |||
| No treatment | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | |||
| IL-1β | 559.6 | 713.1 | 5.91 | 3.7 | 8.99 | 8.84 | 34.96 | 5.93 | |||
| NF-κB-I + IL-1β | 12.7 | 2.95 | 3.39 | 2.34 | 1.18 | 0.37 | 0.15 | 0.79 | |||
Note upregulation of gene expressions by IL-1β and downregulation by addition of NF-κB inhibitor to IL-1β. CXCL5 and CXCL11 were analyzed as unchanged gene expressions and confirmed by quantitative real-time PCR.
NFKB-I = NF-κB inhibitor (10 µM).
IL-1β Activates the NF-κB Pathway and Promotes Nuclear Translocation in HGFs
Western blot analyses, using whole-cell lysates of HGFs treated with IL-1β, revealed phosphorylation of IκBα starting at 30 minutes after treatment with IL-1β; it reached a maximum at 1 hour, followed by downregulation at 2 hours (Fig. 1A). One of the mitogen-activated protein kinases (JNK) and its downstream target (c-Jun transcription factor) were phosphorylated after IL-1β treatment at 15 minutes (Fig. 1A), indicating that at 2 hours, NF-κB and activating protein (AP)-1 transcription factors might play important roles in the transcription of inflammatory genes by IL-1β. Immunostaining with anti–NF-κB p65 antibody showed nuclear translocation of NF-κB in IL-1β–treated cells (Fig. 1B).
Figure 1.
A) Western blot analyses of whole-cell lysates using phosphoantibodies of IκBα, c-Jun, and JNK. B) Immunostaining of HGFs in culture with NF-κB p65 antibody showing nuclear translocation of NF-κB p65 after IL-1β treatment compared to untreated control cells (original magnification ×·10). IL-1β-5ng/ml = test; C = control; m = minutes; h = hours; IL-1β-5ng/ml-1h = test–1 hour.
Cell Growth Rate
Cell proliferation assays did not show any difference in cell growth with IL-1β treatment compared to non-treated cells, but treatment with the NF-κB inhibitor plus IL-1β completely inhibited cell proliferation (Fig. 2A). Treatment of HGFs with IL-1β alone did not induce apoptosis of cultured cells; however, NF-κB suppression augmented apoptosis. Initially, cells treated with NF-κB inhibitor (10 µM) plus IL-1β (5 ng/ml) showed little morphologic change; however, at 6 hours, approximately half of the cells exhibited signs of apoptosis, and, by 24 hours, numerous detached and floating cells and extracellular debris were apparent, suggesting apoptosis (Fig. 2B). Western blot analysis of cell extracts with anti-PARP revealed a high proportion of PARP degradation, confirming apoptosis, whereas no degradation was apparent in untreated and IL-1β–treated cells (Fig. 2B). These results are consistent with a role for NF-κB in stabilizing HGFs by impeding apoptosis.
Figure 2.
A) Growth rates of HGFs after treatment with IL-1β and IL-1β plus NF-κB inhibitor in different concentrations. B) Apoptosis assay. OD = optical density; h = hours; test 1 = IL1β–1 ng/ml; test 2 = IL1β–5 ng/ml; test 3 = IL1β–10 ng/ml; INH1 = NF-κB inhibitor (10 µM) plus 1 ng/ml IL-1β; INH2 = NF-κB inhibitor plus 5 ng/ml IL-1β; INH3 = NF-κB inhibitor plus 10 ng/ml IL-1β; C = control; INH = NF-κB inhibitor plus 5 ng/ml IL-1β.
Therefore, the results indicate that IL-1β is a potent inducer of numerous significant expression changes, many of which are associated with the NF-κB pathway, as judged by specific inhibition studies, and contribute a survival advantage to HGFs via the inhibition of apoptosis.
DISCUSSION
In this study, we used a new model of immortalized HGFs that is particularly relevant to the properties of a major cell mediator of gingivitis, HGFs. We previously developed an immortalized line of HGFs and characterized them with microarray analysis and functional studies. Low-passage immortalized cells provide a stable model for systematic studies. We used these immortalized cells for the present study. It was reported that telomerase-rescued human cells retained all biologic features of their normal progenitors, and hTERT-immortalized cells have normal growth potential.14,15
We observed in the present study that IL-1β could significantly alter the expression of genes in HGFs involved in inflammation. These genes include transcription factors, cytokines, chemokines, extracellular matrix molecules and enzymes, antiapoptotic genes, growth factors, and adhesion molecules (Table 4). The involvement of IL-1β in inflammation has been examined in a large number of studies4,5,7,16 aimed at developing efficacious biologic therapies targeting IL-1β in inflammatory diseases, such as periodontitis and rheumatoid arthritis. Despite the fact that numerous IL-1β target genes have been identified, the broad spectrum of transcriptional effects of IL-1β has not been characterized. We performed gene profiling using expression arrays on IL-1β–stimulated HGFs in an attempt to fully understand the role of proinflammatory cytokine IL-1β in gingival inflammation. This study reveals new IL-1β downstream targets. To the best of our knowledge, this is the first study analyzing IL-1β regulation in HGFs using pangenomic gene expression arrays.
Table 4.
Summary of Significantly Altered Gene Expressions in Groups
| Group | Genes Differentially Expressed by IL-1β |
|---|---|
| NF-κB pathway members | (P <0.001) NFκB1, NFκB2, IκBα, IκB∈, IκBζ, Rel, RELB, TA-NFκBH, MAP3K8 |
| Other transcription factors | ATF3, FOSL1, JUN, JUNB, EGR3, ETS1, STAT5A, IRF1, NFATC1, CEBP, PTTG1 |
| Chemokines | CCL2, CCL5, CCL20, CXCL1, CXCL2, CXCL3, CXCX6, CXCL10 |
| Interferon-induced proteins and receptors | Interferon (INF)-α receptor 2 (INFαR2), INFγR2, IFIH1, IFIT2, IFIT3, GBP1, GBP2 |
| Cytokines, interleukins | IL-6, IL-8, IL-22, IRAK2, CSF1, CSF2 |
| Adhesion molecules and receptors | ICAM1, VCAM1, CD44, CD58, CD83, integrin B8, bradykinin receptor 1 and 2, |
| Extracellular matrix proteins and enzymes | MMP3, MMP12, collagen 3, collagen 1,* HAS2, HAS3, CTGF,* thrombospondin 2,* periostin,* plasminogen activator |
| TNF family members | TNF, TNFAIP2, 3, 6, 8, TNFSF15, FAS, TRAF1, TRAF4, TIFA, C1QTNF1 |
| Antiapoptotic genes and cell cycle regulators | BCL2A1, BCL3, BCOR, BIRC2, BIRC3, BIRC5, CASP8, GADD45A, CCNB1, CCNB1, NEK2, UBE2C, CDCA3 |
| Coagulation factors | Coagulation factor 2 receptor, coagulation factor 3, |
| Growth factors | PDGFA, FGF5, FGF18, HB-EGF |
| Metallothioneins | MT1E, MT1F, MT1H, MT1K, MT1X, MT2A |
Downregulated after IL-1β treatment. All other genes were upregulated by IL-1β.
NF-κB and AP-1 Transcription Factors Are Activated by IL-1β
IL-1β was used as stimulus in this study because it activates NF-κB and AP-1 transcription factors in HGFs, and the expression of these factors is highly correlated with the severity of periodontitis.7,17 Binding of IL-1β to its receptor initiates a signaling cascade leading to the activation of NF-κB and AP-1.18 Ambili et al.19 recently showed that nuclear factor p65 was present in the gingival tissue of 75% of the patients with chronic periodontitis compared to 5% of patients with healthy gingiva. NF-κB activation is mainly regulated by IkB proteins. IκBα is the only inhibitor that dissociates from the NF-κB complex in response to stimulation, such as by LPS and IL-1β.20 In the present study, we showed IκBα phosphorylation and subsequent NF-κB p65 nuclear translocation, suggesting that NF-κB activation is regulated by NF-κB/IκBα dissociation.
Most of the biologic effects take place in cells after nuclear translocation of NF-κB and AP-1, two nuclear factors common to many IL-1–induced genes.12,21 IL-1β increases the nuclear binding of c-Jun and c-fos,21 the two components of AP-1 complex as confirmed in the present study (Table 1;Fig. 1A). Similar to NF-κB, AP-1 sites are present in the promoter regions of many IL-1β–inducible genes. It was shown that NF-κB and AP-1 work together in regulating the transcription of inflammatory genes.7 In the present study, inhibition of NF-κB controlled the expression of a cohort of many important inflammatory genes.
NF-κB Inhibition Induces Apoptosis
It has been suggested that NF-κB activation protects against apoptotic signals.22 Considerable evidence has been presented that NF-κB induces the expression of antiapoptotic gene products,23,24 among them the antiapoptotic regulator Bcl-xL, which is a known NF-κB target gene.25 We observed that inhibition of NF-κB induces apoptosis in cells stimulated by IL-1β. GADD45A and GADD45B are antiapoptotic genes that increase the survival of hematopoietic cells after exposure to ultraviolet irradiation and certain an-ticancer drugs.26 In the present study, IL-1β induced the expression of BCL2A1, BIRC5, and GADD45A genes (Table 1); thereby it might increase the survival of IL-1β–stimulated cells in inflammation. However, addition of the NF-κB inhibitor induced apoptosis of HGFs, suggesting a potential therapeutic effect of NF-κB inhibitors by eliminating IL-1β–affected cells through programmed cell death from inflammatory sites of periodontitis. This study provides a mechanism by which NF-κB inhibitors act in IL-1β–induced inflammation. Similar to our findings, the treatment of synovial cells with TNF-α and IL-1β alone did not induce apoptosis of cultured synovial cells. However, NF-κB suppression augmented apoptosis and cas-pase-3 activity in TNF-α– and IL-1β–stimulated synovial cells compared to unstimulated cells.22
IL-1β Receptor Antagonists as Potential Therapeutic Agents in Periodontal Inflammation
In inflammation, first neutrophils and later mono-cytes/macrophages appear and control aspects of the inflammatory reaction; however, if they cannot limit the immune reaction, they release cytokines to recruit resident cells and immune cells at sites where inflammation is becoming chronic.27 Resident cells play a crucial role in the transition from the acute phase to the chronic phase of inflammation. Our findings reveal many potentially important IL-1β– stimulated genes in gingival resident cells that lead us to consider preventing IL-1β binding to the receptor on HGFs as a possible point of intervention. Research into the use of anticytokine therapies in the treatment of periodontitis is at an early stage, although exogenous soluble IL-1 receptor type I and soluble TNF receptor type II applied to the gingival tissue of primates with experimental periodontitis resulted in the inhibition of inflammatory cell infiltration, alveolar bone loss, and connective tissue break-down.28–30 Based on the number of proinflammatory gene-expression changes observed here, it might be a good strategy to use IL-1β receptor antagonists to prevent IL-1β binding.
NF-κB Inhibition Can Attenuate the Actions of IL-1β
The actions of IL-1β could be attenuated by the addition of an NF-κB inhibitor to cell culture before activation by blocking of IκBα phosphorylation.31 In agreement with that study, we showed IκBα phosphorylation within 1 hour of induction by IL-1β, and we repressed gene expression in a cohort of IL-1β–induced inflammatory genes with IκBα phosphorylation inhibitor (BAY-11 7082), indicating that IκBα is the main inhibitor of NF-κB activation by IL-1β in HGFs. Thus, the NF-κB pathway in HGFs may be an important, if not dominant, pathway that regulates inflammatory gene expression. NF-κB activation by IL-1β in the present study protected against apoptotic signals,32 suggesting a strong involvement of NF-κB in the maintenance of IL-1β–affected cells in the inflammatory environment, thereby promoting the continued release of inflammatory factors that maintain the inflammatory reaction. Thus, NF-κB inhibitors induce apoptosis and clear the inflammatory area. These data strongly support the notion that NF-κB is a potential target for the treatment of inflammatory diseases, such as periodontitis. Further studies are required to confirm these conjectures.
Chemokines are considered key players in the diapedesis of leukocytes from the vasculature into tissues in inflammatory diseases.33–35 Many chemokines were upregulated by IL-1β in HGFs in the present study. Chemokines produced by fibroblasts stimulate the chemotaxis of neutrophils, macrophages, and T lymphocytes; these inflammatory cellsproduce inflammatory cytokines and matrix-degradative enzymes. The released enzymes and oxidative metabolites cause degradation of the extracellular matrix; in turn, the inflammatory cytokines stimulate fibro-blasts to produce more chemokines.16 Upon IL-1β stimulation, HGFs expressed high levels of several chemokines (CCL2, CCL5, CCL20, CXCL1, CXCL2, CXCL3, CXCL6, and CXCL10) (Table 3), suggesting that IL-1β promotes the recruitment of leukocytes to the inflammation sites and, therefore, may play a significant role in the progression to the chronic phase of gingival inflammation.
Metallothioneins (MTs), zinc-binding proteins, as a regulator for intracellular zinc signaling increase in aging and chronic inflammation, thereby allowing the continuous sequestration of intracellular zinc with subsequent low zinc ion availability against inflammation. This phenomenon leads to an impaired inflammatory/immune response.36,37 It was suggested that the major target of zinc is NF-κB, and the effects of zinc on the translocation of NF-κB have been attributed to the suppression of phosphorylation of inhibitory proteins.36 Therefore, zinc deficiency, via MT homeostasis, causes dysregulation of the inflammatory/immune response in chronic inflammation. To the best of our knowledge, this was the first study in which microarray data showed that MTs are upregulated by IL-1β in HGFs, which might be through NF-κB, and might contribute to the process of gingival inflammation. This phenomenon needs to be clarified in future studies to understand the role of MTs and zinc in the pathogenesis of periodontal disease.
The development and application of genome-scale technologies for studying the IL-1β response may help us to understand inflammation and develop new treatment strategies. Targeting the NF-κB family proteins with inhibitors might suggest a significant role for this transcription factor in regulating gingival inflammation; this needs to be verified in future studies.
CONCLUSIONS
IL-1β leads to a large number of significant expression changes consistent with a pathologic role in periodontitis, including enhancement of inflammatory cytokines, chemokines, transcription factors, matrix metalloproteinases, adhesion molecules, and, especially, NF-κB–dependent antiapoptotic genes. NF-κB activation blocks apoptosis, thereby stabilizing the HGF population in inflammation.
ACKNOWLEDGMENTS
This study was supported, in part, by the National Institutes of Health, Bethesda, Maryland (grant U01CA114810-03). The authors report no conflicts of interest related to this study.
Footnotes
American Type Culture Collection CRL-2014, Manassas, VA.
Calbiochem, San Diego, CA.
Calbiochem.
GeneChip U133 plus 2.0 arrays, Affymetrix, Santa Clara, CA.
TRIzol, Gibco BRL Life Technologies, Rockville, MD.
RNeasy spin column, Qiagen, Chatsworth, CA.
Lab-On-A-Chip, Caliper Technologies, Mountain View, CA.
Agilent Bioanalyzer 2100, Agilent Technologies, Palo Alto, CA.
SuperScript Double-Stranded cDNA Synthesis Kit, Invitrogen, Carlsbad, CA.
Affymetrix GeneChip IVT Labeling Kit, Affymetrix.
Affymetrix GeneChip Expression Analysis Technical Manual, Affymetrix.
Affymetrix GeneChip Hybridization Oven 640, Affymetrix.
GeneChip U133 Plus 2.0 arrays, Affymetrix.
Affymetrix Fluidics Station 450, Affymetrix.
GeneChip Scanner 3000, Affymetrix.
CGOS 1.2 software, Affymetrix.
Cyber-t, University of California, Irvine, CA.
TRIzol, Gibco BRL Life Technologies.
RNeasy spin column, Qiagen.
SuperScript III First-Strand Synthesis SuperMix Kit, Invitrogen.
ABI Prism 7900 Sequence Detection System, PE Applied Biosys-tems, Foster City, CA.
2× SYBR green PCR Master Mix, PE Applied Biosystems, Warrington, U.K.
NuPAGE 10% Tris-Acetate Gel, Invitrogen.
Immobilon-P membranes, Millipore, Billerica, MA.
Santa Cruz Biotechnology, Santa Cruz, CA.
ECL Kit, Amersham Biosciences, Piscataway, NJ.
Santa Cruz Biotechnology.
Jackson ImmunoResearch Laboratories, West Grove, PA.
Vector Laboratories, Burlingame, CA.
Roche Applied Bioscience, Mannheim, Germany.
BAY 11-7082, Calbiochem.
Calbiochem.
Pathway Studio, Ariadne Genomics, Rockville, MD.
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