Get 20M+ Full-Text Papers For Less Than $1.50/day. Start a 14-Day Trial for You and Your Team.

Learn More →

Identification of Specific Gene Expression Profiles in Fibroblasts Derived From Middle Ear Cholesteatoma

Identification of Specific Gene Expression Profiles in Fibroblasts Derived From Middle Ear... ObjectiveTo investigate the role of fibroblasts in the pathogenesis of cholesteatoma.DesignTissue specimens were obtained from our patients. Middle ear cholesteatoma–derived fibroblasts (MECFs) and postauricular skin–derived fibroblasts (SFs) as controls were then cultured for a few weeks. These fibroblasts were stimulated with interleukin (IL) 1α and/or IL-1β before gene expression assays. We used the human genome U133A probe array (GeneChip) and real-time polymerase chain reaction to examine and compare the gene expression profiles of the MECFs and SFs.SubjectsSix patients who had undergone tympanoplasty.ResultsThe IL-1α–regulated genes were classified into 4 distinct clusters on the basis of profiles differentially regulated by SF and MECF using a hierarchical clustering analysis. The messenger RNA expressions of LARC(liver and activation-regulated chemokine), GMCSF(granulocyte-macrophage colony-stimulating factor), epiregulin, ICAM1(intercellular adhesion molecule 1), and TGFA(transforming growth factor α) were more strongly up-regulated by IL-1α and/or IL-1β in MECF than in SF, suggesting that these fibroblasts derived from different tissues retained their typical gene expression profiles.ConclusionsFibroblasts may play a role in hyperkeratosis of middle ear cholesteatoma by releasing molecules involved in inflammation and epidermal growth. These fibroblasts may retain tissue-specific characteristics presumably controlled by epigenetic mechanisms.Recently, the interaction between mesenchymal cells such as fibroblasts and epithelial cells or keratinocytes has been proposed to be involved in inflammation, homeostasis, and tissue regeneration.For example, interleukin (IL) 1 produced by keratinocytes induces the release of keratinocyte growth factor (KGF), granulocyte-macrophage colony-stimulating factor (GM-CSF), and transforming growth factor α (TGF-α) from fibroblasts.These fibroblast-derived cytokines support the proliferation and differentiation of keratinocytes. This paracrine loop is thought to be important in repair processes of tissue inflammation. In the study presented herein, we used the human genome U133A probe array (GeneChip; Affymetrix, Inc, Santa Clara, Calif) and real-time polymerase chain reaction (PCR) to examine the gene expression profiles of fibroblasts derived from middle ear cholesteatoma and postauricular skin obtained from patients to determine whether these fibroblasts express some molecules that may interact with keratinocytes and be involved in the pathogenesis of middle ear cholesteatoma.METHODSCELL CULTURESMiddle ear cholesteatoma and normal postauricular skin samples were obtained from patients undergoing tympanoplasty, and single fibroblasts from each sample were cultured in Dulbecco Modified Eagle Medium/F12 (DMEM/F12; Invitrogen Corp, Carlsbad, Calif) with 10% fetal calf serum (JRH Biosciences, Lenexa, Kan) and a combination of 60-μg/mL penicillin and 100-μg/mL streptomycin (Invitrogen Corp) for a few weeks. The cells were incubated at 37°C in a humidified incubator containing 5% carbon dioxide in air and were analyzed after 4 passages. Patient consent and the approval of our university's ethics review board were obtained before the start of the study.CYTOKINE STIMULATION OF FIBROBLASTSMiddle ear cholesteatoma fibroblasts (MECFs) and skin fibroblasts (SFs) were stimulated with 10 ng/mL of IL-1α or 10 ng/mL of IL-1β (both from R&D Systems Inc, Minneapolis, Minn) for 4 hours before messenger RNA (mRNA) extraction.MICROARRAY EXPRESSION ANALYSISHuman genome-wide gene expression was examined using the human genome U133A probe array (GeneChip), which contains the oligonucleotide probe set for approximately 22 000 full-length genes, according to the manufacturer's protocol and previously reported strategies.Total RNA (5 &mgr;g) was extracted from the fibroblasts, and double-stranded complementary DNA (cDNA) was synthesized using a SuperScript Choice system (Invitrogen Corp) and a T7-(dT)24 primer (Amersham Pharmacia Biotech, Buckinghamshire, England). The cDNA was subjected to in vitro transcription in the presence of biotinylated nucleoside triphosphates using a high-yield RNA transcript labeling kit (BioArray; Enzo Diagnostics, Farmingdale, NY). The biotinylated complementary RNA was then hybridized with the probe array for 16 hours at 45°C. After washing, the hybridized biotinylated complementary RNA was stained with streptavidin-phycoerythrin (Molecular Probes, Eugene, Ore) and scanned with a gene array scanner (Hewlett-Packard Company, Palo Alto, Calif). The fluorescence intensity of each probe was quantified using the GeneChip Analysis Suite program, version 5.0 (Affymetrix, Inc). The expression level of single mRNA was determined as the mean fluorescence intensity among the intensities obtained with 11 paired (perfect-matched and single nucleotide–mismatched) probes. If the intensities of the mismatched probes were very high, gene expression was judged to be absent, even if a high mean fluorescence was obtained with the GeneChip Analysis Suite 5.0 program.The resulting data were analyzed using GeneSpring software, version 7.2 (Silicon Genetics, San Carlos, Calif). To normalize the staining intensity variations among the chips, the values for all genes on a given chip were divided by the median of all measurements on that chip. To eliminate changes within the range of background noise and to select the most differentially expressed genes, data were used only if the raw data values were less than 100 and gene expression was judged to be present by a gene expression data analysis. Hierarchical clustering analysis with standard correlation was used to identify gene clusters.QUANTITATIVE REAL-TIME PCRTo confirm the GeneChip microarray expression analysis data, we quantified the gene expression in fibroblasts derived from 5 other patients using quantitative real-time PCR. Total RNA was isolated using an RNA purification kit that included DNase digestion (RNeasy Mini Kit; Qiagen GmbH, Hilden, Germany). The RNA was then transcribed into cDNA using reverse transcriptase (Superscript II: Invitrogen Corp). Quantitative PCR was performed using a sequence detector system (ABI/PRISM 7700; Applied Biosystems, Foster City, Calif) and TaqMan Universal PCR Master Mix (Applied Biosystems), according to the manufacturers’ instructions. The primers and TaqMan probes used for the genes LARC(liver and activation-regulated chemokine), GMCSF(granulocyte-macrophage colony-stimulating factor), ICAM1(intercellular adhesion molecule 1), and TGFA(transforming growth factor-α) were as follows: LARC: forward, 5′-TGTCAGTGCTGCTACTCCACCT-3′; reverse, 5′-CTGTGTATCCAAGACAGCAGTCAA-3′; and TaqMan probe, 5′-TGCGGCGAATCAGAAGCAGCAA-3′; GMCSF: forward, 5′-GCCTCACCAAGCTCAAGGG-3′; reverse, 5′-GGTTGGAGGGCAGTGCTG-3′; and TaqMan probe, 5′-CCCTTGACCATGATGGCCAGCC-3′; ICAM1: forward, 5′-CTGTGTCCCCCTCAAAAGTCA-3′; reverse, 5′-ATACACCTTCCGGTTGTTCCC-3′; and TaqMan probe, 5′-TGCGGCGAATCAGAAGCAGCAA-3′; TGFA: forward, 5′-AGGAGACCCCTGCCCTCTAGT-3′; reverse, 5′-TCTGCAATGTGTTCTTGGTTTTG-3′; and TaqMan probe, 5′-TTCCAACCTGCCCAGTCACAGAAGG-3′. For epiregulin, quantitative PCR was performed using SYBR green PCR master mix (Applied Biosystems). The primers for epiregulin were as follows: forward, 5′-ATCCTGGCATGTGCTAGGGT-3′ and reverse, 5′-GTGCTCCAGAGGTCAGCCAT-3′. The expression levels of mRNA were normalized by the mean expression of a housekeeping gene (glyceraldehyde-3-phosphate dehydrogenase [GAPDH]), which was measured using Pre-Developed Assay Reagents (Applied Biosystems).STATISTICAL ANALYSISData are presented as mean ± SEM. Statistical significance was determined using the paired ttest, and differences were considered significant at P<.05.RESULTSEXPERIMENTAL DESIGNGeneChip was used to identify the gene expression pattern induced by IL-1α in the MECFs (n = 1) and SFs (n = 1). Following the identification of candidate disease–related genes with GeneChip, quantitative real-time PCR was used to confirm the expression of the selected gene (n = 5 for each).To assign a “fold-change cutoff” threshold, we used a GeneSpring analysis for selected genes in which the mean expression level had increased or decreased by more than 2-fold after 4 hours. As shown in Figure 1and Figure 2, IL-1α had an effect on numerous genes in the SF and MECF cultures.Figure 1.Interleukin (IL) 1α–mediated up-regulation of gene clusters in postauricular skin–derived fibroblasts (SFs) and middle ear cholesteatoma–derived fibroblasts (MECFs). Representation of messenger RNA expression levels in spontaneous SF, IL-1α–stimulated SF, spontaneous MECF, and IL-1α–stimulated MECF. The SFs and MECFs were stimulated with IL-1α (10 ng/mL) for 4 hours. The colored bars show the magnitude of the response of each gene, according to the scale of expression level shown. Cluster A contained 164 genes (1) in which expression in SFs increased at least 2-fold after stimulation with IL-1α and (2) in which increased gene expression was more than 2-fold that of IL-1α–stimulated MECFs. Cluster B contained 84 genes (1) in which expression in MECFs increased at least 2-fold after stimulation with IL-1α and (2) in which increased gene expression was more than 2-fold that of IL-1α–stimulated SFs.Figure 2.Interleukin (IL) 1α–mediated down-regulation of gene clusters in postauricular skin-derived fibroblasts (SFs) and middle ear cholesteatoma–derived fibroblasts (MECFs). Representation of messenger RNA expression levels in spontaneous SF, IL-1α–stimulated SF, spontaneous MECF, and IL-1α–stimulated MECF. The SFs and MECFs were stimulated with IL-1α (10 ng/mL) for 4 hours. The colored bars show the magnitude of the response of each gene, according to the scale of the expression level shown. Cluster C contained 65 genes (1) in which expression in SFs decreased by at least half after stimulation with IL-1α and (2) in which expression in spontaneous SFs increased gene expression by more than 2-fold that of spontaneous MECFs. Cluster D contained 69 genes (1) in which expression in MECFs decreased by at least half after stimulation with IL-1α and (2) in which expression in spontaneous MECFs increased gene expression by more than 2-fold that of spontaneous SFs.GENE EXPRESSION DATA ANALYSISUsing hierarchical clustering analysis of the gene expression profiles of 22 283 genes, we identified a cluster containing genes that were up-regulated in SF or MECF after IL-1α stimulation (Figure 1). Visual inspection identified 2 major groups among the up-regulated genes. The first group of 164 genes displayed an increase in gene expression in SF that was more than 2-fold of that in MECF (Figure 1, Table 1), while the second group of 84 genes displayed an increase in gene expression in MECF that was more than 2-fold of that in SF (Figure 1, Table 2).Table 1. Normalized Levels of 45 Genes in Cluster AGenBank Accession No.Gene SymbolGene DescriptionNormalized LevelSFMECFNoneIL-1αNoneIL-1αAW611727GAS1Growth-arrest–specific protein 11.8 P4.7 P0.2 A0.0 ANM_022162NOD2Caspase recruitment domain family, member 150.1 A9.1 P1.8 A0.2 AAI984980MCP2Monocyte chemotactic protein 21.1 A27.3 P0.3 A0.9 ABG166705ENA78Epithelial neutrophil-activating peptide 780.1 A30.3 P0.4 A1.6 ANM_005246TYK3Fer (fps/fes related) tyrosine kinase (phosphoprotein NCP94)1.2 A3.3 P0.8 A0.2 ANM_001250CD40Tumor necrosis factor receptor superfamily, member 51.7 A4.1 P0.1 A0.3 ANM_005761PLXNC1Plexin C11.5 A3.1 P0.5 A0.2 ABC005254CLECSF2C-type lectin, superfamily member 2 (activation induced)1.6 P4.0 P0.0 A0.4 PNM_002068GNA16Guanine nucleotide binding protein, α150.9 A4.4 P1.1 A0.4 ANM_004972JAK2JAK 2 (a protein tyrosine kinase)1.6 A3.5 P0.2 A0.4 ANM_002422MMP3Matrix metalloproteinase 3 (stromelysin 1, progelatinase)1.5 P4.1 P0.1 A0.5 PNM_005562LAMC2Laminin, γ 20.8 P2.5 P1.2 A0.3 ANM_006383KIP2DNA-dependent protein kinase catalytic subunit-interacting0.5 A1.5 P2.8 A0.2 ANM_006273MCP3Monocyte chemotactic protein 30.6 A8.6 P0.9 A1.1 ANM_000759GCSFColony-stimulating factor 3 (granulocyte)0.3 A12.6 P0.3 A1.7 ANM_000064C3Complement component 31.0 A7.0 P0.2 A1.0 AAB005043SSI1JAK binding protein, STAT-induced STAT inhibitor 11.4 A3.7 P0.5 A0.6 AM21121RANTESRegulated on activation normal T cell expressed and secreted0.2 A10.3 P0.1 A1.8 ANM_000880IL7Interleukin 71.1 A5.2 P0.4 A0.9 ANM_001218CA12Carbonic anhydrase XII1.4 P2.9 P0.4 P0.6 PNM_004527MOX1Mesenchyme homeobox 10.9 A5.6 P0.4 A1.1 PNM_002185IL7RInterleukin 7 receptor1.1 P4.8 P0.2 A0.9 PNM_005461KRMLv-mafMusculoaponeurotic fibrosarcoma oncogene homologue B1.4 A2.9 P0.1 A0.6 ANM_002421MMP1Matrix metalloproteinase 1 (interstitial collagenase)1.3 P3.6 P0.3 P0.7 PBC003600LMO4LIM domain only 41.3 P3.4 P0.7 P0.7 PNM_004820CP7BCytochrome P450, subfamily VIIB, polypeptide 10.8 P6.0 P0.4 A1.2 PAI817041RDC1Orphan G protein–coupled receptor with 7 transmembrane domains1.0 P5.0 P0.4 A1.0 PNM_001565IP10Interferon-inducible protein 100.3 A7.9 P0.3 A1.7 PNM_016087WNT16Wingless-type MMTV integration site family, member 160.9 A4.5 P1.0 A1.0 AAF055585SLIT2Slit (Drosophila) homologue 21.3 P3.1 P0.6 P0.7 PBC000388BING4Chromosome 6 open reading frame 110.2 A1.6 P1.8 A0.4 AL27624TFPI2Tissue factor pathway inhibitor 20.4 P5.7 P0.5 P1.5 PU74324RABIFRAB interacting factor0.9 P1.8 P1.1 P0.5 PNM_000585IL15Interleukin 151.1 P3.4 P0.4 P0.9 PNM_001426EN1Engrailed homologue 11.2 A2.8 P0.6 A0.8 AAW083357IL1RNInterleukin 1 receptor antagonist protein precursor0.9 A4.1 P0.7 A1.1 ANM_002426MMP12Matrix metalloproteinase 12 (macrophage elastase)1.0 P3.6 P0.4 M1.0 PM14333FYNFYN oncogene related to SRC, FGR, YES1.1 P3.0 P0.7 P0.9 PU13699ICECaspase 1, apoptosis-related cysteine protease1.0 A2.6 P1.0 A0.8 PNM_001078VCAM1Vascular cell adhesion molecule 10.3 A4.2 P0.5 A1.5 PNM_002009KGFFibroblast growth factor 7 (keratinocyte growth factor)1.0 P2.5 P0.6 P1.0 PD49372EotaxinSmall inducible cytokine subfamily A (cys-cys), member 110.3 A3.9 P0.1 A1.7 PM15330IL1BHuman interleukin 1β (IL-1β) mRNA0.0 A4.3 P0.1 A1.9 PNM_000963PTGS2Prostaglandin-endoperoxide synthase 20.3 M3.8 P0.2 A1.7 PAF030514ITACInterferon-inducible T-cell α chemoattractant0.5 A3.3 P0.4 A1.5 P Abbreviations: A, absence; IL, interleukin; JAK, Janus kinase; M, marginal; MECF, middle ear cholesteatoma fibroblast; MMTV, mouse mammary tumor virus; mRNA, messenger RNA; P, presence; SF, postauricular skin–derived fibroblasts; STAT, signal transducers and activators of transcription.Table 2. The Normalized Levels of 50 Genes in Cluster B*GenBank Accession No.Gene SymbolGene DescriptionNormalized LevelSFMECFNoneIL-1αNoneIL-1αNM_004591LARCLiver and activation-regulated chemokine0.0 A1.9 P0.1 A9.6 PM11734GMCSFColony-stimulating factor 2 (granulocyte-macrophage)0.0 A1.9 P0.1 A5.8 PU12767NR4A3Nuclear receptor subfamily 4, group A, member 30.2 A1.7 A0.3 A10.3 PNM_000201ICAM1Intercellular adhesion molecule 1 (CD54)0.1 A1.9 P0.1 P4.3 PNM_002888TIG1Retinoic acid receptor responder (tazarotene induced) 10.4 A1.6 A0.2 A4.5 PU22386CSF1Colony-stimulating factor 1 (macrophage)0.2 A1.7 P0.3 A4.7 PNM_002173IFNA16Interferon α 161.4 P0.6 M0.1 A1.5 PU37546API2Baculoviral IAP repeat-containing 30.2 A1.2 P0.8 P8.0 PNM_002575SERPINB2Serine proteinase inhibitor, clade B, member 20.1 P1.6 P0.4 P3.8 PM37435MCSFColony-stimulating factor 1 (macrophage)0.3 P1.6 P0.4 P3.6 PNM_012429TAPSEC14 (Saccharomyces cerevisiae)–like 21.1 A0.9 A0.3 A2.7 PNM_004233BL11Activated B lymphocytes, immunoglobulin superfamily0.8 A1.2 P0.6 A4.5 PAI360875SOX11SRY-box 110.3 A1.0 A1.0 A6.5 PNM_002015FKH1Forkhead box O1A (rhabdomyosarcoma)1.1 P0.9 A0.4 A2.7 PNM_006622SNKSerum-inducible kinase0.5 P1.5 P0.5 P3.2 PD89377MSX2Muscle segment homeobox (Msh) (Drosophila) homeobox homologue 20.5 A1.0 A1.0 A5.7 PNM_000029SERPINA8Serine proteinase inhibitor, clade A, member 81.3 M0.7 A0.4 A2.1 PNM_005658EBI6TNF receptor–associated factor 10.1 A1.0 P1.0 A5.1 PNM_003236TGFATransforming growth factor α0.6 A0.9 P1.1 P5.2 PNM_006981CHNNuclear receptor subfamily 4, group A, member 30.4 A1.0 P1.0 P4.5 PD31771HOX8Msh (Drosophila) homeobox homologue 20.8 A0.2 A1.2 A5.4 PAF153882RILLIM domain protein0.1 A1.3 P0.7 P3.0 PD32201ADRA1CAdrenergic, α-1A, receptor1.3 A0.7 A0.7 A3.1 PNM_002309CDFLeukemia inhibitory factor (cholinergic differentiation factor)0.1 A1.0 P1.0 P4.2 PAB044088SHARP-1Basic helix-loop-helix domain containing, class B, 30.3 A1.2 P0.8 A3.0 PU08015NFATCNuclear factor of activated T cells0.4 A1.0 P1.0 P3.8 PD87811GATA6GATA-binding protein 60.4 P0.9 P1.1 P4.2 PNM_002777PR3Proteinase 3 (serine proteinase)0.5 A1.2 A0.8 A2.7 PNM_001432EREpiregulin0.1 A0.1 A1.9 P6.8 PNM_003125SPRR1Small proline-rich protein 1B (cornifin)1.3 A0.7 A0.7 A2.2 PY15014GLCT2Uridine diphosphate (UDP)-galactose0.7 A1.0 A1.0 A3.0 PNM_001945DTRDiphtheria toxin receptor0.1 A0.2 A1.8 A4.8 PM79321JTK8Lyn B protein0.7 P0.9 P1.1 P2.9 PNM_005092TL6Tumor necrosis factor (ligand) superfamily, member 181.0 A0.8 A1.0 A2.7 PNM_006516GLUT1Solute carrier family 2, member 10.6 P0.9 P1.1 P2.8 PZ21533HHEXHematopoietically expressed homeobox0.7 P0.9 P1.1 P2.9 PNM_004362CLGNCalmegin0.7 A0.2 A1.3 A3.2 PNM_014030GIT1G protein–coupled receptor kinase-interactor 11.2 P0.8 P0.8 P1.9 PJ03223PRGProteoglycan 1, secretory granule0.2 A0.7 P1.3 P3.3 PNM_002253VEGFR2Kinase insert domain receptor0.9 A1.0 A1.0 A2.5 PAF009616CFLARCASP8 and FADD-like apoptosis regulator0.7 P1.1 P0.9 P2.2 PNM_001860CTR2Solute carrier family 31 (copper transporters), member 20.4 P0.7 P1.3 P3.0 PNM_002448HOX7Muscle segment homeobox (Msh) (Drosophila) homeobox homologue 10.5 P1.0 P1.0 P2.2 PU26662NPTX2Neuronal pentraxin II1.1 A0.4 A0.9 A2.0 PNM_000047ARSEArylsulfatase E (chondrodysplasia punctata 1)1.2 A0.4 A0.8 A1.8 PNM_001257CDHHCadherin 13, H-cadherin (heart)0.5 M0.4 A1.5 P3.3 PNM_004694SLC16A6Solute carrier family 16, member 60.1 A0.2 A1.8 P3.8 PNM_005860FLRGFollistatinlike 3 (secreted glycoprotein)0.7 P0.7 P1.3 P2.6 PAF001434PASTEH-domain containing 10.8 P0.8 P1.2 P2.5 P Abbreviations: A, absence; CASP8, caspase-8; FADD, Fas-associated death domain; IAP, integrin-associated protein; IL, interleukin; M, marginal; MECF, middle ear cholesteatoma fibroblast; P, presence; SF, postauricular skin–derived fibroblast; SRY, sex-determining region Y; TNF, tumor necrosis factor. *Unshaded cells indicate 5 genes associated with the pathogenesis of middle ear cholesteatoma.Among the genes in Table 1, several were identified whose expressions are generally known to be induced by IL-1α in fibroblasts, including monocyte chemoattractant protein 2 (MCP2); MCP3;granulocyte colony-stimulating factor (GCSF); regulated on activation normal T cell expressed and secreted (RANTES); matrix metalloproteinase 1 (MMP1); interferon-inducible protein 10 (IP10); IL-15 (IL15); vascular cell adhesion molecule 1 (VCAM1); KGF; eotaxin; and interferon-inducible T-cell α chemoattractant (ITAC). Genes for multiple profibrotic cytokines and chemokines that exhibited elevated expressions are shown in Table 2. These cytokines included TGFA, GMCSF, ICAM1, epiregulin, and LARC.Figure 2shows the genes that were down-regulated in SF or MECF after 4 hours of stimulation with IL-1α. Two major groups were identified among these genes: 65 genes showed a decrease in gene expression in SF that was less than half of that in MECF (Figure 2, Table 3), while the other 69 genes displayed a decrease in gene expression in MECF that was less than half of that in SF (Figure 2, Table 4).Table 3. Normalized Levels of 20 Genes in Cluster CGenBank Accession No.Gene SymbolGene DescriptionNormalized LevelSFMECFNoneIL-1αNoneIL-1αNM_004982KCNJ8Potassium inwardly rectifying channel, subfamily J, member 817.6 P1.6 A0.2 A0.4 AM21692ADH2Alcohol dehydrogenase 1B (class I), β polypeptide8.5 P1.8 P0.2 A0.2 ANM_005069SIMSingle-minded (Drosophila) homologue 24.8 P1.8 P0.1 A0.2 ANM_020379HMIC1,2-α-mannosidase3.7 P1.5 P0.1 A0.5 ANM_014421DKK-2Dickkopf (Xenopus laevis) homologue 24.0 P1.5 P0.4 A0.5 AD64137KIP2Human KIP2gene for CDK-inhibitor p57KIP24.1 P1.4 A0.5 A0.6 AAF284095ADRA2AAdrenergic, α-2A-, receptor5.3 P1.2 P0.8 A0.6 ANM_016109ANGPTL4Angiopoietinlike 44.0 P0.9 A0.6 M1.1 ANM_001546ID4Inhibitor of DNA binding 43.7 P0.9 P0.7 A1.1 PNM_018490LGR4G protein–coupled receptor 483.7 P1.3 P0.7 P0.5 PX75208HEK2Homo sapiensHEK2 mRNA for protein tyrosine kinase receptor4.2 P0.5 A0.9 A1.1 ANM_021047BMZF1Zinc finger protein 2531.8 P0.7 A0.5 M1.3 MNM_001611TRAPAcid phosphatase 5, tartrate resistant2.5 P0.5 A0.7 A1.3 ANM_014210EVI2AEcotropic viral integration site 2A2.8 P1.2 P0.8 A0.6 PAK027146RPL5Ribosomal protein L52.6 P1.3 P0.7 P0.6 PU82979HM18Leukocyte immunoglobulinlike receptor, subfamily B, member 42.2 P0.8 P0.6 P1.2 PAF263541DYRK4Dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 42.9 P1.1 P0.9 A0.8 MNM_003426ZNF74Zinc finger protein 742.9 P1.1 A0.9 A0.8 ANM_002612PDK4Pyruvate dehydrogenase kinase, isoenzyme 42.5 P0.8 A0.8 A1.2 ANM_007195POLIPolymerase (DNA-directed) iota1.7 P0.8 P0.6 P1.2 P Abbreviations: A, absence; CDK, cyclin-dependent kinase; IL, interleukin; M, marginal; MECF, middle ear cholesteatoma fibroblast; mRNA, messenger RNA; P, presence; SF, postauricular skin–derived fibroblast.Table 4. Normalized Levels of 20 Genes in Cluster DGenBank Accession No.Gene SymbolGene DescriptionNormalized LevelSFMECFNoneIL-1αNoneIL-1αBF061658TGFB2Transforming growth factor β2 precursor0.0 A0.6 A2.9 P1.4 PAF062006HG38Orphan G protein–coupled receptor HG380.1 A0.0 A4.4 P1.9 PNM_005613RGS4Regulator of G-protein signaling 40.3 A0.0 A8.6 P1.7 PNM_016931NOX4NADPH oxidase 40.4 A0.4 M3.4 P1.6 PNM_002193INHBBInhibin, β B (activin AB β polypeptide)0.6 A0.2 A3.6 P1.4 ANM_014583LMCD1LIM and cysteine-rich domains 10.4 P1.0 P2.5 P1.0 PNM_000908NPRCNatriuretic peptide receptor C/guanylate cyclase C0.6 A0.5 A3.3 P1.4 ANM_001949E2F3E2F transcription factor 30.4 P1.2 P1.9 P0.8 PNM_005912MC4RMelanocortin 4 receptor0.7 A0.8 A3.7 P1.2 AX54559METMet proto-oncogene (hepatocyte growth factor receptor)0.7 P0.7 P3.0 P1.3 PL20966DPDE4Phosphodiesterase 4B0.8 P0.5 P3.7 P1.2 PNM_012219MRASMuscle rasoncogene homologue0.7 A0.9 A2.9 P1.1 ANM_000901NR3C2Nuclear receptor subfamily 3, group C, member 20.8 A0.1 A2.8 P1.2 AX54559METMet proto-oncogene (hepatocyte growth factor receptor)0.8 P0.4 P2.4 P1.2 PNM_002204ITGA3Integrin, α 3 (antigen CD49C, α 3 subunit of VLA-3 receptor)0.8 M0.5 A2.4 P1.2 AAB004903SOCS-2STAT-induced STAT inhibitor 20.9 P0.4 P2.4 P1.1 PNM_000824GLRBGlycine receptor, β0.9 A1.0 P2.2 P1.0 MNM_001955ET1Endothelin 11.1 A0.6 A2.6 P0.9 ANM_000875IGF1RInsulinlike growth factor 1 receptor0.9 P1.0 P2.1 P1.0 PNM_002276KRT19Keratin 190.9 P0.3 A2.1 P1.1 P Abbreviations: A, absence; M, marginal; MECF, middle ear cholesteatoma–derived fibroblast; P, presence; SF, postauricular skin–derived fibroblast; STAT, signal transducers and activators of transcription; VLA, very late antigen.ANALYSIS OF mRNA EXPRESSION BY REAL-TIME PCRWe determined the mRNA levels using real-time PCR to confirm the GeneChip data of cluster B because it was thought that cluster B might contain some of the genes associated with the pathogenesis of middle ear cholesteatoma. Our results showed that the mRNA expression of LARC, GMCSF, epiregulin, ICAM1, and TGFAwas significantly more strongly up-regulated by IL-1α and/or IL-1β in MECF than in SF (Figure 3).Figure 3.Validation of microarray expression levels by real-time polymerase chain reaction (PCR) in fibroblasts. To confirm the human genome U133A probe array (GeneChip; Affymetrix, Inc, Santa Clara, Calif) data, we additionally cultured postauricular skin–derived fibroblasts (SFs) and middle ear cholesteatoma–derived fibroblasts (MECFs) (some cultures stimulated with 10 ng/mL of interleukin [IL] 1α and/or IL-1β) from 5 other patients and determined the messenger RNA (mRNA) levels of liver and activation-regulated chemokine (LARC) (A), granulocyte-macrophage colony-stimulating factor (GM-CSF) (B), epiregulin (C), intercellular adhesion molecule 1 (ICAM-1) (D), and transforming growth factor α (TGF-α) (E) (extracted from Table 2) using real-time PCR analysis. The results are shown as mean ± SEM (*P<.05). The copy number is expressed as the number of transcripts. These data were divided by the expression level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) for background correction.COMMENTThis study is the first report, to our knowledge, of a global gene expression analysis used to identify specific genes in fibroblasts derived from middle ear cholesteatoma and normal postauricular skin in vitro. We identified many differences in the gene expression patterns of SFs and MECFs, although the fibroblasts were cultured and stimulated under the same conditions. Thus, SFs and MECFs appear to have different phenotypes because they were derived from a single genotype. Several reportshave stated that fibroblasts have different phenotypes in lesions and normal tissues. A recent reportfound that the cell-type–specific absence of CCCAT/enhancer-binding protein α (C/EBPα) was responsible for the enhanced proliferation of bronchial smooth muscle cells derived from subjects with asthma, explaining the failure of glucocorticoids to inhibit proliferation in vitro. Our findings suggest that tissue-derived cells may retain their characteristic features even when cultured for long periods. Consequently, these data suggest that fibroblasts derived from different tissues retain their typical gene expression profiles. Furthermore, the differing characteristics might be controlled by epigenetic mechanisms.Real-time PCR analysis showed that the subepidermal fibroblasts obtained from the middle ear cholesteatoma produced much more GMCSF, epiregulin, and TGFAthan did fibroblasts obtained from postauricular skin. Thus, activated MECFs may induce the exuberant growth of keratinocytes, resulting in the production of IL-1α and/or IL-1β in the injured and/or infected tissues. Transforming growth factor α is thought to be the main growth factor influencing keratinocytes via the previously mentioned paracrine loop. Many reports on TGF-α expression in cholesteatoma and the autocrine mechanism of TGF-α have been published.However, this is the first report, to our knowledge, that describes the expression of epiregulin in middle ear cholesteatoma. Epiregulin has been purified from the conditioned media of a mouse fibroblast-derived tumor cell line, NIH3T3/clone T7, and is a member of the epidermal growth factor (EGF) family.In recent studies,epiregulin was shown to be an autocrine growth factor in normal human keratinocytes, organizing the epidermal structure by regulating keratinocyte proliferation and differentiation, as well as the expression of TGF-α, heparin-binding–EGF, and amphiregulin. Consequently, a tendency for these growth factors to be expressed may be the origin of middle ear cholesteatoma. However, many other growth factors actually participate in the growth of keratinocytes. For example, the mRNA expression of KGF was enhanced in SFs after stimulation with IL-1α and/or IL-1β (data not shown). Our results contradict those for GM-CSF, epiregulin, and TGF-α, and future study is needed to determine the cause of this discrepancy.In addition, the mRNA expressions of LARC and ICAM-1 were more strongly up-regulated in MECF than in SF. The chemokine LARC is thought to contribute to the initiation of the immune response of T lymphocytes during the early phase of inflammation because it promotes the migration of immature dendritic cells and memory T lymphocytes to the area of local inflammation.In other words, compared with SFs, MECFs allow more CCR6-positive cells, such as immature dendritic cells and memory T lymphocytes, to accumulate during the early phase of inflammation triggered by a foreign antigen. Thus, the phenotype of activated fibroblasts residing in a given tissue may directly influence the nature and magnitude of leukocyte recruitment. For example, LARC has been previously shown to be related to the onset of rheumatoid arthritis.However, further examination of middle ear cholesteatoma is needed to determine whether fibroblasts are the main producers of LARC. The adhesion molecule ICAM-1 belongs to an immunoglobulin superfamily and is expressed in endothelial cells. ICAM-1 is mainly responsible for the migration of white blood cells to areas of inflammation. In middle ear cholesteatoma, ICAM-1 was shown by immunohistochemical analysis to be present in areas of inflammatory change.The significance of ICAM-1 being expressed on fibroblasts is that the fibroblasts then adhere to inflammatory cells, which migrate from blood vessels and remain in tissue. Consequently, local inflammation depending on the activation of inflammatory cells may be initiated by the binding of inflammatory cells to integrin, such as lymphocyte function–associated antigen 1 (LFA-1) and Mac-1 (CD11b/CD18), on cell surfaces. The present data for LARC and ICAM-1 suggest that MECFs may be able to evoke inflammation and contribute to its persistence more easily than SFs.Because these genes were differentially expressed in IL-1–stimulated MECFs but not in SFs, they seem to be related specifically to local immunity, such as the prominent hyperkeratosis seen in middle ear cholesteatoma. However, because these results were obtained with the use of cultured cells, further investigation is needed to elucidate how they actually contribute to the pathogenesis of middle ear cholesteatoma.CONCLUSIONSWe identified many differences in the gene expression patterns of SFs and MECFs, although the fibroblasts were cultured and stimulated under the same conditions. Thus, SFs and MECFs appear to have different phenotypes, since they were derived from a single genotype. These differential gene expressions suggest that subepidermal fibroblasts may play a role in the hyperkeratosis that occurs during middle ear cholesteatoma by releasing molecules involved in inflammation and epidermal growth; they also suggest that these fibroblasts retain tissue-specific characteristics that are presumably controlled by epigenetic mechanisms. These results may contribute to our understanding of the pathogenesis of middle ear cholesteatoma.Correspondence:Mamoru Yoshikawa, MD, PhD, Department of Otorhinolaryngology, Jikei University School of Medicine, 3-25-8 Nishi-Shimbashi, Minato-ku, Tokyo 105-8461, Japan (yoshikawa@jikei.ac.jp).Submitted for Publication:December 3, 2005; final revision received February 22, 2006; accepted March 15, 2006.Financial Disclosure:None reported.Funding/Support:This study was supported by Grants-in-Aid from the National Institute of Biomedical Innovation, Osaka, Japan.Acknowledgment:We thank Noriko Hashimoto, of the National Research Institute for Child Health and Development, for her skillful technical assistance.REFFRENCESNMaas-SzabowskiAShimotoyodomeNEFusenigKeratinocyte growth regulation in fibroblast cocultures via a double paracrine mechanism.J Cell Sci1999112(pt 12)1843185310341204ASzabowskiNMaas-SzabowskiSAndrechtc-Jun and JunB antagonistically control cytokine-regulated mesenchymal-epidermal interaction in skin.Cell200010374575511114331AEl GhalbzouriELammeMPonecCrucial role of fibroblasts in regulating epidermal morphogenesis.Cell Tissue Res200231018919912397374AEl-GhalbzouriSGibbsELammeCAVan BlitterswijkMPonecEffect of fibroblasts on epidermal regeneration.Br J Dermatol200214723024312174092SWernerHSmolaParacrine regulation of keratinocyte proliferation and differentiation.Trends Cell Biol20011114314611306276VSchillingBNegriJBujiaPSchulzEKastenbauerPossible role of interleukin 1α and interleukin 1β in the pathogenesis of cholesteatoma of the middle ear.Am J Otol1992133503551384343JWChungTHYoonDifferent production of interleukin-1α, interleukin-1β and interleukin-8 from cholesteatomatous and normal epithelium.Acta Otolaryngol19981183863919655214SYetiserBSatarNAydinExpression of epidermal growth factor, tumor necrosis factor-α, and interleukin-1α in chronic otitis media with or without cholesteatoma.Otol Neurotol20022364765212218613RHWaterstonKLindblad-TohEBirneyInitial sequencing and analysis of the human genome [published corrections appear in Nature. 2001;412:565 and Nature. 2001;411:720].Nature200140986092111237011JDrewsDrug discovery: a historical perspective.Science20002871960196410720314ALHopkinsCRGroomThe druggable genome.Nat Rev Drug Discov2002172773012209152MIidaKMatsumotoHTomitaSelective down-regulation of high-affinity IgE receptor (Fc&epsiv;RI) α-chain messenger RNA among transcriptome in cord blood–derived versus adult peripheral blood–derived cultured human mast cells.Blood2001971016102211159531DBrouty-BoyeCPottin-ClemenceauCDoucetCJasminBAzzaroneChemokines and CD40 expression in human fibroblasts.Eur J Immunol20003091491910741409CMHogaboamMLSteinhauserSWChensueSLKunkelNovel roles for chemokines and fibroblasts in interstitial fibrosis.Kidney Int199854215221599853282TPapUMuller-LadnerREGaySGayFibroblast biology.Arthritis Res2000236136711094449CCParkMJBissellMHBarcellos-HoffThe influence of the microenvironment on the malignant phenotype.Mol Med Today2000632432910904250TSilzleMKreutzMADoblerGBrockhoffRKnuechelLAKunz-SchughartTumor-associated fibroblasts recruit blood monocytes into tumor tissue.Eur J Immunol2003331311132012731056RSSmithTJSmithTMBliedenRPPhippsFibroblasts as sentinel cells.Am J Pathol19971513173229250144TSilzleGJRandolphMKreutzLAKunz-SchughartThe fibroblast: sentinel cell and local immune modulator in tumor tissue.Int J Cancer200410817318014639599MRothPRJohnsonPBorgerDysfunctional interaction of C/EBPα and the glucocorticoid receptor in asthmatic bronchial smooth-muscle cells.N Engl J Med200435156057415295049HSudhoffSDazertAMGonzalesAngiogenesis and angiogenic growth factors in middle ear cholesteatoma.Am J Otol20002179379811078065MShiwaHKojimaHMoriyamaExpression of transforming growth factor-α (TGF-α) in cholesteatoma.J Laryngol Otol19981127507549850316YTanakaMShiwaHKojimaHMiyazakiYKamideHMoriyamaA study on epidermal proliferation ability in cholesteatoma.Laryngoscope19981085375429546266SErgunXZhengBCarlsooExpression of transforming growth factor-α and epidermal growth factor receptor in middle ear cholesteatoma.Am J Otol1996173933968817015PSchulzJBujiaAHollyVShillingEKastenbauerPossible autocrine growth stimulation of cholesteatoma epithelium by transforming growth factor α.Am J Otolaryngol19931482878484481HToyodaTKomurasakiDUchidaEpireregulin: a novel epidermal growth factor with mitogenic activity for rat primary hepatocytes.J Biol Chem1995270749575007706296YShirakataTKomurasakiHToyodaEpiregulin, a novel member of the epidermal growth factor family, is an autocrine growth factor in normal human keratinocytes.J Biol Chem20002755748575310681561KHashimotoRegulation of keratinocyte function by growth factors.J Dermatol Sci200024(suppl 1)S46S5011137396DRGreavesWWangDJDairaghiCCR6, a CC chemokine receptor that interacts with macrophage inflammatory protein 3α and is highly expressed in human dendritic cells.J Exp Med19971868378449294138CAPowerDJChurchAMeyerCloning and characterization of a specific receptor for the novel CC chemokine. MIP-3α from lung dendritic cells.J Exp Med19971868258359294137ASCharbonnierNKohrgruberEKriehuberGStinglARotDMaurerMacrophage inflammatory protein 3α is involved in the constitutive trafficking of epidermal Langerhans cells.J Exp Med19991901755176810601351FLiaoRLRabinCSSmithGSharmaTBNutmanJMFarberCC-chemokine receptor 6 is expressed on diverse memory subsets of T cells and determines responsiveness to macrophage inflammatory protein 3α.J Immunol19991621861949886385JHRuthSShahraraCCParkRole of macrophage inflammatory protein-3α and its ligand CCR6 in rheumatoid arthritis.Lab Invest20038357958812695561TMatsuiTAkahoshiRNamaiSelective recruitment of CCR6-expressing cells by increased production of MIP-3α in rheumatoid arthritis.Clin Exp Immunol200112515516111472439RAkimotoRPawankarTYagiSBabaAcquired and congenital cholesteatoma: determination of tumor necrosis factor-alpha, intercellular adhesion molecule-1, interleukin-1-alpha and lymphocyte functional antigen-1 in the inflammatory process.ORL J Otorhinolaryngol Relat Spec20006225726510965261HShinodaCCHuangLocalization of intercellular adhesion molecule-1 in middle ear cholesteatoma.Eur Arch Otorhinolaryngol19952523853908562031JBujiaAHollyCKimNScanadyEKastenbauerExpression of human intercellular adhesion molecules in middle ear cholesteatoma.Am J Otolaryngol1994152712757526720 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png JAMA Otolaryngology - Head & Neck Surgery American Medical Association

Identification of Specific Gene Expression Profiles in Fibroblasts Derived From Middle Ear Cholesteatoma

Loading next page...
 
/lp/american-medical-association/identification-of-specific-gene-expression-profiles-in-fibroblasts-M8K7JyJTZK
Publisher
American Medical Association
Copyright
Copyright 2006 American Medical Association. All Rights Reserved. Applicable FARS/DFARS Restrictions Apply to Government Use.
ISSN
2168-6181
eISSN
2168-619X
DOI
10.1001/archotol.132.7.734
pmid
16847181
Publisher site
See Article on Publisher Site

Abstract

ObjectiveTo investigate the role of fibroblasts in the pathogenesis of cholesteatoma.DesignTissue specimens were obtained from our patients. Middle ear cholesteatoma–derived fibroblasts (MECFs) and postauricular skin–derived fibroblasts (SFs) as controls were then cultured for a few weeks. These fibroblasts were stimulated with interleukin (IL) 1α and/or IL-1β before gene expression assays. We used the human genome U133A probe array (GeneChip) and real-time polymerase chain reaction to examine and compare the gene expression profiles of the MECFs and SFs.SubjectsSix patients who had undergone tympanoplasty.ResultsThe IL-1α–regulated genes were classified into 4 distinct clusters on the basis of profiles differentially regulated by SF and MECF using a hierarchical clustering analysis. The messenger RNA expressions of LARC(liver and activation-regulated chemokine), GMCSF(granulocyte-macrophage colony-stimulating factor), epiregulin, ICAM1(intercellular adhesion molecule 1), and TGFA(transforming growth factor α) were more strongly up-regulated by IL-1α and/or IL-1β in MECF than in SF, suggesting that these fibroblasts derived from different tissues retained their typical gene expression profiles.ConclusionsFibroblasts may play a role in hyperkeratosis of middle ear cholesteatoma by releasing molecules involved in inflammation and epidermal growth. These fibroblasts may retain tissue-specific characteristics presumably controlled by epigenetic mechanisms.Recently, the interaction between mesenchymal cells such as fibroblasts and epithelial cells or keratinocytes has been proposed to be involved in inflammation, homeostasis, and tissue regeneration.For example, interleukin (IL) 1 produced by keratinocytes induces the release of keratinocyte growth factor (KGF), granulocyte-macrophage colony-stimulating factor (GM-CSF), and transforming growth factor α (TGF-α) from fibroblasts.These fibroblast-derived cytokines support the proliferation and differentiation of keratinocytes. This paracrine loop is thought to be important in repair processes of tissue inflammation. In the study presented herein, we used the human genome U133A probe array (GeneChip; Affymetrix, Inc, Santa Clara, Calif) and real-time polymerase chain reaction (PCR) to examine the gene expression profiles of fibroblasts derived from middle ear cholesteatoma and postauricular skin obtained from patients to determine whether these fibroblasts express some molecules that may interact with keratinocytes and be involved in the pathogenesis of middle ear cholesteatoma.METHODSCELL CULTURESMiddle ear cholesteatoma and normal postauricular skin samples were obtained from patients undergoing tympanoplasty, and single fibroblasts from each sample were cultured in Dulbecco Modified Eagle Medium/F12 (DMEM/F12; Invitrogen Corp, Carlsbad, Calif) with 10% fetal calf serum (JRH Biosciences, Lenexa, Kan) and a combination of 60-μg/mL penicillin and 100-μg/mL streptomycin (Invitrogen Corp) for a few weeks. The cells were incubated at 37°C in a humidified incubator containing 5% carbon dioxide in air and were analyzed after 4 passages. Patient consent and the approval of our university's ethics review board were obtained before the start of the study.CYTOKINE STIMULATION OF FIBROBLASTSMiddle ear cholesteatoma fibroblasts (MECFs) and skin fibroblasts (SFs) were stimulated with 10 ng/mL of IL-1α or 10 ng/mL of IL-1β (both from R&D Systems Inc, Minneapolis, Minn) for 4 hours before messenger RNA (mRNA) extraction.MICROARRAY EXPRESSION ANALYSISHuman genome-wide gene expression was examined using the human genome U133A probe array (GeneChip), which contains the oligonucleotide probe set for approximately 22 000 full-length genes, according to the manufacturer's protocol and previously reported strategies.Total RNA (5 &mgr;g) was extracted from the fibroblasts, and double-stranded complementary DNA (cDNA) was synthesized using a SuperScript Choice system (Invitrogen Corp) and a T7-(dT)24 primer (Amersham Pharmacia Biotech, Buckinghamshire, England). The cDNA was subjected to in vitro transcription in the presence of biotinylated nucleoside triphosphates using a high-yield RNA transcript labeling kit (BioArray; Enzo Diagnostics, Farmingdale, NY). The biotinylated complementary RNA was then hybridized with the probe array for 16 hours at 45°C. After washing, the hybridized biotinylated complementary RNA was stained with streptavidin-phycoerythrin (Molecular Probes, Eugene, Ore) and scanned with a gene array scanner (Hewlett-Packard Company, Palo Alto, Calif). The fluorescence intensity of each probe was quantified using the GeneChip Analysis Suite program, version 5.0 (Affymetrix, Inc). The expression level of single mRNA was determined as the mean fluorescence intensity among the intensities obtained with 11 paired (perfect-matched and single nucleotide–mismatched) probes. If the intensities of the mismatched probes were very high, gene expression was judged to be absent, even if a high mean fluorescence was obtained with the GeneChip Analysis Suite 5.0 program.The resulting data were analyzed using GeneSpring software, version 7.2 (Silicon Genetics, San Carlos, Calif). To normalize the staining intensity variations among the chips, the values for all genes on a given chip were divided by the median of all measurements on that chip. To eliminate changes within the range of background noise and to select the most differentially expressed genes, data were used only if the raw data values were less than 100 and gene expression was judged to be present by a gene expression data analysis. Hierarchical clustering analysis with standard correlation was used to identify gene clusters.QUANTITATIVE REAL-TIME PCRTo confirm the GeneChip microarray expression analysis data, we quantified the gene expression in fibroblasts derived from 5 other patients using quantitative real-time PCR. Total RNA was isolated using an RNA purification kit that included DNase digestion (RNeasy Mini Kit; Qiagen GmbH, Hilden, Germany). The RNA was then transcribed into cDNA using reverse transcriptase (Superscript II: Invitrogen Corp). Quantitative PCR was performed using a sequence detector system (ABI/PRISM 7700; Applied Biosystems, Foster City, Calif) and TaqMan Universal PCR Master Mix (Applied Biosystems), according to the manufacturers’ instructions. The primers and TaqMan probes used for the genes LARC(liver and activation-regulated chemokine), GMCSF(granulocyte-macrophage colony-stimulating factor), ICAM1(intercellular adhesion molecule 1), and TGFA(transforming growth factor-α) were as follows: LARC: forward, 5′-TGTCAGTGCTGCTACTCCACCT-3′; reverse, 5′-CTGTGTATCCAAGACAGCAGTCAA-3′; and TaqMan probe, 5′-TGCGGCGAATCAGAAGCAGCAA-3′; GMCSF: forward, 5′-GCCTCACCAAGCTCAAGGG-3′; reverse, 5′-GGTTGGAGGGCAGTGCTG-3′; and TaqMan probe, 5′-CCCTTGACCATGATGGCCAGCC-3′; ICAM1: forward, 5′-CTGTGTCCCCCTCAAAAGTCA-3′; reverse, 5′-ATACACCTTCCGGTTGTTCCC-3′; and TaqMan probe, 5′-TGCGGCGAATCAGAAGCAGCAA-3′; TGFA: forward, 5′-AGGAGACCCCTGCCCTCTAGT-3′; reverse, 5′-TCTGCAATGTGTTCTTGGTTTTG-3′; and TaqMan probe, 5′-TTCCAACCTGCCCAGTCACAGAAGG-3′. For epiregulin, quantitative PCR was performed using SYBR green PCR master mix (Applied Biosystems). The primers for epiregulin were as follows: forward, 5′-ATCCTGGCATGTGCTAGGGT-3′ and reverse, 5′-GTGCTCCAGAGGTCAGCCAT-3′. The expression levels of mRNA were normalized by the mean expression of a housekeeping gene (glyceraldehyde-3-phosphate dehydrogenase [GAPDH]), which was measured using Pre-Developed Assay Reagents (Applied Biosystems).STATISTICAL ANALYSISData are presented as mean ± SEM. Statistical significance was determined using the paired ttest, and differences were considered significant at P<.05.RESULTSEXPERIMENTAL DESIGNGeneChip was used to identify the gene expression pattern induced by IL-1α in the MECFs (n = 1) and SFs (n = 1). Following the identification of candidate disease–related genes with GeneChip, quantitative real-time PCR was used to confirm the expression of the selected gene (n = 5 for each).To assign a “fold-change cutoff” threshold, we used a GeneSpring analysis for selected genes in which the mean expression level had increased or decreased by more than 2-fold after 4 hours. As shown in Figure 1and Figure 2, IL-1α had an effect on numerous genes in the SF and MECF cultures.Figure 1.Interleukin (IL) 1α–mediated up-regulation of gene clusters in postauricular skin–derived fibroblasts (SFs) and middle ear cholesteatoma–derived fibroblasts (MECFs). Representation of messenger RNA expression levels in spontaneous SF, IL-1α–stimulated SF, spontaneous MECF, and IL-1α–stimulated MECF. The SFs and MECFs were stimulated with IL-1α (10 ng/mL) for 4 hours. The colored bars show the magnitude of the response of each gene, according to the scale of expression level shown. Cluster A contained 164 genes (1) in which expression in SFs increased at least 2-fold after stimulation with IL-1α and (2) in which increased gene expression was more than 2-fold that of IL-1α–stimulated MECFs. Cluster B contained 84 genes (1) in which expression in MECFs increased at least 2-fold after stimulation with IL-1α and (2) in which increased gene expression was more than 2-fold that of IL-1α–stimulated SFs.Figure 2.Interleukin (IL) 1α–mediated down-regulation of gene clusters in postauricular skin-derived fibroblasts (SFs) and middle ear cholesteatoma–derived fibroblasts (MECFs). Representation of messenger RNA expression levels in spontaneous SF, IL-1α–stimulated SF, spontaneous MECF, and IL-1α–stimulated MECF. The SFs and MECFs were stimulated with IL-1α (10 ng/mL) for 4 hours. The colored bars show the magnitude of the response of each gene, according to the scale of the expression level shown. Cluster C contained 65 genes (1) in which expression in SFs decreased by at least half after stimulation with IL-1α and (2) in which expression in spontaneous SFs increased gene expression by more than 2-fold that of spontaneous MECFs. Cluster D contained 69 genes (1) in which expression in MECFs decreased by at least half after stimulation with IL-1α and (2) in which expression in spontaneous MECFs increased gene expression by more than 2-fold that of spontaneous SFs.GENE EXPRESSION DATA ANALYSISUsing hierarchical clustering analysis of the gene expression profiles of 22 283 genes, we identified a cluster containing genes that were up-regulated in SF or MECF after IL-1α stimulation (Figure 1). Visual inspection identified 2 major groups among the up-regulated genes. The first group of 164 genes displayed an increase in gene expression in SF that was more than 2-fold of that in MECF (Figure 1, Table 1), while the second group of 84 genes displayed an increase in gene expression in MECF that was more than 2-fold of that in SF (Figure 1, Table 2).Table 1. Normalized Levels of 45 Genes in Cluster AGenBank Accession No.Gene SymbolGene DescriptionNormalized LevelSFMECFNoneIL-1αNoneIL-1αAW611727GAS1Growth-arrest–specific protein 11.8 P4.7 P0.2 A0.0 ANM_022162NOD2Caspase recruitment domain family, member 150.1 A9.1 P1.8 A0.2 AAI984980MCP2Monocyte chemotactic protein 21.1 A27.3 P0.3 A0.9 ABG166705ENA78Epithelial neutrophil-activating peptide 780.1 A30.3 P0.4 A1.6 ANM_005246TYK3Fer (fps/fes related) tyrosine kinase (phosphoprotein NCP94)1.2 A3.3 P0.8 A0.2 ANM_001250CD40Tumor necrosis factor receptor superfamily, member 51.7 A4.1 P0.1 A0.3 ANM_005761PLXNC1Plexin C11.5 A3.1 P0.5 A0.2 ABC005254CLECSF2C-type lectin, superfamily member 2 (activation induced)1.6 P4.0 P0.0 A0.4 PNM_002068GNA16Guanine nucleotide binding protein, α150.9 A4.4 P1.1 A0.4 ANM_004972JAK2JAK 2 (a protein tyrosine kinase)1.6 A3.5 P0.2 A0.4 ANM_002422MMP3Matrix metalloproteinase 3 (stromelysin 1, progelatinase)1.5 P4.1 P0.1 A0.5 PNM_005562LAMC2Laminin, γ 20.8 P2.5 P1.2 A0.3 ANM_006383KIP2DNA-dependent protein kinase catalytic subunit-interacting0.5 A1.5 P2.8 A0.2 ANM_006273MCP3Monocyte chemotactic protein 30.6 A8.6 P0.9 A1.1 ANM_000759GCSFColony-stimulating factor 3 (granulocyte)0.3 A12.6 P0.3 A1.7 ANM_000064C3Complement component 31.0 A7.0 P0.2 A1.0 AAB005043SSI1JAK binding protein, STAT-induced STAT inhibitor 11.4 A3.7 P0.5 A0.6 AM21121RANTESRegulated on activation normal T cell expressed and secreted0.2 A10.3 P0.1 A1.8 ANM_000880IL7Interleukin 71.1 A5.2 P0.4 A0.9 ANM_001218CA12Carbonic anhydrase XII1.4 P2.9 P0.4 P0.6 PNM_004527MOX1Mesenchyme homeobox 10.9 A5.6 P0.4 A1.1 PNM_002185IL7RInterleukin 7 receptor1.1 P4.8 P0.2 A0.9 PNM_005461KRMLv-mafMusculoaponeurotic fibrosarcoma oncogene homologue B1.4 A2.9 P0.1 A0.6 ANM_002421MMP1Matrix metalloproteinase 1 (interstitial collagenase)1.3 P3.6 P0.3 P0.7 PBC003600LMO4LIM domain only 41.3 P3.4 P0.7 P0.7 PNM_004820CP7BCytochrome P450, subfamily VIIB, polypeptide 10.8 P6.0 P0.4 A1.2 PAI817041RDC1Orphan G protein–coupled receptor with 7 transmembrane domains1.0 P5.0 P0.4 A1.0 PNM_001565IP10Interferon-inducible protein 100.3 A7.9 P0.3 A1.7 PNM_016087WNT16Wingless-type MMTV integration site family, member 160.9 A4.5 P1.0 A1.0 AAF055585SLIT2Slit (Drosophila) homologue 21.3 P3.1 P0.6 P0.7 PBC000388BING4Chromosome 6 open reading frame 110.2 A1.6 P1.8 A0.4 AL27624TFPI2Tissue factor pathway inhibitor 20.4 P5.7 P0.5 P1.5 PU74324RABIFRAB interacting factor0.9 P1.8 P1.1 P0.5 PNM_000585IL15Interleukin 151.1 P3.4 P0.4 P0.9 PNM_001426EN1Engrailed homologue 11.2 A2.8 P0.6 A0.8 AAW083357IL1RNInterleukin 1 receptor antagonist protein precursor0.9 A4.1 P0.7 A1.1 ANM_002426MMP12Matrix metalloproteinase 12 (macrophage elastase)1.0 P3.6 P0.4 M1.0 PM14333FYNFYN oncogene related to SRC, FGR, YES1.1 P3.0 P0.7 P0.9 PU13699ICECaspase 1, apoptosis-related cysteine protease1.0 A2.6 P1.0 A0.8 PNM_001078VCAM1Vascular cell adhesion molecule 10.3 A4.2 P0.5 A1.5 PNM_002009KGFFibroblast growth factor 7 (keratinocyte growth factor)1.0 P2.5 P0.6 P1.0 PD49372EotaxinSmall inducible cytokine subfamily A (cys-cys), member 110.3 A3.9 P0.1 A1.7 PM15330IL1BHuman interleukin 1β (IL-1β) mRNA0.0 A4.3 P0.1 A1.9 PNM_000963PTGS2Prostaglandin-endoperoxide synthase 20.3 M3.8 P0.2 A1.7 PAF030514ITACInterferon-inducible T-cell α chemoattractant0.5 A3.3 P0.4 A1.5 P Abbreviations: A, absence; IL, interleukin; JAK, Janus kinase; M, marginal; MECF, middle ear cholesteatoma fibroblast; MMTV, mouse mammary tumor virus; mRNA, messenger RNA; P, presence; SF, postauricular skin–derived fibroblasts; STAT, signal transducers and activators of transcription.Table 2. The Normalized Levels of 50 Genes in Cluster B*GenBank Accession No.Gene SymbolGene DescriptionNormalized LevelSFMECFNoneIL-1αNoneIL-1αNM_004591LARCLiver and activation-regulated chemokine0.0 A1.9 P0.1 A9.6 PM11734GMCSFColony-stimulating factor 2 (granulocyte-macrophage)0.0 A1.9 P0.1 A5.8 PU12767NR4A3Nuclear receptor subfamily 4, group A, member 30.2 A1.7 A0.3 A10.3 PNM_000201ICAM1Intercellular adhesion molecule 1 (CD54)0.1 A1.9 P0.1 P4.3 PNM_002888TIG1Retinoic acid receptor responder (tazarotene induced) 10.4 A1.6 A0.2 A4.5 PU22386CSF1Colony-stimulating factor 1 (macrophage)0.2 A1.7 P0.3 A4.7 PNM_002173IFNA16Interferon α 161.4 P0.6 M0.1 A1.5 PU37546API2Baculoviral IAP repeat-containing 30.2 A1.2 P0.8 P8.0 PNM_002575SERPINB2Serine proteinase inhibitor, clade B, member 20.1 P1.6 P0.4 P3.8 PM37435MCSFColony-stimulating factor 1 (macrophage)0.3 P1.6 P0.4 P3.6 PNM_012429TAPSEC14 (Saccharomyces cerevisiae)–like 21.1 A0.9 A0.3 A2.7 PNM_004233BL11Activated B lymphocytes, immunoglobulin superfamily0.8 A1.2 P0.6 A4.5 PAI360875SOX11SRY-box 110.3 A1.0 A1.0 A6.5 PNM_002015FKH1Forkhead box O1A (rhabdomyosarcoma)1.1 P0.9 A0.4 A2.7 PNM_006622SNKSerum-inducible kinase0.5 P1.5 P0.5 P3.2 PD89377MSX2Muscle segment homeobox (Msh) (Drosophila) homeobox homologue 20.5 A1.0 A1.0 A5.7 PNM_000029SERPINA8Serine proteinase inhibitor, clade A, member 81.3 M0.7 A0.4 A2.1 PNM_005658EBI6TNF receptor–associated factor 10.1 A1.0 P1.0 A5.1 PNM_003236TGFATransforming growth factor α0.6 A0.9 P1.1 P5.2 PNM_006981CHNNuclear receptor subfamily 4, group A, member 30.4 A1.0 P1.0 P4.5 PD31771HOX8Msh (Drosophila) homeobox homologue 20.8 A0.2 A1.2 A5.4 PAF153882RILLIM domain protein0.1 A1.3 P0.7 P3.0 PD32201ADRA1CAdrenergic, α-1A, receptor1.3 A0.7 A0.7 A3.1 PNM_002309CDFLeukemia inhibitory factor (cholinergic differentiation factor)0.1 A1.0 P1.0 P4.2 PAB044088SHARP-1Basic helix-loop-helix domain containing, class B, 30.3 A1.2 P0.8 A3.0 PU08015NFATCNuclear factor of activated T cells0.4 A1.0 P1.0 P3.8 PD87811GATA6GATA-binding protein 60.4 P0.9 P1.1 P4.2 PNM_002777PR3Proteinase 3 (serine proteinase)0.5 A1.2 A0.8 A2.7 PNM_001432EREpiregulin0.1 A0.1 A1.9 P6.8 PNM_003125SPRR1Small proline-rich protein 1B (cornifin)1.3 A0.7 A0.7 A2.2 PY15014GLCT2Uridine diphosphate (UDP)-galactose0.7 A1.0 A1.0 A3.0 PNM_001945DTRDiphtheria toxin receptor0.1 A0.2 A1.8 A4.8 PM79321JTK8Lyn B protein0.7 P0.9 P1.1 P2.9 PNM_005092TL6Tumor necrosis factor (ligand) superfamily, member 181.0 A0.8 A1.0 A2.7 PNM_006516GLUT1Solute carrier family 2, member 10.6 P0.9 P1.1 P2.8 PZ21533HHEXHematopoietically expressed homeobox0.7 P0.9 P1.1 P2.9 PNM_004362CLGNCalmegin0.7 A0.2 A1.3 A3.2 PNM_014030GIT1G protein–coupled receptor kinase-interactor 11.2 P0.8 P0.8 P1.9 PJ03223PRGProteoglycan 1, secretory granule0.2 A0.7 P1.3 P3.3 PNM_002253VEGFR2Kinase insert domain receptor0.9 A1.0 A1.0 A2.5 PAF009616CFLARCASP8 and FADD-like apoptosis regulator0.7 P1.1 P0.9 P2.2 PNM_001860CTR2Solute carrier family 31 (copper transporters), member 20.4 P0.7 P1.3 P3.0 PNM_002448HOX7Muscle segment homeobox (Msh) (Drosophila) homeobox homologue 10.5 P1.0 P1.0 P2.2 PU26662NPTX2Neuronal pentraxin II1.1 A0.4 A0.9 A2.0 PNM_000047ARSEArylsulfatase E (chondrodysplasia punctata 1)1.2 A0.4 A0.8 A1.8 PNM_001257CDHHCadherin 13, H-cadherin (heart)0.5 M0.4 A1.5 P3.3 PNM_004694SLC16A6Solute carrier family 16, member 60.1 A0.2 A1.8 P3.8 PNM_005860FLRGFollistatinlike 3 (secreted glycoprotein)0.7 P0.7 P1.3 P2.6 PAF001434PASTEH-domain containing 10.8 P0.8 P1.2 P2.5 P Abbreviations: A, absence; CASP8, caspase-8; FADD, Fas-associated death domain; IAP, integrin-associated protein; IL, interleukin; M, marginal; MECF, middle ear cholesteatoma fibroblast; P, presence; SF, postauricular skin–derived fibroblast; SRY, sex-determining region Y; TNF, tumor necrosis factor. *Unshaded cells indicate 5 genes associated with the pathogenesis of middle ear cholesteatoma.Among the genes in Table 1, several were identified whose expressions are generally known to be induced by IL-1α in fibroblasts, including monocyte chemoattractant protein 2 (MCP2); MCP3;granulocyte colony-stimulating factor (GCSF); regulated on activation normal T cell expressed and secreted (RANTES); matrix metalloproteinase 1 (MMP1); interferon-inducible protein 10 (IP10); IL-15 (IL15); vascular cell adhesion molecule 1 (VCAM1); KGF; eotaxin; and interferon-inducible T-cell α chemoattractant (ITAC). Genes for multiple profibrotic cytokines and chemokines that exhibited elevated expressions are shown in Table 2. These cytokines included TGFA, GMCSF, ICAM1, epiregulin, and LARC.Figure 2shows the genes that were down-regulated in SF or MECF after 4 hours of stimulation with IL-1α. Two major groups were identified among these genes: 65 genes showed a decrease in gene expression in SF that was less than half of that in MECF (Figure 2, Table 3), while the other 69 genes displayed a decrease in gene expression in MECF that was less than half of that in SF (Figure 2, Table 4).Table 3. Normalized Levels of 20 Genes in Cluster CGenBank Accession No.Gene SymbolGene DescriptionNormalized LevelSFMECFNoneIL-1αNoneIL-1αNM_004982KCNJ8Potassium inwardly rectifying channel, subfamily J, member 817.6 P1.6 A0.2 A0.4 AM21692ADH2Alcohol dehydrogenase 1B (class I), β polypeptide8.5 P1.8 P0.2 A0.2 ANM_005069SIMSingle-minded (Drosophila) homologue 24.8 P1.8 P0.1 A0.2 ANM_020379HMIC1,2-α-mannosidase3.7 P1.5 P0.1 A0.5 ANM_014421DKK-2Dickkopf (Xenopus laevis) homologue 24.0 P1.5 P0.4 A0.5 AD64137KIP2Human KIP2gene for CDK-inhibitor p57KIP24.1 P1.4 A0.5 A0.6 AAF284095ADRA2AAdrenergic, α-2A-, receptor5.3 P1.2 P0.8 A0.6 ANM_016109ANGPTL4Angiopoietinlike 44.0 P0.9 A0.6 M1.1 ANM_001546ID4Inhibitor of DNA binding 43.7 P0.9 P0.7 A1.1 PNM_018490LGR4G protein–coupled receptor 483.7 P1.3 P0.7 P0.5 PX75208HEK2Homo sapiensHEK2 mRNA for protein tyrosine kinase receptor4.2 P0.5 A0.9 A1.1 ANM_021047BMZF1Zinc finger protein 2531.8 P0.7 A0.5 M1.3 MNM_001611TRAPAcid phosphatase 5, tartrate resistant2.5 P0.5 A0.7 A1.3 ANM_014210EVI2AEcotropic viral integration site 2A2.8 P1.2 P0.8 A0.6 PAK027146RPL5Ribosomal protein L52.6 P1.3 P0.7 P0.6 PU82979HM18Leukocyte immunoglobulinlike receptor, subfamily B, member 42.2 P0.8 P0.6 P1.2 PAF263541DYRK4Dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 42.9 P1.1 P0.9 A0.8 MNM_003426ZNF74Zinc finger protein 742.9 P1.1 A0.9 A0.8 ANM_002612PDK4Pyruvate dehydrogenase kinase, isoenzyme 42.5 P0.8 A0.8 A1.2 ANM_007195POLIPolymerase (DNA-directed) iota1.7 P0.8 P0.6 P1.2 P Abbreviations: A, absence; CDK, cyclin-dependent kinase; IL, interleukin; M, marginal; MECF, middle ear cholesteatoma fibroblast; mRNA, messenger RNA; P, presence; SF, postauricular skin–derived fibroblast.Table 4. Normalized Levels of 20 Genes in Cluster DGenBank Accession No.Gene SymbolGene DescriptionNormalized LevelSFMECFNoneIL-1αNoneIL-1αBF061658TGFB2Transforming growth factor β2 precursor0.0 A0.6 A2.9 P1.4 PAF062006HG38Orphan G protein–coupled receptor HG380.1 A0.0 A4.4 P1.9 PNM_005613RGS4Regulator of G-protein signaling 40.3 A0.0 A8.6 P1.7 PNM_016931NOX4NADPH oxidase 40.4 A0.4 M3.4 P1.6 PNM_002193INHBBInhibin, β B (activin AB β polypeptide)0.6 A0.2 A3.6 P1.4 ANM_014583LMCD1LIM and cysteine-rich domains 10.4 P1.0 P2.5 P1.0 PNM_000908NPRCNatriuretic peptide receptor C/guanylate cyclase C0.6 A0.5 A3.3 P1.4 ANM_001949E2F3E2F transcription factor 30.4 P1.2 P1.9 P0.8 PNM_005912MC4RMelanocortin 4 receptor0.7 A0.8 A3.7 P1.2 AX54559METMet proto-oncogene (hepatocyte growth factor receptor)0.7 P0.7 P3.0 P1.3 PL20966DPDE4Phosphodiesterase 4B0.8 P0.5 P3.7 P1.2 PNM_012219MRASMuscle rasoncogene homologue0.7 A0.9 A2.9 P1.1 ANM_000901NR3C2Nuclear receptor subfamily 3, group C, member 20.8 A0.1 A2.8 P1.2 AX54559METMet proto-oncogene (hepatocyte growth factor receptor)0.8 P0.4 P2.4 P1.2 PNM_002204ITGA3Integrin, α 3 (antigen CD49C, α 3 subunit of VLA-3 receptor)0.8 M0.5 A2.4 P1.2 AAB004903SOCS-2STAT-induced STAT inhibitor 20.9 P0.4 P2.4 P1.1 PNM_000824GLRBGlycine receptor, β0.9 A1.0 P2.2 P1.0 MNM_001955ET1Endothelin 11.1 A0.6 A2.6 P0.9 ANM_000875IGF1RInsulinlike growth factor 1 receptor0.9 P1.0 P2.1 P1.0 PNM_002276KRT19Keratin 190.9 P0.3 A2.1 P1.1 P Abbreviations: A, absence; M, marginal; MECF, middle ear cholesteatoma–derived fibroblast; P, presence; SF, postauricular skin–derived fibroblast; STAT, signal transducers and activators of transcription; VLA, very late antigen.ANALYSIS OF mRNA EXPRESSION BY REAL-TIME PCRWe determined the mRNA levels using real-time PCR to confirm the GeneChip data of cluster B because it was thought that cluster B might contain some of the genes associated with the pathogenesis of middle ear cholesteatoma. Our results showed that the mRNA expression of LARC, GMCSF, epiregulin, ICAM1, and TGFAwas significantly more strongly up-regulated by IL-1α and/or IL-1β in MECF than in SF (Figure 3).Figure 3.Validation of microarray expression levels by real-time polymerase chain reaction (PCR) in fibroblasts. To confirm the human genome U133A probe array (GeneChip; Affymetrix, Inc, Santa Clara, Calif) data, we additionally cultured postauricular skin–derived fibroblasts (SFs) and middle ear cholesteatoma–derived fibroblasts (MECFs) (some cultures stimulated with 10 ng/mL of interleukin [IL] 1α and/or IL-1β) from 5 other patients and determined the messenger RNA (mRNA) levels of liver and activation-regulated chemokine (LARC) (A), granulocyte-macrophage colony-stimulating factor (GM-CSF) (B), epiregulin (C), intercellular adhesion molecule 1 (ICAM-1) (D), and transforming growth factor α (TGF-α) (E) (extracted from Table 2) using real-time PCR analysis. The results are shown as mean ± SEM (*P<.05). The copy number is expressed as the number of transcripts. These data were divided by the expression level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) for background correction.COMMENTThis study is the first report, to our knowledge, of a global gene expression analysis used to identify specific genes in fibroblasts derived from middle ear cholesteatoma and normal postauricular skin in vitro. We identified many differences in the gene expression patterns of SFs and MECFs, although the fibroblasts were cultured and stimulated under the same conditions. Thus, SFs and MECFs appear to have different phenotypes because they were derived from a single genotype. Several reportshave stated that fibroblasts have different phenotypes in lesions and normal tissues. A recent reportfound that the cell-type–specific absence of CCCAT/enhancer-binding protein α (C/EBPα) was responsible for the enhanced proliferation of bronchial smooth muscle cells derived from subjects with asthma, explaining the failure of glucocorticoids to inhibit proliferation in vitro. Our findings suggest that tissue-derived cells may retain their characteristic features even when cultured for long periods. Consequently, these data suggest that fibroblasts derived from different tissues retain their typical gene expression profiles. Furthermore, the differing characteristics might be controlled by epigenetic mechanisms.Real-time PCR analysis showed that the subepidermal fibroblasts obtained from the middle ear cholesteatoma produced much more GMCSF, epiregulin, and TGFAthan did fibroblasts obtained from postauricular skin. Thus, activated MECFs may induce the exuberant growth of keratinocytes, resulting in the production of IL-1α and/or IL-1β in the injured and/or infected tissues. Transforming growth factor α is thought to be the main growth factor influencing keratinocytes via the previously mentioned paracrine loop. Many reports on TGF-α expression in cholesteatoma and the autocrine mechanism of TGF-α have been published.However, this is the first report, to our knowledge, that describes the expression of epiregulin in middle ear cholesteatoma. Epiregulin has been purified from the conditioned media of a mouse fibroblast-derived tumor cell line, NIH3T3/clone T7, and is a member of the epidermal growth factor (EGF) family.In recent studies,epiregulin was shown to be an autocrine growth factor in normal human keratinocytes, organizing the epidermal structure by regulating keratinocyte proliferation and differentiation, as well as the expression of TGF-α, heparin-binding–EGF, and amphiregulin. Consequently, a tendency for these growth factors to be expressed may be the origin of middle ear cholesteatoma. However, many other growth factors actually participate in the growth of keratinocytes. For example, the mRNA expression of KGF was enhanced in SFs after stimulation with IL-1α and/or IL-1β (data not shown). Our results contradict those for GM-CSF, epiregulin, and TGF-α, and future study is needed to determine the cause of this discrepancy.In addition, the mRNA expressions of LARC and ICAM-1 were more strongly up-regulated in MECF than in SF. The chemokine LARC is thought to contribute to the initiation of the immune response of T lymphocytes during the early phase of inflammation because it promotes the migration of immature dendritic cells and memory T lymphocytes to the area of local inflammation.In other words, compared with SFs, MECFs allow more CCR6-positive cells, such as immature dendritic cells and memory T lymphocytes, to accumulate during the early phase of inflammation triggered by a foreign antigen. Thus, the phenotype of activated fibroblasts residing in a given tissue may directly influence the nature and magnitude of leukocyte recruitment. For example, LARC has been previously shown to be related to the onset of rheumatoid arthritis.However, further examination of middle ear cholesteatoma is needed to determine whether fibroblasts are the main producers of LARC. The adhesion molecule ICAM-1 belongs to an immunoglobulin superfamily and is expressed in endothelial cells. ICAM-1 is mainly responsible for the migration of white blood cells to areas of inflammation. In middle ear cholesteatoma, ICAM-1 was shown by immunohistochemical analysis to be present in areas of inflammatory change.The significance of ICAM-1 being expressed on fibroblasts is that the fibroblasts then adhere to inflammatory cells, which migrate from blood vessels and remain in tissue. Consequently, local inflammation depending on the activation of inflammatory cells may be initiated by the binding of inflammatory cells to integrin, such as lymphocyte function–associated antigen 1 (LFA-1) and Mac-1 (CD11b/CD18), on cell surfaces. The present data for LARC and ICAM-1 suggest that MECFs may be able to evoke inflammation and contribute to its persistence more easily than SFs.Because these genes were differentially expressed in IL-1–stimulated MECFs but not in SFs, they seem to be related specifically to local immunity, such as the prominent hyperkeratosis seen in middle ear cholesteatoma. However, because these results were obtained with the use of cultured cells, further investigation is needed to elucidate how they actually contribute to the pathogenesis of middle ear cholesteatoma.CONCLUSIONSWe identified many differences in the gene expression patterns of SFs and MECFs, although the fibroblasts were cultured and stimulated under the same conditions. Thus, SFs and MECFs appear to have different phenotypes, since they were derived from a single genotype. These differential gene expressions suggest that subepidermal fibroblasts may play a role in the hyperkeratosis that occurs during middle ear cholesteatoma by releasing molecules involved in inflammation and epidermal growth; they also suggest that these fibroblasts retain tissue-specific characteristics that are presumably controlled by epigenetic mechanisms. These results may contribute to our understanding of the pathogenesis of middle ear cholesteatoma.Correspondence:Mamoru Yoshikawa, MD, PhD, Department of Otorhinolaryngology, Jikei University School of Medicine, 3-25-8 Nishi-Shimbashi, Minato-ku, Tokyo 105-8461, Japan (yoshikawa@jikei.ac.jp).Submitted for Publication:December 3, 2005; final revision received February 22, 2006; accepted March 15, 2006.Financial Disclosure:None reported.Funding/Support:This study was supported by Grants-in-Aid from the National Institute of Biomedical Innovation, Osaka, Japan.Acknowledgment:We thank Noriko Hashimoto, of the National Research Institute for Child Health and Development, for her skillful technical assistance.REFFRENCESNMaas-SzabowskiAShimotoyodomeNEFusenigKeratinocyte growth regulation in fibroblast cocultures via a double paracrine mechanism.J Cell Sci1999112(pt 12)1843185310341204ASzabowskiNMaas-SzabowskiSAndrechtc-Jun and JunB antagonistically control cytokine-regulated mesenchymal-epidermal interaction in skin.Cell200010374575511114331AEl GhalbzouriELammeMPonecCrucial role of fibroblasts in regulating epidermal morphogenesis.Cell Tissue Res200231018919912397374AEl-GhalbzouriSGibbsELammeCAVan BlitterswijkMPonecEffect of fibroblasts on epidermal regeneration.Br J Dermatol200214723024312174092SWernerHSmolaParacrine regulation of keratinocyte proliferation and differentiation.Trends Cell Biol20011114314611306276VSchillingBNegriJBujiaPSchulzEKastenbauerPossible role of interleukin 1α and interleukin 1β in the pathogenesis of cholesteatoma of the middle ear.Am J Otol1992133503551384343JWChungTHYoonDifferent production of interleukin-1α, interleukin-1β and interleukin-8 from cholesteatomatous and normal epithelium.Acta Otolaryngol19981183863919655214SYetiserBSatarNAydinExpression of epidermal growth factor, tumor necrosis factor-α, and interleukin-1α in chronic otitis media with or without cholesteatoma.Otol Neurotol20022364765212218613RHWaterstonKLindblad-TohEBirneyInitial sequencing and analysis of the human genome [published corrections appear in Nature. 2001;412:565 and Nature. 2001;411:720].Nature200140986092111237011JDrewsDrug discovery: a historical perspective.Science20002871960196410720314ALHopkinsCRGroomThe druggable genome.Nat Rev Drug Discov2002172773012209152MIidaKMatsumotoHTomitaSelective down-regulation of high-affinity IgE receptor (Fc&epsiv;RI) α-chain messenger RNA among transcriptome in cord blood–derived versus adult peripheral blood–derived cultured human mast cells.Blood2001971016102211159531DBrouty-BoyeCPottin-ClemenceauCDoucetCJasminBAzzaroneChemokines and CD40 expression in human fibroblasts.Eur J Immunol20003091491910741409CMHogaboamMLSteinhauserSWChensueSLKunkelNovel roles for chemokines and fibroblasts in interstitial fibrosis.Kidney Int199854215221599853282TPapUMuller-LadnerREGaySGayFibroblast biology.Arthritis Res2000236136711094449CCParkMJBissellMHBarcellos-HoffThe influence of the microenvironment on the malignant phenotype.Mol Med Today2000632432910904250TSilzleMKreutzMADoblerGBrockhoffRKnuechelLAKunz-SchughartTumor-associated fibroblasts recruit blood monocytes into tumor tissue.Eur J Immunol2003331311132012731056RSSmithTJSmithTMBliedenRPPhippsFibroblasts as sentinel cells.Am J Pathol19971513173229250144TSilzleGJRandolphMKreutzLAKunz-SchughartThe fibroblast: sentinel cell and local immune modulator in tumor tissue.Int J Cancer200410817318014639599MRothPRJohnsonPBorgerDysfunctional interaction of C/EBPα and the glucocorticoid receptor in asthmatic bronchial smooth-muscle cells.N Engl J Med200435156057415295049HSudhoffSDazertAMGonzalesAngiogenesis and angiogenic growth factors in middle ear cholesteatoma.Am J Otol20002179379811078065MShiwaHKojimaHMoriyamaExpression of transforming growth factor-α (TGF-α) in cholesteatoma.J Laryngol Otol19981127507549850316YTanakaMShiwaHKojimaHMiyazakiYKamideHMoriyamaA study on epidermal proliferation ability in cholesteatoma.Laryngoscope19981085375429546266SErgunXZhengBCarlsooExpression of transforming growth factor-α and epidermal growth factor receptor in middle ear cholesteatoma.Am J Otol1996173933968817015PSchulzJBujiaAHollyVShillingEKastenbauerPossible autocrine growth stimulation of cholesteatoma epithelium by transforming growth factor α.Am J Otolaryngol19931482878484481HToyodaTKomurasakiDUchidaEpireregulin: a novel epidermal growth factor with mitogenic activity for rat primary hepatocytes.J Biol Chem1995270749575007706296YShirakataTKomurasakiHToyodaEpiregulin, a novel member of the epidermal growth factor family, is an autocrine growth factor in normal human keratinocytes.J Biol Chem20002755748575310681561KHashimotoRegulation of keratinocyte function by growth factors.J Dermatol Sci200024(suppl 1)S46S5011137396DRGreavesWWangDJDairaghiCCR6, a CC chemokine receptor that interacts with macrophage inflammatory protein 3α and is highly expressed in human dendritic cells.J Exp Med19971868378449294138CAPowerDJChurchAMeyerCloning and characterization of a specific receptor for the novel CC chemokine. MIP-3α from lung dendritic cells.J Exp Med19971868258359294137ASCharbonnierNKohrgruberEKriehuberGStinglARotDMaurerMacrophage inflammatory protein 3α is involved in the constitutive trafficking of epidermal Langerhans cells.J Exp Med19991901755176810601351FLiaoRLRabinCSSmithGSharmaTBNutmanJMFarberCC-chemokine receptor 6 is expressed on diverse memory subsets of T cells and determines responsiveness to macrophage inflammatory protein 3α.J Immunol19991621861949886385JHRuthSShahraraCCParkRole of macrophage inflammatory protein-3α and its ligand CCR6 in rheumatoid arthritis.Lab Invest20038357958812695561TMatsuiTAkahoshiRNamaiSelective recruitment of CCR6-expressing cells by increased production of MIP-3α in rheumatoid arthritis.Clin Exp Immunol200112515516111472439RAkimotoRPawankarTYagiSBabaAcquired and congenital cholesteatoma: determination of tumor necrosis factor-alpha, intercellular adhesion molecule-1, interleukin-1-alpha and lymphocyte functional antigen-1 in the inflammatory process.ORL J Otorhinolaryngol Relat Spec20006225726510965261HShinodaCCHuangLocalization of intercellular adhesion molecule-1 in middle ear cholesteatoma.Eur Arch Otorhinolaryngol19952523853908562031JBujiaAHollyCKimNScanadyEKastenbauerExpression of human intercellular adhesion molecules in middle ear cholesteatoma.Am J Otolaryngol1994152712757526720

Journal

JAMA Otolaryngology - Head & Neck SurgeryAmerican Medical Association

Published: Jul 1, 2006

References