Identification and Characterization of Cartilage Oligomeric Matrix Protein as a Novel Pathogenic Factor in Keloids
2011; Elsevier BV; Volume: 179; Issue: 4 Linguagem: Inglês
10.1016/j.ajpath.2011.06.034
ISSN1525-2191
AutoresShigeki Inui, Fumie Shono, Takeshi Nakajima, Ko Hosokawa, Satoshi Itami,
Tópico(s)Collagen: Extraction and Characterization
ResumoTo elucidate pathogenic molecules in keloids, microarray analysis was performed using RNAs extracted from keloid-derived fibroblasts and normal skin-derived fibroblasts from the same patient with a typical keloid. Among 11 up-regulated extracellular matrix genes, cartilage oligomeric matrix protein (COMP) was most prominently increased. Up-regulation of COMP mRNA and protein was confirmed in the keloid tissue by quantitative RT-PCR and Western blot. Using immunohistochemistry, we compared 15 keloids and 6 control normal tissues using a COMP-specific antibody and found that COMP stained positively in 10 keloids (66.7%), whereas no staining was observed in normal tissues, demonstrating the ectopic expression of COMP in keloids. Comparing keloids smaller or larger than 10 cm2, the larger keloids were significantly more intensely stained with the COMP-specific antibody. Because COMP reportedly accelerates collagen type I fibril assembly, we examined whether extracellular type I collagen deposition is altered by silencing COMP mRNA by small interfering RNA (siRNA). Immunocytochemistry showed at 96 hours after transfection with COMP siRNA that the extracellular deposition of type I collagen was decreased compared to that observed with control siRNA. Further, COMP knockdown decreased amount collagens type I to V in the medium and on the cell surfaces. Our data suggest that COMP facilitates keloid formation by accelerating collagen deposition, thus providing a new therapeutic target. To elucidate pathogenic molecules in keloids, microarray analysis was performed using RNAs extracted from keloid-derived fibroblasts and normal skin-derived fibroblasts from the same patient with a typical keloid. Among 11 up-regulated extracellular matrix genes, cartilage oligomeric matrix protein (COMP) was most prominently increased. Up-regulation of COMP mRNA and protein was confirmed in the keloid tissue by quantitative RT-PCR and Western blot. Using immunohistochemistry, we compared 15 keloids and 6 control normal tissues using a COMP-specific antibody and found that COMP stained positively in 10 keloids (66.7%), whereas no staining was observed in normal tissues, demonstrating the ectopic expression of COMP in keloids. Comparing keloids smaller or larger than 10 cm2, the larger keloids were significantly more intensely stained with the COMP-specific antibody. Because COMP reportedly accelerates collagen type I fibril assembly, we examined whether extracellular type I collagen deposition is altered by silencing COMP mRNA by small interfering RNA (siRNA). Immunocytochemistry showed at 96 hours after transfection with COMP siRNA that the extracellular deposition of type I collagen was decreased compared to that observed with control siRNA. Further, COMP knockdown decreased amount collagens type I to V in the medium and on the cell surfaces. Our data suggest that COMP facilitates keloid formation by accelerating collagen deposition, thus providing a new therapeutic target. Keloids are raised skin lesions with redness, pain, and itching, often caused by trauma, burns, or surgical invasion. They grow larger beyond the size of the original wounds, causing not only esthetic but also mental distress.1Brown B.C. McKenna S.P. Siddhi K. McGrouther D.A. Bayat A. The hidden cost of skin scars: quality of life after skin scarring.J Plast Reconstr Aesthet Surg. 2008; 61: 1049-1058Abstract Full Text Full Text PDF PubMed Scopus (318) Google Scholar Keloid pathogenesis basically involves excessive wound healing with prolonged inflammation. Histopathologically, the infiltration of inflammatory cells, as well as irregular and excess accumulations of extracellular matrix components (eg, collagen, fibronectin, elastin, and proteoglycans) are observed. The molecular aberrant mechanisms in keloids can be categorized into three groups: i) extracellular matrix proteins and their deposition and degradation, ii) cytokines and growth factors, and iii) apoptotic pathways.2Shih B. Garside E. McGrouther D.A. Bayat A. Molecular dissection of abnormal wound healing processes resulting in keloid disease.Wound Repair Regen. 2010; 18: 139-153Crossref PubMed Scopus (173) Google Scholar To explore keloid pathogenesis, differences between keloid-derived fibroblasts (KDFs) and normal skin-derived fibroblasts (NDFs) have been investigated. KDFs, showing reduced growth-factor requirement,3Russell S.B. Trupin K.M. Rodriguez-Eaton S. Russell J.D. Trupin J.S. Reduced growth-factor requirement of keloid-derived fibroblasts may account for tumor growth.Proc Natl Acad Sci USA. 1988; 85: 587-591Crossref PubMed Scopus (115) Google Scholar proliferate and migrate at a faster rate than NDFs.4Witt E. Maliri A. McGrouther D.A. Bayat A. RAC activity in keloid disease: comparative analysis of fibroblasts from margin of keloid to its surrounding normal skin.Eplasty. 2008; 8: e19PubMed Google Scholar Moreover, KDFs are resistant to corticosteroid in terms of growth response 5Russell J.D. Witt W.S. Cell size and growth characteristics of cultured fibroblasts isolated from normal and keloid tissue.Plast Reconstr Surg. 1976; 57: 207-212Crossref PubMed Scopus (54) Google Scholar and further down-regulation of types I, III, and V collagen6Russell J.D. Russell S.B. Trupin K.M. Differential effects of hydrocortisone on both growth and collagen metabolism of human fibroblasts from normal and keloid tissue.J Cell Physiol. 1978; 97: 221-229Crossref PubMed Scopus (73) Google Scholar, 7Russell S.B. Trupin J.S. Myers J.C. Broquist A.H. Smith J.C. Myles M.E. Russell J.D. Differential glucocorticoid regulation of collagen mRNAs in human dermal fibroblasts Keloid-derived and fetal fibroblasts are refractory to down-regulation.J Biol Chem. 1989; 264: 13730-13735Abstract Full Text PDF PubMed Google Scholar, elastin,8Russell S.B. Trupin J.S. Kennedy R.Z. Russell J.D. Davidson J.M. Glucocorticoid regulation of elastin synthesis in human fibroblasts: down-regulation in fibroblasts from normal dermis but not from keloids.J Invest Dermatol. 1995; 104: 241-245Crossref PubMed Scopus (47) Google Scholar connective tissue growth factor, and insulin-like growth factor-binding protein 39Smith J.C. Boone B.E. Opalenik S.R. Williams S.M. Russell S.B. Gene profiling of keloid fibroblasts shows altered expression in multiple fibrosis-associated pathways.J Invest Dermatol. 2008; 128: 1298-1310Crossref PubMed Scopus (123) Google Scholar gene expression. In addition, KDFs are reportedly refractory to phorbol esters and prostaglandin E2.10Myles M.E. Russell J.D. Trupin J.S. Smith J.C. Russell S.B. Keloid fibroblasts are refractory to inhibition of DNA synthesis by phorbol esters Altered response is accompanied by reduced sensitivity to prostaglandin E2 and altered down-regulation of phorbol ester binding sites.J Biol Chem. 1992; 267: 9014-9020PubMed Google Scholar These basic findings recapitulate well clinical resistance of keloids to various treatments.11Mustoe T.A. Cooter R.D. Gold M.H. Hobbs F.D. Ramelet A.A. Shakespeare P.G. Stella M. Teot L. Wood F.M. Ziegler U.E. International clinical recommendations on scar management.Plast Reconstr Surg. 2002; 110: 560-571Crossref PubMed Scopus (784) Google Scholar, 12Ogawa R. The most current algorithms for the treatment and prevention of hypertrophic scars and keloids.Plast Reconstr Surg. 2010; 125: 557-568Crossref PubMed Scopus (249) Google Scholar Therefore, we consider that research using KDFs and NDFs is a powerful strategy to characterize the pathological mechanism of keloids as a clue to provide new targets for treatment. To explore novel target molecules, microarray analyses of keloids have been performed and reported the up-regulated expression of many genes related to the cell cycle,13Satish L. Lyons-Weiler J. Hebda P.A. Wells A. Gene expression patterns in isolated keloid fibroblasts.Wound Repair Regen. 2006; 14: 463-470Crossref PubMed Scopus (63) Google Scholar intercellular signaling14Nassiri M. Woolery-Lloyd H. Ramos S. Jacob S.E. Gugic D. Viciana A. Romanelli P. Elgart G. Berman B. Vincek V. Gene expression profiling reveals alteration of caspase 6 and 14 transcripts in normal skin of keloid-prone patients.Arch Dermatol Res. 2009; 301: 183-188Crossref PubMed Scopus (19) Google Scholar and the extracellular matrix,9Smith J.C. Boone B.E. Opalenik S.R. Williams S.M. Russell S.B. Gene profiling of keloid fibroblasts shows altered expression in multiple fibrosis-associated pathways.J Invest Dermatol. 2008; 128: 1298-1310Crossref PubMed Scopus (123) Google Scholar, 15Naitoh M. Kubota H. Ikeda M. Tanaka T. Shirane H. Suzuki S. Nagata K. Gene expression in human keloids is altered from dermal to chondrocytic and osteogenic lineage.Genes Cells. 2005; 10: 1081-1091Crossref PubMed Scopus (70) Google Scholar, 16Seifert O. Bayat A. Geffers R. Dienus K. Buer J. Lofgren S. Matussek A. Identification of unique gene expression patterns within different lesional sites of keloids.Wound Repair Regen. 2008; 16: 254-265Crossref PubMed Scopus (82) Google Scholar and the down-regulated expression apoptosis-related genes.14Nassiri M. Woolery-Lloyd H. Ramos S. Jacob S.E. Gugic D. Viciana A. Romanelli P. Elgart G. Berman B. Vincek V. Gene expression profiling reveals alteration of caspase 6 and 14 transcripts in normal skin of keloid-prone patients.Arch Dermatol Res. 2009; 301: 183-188Crossref PubMed Scopus (19) Google Scholar In this study, using microarray analysis of KDFs and NDFs from the same patient, we identify and characterize a new potentially pathogenic gene, which encodes cartilage oligomeric matrix protein (COMP), because most previous studies used keloid and normal fibroblasts or tissues from different persons, our methodology may avoid individual difference and bias. COMP, also referred to as thrombospondin 5, is a 524-kDa homopentameric, noncollagenous, extracellular matrix glycoprotein, which is found in cartilage, tendons, and ligaments and the growth plate.17Hedbom E. Antonsson P. Hjerpe A. Aeschlimann D. Paulsson M. Rosa-Pimentel E. Sommarin Y. Wendel M. Oldberg A. Heinegard D. Cartilage matrix proteins An acidic oligomeric protein (COMP) detected only in cartilage.J Biol Chem. 1992; 267: 6132-6136Abstract Full Text PDF PubMed Google Scholar Its carboxyterminal globular domain binds to type I, II, and IX collagens, fibronectin,18Rosenberg K. Olsson H. Morgelin M. Heinegard D. Cartilage oligomeric matrix protein shows high affinity zinc-dependent interaction with triple helical collagen.J Biol Chem. 1998; 273: 20397-20403Crossref PubMed Scopus (295) Google Scholar, 19Holden P. Meadows R.S. Chapman K.L. Grant M.E. Kadler K.E. Briggs M.D. Cartilage oligomeric matrix protein interacts with type IX collagen, and disruptions to these interactions identify a pathogenetic mechanism in a bone dysplasia family.J Biol Chem. 2001; 276: 6046-6055Crossref PubMed Scopus (175) Google Scholar, 20Thur J. Rosenberg K. Nitsche D.P. Pihlajamaa T. Ala-Kokko L. Heinegard D. Paulsson M. Maurer P. Mutations in cartilage oligomeric matrix protein causing pseudoachondroplasia and multiple epiphyseal dysplasia affect binding of calcium and collagen I, II, and IX.J Biol Chem. 2001; 276: 6083-6092Crossref PubMed Scopus (184) Google Scholar and aggrecan,21Chen F.H. Herndon M.E. Patel N. Hecht J.T. Tuan R.S. Lawler J. Interaction of cartilage oligomeric matrix protein/thrombospondin 5 with aggrecan.J Biol Chem. 2007; 282: 24591-24598Crossref PubMed Scopus (109) Google Scholar and thereby accelerates fibrillogenesis through the promotion of matrix component assembly.22Halasz K. Kassner A. Morgelin M. Heinegard D. COMP acts as a catalyst in collagen fibrillogenesis.J Biol Chem. 2007; 282: 31166-31173Crossref PubMed Scopus (246) Google Scholar In addition, the coiled-coil domain stores and delivers hydrophobic ligands, such as retinoid and vitamin D.23Guo Y. Bozic D. Malashkevich V.N. Kammerer R.A. Schulthess T. Engel J. All-trans retinol, vitamin D and other hydrophobic compounds bind in the axial pore of the five-stranded coiled-coil domain of cartilage oligomeric matrix protein.EMBO J. 1998; 17: 5265-5272Crossref PubMed Scopus (64) Google Scholar, 24Ozbek S. Engel J. Stetefeld J. Storage function of cartilage oligomeric matrix protein: the crystal structure of the coiled-coil domain in complex with vitamin D(3).EMBO J. 2002; 21: 5960-5968Crossref PubMed Scopus (54) Google Scholar These studies suggest that COMP potentially exerts a wide range of biological functions. Furthermore, recent evidences are accumulating that COMP is both a pathogenic factor and biomarker in scleroderma,25Farina G. Lafyatis D. Lemaire R. Lafyatis R. A four-gene biomarker predicts skin disease in patients with diffuse cutaneous systemic sclerosis.Arthritis Rheum. 2010; 62: 580-588Crossref PubMed Scopus (127) Google Scholar, 26Farina G. Lemaire R. Korn J.H. Widom R.L. Cartilage oligomeric matrix protein is overexpressed by scleroderma dermal fibroblasts.Matrix Biol. 2006; 25: 213-222Crossref PubMed Scopus (56) Google Scholar, 27Farina G. Lemaire R. Pancari P. Bayle J. Widom R.L. Lafyatis R. Cartilage oligomeric matrix protein expression in systemic sclerosis reveals heterogeneity of dermal fibroblast responses to transforming growth factor beta.Ann Rheum Dis. 2009; 68: 435-441Crossref PubMed Scopus (51) Google Scholar, 28Hesselstrand R. Kassner A. Heinegard D. Saxne T. COMP: a candidate molecule in the pathogenesis of systemic sclerosis with a potential as a disease marker.Ann Rheum Dis. 2008; 67: 1242-1248Crossref PubMed Scopus (59) Google Scholar, 29Skoumal M. Haberhauer G. Feyertag J. Kittl E.M. Bauer K. Dunky A. Serum levels of cartilage oligomeric matrix protein are elevated in rheumatoid arthritis, but not in inflammatory rheumatic diseases such as psoriatic arthritis, reactive arthritis Raynaud's syndrome, scleroderma, systemic lupus erythematosus, vasculitis and Sjogren's syndrome.Arthritis Res Ther. 2004; 6: 73-74Crossref PubMed Google Scholar, 30Yamamoto M. Takahashi H. Suzuki C. Naishiro Y. Yamamoto H. Imai K. Shinomura Y. Cartilage oligomeric matrix protein in systemic sclerosis.Rheumatology (Oxford). 2007; 46: 1858-1859Crossref PubMed Scopus (7) Google Scholar indicating that this molecule can be involved in pathogenesis of other fibrosing diseases. Additionally, existence of COMP in horse scar was previously reported,31Smith R.K. Zunino L. Webbon P.M. Heinegard D. The distribution of cartilage oligomeric matrix protein (COMP) in tendon and its variation with tendon site, age and load.Matrix Biol. 1997; 16: 255-271Crossref PubMed Scopus (180) Google Scholar but the role of COMP in scar or keloid is still unknown. These results prompted us to study the potential pathogenic role of COMP in keloid formation. Keloid tissues were obtained from 8 males and 7 females with a mean age of 50 years (range, 15 to 80 years) (Table 1). Normal skin as control samples was obtained from four males and two females, with a mean age of 59.2 years (range, 2 to 80 years). Among them, three were the uninvolved normal skin specimens around keloids from patients 1, 2, and 3 in Table 1. The three other normal samples were obtained from nonkeloid patients who underwent benign skin tumor excisions (two, inguinal; one abdomen).Table 1Clinical Characteristics of the Patients from Whom the Samples Were ObtainedPatient no.Age/SexSite of lesion>10 cm2 168 MChest 260 MChest 380 FNeck 471 MInguinal 580 FRetroauricular 50%, ++. Cultured human fibroblasts were grown on Lab-Tek chamber slides (Electron Microscopy Sciences, Hatfield, PA) and at subconfluency the cells were transiently transfected with control and COMP siRNA as mentioned as follows. Twenty-four hours later, the cells were re-fed with fresh 10% FCS DMEM containing 50 mg/mL ascorbic acid. After incubation for 72 hours, the cells were fixed in 4% paraformaldehyde for 10 minutes at room temperature and permeabilized with 0.1% Triton X-100. COMP was detected by incubation with rat anti-human COMP IgG (1:50) (sc-59941, Santa Cruz Biotechnology, Santa Cruz, CA) overnight at 4°C, followed by Alexa fluor 488 conjugated goat anti-rat IgG. Extracellular type I collagen was detected with rabbit anti-human collagen type 1 alpha 1 (1:50) (sc-28657, Santa Cruz Biotechnology) overnight at 4°C, followed by Alexa fluor 594 conjugated donkey anti-rabbit IgG. Nuclei were stained with Hoechst33342. The cells were observed by confocal laser scanning microscope FluoView 1000-D (Olympus, Tokyo, Japan). COMP siRNA and a negative control siRNA were prepared and provided by Ambion 32Gagarina V. Carlberg A.L. Pereira-Mouries L. Hall D.J. Cartilage oligomeric matrix protein protects cells against death by elevating members of the IAP family of survival proteins.J Biol Chem. 2008; 283: 648-659Crossref PubMed Scopus (35) Google Scholar. The nucleotide sequences of sense and antisense COMP siRNAs used were: 5′-GGAGGACUCAGACCACGAUdTdT-3′ and 5′-AUCGUGGUCUGAGUCCUCCdTdG-3′. KDFs were transiently transfected with 100 nmol/L siRNA against COMP using Amaxa nucleofection kits (Lonza Cologne AG, Cologne, Germany) for adult human dermal fibroblasts according to the manufacturer's protocol. After the transfection, cells were cultured in medium. In the preliminary experiments, we confirmed that successful transfection of siRNA is constantly obtained by co-transfection of expression vector of GFP. Total RNAs were isolated from keloid and uninvolved normal tissue from patient 1 in Table 1 using the acid guanidium thiocyanate-phenol-chloroform method and the purity of the extracted RNA was calculated with the optical densities at 260 and 280 nm. The cDNA was reverse-transcribed from 1 mg total RNA from each sample using M-MLV Reverse Transcriptase (Invitrogen, Carlsbad, CA) according to the manufacturer's protocol. The oligonucleotide primers used were as follows: COMP designed by primer 3 software-sense primer 5′-TGGGCCCGCAGATGCTTC-3′ and antisense primer 5′-CCGTCTTCCGTCTGGATG-3′, glyceraldehyde-3-phosphate dehydrogenase (G3PDH) as an internal control-sense primer 5′-CCCATCACCATCTTCCAG-3′ and antisense primer 5′- CCTGCTTCACCACCTTCT-3′. Real-time PCR was conducted using Power SYBR Green PCR Master Mix (Applied Biosystems, Carlsbad, CA), according to the manufacturer's instructions. The final PCR mixture contained 0.5 μL each of forward and reverse primers (final concentration of each, 500 nmol/L), 25 μL of Power SYBR Green PCR Master Mix (Applied Biosystems), and 1 μL of sample (equivalent to 50 ng RNA). Real-time PCR was performed with an ABI Prism 7900 HT machine (Applied Biosystems) and universal cycling conditions (2 minutes at 50°C, 10 minutes at 95°C, 40 cycles of 15 seconds at 95°C, and 1 minute at 60°C). Cycle threshold values were determined by automated threshold analysis with ABI Prism version 1.0 software (Applied Biosystems). The amplification efficiencies were determined by serial dilution and calculated as E = exp-−1/m, where E is the amplification efficiency and m is the slope of the dilution curve. Dissociation curves were recorded after each run. Tissues from keloid and normal skin were thoroughly homogenized in 20 mmol/L HEPES, pH 7.2, containing 1% Nonidet P-40, 0.4 M NaCl and aprotinin and were centrifuged to extract the proteins into supernatants. The protein samples were mixed with 2 × SDS-PAGE buffer, heat denatured with a reducing agent and then loaded onto 4 to 12% Tris-glycine gels. Cultured cells were lysed in 100 μL 2 × SDS-PAGE buffer in reducing conditions. All samples (5 mg/lane) were heated and loaded on 7.5% Tris-glycine gels. After electrophoresis, proteins on the gels were transferred to nitrocellulose membranes. The membranes were briefly stained with Ponceau S, photographed, and blocked in 5% milk in TBST buffer for 1 hour and then incubated for 1 hour with antibodies against COMP (1:100) (sc-59941, Santa Cruz Biotechnology) or β-actin, and then washed 3 times for 5 minutes each with TBST, followed by a 1 hour incubation with horseradish peroxidase conjugated secondary antibody. All membranes were developed using the ECL Plus Western Blotting Detection System (GE Healthcare, Buckinghamshire, England). Quantification and densitometric analysis was performed using Image J software (National Institutes of Health, Bethesda, MD). At 24 hours after transfection of the control and CONP siRNA into the subconfluent KDFs at 6-well plates, we put 1 mL fresh DMEM supplemented with 10% FCS containing 50 mg/mL ascorbic acid. For measuring amount of collagen, at 72 hours after the medium change, the conditioned media were harvested. We used the Sircol Collagen Assay Kit (Biocolor Ltd., Carrickfergus, UK) to measure type I to V collagens according to the manufacturer's instructions. The 1 mL of conditioned media were mixed with 200 mL isolation and concentration reagent in the kit and incubated at 4°C overnight. After centrifugation, pellets were re-dissolved in 1 mL of Sircol dye reagent (Biocolor Ltd.) which specifically binds to collagens. After centrifugation, pellets were suspended in 1 mL of the alkali compound included in the kit and collagen concentration was then determined by spectrophotometric absorbance at 540 nm as compared with a standard curve. After aspiration of the culture supernatant, extracellular matrix formed on the cells was treated with 1 mL 0.5 M cold acetic acid for 24 hours and the amount of collagens in the extract was determined with the Sircol Collagen Assay Kit (Biocolor Ltd) as previously mentioned. The amounts of collagens were determined and expressed as relatives of each control transfected with the negative control siRNA. KDFs were grown in 10% FCS DMEM until subconfluency. Then the media were changed to DMEM without FCS for serum depletion. At 24 hours later, the cells were re-fed with fresh DMEM without FCS containing 1 ng/mL human recombinant transforming growth factor (TGF)-β1 (240B, R&D Systems, Minneapolis, MN) or a mock solution. After incubation for 24 hours, the cells were harvested and subjected to Western blot as previously mentioned. Statistical differences of immunohistochemical staining intensity were determined by the Mann-Whitney U-test; differences with P < 0.05 are considered significant. For other experiments, statistical differences were determined by Student's t-test; differences with P < 0.05 are considered significant. Initially we tried to use RNA from keloid and normal tissues for microarray analyses but we had difficulty producing high quality samples. Moreover, as previously mentioned, the prior reports demonstrated that the differences of KDFs and NDFs recapitulate well in vivo keloid pathogenesis 3 to 10. Therefore, to elucidate potentially pathogenic genes or molecules in keloids, microarray analyses were performed using RNAs extracted from KDFs and NDFs from the same patient (patient 1 in Table 1) with a clinically typical keloid on the chest (Figure 1A). The normal tissue was obtained from the surrounding normal skin. Among the 38,500 genes on the array, 374 were up-regulated (fold value, >2) and 758 were down-regulated (fold value, <0.5) in KDFs compared with NDFs. The most significantly up-regulated 31 genes are listed in Table 2. The differentially expressed genes were classified by molecular functions, possibly related to wound healing and keloid growth (Table 3). More than 10 genes were up-regulated in the molecular functions in the following gene ontology categories: extracellular matrix constituents, growth factors and their receptors, transcription factors and proteins binding with DNA, and apoptosis. In contrast, genes down-regulated in molecular functions overlapped in two categories: growth factors and their receptors and transcription factors and proteins binding with DNA. Shih et al 33Shih B. McGrouther D.A. Bayat A. Identification of novel keloid biomarkers through profiling of tissue biopsies versus cell cultures in keloid margin specimens compared to adjacent normal skin.Eplasty. 2010; 10: e24PubMed Google Scholar summarized previous studies and their results of microarray analyses for keloid tissues and KDFs compared with normal tissues and NDFs. As a result, they bioinformatically selected 19 valid candidate genes. Our microarray data were in agreement on the increase of aggrecan, asporin, pleiotrophin, serpin F1, and LIM and senescent cell antigen-like domains 2, and the decrease of epidermal growth factor receptor. Eleven extracellular matrix genes were up-regulated, and of those, COMP was most prominently up-regulated (Table 4).Table 2Genes Showing the Greatest Increase in Expression in KDFs Compared to NDFs in Microarray AnalysisGene nameGene symbolFold changeUniGene IDMRNA; cDNA DKFZp686A0837 (from clone DKFZp686A0837)—78.793242Hs.638566Cartilage oligomeric matrix proteinCOMP48.50293Hs.1584Secreted frizzled-related protein 2SFRP242.224253Hs.481022v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homologKIT39.396621Hs.479754C-type lectin domain family 12, member ACLEC12A36.758347Hs.190519Fibroblast growth factor receptor 2FGFR234.296751Hs.533683Testis expressed 9TEX925.992077Hs.511476Retinoic acid receptor responder (tazarotene induced) 2RARRES225.992077Hs.647064Transmembrane protein 16ATMEM16A22.627417Hs.503074Transcribed locus—19.698311Hs.662908Armadillo repeat containing, X-linked 4ARMCX418.379174Hs.399873Death-associated protein kinase 1DAPK117.148375Hs.380277cDNA FLJ38181 fis, clone FCBBF1000125—16Hs.143134cDNA clone IMAGE:4830514—16Hs.570820Colony stimulating factor 2 receptor, beta, low-affinity (granulocyte-macrophage)CSF2RB16Hs.592192ChondrolectinCHODL14.928528Hs.283725DermatopontinDPT14.928528Hs.80552Insulin-like growth factor 2 (somatomedin A) /// insulin- insulin-like growth factor 2IGF2 /// INS-IGF213.928809Hs.523414Gap junction protein, alpha 5, 40kDaGJA512.996038Hs.447968Inter-alpha (globulin) inhibitor H5ITIH512.996038Hs.498586cDNA FLJ30757 fis, clone FEBRA2000468—12.996038Hs.662237G protein–coupled receptor 37 (endothelin receptor type B-like)GPR3712.125733Hs.406094Unc-5 homolog B (C. elegans)UNC5B12.125733Hs.585457Nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor-like 1NFKBIL111.313708Hs.2764Hypothetical gene supported by BC008048LOC44093410.556063Hs.238964Dopamine receptor D1DRD110.556063Hs.2624Signal-induced proliferation-associated 1 like 2SIPA1L210.556063Hs.268774Deleted in bladder cancer 1DBC110.556063Hs.532316Calcium/calmodulin-dependent protein kinase ID /// Hypothetical protein LOC283070CAMK1D /// LOC28307010.55606
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