Identification and characterization of cartilage oligomeric matrix protein as a novel pathogenic factor in keloids

Abstract

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 cm(2), 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.

Figure 1

Clinical appearance of the keloid of patient 1 and cartilage oligomeric matrix protein (COMP) mRNA and protein expression in the keloid tissues. Microarray analysis was performed using RNAs extracted from keloid-derived fibroblasts (KDFs) derived from the 23 × 7.5-cm keloid and from NDFs derived from the surrounding uninvolved skin on the chest of a 68-year-old Japanese male (patient 1 in Table 1) (A). (B) COMP mRNA amount was examined by quantitative real-time RT-PCR, as mentioned in Materials and Methods. Statistical difference was determined by the Student's t-test. **P < 0.01 (C) Skin tissue from the keloid and uninvolved (normal) skin from the five patients (patients 1 to 5 in Table 1) were homogenized in 20 mmol/L HEPES, pH 7.2, containing 1% Nonidet P-40, 0.4 M NaCl, and aprotinin and were analyzed for COMP and β-actin levels by Western blot. The lower graph shows mean ± SD of COMP/β-actin protein ratios of patients 1 to 5 (n = 5). Statistical difference was determined by the Student's t-test. *P < 0.05.

Figure 2

Immunohistochemical staining for cartilage oligomeric matrix protein (COMP) expression. COMP was positively stained in keloid lesions in the upper dermis (A) COMP staining; (D) H&E staining, ×200 magnification; and in the lower dermis (B) COMP staining; (E) H&E staining, ×200. COMP was detected on the wavy keloid-derived fibroblasts (KDFs); (C) COMP staining; (F) H&E staining, ×400.

Figure 3

Suppression of cartilage oligomeric matrix protein (COMP) by small interfering RNA (siRNA) reduces levels of extracellular type I collagen. At 96 hours after transient transfection with the COMP siRNA (A–D) or with the control siRNA (E–H) into subconfluent keloid-derived fibroblasts (KDFs), the cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. Extracellular type I collagen was then detected with an anti-type I collagen antibody and was visualized using a red fluorophore (A and E); COMP was detected with a COMP-specific antibody and visualized using a green fluorophore (B and F), and nuclei were stained with Hoechst (C and G). A merged image (A, B, and C) is shown in (D) and an image (E, F, and G) is shown in (H). Transfection of the COMP siRNA reduces levels of extracellular type I collagen and COMP (A versus E, B versus F). Nuclei of fibroblasts were nonspecifically stained in each experiment. The distribution of type I collagen and COMP overlapped closely (H). The cells were observed by confocal laser scanning microscope and all photographs are shown at ×20 magnification. (I) KDFs transiently transfected with the control or COMP siRNA were lysed in 100 μL 2 × SDS-PAGE buffer in reducing conditions and subjected to Western blot for COMP and β-actin. The COMP/β-actin ratios were determined and expressed as the relative of COMP/β-actin of the each control sample transfected with control siRNA. The lower graph shows mean ± SD of COMP/β-actin protein ratios using the KDFs from three different patients (n = 3). Statistical difference was determined by the Student's t-test. **P < 0.01.

Figure 4

Suppression of cartilage oligomeric matrix protein (COMP) by small interfering RNA (siRNA) reduces collagens in the culture medium of keloid fibroblasts and deposited on the cell surfaces. At 24 hours after transfection of the control (lanes 1 and 3) and COMP siRNA (lanes 2 and 4) into the subconfluent keloid-derived fibroblasts (KDFs) at 6-well plates, we put 1 mL fresh Dulbecco's modified Eagle's medium (DMEM) (Nissui Pharmaceutical, Tokyo, Japan) supplemented with 10% fetal calf serum containing 50 mg/mL ascorbic acid. For assay of collagen amount, at 72 hours after the medium change, the conditioned media (lanes 1 and 2) were subjected to the Sircol Collagen Assay Kit (Biocolor Ltd., Carrickfergus, UK) to measure type I to V collagens. Briefly, 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.). After centrifugation, pellets were suspended in 1 mL of the alkali compound and collagen concentration was determined by spectrophotometric absorbance at 540 nm. 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 (lanes 3 and 4). The amounts of collagens were determined using the KDFs from three different patients (n = 3) and expressed as relatives of each control transfected with the negative control siRNA (lanes 1 and 3). Statistical difference was determined by the Student's t-test. *P < 0.05, **P < 0.01, cont, negative control siRNA.

Figure 5

Induction of cartilage oligomeric matrix protein (COMP) by transforming growth factor (TGF)-β1 in keloid fibroblasts. Keloid-derived fibroblasts (KDFs) were grown in 10% fetal calf serum (FCS) Dulbecco's modified Eagle's medium (DMEM) (Nissui Pharmaceutical, Tokyo, Japan) until confluency. Then, the media were changed to DMEM without FCS for serum depletion and the cells were incubated for 24 hours. The cells were re-fed with fresh DMEM (Nissui Pharmacetical) without FCS containing 1 ng/mL human recombinant TGF-β1 or a mock solution. After incubation for 24 hours, the cells were harvested and subjected to Western blot. The COMP/β-actin ratios were determined and expressed as the relative of COMP/β-actin of each control sample (a mock treatment). The lower graph shows mean ± SD of COMP/β-actin protein ratios using the KDFs from three different patients (n = 3). Statistical difference was determined by the Student's t-test. **P < 0.01