title: Cholesteatoma fibroblasts promote epithelial cell proliferation through overexpression of epiregulin.
abstract: To investigate whether keratinocytes proliferate in response to epiregulin produced by subepithelial fibroblasts derived from middle ear cholesteatoma. Tissue samples were obtained from patients undergoing tympanoplasty. The quantitative polymerase chain reaction and immunohistochemistry were performed to examine epiregulin expression and localization in cholesteatoma tissues and retroauricular skin tissues. Fibroblasts were cultured from cholesteatoma tissues and from normal retroauricular skin. These fibroblasts were used as feeder cells for culture with a human keratinocyte cell line (PHK16-0b). To investigate the role of epiregulin in colony formation by PHK16-0b cells, epiregulin mRNA expression was knocked down in fibroblasts by using short interfering RNA and epiregulin protein was blocked with a neutralizing antibody. Epiregulin mRNA expression was significantly elevated in cholesteatoma tissues compared with that in normal retroauricular skin. Staining for epiregulin was more intense in the epithelial cells and subepithelial fibroblasts of cholesteatoma tissues than in retroauricular skin. When PHK16-0b cells were cultured with cholesteatoma fibroblasts, their colony-forming efficiency was 50% higher than when these cells were cultured with normal skin fibroblasts. Also, knockdown of epiregulin mRNA in cholesteatoma fibroblasts led to greater suppression of colony formation than knockdown in skin fibroblasts. Furthermore, the colony-forming efficiency of PHK16-0b cells was significantly reduced after treatment with an epiregulin neutralizing antibody in co-culture with cholesteatoma fibroblasts, but not in co-culture with skin fibroblasts. These results suggest that keratinocyte hyperproliferation in cholesteatoma is promoted through overexpression of epiregulin by subepithelial fibroblasts via epithelial-mesenchymal interactions, which may play a crucial role in the pathogenesis of middle ear cholesteatoma.
authors.affiliation: Department of Otorhinolaryngology, Toho University School of Medicine, Meguro-ku, Tokyo, Japan. mamoru.yoshikawa@med.toho-u.ac.jp
authors.forename: Mamoru
authors.initials: M
authors.lastname: Yoshikawa
authors.forename: Hiromi
authors.initials: H
authors.lastname: Kojima
authors.forename: Yuichiro
authors.initials: Y
authors.lastname: Yaguchi
authors.forename: Naoko
authors.initials: N
authors.lastname: Okada
authors.forename: Hirohisa
authors.initials: H
authors.lastname: Saito
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Moriyama
mesh_terms: D001483:Base Sequence; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D002781:Cholesteatoma / Q000378:metabolism / Q000473:pathology*; D018920:Coculture Techniques; D017931:DNA Primers; D004432:Ear, Middle / Q000378:metabolism; D004815:Epidermal Growth Factor / Q000235:genetics / Q000378:metabolism*; D066260:Epiregulin; D004847:Epithelial Cells / Q000378:metabolism / Q000473:pathology; D006801:Humans; D012333:RNA, Messenger / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction
chemical_list: D017931:DNA Primers; C587880:EREG protein, human; D066260:Epiregulin; D012333:RNA, Messenger; D004815:Epidermal Growth Factor