epidermal-growth-factor and Cholesteatoma

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.

Topics: Base Sequence; Cell Line, Tumor; Cell Proliferation; Cholesteatoma; Coculture Techniques; DNA Primers; Ear, Middle; Epidermal Growth Factor; Epiregulin; Epithelial Cells; Humans; Real-Time Polymerase Chain Reaction; RNA, Messenger

The localization of epidermal growth factor (EGF) in human cholesteatoma tissue was examined immunohistochemically, using sections of formalin-fixed, paraffin-embedded tissue with avidin-biotin peroxidase complex method. Thirty-three cases of active cholesteatoma which had abundant granulation and debris were studied. Out of 33 cases, EGF was positive in 32 cases (97%) in the epidermis, 30 cases (91%) in the fibroblast, and 21 cases (64%) in the endothelial cells. In 9 cases of inactive cholesteatoma, on the other hand, EGF was positive in 5 cases (56%) in the epidermis, no case in the fibroblast, and a case (11%) in the endothelial cells. Active cholesteatoma had higher immunoreactivity in the epidermis than inactive cholesteatoma. This suggests that the activity of cholesteatoma is indicated by the immunoreactivity of EGF. Difference of EGF immunoreactivity between active and inactive cholesteatoma was bigger in the fibroblast in the subcutaneous tissue of cholesteatoma than in the epidermis. This gives the reason that the activity of cholesteatoma exists in the subcutaneous tissue. These results suggest that EGF plays an important role in accelerating the growth of cholesteatoma.

Topics: Adolescent; Adult; Aged; Child; Child, Preschool; Cholesteatoma; Ear Diseases; Ear, Middle; Endothelium, Vascular; Epidermal Growth Factor; Epidermis; Fibroblasts; Humans; Immunoenzyme Techniques; Middle Aged

The localization of epidermal growth factor (EGF) in human cholesteatoma tissue, normal ear drum and external auditory canal skin was examined immunohistochemically, using avidin-biotin peroxidase complex method. Bouin-fixed tissue was stained for investigation of horny layer in the epidermis, because fixation in Bouin's solution provides better preservation of the antigen. In the horny layer of cholesteatoma tissue, 19 out of 24 cases had EGF-positive immunoreactivity (79%). In 2 cases of normal external auditory canal skin, 4 cases of normal ear drum and a case of postauricular skin, no EGF-immunoreactivity was revealed in the horny layer. EGF was assayed in the debris of cholesteatoma and the horny layer of the normal bony external canal with dot blot immunoassay. EGF content of the debris was higher than that of the horny layer of normal skin. The result of the first report suggests the activity of cholesteatoma exists in the subcutaneous tissue (see the previous paper). In this report EGF content of cholesteatoma in the horny layer was found higher than that of normal external skin. This result demonstrates that EGF in the horny layer plays an important role in accelerating the growth and bony destruction in cholesteatoma. To summarize these two reports, the following conclusion was reached. In the epidermis EGF content is equal in cholesteatoma and normal skin. But in the subcutaneous tissue and the horny layer EGF content of cholesteatoma is higher than that of normal skin. EGF in situ may be strongly related to the growth and bony destruction of cholesteatoma.

Topics: Adolescent; Adult; Aged; Child; Cholesteatoma; Ear Diseases; Ear, Middle; Epidermal Growth Factor; Epidermis; Humans; Immunoenzyme Techniques; Middle Aged