Transglutaminase (TGase) isoenzymes are involved in the process of the differentiation and cornification of keratinocytes in the epidermis. This study investigates the presence and localization of three TGase isoenzymes to elucidate the nature and differentiation status of the squamous epithelium in human aural cholesteatoma. Twenty cholesteatoma specimens were used. The presence and localization of three TGase isoenzymes were studied by reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry. mRNA expression of three TGase isoenzymes were detected in the tested cholesteatomas with variable levels. The immunohistochemical staining patterns of three TGase isoenzymes showed variations within specimens, relating to keratinizing activity. TGase K is the most abundant among three isoenzymes. Keratinizing epithelium of cholesteatoma have similar expression profiles of TGase isoenzymes with those of epidermis of the skin. Other areas, particularly those showing non-keratinizing epithelium, showed weak immunostaining of TGase E and C, suggesting its different maturation status from keratinizing epithelium. The results of this study indicate that epithelium of cholesteatoma undergoes same direction of maturation and differentiation characteristics as the epidermis of skin, evidenced by similar expressions of TGases both in mRNA level and immunohistochemistry.
Cell Differentiation ; Cholesteatoma, Middle Ear/genetics ; Cholesteatoma, Middle Ear/enzymology* ; Comparative Study ; Diagnosis, Differential ; Epidermis/enzymology ; Epithelial Cells/enzymology ; Human ; Immunohistochemistry ; Isoenzymes/metabolism ; Isoenzymes/genetics ; Keratinocytes/enzymology ; Protein-Glutamine gamma-Glutamyltransferase/metabolism* ; Protein-Glutamine gamma-Glutamyltransferase/genetics ; RNA, Messenger/metabolism ; Reverse Transcriptase Polymerase Chain Reaction

Calpain I (mu-calpain) and II (m-calpain) are well known calcium-activated neutral cysteine proteases. Many reports have shown that activation of calpain is related to cataract formation, neuronal degeneration, blood clotting, ischemic injuries, muscular dystrophy and cornified cell envelope (CE) formation. Here, we report that insoluble CE formation was reduced after treatment with calpain I inhibitor (N-acetyl-leucyl-leucyl-norleucinal) on normal human epidermal keratinocytes (NHEK), whereas serine and thiol protease inhibitors had no effect on the reduction of CE. When NHEK cells were confluent, keratinocytes were treated with various concentrations (0.5 microM-0.5 mM) of calpain I inhibitor or serine and thiol protease inhibitors under calcium induced differentiation. Insoluble CE formation was reduced about 90% in the 50 microM calpain inhibitor I treated group by day 9 of culture, whereas insoluble CE was reduced only 10% in the same condition. Interestingly TGase activity was blocked by 90% in the 0.5 mM calpain inhibitor treated group within 72 h, whereas TGase activity was retained by 80% in the 0.5 mM serine protease inhibitor treated group at 7 day treatment. Therefore it can be suggested that cysteine protease calpains might be responsible for the activation of the TGase 1 enzyme to complete insoluble CE formation during epidermal differentiation.
Calcium/pharmacology ; Calpain/metabolism* ; Calpain/antagonists & inhibitors* ; Cell Differentiation ; Dose-Response Relationship, Drug ; Epidermis/metabolism ; Human ; In Vitro ; Keratinocytes/metabolism ; Keratinocytes/enzymology ; Protease Inhibitors/pharmacology ; Protein-Glutamine gamma-Glutamyltransferase/metabolism* ; Protein-Glutamine gamma-Glutamyltransferase/antagonists & inhibitors* ; Tissue Culture