Expression of desmosomal proteins in rat keratinocytes during in vitro differentiation

The keratinocyte, the major component of the epidermis, expresses several proteins that characterize the keratinization during the differentiation. Proliferation and differentiation of cultured human keratinocytes are known to be regulated by the Ca2+ concentration in the culture medium. However, informations about the rat keratinocyte are relatively limited and their physiology is still an open question. To elucidate the characteristics of the rat keratinocyte, we established rat keratinocyte culture system and examined effects of extracellular calcium concentration on the expression of differentiation-related proteins. Keratinocytes were isolated from the newborn rat skin with 0.25% trypsin, followed by separation with a Percoll density gradient. The separated cells were grown in MCDB 153 medium containing several growth factors and Ca(2+)-free fetal bovine serum, then stimulated with Ca2+. Immunoblotting demonstrated strong expression of beta1 integrin in unstimulated cells, suggesting that the primary culture of rat keratinocytes was successfully established. Expression of desmoglein and transglutaminase was increased by Ca2+ stimulation, whereas beta1 integrin expression was decreased in response to increasing concentrations of Ca2+. These observations indicate that cultured rat keratinocytes maintain the ability to differentiate in vitro, which is similar to that of the basal keratinocytes in the epidermis.