Keratin 4 upregulation by retinoic acid in vivo: A sensitive marker for retinoid bioactivity in human epidermis

Retinoids affect keratinocyte differentiation and modulate the expression o f many epidermal proteins, among them cellular retinoic acid-binding protei n II and the family of cytokeratins. The upregulation of the former protein is a well-known phenomenon, whereas the retinoid-induced regulation of epi dermal keratin expression is more complex and only partially understood. We studied the effect of topical retinoids on the expression in healthy skin of cellular retinoic acid-binding protein II, tazarotene-induced genes 1 an d 2, several epidermal keratins (K1, K2e, and K10), and two mucous keratins (K4 and K13) known to appear in epidermis under certain abnormal condition s. Reverse transcription-polymerase chain reaction experiments showed that the K4 expression was the one most overtly induced by 2 wk of open treatmen t with 0.05% of retinoic acid and tazarotene. Using real-time quantitative polymerase chain reaction (TaqMan) and normalization of the mRNA values to beta-actin, the increase in K4 was found to be 100-1000-fold. In comparison , the expression of K13 and cellular retinoic acid-binding protein II was i ncreased 10-50-fold, the K1 and K10 mRNA levels remained unchanged, and the K2e level decreased by a factor of 100-1000. In parallel biopsies, immunoh istochemistry showed no change in K1, K2e, or K10 staining, but a strong de novo appearance of K4 in the granular layer after retinoid treatment. In a separate study, occlusive application of 0.025% retinoic acid in four heal thy subjects produced a maximal K4 mRNA signal after 48 h and strong K4 sta ining after 80 h. Finally, a dose-response study showed that the de novo ap pearance of K4 can be utilized as a sensitive test for retinoid bioactivity in epidermis in vivo.