Expression of human cystatin A by keratinocytes is positively regulated via the Ras/MEKK1/MKK7/JNK signal transduction pathway but negatively regulated via the Ras/Raf-1/MEK1/ERK pathway.
Cystatin A, a cysteine proteinase inhibitor, is a cornified cell envelope constituent expressed in the upper epidermis. We previously reported that a potent protein kinase C activator, 12-O-tetradecanoylphorbol-13-acetate, increases human cystatin A expression by the activation of AP-1 proteins. Here, we delineate the signaling cascade responsible for this regulation. Co-transfection of the cystatin A promoter into normal human keratinocytes together with a dominant active form of ras increased the promoter activity by 3-fold. In contrast, a dominant negative form of ras suppressed basal cystatin A promoter activity. Further analyses disclosed that transfection of dominant negative forms of raf-1, MEK1, ERK1, ERK2, or wild-type MEKK1 all increased cystatin A promoter activity in normal human keratinocytes, whereas wild-type raf-1, ERK1, ERK2, or dominant negative forms of MEKK1, MKK7, or JNK1 suppressed the promoter activity. The increased or decreased promoter activity reflected the expression of cystatin A on mRNA and protein levels. These effects were not observed when a cystatin A promoter with a T2 (-272 to -278) deletion was used. In contrast, transfection of dominant negative forms of MKK3, MKK4, or p38 did not affect cystatin A promoter activity. Immunohistochemical analyses revealed that phosphorylated active extracellular signal-regulated kinases and c-Jun N-terminal kinase were expressed in the nuclei of basal cells and cells in the suprabasal-granular cell layer, respectively. These results indicate that the expression of cystatin A is regulated via mitogen-activated protein kinase pathways positively by Ras/MEKK1/MKK7/JNK and negatively by Ras/Raf/MEK1/ERK.