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

Abstract

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.

Dermokine‐β impairs ERK signaling through direct binding to GRP78

“…Future studies should focus on identification of such transcriptional repressors that bind to the DNA sequence within dermokine promoter. A previous study has shown that the phosphorylated forms of ERKs were present in the basal cell layer, but totally disappeared in the granular layer of the normal human epidermis [20]. In the present study, we showed that exposure of human keratinocytes to dermokine-b significantly decreased the phosphorylation level of ERK1/2 by 50% compared to control in proliferating culture conditions.…”

The Mitogen-activated Protein Kinase Kinase Kinase Dual Leucine Zipper-bearing Kinase (DLK) Acts as a Key Regulator of Keratinocyte Terminal Differentiation

“…Evidence supporting the involvement of JNK in keratinocyte differentiation derives from the findings of Takahashi et al (10), who have shown by immunohistochemistry on normal human epidermis that the active phosphorylated form of JNK is present in keratinocytes of the upper spinous and granular layers. Moreover, in studies carried out in vitro with cultured normal human keratinocytes, these authors demonstrated that the expression of cystatin A, a key marker of differentiation, requires the activation of JNK (11). Similarly experiments with cultured keratinocytes have also highlighted the importance of p38 MAPK signaling in the terminal differentiation of these epidermal cells.…”

“…In vitro studies with cultured normal human keratinocytes have clearly demonstrated that JNK, p38, and many of their upstream activators regulate positively the expression of proteins such as involucrin, loricrin, transglutaminase 1, and cystatin A (11,16), which are required during the final stage of the terminal differentiation program. The results presented here show that the MAPKKK DLK preferentially stimulated the JNK subgroup of MAPKs upon expression in poorly differentiated human keratinocytes.…”

“…However, biochemical and genetic evidence has demonstrated that the MAPKK MKK7, which serves as a substrate for DLK (39), is essential for JNK activation (40). Immunohistochemistry experiments with normal human epidermis (11) have shown that the active phosphorylated form of JNK is present in the most differentiated cells of the granular layer like DLK and c-Jun (41, 42), the transcription factor through which JNK activation leads to the upregulation of target genes in a variety of mammalian cells (43). The observation that DLK, active JNK, and c-Jun are concomitantly localized in granular keratinocytes raises the intriguing possibility that DLK may act as an important regulator of the JNK pathway in human skin in vivo.…”

The UV (Ribotoxic) Stress Response of Human Keratinocytes Involves the Unexpected Uncoupling of the Ras-Extracellular Signal-Regulated Kinase Signaling Cascade from the Activated Epidermal Growth Factor Receptor

“…However, indirect evidence strongly suggests that the proliferative state of basal keratinocytes in human skin crucially depends on the activity of the EGFR-Ras-ERK cascade. For instance, phosphorylated ERK has been detected, via immunohistochemical methods, almost exclusively in the stratum basale, the proliferative compartment of the skin harboring the keratinocyte stem cells (see below) (1,70). Furthermore, abnormally increased phosphorylation of ERK in the stratum spinosum has been observed in cases of psoriasis, a disorder that is characterized by a keratinocyte hyperproliferation in the suprabasal layers (24).…”