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

J Biol Chem. 2001 Sep 28;276(39):36632-8. doi: 10.1074/jbc.M102021200. Epub 2001 Jul 12.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenoviridae / genetics
  • Blotting, Northern
  • Blotting, Western
  • Carcinogens
  • Cells, Cultured
  • Chloramphenicol O-Acetyltransferase / metabolism
  • Cystatins / biosynthesis*
  • DNA, Complementary / metabolism
  • Enzyme Inhibitors / pharmacology
  • Flavonoids / pharmacology
  • Gene Expression Regulation*
  • Genes, Dominant
  • Humans
  • Immunohistochemistry
  • Keratinocytes / metabolism*
  • MAP Kinase Kinase 1
  • MAP Kinase Kinase 7
  • MAP Kinase Kinase Kinase 1*
  • Mitogen-Activated Protein Kinase 8
  • Mitogen-Activated Protein Kinase Kinases / metabolism*
  • Mitogen-Activated Protein Kinases / metabolism*
  • Phosphorylation
  • Plasmids / metabolism
  • Promoter Regions, Genetic
  • Protein-Serine-Threonine Kinases / metabolism*
  • Proto-Oncogene Proteins c-raf / metabolism*
  • RNA, Messenger / metabolism
  • Reverse Transcriptase Polymerase Chain Reaction
  • Signal Transduction
  • Tetradecanoylphorbol Acetate
  • Transfection
  • ras Proteins / metabolism*


  • Carcinogens
  • Cystatins
  • DNA, Complementary
  • Enzyme Inhibitors
  • Flavonoids
  • RNA, Messenger
  • Chloramphenicol O-Acetyltransferase
  • Protein-Serine-Threonine Kinases
  • Proto-Oncogene Proteins c-raf
  • Mitogen-Activated Protein Kinase 8
  • Mitogen-Activated Protein Kinases
  • MAP Kinase Kinase Kinase 1
  • MAP3K1 protein, human
  • MAP Kinase Kinase 1
  • MAP Kinase Kinase 7
  • MAP2K1 protein, human
  • MAP2K7 protein, human
  • Mitogen-Activated Protein Kinase Kinases
  • ras Proteins
  • Tetradecanoylphorbol Acetate
  • 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one