Abstract
Involucrin is a marker of keratinocyte terminal differentiation. Our previous studies show that involucrin mRNA levels are increased by the keratinocyte differentiating agent, 12-O-tetradecanoylphorbol-13-acetate (TPA) (Welter, J. F., Crish, J. F., Agarwal, C., and Eckert, R. L. (1995) J. Biol. Chem. 270, 12614-12622). We now study the signaling cascade responsible for this regulation. Protein kinase C and tyrosine kinase inhibitors inhibit both the TPA-dependent mRNA increase and the TPA-dependent increase in hINV promoter activity. The relevant response element is located within the promoter proximal regulatory region and includes an AP1 site, AP1-1. Co-transfection of the hINV promoter with dominant negative forms of Ras, MEKK1, MEK1, MEK7, MEK3, p38/RK, and c-Jun inhibit the TPA-dependent increase. Wild type MEKK1 enhances promoter activity and the activity can be inhibited by dominant negative MEKK1, MEK1, MEK7, MEK3, p38/RK, and c-Jun. In contrast, wild type Raf-1, ERK1, ERK2, MEK4, or JNK1 produced no change in activity and the dominant negative forms of these kinases failed to suppress TPA-dependent transcription. Treatment with an S6 kinase (S6K) inhibitor, or transfection with constitutively active S6K produced relatively minor changes in promoter activity, ruling out a regulatory role for S6K. These results suggest that activation of involucrin transcription involves a pathway that includes protein kinase C, Ras, MEKK1, MEK3, and p38/RK. Additional pathways that transfer MEKK1 activation via MEK1 and MEK7 also may function, but the downstream targets of these kinases need to be identified. AP1 transcription factors appear to be the ultimate target of this regulation.
Highlights
Our results suggest (i) that a PKC 3 Ras 3 MEKK1 3 MEK3 3 p38/RK 3 AP1 pathway is important for regulation of hINV gene expression, (ii) that parallel pathways, possibly involving MEK1 and MEK7 may be important, and (iii) that these pathways target AP1 factors that bind to an element between Ϫ128/Ϫ100 in the hINV promoter proximal regulatory region
Our results suggest that both MEK7 and MEK1 have a role in regulating hINV promoter activity; these kinases do not appear to act via the traditional downstream targets, indicating that different targets may exist in keratinocytes
Summary
Chemicals and Reagents—Keratinocyte serum-free medium (KSFM), gentamicin, trypsin, Hank’s balanced salt solution, and Lipofectin were obtained from Life Technologies, Inc. Genistein, bis-indolylmaleimide (BIS-IM), PD90859, SB203580, and rapamycin were from Calbiochem. Phorbol ester (TPA), anisomycin, and dimethyl sulfoxide were purchased from Sigma. [␣-32P]dCTP was obtained from NEN Life Science Products. The pGL2Basic plasmid, AP1 consensus oligonucleotide (AP1c), and the chemiluminescent luciferase assay system were obtained from Promega. Chemiluminescence was measured using a Berthold luminometer. Oligonucleotides for gel shifts and construction of mutant promoter sequences were synthesized using an Applied Biosystems DNA synthesizer
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