A DOMINANT-NEGATIVE MUTANT OF JUN BLOCKING 12-O-TETRADECANOYLPHORBOL-13-ACETATE-INDUCED INVASION IN MOUSE KERATINOCYTES

We previously reported that induced activator protein-1 (AP-1) transcr iptional activity appears to be required for tumor promoter-induced tr ansformation in mouse epidermal JB6 cells. To extend this investigatio n to a keratinocyte culture model and a transgenic mouse model, we con structed K14TAM67, a keratin 14 promoter-controlled version of the dom inant negative jun mutant to directly block AP-1 activity and possibly indirectly block NF kappa B activity in basal squamous epithelia. Thi s study was directed at characterizing TAM67 expression and biological activity in the mouse cell line 308, a keratinocyte model for studyin g carcinogenesis. Cotransfection of K14TAM67 with luciferase plasmid r eporter DNAs produced inhibition of basal and 12-O-tetradecanoylphorbo l-13-acetate (TPA)-induced AP-1 and NF kappa B activity but had no eff ect on p53-dependent transcriptional activity. In an in vitro invasion assay, stable expression of TAM67 in 308 cells blocked TPA-induced Ma trigel invasion. This suggests that blocking TPA-induced AP-1- or NF k appa B-regulated gene expression by TAM67 inhibits TPA-induced progres sion. Recombinant tissue inhibitor of metalloproteinase 1 reduced TPA- induced in vitro invasion, thus implicating metalloproteinases at leas t in part in the transcription factor-dependent process. Analysis of m RNA levels for members of the matrix metalloproteinase (MMP) family, h owever, revealed that the expression of any single MMP family member d id not correlate with regulation of AP-1 or NF kappa B activity. Howev er, the combination of substantial levels of mRNA for stromelysin-l, s tromelysin-2, collagenase, membrane type 1 MMP, and gelatinase A occur red only in TPA-treated cells in the absence of TAM67. These results s uggest that the action of the dominant negative jun mutant on AP-I and NF kappa B gene regulation results in complex alterations in the leve ls of downstream effector genes, such as the metalloproteinases, that effect TPA-induced cellular invasion. (C) 1997 Wiley-Liss, Inc.(dagger )