A dominant negative mutant of jun blocking 12-O-tetradecanoylphorbol-13-acetate–induced invasion in mouse keratinocytes

Abstract

We previously reported that induced activator protein-1 (AP-1) transcriptional activity appears to be required for tumor promoter-induced transformation in mouse epidermal JB6 cells. To extend this investigation to a keratinocyte culture model and a transgenic mouse model, we constructed K14TAM67, a keratin 14 promoter-controlled version of the dominant negative jun mutant to directly block AP-1 activity and possibly indirectly block NF kappa B activity in basal squamous epithelia. This study was directed at characterizing TAM67 expression and biological activity in the mouse cell line 308, a keratinocyte model for studying carcinogenesis. Cotransfection of K14TAM67 with luciferase plasmid reporter DNAs produced inhibition of basal and 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced AP-1 and NF kappa B activity but had no effect on p53-dependent transcriptional activity. In an in vitro invasion assay, stable expression of TAM67 in 308 cells blocked TPA-induced Matrigel invasion. This suggests that blocking TPA-induced AP-1- or NF kappa B-regulated gene expression by TAM67 inhibits TPA-induced progression. Recombinant tissue inhibitor of metalloproteinase 1 reduced TPA-induced in vitro invasion, thus implicating metalloproteinases at least in part in the transcription factor-dependent process. Analysis of mRNA levels for members of the matrix metalloproteinase (MMP) family, however, revealed that the expression of any single MMP family member did not correlate with regulation of AP-1 or NF kappa B activity. However, the combination of substantial levels of mRNA for stromelysin-1, stromelysin-2, collagenase, membrane type 1 MMP, and gelatinase A occurred only in TPA-treated cells in the absence of TAM67. These results suggest that the action of the dominant negative jun mutant on AP-1 and NF kappa B gene regulation results in complex alterations in the levels of downstream effector genes, such as the metalloproteinases, that effect TPA-induced cellular invasion.

Expression of dominant negative Jun inhibits elevated AP-1 and NF-κB transactivation and suppresses anchorage independent growth of HPV immortalized human keratinocytes

“…Our present data show that Fos family is related to the AP-1 transactivation and a mutant c-jun eectively inhibits both AP-1 and NF-kB transactivation in the more progressed human keratinocytes under the control of a human K14-promoter. Relative low levels of TAM (compared to endogenous Jun) were required for substantial transcription factor transrepression, similar to the observation with mouse keratinocytes (Dong et al, 1997). It is noteworthy that K14-driven TAM expression in the HPV-immortalized keratinocytes did not inhibit the AP-1 and NF-kB transactivation levels seen in the less progressed cell lines but eciently inhibited thè elevated' AP-1 and NF-kB activation seen in the more progressed cell lines, 16RH and 18 v-fos.…”

“…Although AP-1 and NF-kB can synergize in either direction, the fact that NF-kB transactivation occurs ®rst in the JB6 mouse skin cell model, suggests an NF-kB-to-AP-1 direction in tumor promoter induced transformation, but the possibility of separate pathways is not excluded. Implication of either or both transcription factors in keratinocyte progression has been further supported by our observation that dominant negative jun expression in a mouse keratinocyte papilloma cell line blocked not only AP-1 but also NF-kB activation when invasion was also blocked (Dong et al, 1997). Thus alterations in the expression of target genes regulated by AP-1 and/or NF-kB transactivation may determine the phenotypic changes seen in the more progressed cell lines.…”

The Transcription Factor AP-1 Is Required for EGF-induced Activation of Rho-like GTPases, Cytoskeletal Rearrangements, Motility, and In Vitro Invasion of A431 Cells

“…2 b ). As was previously demonstrated for malignant mouse epidermal cell lines (Domann et al, 1994 b ;Dong et al, 1997), introduction of TAM67 did not alter the growth rates of expressing clones (data not shown).…”

The Role of Glycogen Synthase Kinase 3β in the Transformation of Epidermal Cells

“…It has been shown that AP-1 activity is essential for the transformation of JB6 P+ cells (12,13,23). We sought to determine whether GSK3h regulates the activation of AP-1.…”

“…With this carcinogenesis model system, a number of studies show that AP-1 activity is required for skin tumorigenesis as well as malignant transformation (30)(31)(32)(33). These findings are confirmed by in vitro studies that show that AP-1 activity is essential for the transformation of JB6 P+ cells (12,13,23). Three signaling pathways, namely, PI3K/Akt, PKC, and MEK1/Erk, have been shown to regulate EGF-and TPAinduced AP-1 transactivation and transformation of JB6 P+ cells (13,14,23).…”