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A dominant negative mutant of jun blocking 12-O-tetradecanoylphorbol-13-acetate-induced invasion in mouse
Z Dong1, H C Crawford, V Lavrovsky
1Laboratory of Viral Carcinogenesis, National Cancer Institute, Frederick Cancer Research and Development Center, Maryland, USA.
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.
Insights
Blocking activator protein-1 (AP-1) and NF kappa B activity with a dominant-negative jun mutant inhibits tumor promoter-induced keratinocyte invasion. This suggests AP-1 and NF kappa B are key regulators of carcinogenesis progression.
Area of Science:
- Molecular Biology
- Cancer Research
- Dermatology
Background:
- Activator protein-1 (AP-1) transcriptional activity is crucial for tumor promoter-induced transformation in mouse epidermal cells.
- A dominant-negative jun mutant (TAM67) was developed to inhibit AP-1 and potentially NF kappa B activity in squamous epithelia.
Purpose of the Study:
- To characterize the expression and biological activity of TAM67 in a keratinocyte model (308 cells) for carcinogenesis.
- To investigate the role of AP-1 and NF kappa B in 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced keratinocyte invasion.
Main Methods:
- K14TAM67 construct was created for keratin 14 promoter-controlled expression.
- Cotransfection with luciferase reporter plasmids to assess AP-1, NF kappa B, and p53 activity.
- In vitro invasion assays using Matrigel to evaluate TAM67's effect on TPA-induced invasion.
- Analysis of matrix metalloproteinase (MMP) family mRNA levels.
Main Results:
- K14TAM67 expression inhibited both basal and TPA-induced AP-1 and NF kappa B activity, but not p53 activity.
- Stable TAM67 expression in 308 cells blocked TPA-induced Matrigel invasion.
- Recombinant tissue inhibitor of metalloproteinase 1 reduced TPA-induced invasion, implicating metalloproteinases.
- While no single MMP correlated with AP-1/NF kappa B activity, a combination of MMPs was observed in TPA-treated cells without TAM67.
Conclusions:
- Blocking AP-1 and NF kappa B activity via TAM67 inhibits TPA-induced keratinocyte invasion.
- TAM67's action leads to complex alterations in downstream effector genes, including metalloproteinases.
- These findings highlight the role of AP-1 and NF kappa B in regulating genes involved in TPA-induced cellular invasion and carcinogenesis.