Studies of induced transcription in response to an activated Ha-ras, TPA and UV radiation in keratinocytes

Sammanfattning: The mutational activation of the Ha-ras gene in keratinocytes has been shown tooccur during the initiation step in the mouse skin model for multistage carcinogen-esis. Skin tumor promoters such as 12-O-tetradecanoylphorbol-13-acetate (TPA)and ultraviolet radiation (UVR) stimulate a selective outgrowth of initiated kerati-nocytes to form benign tumours (papillomas). The mechanisms that determine thecooperativity between an activated Ha-ras and a tumor promoter is assumed todepend on altered gene expression caused by these two types of transcriptionalactivators. This thesis describes the characterization of response elements, tran-scription factors and signalling pathways activated by an Ha-ras gene and tumourpromoters respectively.Paper III and IV work describes the identification and characterisation of a 20 bpsequence (B10 RRE), present in the LTR of an endogenous VL30 retrotransposon,that mediates induced transcription in response to an activated Ha-ras, Raf-1 andEGF. B10 RRE is a composite responsive element composed of a 5' AP-1-likesequence and a 3' sequence unrelated to previously known transcription factorbinding sites. The AP-1-like motif preferentially binds ATF3/c-Jun and ATF3/JunD heterodimers, whereas the 3 sequence binds a novel nuclear factor (TAR) withan appearent size of 178 kDa in solution. DNA-affinity chromatography andsouthwestern blot analysis revealed that the DNA binding component of TAR is a50 kDa protein.Interestingly, B10 RRE is unresponsive to both TPA and WUVR. Instead, these twostimuli induce transcription through a previously characterised TPA responsiveelement (VLTRE), present in the same LTR as B10 RRE. VLTRE is demonstrated tobe a target for TPA-and UVR-induced binding of Rel/NF-KB proteins. Whencomparing the signalling pathways activated by TPA and UVR respectively, it isshown that TPA and UVB activate different sets of Rel/NF-KB proteins and thatUVB induced transcription is independent of PKC activity. Furthermore, UVBtransactivation is not dependent on a functional Ras protein, and the Raf-1 kinasehas only a permissive role in mediating the UVB response.In conclusion, this work has led to the identification of different response elementsand transcription factors that mediate induced transcription in response to anUVR)(papers I and II) respectively. These results now provide tools to further elucidatedifferences and similarities in intracellular signals activated during the differentphases in mouse skin tumourigenesis.Key words: keratinocytes, Ha-ras, ultraviolet radiation, transcription factor, TPA, NF-kB ISBN 91-628-20834