Regulation of epidermal growth factor receptor by activated H-ras and V-myc oncogenes in mouse Balb/3T3 cells: possible roles of AP-1.

We previously reported that introduction of H-ras oncogene decreases the epidermal growth factor (EGF) binding activity to cell surface EGF receptor in mouse Balb/3T3. In this study, we have further isolated four H-ras transfectants, four v-myc transfectants and three both H-ras and v-myc (H-ras/v-myc) transfectants of mouse Balb/3T3 cells. In comparison with introduction of v-myc alone or both H-ras and v-myc oncogene, introduction of H-ras alone resulted in a loss of [125I]EGF binding activity to the cell surface EGF receptor. RT-PCR analysis also showed much lower levels of EGF receptor gene expression in H-ras transfectants compared to that of parental untransformed cells (Balb-Neo1), v-myc and H-ras/v-myc transfectants. Our results demonstrated the activated binding of a transcription factor, Stat1 p84/p91, which directly interacts with EGF receptor, to c-sis-inducible element (SIE) in both v-myc and H-rs/v-myc transfectants, but not in H-ras transfectants. Among transcription factors which we have analysed, activator protein 1 (AP-1) but not SP-1 was modulated by H-ras. Gel shift assays demonstrated the mobility pattern of TPA-responsive element (TRE) binding complex with AP-1 derived from H-ras transfectants migrated faster than those from Balb-Neo1, v-myc and H-ras/v-myc. Expression of c-Jun and Fra-1 was increased more than threefold in H-ras transfectants compared with Balb-Neo1, v-myc and H-ras/v-myc transfectants, but that of c-Fos, Jun B and SP-1 was unchanged. Both transient and permanent expression of H-ras enhanced AP-1 activity in mouse cells, but further co-introduction of dominant negative c-jun mutant encoding a transcriptionally inactive product inhibited the H-ras dependent AP-1 induction. Transfection of the dominant negative c-jun mutant also restored down-regulation of EGF binding by activated H-ras oncogene. Down-regulation of EGf receptor by activated H-ras and the possible involvement of a transcription factor, AP-1 will be discussed.