IDENTIFICATION OF A RAS-ACTIVATED ENHANCER IN THE MOUSE OSTEOPONTIN PROMOTER AND ITS INTERACTION WITH A PUTATIVE ETS-RELATED TRANSCRIPTION FACTOR WHOSE ACTIVITY CORRELATES WITH THE METASTATIC POTENTIAL OF THE CELL

The role of RAS in transducing signals from an activated receptor into altered gene expression is becoming clear, though some links in the c hain are still missing. Cells possessing activated RAS express higher levels of osteopontin (OPN), an alpha(v) beta(3) integrin-binding secr eted phosphoprotein implicated in a number of developmental, physiolog ical, and pathological processes. We report that in T24 H-ras-transfor med NIH 3T3 cells enhanced transcription contributes to the increased expression of OPN. Transient transfection studies, DNA-protein binding assays, and methylation protection experiments have identified a nove l ras-activated enhancer, distinct from known ras response elements, t hat appears responsible for part of the increase in OPN transcription in cells with an activated RAS. In electrophoretic mobility shift assa ys, the protein-binding motif GGAGGCAGG was found to be essential for the formation of several complexes, one of which (complex A) was gener ated at elevated levels by cell lines that are metastatic. Southwester n blotting and UV light cross-linking studies indicated the presence o f several proteins able to interact with this sequence. The proteins t hat form these complexes have molecular masses estimated at approximat ely 16, 28, 32, 45, 80, and 100 kDa. Because the similar to 16-kDa pro tein was responsible for complex A formation, we have designated it MA TF for metastasis-associated transcription factor. The GGANNNAGG motif is also found in some other promoters, suggesting that they may be si milarly controlled by MATF.