P52(PAI-1) GENE-EXPRESSION IN BUTYRATE-INDUCED FLAT REVERTANTS OF V-RAS-TRANSFORMED RAT-KIDNEY CELLS - MECHANISM OF INDUCTION AND INVOLVEMENT IN THE MORPHOLOGICAL RESPONSE

Sodium n-butyrate-induced flat reversion in v-K-ras oncogene-transform ed rat kidney (KNRK) cells is associated with transcriptional activati on of the p52(PAI-1) gene (which encodes the type-1 inhibitor of plasm inogen activator). Butyrate-initiated expression of p52(PAI-1) mRNA an d protein correlated with induced cell spreading and preceded developm ent of cell-to-substrate focal adhesions. Such undersurface matrix con tact structures, which are absent from parental KNRK cells, require pr oximal PAI-1 deposition for their stabilization. Stimulated p52(PAI-1) expression by flat revertants (approximating 25-fold that of control cells) and the accompanying cytoarchitectural reorganization appeared to be programmed responses to butyrate as both events required de novo RNA and protein synthesis, metabolic characteristics consistent with a secondary pathway of gene regulation. To assess the relevance of p52 (PAI-1) induction to the process of flat reversion more. critically, a molecular genetic approach was designed to maintain high-level consti tutive p52(PAI-1) synthesis in the absence of butyrate. KNRK cells tra nsfected with a Rc/CMVPAI plasmid construct, in which expression of a p52(PAI-1) cDNA insert was driven by enhancer-promoter sequences from the immediate-early gene of human cytomegalovirus, formed colonies com prised of flat-revertant-like cells with a greater frequency than did cells transfected with the Rc/CMV vector alone (24.8% and 1.7% respect ively). Comparative analysis of randomly selected Rc/CMVPAI clones ind icated that a 10-fold increase in immunoreactive p52(PAI-1) protein, r elative to Rc/CMV isolates, correlated with generation of the flat phe notype. These data suggest that induced p52(PAI-1) expression probably functions in the development of morphological revertants in the KNRK cell system.