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-transformed rat kidney (KNRK) cells is associated with transcriptional activation of the p52(PAI-1) gene (which encodes the type-1 inhibitor of plasminogen activator). Butyrate-initiated expression of p52(PAI-1) mRNA and protein correlated with induced cell spreading and preceded development of cell-to-substrate focal adhesions. Such undersurface matrix contact structures, which are absent from parental KNRK cells, require proximal 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 constitutive p52(PAI-1) synthesis in the absence of butyrate. KNRK cells transfected 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 comprised of flat-revertant-like cells with a greater frequency than did cells transfected with the Rc/CMV vector alone (24.8% and 1.7% respectively). Comparative analysis of randomly selected Rc/CMVPAI clones indicated that a 10-fold increase in immunoreactive p52(PAI-1) protein, relative to Rc/CMV isolates, correlated with generation of the flat phenotype. These data suggest that induced p52(PAI-1) expression probably functions in the development of morphological revertants in the KNRK cell system.