P52PAI-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 p52PAI-1 gene (which encodes the type-1 inhibitor of plasminogen activator). Butyrate-initiated expression of p52PAI-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 p52PAI-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 p52PAI-1 induction to the process of flat reversion more critically, a molecular genetic approach was designed to maintain high-level constitutive p52PAI-1 synthesis in the absence of butyrate. KNRK cells transfected with a Rc/CMVPAI plasmid construct, in which expression of a p52PAI-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 p52PAI-1 protein, relative to Rc/CMV isolates, correlated with generation of the flat phenotype. These data suggest that induced p52PAI-1 expression probably functions in the development of morphological revertants in the KNRK cell system.
Higgins
P J
PJ
Department of Microbiology, Immunology and Molecular Genetics, Albany Medical College, NY 12208, USA.
Ryan
M P
MP
Jelley
D M
DM
D000818:Animals; D002087:Butyrates / Q000494:pharmacology*; D020148:Butyric Acid; D002465:Cell Movement / Q000235:genetics; D002472:Cell Transformation, Viral / Q000187:drug effects / Q000235:genetics*; D002478:Cells, Cultured; D015967:Gene Expression Regulation, Viral / Q000187:drug effects*; D011905:Genes, ras; D007668:Kidney / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D017395:Plasminogen Activator Inhibitor 1 / Q000096:biosynthesis* / Q000235:genetics*; D051381:Rats
D002087:Butyrates; D017395:Plasminogen Activator Inhibitor 1; D020148:Butyric Acid