CELL SHAPE-DEPENDENT PATHWAY OF PLASMINOGEN-ACTIVATOR INHIBITOR TYPE-1 GENE-EXPRESSION REQUIRES CYTOSKELETAL REORGANIZATION

Synthesis of plasminogen activator inhibitor type-1 (PAI-1), a major p hysiological modulator of plasmin generation, is regulated by growth f actors and changes in cell shape. To evaluate the specific relationshi p between PAl-7 gene expression and cytoarchitecture, serum-free cultu res of quiescent rat kidney (NRK) cells were exposed to cytochalasin D (CD) at concentrations that disrupt microfilament structure. Treatmen t with 1-10 mu M CD resulted in an increased 1) incidence of rounded c ells, 2) relative PAI-1 mRNA content, and 3) fraction of PAl-1: protei n-expressing cells. Abrupt increases in each response were evident at a final concentration of 5 mu M CD. Maximal levels of induced PAl-7 tr anscripts (18-fold that of control) occurred 4 hours post-CD addition and declined thereafter but remained elevated (by at least tenfold) fo r 24 hours. Assessment of the metabolic requirements for CD-induced PA I-1 expression by using the protein synthesis inhibitors puromycin and cycloheximide indicated that PAI-1 transcripts were regulated in a co mplex manner in response to CD. The predominant mode of induction refl ected secondary (protein synthesis-dependent) metabolic processes, alt hough a minor, albeit significant, primary (protein synthesis-independ ent) pathway was also evident. PAI-1 mRNA levels in NRK cells maintain ed in serum-and CD-free agarose suspension culture were low or undetec table. Relative abundance of PAI-1 transcripts in suspended cells cult ured in the presence of CD, however, closely approximated that of plas tic-adherent, CD-treated cells (13-fold over control). NRK cells in su spension culture with or without CD were morphologically identical, re maining spherical and unattached. It appears, therefore, that cell rou nding alone is not a sufficient stimulus to induce PAI-1 expression in quiescent NRK cells and that perturbation of the actin skeleton as a consequence of CD treatment is a critical event in the inductive respo nse. A protein tyrosine kinase is likely involved in the CD-mediated s ignal-transduction cascade, since induced PAI-1 expression can be down -regulated by genistein and herbimycin A but not by calphostin C or ty rphostin B46. (C) 1998 Wiley-Liss, Inc.