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 physiological modulator of plasmin generation, is regulated by growth factors and changes in cell shape. To evaluate the specific relationship between PAI-1 gene expression and cytoarchitecture, serum-free cultures of quiescent rat kidney (NRK) cells were exposed to cytochalasin D (CD) at concentrations that disrupt microfilament structure. Treatment with 1-10 microM CD resulted in an increased 1) incidence of rounded cells, 2) relative PAI-1 mRNA content, and 3) fraction of PAI-1 protein-expressing cells. Abrupt increases in each response were evident at a final concentration of 5 microM CD. Maximal levels of induced PAI-1 transcripts (18-fold that of control) occurred 4 hours post-CD addition and declined thereafter but remained elevated (by at least tenfold) for 24 hours. Assessment of the metabolic requirements for CD-induced PAI-1 expression by using the protein synthesis inhibitors puromycin and cycloheximide indicated that PAI-1 transcripts were regulated in a complex manner in response to CD. The predominant mode of induction reflected secondary (protein synthesis-dependent) metabolic processes, although a minor, albeit significant, primary (protein synthesis-independent) pathway was also evident. PAI-1 mRNA levels in NRK cells maintained in serum- and CD-free agarose suspension culture were low or undetectable. Relative abundance of PAI-1 transcripts in suspended cells cultured in the presence of CD, however, closely approximated that of plastic-adherent, CD-treated cells (13-fold over control). NRK cells in suspension culture with or without CD were morphologically identical, remaining spherical and unattached. It appears, therefore, that cell rounding 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 response. A protein tyrosine kinase is likely involved in the CD-mediated signal-transduction cascade, since induced PAI-1 expression can be down-regulated by genistein and herbimycin A but not by calphostin C or tyrphostin B46.
Hawks
K
K
Department of Experimental Pathology, Albany Medical College, New York 12208, USA.
Higgins
P J
PJ
D000818:Animals; D016227:Benzoquinones; D002460:Cell Line; D048429:Cell Size / Q000235:genetics; D003513:Cycloheximide / Q000494:pharmacology; D015638:Cytochalasin D / Q000494:pharmacology; D003599:Cytoskeleton / Q000502:physiology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000187:drug effects / Q000502:physiology; D019833:Genistein / Q000494:pharmacology; D007668:Kidney / Q000166:cytology* / Q000201:enzymology; D007700:Kinetics; D047029:Lactams, Macrocyclic; D009281:Naphthalenes / Q000494:pharmacology; D009570:Nitriles / Q000494:pharmacology; D019384:Nucleic Acid Synthesis Inhibitors / Q000494:pharmacology; D017395:Plasminogen Activator Inhibitor 1 / Q000235:genetics*; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011691:Puromycin / Q000494:pharmacology; D011809:Quinones / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017828:Rifabutin / Q000031:analogs & derivatives; D020032:Tyrphostins
D016227:Benzoquinones; D004791:Enzyme Inhibitors; D047029:Lactams, Macrocyclic; D009281:Naphthalenes; D009570:Nitriles; D019384:Nucleic Acid Synthesis Inhibitors; D017395:Plasminogen Activator Inhibitor 1; D011500:Protein Synthesis Inhibitors; D011809:Quinones; D012333:RNA, Messenger; D020032:Tyrphostins; C095713:alpha-cyano-(3,4-dihydroxy)-N-(3-phenylpropyl)cinnamide; D017828:Rifabutin; D015638:Cytochalasin D; D011691:Puromycin; C020754:herbimycin; D003513:Cycloheximide; D019833:Genistein; D011505:Protein-Tyrosine Kinases; C058819:calphostin C