title: Perturbation of the actin cytoskeleton induces PAI-1 gene expression in cultured epithelial cells independent of substrate anchorage.
abstract: Perturbation of cellular architecture with agents that alter cytoskeletal organization provides a means to assess the relationship between cell shape and gene expression. Induced transcription of the plasminogen activator inhibitor type-1 (PAI-1) gene in serum-free cultures of normal rat kidney (NRK-52E) cells following disruption of actin microfilament structures with cytochalasin D (CD) provides a simple model to probe mechanisms underlying shape-related expression control. Transition from the typical flat epithelial cell shape to an "arborized" phenotype was a concomitant of the PAI-1 inductive response. Stimulated expression occurred rapidly (i.e., within 2 h of CD addition), involved increases in both PAI-1 mRNA abundance and de novo protein synthesis, and was dependent upon the concentration of CD used. A series of culture conditions were designed (e.g., use of bacteriological surfaces, poly-HEMA coated surfaces, maintenance in suspension on agarose) to discriminate cell shape from adhesive influences on CD-stimulated PAI-1 expression. Cytoskeletal disruption, and not simply changes in cell shape, was a critical aspect of CD-mediated PAI-1 expression in NRK cells cultured under serum-free conditions; induced expression was independent of substrate anchorage. Low concentrations of CD (1-2 microM) failed to cause cell arborization or increase either relative PAI-1 mRNA/protein abundance levels suggesting, however, that cell rounding may be a necessary but not sufficient aspect in CD-mediated PAI-1 induction. Transfection of PAI-1 promoter-CAT reporter constructs into NRK cells followed by stimulation with CD or serum additionally indicated that CD-induced PAI-1 expression did not utilize the same functional complement of serum-responsive promoter sequences, thus, further defining differences in the growth factor- and cytoskeletal-mediated pathways of PAI-1 gene regulation.
authors: Department of Microbiology, Immunology and Molecular Genetics, Albany Medical College, New York 12208, USA.
authors: K M
authors: KM
authors: Providence
authors: S M
authors: SM
authors: Kutz
authors: P J
authors: PJ
authors: Higgins
mesh_terms: D000199:Actins / Q000378:metabolism*; D000818:Animals; D015152:Blotting, Northern; D015139:Blotting, Southern; D002478:Cells, Cultured; D002701:Chloramphenicol / Q000378:metabolism; D015638:Cytochalasin D / Q000494:pharmacology; D003599:Cytoskeleton / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D004847:Epithelial Cells / Q000378:metabolism*; D015870:Gene Expression; D015151:Immunoblotting; D017395:Plasminogen Activator Inhibitor 1 / Q000235:genetics*; D051381:Rats; D012685:Sepharose / Q000378:metabolism; D013997:Time Factors; D014158:Transcription, Genetic / Q000187:drug effects; D014162:Transfection; D014357:Trypsin / Q000494:pharmacology
chemical_list: D000199:Actins; D017395:Plasminogen Activator Inhibitor 1; D015638:Cytochalasin D; D002701:Chloramphenicol; D012685:Sepharose; D014357:Trypsin