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Perturbation of the actin cytoskeleton induces PAI-1 gene expression in cultured epithelial cells independent of substrate anchorage.

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.