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 o rganization provides a means to assess the relationship between cell shape and ec nl expression. Induced transcription of the plasminogen activator in hibitor type-1 (PAI-1) gene in serum-free cultures of normal rat kidney (NR K-52E) cells following disruption of actin microfilament structures with cy tochalasin D (CD) provides a simple model to probe mechanisms underlying sh ape-related expression control. Transition from the typical flat epithelial cell shape to an "arborized" phenotype was a concomitant of the PAI-1 indu ctive 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 s eries of culture conditions were designed (e.g., use of bacteriological sur faces, poly-HEMA coated surfaces, maintenance in suspension on agarose) to discriminate cell shape from adhesive influences on CD-stimulated PAI-1 exp ression. Cytoskeletal disruption, and not simply changes in cell shape, was a critical aspect of CD-mediated PAI-1 expression in NRK cells cultured un der serum-free conditions: induced expression was independent of substrate anchorage. Low concentrations of CD (1-2 mu M) failed to cause cell arboriz ation or increase either relative PAI-1 mRNA/protein abundance levels sugge sting, however, that cell rounding may be a necessary but not sufficient as pect in CD-mediated PAI-1 induction. Transfection of PAI-1 promoter-CAT rep orter constructs into NRK cells followed by stimulation with CD or serum ad ditionally indicated that CD-induced PAI-1 expression did not utilize the s ame functional complement of serum-responsive promoter sequences, thus, fur ther defining differences in the growth factor- and cytoskeletal-mediated p athways of PAI-1 gene regulation. Cell Motil. Cytoskeleton 42:218-229. 1999 . (C) 1999 Wiley-Liss, Inc.