p52 induction by cytochalasin D in rat kidney fibroblasts: Homologies between p52 and plasminogen activator inhibitor type‐1

Abstract

Normal rat kidney (NRK) fibroblasts respond to the cell shape-modulating chemical agent cytochalasin D (CD) with augmented synthesis of the 52-kDa substrate-associated protein p52. p52 is a complex glycoprotein, existing as 12 different isoforms, which include a 43-kDa "core" protein (p43), four 50-kDa species (p50-0,1,2,3), and at least seven distinct pI variants of the mature 52-kDa protein. A threshold of 2-4 microM CD was found to be necessary to augment p52 deposition into both the secreted protein- and saponin-resistant cytomatrix (SAP) fractions of NRK cells. This concentration of CD was also necessary to initiate significant cell rounding. Augmented p52 production in CD-treated NRK (NRK/CD) cells provided a means to assess the identity of this protein. p52 was found to be identical to rat plasminogen activator inhibitor type-1 (rPAI-1) and to PAI-1-like proteins of other species by comparative immunoprecipitation, 2-D electrophoretic profile, V8 protease digest mapping, and subcellular fractionation criteria. Quantitation of rPAI-1 cytoplasmic mRNA abundance, using the rPAI-1 cDNA probe pSS1-3, revealed an induction of rPAI-1 mRNA in NRK/CD cells which paralleled the increased protein production. CD-augmented p52(rPAI-1) synthesis and SAP deposition was blocked by actinomycin D, implicating a need for RNA synthesis during the period of CD exposure to effect induction. Augmentation of p52 expression in NRK/CD fibroblasts, thus, appears to involve both cell shape-associated metabolic processes and concomitant RNA synthesis.

The urokinase-type plasminogen activator system in cancer metastasis: A review

“…During cell migration, the state of the cytoskeleton changes. The observation that agents causing cytoskeletal disruption lead to increased expression of u-PA and PAI-1, therefore, suggests that these proteins exhibit differential expression at different stages of cell migration (Botteri et al, 1990;Santell et al, 1992;Lee, J.S., et al, 1993;Higgins et al, 1990Higgins et al, , 1994.…”

PAI-1 gene expression is regionally induced in wounded epithelial cell monolayers and required for injury repair

“…The conditioned labeling medium was aspirated and monolayers washed with CMF-PBS prior to extraction with 0.2% (w/v) saponin in CMF-PBS to isolate cell-substratum contact regions and associated undersurface proteins (Higgins et al, 1990(Higgins et al, , 1991. Saponin-resistant (SAP fraction) residues were scraped into sample buffer (50 mM Tris/HCl, pH 6.8, 10% glycerol, 1% SDS, 1% 2-mercaptoethanol) and boiled.…”

“…Labeled protein bands were visualized by fluorography and quantified by computerized densitometry (Smith et al, 1992). Identification of the rat PAI-1 protein in one-dimensional electrophoretic separations utilized criteria described previously (Higgins et al, 1989(Higgins et al, , 1990 as well as by immunochemical reactivity with PAI-1-specific antibodies (Higgins et al, 1990).…”

Growth state-dependent regulation of plasminogen activator inhibitor type-1 gene expression during epithelial cell stimulation by serum and transforming growth factor-?1

“…RNA was immobilized by UV crosslinking and blots incubated at 42°C for 2 h in 5ϫ SSPE-or 5ϫ SSC-based prehybridization buffers (50% formamide, 5ϫ Denhardt's reagent, 1%[w/v] SDS, 100 g/ml sheared/heatdenatured salmon sperm DNA [ssDNA]). Hybridization with random-primed [ 32 P]dCTP-labeled cDNA probes (2-5 ϫ 10 6 cpm) to PAI-1 (Higgins et al, 1990;Zeheb and Gelehrter, 1988), actin, A-50 (Ryan and Higgins, 1993), and SPARC (osteonectin) was for 16 h at 42°C in 50% formamide, 2.5ϫ Denhardt's reagent, 1% SDS, 100 g/ml ssDNA, 5ϫ SSPE (or 5ϫ SSC), and 10% dextran sulfate. Membranes were washed twice in 6ϫ SSPE/0.1% SDS at room temperature for 15 min followed by two 15-min washes in 1ϫ SSPE/0.1% SDS at 37°C or in 0.1ϫ SSC/0.1% SDS for three washes at 42°C and three washes at 55°C.…”

“…Conditioned labeling media were aspirated, clarified at 13,000 ϫ g, and IP of PAI-1 and SPARC was done simultaneously utilizing procedures and antibodies developed previously (Higgins et al, 1990). Immune complexes were collected with Protein G-Sepharose beads (Genex Corp., Gaithersburg, MD), washed three times in IP buffer (10 mM Tris-HCl, pH 8.0, 0.15 M NaCl, 1% Triton-100, 0.1% SDS, 0.5% deoxycholate), and precipitated antigen released by boiling in sample buffer (50 mM Tris-HCl, pH 6.8, 10% glycerol, 1% SDS, 1% 2-mercaptoethanol) prior to electrophoresis on SDS/10% acrylamide gels (Higgins et al, 1991).…”