Biochemical localization of the transformation-sensitive 52 kDa (p52) protein to the substratum contact regions of cultured rat fibroblasts. Butyrate induction, characterization, and quantification of p52 in v-ras transformed cells

Abstract

A 52 kDa protein (p52) was identified, using differential extraction and electrophoretic criteria, as a major extracellular and substrate-associated component of normal rat kidney (NRK) fibroblasts. Cells transformed with Kirsten murine sarcoma virus (KNRK cells) did not express p52 constitutively, but were inducible for both p52 production -and its substrate association during culture in sodium butyrate (NaB)-supplemented growth medium. Comparative analysis of the relative molecular mass, subcellular distribution, and isoelectric complexity (five variants ranging in pl from 5.4 to 6.2) of the 52 kDa species constitutively and inducibly expressed by NRK and KNRK/NaB cells respectively, indicated that they were, indeed, the same protein. p52 selectively localized to cellular fractions enriched in substrate focal contact sites and associated ventral undersurface components. NaB induction of p52 in KNRK cells occurred before cell spreading; other polar compounds, such as dimethyl sulphoxide, which did not induce KNRK cell spreading, similarly failed to elicit p52 production. p52 accumulated more rapidly in (and was quickly released from) the focalcontact-enriched protein fraction of NRK cells compared with its time course of appearance in the medium. These data collectively suggest that p52 is one of a relatively small number of proteins the synthesis of which is either involved in determination of cell shape or regulated as a consequence of cell-shape changes.

PAI‐1 expression is required for epithelial cell migration in two distinct phases of in vitro wound repair

“…Cells were labeled in serum‐/methionine‐free medium containing 50 μCi 35 S‐methionine/ml (specific activity = 1,100 Ci/mmol) prior to extraction with 0.2% saponin (Higgins and Ryan, 1989). The PAI‐1‐enriched saponin fraction was solubilized in 50 mM Tris‐HCl, pH 6.8, 10% glycerol, 1% SDS, 1% 2‐mercaptoethanol, boiled and 25,000 cpm TCA‐insoluble protein separated on SDS/9% acrylamide gels followed by En 3 Hance‐treatment and fluorography.…”

“…Endocytosis of uPAR‐associated uPA/PAI‐1 complexes, moreover, promotes uPAR recycling and, thereby, vitronectin‐dependent cell movement (Mignatti and Rifkin, 2000). PAI‐1 does accumulate specifically in the cellular undersurface region (Higgins and Ryan, 1989; Seiffert et al, 1994; Lawrence et al, 1997; Loskutoff et al, 1999) where it is well‐positioned to modulate integrin‐ECM or uPA/uPAR‐ECM interactions as well as stromal proteolysis (Andreasen et al, 2000; Czekay et al, 2003). Since PAI‐1 is deposited into migration tracks (Pepper et al, 1992; Seebacher et al, 1992; Providence et al, 2002), changes in PAI‐1 expression may influence motile behavior by modulating either uPA‐dependent pericellular proteolysis or cell‐to‐matrix adhesion (Ciambrone and McKeown‐Longo, 1990; Liu et al, 1995; Deng et al, 1996; Stefansson and Lawrence, 1996; Chapman, 1997; Kutz et al, 1997; Loskutoff et al, 1999; Mignatti and Rifkin, 2000; Czekay et al, 2003).…”

Epithelial monolayer wounding stimulates binding of USF-1 to an E-box motif in the plasminogen activator inhibitor type 1 gene

“…De novo synthesized PAI-1 protein accumulates in the cellular undersurface region likely in a complex with matrix vitronectin (Higgins and Ryan, 1989;Seiffert et al, 1994;Lawrence et al, 1997), although PAI-1 has been suggested to also associate with fibronectin and/or laminin deposits in migration tracks (Seebacher et al, 1992). This SERPIN is well-positioned, therefore, to modulate integrin-ECM or uPA/uPAR-ECM interactions as well as ECM barrier proteolysis.…”

“…These findings highlight the complexity of cellular motile controls that collectively reflect the level of expression of participating elements, the nature of the 'matrix' encountered, the system context (i.e., 2D vs 3D migration) and the growth factor environment. The rapid kinetics of wound-stimulated PAI-1 induction and relatively short matrix-associated half-life (Higgins and Ryan, 1989) suggests that this protein may influence cellular adhesive events for a specified duration during injury repair.…”

PAI-1 Expression Is Required for HDACi-Induced Proliferative Arrest inras-Transformed Renal Epithelial Cells

“…The derivation of the PAI-1 functionally null knockdown (PAI-1 KD ) 4HH cell line by transfection of a 2.6 kb rat PAI-1 Eco R1/ Hin dIII cDNA fragment (representing nucleotides −118 to +2572) cloned in anti-sense orientation (Rc/CMVIAP) has been described [34, 35]. v- ras -transformed cells were also transfected with the Rc/CMVPAI sense vector to initiate high-level PAI-1 expression in the absence of NaB or with the empty Rc/CMV construct [32].…”