Induced PAI-1 mRNA expression and targeted protein accumulation are early G1 events in serum-stimulated rat kidney cells

Abstract

Expression of plasminogen activator inhibitor type-1 (PAI-1), a member of the SERPIN gene family that functions to regulate the plasmin-based pericellular proteolytic cascade, is growth state-regulated in normal rat kidney (NRK) cells (Ryan and Higgins, 1990, J. Cell. Physiol., 155:376-384; Ryan et al., 1996, Biochem. J., 314:1041-1046). Comparative analysis of arrest states induced in NRK cells upon exposure to serum-deficient (0.5% FBS) or serum-free culture conditions served to define the kinetics of PAI-1 gene expression and fate of de novo-synthesized PAI-1 protein. While cells rendered quiescent in serum-free or serum-deficient media were equivalent with regard to the time course of PAI-1 mRNA induction, the level of expressed transcripts (27-fold vs. 12-fold) and accumulated saponin fraction PAI-1 protein (12-fold vs. 6-fold) were consistently greater in cells recruited into exponential growth phase from a serum-free as compared to a serum-deficient arrest condition. Relative PAI-1 mRNA abundance increased within 1-2 hr post-serum addition, was maximal at 4 hr, and declined rapidly thereafter; this time course of expression coupled with placement of entry into DNA synthetic phase at approximately 12 hr after stimulation indicates that PAI-1 induction is an early-to-mid G1 phase event. Induced PAI-1 protein was evident immunocytochemically within 2 hr of serum stimulation as a peripheral "rim" of accumulated protein restricted to the cellular ventral surface at the plane of the substrate. No PAI-1 was detected between individual cells suggesting that this protein may be targeted directly to the undersurface region. By 6 hr post-stimulation, the rim of PAI-1 deposition increased in intensity and broadened to occupy approximately 30 to 50% of the total undersurface area. Double-label immunocytochemistry indicated that accumulated PAI-1 was deposited in close proximity to, but not actually within, vinculin-containing focal contact structures. Potential functionality of induced PAI-1 expression to either the initiation or maintenance of the serum-stimulated phenotype was assessed using antibodies to PAI-1. The IgG fractions of two different antisera which neutralize the ability of PAI-1 to complex with and thereby inhibit the catalytic activity of urokinase plasminogen activator significantly reduced (by 25-35%) the incidence of cells displaying the serum-stimulated phenotype; antibodies that bind PAI-1 but do not block PAI-1 inhibitory activity were without effect. In view of the vagaries of antibody accessibility and in situ neutralizing activity (particularly in a region as structurally complex as the focal contact), these data may actually underestimate the importance of PAI-1 in maintaining the activated phenotype.

SERPINE1 (PAI-1) is deposited into keratinocyte migration “trails” and required for optimal monolayer wound repair

“…Collectively, PAI-1 may function within the global program of the tissue repair response to coordinate cycles of cell-to-substrate adhesion/detachment [7,8,10,24,27,29]. The association between the activated phenotype and targeted accumulation of PAI-1 in close proximity to newly formed focal adhesions [20] is consistent with this function. PAI-1 may also influence substrate attachment directly, as one component in the molecular complex that bridges the cell and the ECM [26], additionally suggesting PAI-1 can affect adhesion when presented to cells as both a soluble as well matrix-anchored SERPIN [9,21,24,26].…”

“…Perhaps as importantly, PAI-1 not only stimulated keratinocyte adhesion and wound-initiated planar migration but also rescued keratinocytes from plasminogen-induced substrate detachment/anoikis. These effects may reflect the fact that PAI-1 accumulates specifically in the cellular undersurface region where it is wellpositioned to modulate integrin-ECM or uPA/uPAR-ECM interactions as well as stromal remodeling [2,3,6,[8][9][10]14,15,20,24,29,37,42]. Activation of the PAI-1 gene early after monolayer wounding and its prominence in the injury repair transcriptome are consistent with the present data suggesting a dual function for PAI-1 (i.e., as a keratinocyte survival factor and regulator of epithelial migration) in the cutaneous injury response program.…”

Molecular Disruption of Breast Tumor Angiogenesis

“…The amplitude as well as maintenance of expression through mid-Gj phase is anchorage responsive and this latter adhesion-dependent requirement, unlike initial induction, involves secondary (i.e., protein synthesis-dependent) transcriptional-level events (Ryan et al, 1996). These data provided for a model of PAI-1 gene control in serum-stimulated cells, which incorporates both IER and secondary regulatory influences within an "activated" G 1 state (Kutz et al, 1997;Mu et al, 1998). Such fine control over the kinetics of PAI-1 expression appears to be one modulating aspect in the complexity of G : progression.…”

“…The exact relationship between regenerative growth and trauma repair, however, remains to be defined (Jensen and Lavker, 1996;Zahm et al, 1997). Within the setting of induced cellular proliferation and migration, stimulated expression of PAI-1 appears associated with both processes (e.g., Bade and Feindler, 1988;Pepper et al, 1992;Thornton et al, 1994;Ryan et al, 1996;Kutz et al, 1997). Transcriptional regulation of the PAI-1 gene, moreover, is superimposed on a growth state-dependent program, which culminates in a proliferative response (Ryan et al, 1996;Kutz et al, 1997).…”

“…Within the setting of induced cellular proliferation and migration, stimulated expression of PAI-1 appears associated with both processes (e.g., Bade and Feindler, 1988;Pepper et al, 1992;Thornton et al, 1994;Ryan et al, 1996;Kutz et al, 1997). Transcriptional regulation of the PAI-1 gene, moreover, is superimposed on a growth state-dependent program, which culminates in a proliferative response (Ryan et al, 1996;Kutz et al, 1997). Kinetic assessments indicate that PAI-1 transcription and mRNA expression, similar to that of uPA (Grimaldi et al, 1986), occur early and in immediate-early response (IER) fashion on addition of serum to quiescent cells (Ryan and Higgins, 1993;Ryan et al, 1996;Uno et al, 1997), thereby mimicking regenerative events in vivo (Schneiderman et al, 1993;Thornton et al, 1994).…”