Induced PAI-1 mRNA expression and targeted protein accumulation are early G1 events in serum-stimulated rat kidney cells.
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.
Kutz
S M
SM
Department of Microbiology, Immunology and Molecular Genetics, Albany Medical College, New York 12208, USA.
Nickey
S A
SA
White
L A
LA
Higgins
P J
PJ
D000818:Animals; D001345:Autoradiography; D001798:Blood Proteins / Q000494:pharmacology*; D015152:Blotting, Northern; D002455:Cell Division / Q000187:drug effects / Q000502:physiology; D002460:Cell Line / Q000737:chemistry / Q000166:cytology / Q000502:physiology; D005455:Fluorescent Antibody Technique; D016193:G1 Phase / Q000502:physiology*; D005786:Gene Expression Regulation / Q000187:drug effects / Q000502:physiology; D007668:Kidney / Q000166:cytology; D007700:Kinetics; D010641:Phenotype; D017395:Plasminogen Activator Inhibitor 1 / Q000032:analysis / Q000235:genetics*; D011506:Proteins / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats
D001798:Blood Proteins; D017395:Plasminogen Activator Inhibitor 1; D011506:Proteins; D012333:RNA, Messenger