Stacie M. Kutz

Plasminogen activator inhibitor type-1 gene expression and induced migration in TGF-β1-stimulated smooth muscle cells is pp60c-src/MEK-dependent

Transforming growth factor‐β1 (TGF‐β1) stimulates expression of plasminogen activator inhibitor type‐1 (PAI‐1), a serine protease inhibitor (SERPIN) important in the control of stromal barrier proteolysis and cell‐to‐matrix adhesion. Pharmacologic agents that target MEK (PD98059, U0126) or src family (PP1) kinases attenuated TGF‐β1‐dependent PAI‐1 transcription in R22 aortic smooth muscle cells. Pretreatment with PP1 at concentrations that inhibited TGF‐β1‐dependent PAI‐1 expression also blocked ERK1/2 activation/nuclear accumulation suggesting that the required src kinase activity is upstream of ERK1/2 in the TGF‐β1‐initiated signaling cascade. The IC50 of the PP1‐sensitive kinase, furthermore, specifically implied involvement of pp60c‐src in PAI‐1 induction. Indeed, addition of TGF‐β1 to quiescent R22 cells resulted in a 3‐fold increase in pp60c‐src autophosphorylation and kinase activity. Transfection of a dominant‐negative pp60c‐src construct, moreover, reduced TGF‐β1‐induced PAI‐1 expression levels to that of unstimulated controls or PP1‐pretreated cells. A ≥170 kDa protein that co‐immunoprecipitated with TGF‐β1‐activated pp60c‐src was also phosphorylated transiently in response to TGF‐β1. TGF‐β1 is known to transactivate the 170 kDa EGF receptor (EGFR) by autocrine HB‐EGF or TGF‐α mechanisms suggesting involvement of EGFR activation in certain TGF‐β1‐initiated responses. Incubation of quiescent R22 cells with the EGFR‐specific inhibitor AG1478 prior to growth factor (EGF or TGF‐β1) addition effectively blocked EGFR activation as determined by direct visualization of receptor internalization. AG1478 suppressed (in a dose‐dependent fashion) EGF‐induced PAI‐1 protein levels and, at a final concentration of 2.5 μM, virtually eliminated EGF‐dependent PAI‐1 synthesis. More importantly, AG1478 similarly repressed inducible PAI‐1 levels in TGF‐β1‐stimulated R22 cultures. PP1, PD98059, and U0126 also inhibited TGF‐β1‐dependent cell motility at concentrations that significantly attenuated PAI‐1 expression. Consistent with the AG1478‐associated reductions in EGF‐ and TGF‐β1‐stimulated PAI‐1 expression, pretreatment of R22 cell cultures with AG1478 effectively suppressed growth factor‐stimulated cell motility. These data indicate that two major phenotypic characteristics of TGF‐β1‐exposure (i.e., transcription of specific target genes [e.g., PAI‐1], increased cell motility) are linked in the R22 vascular smooth muscle cell system, require pp60c‐src kinase activity and MEK signaling and involve activation of an AG1478‐sensitive (likely EGFR‐dependent) pathway.

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

Induced expression of plasminogen activator inhibitor type‐1 (PAI‐1), a major negative regulator of pericellular plasmin generation, accompanies wound repair in vitro and in vivo. Since transcriptional control of the PAI‐1 gene is superimposed on a growth state–dependent program of cell activation (Kutz et al., 1997, J Cell Physiol 170:8–18), it was important to define potentially functional relationships between PAI‐1 synthesis and subpopulations of cells that emerge during the process of injury repair in T2 renal epithelial cells. Specific cohorts of migratory and proliferating cells induced in response to monolayer trauma were spatially as well as temporally distinct. Migrating cells did not divide in the initial 12 to 20 h postinjury. After 24 h, S‐phase cells were generally restricted to a region 1 to 2 mm from, and parallel to, the wound edge. Proliferation of wound bed cells occurred subsequent to wound closure, whereas the distal contact‐inhibited monolayer remained generally quiescent. Hydroxyurea blockade indicated, however, that proliferation (most likely of cells immediately behind the motile “tongue”) was necessary for maintenance of cell‐to‐cell cohesiveness in the advancing front, although the ability to migrate was independent of proliferation. PAI‐1 mRNA expression was rapidly up‐regulated in response to wounding with inductive kinetics approximating that of serum‐stimulated cultures. Differential harvesting of T2 cell subpopulations, based on proximity to the injury site, prior to Northern assessments of PAI‐1 mRNA abundance indicated that PAI‐1 transcripts were restricted to cells immediately bordering the wound or actively migrating and not expressed by cells in the distal contact‐inhibited monolayer regions. Such cell location–specific distribution of PAI‐1‐producing cells was confirmed by immunocytochemistry. PAI‐1 synthesis in cells that locomoted into the wound field continued until injury closure. Down‐regulation of PAI‐1 synthesis and matrix deposition in renal epithelial cells, stably transfected with a PAI‐1 antisense expression vector, significantly impaired wound closure. Transfection of the wound repair–deficient R/A epithelial line with a sense PAI‐1 expression construct restored both approximately normal levels of PAI‐1 synthesis and repair ability. These data indicate that PAI‐1 induction is an early event in creation of the wound‐activated phenotype and appears to participate in the regulation of renal epithelial cell motility during in vitro injury resolution. J. Cell. Physiol. 182:269–280, 2000.

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

Transcription of the plasminogen activator inhibitor type‐1 (PAI‐1) gene appears to be growth state regulated in several cell types (e.g., Ryan and Higgins, 1993, J Cell Physiol 155:376–384; Mu et al., 1998, J Cell Physiol 174:90–98). Transit of serum‐stimulated normal rat kidney (NRK) epthelial cells through the first division cycle after release from quiescence (G0) provided a model system to assess the kinetics and mechanisms underlying PAI‐1 expression in a growth “activated” phenotype. PAI‐1 mRNA transcripts increased by more than 20‐fold during the G0→G1 transition; induced expression had immediate‐early response characteristics and abruptly declined prior to the onset of DNA synthesis. Transcriptional activity of the PAI‐1 gene paralleled the steady‐state mRNA abundance profile during this first synchronized growth cycle after release from quiescence. Although PAI‐1 mRNA levels were up‐regulated (approximately threefold) upon exposure to several different growth factors, neutralizing antibodies to transforming growth factor‐β1 (TGF‐β1) effectively attenuated the more than ninefold serum‐associated PAI‐1 inductive response by more than 70% (at both the mRNA transcript and protein levels). Similar to the metabolic requirements for serum‐mediated PAI‐1 transcription, PAI‐1 induction upon addition of TGF‐β1 to quiescent NRK cell cultures was actinomycin D sensitive and resistant to cyclohexamide and puromycin, suggesting a primary mode of transcript control. The response to protein synthesis inhibitors, however, was complex. While cyclohexamide appeared to stabilize, or at least maintain, fetal bovine serum (FBS)‐ or TGF‐β1‐stimulated PAI‐1 mRNA levels, puromycin had no such affect. The amplitude and duration of induced PAI‐1 expression were the same in either the presence or absence of puromycin. Cyclohexamide when used alone (i.e., in non‐FBS‐ or TGF‐β1‐treated cultures), moreover, effectively stimulated PAI‐1 induction whereas puromycin was ineffective. Although TGF‐β1 was not a complete mitogen in the NRK cell system, incubation of quiescent renal cell cultures with TGF‐β1, prior to serum stimulation, resulted in a 10‐ to 12‐fold increase in PAI‐1 expression coincident with exit out of G0. These data support a model in which PAI‐1 gene expression is closely associated with creation of the growth‐activated state and that cell cycle controls appear to be superimposed on the time course of the serum‐induced expression of the PAI‐1 gene. J. Cell. Physiol. 181:96–106, 1999.

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. J Cell Physiol 170:8–18, 1997

Novel Combinatorial Therapeutic Targeting of PAI-1(SERPINE1) Gene Expression in Alzheimer’s Disease

Accumulation of neurotoxic amyloid peptides (Aβ) in the brain, generated by β-site proteolytic processing of the amyloid precursor protein (APP), is the hallmark pathophysiologic feature of Alzheimers disease. The plasmin-activating cascade, in which urokinase (uPA) and tissue-type (tPA) plasminogen activators convert plasminogen to the broad-spectrum protease plasmin, appears to serve a protective, Aβ-clearing, role in the central nervous system. Plasmin degrades Aβ and catalyzes α- site APP proteolysis generating nontoxic peptides. Plasmin activation in the brain is negatively regulated by the fast-acting clade E serine protease inhibitor (SERPIN) plasminogen activator inhibitor type-1 (PAI-1; SERPINE1) resulting in Aβ accumulation. PAI-1 and its major physiological inducer TGF-β1, moreover, are both increased in Alzheimers disease models and implicated in the etiology and progression of human neurodegenerative disorders. Current findings support the hypothesis that targeting of PAI-1 function (by small molecule drugs) and/or gene expression (by histone deacetylase inhibitors) may constitute a clinically-relevant molecular approach to the therapy of neurodegenerative diseases associated with increased PAI-1 levels.