Plasminogen Activator Inhibitor Type 1 mRNA Levels Research Articles

Changes in ovarian expression of tissue-type plasminogen activator and plasminogen activator inhibitor type-1 messenger ribonucleic acids during ovulation in rat.

We investigated the time course and localization of ovarian tissue-type plasminogen activator (tPA) and plasminogen activator inhibitor type-1 (PAI-1) expression during the ovulatory period in rat by RNase protection assay and in situ hybridization. Immature female Wistar rats were injected with 25 IU pregnant mare serum gonadotropin (PMSG), followed 50 h later by 25 IU human chorionic gonadotropin (hCG). Levels of tPA mRNA were low before hormone treatment and after PMSG treatment. After hCG treatment, tPA mRNA levels increased rapidly, the first peak at 4 h after hCG treatment and reached a maximum just prior to ovulation, 12 h later, before declining again. PAI-1 mRNA was barely detectable before hormone treatment but was transiently induced by hCG treatment, reaching peak levels after 4 h. Subsequently, PAI-1 mRNA levels decreased until early luteinization. The expression of tPA mRNA 4 h after hCG treatment occurred mainly in the follicular thecal-interstitial cells, but was barely detectable in the granulosa cells, whereas 12 h after hCG treatment it was maximal in the granulosa cells of the large follicles destined to ovulate. PAI-1 mRNA was expressed mainly in ovarian stromal tissue and in the thecal external interstitial cells encapsulating the follicles at 4 h after hCG treatment. These results suggest that the temporal regulation of tPA biosynthesis after hCG induction depends on the cell types and size classes in the various ovarian compartments. PAI-1 may be produced by the stormal tissue and the thecal external interstitial cells and is perhaps implicated in structural changes during follicular growth, ovulation and luteinization.

Complex regulation of plasminogen activator inhibitor type-1 (PAI-1) gene expression by serum and substrate adhesion

Expression of plasminogen activator inhibitor type-1 (PAI-1), a member of the serine protease inhibitor (SERPIN) superfamily that functions to negatively regulate the plasmin-based pericellular proteolytic cascade, was induced early after exposure of growth-arrested normal rat kidney (NRK) cells to serum-containing medium. Increased PAI-1 transcription was rapid (evident within 10 min of serum addition) and involved immediate-early response kinetics. [3H]Thymidine autoradiography was used to map the time frame of PAI-1 expression during a synchronous growth cycle. PAI-1 transcript accumulation peaked in mid-G1 phase (approx. 4-6 h post-stimulation) and declined prior to, or concomitant with, the onset of DNA synthetic phase. Serum increased PAI-1 expression in NRK cells in agarose suspension, as well as monolayer, culture; induction in suspended cells (similar to monolayer culture conditions) also occurred in the presence of cyclohexamide or puromycin. The serum-inductive pathway leading to PAI-1 gene activation is thus functional regardless of adhesive conditions or capacity for de novo protein synthesis. The amplitude of induction and maintenance of expression in later stages of G1, however, were subject to adhesive influences. PAI-1 transcript accumulation at 4 and 8 h post-stimulation in newly adherent cells, moreover, was blocked by puromycin, indicating that both immediate-early and secondary mechanisms regulate PAI-1 mRNA levels during progression of NRK cells through an 'activated' G1 growth phase.

Leukoregulin induces plasminogen activator inhibitor type 1 in human orbital fibroblasts

Leukoregulin, a 50-kDa T lymphocyte-derived cytokine, influences the synthesis of collagenase, stromelysin-1, collagen, and hyaluronan in human fibroblasts and is thus a determinant of extracellular matrix economy. We studied the effect of leukoregulin on the expression of plasminogen activator inhibitor type 1 (PAI-1) in human orbital and dermal fibroblasts. The lymphokine upregulated 35S-labeled PAI-1 protein expression in orbital fibroblasts in dose-dependent manner. The effect on extracellular matrix-associated PAI-1 evolved over several hours and was maximal at 10 h, when levels were 75-fold higher than controls, and then fell by 24 h. Leukoregulin treatment increased prostaglandin E2 production in orbital cultures after 24 h. When this increase was blocked with indomethacin, peak PAI-1 levels were maintained. Northern analysis demonstrated a substantial induction of steady-state PAI-1 mRNA levels within 6 h of treatment in orbital cultures. In contrast, leukoregulin lowered PAI-1 protein levels dramatically in skin fibroblasts from the abdominal wall. With regard to PAI-1 expression, it would appear that the anatomic site of origin of fibroblasts is a crucial determinant of the cellular response to leukoregulin.

Stimulatory effect of unsaturated fatty acids on the level of plasminogen activator inhibitor-1 mRNA in cultured human endothelial cells

To determine whether unsaturated fatty acids induce changes in the mRNA level of plasminogen activator inhibitor type-1 (PAI-1), Northern analyses were performed on human umbilical vein endothelial cells (HUVEC) and vascular smooth muscle cells that were treated with two common fatty acids. Supplementation of cultured HUVEC with docosahexanoic acid (DHA) or with dihomogamma linolenic acid (DGLA), resulted in a concentration dependent, specific increase of the PAI-1 transcript levels, which was detectable within 2 h. DHA and DGLA treatment of smooth muscle cells did not result in changes in the PAI-1 mRNA levels. Homology search of the upstream regulatory region of the PAI-1 gene sequences identified a consensus nucleotide sequence for a fatty acid-responsive element. Our results indicate that unsaturated fatty acids selectively increase PAI-1 mRNA levels in endothelial cells, the primary source of circulating PAI-1 in vivo.

Overexpression of plasminogen activator inhibitor type‐1 in senescent fibroblasts from normal subjects and those with Werner syndrome

We previously reported that plasminogen activator inhibitor type-1 (PAI-1) mRNA was present at higher steady-state levels in prematurely senescent fibroblasts derived from a subject with Werner syndrome (WS) compared to early passage (EP) fibroblasts from an age-matched normal subject (Murano et al., 1991, Mol. Cell. Biol. 11:3905-3914). To explore the generally of this phenomenon with respect to chronological age of donor (in vivo aging) and the late-passage (LP) or senescent phase of the fibroblast replicative lifespan, we assayed PAI-1 mRNA in cells and PAI-1 antigen in medium conditioned by 20 normal fibroblast strains at EP and LP and six WS strains during their curtailed replicative lifespans. The lowest accumulations of PAI-1 were found in medium conditioned by fetal and newborn cells with a shallow but progressive rise seen in postnatal cells from normal donors of increasing chronological age. With few exceptions, normal LP fibroblasts showed increased PAI-1 accumulations in medium compared to their EP counterparts. Conditioned medium from four of the six WS strains showed PAI-1 accumulations that were significantly higher than the media of any normal controls at EP and LP. PAI-1 mRNa levels were generally commensurate with the cumulative amount of PAI-1 in the medium but the frequent exceptions indicate that translational and post-translational mechanisms also regulate PAI-1 output. The augmentation in PAI-1 output of fibroblasts as a direct function of chronological age and during in vitro senescence suggests that PAI-1 may play an important role in the reduced capacity for wound healing and the increasing tendency to thrombogenesis and atherogenesis seen during biological aging and in particular in persons with Werner syndrome.

Sodium butyrate differentially modulates plasminogen activator inhibitor type-1, urokinase plasminogen activator, and its receptor in a human colon carcinoma cell.

Human colonic epithelium is exposed to varying levels of sodium butyrate, which is derived from the bacterial fermentation of dietary carbohydrate. Sodium butyrate has several effects on colonic tumor cells in vitro, including arrest of cell growth and differentiation. In the present study we have found that, in addition to a reduction in cellular proliferation, sodium butyrate induces the transient expression of plasminogen activator inhibitor type-1 (PAI-1) in the LIM 2405 human colonic tumor cell. Approximately 40% of the PAI-1 secreted is biologically active as judged by the formation of higher molecular weight, SDS-resistant complexes with urokinase plasminogen activator (uPA). The enhanced PAI-1 biosynthesis was accompanied by an increase in PAI-1 mRNA levels. During the same time period, the amount of secreted uPA remained relatively constant, but the level of cell associated uPA decreased slowly and was accompanied by a decrease in uPA mRNA levels. The uPA receptor is synthesized constitutively by these cells, and was down-regulated at both the protein and mRNA levels in response to sodium butyrate. The results demonstrate that sodium butyrate can alter the balance of components of the plasminogen activator system in a manner which favours net decreased plasminogen activator activity and suggests a role for sodium butyrate in the regulation of extracellular proteolysis.

Induction of endothelial cell expression of the plasminogen activator inhibitor type 1 gene by thrombosis in vivo.

We have shown previously that products from activated platelets can augment synthesis of plasminogen activator inhibitor type 1 (PAI-1) in cultured endothelial and hepatoma (Hep G2) cells in vitro and increase plasma PAI-1 activity in vivo in rabbits. Accordingly, the effects of activation of platelets associated with thrombosis and thrombolysis in vivo on plasma PAI-1 activity and expression of the PAI-1 gene in endothelium, liver, and other organs were characterized. Endothelial injury giving rise to platelet-rich thrombi was induced with electrical stimulation in carotid arteries in rabbits. Clot lysis and recanalization were induced subsequently with intravenous tissue-type plasminogen activator (t-PA) and verified with Doppler flow probes. Plasma PAI-1 activity (mean +/- SD) increased from 6 +/- 2 arbitrary units (AU)/ml to 129 +/- 48 AU/ml (n = 15) within several hours after recanalization. When t-PA had failed to induce recanalization, the increase was much less (from 7 +/- 2 to 42 +/- 23 AU/ml, n = 11). To define mechanisms responsible for these changes, PAI-1 messenger RNA (mRNA) was evaluated by Northern blot analysis and localized in tissues by in situ hybridization. Strong and consistent induction of PAI-1 mRNA was evident in aorta, heart, and liver of animals subjected to thrombosis (twofold to threefold increases compared with values in controls), particularly in those in which thrombolysis had been induced (fourfold to sixfold). After thrombolysis, an intense, PAI-1 mRNA-specific signal was detected in endothelium of aorta, liver, and heart, with less intense signals in endothelium of lung, adrenals, and kidneys. The increases in plasma PAI-1 activity follow a preceding increase in endothelial cell expression of the PAI-1 gene as reflected by PAI-1 mRNA levels. Thus, increased synthesis of endothelial cell PAI-1 after thrombosis and thrombolysis may attenuate endogenous fibrinolysis early after coronary thrombolysis, thereby potentiating early, thrombotic reocclusion.

Glucocorticoid and Cyclic Nucleotide Regulation of Plasminogen Activator and Plasminogen Activator-Inhibitor Gene Expression in Primary Cultures of Rat Hepatocytes

Primary cultures of rat hepatocytes produce tissue-type plasminogen activator (tPA) and plasminogen activator-inhibitor type 1 (PAI-1). Incubation of hepatocytes with 50 microM 8-(4-chlorophenylthio)cAMP (CPT-cAMP) results in a 4-fold increase in tPA activity, whereas the synthetic glucocorticoid dexamethasone (1 microM) causes a more than 90% decrease. In combination, dexamethasone completely overcomes the CPT-cAMP effect and markedly decreases PA activity. PAI-1 is induced by both CPT-cAMP and dexamethasone, and the effects of these agents are additive. Accumulation of tPA mRNA is increased more than 4-fold by CPT-cAMP and is greatly decreased by incubation with dexamethasone. Dexamethasone in combination with CPT-cAMP totally blocks this cAMP effect. The protein synthesis inhibitor cycloheximide does not prevent either the dexamethasone-induced decrease or the CPT-cAMP-induced increase in tPA message and, in fact, augments the cAMP-induced increase in tPA mRNA. Hepatocyte PAI-1 mRNA levels are increased 2-fold by incubation with either CPT-cAMP or dexamethasone; in combination, these effectors cause a 4-fold increase in PAI-1 mRNA. Cycloheximide alone causes a marked increase in PAI-1 mRNA, but does not block the induction by either CPT-cAMP or dexamethasone. We conclude that incubation of hepatocytes with CPT-cAMP induces tPA activity by increasing tPA mRNA accumulation and that dexamethasone causes a decrease in tPA activity by both decreasing tPA mRNA and increasing PAI-1 mRNA and activity. Concomitant protein synthesis is not required for the regulation of tPA or PAI-1 mRNA by either CPT-cAMP or dexamethasone, indicating a primary effect of these agents on gene transcription or mRNA stability.