Plasminogen activator inhibitor 1: mechanisms of its synergistic regulation by growth factors

As the major physiological inhibitor of plasminogen activator (PA), the type I plasminogen activator inhibitor (PAI-1) is important for controlling blood clotting and tissue remodeling events such as those that involve cell migration. The changing needs for protease activity under various physiological conditions necessitates a tight cellular control of gene expression to enable rapid changes in the levels of PAI-1 and PA activity. Cooperation between epidermal growth factor (EGF) and transforming growth factor-β (TGF-β) dramatically increase PAI-1 protein level. In this study, we found activators of tyrosine kinase (EGF, FGF, IGF-I and TNF α) or an activator of PKC (PMA) synergize with TGF- β in stimulation of PAI-1. The mechanism by which EGF and TGFyβ synergistically increase PAI-1 expression was explored. TGF-β increases the sensitivity of the cells to EGF, thereby recruiting EGF at suboptimal concentrations to contribute to the synergistic activation of PAI-1. The contribution of EGF to the regulation of PAI involves the MAPK pathway and the synergistic interface with the TGF-β pathway is downstream of MEK and neither involves phosphorylation of Erk1/2 nor Smad2/3. EGF and TGF-β synergistically increase PAI-1 transcription that involves cooperation between transcription factors Smad and AP-1. This increase of PAI-1 mRNA is further amplified by a decrease in the rate of mRNA degradation, the latter being regulated only by EGF. This work demonstrates the existence of a multidimensional cellular mechan€ism by which EGF and TGF-β are able to promote large and rapid changes in PAI-1 expression. Identification of regions on mRNAs that are accessible for hybridization in the living cell is required for antisense and RNAi technologies and for accurate prediction of authentic targets of microRNAs. Although the sequence of an mRNA holds some useful information, the secondary and tertiary RNA structure determines access for hybridization. Because in vivo assays are very time consuming and expensive, computational and cell culture analysis have been utilized with varying success for identifying effective oligonucleotide inhibitors. With the advent of microarray spotters and readers as standard equipment in many research facilities, this approach to analyze RNA for accessible regions is worthy of investigation for its application to identify antisense and RNAi targets. We have utilized a tiled microarray format to investigate the accessible regions in two mRNAs (Bcl2 and Lcn2) and compared this with cell culture and computational approaches for predicting accessible targets for antisense oligodeoxynucleotides (ODNs) and siRNAs. Our results suggest that a tiled microarray is an effective means of identifying the mRNA regions that are accessible to antisense ODNs and RNAi

The role of the plasminogen activator system in the etiology of ovarian follicular cysts in dairy cattle

Cystic ovarian disease (COD) is an anovulatory condition in cattle that afflicts between 6-18% of all dairy cows in the US. Ovulation is dependent on the plasminogen activator (PA) family of proteases and protease inhibitor for proteolysis, culminating in follicular rupture. Failure of the follicle to ovulate suggests an aberration in proteolysis in cystic follicles. Polycystic ovarian disease in humans is phenotypically similar to COD is associated with elevated plasma PAI-1. PAI-1 regulates the proteolytic activity responsible for ovulation. Vitamin E supplementation reduces plasma PAI-1 and reduces the incidence of COD. Four experiments were conducted to elucidate the role of the plasminogen activator system in the etiology of COD and the ability of vitamin E supplementation to modulate the PA system in cattle. In experiment one, blood samples were collected from lactating dairy cows upon diagnosis of a follicular cyst at least 3.0 cm in diameter by a licensed veterinarian. Cows were classified based upon their history of follicular cysts. Tissue-type plasminogen activator (tPA) and plasminogen activator inhibitor-1 (PAI-1) concentrations and activities were measured using commercially available ELISA and chromogenic reagents. No differences were detected between cystic and non-cystic dairy cows. In experiment two, six non-lactating beef cows were supplemented with 2750 IU of α-tocopherol every four days for 24 days. Blood samples were collected every two days and assayed for tPA and PAI-1 concentrations and activities. Supplementation with α- tocopherol decreased the ratio of PAI-1 to tPA activities adjusted to the Day 0 measurement (P=0.097). In experiment three, mRNA was isolated from follicular cysts and preovulatory follicles and relative quantitative RT-PCR was performed for tPA, urokinase-type plasminogen activator (uPA), urokinase-type plasminogen activator receptor (uPAR), PAI-1, and β-actin. uPA expression was reduced (P<0.1) and uPAR expression was greater (P<0.1) in follicular cysts compared to preovulatory follicles. Lastly, in experiment four DNA was extracted from whole blood obtained from cystic and normal dairy cows and the promoter region of the PAI-1 gene was sequenced. More Jersey cows with COD possessed a four basepair deletion polymorphism than normal cows (P<0.1) and more Jerseys possessed the deletion polymorphism than Holsteins (P<0.01)

The effect of exercise on PAI-1 and other markers of the insulin resistance syndrome in overweight & obese individuals : the impact of work stressors and other predictors

Introduction: Obesity, and in particular central fat accumulation, is associated with a number of metabolic disturbances such as dyslipidaemia and insulin resistance. Such `clustering' of factors is known as the Insulin ResistanceS yndrome( IRS). More recently, hypofibrinolysisa s a resulto f elevated concentrations of PAI-1 at rest has been included in the IRS. Acute exercise in normal weight individuals results in an increase in fibrinolytic capacity due to a rise in t-PA and a reduction in PAI-1 concentrations. The primary aim of the following studies therefore was to determine the effect of acute exercise and exercise training on fibrinolytic markers in obese populations. The secondary aim of this work was to examine the relationships between PAI-1 concentrations and other markers of the IRS to determine a potential role for these factors in the short-term regulation of plasma PAI-1 concentrations. Methods: Premenopausal female and male overweight and obese volunteers underwent acute bouts of exercise at intensities ranging from 50% to 100% V02 max A group of obese premenopausal omen also underwent a graded maximal exerciset est to exhaustionb efore and after 12 weeks of exercise training at 50% or 70% VO2 max. Venous blood samples were taken at rest, immediately post exercise and up to 2 hours post exercise. Samples were analysed for fibrinolytic factors (t-PA, total PAI-1 & active PAI-1), markers of endothelial damage (vWF) as well as other components of the IRS including lipid profiles, insulin and leptin. Results: Plasma t-PA concentrations rose with acute exercise at intensities greater than 50% V02 max in all study populations with the exception of obese sedentary males. In all cases t-PA returned to baseline levels 30 minutes post exercise. None of the protocols administered were sufficient to lower total PAI-1 concentrations immediately post exercise but exercise at an intensity of 70% V02 max and a duration of greater than 30 minutes resulted in elevated PAI-1 concentrations 30 minutes post exercise in the overweight and obese populations. Active PAI-1 concentrations decreased with exercise either immediately or within 30 minutes post exercise at an intensity of 70% V02 max and durations greater than 30 minutes. Exercise training at both 50% and 70% V02 max increased the t-PA response to maximal exercise but only exercise training at 70% V02 max resulted in greater decrease in active PAI-1 with exercise. No factors were consistently associated with PAI-1 throughout the studies although anthropometric measures and blood pressure were regularly associated with PAI-1 at rest. Conclusions: Overall, exercise at an intensity of 70% V02 max for duration of at least 30 minutes in obese populations results in an increased fibrinolytic capacity as shown by elevated t- PA concentrations and decreased active PAI-1 concentrations. It is important to remember however that obese populations still remain hypofibrinolytic with respect to non-obese populations at rest, during exercise and in the recovery period

THE ROLE OF MIR-340 IN POST-TRANSCRIPTIONAL REGULATION OF THE UPA-SYSTEM IN BREAST CANCER

The urokinase-type plasminogen activator system (uPA-system), whose main components are the serine protease uPA (PLAU), the cell surface receptor uPAR (PLAUR) and the uPA inhibitor PAI-1 (SERPINE1), has been extensively studied for its involvement in cancer pathogenesis. Specifically, nowadays the components of the uPA-system are well-characterised determinants for the prognosis of breast cancer. The regulation of the gene expression of the uPA-system components is very complex and depends on a plethora of stimuli acting both at transcriptional and post-transcriptional level. The uPA-system components are often over expressed in breast cancer but the detailed molecular mechanisms regulating the expression are still to uncover. In an expression analysis conducted on a cohort of unselected breast cancer patients, we found that the expression of PLAU and PLAUR is highly correlated. Meta-analyses of published experimental data and in silico studies pointed out the possibility that PLAU, PLAUR and also SERPINE1 might be negatively regulated at post-transcriptional level by a microRNA, the miR-340. We experimentally validated the role of miR-340 as negative regulator of the expression of the three uPA-system components using MDA-MB-231, a triple negative breast cancer cell line. Microarray experiments, performed to characterise the global transcriptome changes induced by miR-340 in MDA-MB-231 cells, showed that miR-340 down regulates also the expression of desmoplastic reaction-related genes underlining a possible role of miR-340 in regulating tumour-associated genes. Notably, most of the identified miR-340 target genes were found indeed to be associated with poor clinical outcome in breast cancer. Functional studies carried out in MDA-MB-231 cells suggested that miR-340 might modulate cell proliferation, even if this effect was not confirmed in vivo. In order to better define the functional role of miR-340, we generated a miR-340 deficient mouse model, taken advantage of the zinc finger nuclease technology. Overall these data identify, for the first time, a single microRNA that is able to down regulate the expression of the three main components of the uPA-system together with desmoplastic reaction and breast cancer prognosis-related genes, thus representing a new potential player in the pathogenesis of breast cancer