Posttranscriptional Regulation of the Plasminogen Activation System by Non-Coding RNA in Cancer.

Various species of non-coding RNAs (ncRNAs) may act as functional molecules regulating diverse biological processes. In cancer cell biology, ncRNAs include RNAs that regulate the expression of oncogenes and tumor suppressor genes through various mechanisms. The urokinase (uPA)-mediated plasminogen activation system (PAS) includes uPA, its inhibitors PAI-1 and PAI-2 and its specific cellular receptor uPAR; their increased expression represents a negative prognostic factor in several cancers. Here, we will briefly describe the main uPA-mediated PAS components and ncRNA species; then, we will review more recent evidence of the roles that ncRNAs may play in regulating the expression and functions of uPA-mediated PAS components in cancer.

Simulated in vitro hypoxic conditions from psoriatic arthritis cartilage change plasminogen activating system urokinase and serpine functionality. Reversal of antiapoptotic protection suggests common homeostatic buffering.

Introduction: Rheumatoid and psoriatic arthritis are both characterised by synovial destruction associated with a higher turnover of the extracellular matrix. In both conditions, inflammatory processes create hypoxic environments which destabilise members of the plasminogen activating system. Aim: Comparing the effect of bioactive concentrations of urokinase (uPA) and serpine (PAI-1) on cellular survival of human fibroblast-like-synoviocytes (HFLS) in rich and hypoxic growth media. Material and methods: Monocultures of HFLS were exposed to bioactive uPA and PAI-1 concentrations in both media conditions for 24, 48 and 72 h. Cellular survival was evaluated with a cell viability assay by spectrum absorbance of formazan reduced WST-8. Results: PAI-1 at 0.1 and 1 µg/ml was found to stimulate cell viability under hypoxic stress at 48 and 72 h of incubation, with the effect increasing from 48 to 72 h. uPA increased cell viability in rich medium at 48 and 72 h of incubation between 5 and 40 ng/l, but was found to reduce viability at 80 ng/l at 24 and 48 h. PAI-1 increased cell viability in the hypoxic stress model, while high concentrations of uPA decreased cell viability in rich medium. Conclusions: The alternative modes of function at extreme concentrations provide a novel description of PAI-1 and uPA activity based on their colocalization and mutual buffering capacity, helping to place these molecules more accurately in the context of arthritic synovial deterioration.

bFGF stimulated plasminogen activation factors, but inhibited alkaline phosphatase and SPARC in stem cells from apical Papilla: Involvement of MEK/ERK, TAK1 and p38 signaling.

INTRODUCTION: Basic fibroblast growth factor (bFGF) plays a critical role in odontoblast differentiation and dentin matrix deposition, thereby aiding pulpo-dentin repair and regeneration. OBJECTIVES: The purpose of this study was to clarify the effects of bFGF on plasminogen activation factors, TIMP-1), ALP; and SPARC (osteonectin) expression/production of stem cells from apical papilla (SCAP) in vitro; and the involvement of MEK/ERK, p38, Akt, and TAK1 signaling. METHODS: SCAP were exposed to bFGF with/without pretreatment and co-incubation with various signal transduction inhibitors (U0126, SB203580, LY294002, and 5Z-7-oxozeaenol). The expression of FGF receptors (FGFRs), PAI-1, uPA, p-ERK, p-TAK1, and p-p38 was analyzed via immunofluorescent staining. The gene expression and protein secretion of SCAP were determined via real-time PCR and ELISA. ALP activity was evaluated via ALP staining. RESULTS: SCAP expressed FGFR1, 2, 3, and 4. bFGF stimulated the PAI-1, uPA, uPAR, and TIMP-1 mRNA expression (p < 0.05). bFGF induced PAI-1, uPA, and soluble uPAR production (p < 0.05) but suppressed the ALP activity and SPARC production (p < 0.05) of SCAP. bFGF stimulated ERK, TAK1, and p38 phosphorylation of SCAP. U0126 (a MEK/ERK inhibitor) and 5Z-7-oxozeaenol (a TAK1 inhibitor) attenuated the bFGF-induced PAI-1, uPA, uPAR, and TIMP-1 expression and production of SCAP, but SB203580 (a p38 inhibitor) did not. LY294002, SB203580, and 5Z-7oxozeaenol could not reverse the inhibition of ALP activity caused by bFGF. Interestingly, U0126 and 5Z-7-oxozeaenol prevented the bFGF-induced decline of SPARC production (p < 0.05). CONCLUSION: bFGF may regulate fibrinolysis and matrix turnover via modulation of PAI-1, uPA, uPAR, and TIMP-1, but bFGF inhibited the differentiation (ALP, SPARC) of SCAP. These events are mainly regulated by MEK/ERK, p38, and TAK1. Combined use of bFGF and SCAP may facilitate pulpal/root repair and regeneration via regulation of the plasminogen activation system, migration, matrix turnover, and differentiation of SCAP.

Role of Plasminogen Activation System in Platelet Pathophysiology: Emerging Concepts for Translational Applications.

Traditionally, platelets have been exclusively considered for their procoagulant and antifibrinolytic effects during normal activation of hemostasis. Effectively, activated platelets secrete coagulation factors, expose phosphatidylserine, and promote thrombin and fibrin production. In addition to procoagulant activities, platelets confer resistance of thrombi to fibrinolysis by inducing clot retraction of the fibrin network and release of huge amounts of plasminogen activator inhibitor-1, which is the major physiologic inhibitor of the fibrinolytic cascade. However, the discovery of multiple relations with the fibrinolytic system, also termed Plasminogen Activation System (PAS), has introduced new perspectives on the platelet role in fibrinolysis. Indeed, the activated membrane surface of platelets provides binding sites on which fibrinolytic enzymes can be activated. This review discusses the evidence of the profibrinolytic properties of platelets through the description of PAS components and related proteins that are contained in or bind to platelets. Our analyses of literature data lead to the conclusion that in the initial phase of the hemostatic process, antifibrinolytic effects prevail over profibrinolytic activity, but at later stages, platelets might enhance fibrinolysis through the engagement of PAS components. A better understanding of spatial and temporal characteristics of platelet-mediated fibrinolysis during normal hemostasis could improve therapeutic options for bleeding and thrombotic disorders.

Roles of the tissue-type plasminogen activator in immune response.

The COVID-19 pandemic has once again brought to the forefront the existence of a tight link between the coagulation/fibrinolytic system and the immunologic processes. Tissue-type plasminogen activator (tPA) is a serine protease with a key role in fibrinolysis by converting plasminogen into plasmin that can finally degrade fibrin clots. tPA is released in the blood by endothelial cells and hepatocytes but is also produced by various types of immune cells including T cells and monocytes. Beyond its role on hemostasis, tPA is also a potent modulator of inflammation and is involved in the regulation of several inflammatory diseases. Here, after a brief description of tPA structure, we review its new functions in adaptive immunity focusing on T cells and antigen presenting cells. We intend to synthesize the recent knowledge on proteolysis- and receptor-mediated effects of tPA on immune response in physiological and pathological context.

Origin and diversification of the plasminogen activation system among chordates.

BACKGROUND: The plasminogen (PLG) activation system is composed by a series of serine proteases, inhibitors and several binding proteins, which together control the temporal and spatial generation of the active serine protease plasmin. As this proteolytic system plays a central role in human physiology and pathophysiology it has been extensively studied in mammals. The serine proteases of this system are believed to originate from an ancestral gene by gene duplications followed by domain gains and deletions. However, the identification of ancestral forms in primitive chordates supporting these theories remains elusive. In addition, evolutionary studies of the non-proteolytic members of this system are scarce. RESULTS: Our phylogenetic analyses place lamprey PLG at the root of the vertebrate PLG-group, while lamprey PLG-related growth factors represent the ancestral forms of the jawed-vertebrate orthologues. Furthermore, we find that the earliest putative orthologue of the PLG activator group is the hyaluronan binding protein 2 (HABP2) gene found in lampreys. The prime plasminogen activators (tissue- and urokinase-type plasminogen activator, tPA and uPA) first occur in cartilaginous fish and phylogenetic analyses confirm that all orthologues identified compose monophyletic groups to their mammalian counterparts. Cartilaginous fishes exhibit the most ancient vitronectin of all vertebrates, while plasminogen activator inhibitor 1 (PAI-1) appears for the first time in cartilaginous fishes and is conserved in the rest of jawed vertebrate clades. PAI-2 appears for the first time in the common ancestor of reptiles and mammals, and represents the latest appearing plasminogen activator inhibitor. Finally, we noted that the urokinase-type plasminogen activator receptor (uPAR)-and three-LU domain containing genes in general-occurred later in evolution and was first detectable after coelacanths. CONCLUSIONS: This study identifies several primitive orthologues of the mammalian plasminogen activation system. These ancestral forms provide clues to the origin and diversification of this enzyme system. Further, the discovery of several members-hitherto unknown in mammals-provide new perspectives on the evolution of this important enzyme system.

Characterising the Subsite Specificity of Urokinase-Type Plasminogen Activator and Tissue-Type Plasminogen Activator using a Sequence-Defined Peptide Aldehyde Library.

Urokinase-type plasminogen activator (uPA) and tissue-type plasminogen activator (tPA) are two serine proteases that contribute to initiating fibrinolysis by activating plasminogen. uPA is also an important tumour-associated protease due to its role in extracellular matrix remodelling. Overexpression of uPA has been identified in several different cancers and uPA inhibition has been reported as a promising therapeutic strategy. Although several peptide-based uPA inhibitors have been developed, the extent to which uPA tolerates different tetrapeptide sequences that span the P1-P4 positions remains to be thoroughly explored. In this study, we screened a sequence-defined peptide aldehyde library against uPA and tPA. Preferred sequences from the library screen yielded potent inhibitors for uPA, led by Ac-GTAR-H (Ki =18 nm), but not for tPA. Additionally, synthetic peptide substrates corresponding to preferred inhibitor sequences were cleaved with high catalytic efficiency by uPA but not by tPA. These findings provide new insights into the binding specificity of uPA and tPA and the relative activity of tetrapeptide inhibitors and substrates against these enzymes.

Priming the seed: Helicobacter pylori alters epithelial cell invasiveness in early gastric carcinogenesis.

Helicobacter pylori (H. pylori) infection is a well-established risk factor for the development of gastric cancer (GC), one of the most common and deadliest neoplasms worldwide. H. pylori infection induces chronic inflammation in the gastric mucosa that, in the absence of treatment, may progress through a series of steps to GC. GC is only one of several clinical outcomes associated with this bacterial infection, which may be at least partially attributed to the high genetic variability of H. pylori. The biological mechanisms underlying how and under what circumstances H. pylori alters normal physiological processes remain enigmatic. A key aspect of carcinogenesis is the acquisition of traits that equip preneoplastic cells with the ability to invade. Accumulating evidence implicates H. pylori in the manipulation of cellular and molecular programs that are crucial for conferring cells with invasive capabilities. We present here an overview of the main findings about the involvement of H. pylori in the acquisition of cell invasive behavior, specifically focusing on the epithelial-to-mesenchymal transition, changes in cell polarity, and deregulation of molecules that control extracellular matrix remodeling.

Relevance of the plasminogen activation system in the pathogenesis and progression of gastric cancer / La importancia del sistema activador de plasminógeno en la patogénesis y progresión del cáncer gástrico

Abstract Gastric cancer is ranked as the third death-causing cancer and one of the most incident malignancies worldwide. AlthoughHelicobacter pyloriis the most well-established risk factor for the development of this neoplasm, most of the infected individuals do not develop gastric cancer. Two of the main challenges faced by the world's scientific community in the combat against gastric cancer are the unraveling of its pathogenesis and the identification of novel ways to bring down the mortality. Malignant cell invasion of the non-neoplastic adjacent tissue and metastasis are pivotal events during cancer development and progression. Both processes are facilitated by proteases capable of degrading components of the extracellular matrix, some of which have been associated to clinic-pathological aspects of the disease. Recent studies have suggested the possible connection betweenH. pyloriand the expression of some of these proteases in gastric mucosa. This review summarizes the current knowledge about epidemiological, clinical and biological aspects of gastric cancer; it also discusses the main findings about the involvement of the plasminogen activation system in the development and progression of this disease, as well as its potential repercussions in the clinical setting. Rev. Biol. Trop. 66(1): 28-47. Epub 2018 March 01.

Resumen El cáncer gástrico es la tercera causa de muerte por cáncer a nivel mundial y uno de los más incidentes. A pesar de que la infección porHelicobacter pylories el factor de riesgo más reconocido para el desarrollo de esta neoplasia, la mayoría de personas infectadas con la bacteria no desarrolla la enfermedad. Dos de los principales desafíos a los que actualmente se enfrenta la comunidad científica mundial en la lucha contra el cáncer gástrico son el esclarecimiento de la patogénesis y la identificación de nuevos parámetros que contribuyan a disminuir la mortalidad. La invasión de las células malignas al tejido no neoplásico adyacente y la metástasis son eventos claves durante el desarrollo y progresión del cáncer. Lo anterior es facilitado por proteasas capaces de degradar los componentes de la matriz extracelular, algunas de las cuales han sido asociadas con aspectos clínico-patológicos de la enfermedad. Estudios recientes han sugerido la posible relación entre la bacteriaH. pyloriy la inducción en mucosa gástrica de algunas de estas proteasas. Esta revisión resume conocimientos actuales sobre aspectos epidemiológicos, clínicos y biológicos del cáncer gástrico; también discute los principales hallazgos en torno a la participación del sistema activador de plasminógeno en el desarrollo y progresión del mismo, así como sus potenciales repercusiones en la práctica clínica.

Amorphous protein aggregates stimulate plasminogen activation, leading to release of cytotoxic fragments that are clients for extracellular chaperones.

The misfolding of proteins and their accumulation in extracellular tissue compartments as insoluble amyloid or amorphous protein aggregates are a hallmark feature of many debilitating protein deposition diseases such as Alzheimer's disease, prion diseases, and type II diabetes. The plasminogen activation system is best known as an extracellular fibrinolytic system but was previously reported to also be capable of degrading amyloid fibrils. Here we show that amorphous protein aggregates interact with tissue-type plasminogen activator and plasminogen, via an exposed lysine-dependent mechanism, to efficiently generate plasmin. The insoluble aggregate-bound plasmin is shielded from inhibition by α2-antiplasmin and degrades amorphous protein aggregates to release smaller, soluble but relatively hydrophobic fragments of protein (plasmin-generated protein fragments (PGPFs)) that are cytotoxic. In vitro, both endothelial and microglial cells bound and internalized PGPFs before trafficking them to lysosomes. Clusterin and α2-macroglobulin bound to PGPFs to significantly ameliorate their toxicity. On the basis of these findings, we hypothesize that, as part of the in vivo extracellular proteostasis system, the plasminogen activation system may work synergistically with extracellular chaperones to safely clear large and otherwise pathological protein aggregates from the body.

Leucocyte expression of genes implicated in the plasminogen activation cascade is modulated by yoghurt peptides.

The urokinase-plasminogen activator (u-PA), its receptor (u-PAR) and the inhibitors of u-PA (PAI-1 and PAI-2) provide a multi-molecular system in leucocytes that exerts pleiotropic functions influencing the development of inflammatory and immune responses. The objective of the present study was to examine the ability of water soluble extracts (WSE) obtained from traditional Greek yoghurt made from bovine or ovine milk to modulate the expression of u-PA, u-PAR, PAI-1 and PAI-2 in ovine monocytes and neutrophils. WSE were obtained from 8 commercial traditional type Greek yoghurts made from ovine or bovine milk. WSE upregulated the expression of all 4 u-PA related genes in monocytes but the upregulation was much higher in the PAI-1 (10-fold) than in u-PA and u-PAR (3-4 fold) thus, shifting the system towards inhibition. In line with this observation, WSE reduced total and membrane-bound u-PA activity in monocytes. In neutrophils, WSE caused small (50-60%) but significant (P < 0·05) reductions in expression of u-PAR and PAI-2 but had no effect on expression of u-PA, PAI-1 and on total cell-associated and membrane-bound u-PA activity. WSE from yoghurts made from bovine or ovine milk were essentially equally effective in affecting the u-PA system except for the u-PAR gene in ovine neutrophils that was affected (reduced) by the ovine and not the bovine WSE. In conclusion, peptides present in WSE modulated the expression of u-PA related genes but the effect was much more prominent in monocytes than in neutrophils.

Plasminogen Activation System in Rectal Adenocarcinoma.

BACKGROUND: The purpose of the present study was to determine the extent of expression of proteins that are the elements of the plasminogen activation system, i.e. urokinase plasminogen activator (uPA), receptor of uPA (uPAR), plasminogen activating inhibitor-1 (PAI-1) and tissue plasminogen activator (tPA) in the primary site of rectal adenocarcinoma, as well as in lymph node metastases, and their correlation with clinical and pathological data. MATERIALS AND METHODS: The study material consisted of archival paraffin blocks from 108 patients with confirmed oncological diagnosis, treated in 2002 and 2003 in the Lower Silesian Oncology Centre in Wroclaw. This material was used for immunohistochemical reactions with antibodies against uPA, uPAR, PAI-1 and tPA. The extent of expression was evaluated semi-quantitatively based on the immunoreactive score according to Remmele and Stegner. The obtained results were correlated with clinical and pathological data: stage of the disease (modified Dukes' classification of Astler and Coller), grade of histological malignancy, event-free survival and overall survival. RESULTS: It was found that cytoplasmic expression of uPA, uPAR, PAI-1 and tPA was higher in cells of tumor and metastases of perienteric lymph nodes in comparison to normal rectal tissues. Positive correlation was shown between expression of PAI-1 and tPA, uPA and uPAR, as well uPA and PAI-1 (r=0.70, r=0.77 and r=0.34, respectively; p<0.05 for all). Additionally, in patients with low and moderate expression of uPA and uPAR, the overall survival rate was higher in comparison to patients with high expression of the studied markers. CONCLUSION: The intensity of expression of uPA and uPAR might be a prognostic factor of survival time for patients with primary rectal adenocarcinomas.

SerpinB2 mediated regulation of macrophage function during enteric infection.

Host defense is an orchestrated response involving changes in the expression of receptors and release of mediators from both immune and structural cells. There is a growing recognition of the important role of proteolytic pathways for the protective immune response to enteric pathogens. Enteric nematode infection induces a type 2 immune response with polarization of macrophages toward the alternatively activated phenotype (M2). The Th2 cytokines, IL-4, and IL-13, induce a STAT6-dependent upregulation of the expression of the protease inhibitor, serpinB2, which protects macrophages from apoptosis. M2 are critical to worm clearance and a novel role for serpinB2 is its regulation of the chemokine, CCL2, which is necessary for monocyte and/or macrophage influx into small intestine during infection. There is a growing list of factors including immune (LPS, Th2 cytokines) as well as hormonal (gastrin, 5-HT) that are linked to increased expression of serpinB2. Thus, serpinB2 represents an immune regulated factor that has multiple roles in the intestinal mucosa.

Angiogenin interacts with the plasminogen activation system at the cell surface of breast cancer cells to regulate plasmin formation and cell migration.

Angiogenin (ANG), a 14-kDa pro-angiogenic secreted protein, has been shown to play a role in cell migration and tumor invasion, which involve proteolytic cleavage of plasminogen to generate plasmin. However, the mechanism by which ANG regulates plasmin formation and cell migration was not known. Our studies here detected elevated levels of secreted and cell surface-bound ANG in highly invasive metastatic breast cancer cells. ANG was also detected at very high levels in the tumor cells in infiltrating ductal carcinomas. By immunofluorescence and immunoprecipitation analysis, ANG was detected at the leading edges of the cell surfaces where it colocalized and interacted with members of the plasminogen activation system (PAS) such as annexin A2 (A2), calpactin (S100-A10) and urokinase plasminogen activator receptor (uPAR). Analysis of lipid raft (LR) and non-lipid raft (NLR) regions of the cell membranes showed the predominance of ANG, A2 and S100-A10 in the LR regions. In contrast, uPAR was detected predominantly in the NLR fractions, suggesting that ANG interacts with uPAR at the junctions of LR and NLR regions. ANG knockdown in T47D and MDA-MB-231 breast cancer cell lines did not affect the cellular expression of A2, S100-A10 and uPAR but decreased cell migration and plasmin formation. Neutralization of ANG with monoclonal antibodies similarly decreased the migration of MDA-MB-231 cells. In the presence of ANG, uPAR was observed to interact with uPA, which is necessary for plasmin formation. Conversely, in the absence of ANG, uPAR did not interact with uPA and FAK and Src kinases were observed to be dephosphorylated. Exogenous addition of recombinant ANG to ANG knocked down MDA-MB-231 cells restored FAK phosphorylation, uPAR interactions with uPA, plasmin formation as well as migration of these cells. Taken together, our results identified a novel role for ANG as a member of the uPAR interactome that facilitates the interaction of uPAR with uPA, leading to plasmin formation and cell migration necessary for tumor invasion and metastasis of breast cancer cells.

High concentration of urokinase-type plasminogen activator receptor in the serum of women with primary breast cancer.

AIM OF THE STUDY: The purpose of this study was to assess the concentration of urokinase-type plasminogen activator receptor (uPAR) in the serum of 103 women with breast cancer. Commonly recognized prognostic factors were taken into account, including age, histological grade of malignancy, stage of clinical advancement of the disease, status of local axillary lymph nodes and the size of the primary tumour. MATERIAL AND METHODS: The concentration of uPAR was assessed using an enzyme-linked immunosorbent assay (R&D Systems). RESULTS: The concentration of uPAR in women with breast cancer was found to be higher than in a control group and the difference was statistically significant. The concentration of uPAR was found to increase in line with increasing disease stage and this too was of statistical significance. Raised levels of uPAR were found in women with breast cancer both with and without metastases to the lymph nodes of the axilla. A positive relationship was also found between the concentration of the tested receptor and the size of the primary tumour. No significant relationship, however, was found between the concentration of uPAR and the histological grade of malignancy of the tumour. No statistically significant results were obtained regarding the menopausal status of the women, that is, whether they were pre- or post-menopausal. CONCLUSIONS: Concentration of uPAR in serum of women with breast cancer is positively correlated with the stage of advancement of the disease. Thus, the assessment of this parameter can be useful in the clinical evaluation of women with breast cancer.