uPA-deficient Mice Research Articles

Urokinase-type plasminogen activator promotes corneal epithelial migration and nerve regeneration

Urokinase-type plasminogen activator (uPA) is a serine protease that plays a central role in the pericellular fibrinolytic system, mediates the degradation of extracellular matrix proteins and activation of growth factors, and contributes to the regulation of various cellular processes including cell migration and adhesion, chemotaxis, and angiogenesis. The corneal epithelium responds rapidly to injury by initiating a wound healing process that involves cell migration, cell proliferation, and tissue remodeling. It is innervated by sensory nerve endings that play an important role in the maintenance of corneal epithelial homeostasis and in the wound healing response. We here investigated the role of uPA in corneal nerve regeneration and epithelial resurfacing after corneal injury with the use of uPA-deficient mice. Both the structure of the corneal epithelium and the pattern of corneal innervation in uPA−/− mice appeared indistinguishable from those in uPA+/+ mice. Whereas the cornea was completely resurfaced by 36–48 h after epithelial scraping in uPA+/+ mice, however, such resurfacing required at least 72 h in uPA−/− mice. Restoration of epithelial stratification was also impaired in the mutant mice. Fibrin zymography revealed that the expression of uPA increased after corneal epithelial scraping and returned to basal levels in association with completion of re-epithelialization in wild-type animals. Staining of corneal whole-mount preparations for βIII-tubulin also revealed that the regeneration of corneal nerves after injury was markedly delayed in uPA−/− mice compared with uPA+/+ mice. Our results thus demonstrate an important role for uPA in both corneal nerve regeneration and epithelial migration after epithelial debridement, and they may provide a basis for the development of new treatments for neurotrophic keratopathy.

MO266: Deficiency of Urokinase-Type Plasminogen Activator Exacerbates CBSA-Induced Membranous Nephropathy in Mice

Abstract BACKGROUND AND AIMS In nondiabetic population, primary membranous nephropathy (PMN) is the most common cause of idiopathic nephrotic syndrome in the world. Urokinase-type plasminogen activator (uPA) is one of the key members of fibrinolysis system, which functions as dissolving blood clots to prevent obstruction of blood vessels. Currently, the roles of uPA in PMN remain undefined. METHOD Six-week-old male mice with Plau wild-type and knockout in BALB/c strain were used to induce MN by cationic bovine serum albumin (cBSA). Biochemistry assays of blood and urine were measured by a Fuji Dri-Chem FDC NX 500 machine. Glomerular polyanions were determined by using Colloidal Iron Stain Kit. Reactive oxygen species (ROS) was detected by immunohistochemistry stained by 4-HNE antibody. TUNEL positivity in the kidneys of mice was determined using an ApopTag® Plus Peroxidase In Situ Apoptosis Kit. The deposition of immune complex in glomeruli was examined by a transmission electron microscope. Lymphocyte subsets were determined by flow cytometry. Serum concentrations of IgG1 and IgG2a were measured by ELISA. RESULTS It has been reported that BALB/c mice is an ideal strain for mimicking human PMN. Thus, we generated uPA deficient mice (Plau-/-) in BALB/c strain by backcrossing C.129S2-Plautm1Mlg/J mice with BALB/c wild-type mice to N10. Plau-/- mice exhibited severe urinary albumin-to-creatinine ratio significantly when compared with wild-type mice at 2 weeks post-cBSA administration. Consistently, cBSA-induced Plau-/- mice displayed higher serum creatinine, hypoalbuminemia and hypercholesterolemia than wild-type mice. Histologically, Plau-/- mice presented more severed glomerular basement thickening, IgG granular deposition, intensified podocyte effacement, irregular thickening of glomerular basement membrane and subepithelial deposits than wild-type mice. Attenuated colloidal iron stain was observed in Plau-/- mice when compared with wild-type mice. Moreover, elevated ROS and apoptosis in the kidneys of Plau-/- mice were observed. B lymphocyte subsets were enriched significantly in the Plau-/- mice with MN, suggesting that pathological aggravation of MN is associated with dominant B lymphocyte population. The enriched ratio of IgG1 to IgG2a further confirmed predominant Th2 subset in Plau-/- mice. CONCLUSION uPA deficiency enhances Th2 dominant immune response, leading to enhancement of ROS and apoptosis in the kidneys, which subsequently exacerbates the progression of MN. Maintenance of uPA homeostasis may be a promising therapeutic approach for MN management.

Specificity of aminergic status in skin and tumor of C57BL/6-PlautmI.IBug-This Plau6FDhu/GFDhu mice with B16/F10 melanoma

Background. Systems of plasminogen activation and biogenic amines are involved in carcinogenesis, but their relationship has not been established.
 Aim – study of the quantitative specificity of biogenic amines in the skin and melanoma in urokinase-gene knockout mice.
 Materials and methods. Levels of catecholamines, histamine, serotonin and 5-hydroxyindoleacetic acid (HIAA) were determined by ELISA in the skin and В16/F10 melanoma of urokinase (uPA) -gene knockout mice of both genders (n=24); С57ВL/6 mice (n=64) were controls (C).
 Results. Differential characteristics of melanoma development in uPA-deficient mice included: an earlier onset of the primary tumor and its slow growth, more pronounced in females, in combination with hemorrhages in the lungs of males and solitary metastases in the lungs of females. This was facilitated by higher levels of norepinephrine in the skin of males/females – by 4.8/4.9 times, histamine – by 3.6/1.7 (p<0.05) times and serotonin – by 3.4/8.3 times. Dopamine accumulated in melanoma in all uPA-gene knockout mice: in females –1.6 times (p<0.05), in males – 2.1 times higher than in intact skin, with a 2.5 times reduction of norepinephrine in females. Levels of histamine decreased, but exceeded controls: in females – by 1.8 times (p<0.05), in males –by 3.5 times. Levels of serotonin in uPA-deficient females were as high, while in males they were 3.4 times lower than in intact skin.
 Conclusions. The specificity of the aminergic system in the skin of uPA-gene knockout mice demonstrated the inhibition of local stress and contributed to the reduction of malignant potential of melanoma by increasing immune properties of the skin.

System of plasminogen regulators in the skin of intact uPA gene-knockout C57BL/6-PlautmI.IBug-This Plau6FDhu/GFDhu mice and in a model of chronic neurogenic pain

Purpose of the study . Studying the components of the cascade of plasminogen (PG) regulator in the skin of intact uPA (urokinase plasminogen activator) gene-knockout mice and in a model of chronic neurogenic pain (CNP). Materials and methods . The study included 76 mice divided in groups depending on the gender, presence or absence of uPA gene knockout and CNP. A CNP model was created by sciatic nerve ligation; levels and activity of components of PG activation cascade were studied by ELISA using standard test systems. Results . Intact uPA -deficient C57BL/6-PlautmI.IBug-ThisPlau6FDhu/GFDhu mice differed from C57BL/6 mice by a decreased level of PG regulators, excluding plasmin in all mice, PG in males and urokinase receptor (uPAR). uPA-deficient mice with CNP showed continuing decrease in levels of plasminogen regulators, except for uPAR in all mice, PG in males and the activity of plasminogen activator inhibitor-1 (PAI-1-act) in females. Conclusion . Almost the entire cascade of PG regulators was inhibited in intact uPA-deficient mice of both genders, compared to C57BL/6 animals, except for uPAR in females, PG in males and plasmin in all animals. In CNP, PG regulators in uPA-deficient mice were inhibited compared to the background, excluding uPAR in males and females. The comparison with C57BL/6 mice demonstrated that the CNP model resulted in the uPA system state very similar to that in intact uPA-deficient C57BL/6-PlautmI.IBug-ThisPlau6FDhu/GFDhu animals. In C57BL/6 mice with CNP, levels of uPA were reduced by 2 orders of magnitude, relative to the norm, with the almost complete depletion of uPA activity. uPA gene knockout results in a persistent suppression of the system of PG regulators which is practically not affected by CNP, except for the uPAR level, and causes changes similar to that in the CNP modeling in normal mice.

The urokinase gene knockout effects on growth factor dynamics in mice with melanoma, developing on the background of chronic neurogenic pain

Purpose of the study . Studying the dynamics of growth factors (GF) in urokinase (uPA)-deficient mice with chronic neurogenic pain (CNP) and B16/F10 melanoma. Materials and methods . Levels of VEGFA, VEGFC, sVEGFRl, sVEGFR3, IGF1, IGF2, TGFp1 and FGF21 were determined by ELISA in tumors, perifocal tissues (PT) and the skin of male and female С 57 BL/6 mice (with a normal genome, n = 75) and C57BL/6-Plautm1.1BugThisPlauGFDhu/GFDhu mice (uPA-deficient animals, n = 46) at 3nd week of the carcinogenesis and CNP. Results . The skin of intact uPA-deficient mice demonstrated higher GF levels than in C57BL/6 mice, but VEGF- А and TGF-p1 in males (unlike females) were 4.4 and 5 times lower than in C57BL/6 males. This changes were generally similar in the skin of C57BL/6 mice with CNP. uPA-deficient females showed elevated GF in PT, especially VEGF- А and IGF1 — by 21.5 and 8.1 times, respectively in simultaneously CNP and growth of the melanoma. uPA gene-knockout males had similar changes in GF, although less marked. The levels of all studied GF in tumor tissue were lower than levels in PT in both males and females, except for VEGFA in males — 5.6 times higher in tumor tissue. Changes in PT of C57BL/6 mice were similar: maximally increased levels of all GF, especially VEGF, IGF and TGF-p1 — in females on average by 6.2, 15.9 and 5.5 times, respectively, in males by 9.4, 5.9 and 6.7 times, respectively, compared to the skin levels. While the absolute values of GF concentrations and the intensity of changes were higher than in uPA-deficient mice. Conclusion . In general, skin levels of GF in intact uPA-deficient mice were similar to the levels in mice without uPA-deficient with CNP. The GF dynamics was analogous in mice of both lines at simultaneously CNP and growth of the melanoma, but the intensity of changes in mice without uPA-deficient was significantly higher implying a synergic effect of CNP and paracrine influence of melanoma on the GF levels.

Inflammation-induced colon cancer in uPA-deficient mice is associated with a deregulated expression of Notch signaling pathway components.

Notch is an evolutionarily conserved signaling pathway with an important role in development and cell fate determination. Deregulation of Notch signaling has been associated with several pathological conditions, including cancer. Acting as an oncogene in some types of cancers and as a tumor suppressor in other, Notch effects seem to be highly context-dependent in solid tumors. In the present study, we aimed to investigate gene expression levels of Notch pathway constituents, including ligands, receptors, and target genes, during the early stages of inflammation-associated intestinal carcinogenesis. To achieve so, we used our recently developed mouse model, in which colon cancer arises in the absence of urokinase-type plasminogen activator (uPA) due to colitis induced by dextran sodium sulfate (DSS) treatment. Among the cell surface components, ligands Jag1/Jag2 and receptors Notch1/Notch2 were found to be significantly upregulated in the uPA-deficient protumorigenic inflammatory microenvironment. Moreover, several intracellular Notch modulators, i.e. Hes1, Hey1, and Klf4, were also shown to be deregulated with inflammation, yet irrespective of uPA status. Sox9 transcription factor, however, was significantly downregulated in the uPA-deficient/DSS-treated mice that developed colon adenomas as compared to the wild-type/DSS-treated group with no neoplasia identified. The latter finding supports a tumor suppressive role of Sox9 in intestinal carcinogenesis. Our results point towards an early activation of Notch signaling pathway at the receptor-ligand level in inflammation-associated colon neoplasmatogenesis developed in the absence of uPA. Interestingly, such activation may not be accompanied by deregulation of downstream Notch-target genes, possibly due to the effects of other inter-related signaling pathways.

Urokinase-type plasminogen activator (uPA) is not essential for epithelial sodium channel (ENaC)-mediated sodium retention in experimental nephrotic syndrome.

In nephrotic syndrome, aberrantly filtered plasminogen (plg) is converted to active plasmin by tubular urokinase-type plasminogen activator (uPA) and thought to lead to sodium retention by proteolytic activation of the epithelial sodium channel (ENaC). This concept predicts that uPA is an important factor for sodium retention and that inhibition of uPA might be protective in nephrotic syndrome. Activation of amiloride-sensitive currents by uPA and plg were studied in Xenopus laevis oocytes expressing murine ENaC. In doxorubicin-induced nephrotic mice, uPA was inhibited pharmacologically by amiloride and genetically by the use of uPA-deficient mice (uPA-/- ). Experiments in Xenopus laevis oocytes expressing murine ENaC confirmed proteolytic ENaC activation by a combination of plg and uPA which stimulated amiloride-sensitive currents with concomitant cleavage of the ENaC γ-subunit at the cell surface. Treatment of nephrotic wild-type mice with amiloride inhibited urinary uPA activity, prevented urinary plasmin formation and sodium retention. In nephrotic mice lacking uPA (uPA-/- ), urinary plasmin formation from plg was suppressed and urinary uPA activity absent. However, in nephrotic uPA-/- mice, sodium retention was not reduced compared to nephrotic uPA+/+ mice. Amiloride prevented sodium retention in nephrotic uPA-/- mice which confirmed the critical role of ENaC in sodium retention. uPA is responsible for the conversion of aberrantly filtered plasminogen to plasmin in the tubular lumen in vivo. However, uPA-dependent plasmin generation is not essential for ENaC-mediated sodium retention in experimental nephrotic syndrome.

Inflammation-driven colon neoplasmatogenesis in uPA-deficient mice is associated with an increased expression of Runx transcriptional regulators

Deregulation of the bone morphogenetic protein (BMP) pathway has been documented in colorectal cancer (CRC). Previously, we investigated possible associations between urokinase-type plasminogen activator (uPA) deficiency and expression of extracellular constituents of BMP signaling in a newly developed mouse model of inflammation-driven intestinal neoplasmatogenesis, in which chronic colitis and CRC are induced using dextran sodium sulfate (DSS). In this report, we explored the contribution of intracellular components of Smad-mediated BMP signal transduction using the same model. Interestingly, upon DSS treatment, we noticed an overexpression of Runx1/2/3 transcription factors in both wild-type and uPA-deficient mice. Moreover, Runx1 and Runx2 expression levels exhibited an even higher increase in DSS-treated/uPA-deficient mice as compared to DSS-treated/wild-type animals. In all experimental conditions, in situ investigation of Runx-expressing cell types, revealed detection of all three Runx in the immune cells, yet in the DSS-treated/uPA-deficient mice Runx1 and Runx2 were also identified in the preneoplastic epithelium of advanced high-grade dysplasia and carcinoma in-situ colonic lesions. Finally, the uPA-deficient pro-tumorigenic colitic microenvironment exhibited increased levels of the Runx-induced target genes Snai2, Bim and Claudin1, known to have a role in tumor development and progression. These findings suggest that the absence of uPA correlates with increased levels of Runx transcriptional regulators in a way that promotes inflammation-associated carcinogenesis.

Regulation of p53-mediated changes in the uPA-fibrinolytic system and in lung injury by loss of surfactant protein C expression in alveolar epithelial cells.

Pulmonary surfactant protein C (SP-C) expression by type II alveolar epithelial cells (AECs) is markedly reduced in diverse types of lung injuries and is often associated with AEC apoptosis. It is unclear whether loss of SP-C contributes to the increased p53 and urokinase-type plasminogen activator (uPA) system cross-talk and apoptosis of AECs. Therefore, we inhibited SP-C expression in human and murine AECs using lentivirus vector expressing shRNA and tested p53 and downstream changes in the uPA-fibrinolytic system. Inhibition of SP-C expression in AECs induced p53 and activated caspase-3, indicating AEC apoptosis. We also found that bleomycin or cigarette smoke exposure failed to inhibit SP-C expression or apoptosis in AECs in p53- and plasminogen activator inhibitor-1 (PAI-1)-deficient mice. Depletion of SP-C expression by lentiviral SP-C shRNA in PAI-1-deficient mice failed to induce p53 or apoptosis in AECs, whereas it increased both AEC p53 and apoptosis in wild-type and uPA-deficient mice. SP-C inhibition in AECs also increased in CXCL1 and CXCL2 and their receptor CXCR2 as well as ICAM-1 expression, which is indicative of a proinflammatory response. Overexpression of p53-binding 3'-UTR sequences in AECs inhibited PAI-1 induction while maintaining uPA and uPAR protein and mRNA expression. Furthermore, caveolin-1 expression and phosphorylation were increased in AECs, indicating an intricate link between caveolin-1 and Src kinase-mediated cell signaling and AEC apoptosis due to loss of SP-C expression through p53 and uPA system-mediated cross-talk. The role of uPA, PAI-1, and p53 in the regulation of AEC apoptosis after injury was also determined in knockout mice.

P53 and miR-34a Feedback Promotes Lung Epithelial Injury and Pulmonary Fibrosis

Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal interstitial lung disease. The pathogenesis of interstitial lung diseases, including its most common form, IPF, remains poorly understood. Alveolar epithelial cell (AEC) apoptosis, proliferation, and accumulation of myofibroblasts and extracellular matrix deposition results in progressive loss of lung function in IPF. We found induction of tumor suppressor protein, p53, and apoptosis with suppression of urokinase-type plasminogen activator (uPA) and the uPA receptor in AECs from the lungs of IPF patients, and in mice with bleomycin, cigarette smoke, silica, or sepsis-induced lung injury. Treatment with the caveolin-1 scaffolding domain peptide (CSP) reversed these effects. Consistent with induction of p53, AECs from IPF lungs or mice with diverse types of lung injuries showed increased p53 acetylation and miR-34a expression with reduction in Sirt1. This was significantly reduced after treatment of wild-type mice with CSP, and uPA-deficient mice were unresponsive. Bleomycin failed to induce miR-34a in p53- or plasminogen activator inhibitor-1 (PAI-1)-deficient mice. CSP-mediated inhibition of miR-34a restored Sirt1, suppressed p53 acetylation and apoptosis in injured AECs, and prevented pulmonary fibrosis (PF). AEC-specific suppression of miR-34a inhibited bleomycin-induced p53, PAI-1, and apoptosis and prevented PF, whereas overexpression of precursor-miR-34a increased p53, PAI-1, and apoptosis in AECs of mice unexposed to bleomycin. Our study validates p53-miR-34a feedback as a potential therapeutic target in PF.

BMP pathway suppression is an early event in inflammation-driven colon neoplasmatogenesis of uPA-deficient mice.

The suppression of the bone morphogenetic protein (BMP) signaling pathway has been recently shown to promote adenoma-to-carcinoma transition in sporadic colon cancer. However, its role in the evolution of early preneoplastic changes to neoplasia remains elusive. In the present study, we aimed to investigate the gene expression levels of multiple extracellular BMP family constituents, including BMP ligands/receptors and inhibitors, during the early stages of inflammation-associated colon carcinogenesis. For that, we used the recently developed urokinase-type plasminogen activator (uPA)-deficient mouse model of colonic polypoidogenesis, in which adenomatous polyps arise several months after the induction of dextran sodium sulfate (DSS) colitis. In DSS-treated wild-type mice, the preneoplastic lesions which did not eventually evolve to adenomas resided in a colitic microenvironment characterized by a balanced upregulation of both BMP ligands, i.e., Bmp4/7 and BMP inhibitors, such as chordin, noggin, and gremlin-1. In the uPA-deficient tumor-promoting inflammatory microenvironment, however, there was a clear evidence for BMP pathway suppression. By contrast to DSS-treated wild-type controls, the inflammation-associated Bmp4 upregulation was abolished, and the BMP signaling suppression was further enhanced by a particularly high increase of gremlin-1 expression. These findings propose that BMP pathway suppression in colon cancer could be associated with very early stages of the preneoplasia-to-neoplasia sequence of events.

Spontaneous lung and lymph node metastasis in transgenic breast cancer is independent of the urokinase receptor uPAR.

Urokinase-type plasminogen activator (uPA) is an extracellular protease that plays a pivotal role in tumor progression. uPA activity is spatially restricted by its anchorage to high-affinity uPA receptors (uPAR) at the cell surface. High tumor tissue expression of uPA and uPAR is associated with poor prognosis in lung, breast, and colon cancer patients in clinical studies. Genetic deficiency of uPA leads to a significant reduction in metastases in the murine transgenic MMTV-PyMT breast cancer model, demonstrating a causal role for uPA in cancer dissemination. To investigate the role of uPAR in cancer progression, we analyze the effect of uPAR deficiency in the same cancer model. uPAR is predominantly expressed in stromal cells in the mouse primary tumors, similar to human breast cancer. In a cohort of MMTV-PyMT mice [uPAR-deficient (n = 31) or wild type controls (n = 33)], tumorigenesis, tumor growth, and tumor histopathology were not significantly affected by uPAR deficiency. Lung and lymph node metastases were also not significantly affected by uPAR deficiency, in contrast to the significant reduction seen in uPA-deficient mice. Taken together, our data show that the genetic absence of uPAR does not influence the outcome of the MMTV-PyMT cancer model.

Promotion of Wound Healing by an Agonist of Adenosine A2A Receptor Is Dependent on Tissue Plasminogen Activator.

Impaired wound healing, as it occurs in diabetes mellitus or long-term corticoid treatment, is commonly associated with disability, diminished quality of life, and high economic costs. Selective agonists of the A2A receptor subtype of adenosine, an endogenous regulator of inflammation, promote tissue repair in animal models, both healthy and with impaired healing. Plasmin-mediated proteolysis of fibrin and other matrix proteins is essential for cell migration at sites of injury. Since adenosine A2A receptor activation increases plasminogen activator release from macrophages and mast cells, we studied the effect of a selective agonist, CGS-21680, on full-thickness excisional wound closure in wild-type, urokinase plasminogen activator (uPA)-deficient, and tissue plasminogen activator (tPA)-deficient mice. Wound closure was impaired in tPA- and uPA-deficient mice as compared with wild-type mice, and topical application of CGS-21680 significantly increased the rate at which wounds closed in wild-type mice and uPA-deficient mice, but not in tPA-deficient mice. Immunostaining of tissue sections showed that tPA was present in endothelial cells and histiocytes by day 3 post-wound and also by day 6. In contrast, uPA was more prominent in these cell types only by day 6 post-wound. Our results confirm that plasminogen activation contributes to wound repair and are consistent with the hypothesis that adenosine A2A receptor activation promotes wound closure by a mechanism that depends upon tPA, but not uPA. Moreover, our results suggest that topical adenosine A2A receptor agonists may be useful in promotion of wound closure in patients with impaired wound healing.

Mice Deficient in Urokinase-Type Plasminogen Activator (uPA) or uPA Receptor Develop Significantly Diminished Collagen-Induced Arthritis

Rheumatoid arthritis (RA) is a common and debilitating autoimmune disease characterized by chronic inflammation, synovial hyperplasia, edema, cartilage and bone erosion and loss of joint function. Increasing evidence suggests that the plasminogen activation (PA) system plays a fundamental role in the mechanisms mediating inflammatory joint disease pathogenesis. However, analysis of the precise contribution of PA system components to arthritis pathogenesis has been complicated by the use of gene-targeted mice on non-susceptible genetic backgrounds or experimental models that simultaneously induce wound trauma in conjunction with arthritis induction. To rigorously define the contribution of the urokinase-type plasminogen activator system to arthritis pathogenesis, previously generated genetic deficiencies in both uPA and uPA receptor (uPAR) were inbred for 7 generations (99% inbred) to the well-characterized, collagen-induced arthritis (CIA)-susceptible strain, DBA/1J. Our results indicate a near complete amelioration of joint disease in uPA-deficient mice that was also observed in uPAR-deficient mice. Limited disease development in both uPA- and uPAR-deficient mice correlated with significantly reduced local mRNA levels of key inflammatory mediators (e.g., TNFα, IL-1β, and IL-6) in these animals. To determine if development of inflammatory joint disease in CIA-challenged mice was dependent on the expression of uPAR by non-hematopoietic- or hematopoietic-derived cells, reciprocal bone marrow transplant studies were performed. Mice in which uPAR deficiency was limited to the bone marrow compartment elicited significantly reduced macroscopic and histopathological disease in the paws and knees compared to wild-type mice or mice in which only hematopoietic-derived cells express uPAR. Our results are the first to report in the context of the highly CIA susceptible DBA/1 background that both uPA and uPAR are key determinants of inflammatory joint disease pathogenesis. Furthermore, our findings indicate a fundamental role for uPAR expression by hematopoietic cells in driving arthritis incidence and progression. Thus, these findings suggest that cell-surface associated uPA/uPAR-mediated proteolysis and/or uPAR-mediated signaling events from bone-marrow derived cells are important in promoting inflammatory joint disease, and that disrupting this key proteolytic/signaling system may provide a novel therapeutic strategy to limit clinical arthritis. DisclosuresNo relevant conflicts of interest to declare.

Regulation of epithelial sodium channels in urokinase plasminogen activator deficiency.

Epithelial sodium channels (ENaC) govern transepithelial salt and fluid homeostasis. ENaC contributes to polarization, apoptosis, epithelial-mesenchymal transformation, etc. Fibrinolytic proteases play a crucial role in virtually all of these processes and are elaborated by the airway epithelium. We hypothesized that urokinase-like plasminogen activator (uPA) regulates ENaC function in airway epithelial cells and tested that possibility in primary murine tracheal epithelial cells (MTE). Both basal and cAMP-activated Na(+) flow through ENaC were significantly reduced in monolayers of uPA-deficient cells. The reduction in ENaC activity was further confirmed in basolateral membrane-permeabilized cells. A decrease in the Na(+)-K(+)-ATPase activity in the basolateral membrane could contribute to the attenuation of ENaC function in intact monolayer cells. Dysfunctional fluid resolution was seen in uPA-disrupted cells. Administration of uPA and plasmin partially restores ENaC activity and fluid reabsorption by MTEs. ERK1/2, but not Akt, phosphorylation was observed in the cells and lungs of uPA-deficient mice. On the other hand, cleavage of γ ENaC is significantly depressed in the lungs of uPA knockout mice vs. those of wild-type controls. Expression of caspase 8, however, did not differ between wild-type and uPA(-/-) mice. In addition, uPA deficiency did not alter transepithelial resistance. Taken together, the mechanisms for the regulation of ENaC by uPA in MTEs include augmentation of Na(+)-K(+)-ATPase, proteolysis, and restriction of ERK1/2 phosphorylation. We demonstrate for the first time that ENaC may serve as a downstream signaling target by which uPA controls the biophysical profiles of airway fluid and epithelial function.

Urokinase plasminogen activator gene deficiency inhibits fracture cartilage remodeling

Urokinase plasminogen activator (uPA) regulates a proteolytic cascade of extracellular matrix degradation that functions in tissue development and tissue repair. The development and remodeling of the skeletal extracellular matrix during wound healing suggests that uPA might regulate bone development and repair. To determine whether uPA functions regulate bone development and repair, we examined the basal skeletal phenotype and endochondral bone fracture repair in uPA-deficient mice. The skeletal phenotype of uPA knockout mice was compared with that of control mice under basal conditions by dual-energy X-ray absorptiometry and micro-CT analysis, and during femur fracture repair by micro-CT and histological examination of the fracture callus. No effects of uPA gene deficiency were observed in the basal skeletal phenotype of the whole body or the femur. However, uPA gene deficiency resulted in increased fracture callus cartilage abundance during femur fracture repair at 14days healing. The increase in cartilage corresponded to reduced tartrate-resistant acid phosphatase (TRAP) staining for osteoclasts in the uPA knockout fracture callus at this time, consistent with impaired osteoclast-mediated remodeling of the fracture cartilage. CD31 staining was reduced in the knockout fracture tissues at this time, suggesting that angiogenesis was also reduced. Osteoclasts also colocalized with CD31 expression in the endothelial cells of the fracture tissues during callus remodeling. These results indicate that uPA promotes remodeling of the fracture cartilage by osteoclasts that are associated with angiogenesis and suggest that uPA promotes angiogenesis and remodeling of the fracture cartilage at this time of bone fracture repair.

Regulation of alveolar epithelial cell apoptosis and pulmonary fibrosis by coordinate expression of components of the fibrinolytic system

Alveolar type II (ATII) cell apoptosis and depressed fibrinolysis that promotes alveolar fibrin deposition are associated with acute lung injury (ALI) and the development of pulmonary fibrosis (PF). We therefore sought to determine whether p53-mediated inhibition of urokinase-type plasminogen activator (uPA) and induction of plasminogen activator inhibitor-1 (PAI-1) contribute to ATII cell apoptosis that precedes the development of PF. We also sought to determine whether caveolin-1 scaffolding domain peptide (CSP) reverses these changes to protect against ALI and PF. Tissues as well as isolated ATII cells from the lungs of wild-type (WT) mice with BLM injury show increased apoptosis, p53, and PAI-1, and reciprocal suppression of uPA and uPA receptor (uPAR) protein expression. Treatment of WT mice with CSP reverses these effects and protects ATII cells against bleomycin (BLM)-induced apoptosis whereas CSP fails to attenuate ATII cell apoptosis or decrease p53 or PAI-1 in uPA-deficient mice. These mice demonstrate more severe PF. Thus p53 is increased and inhibits expression of uPA and uPAR while increasing PAI-1, changes that promote ATII cell apoptosis in mice with BLM-induced ALI. We show that CSP, an intervention targeting this pathway, protects the lung epithelium from apoptosis and prevents PF in BLM-induced lung injury via uPA-mediated inhibition of p53 and PAI-1.

Urokinase-Type Plasminogen Activator Promotes Paracellular Transmigration of Neutrophils Via Mac-1, But Independently of Urokinase-Type Plasminogen Activator Receptor

Urokinase-type plasminogen activator (uPA) has recently been implicated in the pathogenesis of ischemia-reperfusion (I/R) injury. The underlying mechanisms remain largely unclear. Using in vivo microscopy on the mouse cremaster muscle, I/R-elicited firm adherence and transmigration of neutrophils were found to be significantly diminished in uPA-deficient mice and in mice treated with the uPA inhibitor WX-340, but not in uPA receptor (uPAR)-deficient mice. Interestingly, postischemic leukocyte responses were significantly reduced on blockade of the integrin CD11b/Mac-1, which also serves as uPAR receptor. Using a cell transfer technique, postischemic adherence and transmigration of wild-type leukocytes were significantly decreased in uPA-deficient animals, whereas uPA-deficient leukocytes exhibited a selectively reduced transmigration in wild-type animals. On I/R or stimulation with recombinant uPA, >90% of firmly adherent leukocytes colocalized with CD31-immunoreactive endothelial junctions as detected by in vivo fluorescence microscopy. In a model of hepatic I/R, treatment with WX-340 significantly attenuated postischemic neutrophil infiltration and tissue injury. Our data suggest that endothelial uPA promotes intravascular adherence, whereas leukocyte uPA facilitates the subsequent paracellular transmigration of neutrophils during I/R. This process is regulated via CD11b/Mac-1, and does not require uPAR. Pharmacological blockade of uPA interferes with these events and effectively attenuates postischemic tissue injury.

Abstract 4401: Antibody-mediated targeting of the uPA proteolytic activity neutralizes uPA functions in vivo

Abstract Urokinase-type plasminogen activator (uPA) plays a central role in tissue remodeling processes, during both normal physiological conditions and cancer progression. Most of our understanding of the role of uPA in vivo is derived from studies using gene-targeted uPA-deficient mice. To enable in vivo studies on the acute and specific interference with uPA functionality in adult mice, we have now developed murine monoclonal antibodies (mAbs) directed against murine uPA by immunization of uPA-deficient mice with the recombinant protein. Guided by ELISA, Western blotting, SPR and enzyme kinetic analyses, we have selected two highly potent and inhibitory anti-uPA mAbs (mU1 and mU3). Both mAbs recognize epitopes located on the B-chain of uPA that encompasses the catalytic site. In activity assays in vitro, mU1 blocked uPA-catalyzed plasminogen activation as well as plasmin-mediated pro-uPA activation, whereas mU3 only was directed against the first of these reactions. We provide evidence that acute disruption of uPA activity in the adult mouse was effective with mU1, but not mU3, because only mU1 rescued mice treated with a uPA-activable anthrax pro-toxin. Moreover, systemic administration of mU1 (i) delayed wound healing in tissue-type plasminogen activator (tPA)-deficient mice, and (ii) impaired uPA-mediated hepatic fibrinolysis in tPA-deficient mice, resulting in a phenotype mimicking that of uPA;tPA double-deficient mice. Importantly, these results demonstrate the specific antagonist-directed targeting of mouse uPA at the enzyme activity level in normal physiological processes in vivo. Preliminary results indicate that wild-type mice, which have been transplanted with murine Lewis lung carcinoma cells, display significant reduction in primary tumour growth, when treated with mU1 as compared to placebo-treated (i.e. PBS-treated) littermates. We thus suggest that mU1 is an excellent mAb candidate for therapeutic intervention studies in mouse cancer models. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4401.

Induction of Tissue Factor by Urokinase in Lung Epithelial Cells and in the Lungs

Urokinase-type plasminogen activator (uPA) regulates extracellular proteolysis in lung injury and repair. Although alveolar expression of uPA increases, procoagulant activity predominates. This study was designed to investigate whether uPA alters the expression of tissue factor (TF), the major initiator of the coagulation cascade, in lung epithelial cells (ECs). Bronchial, primary airway ECs and C57B6 wild-type, uPA-deficient (uPA(-/-)) mice were exposed to phosphate-buffered saline, uPA, or LPS. Immunohistochemistry, protein, cellular, and molecular techniques were used to assess TF expression and activity. uPA enhanced TF mRNA and protein expression, and TF-dependent coagulation in lung ECs. uPA-induced expression of TF involves both increased synthesis and enhanced stabilization of TF mRNA. uPA catalytic activity had little effect on induction of TF. By contrast, deletion of the uPA receptor binding growth factor domain from uPA markedly attenuated the induction of TF, suggesting that uPA receptor binding is sufficient for TF induction. Lung tissues of uPA-deficient mice expressed less TF protein and mRNA compared with wild-type mice. In addition, intratracheal instillation of mouse uPA increased TF mRNA and protein expression and accelerated coagulation in lung tissues. uPA(-/-) mice exposed to LPS failed to induce TF. uPA increased TF expression and TF-dependent coagulation in the lungs of mice. We hypothesize that uPA-mediated induction of TF occurs in lung ECs to promote increased fibrin deposition in the airways during acute lung injury.