Regulation of Airway and Alveolar Epithelial Cell Apoptosis by p53-Induced Plasminogen Activator Inhibitor-1 during Cigarette Smoke Exposure Injury

Increased expression of tumor suppressor protein p53 and of plasminogen activator inhibitor (PAI)-1 is associated with cigarette smoke (CS) exposure-induced lung epithelial injury. p53 induces PAI-1 through mRNA stabilization in lung epithelial cells. However, it is unclear how this process affects lung epithelial damage. Here, we show that CS induces p53 and PAI-1 expression and apoptosis in cultured Beas2B and primary alveolar type (AT)II cells. CS exposure augmented binding of p53 protein with PAI-1 mRNA. Inhibition of p53 from binding to PAI-1 mRNA through expression of p53-binding 70 nt PAI-1 mRNA 3'UTR sequences suppressed CS-induced PAI-1 expression. Treatment of Beas2B cells with caveolin-1 scaffolding domain peptide (CSP) suppressed p53 expression and p53-PAI-1 mRNA interaction. These changes were associated with parallel inhibition of CS-induced PAI-1 expression and apoptosis in Beas2B cells. Wild-type mice exposed to passive CS likewise show augmented p53 and PAI-1 with parallel induction of ATII cell apoptosis, whereas mice deficient for p53 or PAI-1 expression resisted apoptosis of ATII cells. CSP suppressed CS-induced ATII cell apoptosis in wild-type mice and abrogated p53-PAI-1 mRNA interaction with parallel inhibition of p53 and PAI-1 expression. The protection against ATII cell apoptosis by CSP involves inhibition of passive CS-induced proapoptotic Bax and Bak expression and restoration of the prosurvival proteins Bcl-X(L). These observations demonstrate that inhibition of p53 binding to PAI-1 mRNA 3'UTR attenuates CS-induced ATII cell apoptosis. This presents a novel link between p53-mediated PAI-1 expression and CS-induced ATII cell apoptosis.     

Urokinase Plasminogen Activator Receptor-Deficient Mice Demonstrate Reduced Hyperoxia-Induced Lung Injury

Patients with respiratory failure often require supplemental oxygen therapy and mechanical ventilation. Although both supportive measures are necessary to guarantee adequate oxygen uptake, they can also cause or worsen lung inflammation and injury. Hyperoxia-induced lung injury is characterized by neutrophil infiltration into the lungs. The urokinase plasminogen activator receptor (uPAR) has been deemed important for leukocyte trafficking. To determine the expression and function of neutrophil uPAR during hyperoxia-induced lung injury, uPAR expression was determined on pulmonary neutrophils of mice exposed to hyperoxia. Hyperoxia exposure (O₂>80%) for 4 days elicited a pulmonary inflammatory response as reflected by a profound rise in the number of neutrophils that were recovered from bronchoalveolar lavage fluid and lung cell suspensions, as well as increased bronchoalveolar keratinocyte-derived chemokine, interleukin-6, total protein, and alkaline phosphatase levels. In addition, hyperoxia induced the migration of uPAR-positive granulocytes into lungs from wild-type mice compared with healthy control mice (exposed to room air). uPAR deficiency was associated with diminished neutrophil influx into both lung tissues and bronchoalveolar spaces, which was accompanied by a strong reduction in lung injury. Furthermore, in uPAR^−/− mice, activation of coagulation was diminished. These data suggest that uPAR plays a detrimental role in hyperoxia-induced lung injury and that uPAR deficiency is associated with diminished neutrophil influx into both lung tissues and bronchoalveolar spaces, accompanied by decreased pulmonary injury.Patients with acute lung injury or the acute respiratory distress syndrome commonly receive supportive care consisting of low tidal volume protective ventilation and the administration of high concentrations of inspired oxygen.^1,2 Prolonged exposure to high oxygen concentrations, however, can worsen or induce lung damage in already injured or previously healthy lungs.³ The mechanisms underlying hyperoxia-induced lung injury are beginning to be understood, despite difficulties in distinguishing the effects of exposure to hyperoxia from those of the inciting disease for which oxygen therapy was started. Insight into factors involved in hyperoxia-induced lung injury could help identify possible new targets for improving the management of patients who require supplemental oxygen.The inflammatory response to hyperoxia is dominated by neutrophils and likely mediated by the generation of reactive oxygen species.^1,3 The recruitment of neutrophils to the lungs on exposure to hyperoxia has been linked to the local production of CXC chemokines and the expression of CXCR2, the predominant neutrophil receptor for CXC chemokines in rodents.^4,5,6 In addition, increased expression of adhesion molecules on the pulmonary endothelial cell surface has been found to contribute to neutrophil trafficking into the bronchoalveolar compartment during hyperoxia.⁷ We and others recently identified a pivotal role for the urokinase plasminogen activator receptor (uPAR, CD87) in neutrophil migration to the lungs during bacterial pneumonia.^8,9 uPAR is a glycosylphosphatidylinositol-linked receptor expressed by a variety of cell types, including hemopoietic cells, endothelial cells and many different neoplastic cells, that has been implicated as an important player in leukocyte trafficking.^10,11 At present it is unknown whether uPAR is involved in hyperoxia-induced neutrophil migration and lung injury. Therefore, in the present study we sought to investigate the expression and function of neutrophil uPAR during hyperoxia-induced lung inflammation.     

Accelerated Skin Wound Healing in Plasminogen Activator Inhibitor-1-Deficient Mice

Components of the fibrinolytic system have been implicated in cell migratory events associated with tissue remodeling. Studies in plasminogen-deficient mice (PG(-/-)) indicated that skin wound healing is impaired, but is resolved with an additional fibrinogen deficiency. Plasminogen activator inhibitor-1 (PAI-1) expression by keratinocytes has been identified shortly after wound injury. PAI-1 expression could affect wound healing by regulating the fibrinolytic environment of the wounded area, as well as influencing events associated with cell attachment and detachment through interactions with matrix proteins. The present study directly assesses PAI-1 involvement in skin wound healing through analyses of a dermal biopsy punch model in PAI-1-deficient (PAI-1(-/-) mice. While the cellular events associated with the healing process are similar between wild-type (WT) and PAI-1(-/-) mice, the rate of wound closure is significantly accelerated in PAI-1(-/-) mice.     

Neuroprotective Effect of Plasminogen Activator Inhibitor-1 Antagonist in the Rat Model of Mild Traumatic Brain Injury

Inflammation - Plasminogen activator inhibitor-1 (PAI-1) antagonists are known for their neuroprotective effects. In this study, it was aimed to investigate the possible protective effects of PAI-1...     

尿激酶型纤溶酶原激活因子cDNA的克隆与原核表达

目的构建尿激酶型纤溶酶原激活因子（uPA）原核表达质粒,并在大肠埃希菌BL21中表达,为进一步研究uPA奠定基础。方法应用逆转录RT—PCR,从人肝细胞cDNA中扩增人尿激酶型纤溶酶原激活因子基因序列,与原核表达质粒pET32a重组,获得表达质粒uPA—pET32a。用氨苄青霉素平板筛选转化子。双酶切与DNA测序进行鉴定,用IPTG诱导表达,并用Westernblotting进行鉴定。结果从肝细胞cDNA中扩增的uPA基因片段长1296bp,酶切及DNA测序证实uPA-pET32a重组质粒构建正确,表达融合蛋白分子量约为68900Mr,经Western blotting证实为目的蛋白。结论成功构建了重组原核表达质粒uPA—pET32a。并能在大肠埃希菌内表达,所表达的融合蛋白分子量大小与预期的相一致．为进一步的研究奠定了基础。     

Promigratory Effect of Plasminogen Activator Inhibitor-1 on Invasive Breast Cancer Cell Populations

The urokinase-type plasminogen activator (uPA) system is a dynamic complex in which the membrane receptor uPAR binds uPA that binds the plasminogen activator inhibitor (PAI)-1 localized in the extracellular matrix, resulting in endocytosis of the whole complex by the low-density lipoprotein receptor-related protein (LRP). High expression of PAI-1 is paradoxically associated with marked tumor spreading and poor prognosis. We previously reported a nonproteolytic role of the [uPAR:uPA:PAI-1:LRP] complex operative in cell migration. Here we explored whether matrix PAI-1 could be used as a migration support by human breast cancer cells. We showed that the uPA system and LRP are localized at filopodia of invasive cells, and that formation/internalization of the [uPAR:uPA:PAI-1:LRP] complex is required for attachment and migration of cancer cells on plastic and on a PAI-1 coat. PAI-1 increased both filopodia formation and migration of cancer cells suggesting a chemokine-like activity. Migration velocity, expression of the uPA system, use of the [uPAR:uPA:PAI-1:LRP] complex to migrate, and promigratory effects of PAI-1 paralleled cancer cell invasiveness. Phenotyping and functional analysis of invasive cancer cell subclones indicated that different cell subpopulations may use different strategies to migrate depending on both the environment and their expression of the uPA system, some of them taking advantage of abundant available PAI-1.     

PAI-1反义寡核苷酸对肾间质细胞抑制作用的实验研究

目的研究纤维蛋白溶解酶原激活物抑制因子-1(PAI-1)反义寡核苷酸(antisense oligonucleotides,ASODN)对大鼠肾间质细胞(NEK293)的抑制作用。方法采用RT-PCR、Western blot检测脂质体介导的PAI-1ASODN目的基因及蛋白的表达;通过细胞生长试验、流式细胞仪检测PAI-1 ASODN转染前后NEK293细胞增殖和凋亡的变化。结果PAI-1ASODN转染后可见NEK293中PAI-1mRNA及蛋白表达明显下调,与对照组相比较,可明显抑制细胞的增殖。诱导其凋亡(P<0.05)。结论PAI-1反义寡核苷酸具有明显抑制肾间质细胞增殖和诱导其凋亡的作用,转染脂质体介导的PAI-1 ASODN有望成为治疗肾纤维化的有效方法。     

雷公藤甲素对血管生成的抑制作用

利用体外培养人脐静脉内皮细胞,经不同浓度的雷公藤甲素(0、5、10、20、30μg/L)处理后,MTT法显示雷公藤甲素可抑制内皮细胞的增殖,5μg/L雷公藤甲素的抑制率达29.15%;琼脂凝胶立体细胞培养系统检测发现内皮细胞经雷公藤甲素作用后,其游走能力降低;鸡胚尿囊膜试验观察到雷公藤甲素可有效抑制血管的生成;荧光定量RT-PCR检测发现雷公藤甲素可下调内皮细胞u-PAmRNA的表达。因此认为,雷公藤甲素可能在基因水平上干扰内皮细胞u-PAmRNA的表达,减少u-PA蛋白的生成,从而有效地抑制血管内皮细胞的增殖和移行,这可能是雷公藤甲素抑制血管生成的主要机制之一。     

Directed Gp130-Mediated Signaling Dissociates Inflammation from Fibrosis in Bleomycin Induced Lung Injury

Inflammation Research -     

The Urokinase Receptor Promotes Cancer Metastasis Independently of Urokinase-Type Plasminogen Activator in Mice

The urokinase receptor (uPAR) promotes metastasis of human malignancies; however, its mechanism of action remains incompletely understood. Established models focus on the ability of uPAR to bind urokinase-type plasminogen activator (uPA) and promote protease activation in the tumor cell microenvironment; however, uPAR also regulates cell signaling and migration by both uPA-dependent and -independent mechanisms in vitro. The significance of uPAR as a cell-signaling receptor in vivo remains unclear. In this study, we expressed either human or mouse uPAR in human embryonic kidney (HEK-293) cells. We selected HEK-293 cells because, unlike most cancer cells, they do not express uPA or uPAR endogenously. Both mouse and human uPAR increased cell adhesion and migration on vitronectin. Rac1 was activated and responsible for the increase in cell migration. HEK-293 cells that did not express uPAR formed palpable tumors in severe combined immunodeficient mice; however, metastases were exceedingly rare. The xenografts contained abundant mouse uPA, produced by infiltrating mouse cells, but no human uPA. Mouse uPA bound only to mouse uPAR and not human uPAR and, thus, could not interact with human uPAR-expressing HEK-293 cells in xenografts. Nevertheless, both mouse and human uPAR significantly increased HEK-293 cell metastasis into the lungs. The activity of human uPAR suggests that uPAR may promote cancer metastasis independent of uPA. Candidate mechanisms include its effects on adhesion, migration, and Rac1 activation.The urokinase receptor (uPAR) is a glycosyl phosphatidylinositol-anchored membrane protein that functions as a biomarker for cancer progression and metastasis in many forms of human malignancy.¹ In the prostate, malignant epithelial cells are uPAR-positive, whereas benign epithelia are uPAR-negative.² The degree of uPAR-positivity in malignant prostate epithelial cells correlates with the Gleason grade of the tumor.³ Similarly, in endometrial cancer, uPAR expression in tumor cells correlates with tumor grade, recurrence, and mortality.⁴ Breast cancer may be more complex because although uPAR expression correlates with disease severity, in many cases, uPAR is expressed by both malignant epithelial cells and nonmalignant stromal cells in the tumor microenvironment.^5,6,7,8 uPAR expression by normal cells in the adult is limited⁵; however, this receptor may play an important role in diseases in addition to cancer, such as rheumatoid arthritis⁹ and infectious lung diseases.¹⁰Binding of urokinase-type plasminogen activator (uPA) to uPAR substantially increases the catalytic efficiency for plasminogen activation.¹¹ Plasmin, which is generated near the cell surface, may participate in the activation of metalloproteases.¹² Based on these findings, the first models developed to explain the cancer-promoting activity of uPAR focused on its ability to support protease activation and remodeling of the extracellular matrix (ECM).^13,14 Activated proteases may facilitate cellular penetration of tissue boundaries such as basement membranes.¹⁵ In migrating cells, uPAR localizes to leading lamellipodia,¹⁶ supporting the hypothesis that remodeling of ECM is an important uPAR activity in cell migration and invasion.uPAR regulates cell adhesion by binding directly to vitronectin and by lateral association with integrins in the plasma membrane.^17,18 Although the effects of uPAR on cell adhesion may be regulated by uPA, there is no strict requirement for uPA. uPAR activates cell-signaling to Rac1 by a pathway that is dependent on vitronectin but not uPA.^19,20,21 Cell-signaling factors that are activated downstream of uPAR but typically in response to uPA-binding include H-Ras, ERK/MAP kinase, and Akt.^22,23,24 uPAR-initiated cell-signaling controls cell survival, proliferation, and cell migration in vitro.^25,26 uPAR also may regulate cell-signaling in response to ligands for receptor tyrosine kinases, such as epidermal growth factor.²⁷In animal model studies, uPAR expression has been correlated with cancer progression and metastasis^28,29,30; however, the mechanisms by which uPAR functions in vivo remain elusive. Recently identified uPAR activities in cell signaling and adhesion remain largely untested in in vivo model systems. To address this problem, we took advantage of the strict species specificity in uPA-binding to uPAR; mouse uPA does not bind with significant affinity to human uPAR.³¹ Xenografts were developed in severe combined immunodeficient (SCID) mice using human embryonic kidney (HEK-293) cells, which do not express uPA or uPAR endogenously,³² but form tumors because of integrated adenovirus genes.³³ We show that nonmalignant mouse cells, which infiltrate HEK-293 cell xenografts, generate abundant mouse uPA. When HEK-293 cells were transfected for stable expression of mouse uPAR, metastasis to the lungs was significantly increased, as anticipated because of the availability of mouse uPA in the tumors. Surprisingly, HEK-293 cells that express human uPAR also demonstrated significantly increased metastasis despite the absence of human uPA. The ability of human uPAR to promote metastasis in mice rules out mechanisms involving activation of proteases, and instead, supports the hypothesis that uPAR may promote metastasis independently of uPA. Candidate mechanisms identified here include regulation of cell adhesion, migration, and Rac1 activation.     

Soluble Urokinase Plasminogen Activator Receptor (suPAR) is Associated with Metabolic Changes in HIV-1-Infected Africans: A Prospective Study

Soluble urokinase plasminogen activator receptor (suPAR) is associated with inflammation and may predict lipodystrophy and dysmetabolism in human immunodeficiency virus (HIV)-infected individuals on antiretroviral therapy. We aimed to assess firstly, whether suPAR levels are elevated in treated and untreated HIV-1-infected Africans compared to uninfected controls at baseline and at a 3-year follow-up, and secondly whether suPAR levels are correlated with cardiovascular and/or metabolic changes. SuPAR, cardiovascular, and metabolic variables were assessed and the percentage change was determined. HIV-1-infected black South Africans had significantly higher suPAR levels than uninfected controls at baseline and at follow-up 3 years later. However, only the treated HIV-1-infected participants showed an increase in suPAR levels at follow-up. The treated group also showed signs of lipodystrophy and their baseline suPAR levels correlated positively with an increased waist circumference. This study indicates that suPAR levels increase and that baseline suPAR is associated with an increase in abdominal fat distribution in HIV-infected black Africans on antiretroviral therapy.     

Porphyromonas gingivalis Gingipains-Mediated Degradation of Plasminogen Activator Inhibitor-1 Leads to Delayed Wound Healing Responses in Human Endothelial Cells

Plasminogen activator inhibitor-1 (PAI-1), a serine protease inhibitor, is constitutively produced by endothelial cells and plays a vital role in maintaining vascular homeostasis. Chronic periodontitis is an inflammatory disease characterized by bleeding of periodontal tissues that support the tooth. In this study, we aimed to determine the role of PAI-1 produced by endothelial cells in response to infections caused by the primary periodontal pathogen Porphyromonas gingivalis. We demonstrated that P. gingivalis infection resulted in significantly reduced PAI-1 levels in human endothelial cells. This reduction in PAI-1 levels could be attributed to the proteolysis of PAI-1 by P. gingivalis proteinases, especially lysine-specific gingipain-K (Kgp). We demonstrated the roles of these degradative enzymes in the endothelial cells using a Kgp-specific inhibitor and P. gingivalis gingipain-null mutants, in which the lack of the proteinases resulted in the absence of PAI-1 degradation. The degradation of PAI-1 by P. gingivalis induced a delayed wound healing response in endothelial cell layers via the low-density lipoprotein receptor-related protein. Our results collectively suggested that the proteolysis of PAI-1 in endothelial cells by gingipains of P. gingivalis might lead to the deregulation of endothelial homeostasis, thereby contributing to the permeabilization and dysfunction of the vascular endothelial barrier.     

Concurrent Upregulation of Urokinase Plasminogen Activator Receptor and CD11b during Tuberculosis and Experimental Endotoxemia

Patients with tuberculosis had higher expression of monocyte urokinase receptor (uPAR) and CD11b than controls. In vitro, lipoarabinomannan and lipopolysaccharide (LPS) from Escherichia coli shared the ability to enhance uPAR and CD11b expression on monocytes and granulocytes. In healthy volunteers, LPS induced increases in monocyte and granulocyte uPAR and CD11b.