Urokinase Expression by Tumor Suppressor Protein p53 (original) (raw)
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Induction of p53 by Urokinase in Lung Epithelial Cells
Journal of Biological Chemistry, 2005
Urokinase plasminogen activator (uPA) is a serine protease that catalyzes the conversion of plasminogen to plasmin. The plasminogen/plasmin system includes the uPA, its receptor, and its inhibitor (plasminogen activator inhibitor-1). Interactions between these molecules regulate cellular proteolysis as well as adhesion, cellular proliferation, and migration, processes germane to the pathogenesis of lung injury and neoplasia. In previous studies, we found that uPA regulates cell surface fibrinolysis by regulating its own expression as well as that of the uPA receptor and plasminogen activator inhibitor-1. In this study, we found that uPA alters expression of the tumor suppressor protein p53 in Beas2B airway epithelial cells in both a time-and concentration-dependent manner. These effects do not require uPA catalytic activity because the amino-terminal fragment of uPA lacking catalytic activity was as potent as two chain active uPA. Single chain uPA also enhanced p53 expression to the same extent as intact two chain active uPA and the amino-terminal fragment. Pretreatment of cells with anti-1 integrin antibody blocked uPA-induced p53 expression. uPA-induced p53 expression occurs without increased p53 mRNA expression. However, uPA induced oncoprotein MDM2 in a concentration-dependent manner. uPA-induced p53 expression does not require activation of tyrosine kinases. Inactivation of protein-tyrosine phosphatase SHP-2 inhibits both basal and uPA-induced p53 expression. Plasmin did not alter uPA-mediated p53 expression. The induction of p53 expression by exposure of lung epithelial cells to uPA is a newly recognized pathway by which urokinase may influence the proliferation of lung epithelial cells. This pathway could regulate pathophysiologic alterations of p53 expression in the setting of lung inflammation or neoplasia.
The American Journal of Pathology, 2013
Alveolar type II epithelial cell (ATII) apoptosis and proliferation of mesenchymal cells are the hallmarks of idiopathic pulmonary fibrosis, a devastating disease of unknown cause characterized by alveolar epithelial injury and progressive fibrosis. We used a mouse model of bleomycin (BLM)einduced lung injury to understand the involvement of p53-mediated changes in urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor-1 (PAI-1) levels in the regulation of alveolar epithelial injury. We found marked induction of p53 in ATII cells from mice exposed to BLM. Transgenic mice expressing transcriptionally inactive dominant negative p53 in ATII cells showed augmented apoptosis, whereas those deficient in p53 resisted BLM-induced ATII cell apoptosis. Inhibition of p53 transcription failed to suppress PAI-1 or induce uPA mRNA in BLM-treated ATII cells. ATII cells from mice with BLM injury showed augmented binding of p53 to uPA, uPA receptor (uPAR), and PAI-1 mRNA. p53-binding sequences from uPA, uPAR, and PAI-1 mRNA 3 0 untranslated regions neither interfered with p53 DNA binding activity nor p53-mediated promoter transactivation. However, increased expression of p53binding sequences from uPA, uPAR, and PAI-1 mRNA 3 0 untranslated regions in ATII cells suppressed PAI-1 and induced uPA after BLM treatment, leading to inhibition of ATII cell apoptosis and pulmonary fibrosis. Our findings indicate that disruption of p53efibrinolytic system cross talk may serve as a novel intervention strategy to prevent lung injury and pulmonary fibrosis. (Am J Pathol 2013, 183: 131e143; http://dx.Idiopathic pulmonary fibrosis is a progressive and fatal lung disease that is refractory to current therapy. A better understanding of the underlying mechanisms is necessary for development of novel treatments. Dysregulated fibrinolysis and induction of p53 are often associated with lung injury and precede development of pulmonary fibrosis. 1 These changes occur in a mouse model of bleomycin (BLM)einduced lung injury and accelerated pulmonary fibrosis. 1 p53 Expression increases substantially in type II alveolar epithelial (ATII) cells after BLM-or cigarette smokeeinduced lung injury, 1e3 in association with induction of plasminogen activator inhibitor-1 (PAI-1) and suppression of urokinase-type plasminogen activator (uPA) and uPA receptor (uPAR) expression.
Regulation of Urokinase Expression at the Posttranscription Level by Lung Epithelial Cells
Biochemistry, 2011
Urokinase-type plasminogen activator (uPA) is expressed by lung epithelial cells and regulates fibrin turnover and epithelial cell viability. PMA, LPS, and TNF-alpha, as well as uPA itself, induce uPA expression in lung epithelial cells. PMA, LPS, and TNF-alpha induce uPA expression through increased synthesis as well as stabilization of uPA mRNA, while uPA increases its own expression solely through uPA mRNA stabilization. The mechanism by which lung epithelial cells regulate uPA expression at the level of mRNA stability is unclear. To elucidate this process, we sought to characterize protein-uPA mRNA interactions that regulate uPA expression. Regulation of uPA at the level of mRNA stability involves the interaction of a ~40 kDa cytoplasmic-nuclear shuttling protein with a 66 nt uPA mRNA 3′UTR sequence. We purified the uPA mRNA 3′UTR binding protein and identified it as ribonucleotide reductase M2 (RRM2). We expressed recombinant RRM2 and confirmed its interaction with a specific 66 nt uPA 3′UTR sequence. Immunoprecipitation of cell lysates with anti-RRM2 antibody and RT-PCR for uPA mRNA confirmed that RRM2 binds to uPA mRNA. Treatment of Beas2B cells with uPA or LPS attenuated RRM2-endogenous uPA mRNA interactions, while overexpression of RRM2 inhibited uPA protein and mRNA expression through destabilization of uPA mRNA. LPS exposure of lung epithelial cells translocates RRM2 from the cytoplasm to the nucleus in a time-dependent manner leading to stabilization of uPA mRNA. This newly recognized pathway could influence uPA expression and a broad range of uPA-dependent functions in lung epithelial cells in the context of lung inflammation and repair.
Urokinase induces its own expression in Beas2B lung epithelial cells
American Journal of Physiology - Lung Cellular and Molecular Physiology, 2002
The urokinase-type plasminogen activator (uPA) interacts with its receptor (uPAR) to promote local proteolysis as well as cellular proliferation and migration. These functions contribute to the pathogenesis of lung inflammation and remodeling as well as the growth and invasiveness of lung neoplasms. In this study, we sought to determine if uPA alters its own expression in lung epithelial cells. Using immunoprecipitation and Western and Northern blotting techniques, we found that uPA treatment enhanced uPA expression in Beas2B lung epithelial cells in a time- and concentration-dependent manner. The induction of uPA expression is mediated through its cell surface receptor uPAR and does not require uPA enzymatic activity. The amino-terminal fragment of uPA, lacking the catalytic domain, is sufficient to induce uPA expression. The serine protease plasmin and the protease inhibitor aprotinin failed to alter uPA-mediated uPA expression, whereas α-thrombin potentiated the response. Pretrea...
Urokinase Receptor mRNA Stability Involves Tyrosine Phosphorylation in Lung Epithelial Cells
American Journal of Respiratory Cell and Molecular Biology, 2004
Interaction between urokinase-type plasminogen activator (uPA) and its receptor (uPAR) localizes cellular proteolysis and promotes cellular proliferation and migration, effects that may contribute to the pathogenesis of lung inflammation and neoplasia. Enhanced uPAR expression as well as stabilization of uPAR mRNA by transforming growth factor- and phorbol myristate acetate (PMA) shares a common mechanism involving phosphorylation and dephosphorylation of a uPAR mRNA-binding protein (uPAR mRNABp). PMA-induced tyrosine phosphorylation of the uPAR mRNABp inhibited the uPAR mRNA-uPAR mRNABp interaction, stabilized uPAR mRNA and enhanced uPAR protein expression. Downregulation of the uPAR mRNA and uPAR mRNABp interaction by PMA and transforming growth factor- can be reversed by pretreatment of cells with herbimycin which in turn inhibits expression of uPAR protein via a decrease in uPAR mRNA stability. Our experiments indicate that post-transcriptional regulation of uPAR expression requires activation of tyrosine kinases. Cytokines can regulate uPAR expression of lung-derived epithelial cells at the post-transcriptional level by tyrosine phosphorylation of the uPAR mRNA binding protein and may thereby influence tissue remodeling in lung injury or neoplasia.
Urokinase Induces Expression of Its Own Receptor in Beas2B Lung Epithelial Cells
Journal of Biological Chemistry, 2001
Interaction between the urokinase-type plasminogen activator (uPA) and its receptor (uPAR) localizes cellular proteolysis and promotes cellular proliferation and migration. The interaction between uPA and uPAR at the surface of epithelial cells thereby contributes to the pathogenesis of lung inflammation and neoplasia. In this study, we sought to determine if uPA itself alters uPAR expression by lung epithelial cells. uPA enhanced uPAR expression as well as 125 I-uPA binding in Beas2B lung epithelial cells in a time-and concentration-dependent manner. The uPA-mediated induction of uPAR is not accomplished through its receptor and requires enzymatic activity. The low molecular weight fragment of uPA, lacking the receptor binding domain, was as potent as intact two-chain uPA in inducing expression of uPAR at the cell surface. Plasmin, the end product of plasminogen activation, did not alter uPA-mediated uPAR expression. Induction of uPAR by uPA represents a novel pathway by which epithelial cells can regulate uPAR-dependent cellular responses that may contribute to stromal remodeling in lung injury or neoplasia.
Cancer research, 1999
MDA-MB-231 cells are highly metastatic breast tumor cells. Their high invasiveness is thought to be due to constitutively high levels of urokinase-type plasminogen activator (uPA) and its receptor. Previously (R. Nanbu et al., C. Eur. J. Biochem., 247: 169-174, 1997), we showed that uPA mRNA in these cells is stable and that mRNA degradation mediated by an AU-rich element (ARE) is impaired. Here we report that treatment of MDA-MB-231 cells with SB203580, an inhibitor of the stress-activated p38 mitogen-activated protein (MAP) kinase, strongly destabilized uPA mRNA in an ARE-dependent manner. In contrast, in LLC-PK1 and HeLa cells, uPA mRNA is unstable, and an ARE present in the 3' untranslated region plays a role in its degradation. Enhanced ARE-mediated mRNA destabilization induced by SB203580 was also observed in both LLC-PK1 and HeLa cells with a globin chimeric mRNA harboring two copies of the ARE (globin-2ARE) from uPA mRNA. Overexpression of constitutively active MKK6, a p...
The urokinase receptor: Structure, regulation and inhibitor-mediated internalization
Fibrinolysis & Proteolysis, 1994
The receptor for urokinase plasminogen activator (uPAR) acts as an anchorage site for UPA on the cell surface where it stimulates pro-uPA activation, allows the internalization of uPA:inhibitor and other complexes and sends directly or indirectly signals into the cell that may promote migration, adhesion and growth. It is a GPI-anchored, three-domain protein that belongs to the Ly6 family and is present at the focal and cell-to-cell contacts, where it concentrates UPA activity.
European Journal of Biochemistry, 1997
In LLC-PK, cells, urokinase-type plasminogen activator (uPA) mRNA has a short half-life of 70 min. We have previously demonstrated that most of the regulatory regions responsible for the rapid turnover of uPA mRNA in LLC-PK, cells reside in its 3' untranslated region (3' UTR), where there are at least three regulatory sites, one of which is A+U-rich. This A+U-rich sequence mediates uPA mRNA stabilization induced by protein kinase C (PKC) down-regulation. In this work, we found that uPA mRNA is rather stable in MDA-MB-231 cells with a half-life of 17 h. We compared the stability of hybrid globin mRNA containing different parts of uPA mRNA in its 3' UTR and found that the A+U-rich sequence of uPA mRNA renders otherwise stable globin mRNA unstable in LLC-PK, cells but not in MDA-MB-231 cells. We identified a cytoplasmic protein of 40 kDa (p40) which specifically interacts with the A+Urich sequence. Levels of p40 activity as detected by ultraviolet cross-linking were higher in MDA-MB-231 and PKC-down-regulated LLC-PK, cells than in untreated LLC-PK, cells. Prior treatment of the cytoplasm with a specific antibody against heterogeneous nuclear ribonucleoprotein C (hnRNP C) significantly reduced p40 activity. These results suggest a correlation between the A+U-rich sequence-dependent uPA mRNA stabilization in vivo and the binding of hnRNP C to the A+U-rich sequence in vitro. Keywords; 3' untranslated region; A+U-rich sequence ; hnRNP C ; mRNA stability; plasminogen activator. The regulation of mRNA stability is thought to play an important role in eukaryotic gene expression, because different mRNAs have different half-lives and various extracellular signals alter their stability (see reviews [l-31). Specific regulation of mRNA stability infers the presence of regulatory sequences in the mRNA molecule responsible for its metabolism. Several instability-determining sequences have been identified in the 3' untranslated region (3' UTR) as well as within the protein-coding region of many mRNAs. The most prominent is an A+Urich element (ARE), present in the 3' UTR of almost all unstable mRNAs, including various cytokine and oncogene mRNAs (see references in [4, 51). The minimum consensus sequence for ARES is UUAUUUA(A/U)(A/U)[6]. A number of proteins that interact with ARE of the 3' UTR of these short-lived mRNAs have been identified as potential transacting factors responsible for ARE-dependent mRNA metabolism [7-131. Three of the