Regulation of urokinase expression at the posttranscription level by lung epithelial cells

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

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 alpha-thrombin potentiated the response. Pretreatment of Beas2B cells with a tyrosine kinase inhibitor, herbimycin, suggests that activation of tyrosine kinase(s) is involved in the uPA-mediated uPA expression. Induction of uPA expression by exposure of lung-derived epithelial cells to uPA is a newly defined pathway by which this protease could influence expression of local fibrinolytic activity and other uPA-dependent cellular responses germane to lung inflammation or neoplasia.     

Induction of p53 by urokinase in lung epithelial cells

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-beta1 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.     

Urokinase induces expression of its own receptor in Beas2B lung epithelial cells

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.     

Regulation of urokinase receptor expression by p53: novel role in stabilization of uPAR mRNA

We found that p53-deficient (p53(-/-)) lung carcinoma (H1299) cells express robust levels of cell surface uPAR and uPAR mRNA. Expression of p53 protein in p53(-/-) cells suppressed basal and urokinase (uPA)-induced cell surface uPAR protein and increased uPAR mRNA degradation. Inhibition of p53 by RNA silencing in Beas2B human airway epithelial cells conversely increased basal as well as uPA-mediated uPAR expression and stabilized uPAR mRNA. Purified p53 protein specifically binds to the uPAR mRNA 3' untranslated region (3'UTR), and endogenous uPAR mRNA associates with p53. The p53 binding region involves a 37-nucleotide uPAR 3'UTR sequence, and insertion of the p53 binding sequence into beta-globin mRNA destabilized beta-globin mRNA. Inhibition of p53 expression in these cells reverses decay of chimeric beta-globin-uPAR mRNA. These observations demonstrate a novel regulatory role for p53 as a uPAR mRNA binding protein that down-regulates uPAR expression, destabilizes uPAR mRNA, and thereby contributes to the viability of human airway epithelial or lung carcinoma cells.     

Regulated poly(A) tail shortening in somatic cells mediated by cap-proximal translational repressor proteins and ribosome association.

The poly(A) tail plays an important role in translation initiation. We report the identification of a mechanism that operates in mammalian somatic cells, and couples mRNA poly(A) tail length with its translation state. The regulation of human ferritin L-chain mRNA by iron-responsive elements (IREs) and iron regulatory proteins (IRPs) is subject to this mechanism: translational repression imposed by IRP binding to the IRE of ferritin L-chain mRNA induces poly(A) tail shortening. For the accumulation of mRNAs with short poly(A) tails, IRP binding to an IRE per se is not sufficient, but must cause translational repression. Interestingly, puromycin and verrucarin (general translation inhibitors that dissociate mRNAs from ribosomes) mimick the negative effect of the specific translational repressor proteins on poly(A) tail length, whereas cycloheximide and anisomycin (general translation inhibitors that maintain the association between mRNAs and ribosomes) preserve long poly(A) tails. Thus, the ribosome association of the mRNA appears to represent the critical determinant. These findings identify a novel mechanism of regulated polyadenylation as a consequence of translational control. They reveal differences in poly(A) tail metabolism between polysomal and mRNP-associated mRNAs. A possible role of this mechanism in the maintenance of translational repression is discussed.     

ANISOMYCIN,ACETOXYCYCLOHEXIMIDE, CYCLOHEXIMIDE,AND PUROMYCIN AS INHIBITORS OF RAT BRAIN ACETYLCHOLINESTERASE IN VITRO1

A kinetic analysis of the interaction of anisomycin, acetoxycycloheximide, cycloheximide, and puromycin with acetylcholinesterase (acetylcholine acetyl-hydrolase, EC 3.1.1.7) in rat brain homogenate shows that all of these protein synthesis inhibitors are also inhibitors or this enzyme. Puromycitl aminonucleoside, a puromycin analog without antibiotic activity, was also found to be an inhibitor of acetylcholinesterase activity much like puromycin. Anisomycin appeared to be a competitive inhibitor whereas all of the other compounds showed mixed inhibition. The apparent 10.5 values for inhibition of rat brain acetylcholinesterase at 50 μM substrate were: anisomycin, 3 mM; acetoxycycloheximide, 1 mM; cycloheximide, 2.2 mM; puromycin, 0.5 mM and puromycin aminonucleoside, 0.6 mM.