uPA-mediated plasminogen activation is involved in pericellular proteolysis, remodeling of extracellular matrix, and a variety of nonproteolytic functions, such as proliferation, adhesion, migration, cell signaling, and gene expression (7
). These functions of uPA may contribute to remodeling of the lung, which occurs in acute respiratory distress syndrome or the interstitial and neoplastic lung diseases (29
The tumor suppressor protein p53 is induced and activated in response to different forms of stress and regulates cellular repair or apoptosis. In addition, p53 normally transactivates a variety of target genes that contain one or more p53 binding sites in their promoter regions (19
). Stabilization and activation of p53 are achieved by protein–protein interactions (e.g., MDM2 ubiquitin ligase) (36
) and post-translational modification such as phosphorylation, acetylation, and sumolation. In most tumor cells, the p53 function is reduced or abolished, which results in uncontrolled proliferative potential in these cells. Inhibition of p53 function is caused by either lack of p53 expression or inactive mutation of the p53 gene. Altered p53 expression may elicit epithelial cell transformation and control the potential of these cells to degrade the extracellular matrix. Most tumor cells that lack p53 exhibit increased cell surface plasminogen activation and uPA-induced proliferation, migration, and invasiveness (37
). We therefore sought to unravel the mechanism by which uPA and p53 interact and to determine how p53 and uPA expression in lung epithelial and carcinoma cells is regulated by uPA.
p53 suppresses cell surface fibrinolysis by inhibition of uPA-mediated plasminogen activation via induction of plasminogen activator inhibitor (PAI)-1 expression through promoter transactivation. The PAI-1 promoter exhibits a p53 binding sequence (25
). Unlike PAI-1 promoter, the uPA promoter lacks a p53 binding site. However, p53 represses uPA expression most likely through TATA-binding protein-associated factors (25
uPA induces its own expression in a concentration-dependent manner (9
), and at a low concentration of approximately 1 nM, uPA induces p53. However, at concentrations greater than 10 nM, uPA inhibits cellular p53 expression. The process involves both Ser15 phosphorylation of p53 as well as MDM2-mediated p53 ubiquitination (13
). Our findings indicate the existence of an inverse relationship between p53 and uPA expression in lung epithelium–derived cells, which is contradictory to the earlier reports in which both p53 and uPA levels are decreased in breast cancer cells (40
). This discrepancy could be due to tissue origin and tumor metastasis stages. These observations support the possibility for cross-talk between p53 and uPA expression. However, a direct link between p53 and uPA expression has been lacking. Here we show for the first time that expression of the uPA gene is regulated by p53. Our results indicate that basal uPA expression is elevated in p53−/−
cells and uPA further increases uPA expression.
We recently reported that maximum uPA expression was observed when lung epithelial cells were treated with a uPA concentration greater than 10 nM (500 ng/ml). We also found that uPA regulates p53 expression in a concentration-dependent manner and that uPA induces its own expression at a concentration at which p53 expression is totally suppressed. Unlike uPA-mediated Beas2B cell p53 expression (13
), uPA and uPAR levels are induced by inhibition of protein tyrosine phosphatase and suppressed by blockade of tyrosine kinase activity (7
). Furthermore, β1
-integrin activation by treatment with anti–β1
-integrin antibody as well as overexpression of protein tyrosine phosphatase SHP2 alters p53 expression (13
). Hence, it is likely that signal transduction through these proteins could contribute to uPA expression. We now show that cells lacking p53 express elevated levels of uPA. We interpret the results to indicate that p53 affects uPA expression and that dose-dependent induction of uPA in lung epithelial cells is due to inhibition of p53. The role of p53 in uPA expression is further supported by the fact that restoration of p53 in p53−/−
cells suppressed basal uPA expression. Interestingly, uPA failed to induce uPA expression in these cells. Furthermore, induction of basal uPA expression as well as the added effect of uPA in p53-inhibited (SiRNA-treated) cells shows that p53 regulates cellular uPA expression.
Although the uPA promoter lacks the p53 binding sequence, it has previously been reported that p53 represses uPA mRNA synthesis (25
). Our results show that expression of p53 in p53−/−
cells failed to induce uPA mRNA synthesis, indicating that inhibition of uPA mRNA synthesis by p53 may not be responsible for the observed effect. This interpretation is further supported by our earlier observation that uPA stabilizes uPA and uPAR mRNA (7
). We recently found that p53 inhibits cell surface uPAR expression and uPA-mediated lung cancer cell proliferation (42
), which suggests that induction of uPAR may be in part due to suppression of p53-mediated uPA mRNA destabilization. Decay of uPA mRNA in H1299 cells transfected with p53 cDNA further demonstrates the involvement of the role of p53 in the destabilization of uPA mRNA.
We hypothesized that p53 directly interacts with uPA mRNA to regulate its stability. We confirmed this assumption by showing that rp53 interacts with the uPA mRNA 3′UTR by gel shift assay. Coprecipitation of uPA mRNA with p53 from Beas2B CLs substantiated the existence of such an interaction in vivo. The p53–uPA mRNA 3′UTR complex was resistant to both RNase A and T1 digestion. The specificity of p53 interaction with the uPA mRNA 3′UTR was assessed by self-competition experiments in which a labeled sense probe was successfully competed by its unlabeled analog. An unlabeled antisense probe failed to compete for specific binding. Furthermore, addition of a molar excess of a homopolyribonucleotide poly(A), (C), (G), or (U) had no effect on p53 protein binding to 32P-labeled uPA mRNA 3′UTR, indicating that p53 binding requires unique sequence. The specificity of p53 binding to the uPA 3′UTR mRNA is also supported by the finding that pretreatment of p53 by proteinase K or SDS or predigestion of uPA mRNA probe with RNAase A and RNAase T1 totally abolished the p53–uPA mRNA 3′UTR complex.
Deletion experiments showed that rp53 specifically binds to a 35-nt uPA mRNA 3′UTR sequence and that the binding region is located upstream of the 66-nt 30-kD uPA mRNA binding protein (mRNABp) binding sequence. Our studies indicate that there is no other p53 binding sequence in uPA CDR or 3′UTR other than this 35-nt region, which corresponds to the uPA cDNA sequence from 1585 to 1620. We found that insertion of the 35-nt p53 binding sequence into β-globin mRNA destabilized the chimeric transcript in Beas2B cells, indicating that the p53 binding sequence contains information for mRNA destabilization. Transfection of cells with this sequence induced uPA expression in p53-expressing H1299 cells by competing with endogenous mRNAs, further confirming that the p53 interaction with the 35-nt uPA mRNA 3′UTR sequence destabilizes uPA mRNA. Our observations show that p53 interacts with the 35-nt uPA mRNA destabilizing element and that increased expression of uPA occurs in cells where expression of wild-type p53 is reduced, delineating a novel regulatory mechanism. Transfection of p53 inhibits uPA expression through down-regulation of uPA mRNA. In all, these studies demonstrate a novel function of p53 as an mRNA binding protein that regulates the expression of uPA mRNA by altering its stability. The pathophysiologic consequences of this pathway on clinical lung diseases, including acute lung injury, pulmonary fibrosis, or lung neoplasia, are currently unclear. However, these diverse diseases are characterized by disordered epithelial viability and disordered alveolar fibrin turnover (2
). Our results strongly suggest that this newly identified p53-mediated regulatory function could play a role in epithelial cell survival and uPA expression in these contexts.
In summary, we confirmed that the p53–uPA mRNA 3′UTR interaction regulates uPA mRNA stability in lung epithelial cells. To our knowledge, this newly identified pathway is the first description of the capability of p53 to interact with uPA mRNA and control its expression at the post-transcriptional level. This pathway adds a new dimension to the p53 functional repertoire and represents a new link between viability of lung epithelial cells and pericellular proteolysis, as occurs in neoplastic transformation, growth, and spread of lung neoplasms. This pathway may likewise contribute to the regulation of uPA-dependent responses of the lung epithelium in the context of lung injury and repair.