Local generation of plasmin by uPA is central to the process by which lung epithelial cells degrade fibrin and extracellular matrices as a consequence of lung injury and repair. Expression of uPA and its cell surface receptor uPAR is germane to a variety of cellular responses involved in the pathogenesis of inflammation. The uPAR focuses uPA-dependent pericellular proteolysis. In addition, interaction of uPA and uPAR facilitates cellular proteolysis, cell proliferation, adhesion, and migration in diverse cell types of many organs, including lung, during infection or injury (12
Bacterial endotoxins are well recognized for their ability to induce pulmonary inflammation in animal models of ALI, but their effect on uPA and uPAR expression is only partially understood (42
). It has previously been reported that expression of uPA and uPAR, as well as their specific interaction, is enhanced by proinflammatory agents, such as LPS, TGF-β, and TNF-α in various cell lines (12
). These agents intensely and rapidly induce cell surface uPAR expression and the effect that can be traced back to a rapid, antecedent increase in the cellular level of uPAR mRNA.
In the present study, we used LPS to stimulate lung epithelial response in ALI in vitro
, and noticed a pronounced effect in uPAR expression on lung epithelial cells. Induction of uPAR was time-dependent, reaching its maximum effect at 24 hours, a phenomenon that is consistent with previously reported findings for PMA in Beas2B and MS-1 cells (23
). LPS similarly increased uPAR mRNA by stabilizing uPAR mRNA in Beas2B cells, indicating that post-transcriptional regulation is involved in endotoxin-induced uPAR expression.
Earlier reports have shown that post-transcriptional regulation of uPAR expression is mediated by the binding of PGK with cis
elements within a 51-nt sequence of the uPAR mRNA CDR (24
). uPAR expression is also increased through mRNA stabilization by hnRNPC binding to a 110-nt uPAR mRNA 3′ UTR determinant. Agents such as PMA, TGF-β, and TNF-α are known to induce uPAR expression via
mRNA stabilization and suppress binding of PGK to uPAR mRNA (23
). These agents also enhance uPAR mRNA stability by augmenting hnRNPC–uPAR 3′ UTR interaction (13
). Therefore, we hypothesized that the induction of uPAR expression by LPS in these cells would be regulated by post-transcriptional mechanisms that involve the interaction between uPAR mRNA and PGK and/or hnRNPC proteins.
LPS alters the interaction of PGK and hnRNPC with uPAR mRNA CDR and 3′ UTR in a coordinated manner that enhances message stability. The mechanism involves tyrosine phosphorylation of both PGK and hnRNPC without altering their basal expression. LPS treatment down-regulated PGK binding to uPAR mRNA CDR in a time-dependent manner, while simultaneously increasing the binding of hnRNPC to the uPAR mRNA 3′ UTR.
Increased uPA and plasminogen activator inhibitor (PAI)-1 are often encountered in severe infections. Bacterial endotoxin and inflammatory cytokines released during infection induce uPA expression (4
). Therefore, we asked whether LPS likewise induce uPAR expression in vivo
using a murine model of endotoxin injury and whether this up-regulation requires the presence of uPA. AT II cells isolated from LPS-treated WT mice showed increased uPAR expression when compared with those obtained from PBS-treated control mice. No significant difference in uPAR expression was detected in AT II cells from LPS- and PBS-treated uPA−/−
mice. uPAR analyses of lung homogenates from LPS-treated WT mice likewise exhibited augmented expression when compared with PBS-treated control mice. These observations demonstrate that LPS induces uPAR expression in the lung, and that AT II cells contribute to the increase. The finding that LPS did not induce uPAR protein or mRNA in uPA-deficient mice further confirms that uPA expression is critical to the process. This is consistent with earlier in vitro
observations that uPA induces uPAR expression (28
). Increased uPA in the circulation during life-threatening infections (6
) and its ability to induce PAI-1 expression in lung epithelial and smooth muscle cells by binding to uPAR via the amino-terminal fragment (47
) indicate that both uPA and uPAR are involved in the net increase in the expression of PAI-1 during infection.
mice are protected from endotoxemia-induced development of lung edema, pulmonary neutrophil accumulation, and increase in lung IL-1β, macrophage inflammatory protein 2 and TNF-α cytokine levels (6
), and neutrophil migration is inhibited in uPAR−/−
mice during lung infection (11
). Taken together, these observations strongly suggest the involvement of uPA and uPAR in lung injury caused by infection. Although LPS induces uPA and uPAR expression at the transcriptional and post-transcriptional level (28
), the finding that LPS does not induce uPAR in uPA−/−
mice, and the ability of uPA to induce uPAR expression only through uPAR mRNA stabilization (28
), implicates post-transcriptional regulation in LPS-induced uPAR expression. Stabilization of uPAR mRNA by LPS in Beas2B cells provides additional support for this inference.
These observations lead to the conclusion that increased uPAR mRNA in cells exposed to LPS is due to decreased post-transcriptional turnover. Prior in vitro
analyses have shown the involvement of uPAR mRNA binding proteins, PGK and hnRNPC, in post-transcriptional regulation of uPAR mRNA expression (13
). Regulation involves tyrosine phosphorylation of both PGK and hnRNPC (24
). This was further supported by the fact that LPS strongly induces p38 mitogen-activated protein kinase (MAPK) phosphorylation (6
), and inhibition of p38 MAPK blocks uPAR mRNA stabilization (49
). Furthermore, PMA, TGF-β, and TNF-α influence the phosphorylation status of PGK, which determines its binding efficiency to uPAR mRNA (23
In the present study, we extended the observations to unravel the regulatory mechanism by which LPS induces uPAR expression in vivo. Our findings show that the mechanism involves the interaction of PGK and hnRNPC with uPAR mRNA at the post-transcriptional level, consistent with in vitro findings. LPS neither induces PGK or hnRNPC protein, nor mRNA expression in either saline (PBS)-treated control or LPS-treated WT mice or uPA−/− mice. However, LPS induces tyrosine phosphorylation of PGK and hnRNPC in LPS-challenged WT mice. LPS-induced tyrosine phosphorylation of PGK prolongs the half-life of uPAR mRNA by down-regulating the binding of PGK protein to uPAR mRNA. LPS also activates hnRNPC via tyrosine phopshorylation, which further stabilizes uPAR mRNA through increased hnRNPC binding to its cognate 3′ UTR determinant. However, additional studies are needed to determine if p38 MAPK activated by LPS is involved in the tyrosine phosphorylation of either PGK or hnRNPC.
We observed greater binding of PGK to uPAR mRNA CDR in saline (PBS)- and LPS -exposed uPA−/−
mice compared with LPS-challenged WT mice. In contrast, hnRNPC showed less binding to uPAR mRNA 3′ UTR. LPS failed to induce tyrosine phosphorylation of either PGK or hnRNPC in uPA−/−
mice. Overexpression of the protein tyrosine phosphatase, SHP2, dephosphorylates PGK and increases its binding affinity for uPAR mRNA CDR, while inhibiting the binding of hnRNPC to uPAR mRNA 3′ UTR (29
), affirming the importance of tyrosine phosphorylation. The opposing effects of PGK and hnRNPC on uPAR mRNA were found to act in concert to enhance uPAR expression.
In conclusion, our study shows, for the first time, that regulation of LPS-mediated uPAR expression is mediated through tyrosine phosphorylation of PGK and hnRNPC. These proteins regulate expression of cell surface uPAR and uPAR mRNA stability in mouse lung tissues. The process involves expression of uPA as an obligate intermediary. This newly recognized mechanism and novel pathway provides opportunities to regulate uPAR-dependent fibrinolysis and signal transduction in LPS-induced ALI.