In this study, we show that uPA increases the expression of TF and TF procoagulant activity in human lung ECs. This response is germane to homeostatic control of fibrin turnover in the normal lung. TF and uPA are both induced by diverse hormones, cytokines, and growth factors. In the lung, TF is expressed at the surface of injured lung ECs, which also elaborate uPA. In the context of lung injury and its repair, this pathway may also provide a feedback mechanism to regulate the extent of fibrin accretion in the airspaces and interstitium. Extravascular fibrin serves as a provisional matrix promoting leukocyte migration, in part mediated through uPA/uPAR–integrin interactions (10
) that help to confront microbial and other provocative stimuli. The fibrin matrix also supports physiological small airway and alveolar remodeling (6
Although excess fibrinolysis can damage and weaken lung ultrastructure, excess coagulation can lead to alveolar fibrin deposition, thus resulting in stiffening of the lung and impairment of function. Excessive fibrin deposition impairs gas exchange in alveoli, as in ARDS, and promotes bronchospasm (22
) and interstitial lung diseases (1
). Therefore, regulation of the delicate balance between coagulation and fibrinolysis is crucial for maintaining proper lung structure and function, and a slight alteration in this balance can dictate whether observed fibrin turnover in the alveolar compartment is homeostatic or aberrant (1
). The balance between enzymes involved in proteolysis and coagulation is also critical in the regulation of tissue remodeling and normal angiogenesis (6
). Thus, the temporal and geographic balance and interplay between the procoagulant and fibrinolytic systems is critical for the lung to adapt to diverse forms of environmental injury. The possibility that this balance is achieved through a process that is initiated by uPA has not been previously addressed, to our knowledge. Therefore, we sought to determine whether uPA contributed to coagulation by regulating TF expression by lung epithelial cells.
uPA-dependent proteolysis contributes to tissue remodeling in inflammation (23
). Binding of uPA to uPAR mediates proteolysis locally and thereby assists in remodeling transitional fibrin in the airspaces (10
). This interaction also regulates the viability of lung ECs (14
), representing a potentially important mechanism that can help to restore damaged airway epithelium. Our data show that the system contributes to homeostatic fibrin turnover in the uninjured lung. Thus, the uPA-TF induction pathway we describe may provide a versatile regulatory system through which the airway epithelium might be protected from the potentially injurious effects of excessive pericellular proteolysis caused by high local concentrations of uPA in the lung microenvironment mediated by cytokines such as tumor necrosis factor-α (18
) and other stimuli (24
). However, the key independent contributions of uPA and TF to the pathogenesis of lung injury and repair suggest that this interaction could likewise limit clearance of aberrant fibrin deposition after acute lung injury.
In the normal lung, uPA is present in BAL fluids and could induce TF, but in the absence of coagulation substrates, activation of coagulation does not occur. In the setting of ALI, uPA-mediated induction of TF could restrict excessive clearance of fibrin at the sites of injury. The concentrations of uPA we studied are biologically relevant and are within the range of those found in the BAL of healthy individuals and patients with ARDS (26
). In fact, as the concentrations of uPA in BAL may actually be higher than reported because of dilution by saline during recovery, the effects we observed may in fact be enhanced in pathophysiological settings. In addition, diverse types of pulmonary cells may be stimulated to express uPA during sepsis and pneumonia and expression of uPAR and other receptors likely enhances the concentrations of uPA at the epithelial surface during resolution of ALI.
Multiple reports have indicated that administration of uPA mitigates bleomycin-induced lung fibrosis (32
). We speculate that under such scenarios the relatively high level of exogenous uPA that is administered readily overwhelms the reciprocal effect of TF induction that occurs locally at the epithelial surface, thus favoring fibrinolysis. The inability of LPS to markedly induce TF expression in uPA−/−
mice and the resistance exhibited by these mice to ALI caused by exposure to bacterial endotoxin (1
) demonstrate an intricate link between procoagulant and fibrinolytic pathways in vivo
and potentially substantial pathophysiological significance. On the other hand, it is interesting to note that uPA-deficient mice develop worse fibrosis after bleomycin-induced injury. These contrasting responses by uPA−/−
mice to acute injury induced by LPS and chronic injury caused by the profibrogenic bleomycin probably involve differences in neutrophil activation and cytokine production by macrophages in the two models (1
) and are worthy of continued study.
IHC and morphometric analyses of lung sections for SPC and TF expression indicate that ATII cells are primarily responsive to uPA. Western blot analyses for TF and SPC antigens of freshly isolated ATII cells from wt and uPA-deficient mice treated with LPS further demonstrate that uPA expression is crucial for induction of TF. Potentiation of LPS-induced neutrophil activation by uPA administration and the early (within 6 h) resistance of uPA−/−
mice to neutrophil influx and edema formation in the lungs (1
) suggested that the differences in TF expression between uPA−/−
and wt mice could have caused by reduced influx of inflammatory cells in the uPA-deficient mice. Although the present study does not exclude this possibility, induction of TF protein and mRNA by uPA in cultured ECs and comparable amounts of focal neutrophil influx, edema formation, and fibrin accumulation in LPS-treated wt or uPA-deficient mice demonstrate that it is unlikely that cytokines released from the inflammatory cells are solely responsible for the up-regulation of TF in the EC. We attribute the focal distribution of ALI we identified in this study to the low concentrations of LPS that were administered intratracheally, with assessment of the lesions 24 hours later.
Lung epithelial cells express increased levels of TF in response to cytokines implicated in the pathogenesis of ALI and its repair (31
). The expression of TF in lower respiratory tract fluids under control conditions and in ALI is likely to derive from multiple resident lung cell populations, including the airway and alveolar epithelium and alveolar macrophages (6
). Our study extends these observations and demonstrates that lung EC expression of TF is also subject to induction by uPA. uPA could therefore regulate TF expression and initiation of coagulation at sites of epithelial injury. uPA could otherwise modulate repair after ALI via TF-mediated signaling through protease activated receptors (PARs) cleaved by Xa, thrombin, and plasmin at the epithelial cell surface (34
Identification of the signaling pathway responsible for uPA-mediated TF expression is currently in progress. Involvement of tyrosine phosphorylation in TF induction by uPA is supported by the observation that inhibition of tyrosine kinases abolishes uPA-mediated TF expression. Conversely, suppression of tyrosine phosphatase activity augments TF expression, indicating that the uPA-mediated effect could involve phosphorylation of other intermediaries, including mRNABps and/or transcription factor(s), consistent with uPA-mediated uPAR expression (17
LPS induces TF expression through both transcriptional and posttranscriptional mechanisms in THP-1 cells (20
). Transcriptional regulation of TF has been studied extensively and involves several transcription factors, including activated protein-1 (AP-1), nuclear factor (NF)-κB, and Egr-1 (20
). By contrast, the mechanisms that govern post-transcriptional regulation of TF expression have not been studied. Our findings suggest that the uPA GFD, which lacks catalytic activity, mimics tcuPA in terms of inducing TF expression. Furthermore, and unlike uPA-mediated uPA–uPAR–PAI-1 expression, induction of TF expression by uPA is mediated at both the transcriptional and post-transcriptional levels.
In previous studies, we found that a post-transcriptional pathway influences levels of uPA, uPAR, and PAI-1 mRNA in lung cancer–derived cell lines and nonmalignant lung ECs (38
). Similar findings were previously reported in cells exposed to phorbol 12-myristate 13-acetate, insulin, insulin-like growth factor, and cyclic nucleotide analogs (40
). Cytokines expressed in the setting of ALI or in the tumor microenvironment increase TF expression (31
). Previous studies have indicated that TF 3′UTR contains several AU-rich elements, including AUUUA, poly U tracks, and UUAUUUAAU, that are known to control the decay of several mRNA species (7
). Studies are in progress to determine the responsible mechanism and to identify the responsible regulatory factors involved in the induction of TF by uPA.
Based on this information, we confirmed by gel shift assay that specific mRNA binding protein(s) interact directly with TF mRNA 3′UTR to regulate its stability. Northwestern assay indicated involvement of two specific protein species, with approximate molecular weights of 80 kD and 37 kD. uPA increased the interaction of 80-kD protein with TF 3′UTR, indicating that this protein may be involved in the stabilization. Interaction of the 37-kD protein was attenuated in lung epithelial cells in a time-dependent manner after uPA exposure, indicating this protein may be involved in the destabilization of TF mRNA. Specificity and competition experiments indicate that the interaction of TF mRNABp with TF mRNA 3′UTR requires a unique A-rich sequence. Purification and characterization of the TF 3′UTR mRNA binding proteins are underway to define its role in the regulatory mechanism.
The mechanism by which tcuPA induces TF in Beas2B cells appears to be largely independent of its proteolytic activity. Inhibitors of the uPA catalytic site or its proteolytic inactivation caused little loss of TF-inducing activity. Moreover, essentially identical amounts of TF were induced by full-length single- and two-chain uPA and by a kringle domain deletion variant. In contrast, little or no TF was induced when the growth factor domain was deleted from the full-length molecule. Thus, binding of the GFD to uPAR appears to be necessary and sufficient to induce TF. This also implies that inactive uPA–PAI-1 complexes that retain interaction with uPAR could signal increased TF expression. This is important because tumor cells express high levels of uPA, uPAR, and PAI-1, and increased circulating and tissue concentrations of PAI-1 are present during severe infection and other causes of lung inflammation, accompanied by other signals that induce TF, resulting in conditions that may augment fibrin deposition by stimulating this TF-mediated pathway and thereby exacerbate lung injury.
In summary, we demonstrate that uPA stimulates expression of TF by lung ECs in culture and in vivo
. This newly identified pathway is, to our knowledge, the first observation that uPA regulates the expression of components involved in coagulation in any cell type. The physiological and pathological implications of uPA-mediated TF expression will require additional study, but our data show that the pathway is operative in normal lung tissue. Induction of TF by uPA regulates fibrin turnover in the alveolar compartment of the normal lung and might contribute to local overexpression of TF associated with inflammatory lung diseases (1
). Identification of the intracellular mediators of uPA-mediated TF expression may provide a new understanding of how regulation of this pathway may help to limit aberrant fibrin turnover associated with lung injury.