The literature strongly supports the participation of LRP-1 in numerous cellular processes, including the regulation of cellular fibrinolytic activity and migration (3
). In this study, we investigated the contribution of LRP-1 to a range of responses of PMCs germane to pleural inflammation and remodeling. We found that uPAR and LRP-1 are expressed by the human pleural mesothelium in normalcy and after injury. We also found that LRP-1 is expressed by PMCs and determined, for the first time, that LRP-1 is suppressed in these cells by TNF-α and IL-1β. Lastly, we discovered that LRP-1 is the only member of the LDL superfamily represented in HPMCs, in which it regulates collagen expression induced by uPA, uPAR stability at the cell surface, PMC proteolysis, and cellular migration.
We and others showed that TGF-β and TNF-α increase expression of uPAR through transcriptional and posttranscriptional mechanisms (15
). Because these cytokines have also been reported to regulate LRP-1 expression in other cell types (28
), we sought to determine the role of TNF-α, TGF-β, and IL-1β on LRP-1 expression in PMCs. As expected, TNF-α, TGF-β, and IL-1β increased uPAR expression in MeT5A cells and HPMCs. However, TNF-α and IL-1β increased uPAR expression to a greater extent while reducing LRP-1. Further, TNF-α and IL-1β reduced uPA-mediated uPAR internalization by 80%. These changes were not observed in peritoneal mononuclear cells in a previous study (35
), perhaps relating to topologic heterogeneity between PMCs and peritoneal mononuclear cells or to the enhanced ability of relatively unstimulated PMCs from patients with CHF to respond to proinflammatory mediators. Because we cannot harvest normal HPMCs from subjects without pleural disease, HPMCs from patients with hydrostatic, relatively noninflammatory pleural effusions represent a relatively quiescent source of human cells. The findings recapitulate the responses of LRP-1 to TNF-α and IL-1β in MeT5A cells and, in the aggregate, represent a newly recognized mechanism by which the selected proinflammatory mediators can regulate uPAR and its functional repertoire in HPMCs.
The role of LRP-1 in uPAR processing in PMCs was confirmed by neutralizing cell surface LRP-1 in MeT5A cells and HPMCs with RAP in immunofluorescence localizations. uPA drives LRP-1 and uPAR to a primarily perinuclear distribution in these cells. RAP promotes uPAR clustering at the cell surface, suggesting that LRP-1 contributes to uPAR localization in PMCs. uPAR clustering was once believed to primarily localize cellular proteolytic activity (16
). However, recent studies have shown that uPAR clustering at the cell surface occurs at the leading edge of migrating cells and influences cellular signaling (36
). Our immunofluorescence results are supported by parallel studies showing that uPA-mediated uPAR internalization decreased by 80% (from 25% to < 5%; P
< 0.05) in the presence of RAP. Down-regulation of LRP-1 with siRNA reduced uPA-dependent uPAR internalization by approximately 50% but did not reach the inhibition seen with RAP. This effect may have been due to the relatively incomplete down-regulation of LRP-1 by siRNA (70%). Further, RAP treatment extended the t1/2
of uPAR in PMCs from 2.5 to 6 hours in the presence of uPA. The findings indicate that complexes of uPAR with exogenous uPA and PAI-1 expressed by the PMC are internalized via interaction with LRP-1. We attempted to stably express the complete LRP-1 construct in the deficient REN cells (data not shown) to test how gain of LRP functionality affects uPAR processing; however, this approach was problematic given the size of the cDNA (> 15 kb) and our inability to confirm cell surface localization of the construct.
Flow cytometry studies indicated that LRP-1 expression correlated with uPAR surface expression in PMCs. Previous studies have shown that LRP-1 function directly affects expression of uPA, PAI-1, and uPAR (39
). In this study, LRP-1–expressing MeT5A PMCs and LRP-1–deficient REN were used in internalization assays. uPA enhances uPAR internalization in MeT5A but not in REN cells. Because these cells express PAI-1, we postulate that endogenously produced PAI-1 forms an inhibitory complex with exogenously added uPA. This newly formed complex binds uPAR and drives internalization of uPAR through interaction with LRP-1 in MeT5A cells. To determine the role of uPA fibrinolytic activity in uPAR catabolism and internalization, preformed uPA/PAI-1 complexes were also used in t1/2
and internalization assays. uPA activity was not required to reduce uPAR t1/2
or to drive uPAR internalization in MeT5A cells (data not shown). However, uPA activity was required to cleave uPAR in the REN line (data not shown). In REN internalization assays, a small but quantifiable amount of uPAR was detected in the glutathione treatment control. This is most likely due to the inability to efficiently remove all biotin from the REN cell surface as a consequence of robust uPAR expression. It is conceivable that basal internalization of biotinylated surface uPAR could contribute to this result.
Because uPAR internalization was found to depend on the expression and function of LRP-1, uPAR half-life studies were performed in LRP-1–expressing and LRP-1–deficient cells. In surface biotinylation assays, we found that uPA treatment reduced uPAR t1/2
in MeT5A, MS-1, and M9K cells by almost 50%, which is consistent with previously published observations in other cell types (11
). The basal uPAR t1/2
, in the absence of supplemental uPA, in MS-1 and M9K was found to be around 4.5 hours (data not shown), similar to that found in the REN and MeT5A cells (5 h). Only REN cells exhibited the cleaved form of uPAR (D2D3) in the presence of uPA throughout the 24-hour time course. The lack of LRP-1 in REN cells may explain the increment of D2D3-uPAR in the presence of uPA. The D2D3 domain of uPAR is believed to play a role in enhanced cell migration and mesenchymal transition (40
). The increased uPAR t1/2
and the abundance of D2D3-uPAR found in the REN cells may contribute to their greater aggressiveness and invasiveness in vivo
, as we previously reported (18
). This aspect of uPAR processing is being evaluated in ongoing independent analyses.
Although LRP-1 neutralization blocked uPAR internalization and extended uPAR t1/2 in PMCs, uPAR was steadily catabolized throughout the time course in the presence or absence of uPA. Further, LRP-1 neutralization did not foster accumulation of cleaved D2D3-uPAR in MeT5A cells, as found in REN cells. These data suggest that LRP-1 mediates uPA-dependent uPAR down-regulation and internalization but that selective cleavage of uPAR by uPA in REN cells may involve alternative form of receptor–protease interaction.
Due to the stabilized expression of uPAR in LRP-deficient REN cells, we hypothesized that RAP treatment would extend the activity of exogenous uPA over a 12-hour time course. MeT5A and REN cells were selected for these analyses because they do not express levels of endogenous uPA that could confound the findings using fibrin enzymography (18
). Because uPA has been previously reported to bind cells through receptors other than uPAR (43
), we first confirmed that uPA-associated with the PMC surface through interactions with uPAR. Because uPA activity was not detected in ATN-617–treated MeT5A cells, we infer that most, but likely not all, of the binding of uPA was to uPAR at the cell surface. In uPA-treated REN cells, the durability of uPA activity is most likely attributable to the lack of LRP-1 and overexpression of uPAR. This conclusion is buttressed by the ability of RAP to sustain uPA activity associated with MeT5A cells, most likely through the stabilization of uPAR at the cell surface. Comparable responses were observed in HPMCs. These data show that LRP-1 regulates cell-associated uPA activity in MeT5A cells and in primary HPMCs.
Previous studies have shown that uPA can also potentiate migration in a uPA/uPAR-dependent manner (23
). Our studies demonstrate that the addition of uPA as a chemo-attractant potentiates PMC migration across a vitronectin-coated filter insert. Further, LRP-1 neutralization with RAP potentiates the promigratory effect of uPA. Although the FBS used in these assays may contain bovine uPA, the species-specific nature of the uPA and uPAR interaction makes a confounding effect unlikely (46
). These studies show that neutralization of LRP-1 potentiates uPA-mediated migration in MeT5A cells and HPMCs.
We also report the novel observation that uPA and LRP-1 can regulate PMC collagen 1 expression. Because the MeT5A line does not produce collagen 1 in response to TGF-β1, primary PMCs were used in these analyses. TGF-β has been reported to stimulate collagen 1 expression in murine and rat PMCs in vitro
and in vivo
). We confirmed that TGF-β induced collagen 1 in RPMCs and HPMCs. We also found that uPA stimulates collagen 1 expression in both these primary cell types. Further, the combination of uPA and TGF-β enhanced collagen 1 expression. Although RAP did not enhance the TGF-β effect, the combination of RAP with uPA increased collagen 1 expression. Hydroxyproline analysis confirmed that total collagen expression was also increased by uPA in RPMCs and HPMCs and that these effects were potentiated by RAP. The data show that LRP-1 can influence uPA-mediated collagen 1 expression.
Because previous studies have shown that plasmin can convert latent TGF-β to its active form (50
), we assayed the potential of uPA or RAP to activate latent TGF-β. uPA did not increase the levels of active TGF-β in HPMCs, indicating that uPA-mediated induction of collagen by HPMCs under the conditions we used may involve alternative intermediaries. RAP alone or with uPA did not change active TGF-β levels in HPMCs, although there was a trend toward increased levels. However, total levels of TGF-β were increased by these conditions as well as by uPA alone. We therefore posit that blockade of LRP-1 by RAP may hinder the internalization and degradation of TGF-β in PMCs and facilitate its accumulation, as reported previously in other cell systems (52
). On the other hand, our data clearly show that RAP alone did not induce collagen in HPMCs, strongly suggesting that levels of induction of activated TGF-β were likely insufficient to recapitulate the effects of the exogenous TGF-β we used. Alternatively, it is possible that the processing of activated TGF-β may be accelerated in the presence of RAP, consistent with its induction of total TGF-β in HPMCs. Although the data do not exclude a potential contribution of TGF-β in uPA-mediated induction of HPMC collagen, its processing by PMCs stimulated by uPA with or without RAP may be complex. Full elucidation of the mechanism by which uPA induces collagen expression by HPMCs requires comprehensive studies that extend this work. Although the precise mechanism is unknown, we posit that uPA may cleave uPAR and thereby initiate mesenchymal transition, as reported by other groups (42
) and will pursue this possibility in an extension of this work.
In summary, the ability of inflammatory cytokines to alter expression of LRP-1 in HPMCs is a novel observation, as is the induction of collagen 1 in primary HPMCs by uPA and potentiation of the effect by RAP. Our results show that TNF-α and IL-1β down-regulate LRP-1 expression at the mRNA and protein levels. Exposure of PMCs to these agents or uPA can thereby influence a broad functional repertoire subject to regulation via LRP-1, including cellular proteolysis, migration, and collagen expression.