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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online July 1, 2004 as doi:10.1096/fj.03-1123fje. |
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Inserm U-466, CHU Rangueil, Toulouse, France;
* Laboratoire d'Hematologie, CHU Rangueil, Toulouse, France; and
Microbiology Department and Cancer Center, University of Virginia, Charlottesville, Virginia, USA
1Correspondence: Biochimie-INSERM U466, IFR-31, CHU Rangueil 1, avenue Jean Poulhès-TSA-50032, 31059 Toulouse Cedex 9, France. E-mail: anesalv{at}rangueil.inserm.fr or salvayre{at}rangueil.inserm.fr
SPECIFIC AIMS
Plasminogen activators urokinase plasminogen activator (uPA) and tissue-type plasminogen activator (tPA) and their receptor, uPAR, have been implicated in various physiological processes such as clot lysis, extracellular proteolysis, matrix remodeling, and wound healing. This system is also involved in the regulation of cell migration, adhesion, and proliferation, with major implications for cancer dissemination and cardiovascular diseases. So far, the nature and interactions between the different signaling pathways elicited by the plasminogen system are only partly understood. On the other hand, the sphingomyelin/ceramide/sphingosine-1-phosphate (Spm/Cer/S1P) pathway has recently emerged as a major signaling system that regulates cellular responses such as growth, survival, and migration and is implicated in pathophysiology of cancer or atherosclerosis.
The aim of present work was to investigate the existence of links between plasminogen activators and the Spm/Cer/S1P pathway, how and when they interact, and the potential implications for cell proliferation elicited by the plasminogen system.
PRINCIPAL FINDINGS
1. The mitogenic effect of tPA and ATF is mediated by the Spm/Cer/S1P pathway
Preliminary experiments indicated that 50 ng/mL of tPA are mitogenic for bladder carcinoma ECV304 and for vascular smooth muscle cells (SMC). Similar results were obtained with ATF (the amino-terminal catalytically inactive fragment of uPA), suggesting that the proteolytic activity of tPA is not required for mitogenic activity.
tPA triggered ERK1/2 activation exhibiting a biphasic time course, the early component peaking at 15 min and the late component at 2 h. Different signaling pathways mediate the two phases of ERK activation; conversely, each phase of ERK activation plays a different role in the mitogenic signaling.
tPA and ATF signaling is associated with the activation of the Spm/Cer/S1P pathway, characterized by a rise of a neutral sphingomyelinase activity (peaking at 45 min), of sphingomyelin hydrolysis and of sphingosine kinase activity (Fig. 1
A–C). As D-erythro-2-(N-myristoylamino)-1-phenyl-propanol (D-MAPP) and dimethyl-sphingosine (DMS), two classical inhibitors of the Spm/Cer/S1P pathway, inhibited tPA-induced DNA synthesis, it is suggested that the Spm/Cer/S1P pathway is implicated in the mitogenic effect.
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Classical inhibitors of the tPA signaling pathway inhibited tPA-induced cell proliferation. Pertussis toxin (PTX, a Gi inhibitor known to block signal transduction triggered by tPA), PAI-1 (the natural inhibitor of the plasminogen system), herbimycin A (a broad range specificity inhibitor of tyrosine kinase, including Src), and the MEK-1 inhibitor PD98059 inhibited DNA synthesis. This led us to investigate more precisely the cross-talk and role of the Spm/Cer/S1P pathway in the complex concert of tPA-induced cell signaling.
2. Cell signaling implicated in the tPA-induced activation of the Spm/Cer/S1P pathway
The early (0–15 min) tPA-induced cell signaling regulates two key enzymes of the Spm/Cer/S1P pathway: neutral sphingomyelinase and sphingosine kinase-1. tPA-induced activation of sphingomyelinase was inhibited by PTX, hence suggesting the involvement of a (PTX-sensitive) Gi-mediated pathway. In contrast, PD98059 and herbimycin A did not block sphingomyelinase activation but inhibited sphingosine kinase-1 activation (Fig. 1D, E
), suggesting that sphingomyelinase and sphingosine kinase-1 are regulated by (partly) separate signaling pathways.
Src and ERK1/2 are required for the tPA-induced sphingosine kinase-1 activation. Src was rapidly activated by tPA in ECV304 cells via a PTX- and herbimycin A-sensitive mechanism but independent of the MEK1-ERK1/2 pathway (as shown by the lack of effect of PD98059). The crucial role of Src in sphingosine kinase-1 activation was demonstrated in fibroblasts overexpressing a kinase defective form of Src (SrcK–) or wild-type Src (SrcK+). tPA elicited sphingomyelin hydrolysis in both SrcK– and SrcK+ cells, but sphingosine kinase-1 and DNA synthesis were not activated in SrcK– (in contrast to SrcK+). The tPA-induced activation of sphingosine kinase was inhibited by PD98059, thereby suggesting a role for the early peak of tPA-induced MEK-1/ERK1/2 pathway.
Our data suggest that the Spm/Cer/S1P pathway plays a central role in the tPA-induced cell proliferation. We identified two signaling pathways implicated in the activation of key enzymes of the Spm/Cer/S1P pathway. The neutral sphingomyelinase was activated by tPA through a Gi-dependent mechanism, whereas sphingosine kinase activation was mediated by Src and ERK1/2 (early peak).
3. The Spm/Cer/S1P pathway is required for the late sustained wave of ERK1/2 activation
A relationship between the Spm/Cer/S1P pathway and late (2 h) ERK1/2 signaling was suggested by the use of inhibitors DMS and D-MAPP. Spm hydrolysis and sphingomyelinase activation occurred after (and was therefore not involved in) the early peak of tPA-induced ERK1/2 activation. In contrast, the late ERK1/2 peak triggered by tPA was strongly inhibited by DMS and D-MAPP, suggesting it is dependent on S1P, the end product of the Spm/Cer pathway. Consistently, this second wave of the tPA-induced ERK activation was severely reduced in SrcK– cells or in cells treated by the Src inhibitor herbimycin, in which sphingosine kinase was not activated by tPA. Altogether, these data suggest that the tPA-induced ERK1/2 activation requires sphingosine kinase activation and S1P synthesis.
S1P is able to activate several signaling pathways, including ERK1/2, by acting as either an intracellular mediator or an auto/paracrine agonist for specific EDG/S1P receptors. S1P is an auto/paracrine mediator that binds EDG/S1P receptors, a novel family of G-protein-coupled receptors, and thereby activates various signaling pathways, including ERK1/2 via Gi (this explains in part the inhibitory effect of PTX on tPA-induced ERK activation and cell proliferation). S1P has also been considered as a second messenger, but this role of intracellular mediator is a subject of controversy because intracellular targets of S1P have not been identified. In our experiments, the mitogenic effect was blocked by anti-EDG-1/S1P1 antibodies. This suggests that, in these cells, S1P is mainly an auto/paracrine mediator acting through the EDG-1/S1P1 receptor-mediated pathway.
4. The Spm/Cer/S1P pathway is involved in tPA-induced uPAR overexpression
As the PAs can up-regulate the expression of their own receptor, uPAR, in several cell types, we investigated whether the Spm/Cer/S1P signaling pathway is involved in regulating the level of uPAR. tPA-induced uPAR overexpression was severely reduced by DMS and to a lesser extent by PTX and PD098059. This is consistent with the above-reported results that show the crucial role of S1P (and sphingosine kinase activation) in ERK1/2 activation. This important set of data suggests that the signaling cascade involving Spm/Cer/S1P pathway activation, sphingosine kinase regulation by ERK1/2 and Src, and leading to the sustained second wave of ERK1/2 activation is required for uPAR overexpression induced by tPA. The role of the Spm/Cer/S1P and ERK1/2 pathways in the induction of uPAR expression is consistent with a ERK1-mediated trans-activation of c-jun and the regulatory role of two AP-1 sites in the uPAR gene promoter. The Spm/Cer/S1P pathway represents a novel pathway that plays a crucial role in the positive feedback loop (tPA-induced uPAR expression) of the tPA/uPA-uPAR system and may help deregulate cell proliferation and stromal remodeling in neoplasia and atherosclerosis.
CONCLUSIONS AND SIGNIFICANCE
The tPA-uPAR system elicits various cellular responses implicated in the pathophysiology and evolution of diseases, but the nature and interactions between the different signaling pathways are only partly understood. The findings of our paper point out the involvement of the Spm/Cer/S1P pathway in mitogenic signaling of the tPA-uPAR system and in uPAR overexpression in vascular and tumoral cells. This work helps clarify the mechanism of sphingosine kinase activation through a Src- and ERK1/2-dependent pathway.
Our data show that the Spm/Cer/S1P pathway is regulated by tPA (independent of its proteolytic activity) through two separate sequences of signaling events: a PTX-sensitive Gi/G0 is implicated in the activation of a neutral sphingomyelinase (initial step of the Spm/Cer/S1P pathway) whereas Src and ERK1/2 are required for sphingosine kinase-1 activation. Sphingosine kinase-1 in turn generates S1P, which binds the EDG-1/S1P1 receptor and activates the Gi-dependent (PTX-sensitive) second step of ERK1/2 phosphorylation that appears to be essential for cell proliferation and uPAR overexpression (Fig. 2
).
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A role for the Spm/Cer/S1P pathway in the tPA-uPAR-dependent signaling pathway leading to cell proliferation has not been known. Sphingosine kinase plays a key role in this mitogenic signaling because S1P 1) activates ERK1/2 phosphorylation and survival signaling pathways and 2) takes part in the positive feedback loop (tPA-induced uPAR expression) of the tPA/uPA-uPAR system (Fig. 2)
.
The link between sphingosine kinase and the uPA/uPAR system is of great interest because it gives a more integrated view of the "how and when" two pro-oncogenic pathways already known to play a major role in cell migration, adhesion, and proliferation can interact to induce cellular responses (cell migration, adhesion, and proliferation) potentially involved in the pathophysiology of diseases. These findings provide new insight on potential therapeutic tools (such as inhibitors of sphingosine kinase-1 and of EDG/S1P receptors) useful to limit uPAR signaling and its consequences in disease progression.
FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-1123fje; doi: 10.1096/fj.03-1123fje
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