PGE 2 INDUCES ANGIOGENESIS VIA MT1-MMP-MEDIATED ACTIVATION OF THE TGF β /ALK5 SIGNALLING PATHWAY.

The development of a new vascular network is essential for the onset and progression of many pathophysiological processes. Cyclooxygenase-2 displays a proangiogenic activity in in vitro and in vivo models, mediated principally through its metabolite PGE 2 . Here we provide evidence for a novel signalling route through which PGE 2 activates the Alk5-Smad3 pathway in endothelial cells. PGE 2 induces Alk5-dependent Smad3 nuclear translocation and DNA binding, and the activation of this pathway involves the release of active TGF β from its latent form through a process mediated by the metalloproteinase MT1-MMP, whose membrane clustering is promoted by PGE 2 . MT1-MMP-dependent TGF β signalling through Alk5 is also required for PGE 2 -induced endothelial cord formation in vitro ; and Alk5 kinase activity is required for PGE 2 -induced neovascularisation in vivo . These findings identify a novel signalling pathway linking PGE 2 and TGF β , two effectors involved in tumour growth and angiogenesis, and reveal potential targets for the treatment of angiogenesis-related disorders. main mediator of COX-2 proangiogenic effects. The phenotype obtained by deleting the EP2 gene in Apc ∆ 716 mice, a model of familiar polyposis, clearly resembles that of COX-2 deficient mice, with diminished vascularisation and reduced expression of proangiogenic factors in the polyps 6 . Similarly, EP3 KO mice show decreased tumour-associated angiogenesis 7 . Explanations offered for the proangiogenic properties of PGE 2 in vitro include increased expression of endothelial genes such as VEGF or CXCR4 8 , activation of the Rac and NO/cGMP pathways 9 , and EP2-mediated EC migration and survival 10 . in intracellular cAMP 19 . In contrast, Smad pathways are activated by molecules such as IGFBP-3, NO donors, and advanced glycation end products, probably through the transactivation of TGF β type I receptor Finally, there are conflicting data on the possible regulatory action of ERK-1,2; these kinases are known to phosphorylate certain R-Smads at their linker regions, and positive and negative regulatory effects on the Smad pathway have been reported 21 . In the present study, we analyze the signal transduction pathways activated by PGE 2 in ECs. We show that PGE 2 is able to signal through the Smad pathway, and that PGE 2 -induced Smad3 activation requires Alk5 transactivation, probably mediated by the MT1-MMP-induced release of bioactive TGF β . Furthermore, we show that Alk5 and MT1-MMP activities are both required for angiogenesis mediated by PGE 2 , underscoring the functional importance of this novel pathway. In this study, we have identified the activation of the Alk5-Smad3 pathway in ECs as an important mediator of PGE 2 -promoted neovascularisation in vitro and in vivo . Activation of Smad3 nuclear translocation and DNA binding by PGE 2 requires the kinase activity of the type I TGF β receptor Alk5, and is prevented by neutralizing antibodies to TGF β and MT1-MMP. TGF β , MT1-MMP and Alk5 kinase activity are all required for PGE 2 -mediated cord formation in vitro ; and inhibition of Alk5 also prevents neovascularisation in an in vivo angiogenesis model. Our findings thus identify a novel transactivation signalling pathway via which PGE 2 triggers the activation of MT1-MMP, which releases TGF β from its latent complex so that it can bind its receptor Alk5, activating the Smad3 pathway and promoting angiogenesis. A. Alfranca designed and performed research, analysed data and wrote the paper. JM López-Oliva, L. Genís, D. López-Maderuelo, I. Mirones, D. Salvado and AJ Quesada performed research. A.G. Arroyo designed research and analysed data. JM Redondo designed and supervised research, and analysed data. The authors have no conflicting financial interests.