Intracellular signaling pathways involved in the release of IL-4 and VEGF from human keratinocytes by activation of kinin B 1 receptor: functional relevance to angiogenesis

The injured skin produces a number of mediators that directly or indirectly modulate cell chemotaxis, migration, proliferation, and angiogenesis. Components of the kinin pathway including the kinin B 1 receptor (B 1 R) have been found to occur in the human skin, but information about its role on keratinocyte biology is still scarce. Our aim was to determine whether stimulation of B 1 R causes the secretion of IL-4 and/or VEGF from human keratinocytes and to evaluate the role of the B 1 R agonist Lys-des[Arg 9 ]bradykinin and IL-4 on various stages of angiogenesis, such as cell migration, proliferation, and release of metalloproteases. By using ELISA and Western blotting, we showed that HaCaT keratinocytes stimulated with the B 1 R agonist release IL-4 and VEGF. Stimulation of B 1 R also caused transient c-JunN-terminal kinase phosphorylation and JunB nuclear translocation, transcription factor that regulates IL-4 expression. The 3D-angiogenesis assay, performed on spheroids of EA.hy923 endothelial cells embedded in a collagen matrix, showed that their cumulative sprout area increased significantly following stimulation with either IL-4 or B 1 R agonist. Furthermore, these ligands produced significant endothelial cell migration and release of metalloproteases 2 and 9, but did not increase endothelial cell proliferation as measured by 5-bromo-2′-deoxyuridine incorporation. Our results provide experimental evidence that establishes IL-4 and B 1 R agonist as important angiogenic factors of relevance for skin repair.