Elastin peptides activate extracellular signal-regulated kinase 1/2 via a Ras-independent mechanism requiring both p110γ/Raf-1 and protein kinase A/B-Raf signaling in human skin fibroblasts

Elastin peptides (EPs) produced during cancer progression bind to the elastin binding protein (EBP) found at the surface of dermal fibroblasts, leading to the expression of collagenase-1 gene. The production of this enzyme involved in stromal reaction is caused by the sustained activation of the extracellular signal-regulated kinases 1/2 (ERK1/2) pathway via cAMP/protein kinase A (PKA) and phosphatidylinositol 3-kinase (PI3K). However, the mechanism of these signaling events remains unknown. We show that kappa-elastin (kappa E), a commonly used EP, induces maximum phosphorylation of mitogen-activated protein kinase/extracellular signal-regulated kinase (MEK)1/2 and ERK1/2 after 30 min. The simultaneous inhibition of PKA and PI3K, by N-(2-(p-bromocinnamylamino)ethyl)-5-isoquinolinesulfonamide (H89) and 2-(4-morpholynil)-8-phenyl-4H-1-bemzopyran-4-one (LY294002), respectively, blocked MEK1/2 and ERK1/2 phosphorylation, as did lactose, an EBP antagonist. kappa E induced Raf-1 phosphorylation and activation in a PI3K-dependent manner. In our system, the PI3K p110 gamma is expressed and activated by beta gamma-derived subunits from a pertussis toxin-sensitive G protein after fibroblast stimulation. Pertussis toxin also blocks the Raf-1/MEK1/2/ERK1/2 phosphorylation cascade. In addition, we found that B-Raf is expressed in dermal fibroblasts and activated in a PKA-dependent manner after kappa E treatment, thereby integrating PKA signals to MEK1/2. It is noteworthy that Ras involvement was excluded because ERK1/2 activation by kappa E was not blocked in RasN17-transfected fibroblasts. Together, our results identify a novel Ras-independent ERK1/2 activation system in which p110 gamma/Raf-1/MEK1/2 and PKA/B-Raf/MEK1/2 cooperate to activate ERK1/2. Thus, p110 gamma and B-Raf seem to be important modulators of dermal fibroblasts physiology and should now qualify as therapeutic targets in strategies aiming at limiting elastin degradation contribution to cancer progression.