Received for publication May 14, 2004.
Revised January 4, 2005.
Accepted for publication January 12, 2005.

Elastin peptides activate ERK1/2 via a Ras-independent mechanism requiring both p110/Raf-1 and PKA/B-Raf signaling in human skin fibroblasts

Abstract

Elastin peptides (EP) 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 due to 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 (kE), a commonly used EP, induces maximum phosphorylation of MEK1/2 and ERK1/2 after 30 min. The simultaneous inhibition of PKA and PI3K, by N-(2-(p-bromocinnamylamino)ethyl)-5-isoquinolinesulfonamide (H-89) and 2-(4-morpholynil)-8-phenyl-4H-1-benzopyran-4-one (LY294002) respectively, blocked MEK1/2 and ERK1/2 phosphorylation, as did lactose, an EBP antagonist. kE induced Raf-1 phosphorylation and activation in a PI3K-dependent manner. In our system, the PI3K p110 is expressed and activated by -derived subunits from a pertussis toxin-sensitive G protein following fibroblast stimulation. Pertussis toxin also blocks the Raf-1/MEK1/2/ERK1/2 phosphorylation cascade. Additionally, we found that B-Raf is expressed in dermal fibroblasts and activated in a PKA-dependent manner following kE treatment, thereby integrating PKA signals to MEK1/2. Importantly, Ras involvement was excluded as ERK1/2 activation by kE was not blocked in RasN17-transfected fibroblasts. Collectively, our results identify a novel Ras-independent ERK1/2 activation system in which p110/Raf-1/MEK1/2 and PKA/B-Raf/MEK1/2 cooperate to activate ERK1/2. Thus, p110 and B-Raf appear as important modulators of dermal fibroblasts physiology and should now qualify as therapeutic targets in strategies aiming at limiting elastin degradation contribution to cancer progression.

Key words: Protein Kinase A, Protein Kinases (other), Ras, Raf family, MAP Kinase