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

Abstract

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 -elastin (E), a commonly used EP, induces maximum phosphorylation of mitogen-activated protein kinase/extracellular signalregulated 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. E 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 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 E treatment, thereby integrating PKA signals to MEK1/2. It is noteworthy that Ras involvement was excluded because ERK1/2 activation by E was not blocked in RasN17-transfected fibroblasts. Together, our results identify a novel Rasindependent 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 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.

Regeneration of Human Dermis by a Multi-Headed Peptide

“…TFP binding to S-Gal conducted the activation of signaling pathways such as ERK1/2 and AKT. This activation was in accordance with previous reports showing that occupancy of S-Gal by elastokines triggered ERK activation in fibroblasts, cardiomyocytes, or endothelial cells (Duca et al, 2012;Hausenloy and Yellon, 2004;Duca et al, 2005). The relationship between TFP stimulation and matrix constituent upregulation was determined by activation with a chemical inhibitor of PI3K/AKT and ERK1/2 that resulted in drastic downregulation of TFP's effects.…”

Regeneration of Human Dermis by a Multi-Headed Peptide

“…TFP binding to S-Gal conducted the activation of signaling pathways such as ERK1/2 and AKT. This activation was in accordance with previous reports showing that occupancy of S-Gal by elastokines triggered ERK activation in fibroblasts, cardiomyocytes, or endothelial cells (Duca et al, 2012;Hausenloy and Yellon, 2004;Duca et al, 2005). The relationship between TFP stimulation and matrix constituent upregulation was determined by activation with a chemical inhibitor of PI3K/AKT and ERK1/2 that resulted in drastic downregulation of TFP's effects.…”

Elastin Peptides Signaling Relies on Neuraminidase-1-Dependent Lactosylceramide Generation

“…This effect was maximum at 30 min (Fig. 4A and 5A), the time at which maximum elastin peptide-induced ERK 1/2 activation is observed [10]. This finding supported our hypothesis that kE treatment leads to GM 3 /LacCer conversion eventually resulting in ERK 1/2 induction.…”

Elastin peptides antagonize ceramide‐induced apoptosis

“…This PI3 K is a major actor of chemotaxis [29], a phenomenon that can be promoted by EP. Additionally, it is involved in angiogenesis [30], ischemia reperfusion injury recovery [28] and proteases production by EP [18]. Our results support the fact that this kinase holds a pivotal role in EP biology.…”

Regeneration of Human Dermis by a Multi-Headed Peptide

“…TFP binding to S-Gal conducted the activation of signaling pathways such as ERK1/2 and AKT. This activation was in accordance with previous reports showing that occupancy of S-Gal by elastokines triggered ERK activation in fibroblasts, cardiomyocytes, or endothelial cells (Duca et al, 2012;Hausenloy and Yellon, 2004;Duca et al, 2005). The relationship between TFP stimulation and matrix constituent upregulation was determined by activation with a chemical inhibitor of PI3K/AKT and ERK1/2 that resulted in drastic downregulation of TFP's effects.…”

Elastin Peptides Signaling Relies on Neuraminidase-1-Dependent Lactosylceramide Generation

“…This effect was maximum at 30 min (Fig. 4A and 5A), the time at which maximum elastin peptide-induced ERK 1/2 activation is observed [10]. This finding supported our hypothesis that kE treatment leads to GM 3 /LacCer conversion eventually resulting in ERK 1/2 induction.…”

Elastin peptides antagonize ceramide‐induced apoptosis

“…This PI3 K is a major actor of chemotaxis [29], a phenomenon that can be promoted by EP. Additionally, it is involved in angiogenesis [30], ischemia reperfusion injury recovery [28] and proteases production by EP [18]. Our results support the fact that this kinase holds a pivotal role in EP biology.…”

Regeneration of Human Dermis by a Multi-Headed Peptide

“…TFP binding to S-Gal conducted the activation of signaling pathways such as ERK1/2 and AKT. This activation was in accordance with previous reports showing that occupancy of S-Gal by elastokines triggered ERK activation in fibroblasts, cardiomyocytes, or endothelial cells (Duca et al, 2012;Hausenloy and Yellon, 2004;Duca et al, 2005). The relationship between TFP stimulation and matrix constituent upregulation was determined by activation with a chemical inhibitor of PI3K/AKT and ERK1/2 that resulted in drastic downregulation of TFP's effects.…”

Elastin Peptides Signaling Relies on Neuraminidase-1-Dependent Lactosylceramide Generation

“…This effect was maximum at 30 min (Fig. 4A and 5A), the time at which maximum elastin peptide-induced ERK 1/2 activation is observed [10]. This finding supported our hypothesis that kE treatment leads to GM 3 /LacCer conversion eventually resulting in ERK 1/2 induction.…”

Elastin peptides antagonize ceramide‐induced apoptosis

“…This PI3 K is a major actor of chemotaxis [29], a phenomenon that can be promoted by EP. Additionally, it is involved in angiogenesis [30], ischemia reperfusion injury recovery [28] and proteases production by EP [18]. Our results support the fact that this kinase holds a pivotal role in EP biology.…”