Interaction between the elastin peptide VGVAPG and human elastin binding protein.

The elastin binding protein (EBP), a spliced variant of lysosomal ß-galactosidase, is the primary receptor of elastin peptides that have been linked to emphysema, aneurysm and cancer progression. The sequences recognized by EBP share the XGXXPG consensus pattern found in numerous matrix proteins, notably in elastin where the VGVAPG motif is repeated. To delineate the elastin binding site of human EBP, we built a homology model of this protein and docked VGVAPG on its surface. Analysis of this model suggested that Gln-97 and Asp-98 were required for interaction with VGVAPG because they contribute to the definition of a pocket thought to represent the elastin binding site of EBP. Additionally, we proposed that Leu-103, Arg-107, and Glu-137 were essential residues because they could interact with VGVAPG itself. Site-directed mutagenesis experiments at these key positions validated our model. This work therefore provides the first structural data concerning the interaction of the VGVAPG with its cognate receptor. The present structural data should now allow the development of EBP-specific antagonists.

Elastin peptides antagonize ceramide-induced apoptosis.

Elastin peptides regulate proliferation, chemotaxis and protease expression. The aim of this work was to assess their influence on apoptosis. Human skin fibroblast cell death was induced using C(2)-ceramide in the presence or absence of elastin peptides. We show that ceramide-induced apoptosis could be blocked by elastin peptides. Using pharmacological inhibitors, we show that elastin peptide treatment leads to activation of the anti-apoptotic protein Akt that phosphorylates the pro-apoptotic protein Bad, the Foxo3a transcription factor and caspase-9. Importantly, the anti-apoptotic effects of elastin peptides were persistent in time. Our results suggest that elastin peptides could be potent cell survival factors.

Involvement of lysine 1047 in type I collagen-mediated activation of polymorphonuclear neutrophils.

Oxidative functions of polymorphonuclear neutrophils (PMNs), which play a deciding role in the phagocytosis process, are stimulated by extracellular matrix proteins such as type I collagen. Previous studies have demonstrated the involvement of a DGGRYY sequence located within the alpha(1) chain C-terminal telopeptide in type I collagen-induced PMN activation, but so far the mechanism has not been completely elucidated. We have recently demonstrated that collagen carbamylation (i.e. post-translational binding of cyanate to lysine epsilon-NH(2) groups) impairs PMN oxidative functions, suggesting the potential involvement of lysine residues in this process. The present study was devoted to the identification of lysine residues involved in the collagen-induced activation of PMNs. The inhibition of PMN activation by collagen in the presence of 6-amino-hexanoic acid, a structural analogue of lysine residues, confirmed the involvement of specific lysine residues. Modification of lysine residues by carbamylation demonstrated that only one residue, located within the alpha(1)CB6 collagen peptide, was involved in this mechanism. A recombinant alpha(1)CB6 peptide, designed for the substitution of lysine 1047 by glycine, exhibited decreased activity, demonstrating that the lysine residue at position 1047 within the collagen molecule played a significant role in the mechanism of activation. These results help to understand in more detail the collagen-mediated PMN activation mechanism and confirm the prominent involvement of lysine residues in interactions between extracellular matrix proteins and inflammatory cells.

Activating transcription factor-1-mediated hepatocyte growth factor-induced down-regulation of thrombospondin-1 expression leads to thyroid cancer cell invasion.

Hepatocyte growth factor (HGF) plays a major role in the pathogenesis of a variety of human epithelial tumors including papillary carcinoma of the thyroid. Previous reports demonstrated that HGF, acting through the Met receptor, repressed thrombospondin-1 (TSP-1) expression. To study the mechanisms by which HGF down-regulated TSP-1 expression, we transiently transfected a panel of deleted human TSP-1 promoter reporter plasmids into papillary thyroid carcinoma cells. We identified a region between -1210 and -1123 bp relative to the transcription start site that is responsive to HGF treatment and harbors a cAMP-responsive element (CRE) at position -1199 (TGACGTCC). Overexpression of various members of the CRE-binding protein family identified activating transcription factor-1 (ATF-1) as the transcription factor responsible for HGF-induced repression of TSP-1 promoter activity. This inhibition was associated with a concomitant increase in the abundance of nuclear ATF-1 protein. Gel shift and antibody supershift studies indicated that ATF-1 was involved in DNA binding to the TSP-1-CRE site. Finally, we utilized small hairpin RNA to target ATF-1 and showed that these small interfering RNA constructs significantly inhibited ATF-1 expression at both the RNA and the protein level. ATF-1 knockdown prevented HGF-induced down-regulation of TSP-1 promoter activity and protein expression and also reduced HGF-dependent tumor cell invasion. Taken together, our results indicate that HGF-induced down-regulation of TSP-1 expression is mediated by the interaction of ATF-1 with the CRE binding site in the TSP-1 promoter and that this transcription factor plays a crucial role for tumor invasiveness in papillary carcinoma of the thyroid triggered by HGF.

The elastin receptor complex transduces signals through the catalytic activity of its Neu-1 subunit.

The binding of elastin peptides on the elastin receptor complex leads to the formation of intracellular signals but how this is achieved remains totally unknown. Using pharmacological inhibitors of the enzymatic activities of its subunits, we show here that the elastin peptide-driven ERK1/2 activation and subsequent pro-MMP-1 production, observed in skin fibroblasts when they are cultured in the presence of these peptides, rely on a membrane-bound sialidase activity. As lactose blocked this effect, the elastin receptor sialidase subunit, Neu-1, seemed to be involved. The use of a catalytically inactive form of Neu-1 and the small interfering RNA-mediated decrease of Neu-1 expression strongly support this view. Finally, we report that N-acetyl neuraminic acid can reproduce the effects of elastin peptides on both ERK1/2 activation and pro-MMP-1 production. Altogether, our results indicate that the enzymatic activity of the Neu-1 subunit of the elastin receptor complex is responsible for its signal transduction, presumably through sialic acid generation from undetermined substrates.

Elastin peptides activate extracellular signal-regulated kinase 1/2 via a Ras-independent mechanism requiring both p110gamma/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 (kappaE), 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. kappaE induced Raf-1 phosphorylation and activation in a PI3K-dependent manner. In our system, the PI3K p110gamma is expressed and activated by betagamma-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 kappaE treatment, thereby integrating PKA signals to MEK1/2. It is noteworthy that Ras involvement was excluded because ERK1/2 activation by kappaE was not blocked in RasN17-transfected fibroblasts. Together, our results identify a novel Ras-independent ERK1/2 activation system in which p110gamma/Raf-1/MEK1/2 and PKA/B-Raf/MEK1/2 cooperate to activate ERK1/2. Thus, p110gamma 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.