Laurent Duca

Annexin A1 processing is associated with caspase-dependent apoptosis in BZR cells

Annexins are widely distributed and have been described in lung as well as in other cells and tissues. Annexin I (ANX AI) is a member of the calcium‐dependent phospholipid binding protein family. Besides its anti‐inflammatory function, ANX AI has been involved in several mechanisms such as the Erk repression pathway or apoptosis. To investigate the role of ANX AI on apoptosis in broncho‐alveolar cells, we have constructed a plasmid containing the ANX AI full length cDNA. Transfected BZR cells displayed a higher level of both forms of ANX AI (37 and 33 kDa) as well as a decrease in cell viability (two‐fold versus cells transfected with an empty vector). In order to analyse the endogenous ANX AI processing during stimulus‐induced apoptosis, BZR cells were treated with a commonly used inducer, i.e. C2 ceramides. In these conditions, microscopic analysis revealed chromatin condensation in dying cells and the Bcl‐2, Bcl‐xL/Bax mRNA balance was altered. Caspase‐3 is one of the key executioners of apoptosis, being responsible for the cleavage of many proteins such as the nuclear enzyme poly(ADP‐ribose) polymerase (PARP). We demonstrate that caspase‐3 was activated after 4 h treatment in the presence of ceramide leading to the cleavage of PARP. Dose–response experiments revealed that cell morphology and viability modifications following ceramide treatment were accompanied by an increase in endogenous ANX AI processing. Interestingly, in both ceramide and transfection experiments, the ANX AI cleaved form was enhanced whereas pre‐treatment with the caspase inhibitor Z‐VAD‐fmk abolished ANX AI cleavage. In conclusion, this study demonstrates a complex regulatory role of caspase‐dependent apoptosis where ANX AI is processed at the N‐terminal region which could give susceptibility to apoptosis upon ceramide treatment.

Degradation of tropoelastin by matrix metalloproteinases - cleavage site specificities and release of matrikines

To provide a basis for the development of approaches to treat elastin‐degrading diseases, the aim of this study was to investigate the degradation of the natural substrate tropoelastin by the elastinolytic matrix metalloproteinases MMP‐7, MMP‐9, and MMP‐12 and to compare the cleavage site specificities of the enzymes using complementary MS techniques and molecular modeling. Furthermore, the ability of the three proteases to release bioactive peptides was studied. Tropoelastin was readily degraded by all three MMPs. Eighty‐nine cleavage sites in tropoelastin were identified for MMP‐12, whereas MMP‐7 and MMP‐9 were found to cleave at only 58 and 63 sites, respectively. Cleavages occurred predominantly in the N‐terminal and C‐terminal regions of tropoelastin. With respect to the cleavage site specificities, the study revealed that all three MMPs similarly tolerate hydrophobic and/or aliphatic amino acids, including Pro, Gly, Ile, and Val, at P1′. MMP‐7 shows a strong preference for Leu at P1′, which is also well accepted by MMP‐9 and MMP‐12. Of all three MMPs, MMP‐12 best tolerates bulky charged and aromatic amino acids at P1′. All three MMPs showed a clear preference for Pro at P3 that could be structurally explained by molecular modeling. Analysis of the generated peptides revealed that all three MMPs show a similar ability to release bioactive sequences, with MMP‐12 producing the highest number of these peptides. Furthermore, the generated peptides YTTGKLPYGYGPGG, YGARPGVGVGGIP, and PGFGAVPGA, containing GxxPG motifs that have not yet been proven to be bioactive, were identified as new matrikines upon biological activity testing.

Protein carbamylation is a hallmark of aging

Significance Human longevity is increasing worldwide because of the advances in scientific knowledge and patient care, which leads to the frequent development of age-related pathologies. Aging remains an elusive process associated with genetic and environmental features, and better understanding would promote sustained wellbeing. We show here for the first time, to our knowledge, that carbamylation, a nonenzymatic posttranslational modification of proteins characterized by the spontaneous binding of isocyanic acid mainly derived from urea, is highly associated with aging and life expectancy in three mammalian species. Carbamylation promotes molecular aging through alteration of protein functions, especially long-lived extracellular matrix proteins. Tissue accumulation of carbamylated proteins may be considered a general hallmark of aging, enabling us to establish a link between cumulative metabolic alterations and age-related complications. Aging is a progressive process determined by genetic and acquired factors. Among the latter are the chemical reactions referred to as nonenzymatic posttranslational modifications (NEPTMs), such as glycoxidation, which are responsible for protein molecular aging. Carbamylation is a more recently described NEPTM that is caused by the nonenzymatic binding of isocyanate derived from urea dissociation or myeloperoxidase-mediated catabolism of thiocyanate to free amino groups of proteins. This modification is considered an adverse reaction, because it induces alterations of protein and cell properties. It has been shown that carbamylated proteins increase in plasma and tissues during chronic kidney disease and are associated with deleterious clinical outcomes, but nothing is known to date about tissue protein carbamylation during aging. To address this issue, we evaluated homocitrulline rate, the most characteristic carbamylation-derived product (CDP), over time in skin of mammalian species with different life expectancies. Our results show that carbamylation occurs throughout the whole lifespan and leads to tissue accumulation of carbamylated proteins. Because of their remarkably long half-life, matrix proteins, like type I collagen and elastin, are preferential targets. Interestingly, the accumulation rate of CDPs is inversely correlated with longevity, suggesting the occurrence of still unidentified protective mechanisms. In addition, homocitrulline accumulates more intensely than carboxymethyl-lysine, one of the major advanced glycation end products, suggesting the prominent role of carbamylation over glycoxidation reactions in age-related tissue alterations. Thus, protein carbamylation may be considered a hallmark of aging in mammalian species that may significantly contribute in the structural and functional tissue damages encountered during aging.

Elastin fragmentation and atherosclerosis progression: The elastokine concept

Atherosclerosis is a progressive multifaceted inflammatory disease affecting large- and medium-sized arteries. Typical feature of this disease is the formation and build-up of atherosclerotic plaques characterized by vascular extracellular matrix degradation and remodeling. Many studies have documented degradation of native elastin, the main extracellular matrix protein responsible for resilience and elasticity of arteries, by local release of elastases, leading to the production of elastin-derived peptides (EDP). These peptides have been proposed to actively participate in the progression of the disease by accelerating different biological processes, such as LDL oxidation and calcification of the vascular wall. These pathophysiological effects are mediated by the binding of EDP on a peculiar heterotrimeric receptor named elastin receptor complex (ERC). In this article, we review the contribution of elastin in biological processes involved in atherosclerosis progression from its initial elastase-driven degradation to its ultimate cellular effects. Finally, we discuss the ERC and its derived signaling pathways as promising therapeutic targets.

The elastin peptides-mediated induction of pro-collagenase-1 production by human fibroblasts involves activation of MEK/ERK pathway via PKA- and PI3K-dependent signaling

Elastin peptides, such as κ‐elastin (kE), bind to the elastin receptor at the cell surface of human dermal fibroblasts and stimulate collagenase‐1 expression at the gene and protein levels. Using specific inhibitors and phosphospecific antibodies, we show here that the binding of elastin peptides to their receptor activates the extracellular signal‐regulated kinase (ERK) pathway; this activation is essential for the induction of pro‐collagenase‐1 production. Moreover, protein kinase A (PKA) and phosphatidylinositol 3‐kinase (PI3K) signaling were found to participate in ERK activation. Concomitantly, we demonstrate that stimulation by elastin peptides leads to enhanced DNA binding of activator protein‐1 (AP‐1). Our data indicate that the up‐regulation of collagenase‐1 following treatment of fibroblasts with elastin peptides results from a cross‐talk between PKA, PI3K and the ERK signaling pathways and that this regulation is accompanied by activation of AP‐1 transcription factors.

Elastin-Derived Peptides Are New Regulators of Insulin Resistance Development in Mice

Although it has long been established that the extracellular matrix acts as a mechanical support, its degradation products, which mainly accumulate during aging, have also been demonstrated to play an important role in cell physiology and the development of cardiovascular and metabolic diseases. In the current study, we show that elastin-derived peptides (EDPs) may be involved in the development of insulin resistance (IRES) in mice. In chow-fed mice, acute or chronic intravenous injections of EDPs induced hyperglycemic effects associated with glucose uptake reduction and IRES in skeletal muscle, liver, and adipose tissue. Based on in vivo, in vitro, and in silico approaches, we propose that this IRES is due to interaction between the insulin receptor (IR) and the neuraminidase-1 subunit of the elastin receptor complex triggered by EDPs. This interplay was correlated with decreased sialic acid levels on the β-chain of the IR and reduction of IR signaling. In conclusion, this is the first study to demonstrate that EDPs, which mainly accumulate with aging, may be involved in the insidious development of IRES.

Elastin-derived peptides potentiate atherosclerosis through the immune Neu1-PI3Kγ pathway.

AIMS Elastin is degraded during vascular ageing and its products, elastin-derived peptides (EP), are present in the human blood circulation. EP binds to the elastin receptor complex (ERC) at the cell surface, composed of elastin-binding protein (EBP), a cathepsin A and a neuraminidase 1. Some in vitro functions have clearly been attributed to this binding, but the in vivo implications for arterial diseases have never been clearly investigated. METHODS AND RESULTS Here, we demonstrate that chronic doses of EP injected into mouse models of atherosclerosis increase atherosclerotic plaque size formation. Similar effects were observed following an injection of a VGVAPG peptide, suggesting that the ERC mediates these effects. The absence of phosphoinositide 3-kinase γ (PI3Kγ) in bone marrow-derived cells prevented EP-induced atherosclerosis development, demonstrating that PI3Kγ drive EP-induced arterial lesions. Accordingly, in vitro studies showed that PI3Kγ was required for EP-induced monocyte migration and ROS production and that this effect was dependent upon neuraminidase activity. Finally, we showed that degradation of elastic lamellae in LDLR(-/-) mice fed an atherogenic diet correlated with atherosclerotic plaque formation. At the same time, the absence of the cathepsin A-neuraminidase 1 complex in cells of the haematopoietic lineage abolished atheroma plaque size progression and decreased leucocytes infiltration, clearly demonstrating the role of this complex in atherogenesis and suggesting the involvement of endogenous EP. CONCLUSION Altogether, this work identifies EP as an enhancer of atherogenesis and defines the Neuraminidase 1/PI3Kγ signalling pathway as a key mediator of this function in vitro and in vivo.

TIMP-1 binding to proMMP-9/CD44 complex localized at the cell surface promotes erythroid cell survival

Besides its ability to inhibit MMP activity, TIMP-1 exhibits other biological functions. We earlier reported that TIMP-1 induced UT-7 erythroid cell survival through activation of the JAK2/PI 3-kinase/Akt pathway and we now aim to determine whether the TIMP-1 anti-apoptotic effect requires MMP involvement. We first show that proMMP-9 was expressed in UT-7 cells and associated with the cell plasma membrane. Such proMMP-9 localization was crucial for TIMP-1 intracellular signalling since (i) TIMP-1 specifically bound to proMMP-9 and (ii) proMMP-9 silencing abrogated the TIMP-1 effect. We also demonstrated that TIMP-1 anti-apoptotic effect was independent on MMP inhibition since MMP-9 function blocking antibodies as well as a synthetic MMP inhibitor were unable to reproduce TIMP-1 effect. Nevertheless, these compounds prevented TIMP-1 binding to proMMP-9 and subsequently abolished TIMP-1-induced cell survival. We finally demonstrated that CD44 anchored proMMP-9 to the plasma membrane and enabled TIMP-1-mediated signal transduction. Therefore, our results indicate that the anti-apoptotic signalling of TIMP-1 depends on the formation of a ternary complex between TIMP-1, proMMP-9 and CD44 at the UT-7 erythroid cell surface.

Matrix ageing and vascular impacts: focus on elastin fragmentation

Cardiovascular diseases (CVDs) are the leading cause of death worldwide and represent a major problem of public health. Over the years, life expectancy has considerably increased throughout the world, and the prevalence of CVD is inevitably rising with the growing ageing of the population. The normal process of ageing is associated with progressive deterioration in structure and function of the vasculature, commonly called vascular ageing. At the vascular level, extracellular matrix (ECM) ageing leads to molecular alterations in long half-life proteins, such as elastin and collagen, and have critical effects on vascular diseases. This review highlights ECM alterations occurring during vascular ageing with a specific focus on elastin fragmentation and also the contribution of elastin-derived peptides (EDP) in age-related vascular complications. Moreover, current and new pharmacological strategies aiming at minimizing elastin degradation, EDP generation, and associated biological effects are discussed. These strategies may be of major relevance for preventing and/or delaying vascular ageing and its complications.

Interaction between the elastin peptide VGVAPG and human elastin binding protein

Background: The interaction of the peptide VGVAPG with the elastin binding protein is critically involved in aneurysm progression. Results: A molecular model of this interaction is proposed and explored using a site-directed mutagenesis strategy. Conclusion: Three residues, Leu-103, Arg-107, and Glu-137, of elastin binding protein are critical players in this interaction. Significance: Our data now allow the design of antagonists of VGVAPG. The elastin binding protein (EBP), a spliced variant of lysosomal β-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.