In vitro degradation of human tropoelastin by MMP-12 and the generation of matrikines from domain 24

Abstract

Degradation of elastic fibers in tissues can result in the development of disorders that include aneurysms, atherosclerosis, and loss of skin elasticity. Tropoelastin is the precursor of the cross-linked elastin and its expression is triggered by elastin-degrading factors as a response to damage. Factors like UV radiation not only increase the expression of tropoelastin but also potent metalloelastases such as macrophage elastase (MMP-12). The development of elastin-degrading diseases, moreover, is a chronic process during which elastin and tropoelastin are repeatedly exposed to attacks by MMP-12. Hence, in this work we report the in vitro susceptibility of tropoelastin and the potential of MMP-12 to generate matrikines. This work provides evidence that tropoelastin is substantially and rapidly degraded by MMP-12 even at very dilute enzyme concentrations. MMP-12 cleaves at least 86 sites in tropoelastin. Analysis of the generated peptides revealed that some small peptides contained the motif GXXPG that may enable them to bind with the elastin binding protein (EBP). Furthermore, using synthesized peptides it was confirmed that several sites in the sequence encoded by exon 24 which contains repetitive units of biologically active VGVAPG domains are susceptible to attack by MMP-12, provided that the active subsites in MMP-12 (S₄ to S₄′) are occupied. Such cleavage events have lead to the generation of ligands that may bind to EBP.

Introduction

Elastic fibers are responsible for elasticity and resilience of various tissues including arterial blood vessels, lung, and the skin. Once synthesized during early development, elastin undergoes very little turnover in normal adult tissues, owing to its high resistance to proteolytic degradation (Brown-Augsburger et al., 1996, Vrhovski and Weiss, 1998, Li and Daggett, 2002, Kozel et al., 2006). Nevertheless, damage of elastic fibers is usually seen associated with disorders such as atherosclerosis (Robert et al., 1998), emphysema (Hautamaki et al., 1997), arthritis (Liu et al., 2004), aneurysms (Curci et al., 1998), and also with photoaging in the skin (Waller and Maibach, 2005). Aberrant expression of many proteolytic enzymes is claimed to be responsible for the fiber destruction. One such enzyme is MMP-12, a member of the family of matrix degrading metalloproteinases (MMPs) (Senior et al., 1989). MMPs are tightly regulated zinc- and calcium-dependent enzymes that play a major role in extracellular matrix (ECM) turnover (Kähäri and Saarialho-Kere, 1997, Sternlicht and Werb, 2001). They are synthesized as prepro-enzymes and secreted in most cases as inactive pro-MMPs. Their activation essentially requires the disruption of a specific Cys-Zn²⁺ interaction (cysteine switch) and the removal of the propeptide which often proceeds in a stepwise manner (Nagase, 1997).

MMP-12 is expressed mainly in macrophages and is associated with the development of many of the aforementioned diseases (Hautamaki et al., 1997, Curci et al., 1998, Liu et al., 2004). In the skin, for example, MMP-12 is a contributing factor to photoaging (Saarialho-Kere et al., 1999). Chronic exposure to UV radiation induced the expression of MMP-12 mRNA and the enzyme was detected in areas where elastic damage was high (Chung et al., 2002). Photoaging is a chronic and complex process taking place over a longer period of time, during which there may be continuous destruction and repair of the damaged fibers and also synthesis of new ones. Repair and new synthesis involve tropoelastin alongside the reutilization of peptides in the elastic fibers (Morris et al., 1998, Vrhovski and Weiss, 1998). Moreover, exposure to UV not only increases the accumulation of MMP-12 but also stimulates the expression of tropoelastin in the skin (Schwartz et al., 1995). However, as compared to the insoluble elastin, tropoelastin is more susceptible to degradation by MMP-12. Hence, MMP-12 is not only involved in the destruction of elastic fibers, but also undermines the repair process by degrading the expressed tropoelastin.

Elastin degradation, besides altering the mechanical properties of tissues, might also interfere strongly with tissue homeostasis through the generation of elastin-derived peptides (EDPs). Matrikines is a general term to describe bioactive ligands that exist as a domain within an ECM protein. Natural matrikines are those that signal directly from the ECM, and cryptic matrikines such as EDPs are those that require proteolytic breakdown for the ligand to be revealed (Ludwig, 2007). EDPs interact with cells via a cell surface protein known as EBP. Through their interaction EDPs display a wide range of biological activities in a number of normal and transformed cells. For example, they are chemotactic for cells such as monocytes, neutrophils, and fibroblasts, they induce vasorelaxation, proliferation of arterial smooth muscle cells, and up-regulation of metalloproteinases (Lapis and Timar, 2002, Maquart et al., 2005, Moroy et al., 2005, Robinet et al., 2005, Lombard et al., 2006). EDPs have been identified in the circulation in the ng mL^− 1 to μg mL^− 1 range (Faury et al., 1998). The potential of some elastases in degrading and generating bioactive peptides from elastin and tropoelastin has been defined (Christner et al., 1978, Mecham et al., 1997, Lombard et al., 2006). In this study, the profiles of degradation and susceptibility of tropoelastin to MMP-12 activity are delineated and sequences of the generated peptides determined with regard to their biological relevance. Moreover, we establish the potential of MMP-12 in generating bioactive peptides especially from exon 24-encoded amino acid sequences.