Original articleA fibrillin-1-fragment containing the elastin-binding-protein GxxPG consensus sequence upregulates matrix metalloproteinase-1: biochemical and computational analysis
Introduction
The Marfan syndrome (MFS) is a relatively common hereditary disorder of connective tissue with prominent manifestations in the cardiovascular, ocular, and skeletal systems [1], [2]. The major cause of death in untreated persons with MFS is aortic dissection, which generally occurs following gradual dilatation of the aortic root over a period of many years. Mutations in the gene for fibrillin-1, FBN1, were shown to be the cause of MFS in 1991 [3]. However, the pathogenetic mechanisms leading from a FBN1 mutation to clinical disease have not been completely elucidated. Several mechanisms have been proposed, ranging from a dominant negative effect of mutant monomers on the highly polymeric fibrillin-rich microfibrils [4], disturbances of tissue homeostasis [5], [6], and alterations in TGFβ signaling [7], [8]. We and others have shown that FBN1 mutations identified in MFS patients can destabilize recombinant fibrillin-1 fragments with respect to proteolysis in vitro [9], [10], [11], [12], [13], [14], [15]. In light of these results together with observations of fibrillin fragmentation in tissues of persons with MFS [16], and reports of increased levels of matrix metalloproteinases in tissues of persons with MFS [17], [18], it is plausible that proteolytic degradation of microfibrils containing mutated fibrillin-1 monomers may be one component of the pathogenesis of MFS.
Extracellular matrix (ECM) proteins often have several functional roles in addition to being structure proteins. In particular, many ECM proteins can couple to cell membrane receptors and thereby trigger intracellular signaling mechanisms. The integrin family is the best-known class of cell surface molecules that mediate such signals [19]. In the present work, we have concentrated on the elastin–laminin binding protein (EBP), which is one of three protein subunits making up the elastin–laminin receptor (ELR) [20]. The EBP is a 67-kDa, enzymatically inactive alternative splice variant of β-galactosidase [21]. The EBP binds to tropoelastin intracellularly, blocking exposed hydrophobic VGVAPG-like domains2 and thereby acting as a molecular chaperone by preventing premature self-aggregation and resultant proteolytic degradation of the highly hydrophobic tropoelastin monomers during transit through the secretory pathway [22]. The EBP–tropoelastin complex reaches the cell surface where tropoelastin is released from the EBP after the latter is engaged by galactosugars protruding from microfibrillar glycoproteins associated with the elastic-fiber scaffold [20], [23]. EBP can act as a shuttle protein and is recycled intracellularly by endocytosis [24].
In addition to these functions, the EBP has been implicated in signaling processes mediated by EBP-binding of elastin's VGVAPG motif and related GxxPG motifs (where x is any amino acid). For instance, the ELR can function as a mechanotransducer in vascular smooth muscle [25], [26]. EBP-transduced signals can cause proliferation of arterial smooth muscle cells [27]. VGVAPG and related hexapeptides in tropoelastin act as chemoattractants for neutrophils [28], [29], [30]. Finally, elastin-derived fragments and VGVAPG are able to upregulate MMP-2, MT1-MMP, and TIMP-2 production in HT-1080 cells [31], and xGxxPG hexapeptides including VGVAPG can upregulate MMP-1 and MMP-3 in dermal fibroblasts [32].
In the present study, we present evidence that a fibrillin-1 fragment containing a putative GxxPG EBP recognition sequence increases MMP-1 expression. Together with our recent finding that fibrillin-1 RGD-containing fragments can increase MMP-1 and MMP-3 production [33], our present findings add fibrillin-1 to a growing list of matrix proteins with multiple motifs that are capable of “out-to-in” signaling to the cell, such as elastin, laminin, and fibronectin. They provide a plausible contributory mechanism for microfibrillar fragmentation and increased MMP concentrations in tissues of Marfan patients. Our Gene-Ontology analysis identified multiple GxxPG motifs in numerous other ECM proteins and suggests a testable hypothesis for previously published observations of increased MMP activity in a number of disorders involving these proteins.
Section snippets
Fibrillin-1 constructs and in vitro mutagenesis
A recombinant construct, rFib47wt, was generated to correspond to amino acids I1929 to E2205 (nt 5785–6615, encoded by FBN1 exons 47–53). This region encompasses cbEGF-like motifs 29, 30, 31, TGFβ1bp-like (8-cys) motif 6, followed by cbEGF-like motifs 32 and 33. rFib47wt contains a putative EBP recognition motif EGFEPG at amino acids 2194–2199 (Fig. 1). Wild-type FBN1 cDNA fragments were generated by reverse transcriptase-polymerase chain reaction (PCR) with primers designed such that the
A fibrillin-1 fragment containing the EBP consensus sequence GxxPG upregulates MMP-1 expression and production
In this work we investigated the influence of a recombinant fibrillin-1 fragment containing a putative EBP binding site on the expression and production of MMP-1. Semiconfluent dermal fibroblasts from normal controls were incubated with 0, 0.1, and 0.2 μM recombinant rFib47wt, which contains the amino acid sequence EGFEPG that conforms to the GxxPG consensus sequence for binding to the EBP [32]. After 48 h incubation, total RNA was isolated for RT-PCR and medium was isolated for determination
Discussion
The molecular pathogenesis of MFS remains elusive despite over a decade of research following the initial discovery of mutations in the FBN1 gene in 1991. Initially, it was hypothesized that mutations in fibrillin would result in a structural weakness in the fibrillin-rich microfibrils according to a dominant negative paradigm [58]. More recently, it has been shown that fibrillin-1 mutations can have other secondary effects above and beyond effects on the structural integrity of tissue. For
Conclusion
In summary, the present study has demonstrated that a polypeptide fragment of fibrillin-1 containing a GxxPG site increases MMP-1 expression and production in cell culture. Together with the integrin binding RGD site of fibrillin-1 [33], this is the second motif in fibrillin-1 that is capable of influencing MMP expression, placing fibrillin in a growing list of ECM proteins with multiple signaling interactions, including fibronectin and laminin. VGVAPG and related GxxPG peptides in elastin have
Acknowledgements
This work was supported in part by the Deutsche Forschungsgemeinschaft, Ro-2005/3. We are grateful to the Temerty Family Foundation and the Canadian Marfan Association for additional support.
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The first two authors contributed equally to this work.