Distinct biological events generated by ECM proteolysis by two homologous collagenases (original) (raw)
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Collagens as organizers of extracellular matrix during morphogenesis
Seminars in Cell & Developmental Biology, 1996
The composition of the extracellular matrix (ECM) varies depending on tissue location and developmental stage. Collagens as the main constituents determine its structure and play a major role in determining its function. Polymers of fibrillar collagens (I,II) form the backbone of many ECMs, whereas many minor collagens regulate or stabilize their structural properties. Changes in the minor collagens introduce subtle modifications in intermolecular interactions, which can modulate the morphology of the ECM and responses of the underlying or embedded cells. The functions of these modulating collagens is now being investigated by a number of biochemical, genetic and molecular approaches.
Matrix biology plus, 2019
Lung fibrosis is characterized by excessive deposition of extracellular matrix (ECM), in particular collagens, by fibroblasts in the interstitium. Transforming growth factor-β1 (TGF-β1) alters the expression of many extracellular matrix (ECM) components produced by fibroblasts, but such changes in ECM composition as well as modulation of collagen post-translational modification (PTM) levels have not been comprehensively investigated. Here, we performed mass spectrometry (MS)-based proteomics analyses to assess changes in the ECM deposited by cultured lung fibroblasts from idiopathic pulmonary fibrosis (IPF) patients upon stimulation with transforming growth factor β1 (TGF-β1). In addition to the ECM changes commonly associated with lung fibrosis, MS-based label-free quantification revealed profound effects on enzymes involved in ECM crosslinking and turnover as well as multiple positive and negative feedback mechanisms of TGF-β1 signaling. Notably, the ECM changes observed in this in vitro model correlated significantly with ECM changes observed in patient samples. Because collagens are subject to multiple PTMs with major implications in disease, we implemented a new bioinformatic platform to analyze MS data that allows for the comprehensive mapping and site-specific quantitation of collagen PTMs in crude ECM preparations. These analyses yielded a comprehensive map of prolyl and lysyl hydroxylations as well as lysyl glycosylations for 15 collagen chains. In addition, site-specific PTM analysis revealed novel sites of prolyl-3hydroxylation and lysyl glycosylation in type I collagen. Interestingly, the results show, for the first time, that TGF-β1 can modulate prolyl-3-hydroxylation and glycosylation in a site-specific manner. Taken together, this proof of concept study not only reveals unanticipated TGF-β1 mediated regulation of collagen PTMs and other ECM components but also lays the foundation for dissecting their key roles in health and disease. The proteomic data has been deposited to the ProteomeXchange Consortium via the MassIVE partner repository with the data set identifier MSV000082958.
Matrix-mediated regulation of type 1 collagen synthesis and degradation in cultured fibroblasts
2009
2.2 Results 2.2.1 Effect of fibroblast-derived ECM on cell proliferation….………………...37 2.2.2 Effect of fibroblast-derived ECM on cell morphology…..………………..38 2.2.3 Downregulation of type I collagen protein levels by the fibroblast-derived ECM…..……………………………………………………………………………..39 2.2.4 Downregulation of collagen α2 (1) mRNA levels by the fibroblast-derived ECM……………………………….……………………………………………...…39 2.2.5 Type I collagen mRNA stability ……………………………………………42 2.2.6 Analysis of type I collagen degradation and matrix metalloproteinase..42
2021
Collagen, the most abundant protein in mammals, contributes to the physical properties of different tissues during development, homeostasis, and disease. The adaptation of physical properties of tissues to mechanical stimuli is thus dependent on the control of tissue collagen levels by well-regulated synthesis and degradation of collagen. Importantly, how various molecular-level events within a tissue sustaining a range of mechanical strains contribute towards maintaining its collagen levels, remains unclear to date. Such molecular level processes in tissues are studied here in the case of isolated tendons consisting of collagen fibrils oriented along tissue loading-axis and beating embryonic hearts to gain understanding of mechanical load dependent tissue sculpting. Using a novel bioreactor design, starved mice tail tendon fascicles were used as a “cell-free” model and were subjected to heterogeneous and uniaxial deformation modes. Patterned photobleaching of fluorescent probes, a ...
Intrinsic fibroblast-mediated remodeling of damaged collagenous matrices in vivo
Matrix Biology, 2005
Numerous studies have examined wound healing and tissue repair after a complete tissue rupture and reported provisional matrix and scar tissue formation in the injury gap. The initial phases of the repair are largely mediated by the coagulation response and a principally extrinsic inflammatory response followed by type III collagen deposition to form scar tissue that may be later remodeled. In this study, we examine subfailure (Grade II sprain) damage to collagenous matrices in which no gross tissue gap is present and a localized concentration of provisional matrix or scar tissue does not form. This results in extracellular matrix remodeling that relies heavily upon type I collagen, and associated proteoglycans, and less heavily on type III scar tissue collagen. For instance, following subfailure tissue damage, collagen I and III expression was suppressed after 1 day, but by day 7 expression of both genes was significantly increased over controls, with collagen I expression significantly larger than type III expression. Concurrent with increased collagen expression were significantly increased expression of the collagen fibrillogenesis supporting proteoglycans fibromodulin, lumican, decorin, the large aggregating proteoglycan versican, and proteases cathepsin K and L. Interestingly, this remodeling process appears intrinsic with little or no inflammation response as damaged tissues show no changes in macrophage or neutrophils levels following injury and expression of the inflammatory markers, tumor necrosis factor-a and tartrate-resistant acid phosphatase were unchanged. Hence, since inflammation plays a large role in wound healing by inducing cell migration and proliferation, and controlling extracellular matrix scar formation, its absence leaves fibroblasts to principally direct tissue remodeling. Therefore, following a Grade II subfailure injury to the collagen matrix, we conclude that tissue remodeling is fibroblast-mediated and occurs without scar tissue formation, but instead with type I collagen fibrillogenesis to repair the tissue. As such, this system provides unique insight into acute tissue damage and offers a potentially powerful model to examine fibroblast behavior. D
Extracellular matrix remodelling properties of human fibrocytes
Journal of Cellular and Molecular Medicine, 2012
The fibrocytes are thought to serve as a source of newly deposited collagens I and III during reparative processes and in certain fibrotic disorders, but their matrix remodelling properties are incompletely understood. We evaluated their ability to produce several extracellular matrix (ECM) components, in comparison with fibroblasts, and to participate in collagen turnover. The collagen gene expression profile of fibrocytes differed from that of fibroblasts because fibrocytes constitutively expressed relatively high levels of the mRNA encoding collagen VI and significantly lower levels of the mRNA encoding collagens I, III and V. The proteoglycan (PG) gene expression profile was also different in fibrocytes and fibroblasts because fibrocytes constitutively expressed the mRNA encoding perlecan and versican at relatively high levels and the mRNA encoding biglycan and decorin at low and very low levels, respectively. Moreover, fibrocytes expressed the mRNA for hyaluronan synthase 2 at higher level than fibroblasts. Significant differences between the two cell populations were also demonstrated by metabolic labelling and analysis of the secreted collagenous proteins, PGs and hyaluronan. Fibrocytes constitutively expressed the scavenger receptors CD163 and CD204 as well as the mannose receptors CD206 and Endo180, and internalized and degraded collagen fragments through an Endo180-mediated mechanism. The results of this study demonstrate that human fibrocytes exhibit ECM remodelling properties previously unexplored, including the ability to participate in collagen turnover. The observed differences in collagen and PG expression profile between fibrocytes and fibroblasts suggest that fibrocytes may predominantly have a matrix-stabilizing function.
Molecular and tissue alterations of collagens in fibrosis
Matrix Biology, 2018
The collagen network is altered in fibrotic diseases associated with extracellular matrix (ECM) biosynthesis and remodeling. This mini-review focuses on the quantitative and qualitative modifications of collagens occurring at the molecular and tissue levels in fibrosis. They result from changes in collagen expression, biosynthesis, enzymatic cross-linking and degradation by several protease families. These molecular modifications, which are mostly regulated by TGF-, are associated with altered collagen organization at the tissue level, leading to a fibrotic signature that can be analyzed by Second Harmonic Generation (SHG) microscopy.
Journal of Biological Chemistry, 1995
72-kI)a gelatinase/type lv collagenase is an important matrix metalloproteinase in the degradation of basement membranes and denatured collagens (gelatin). These proteolytic processes are required for pathologic tissue destruction and physiologic tissue remodeling. To investigate the molecular determinants of substrate specificity of this enzyme, a 21-kDa domain of 72-kDa gelatinase, consisting of three tandem fibronectin type Il-like modtiles, was expressed in Escherichia coil. Similar to fulllength 72-kDa gelatinase and the type II modules in fibronectin, the recombinant (r) fibronectin-like domain of this proteinase bound denatured type I collagen with an apparent Kd in the micromolar range. This domain, designated the collagen-binding domain (rCBD123), possesses at least two collagen-binding sites that can each be simultaneously occupied. rCBD123 also avidly bound elastin and denatured types LV and V collagens, but neither native types lv and V collagens nor fibronectin, all of which are substrates of the enzyme. Although 72-kDa gelatinase is involved in basement membrane degradation, rCBD123 also did not bind reconstituted basement membrane, laminin, or SPARC. Native type I collagen, which is not degraded by 72-kDa gelatinase, competed with gelatin for a shared binding site on rCBD123. rCBD123 also displaced full-length 72-kDa gelatinase bound to native type I collagen, further demonstrating that the collagen binding properties of the recombinant domain closely mimicked those of the full-length enzyme. Since rCBD123 showed reduced binding to pepsin-cleaved type I collagen, either or both of the collagen telopeptide ends contain recognition sites for the 72-k.Da gelatinase fibronectin-like domain. This was confirmed by the avid binding of rCBD 123 to the al(I) collagen cyanogen bromide fragment CB2 from the NH 2-terminal telopeptide. rCBD123 also bound al(I)-CB7, which encompasses the fibronectinbinding site, and to al(I)-CB8, a fragment not bound by fibronectin Thus, type I collagen contains multiple binding sites for rCBD123 which are partially masked by the triple helical conformation of native collagen and fully exposed upon unfolding of the triple helix. The potential of the fibronectin-like collagen binding domain of 72-kDa gelatinase to bind extracellular matrix proteins may fa-cilitate enzyme localization in connective tissue matrices.
Cell-collagen networks breakdown by collagen remodelling
hal.archives-ouvertes.fr
Collagen model tissues, consisting of cells embedded in a collagen matrix at different concentrations (of cells and collagen) were analyzed. Rheological properties were measured and complementary confocal microscopy analysis carried out. An important feature, corresponding to the breakdown of the collagen network (i.e., decrease in network elasticity) was observed at high collagen concentrations, due to the presence of cells. Thanks to confocal microscopy, we showed that cells elongated within the gel and could remodel it, this being a concentration-dependent feature. A careful analysis of the remodeling process showed that cells can attract collagen in their close neighborhood, this being an irreversible process and that migrating cells create collagen-depleted regions behind them.