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Distinct Biological Events Generated by ECM Proteolysis by Two Homologous Collagenases

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Distinct biological events generated by ECM proteolysis by two homologous collagenases Inna Solomonova, Eldar Zehoraia, Dalit Talmi-Franka,b, Sharon G. Wolfc, Alla Shainskayad, Alina Zhuravleva, Elena Kartvelishvilyc, Robert Vissee, Yishai Levind, Nir Kampff, Diego Adhemar Jaitinb, Eyal Davidb, Ido Amitb, Hideaki Nagasee, and Irit Sagia,1 aDepartment of Biological Regulation, Weizmann Institute of Science, Rehovot 76100, Israel; bDepartment of Immunology, Weizmann Institute of Science, Rehovot 76100, Israel; cDepartment of Chemical Research Support, Weizmann Institute of Science, Rehovot 76100, Israel; dNancy and Stephen Grand Israel National Center for Personalized Medicine, Weizmann Institute of Science, Rehovot 76100, Israel; eKennedy Institute of Rheumatology, Nuffield Department of Orthopaedics, Rheumatology, and Musculoskeletal Sciences, University of Oxford, Oxford, OX3 7FY, United Kingdom; and fDepartment of Materials and Interfaces, Weizmann Institute of Science, Rehovot 76100, Israel Edited by Christopher M. Overall, University of British Columbia, Vancouver, Canada, and accepted by Editorial Board Member Rakesh K. Jain August 8, 2016 (received for review October 4, 2015) It is well established that the expression profiles of multiple and to unique ECM–cell crosstalk. We reveal that MMP-1 and MMP-13 possibly redundant matrix-remodeling proteases (e.g., collagenases) cause distinct ECM degradation, bringing about significantly distinct differ strongly in health, disease, and development. Although cellular phenotypes. Our findings show the complexity and selectivity enzymatic redundancy might be inferred from their close similarity in of collagenase-associated degradation mechanisms during tissue structure, their in vivo activity can lead to extremely diverse tissue- remodeling; these mechanisms could be used as a tool for future remodeling outcomes. We observed that proteolysis of collagen-rich therapeutic interventions. natural extracellular matrix (ECM), performed uniquely by individual homologous proteases, leads to distinct events that eventually affect Results overall ECM morphology, viscoelastic properties, and molecular Selective Degradation of ECM by Collagenases Determines Fibroblast composition. We revealed striking differences in the motility and Behavior. We set out to characterize the specific influences of the signaling patterns, morphology, and gene-expression profiles of cells – highly abundant collagenases on fibroblast ECM crosstalk. In this BIOCHEMISTRY interacting with natural collagen-rich ECM degraded by different study we used natural collagen fascicles (termed “ECM”)from collagenases. Thus, in contrast to previous notions, matrix-remodeling tendons of 6-mo-old rats (22, 23). We examined the effect of this systems are not redundant and give rise to precise ECM–cell crosstalk. ECMonrat-1fibroblasts,whichareknowntobesensitivetoECM Because ECM proteolysis is an abundant biochemical process that is composition and rigidity. Moreover, these cells, much like epithe- critical for tissue homoeostasis, these results improve our fundamen- lial cells, can exhibit different modes of migration toward different tal understanding its complexity and its impact on cell behavior. substrates and engage in cell–cell interactions (24, 25). Because fibroblasts inherently express ECM proteins and remodeling en- ECM | MMP | proteolysis zymes, we conducted our experiments at early stages of interaction he function and integrity of the extracellular matrix (ECM) is Significance Tvital for cell behavior as well as for whole-tissue homeostasis – (1 4). The ECM undergoes constant remodeling during health Extracellular matrix (ECM) proteolysis is an abundant bio- and disease states. Its components are regularly being deposited, chemical process. Here we describe the multilayered biological degraded, or otherwise modified. The highly stable fibrillar col- complexity generated by structurally homologous collagenases lagen type I (Col I) is abundant in many organ-derived ECMs (matrix metalloproteinase-1 and matrix metalloproteinase-13) in and connective tissues (5, 6); it serves as a tissue scaffold, deter- collagen-rich, native ECM, that may prove central to tissue ho- mining ECM mechanical properties and anchoring other ECM meostasis and pathology. The events induced by these two col- proteins necessary for cell function (7). These processes are or- lagenases generate microenvironments characterized by distinct chestrated by multiple remodeling enzymes, among which the ma- chemical, biomechanical, and morphological ECM properties that trix metalloproteinase (MMP) family plays an important role. Only lead to different cellular behaviors. Our findings improve the a few members of this proteinase family, the collagenases, are able fundamental understanding of selective ECM degradation by to degrade the resistant fibrillar collagens, i.e., Col I, as well as other homologous collagenases and its impact on cell behavior. ECM molecules (8, 9). The collagenases have conserved amino acids in their zinc-containing catalytic domain (8, 10) and display Author contributions: I. Solomonov, E.Z., I.A., H.N., and I. Sagi designed research; I. Solomonov, high structural similarities, as reflected by their functional domain E.Z., S.G.W., A.S., A.Z., E.K., Y.L., N.K., and D.A.J. performed research; R.V. and H.N. contrib- organization. Nevertheless, the complex effects exerted by different uted new reagents/analytic tools; I. Solomonov, E.Z., D.T.-F., A.S., Y.L., D.A.J., E.D., and MMPs on ECM and cells in vivo remain poorly understood. I. Sagi analyzed data; and I. Solomonov, E.Z., D.T.-F., S.G.W., and I. Sagi wrote the paper. The enzymatic activity of MMPs, and specifically of collage- The authors declare no conflict of interest. nases, in vivo is tightly regulated (11), with enzymatic dysregu- This article is a PNAS Direct Submission. C.M.O. is a Guest Editor invited by the Editorial lation causing irreversible damage associated with a variety of Board. diseases. Abnormally elevated levels of MMP-1 or of both MMP-1 Data deposition: MS data and protein/peptide identifications from in-gel analysis have been uploaded to the ProteomeXchange Consortium via the PRIDE repository, www.ebi. and MMP-13 have been associated with different types of cancers ac.uk/pride/archive/login (project accession no. PXD003796; username: reviewer59836@ andwithinflammatorydiseases(12–21). ebi.ac.uk; password: sTHj8wE8). Mass spectrometry data and protein/peptide identifica- Here, we explored the degradation patterns of natural collagen- tions from in-solution digestion have been uploaded to the ProteomeXchange Consor- rich ECM by two homologous MMPs and tested the response of tium via the PRIDE repository, www.ebi.ac.uk/pride/archive/login (project accession no. PXD003553; username: [email protected]; password: cJGfrm0Z). The RNA- cells to intact vs. selectively degraded ECM. We collectively pro- sequencing dataset has been deposited in the Gene Expression Omnibus database (acces- filed the unique remodeling events caused by two secreted colla- sion no. GSE79749). genases, MMP-1 and MMP-13, by using biochemical, biophysical, 1To whom correspondence should be addressed. Email: [email protected]. and proteomics tools. We show that these proteases drive morpho- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. logical, biochemical, and viscoelastic changes in the ECM leading 1073/pnas.1519676113/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1519676113 PNAS Early Edition | 1of6 Downloaded by guest on September 23, 2021 (up to 4 h); in this time frame, no collagen deposition or MMP-1 adopted round, symmetrical morphologies. To validate these and MMP-13 secretion was detected (Fig. S1 A and B). morphological changes, we further quantified the aspect ratios of Cell motility was characterized using real-time optical imaging. cells that adhered to differentially degraded ECM using our Although fibroblasts demonstrated a clustered, directional motil- SEM images at 4 h after seeding (Fig. 1 D–F). This analysis ity toward native ECM (control) with a measured velocity of 40 ± yielded a trend in cell aspect ratio similar to that obtained with 14 μm/h, they showed individual modes of motility with a velocity live-cell imaging at the measured time point (Fig. 1H). Overall, of 37 ± 5 μm/h when moving toward MMP-1–degraded ECM. In these analyses provide cell-shape statistics indicating that rat-1 the presence of MMP-13–degraded ECM, the cells exhibited re- cells adopt different morphologies upon interaction with differ- duced or arrested motility with a measured velocity of 2 ± 2 μm/h entially degraded Col I-rich ECM. (Fig. 1 A–C, Fig. S1C,andMovies S1–S3). The differential phenotypes acquired by fibroblasts interacting To analyze the overall shape of the cells, we quantified the cell with degraded ECM suggested a typical and unique cellular re- aspect ratio (the length divided by the width) as a function of sponse driven by signal modulations transmitted by the ECM time using our live-cell imaging data (Fig. 1G). This analysis (26, 27). Because cells regulate migration, proliferation, and allowed us to compare precisely the shape of cells migrating on adhesion mainly through the activation of the ERK1/2 cascade differentially remodeled collagen-rich ECM. Cells imaged on (28), we examined the effect of ECM remodeling on this cas- control ECM showed aspect ratios ranging from 1.36 to 1.42, cade. Fibroblasts adhering to native
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