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Mechanical Tension and a2b1 Regulate Fibroblast Functions Beate Eckes1, Manon C. Zweers1, Zhi Gang Zhang1,2, Ralf Hallinger1, Cornelia Mauch1,3, Monique Aumailley2,3 and Thomas Krieg1,3

The extracellular matrix (ECM) environment in connective tissues provides fibroblasts with a structural scaffold and modulates cell shape, but it also profoundly influences the fibroblast phenotype. Here we studied fibroblasts cultured in a three-dimensional network of native collagen, which was either mechanically stressed or relaxed. Mechanical load induces fibroblasts that synthesize abundant ECM and a characteristic array of cytokines/chemokines. This phenotype is reminiscent of late granulation tissue or scleroderma fibroblasts. By contrast, relaxed fibroblasts are characterized by induction of proteases and a subset of cytokines that does not overlap with that of mechanically stimulated cells. Thus, the biochemical composition and physical nature of the ECM exert powerful control over the phenotypes of fibroblasts, ranging from ‘‘synthetic’’ to ‘‘inflammatory’’ phenotypes. Interactions between fibroblasts and collagen fibrils are mostly mediated by a subset of b1 integrin receptors. Fibroblasts utilize a1b1, a2b1, and a11b1 for establishing collagen contacts and transducing signals. In vitro assays and mouse genetics have demonstrated individual tasks served by each receptor, but also functional redundancy. Unraveling the integrated functions of fibroblasts, collagen integrin receptors, collagen fibrils, and mechanical tension will be important to understand the molecular mechanisms underlying tissue repair and fibrosis. Journal of Investigative Dermatology Symposium Proceedings (2006) 11, 66–72. doi:10.1038/sj.jidsymp.5650003

FIBROBLASTS IN RELAXED VERSUS STRESSED In this setting, the cells are surrounded on all surfaces by COLLAGEN LATTICES collagen fibrils, in contrast to monolayer cultures where one Tissue homeostasis is controlled by the interaction of resident large part of the polarized cell is in contact with a rigid cells with each other and with their extracellular environ- substrate of collagen monomers (no fibrils) and the other with ment. Cell–cell communication is achieved either by culture medium. Casting the lattices in bacteriological dishes, receptor–counter receptor binding or by the activity of which are not adhesive for either collagen fibrils nor cells, diffusible mediators, for example growth factor, cytokines leads to relaxed lattices that float freely in the medium (Figure and chemokines, which act in paracrine or autocrine 2a) and shrink over time (Bell et al., 1979). Here, fibroblasts regulatory loops (Figure 1). These interactions are important attach to collagen fibrils using mostly integrin a2b1 (Klein and have been well characterized, but in this overview we et al., 1991; Schiro et al., 1991; Langholz et al., 1995) and will concentrate on the important regulatory functions of remodel the fibrils by traction (Stopak and Harris, 1982). The components of the extracellular matrix (ECM) and on the role result after 1 day is a compact disk-shaped tissue-like of mechanical tension exerting stress on matrix structures and structure of about 1/10 of the original diameter with cells embedded therein. We will focus on dermal fibroblasts fibroblasts being densely packed in this reduced volume interacting with fibrillar type I collagen, which is the most (Figure 2b). abundant ECM component in connective tissues, and high- In contrast, fibroblasts are subjected to mechanical tension how this interaction influences the genetic program of when the matrix to which they adhere and on which they fibroblasts. exert tractional forces, cannot be contracted, because it is To study fibroblast–collagen interactions in close to in vivo tethered to a nylon ring placed inside the dish and lining the conditions, we as many other groups of researchers seed pri- periphery (Figure 2c) (Delvoye et al., 1986; Lambert et al., mary skin fibroblasts in three-dimensional collagen lattices. 1992). In another model, the lattice is cast between two

1Department of Dermatology, University of Cologne, Cologne, Germany; 2Center for Biochemistry, University of Cologne, Cologne, Germany and 3Center for Molecular Medicine, University of Cologne, Cologne, Germany Correspondence: Professor Thomas Krieg, Department of Dermatology, University of Cologne, Kerpener Str. 62, D-50937 Cologne, Germany. E-mail: [email protected] Abbreviations: COX-2, cyclooxygenase-2; ECM, extracellular matrix; MCP-1/-3, monocyte chemoattractant -1/-3; MMP, matrix metalloproteinase; PAI, plasminogen activator inhibitor Received 12 January 2006; accepted 26 January 2006

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Interaction with rectangular bars to which it adheres. One bar is fixed, while the ECM the other one is connected to a force transducer capable of picking up the force generated by fibroblasts within the collagen matrix (Elsdale and Bard, 1972; Kolodney and Wysolmerski, 1992; Brown et al., 1998). We used the circular system (Figure 2c) to monitor changes in the expression profile and fibroblast morphology in comparison Soluble Mechanical to the relaxed system (Figure 2b), and the culture force molecules tension monitor (Figure 2d) to assess magnitude of forces generated (Kessler et al., 2001). To obtain a comprehensive list of activated by Tissue homeostasis mechanical stress, we performed a cDNA microarray screen Figure 1. Factors influencing tissue homeostasis. Cell phenotypes and of fibroblasts cultured in stressed collagen lattices or in biological responses are controlled by the interaction of one or several cell relaxed collagen lattices at 20 hours, when force generation types via direct interaction or via soluble mediators and growth factors. in stressed fibroblasts had reached plateau levels and Furthermore, some cell types, mainly mesenchymal in nature, interact with contraction in the relaxed system was maximal (Kessler components of the ECM by way of specialized receptors, which are activated et al., 2001). to elicit signal cascades that are particular to the individual ligand–receptor interaction; for example cell binding to collagen by integrin a1b1 elicits different responses than binding of collagen by a2b1 (see text). A further level INDUCTION OF AN ‘‘INFLAMMATORY’’ FIBROBLAST of regulation is introduced by the physical nature of the ECM, for example by PHENOTYPE IN A MECHANICALLY RELAXED the mechanical load across the cell membrane. ECM ENVIRONMENT Since long we know that taking fibroblasts from monolayer cultures to freely contracting, relaxed collagen lattices induces a pronounced change in the expression of selected genes. Cells undergo apoptosis by about the time when contraction is macroscopically terminated (Fluck et al., a b 1998), and synthesis of fibrillar collagens is downregulated to approximately 5% of levels produced in monolayers (Mauch et al., 1988). Downregulation involves transcrip- tional as well as post-transcriptional control via collagen transcript destabilization (Eckes et al., 1993, 1996). However, cells do not go into general quiescence, as seen by more than 20-fold induced synthesis and acitvation of matrix metallo- proteinase (MMP)-1 (Mauch et al., 1989) and activation of pro-MMP-2 (Zigrino et al., 2001). The microarray analysis confirmed downregulation of c d collagen, induction of MMPs as well as of genes related to apoptosis, and furthermore indicated induction of transcripts associated with inhibition of proliferation and of a distinct subset of growth factors and stress response genes (Kessler- Becker et al., 2004a). Among the latter were cyclooxygenase- 2 (COX-2), IL-1 and IL-6, ICAM-1, keratinocyte growth factor and the mitochondrial superoxide dismutase. COX-2 was further studied because its relative induction was the highest in the entire screen, suggesting high prostaglandin synthesis in response to inflammatory mediators in this system. In a Figure 2. Fibroblasts in freely contracting and tethered collagen lattices. time course study, interestingly, COX-2 expression was Shown are collagen lattice systems that either lack (b) or generate mechanical similar to that of IL-1, and blocking IL-1 expression by IL-1 tension (c and d). Fibroblasts are lifted off the culture dish and mixed with neutralized collagen solution and fetal calf serum. (a) This mixture is cast in receptor antagonist abrogated COX-2 induction. This result bacteriological (non-adhesive) dishes, resulting in a gel with collagen fibrils suggested an autocrine IL-1 loop in fibroblasts in the after about 30 minutes at 371C. (b) Within 1 day, the cells attach to the fibrils relaxed system which is causally involved in COX-2 and remodel the lattice to form a dense, tissue-like structure. This is a model induction. From these and other data, we concluded that a system largely devoid of mechanical tension acting on the cells. (c) Shows a mechanically relaxed collagen environment induces a lattice that is identical in composition to (b), however, in (c) tension is phenotype in fibroblasts that is characterized by production generated by cells in the lattice being held attached to the nylon thread, of high IL-1 levels, which stimulate synthesis of IL-6 and which is placed at the inner perimeter of the dish. (d) The culture force monitor system is depicted. The rectangular lattice is seen between two bars COX-2 (Figure 3). These cells are clearly not the quiescent on either long side of the gel. One of these bars is fixed, while the other one is cell type often pictured, but by releasing immunomodulatory connected to a force transducer, which records the forces over time. substances, they are active players in an inflammatory setting.

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Resting fibroblast

+ Tension – Tension Interleukin-1

Interleukin-6 COX-2 Activated Inflammatory phenotype phenotype Modulation of inflammation

Figure 3. Fibroblasts are key modulators of inflammatory responses. Matrix Fibrogenic Matrix Inflammatory synthesis cytokines degradation mediators Fibroblasts in contact with a relaxed three-dimensional collagen network Proliferation Inhibition of produce abundant IL-1, which upregulates the production of further proliferation inflammatory mediators, for example IL-6 and COX-2, key mediators of inflammatory reactions. COX-2 induction is abrogated by inhibition of IL-1, Figure 4. Induction of different fibroblast phenotypes by mechanical indicating a hierarchy of cytokine action. tension. Fibroblasts interact with surrounding collagen fibrils using mainly receptors of the b1 family of integrins. Lack of mechanical load in this system produces an ‘‘inflammatory’’ phenotype that is characterized by high Probably this phenotype is required during early phases of expression of inflammatory mediators and proteases along with low tissue repair. proliferation. In contrast, mechanical load induces an ‘‘activated’’ prolifer- ating fibroblast phenotype that synthesizes abundant ECM and protease inhibitors as well as fibrogenic mediators. Presumably, the relaxed fibroblast INDUCTION OF A ‘‘SYNTHETIC’’ FIBROBLAST is encountered in early wound in loose forming granulation tissue. The latter, PHENOTYPE IN AN ECM ENVIRONMENT UNDER activated type is thought to reside in later stage wounds and in fibrotic skin. MECHANICAL TENSION Fibroblasts cultured in a collagen lattice that is identical in composition but differs in that cells cannot contract the Since the circular lattice system is not suited to measure matrix because it is tethered to a ring placed at the periphery the magnitude of forces that the cells generate in this system, of the dish (Figure 2c) assume a different phenotype from we used a culture force monitor with a force transducer those cultured in contracting lattices (Lambert et al., 1992; connected to one of the long sides of the rectangular lattice Kessler et al., 2001). Phase contrast inspection shows bipolar, (Figure 2d). This detects reduction in gel width due to elongated cells of almost 100 mm in length in contrast to the fibroblast-mediated lattice shrinkage, while the length stellate appearance of fibroblasts in relaxed lattices. Most of remains constant (Kessler et al., 2001). The forces produced these cells express a-smooth muscle actin-positive stress by primary human skin fibroblasts were on the order of 1 mN fibers that terminate in well-defined focal adhesions (Kessler (using 2.5 Â 105 cells per ml and 1.75 mg collagen per ml), et al., 2001) and are thought to represent the contractile which is less than the forces developed by smooth muscle myofibroblast phenotype seen in fibrotic tissues (Tomasek cells but more than mouse skin fibroblasts can mount. et al., 2002). This differentiated, specialized fibroblast cell In summary, fibroblasts in a three-dimensional collagen type is not observed in contracting lattices, but is a result of network under mechanical tension turn into an activated, forces acting across the cell membrane via transmembrane matrix producing cell, a phenotype, which is required at later receptors, for example integrins. stages of wound repair. This phenotype could also be In accordance with the morphology, the comparative responsible for excessive matrix deposition in fibrotic microarray analysis revealed upregulation of a-smooth diseases. It is further characterized by a-smooth muscle muscle actin and vinculin and further cytoskeletal and focal actin-positive stress fibers, reminiscent of the specialized adhesion components. In agreement with previous findings myofibroblast, which is also seen in mid to late phases of (Lambert et al., 1992), fibroblasts in stressed conditions healing wounds and in some fibrotic tissues (Gabbiani, proliferate and express proliferation-associated genes, and 1992). they synthesize elevated levels of numerous ECM compo- nents, including collagens, tenascin, and hyaluronan among SIMILAR CHARACTERISTIC PROPERTIES IN others. In addition, protease inhibitors, such as plasminogen MECHANICALLY STIMULATED FIBROBLASTS AND activator inhibitor (PAI)-1 and -2, some of the tissue inhibitors SCLERODERMA FIBROBLASTS of matrix metalloproteinases and others were distinctly Scleroderma is a chronic fibrosing disease that involves the upregulated, creating an environment of ECM accumulation. skin but also many internal organs. It is characterized by In line with this observation, we detected upregulation of excessive deposition of ECM, mainly collagen, throughout mediators that are known to stimulate ECM production, for the entire dermis including the subcutaneous tissue (Denton example transforming growth factor-b1 and -b3, connective and Abraham, 2004). Next to vascular abnormalities and tissue growth factor and the closely related Cyr61, and dysregulated immune responses, fibroblasts become acti- monocyte chemoattractant protein-1 (MCP-1) (Figure 4). vated (Kissin and Korn, 2003), presumably by a combination

68 Journal of Investigative Dermatology Symposium Proceedings (2006), Volume 11 B Eckes et al. Mechanical Tension and Integrin a2b1

of cell–cell contact with endothelial and immune cells and cytoplasmic PAI-2. Possibly it might be involved in the fibroproliferative, and fibrogenic mediators released by the regulation of mRNA stability. environment. Our question was whether these activated scleroderma fibroblasts share phenotypic similarities with the ROLE OF COLLAGEN-BINDING INTEGRIN RECEPTORS mechanically stimulated fibroblasts seen in tethered collagen IN FIBROBLAST–COLLAGEN INTERACTIONS lattices. In the following, three examples will be outlined that We think that the contact of cells with surrounding collagen confirm such similarities. is critical for the generation of the different phenotypes First, we demonstrated that connective tissue growth factor discussed above, and that this interaction is mediated by is abundantly expressed by fibroblasts in lesional skin, in receptors of the b1 integrin family. Native collagens are particular by fibroblasts which also overexpress collagen type recognized by integrins a1b1, a2b1, a10b1, and a11b1 I (Scharffetter et al., 1988; Querfeld et al., 2000). Connective (White et al., 2004). Of these four, fibroblasts express a2b1 tissue growth factor is a chemotactic, fibroproliferative and and a11b1, which bind with relatively high affinity to fibrogenic mediator, that strongly upregulates collagen collagen type I, and a1b1, which has a preference for production in fibrotic skin either following induction by basement membrane collagen IV (Table 1), while a10b1is transforming growth factor-b or on its own. Similar findings expressed in almost undetectable amounts. A combination of were reported by others (Igarashi et al., 1996; Leask et al., experimental approaches involving antibody perturbation 2004). (Klein et al., 1991; Langholz et al., 1995), cells expressing Second, both types of activated fibroblasts express high only a1b1ora2b1 (Riikonen et al., 1995) and genetic levels of MCP-1 (Yamamoto et al., 2001a, b). MCP-1 belongs ablation of a1b1 (Gardner et al., 1996) or a2b1 (Chen et al., to the family of C–C chemokines and is produced by a wide 2002; Holtkotter et al., 2002) showed that these two variety of cell types. It is a potent chemoattractant for receptors are involved in different processes, sometimes monocytes and macrophages, which are recruited in vivo to acting antagonistically (Table 1). Integrin a1b1 is required for sites of inflammation in many pathological conditions. We optimal fibroblast proliferation in culture (Pozzi et al., 1998) found abundant MCP-1 signals in scleroderma, but not in and for reduction of collagen synthesis in vitro and in vivo normal skin and moreover demonstrated that cultured (Langholz et al., 1995; Gardner et al., 1999), while a2b1is scleroderma fibroblasts are more sensitive to added MCP-1 needed for contraction of collagen networks (Schiro et al., than control fibroblasts. Thus it is conceivable that in 1991; Langholz et al., 1995), induction and activation of scleroderma, activated fibroblasts release high levels of MMPs (Langholz et al., 1995; Riikonen et al., 1995; Zigrino MCP-1, to which they react in an autocrine fashion. In et al., 2001). Further roles for a2b1 have been proposed in addition, MCP-1 recruits monocytes/macrophages, which focal adhesion formation and turnover (Zhang et al., further activate fibroblasts, for example by transforming 2006a, b) and collagen fibril formation (Velling et al., 2002; growth factor-b, to produce more ECM. In this way, Li et al., 2003; Jokinen et al., 2004). The phenotype of both fibroblasts are thought to play an important role in modulat- knockout mouse models is rather mild, indicating a backup ing the inflammatory response in early phases of the disease, system of functional compensation by other collagen-binding and that they contribute significantly to the sustained fibrotic integrins. Functions of a11b1 are only recently emerging with tissue response. MCP-1 was also detected at elevated levels roles in cell migration (Tiger et al., 2001; Popova et al., 2004) in the serum of scleroderma patients (Hasegawa et al., 1999), and collagen fibril assembly (Velling et al., 2002). and later also MCP-3 was found increased in early-stage Mice completely deficient in a2b1 live and reproduce, scleroderma as well as in the skin of tight skin-1 mice (Ong which came as a surprise in view of the manifold in vitro et al., 2003). functions ascribed to this receptor. We therefore analyzed The third example is PAI-2, which was among the three different cell types in functional in vitro assays and in transcripts most strongly induced by mechanical forces. It vivo. These are , keratinocytes, and fibroblasts, blocks the conversion of plasminogen to plasmin and thereby which express abundant a2b1 and the function of which indirectly inhibits the activity of plasmin-activated MMPs in was thought to rely on a2b1. the extracellular space, resulting in decreased matrix function appeared normal, as no prolonged degradation. Probably it is also involved in regulating the bleeding was observed in a2-deficient mice. Platelet counts activity of matrix-bound cytokines. By regulating protease were normal, but in vitro aggregation of platelets in response activity, PAI-2 is thought to modulate ECM metabolism and in to collagen was delayed (Holtkotter et al., 2002). However, particular to favor ECM accumulation. Interestingly, a similar in vivo, thrombus formation was normal following mechan- plasmin inhibitor was detected at elevated levels in scler- ical injury to the carotid artery, demonstrating functional oderma fibroblasts, protease nexin-1, which, due to binding backup (Gruner et al., 2003). Prolonged bleeding did, plasmin, may also inhibit MMP activation (Strehlow et al., however, occur in mice which were simultaneously deficient 1999). Our findings demonstrated high PAI-2 RNA and for the collagen receptors a2b1 and GPVI (Gruner et al., protein levels in scleroderma fibroblasts, but immunoblotting 2004). This observation could be of clinical importance, if and staining clearly showed that PAI-2 was confined to the GPVI is targeted by antithrombotic therapy. Given that a2b1 cell cytoplasm (Kessler-Becker et al., 2004b). This result levels vary considerably in healthy humans (Kunicki et al., speaks against an extracellular MMP inhibitory activity, and 1997), such anti-GPVI treatment could have serious side at present it is unclear which function could be fulfilled by effects in patients with low a2b1 expression.

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Table 1. Functions proposed for the collagen-binding integrin receptors expressed by fibroblasts Receptor Proposed function References

a1b1 Adhesion to collagen IVbcollagen I Kern et al. (1993); Tulla et al. (2001) Regulation of collagen synthesis Langholz et al. (1995); Gardner et al. (1999) Fibroblast proliferation Pozzi et al. (1998)

a2b1 Adhesion to collagen Ibcollagen IV Kern et al. (1993); Tiger et al. (2001); Tulla et al. (2001); Zhang et al. (2006b) MMP induction Langholz et al. (1995); Riikonen et al. (1995); Zigrino et al. (2001) Contraction of collagen lattices Klein et al. (1991); Schiro et al. (1991); Langholz et al. (1995); Zhang et al. (2006b) Migration Pilcher et al. (1997) Collagen fibril formation Velling et al. (2002); Li et al. (2003); Jokinen et al. (2004)

a11b1 Adhesion to collagen Ibcollagen IV Tiger et al. (2001); Zhang et al. (2006b) Contraction of collagen lattices Tiger et al. (2001) Migration Tiger et al. (2001); Popova et al. (2004) Collagen fibril formation Velling et al. (2002)

In contrast to platelets and fibroblasts, epidermal keratino- Table 2. Phenotype of primary fibroblasts derived cytes express a2b1 as sole collagen receptor. Accordingly, from integrin a2b1-deficient mice primary keratinocytes isolated from a2b1 knockout mice clearly failed to adhere to either collagen I or IV in vitro Overall morphology Unchanged (Zhang et al., 2006b). Surprisingly, closure of skin wounds in Adhesion to collagen I k these mice proceeded at rates comparable to wild-type Adhesion to collagen IV Unchanged wounds. In addition, rates of wound re-epithelialization were not significantly altered, indicating that wound keratinocytes Adhesion to laminins Unchanged either up-regulate a collagen receptor that is normally not Migration on collagen I Unchanged expressed, or that they do not migrate on collagens in the Contraction of collagen lattices k wound bed (M. Zweers, T. Krieg and B. Eckes, manuscript in Development of tractional forces k preparation). In contrast to keratinocytes, adhesion to collagen I by Activation of Rho GTPases Altered fibroblasts lacking a2b1 was slightly reduced in long-term Distribution of focal contacts Altered assays, but short-term adhesion clearly required a2b1 (Zhang Wound closure Unchanged et al., 2006b) (Table 2). Blocking b1 subunit function in a2-deficient fibroblasts abrogated adhesion, while blocking a1 in these cells had no major effect. These results point to a11b1 as a compensatory receptor for collagen I in these assays. Interestingly, adhesion to collagen IV was found to require not only a1b1, but also a2b1. Thus, binding of wild type. A similar correlation was previously observed in fibroblasts to collagen I can proceed to some extent in the fibroblasts expressing constitutively active RhoA, which were absence of the a2b1 that was so far considered the major equally defective in collagen lattice contraction (Zhang et al., receptor mediating this interaction. Can other fibroblast 2006a). We hypothesize that high levels of active RhoA functions that were thought to require a2b1 be equally inhibit regular focal adhesion turnover, resulting in impaired fulfilled without this receptor? We found that collagen lattice dynamic fibroblast functions which rely on fast formation and contraction by a2 null fibroblasts was delayed and impaired resolution of matrix attachment points. (Table 2), similar to contraction by fibroblasts treated with Comparing mice lacking integrin a2b1ora1b1 clearly antibodies blocking a2 and b1 (Langholz et al., 1995). demonstrates that both receptors serve different functions, Moreover, forces generated by a2b1-deficient fibroblasts in a some of which can be compensated by each other or by culture force monitor were significantly lower than by wild- a11b1. Analysis of mutants deficient in two a-subunits or type fibroblasts (Zhang et al., 2006b). We think that these even all three will enable to delineate the precise contribu- dynamic functions are impaired in fibroblasts lacking a2b1, tion of the individual receptors of the collagen-binding b1 because these cells display higher levels of active RhoA than integrins.

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CONFLICT OF INTEREST Igarashi A, Nashiro K, Kikuchi K, Sato S, Ihn H, Fujimoto M et al. (1996) The authors state no conflict of interest. Connective tissue growth factor gene expression in tissue sections from localized scleroderma, keloid, and other fibrotic skin disorders. J Invest Dermatol 106:729–33 ACKNOWLEDGMENTS We thank Daniela Kessler-Becker, Olaf Holtko¨tter, Frank Hirche, Toshiyuki Jokinen J, Dadu E, Nykvist P, Kapyla J, White DJ, Ivaska J et al. (2004) Integrin- Yamamoto, and Bernhard Nieswandt for fruitful discussion, Gabriele Scherr, mediated to type I collagen fibrils. J Biol Chem Nicole Bru¨ggenolte, and Monika Pesch for excellent technical assistance. 279:31956–63 These studies were supported by the Deutsche Forschungsgemeinschaft (SFB Kern A, Eble J, Golbik R, Kuhn K (1993) Interaction of type IV collagen with 589 and KR558/13), the Deutsche Zentrum fu¨r Luft- und Raumfahrt the isolated integrins a1b1anda2b1. 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