DOI: 10.1111/exd.12237 www.wileyonlinelibrary.com/journal/EXD
Commentary: My Favourite Historical Paper
Deciphering the pro-fibrotic phenotype of fibroblasts in systemic sclerosis € rg H. W. Distler and Christian Beyer Christiane Maier, Jo Department of Internal Medicine 3 and Institute for Clinical Immunology, University of Erlangen-Nuremberg, Erlangen, Germany Correspondence: Christian Beyer, MD, Department of Medicine 3 and Institute for Clinical Immunology, University of Erlangen-Nuremberg, Ulmenweg 18, D-91054 Erlangen, Germany, Tel.: +49-9131-8543023, Fax: +49-9131-8534665, e-mail: [email protected] Abstract: LeRoy’s seminal work on the phenotypic features of scleroderma fibroblasts has been directing fibrosis research in the field of systemic sclerosis (SSc, scleroderma) for the past 30 years. His principal experiment, to culture skin fibroblasts from patients with SSc and study their pro-fibrotic phenotype in comparison with skin fibroblasts from healthy individuals, has been used by most basic and translational fibrosis studies in SSc. LeRoy’s
findings have revolutionized our understanding of the disease pathogenesis and guided the development of novel antifibrotic therapies towards fibroblast-specific approaches.
More than 30 years ago, a pioneering observation shed first light on the key role fibroblasts play in the pathogenesis of systemic sclerosis (SSc, scleroderma). In a series of experiments, LeRoy (1) isolated skin fibroblasts from patients with SSc, kept them in in vitro culture systems and discovered an increased release of collagens and glycoproteins compared with skin fibroblasts from healthy individuals. Most importantly, LeRoy (2) observed that these pro-fibrotic features of SSc fibroblasts persisted for several passages in vitro, even in the absence of any other cell type or constituent of the SSc in vivo milieu. Despite other assumptions that have attributed the key pathogenic role to immune or endothelial cells, there appeared to be only one plausible conclusion from these fascinating observations: SSc fibroblasts not only did execute pro-fibrotic signals from other cells but themselves played a very active role in the progression of fibrotic disease. While we are still marvelled by LeRoy’s findings, our field is deciphering this intrinsic pro-fibrotic phenotype of SSc fibroblasts step by step. Recent studies discovered autocrine signalling loops, epigenetic modifications, microRNA signatures and mechanical stress as important determinants of the SSc fibroblast phenotype first identified by LeRoy. Among the multiple cytokines implicated in the origin of the SSc fibroblast phenotype, transforming growth factor-b (TGF-b) is considered one of the key regulators (3). Stimulation of resting fibroblasts from healthy individuals can induce most of the phenotypic characteristics of SSc fibroblasts, including the increased collagen release. These observations led to the hypothesis that the SSc fibroblast phenotype may result from aberrant autocrine TGFb signalling (4). Indeed, SSc fibroblasts express increased levels of TGF-b receptors, which permit them to mount a robust response to endogenously produced TGF-b (5,6). Furthermore, thrombospondin and avb3 integrins, which mediate latent TGF-b activation, are elevated on SSc fibroblasts (5), again favouring autocrine TGF-b signalling. Nevertheless, autocrine TGF-b signalling cannot account for all of the phenotypic features of SSc fibroblasts, such as constitutive production of connective tissue growth factor
(CTGF) and endothelin-1 (ET-1) (7). Moreover, small molecule inhibitors of TGF-b signalling fail to completely normalize the SSc fibroblast phenotype, so that abnormally regulated TGF-b signalling or increased sensitivity to TGF-b is likely not to be the only underlying cause for all phenotypic features of SSc fibroblasts (8,9). In addition to TGF-b, pro-fibrotic autocrine signalling by CTGF and platelet-derived growth factor (PDGF) might contribute to the SSc fibroblast phenotype (10), although further evidence is needed to demonstrate enhanced responsiveness of SSc fibroblasts to PDGF and CTGF. In addition to autocrine growth factor signalling, epigenetic modifications may explain the pro-fibrotic phenotype of SSc fibroblasts. In this context, histone modifications regulated by the histone deacetylase 7 may play a central role in the SSc fibroblast as highlighted indirectly by blockade of this enzyme: inhibition of histone deacetylase 7 reduces the release of extracellular matrix components, including collagens and fibronectin, from fibroblasts (11). Apart from histone modifications, specific DNA methylation patterns may govern the SSc fibroblast phenotype. As we observed, hypermethylation through DNA methyltransferase 1 (Dnmt1) inactivates the endogenous Wnt antagonists Dickkopf-1 and secreted frizzled-related protein-1 in SSc fibroblasts. Inactivation of endogenous Wnt inhibitors by DNA methylation releases the potent pro-fibrotic Wnt signalling in SSc fibroblasts, resulting in enhanced collagen release and fibrosis. Interestingly, demethylation of the both gene regions by 5′-aza-2′-deoxycytidine, a DNA methyltransferase inhibitor, can normalize gene expression and reverse pro-fibrotic changes in SSc fibroblasts (12,13). Further yet indirect evidence for a central role of epigenetics in the pro-fibrotic phenotype of SSc fibroblasts comes from a seminal study in kidney fibrosis. Dnmt1-dependent hypermethylation of Rasal1, encoding an inhibitor of the Ras oncoprotein, leads to persistent fibroblast activation and fibrogenesis in the kidney (14). While similar mechanisms might be active in SSc, future studies will more clearly define the epigenetic landscape of the pro-fibrotic SSc fibroblast phenotype (15).
ª 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Experimental Dermatology, 2014, 23, 99–100
Key words: fibroblast – fibrosis – pathogenesis – systemic sclerosis – treatment
Accepted for publication 4 September 2013
99
Commentary: My Favourite Historical Paper
Specific microRNA signatures may also determine the profibrotic gene expression profiles of SSc fibroblasts (16). We found that miR-29 is downregulated in SSc fibroblasts and demonstrated in functional experiments a protective role of microRNA-29 in SSc: while experimental knockdown of microRNA-29 exacerbated pro-fibrotic processes, overexpression reversed pro-fibrotic changes (17). Since then, additional microRNAs, including miR-let7a miR-21, miR-29b, miR-145, miR-150 and miR-196a (18–21), have been identified as potential key regulators of the pro-fibrotic activities of SSc ‘fibroblasts’. Fibroblasts produce the beds in which they lie. Embedded in the collagen matrix, fibroblasts meet various biological obligations. Of note, the interaction with the extracellular collagen tissue appears vivid and determines the outcome of many processes. Intriguing studies suggest that the stiff fibrotic scar tissue, as observed in SSc skin, may induce pro-fibrotic phenotypic features in fibroblasts, for example through modulating TGF-b signalling (22,23). This may lead to a vicious circle of excessive collagen release, tissue scaring and fibroblast activation both in SSc skin and in in vitro culture systems. Thus, tissue mechanics and the interaction with the neighbouring extracellular components may further determine the phenotypic features of SSc fibroblasts (24). Bringing fibroblasts into the focus of SSc research, LeRoy helped us to revolutionize our understanding of SSc pathogenesis, disease course and treatment outcomes. Based on his findings, we
References 1 Leroy E C. J Exp Med 1972: 135: 1351–1362. 2 LeRoy E C. J Clin Invest 1974: 54: 880–889. 3 Varga J, Abraham D. J Clin Invest 2007: 117: 557–567. 4 Pannu J, Trojanowska M. Curr Opin Rheumatol 2004: 16: 739–745. 5 Mimura Y, Ihn H, Jinnin M et al. J Invest Dermatol 2005: 124: 886–892. 6 Asano Y, Ihn H, Yamane K et al. Arthritis Rheum 2005: 52: 2897–2905. 7 Shi-Wen X, Rodriguez-Pascual F, Lamas S et al. Mol Cell Biol 2006: 26: 5518–5527. 8 Ishida W, Mori Y, Lakos G et al. J Invest Dermatol 2006: 126: 1733–1744. 9 Chen Y, Shi-wen X, Eastwood M et al. Arthritis Rheum 2006: 54: 1309–1316. 10 Gay S, Jones R E Jr, Huang G Q et al. J Invest Dermatol 1989: 92: 301–303. 11 Hemmatazad H, Rodrigues H M, Maurer B et al. Arthritis Rheum 2009: 60: 1519–1529.
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assume that once triggered by external stimuli, the endogenous pro-fibrotic activity of SSc fibroblasts is the key driving factor in fibrosis. While the pro-fibrotic SSc fibroblasts release massive amounts of collagens to form scar tissue, they also maintain their phenotypic characteristics by intrinsic activity. The concept of the pro-fibrotic SSc fibroblast phenotype has major implications in the development of novel treatment strategies. While anti-inflammatory therapies may fail to modulate fibroblast activity in many clinical scenarios, novel therapies that deactivate the pro-fibrotic SSc fibroblast phenotype are most promising to block collagen release from fibroblasts and treat fibrosis in SSc (25,26). Assuming that LeRoy’s concept of intrinsic fibroblast activation in SSc may also hold true for other fibrotic conditions, fibroblasts may be the key targets of future antifibrotic therapies.
Author contributions CM and CB carried out literature research and data acquisition. CM, JD and CB drafted and revised the manuscript. CM, JD and CB approved the final version of the manuscript.
Ethical approval Not applicable.
Conflict of interests J.H.W. Distler has consultancy relationships and/or has received research funding from Actelion, Pfizer, Ergonex, BMS, Celgene, Bayer Pharma, JB Therapeutics, Sanofi-Aventis, Novartis, Array Biopharma and Active Biotec in the area of potential treatments of SSc and is stock owner of 4D Science. C. Maier and C. Beyer have no conflict of interests.
12 Dees C, Schlottmann I, Funke R et al. Ann Rheum Dis 2013 [Epub ahead of print]. 13 Akhmetshina A, Palumbo K, Dees C et al. Nat Commun 2012: 3: 735. 14 Bechtel W, McGoohan S, Zeisberg E M et al. Nat Med 2010: 16: 544–550. 15 Jungel A, Distler J H, Gay S et al. Expert Rev Clin Immunol 2011: 7: 475–480. 16 Vettori S, Gay S, Distler O. Open Rheumatol J 2012: 6: 130–139. 17 Maurer B, Stanczyk J, Jungel A et al. Arthritis Rheum 2010: 62: 1733–1743. 18 Makino K, Jinnin M, Hirano A et al. J Immunol 2013: 190: 3905–3915. 19 Honda N, Jinnin M, Kira-Etoh T et al. Am J Pathol 2013: 182: 206–216. 20 Honda N, Jinnin M, Kajihara I et al. J Immunol 2012: 188: 3323–3331. 21 Zhu H, Li Y, Qu S et al. J Clin Immunol 2012: 32: 514–522.
22 Wipff P J, Rifkin D B, Meister J J et al. J Cell Biol 2007: 179: 1311–1323. 23 Hinz B, Mastrangelo D, Iselin C E et al. Am J Pathol 2001: 159: 1009–1020. 24 Hinz B. Curr Rheumatol Rep 2009: 11: 120–126. 25 Beyer C, Distler J H. F1000 Med Rep 2009: 1, doi: 10.3410/M1-95. 26 Beyer C, Distler O, Distler J H. Curr Opin Rheumatol 2012: 24: 274–280.
Supporting Information Additional Supporting Information may be found in the online version of this article: Data S1. The editors and publisher gratefully acknowledge Rockefeller University Press for kindly having granted permission to reprint the original article discussed in this Commentary: Leroy E C. Connective tissue synthesis by scleroderma skin fibroblasts in cell culture. J Exp Med 1972: 135: 1351–1362. © 1972 Rockefeller University Press.
ª 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Experimental Dermatology, 2014, 23, 99–100
Commentary: My Favourite Historical Paper
Deciphering the pro-fibrotic phenotype of fibroblasts in systemic sclerosis € rg H. W. Distler and Christian Beyer Christiane Maier, Jo Department of Internal Medicine 3 and Institute for Clinical Immunology, University of Erlangen-Nuremberg, Erlangen, Germany Correspondence: Christian Beyer, MD, Department of Medicine 3 and Institute for Clinical Immunology, University of Erlangen-Nuremberg, Ulmenweg 18, D-91054 Erlangen, Germany, Tel.: +49-9131-8543023, Fax: +49-9131-8534665, e-mail: [email protected] Abstract: LeRoy’s seminal work on the phenotypic features of scleroderma fibroblasts has been directing fibrosis research in the field of systemic sclerosis (SSc, scleroderma) for the past 30 years. His principal experiment, to culture skin fibroblasts from patients with SSc and study their pro-fibrotic phenotype in comparison with skin fibroblasts from healthy individuals, has been used by most basic and translational fibrosis studies in SSc. LeRoy’s
findings have revolutionized our understanding of the disease pathogenesis and guided the development of novel antifibrotic therapies towards fibroblast-specific approaches.
More than 30 years ago, a pioneering observation shed first light on the key role fibroblasts play in the pathogenesis of systemic sclerosis (SSc, scleroderma). In a series of experiments, LeRoy (1) isolated skin fibroblasts from patients with SSc, kept them in in vitro culture systems and discovered an increased release of collagens and glycoproteins compared with skin fibroblasts from healthy individuals. Most importantly, LeRoy (2) observed that these pro-fibrotic features of SSc fibroblasts persisted for several passages in vitro, even in the absence of any other cell type or constituent of the SSc in vivo milieu. Despite other assumptions that have attributed the key pathogenic role to immune or endothelial cells, there appeared to be only one plausible conclusion from these fascinating observations: SSc fibroblasts not only did execute pro-fibrotic signals from other cells but themselves played a very active role in the progression of fibrotic disease. While we are still marvelled by LeRoy’s findings, our field is deciphering this intrinsic pro-fibrotic phenotype of SSc fibroblasts step by step. Recent studies discovered autocrine signalling loops, epigenetic modifications, microRNA signatures and mechanical stress as important determinants of the SSc fibroblast phenotype first identified by LeRoy. Among the multiple cytokines implicated in the origin of the SSc fibroblast phenotype, transforming growth factor-b (TGF-b) is considered one of the key regulators (3). Stimulation of resting fibroblasts from healthy individuals can induce most of the phenotypic characteristics of SSc fibroblasts, including the increased collagen release. These observations led to the hypothesis that the SSc fibroblast phenotype may result from aberrant autocrine TGFb signalling (4). Indeed, SSc fibroblasts express increased levels of TGF-b receptors, which permit them to mount a robust response to endogenously produced TGF-b (5,6). Furthermore, thrombospondin and avb3 integrins, which mediate latent TGF-b activation, are elevated on SSc fibroblasts (5), again favouring autocrine TGF-b signalling. Nevertheless, autocrine TGF-b signalling cannot account for all of the phenotypic features of SSc fibroblasts, such as constitutive production of connective tissue growth factor
(CTGF) and endothelin-1 (ET-1) (7). Moreover, small molecule inhibitors of TGF-b signalling fail to completely normalize the SSc fibroblast phenotype, so that abnormally regulated TGF-b signalling or increased sensitivity to TGF-b is likely not to be the only underlying cause for all phenotypic features of SSc fibroblasts (8,9). In addition to TGF-b, pro-fibrotic autocrine signalling by CTGF and platelet-derived growth factor (PDGF) might contribute to the SSc fibroblast phenotype (10), although further evidence is needed to demonstrate enhanced responsiveness of SSc fibroblasts to PDGF and CTGF. In addition to autocrine growth factor signalling, epigenetic modifications may explain the pro-fibrotic phenotype of SSc fibroblasts. In this context, histone modifications regulated by the histone deacetylase 7 may play a central role in the SSc fibroblast as highlighted indirectly by blockade of this enzyme: inhibition of histone deacetylase 7 reduces the release of extracellular matrix components, including collagens and fibronectin, from fibroblasts (11). Apart from histone modifications, specific DNA methylation patterns may govern the SSc fibroblast phenotype. As we observed, hypermethylation through DNA methyltransferase 1 (Dnmt1) inactivates the endogenous Wnt antagonists Dickkopf-1 and secreted frizzled-related protein-1 in SSc fibroblasts. Inactivation of endogenous Wnt inhibitors by DNA methylation releases the potent pro-fibrotic Wnt signalling in SSc fibroblasts, resulting in enhanced collagen release and fibrosis. Interestingly, demethylation of the both gene regions by 5′-aza-2′-deoxycytidine, a DNA methyltransferase inhibitor, can normalize gene expression and reverse pro-fibrotic changes in SSc fibroblasts (12,13). Further yet indirect evidence for a central role of epigenetics in the pro-fibrotic phenotype of SSc fibroblasts comes from a seminal study in kidney fibrosis. Dnmt1-dependent hypermethylation of Rasal1, encoding an inhibitor of the Ras oncoprotein, leads to persistent fibroblast activation and fibrogenesis in the kidney (14). While similar mechanisms might be active in SSc, future studies will more clearly define the epigenetic landscape of the pro-fibrotic SSc fibroblast phenotype (15).
ª 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Experimental Dermatology, 2014, 23, 99–100
Key words: fibroblast – fibrosis – pathogenesis – systemic sclerosis – treatment
Accepted for publication 4 September 2013
99
Commentary: My Favourite Historical Paper
Specific microRNA signatures may also determine the profibrotic gene expression profiles of SSc fibroblasts (16). We found that miR-29 is downregulated in SSc fibroblasts and demonstrated in functional experiments a protective role of microRNA-29 in SSc: while experimental knockdown of microRNA-29 exacerbated pro-fibrotic processes, overexpression reversed pro-fibrotic changes (17). Since then, additional microRNAs, including miR-let7a miR-21, miR-29b, miR-145, miR-150 and miR-196a (18–21), have been identified as potential key regulators of the pro-fibrotic activities of SSc ‘fibroblasts’. Fibroblasts produce the beds in which they lie. Embedded in the collagen matrix, fibroblasts meet various biological obligations. Of note, the interaction with the extracellular collagen tissue appears vivid and determines the outcome of many processes. Intriguing studies suggest that the stiff fibrotic scar tissue, as observed in SSc skin, may induce pro-fibrotic phenotypic features in fibroblasts, for example through modulating TGF-b signalling (22,23). This may lead to a vicious circle of excessive collagen release, tissue scaring and fibroblast activation both in SSc skin and in in vitro culture systems. Thus, tissue mechanics and the interaction with the neighbouring extracellular components may further determine the phenotypic features of SSc fibroblasts (24). Bringing fibroblasts into the focus of SSc research, LeRoy helped us to revolutionize our understanding of SSc pathogenesis, disease course and treatment outcomes. Based on his findings, we
References 1 Leroy E C. J Exp Med 1972: 135: 1351–1362. 2 LeRoy E C. J Clin Invest 1974: 54: 880–889. 3 Varga J, Abraham D. J Clin Invest 2007: 117: 557–567. 4 Pannu J, Trojanowska M. Curr Opin Rheumatol 2004: 16: 739–745. 5 Mimura Y, Ihn H, Jinnin M et al. J Invest Dermatol 2005: 124: 886–892. 6 Asano Y, Ihn H, Yamane K et al. Arthritis Rheum 2005: 52: 2897–2905. 7 Shi-Wen X, Rodriguez-Pascual F, Lamas S et al. Mol Cell Biol 2006: 26: 5518–5527. 8 Ishida W, Mori Y, Lakos G et al. J Invest Dermatol 2006: 126: 1733–1744. 9 Chen Y, Shi-wen X, Eastwood M et al. Arthritis Rheum 2006: 54: 1309–1316. 10 Gay S, Jones R E Jr, Huang G Q et al. J Invest Dermatol 1989: 92: 301–303. 11 Hemmatazad H, Rodrigues H M, Maurer B et al. Arthritis Rheum 2009: 60: 1519–1529.
100
assume that once triggered by external stimuli, the endogenous pro-fibrotic activity of SSc fibroblasts is the key driving factor in fibrosis. While the pro-fibrotic SSc fibroblasts release massive amounts of collagens to form scar tissue, they also maintain their phenotypic characteristics by intrinsic activity. The concept of the pro-fibrotic SSc fibroblast phenotype has major implications in the development of novel treatment strategies. While anti-inflammatory therapies may fail to modulate fibroblast activity in many clinical scenarios, novel therapies that deactivate the pro-fibrotic SSc fibroblast phenotype are most promising to block collagen release from fibroblasts and treat fibrosis in SSc (25,26). Assuming that LeRoy’s concept of intrinsic fibroblast activation in SSc may also hold true for other fibrotic conditions, fibroblasts may be the key targets of future antifibrotic therapies.
Author contributions CM and CB carried out literature research and data acquisition. CM, JD and CB drafted and revised the manuscript. CM, JD and CB approved the final version of the manuscript.
Ethical approval Not applicable.
Conflict of interests J.H.W. Distler has consultancy relationships and/or has received research funding from Actelion, Pfizer, Ergonex, BMS, Celgene, Bayer Pharma, JB Therapeutics, Sanofi-Aventis, Novartis, Array Biopharma and Active Biotec in the area of potential treatments of SSc and is stock owner of 4D Science. C. Maier and C. Beyer have no conflict of interests.
12 Dees C, Schlottmann I, Funke R et al. Ann Rheum Dis 2013 [Epub ahead of print]. 13 Akhmetshina A, Palumbo K, Dees C et al. Nat Commun 2012: 3: 735. 14 Bechtel W, McGoohan S, Zeisberg E M et al. Nat Med 2010: 16: 544–550. 15 Jungel A, Distler J H, Gay S et al. Expert Rev Clin Immunol 2011: 7: 475–480. 16 Vettori S, Gay S, Distler O. Open Rheumatol J 2012: 6: 130–139. 17 Maurer B, Stanczyk J, Jungel A et al. Arthritis Rheum 2010: 62: 1733–1743. 18 Makino K, Jinnin M, Hirano A et al. J Immunol 2013: 190: 3905–3915. 19 Honda N, Jinnin M, Kira-Etoh T et al. Am J Pathol 2013: 182: 206–216. 20 Honda N, Jinnin M, Kajihara I et al. J Immunol 2012: 188: 3323–3331. 21 Zhu H, Li Y, Qu S et al. J Clin Immunol 2012: 32: 514–522.
22 Wipff P J, Rifkin D B, Meister J J et al. J Cell Biol 2007: 179: 1311–1323. 23 Hinz B, Mastrangelo D, Iselin C E et al. Am J Pathol 2001: 159: 1009–1020. 24 Hinz B. Curr Rheumatol Rep 2009: 11: 120–126. 25 Beyer C, Distler J H. F1000 Med Rep 2009: 1, doi: 10.3410/M1-95. 26 Beyer C, Distler O, Distler J H. Curr Opin Rheumatol 2012: 24: 274–280.
Supporting Information Additional Supporting Information may be found in the online version of this article: Data S1. The editors and publisher gratefully acknowledge Rockefeller University Press for kindly having granted permission to reprint the original article discussed in this Commentary: Leroy E C. Connective tissue synthesis by scleroderma skin fibroblasts in cell culture. J Exp Med 1972: 135: 1351–1362. © 1972 Rockefeller University Press.
ª 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Experimental Dermatology, 2014, 23, 99–100
