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ARD Online First, published on January 14, 2015 as 10.1136/annrheumdis-2014-206214 Basic and translational research
EXTENDED REPORT
Activating transcription factor 3 regulates canonical TGFβ signalling in systemic sclerosis Tatjana Mallano,1 Katrin Palumbo-Zerr,1 Pawel Zerr,1 Andreas Ramming,1 Barbara Zeller,1 Christian Beyer,1 Clara Dees,1 Jingang Huang,1 Tsonwin Hai,2 Oliver Distler,3 Georg Schett,1 Jörg H W Distler1 Handling editor Tore K Kvien 1
Department of Internal Medicine 3, Institute for Clinical Immunology, University of Erlangen-Nuremberg, Erlangen, Germany 2 Department of Molecular and Cellular Biochemistry, The Ohio State University, Columbus, Ohio, USA 3 Rheumaklinik, University Hospital Zurich, Zurich, Switzerland Correspondence to Dr Jörg H W Distler, Department of Medicine 3, Institute for Clinical Immunology, University of Erlangen-Nuremberg, Ulmenweg 18, D-91054 Erlangen, Germany; [email protected] Received 4 July 2014 Revised 4 November 2014 Accepted 20 December 2014
ABSTRACT Background Activating transcription factor 3 (ATF3), a member of the ATF/cAMP-responsive element binding (CREB) family of transcription factors, regulates cellular response to stress including oxidative stress. The aim of this study was to analyse the role of ATF3 in fibroblast activation in systemic sclerosis (SSc). Methods ATF3 was analysed by reverse transcription quantitative PCR, western blot and immunohistochemistry. ATF3 knockout fibroblasts and mice were used to study the functional role of ATF3. Knockdown experiments, reporter assays and coimmunoprecipitation were performed to study the effects of ATF3 on Smad and activation protein 1 (AP-1) signalling. The role of c-Jun was analysed by costaining, specific inactivation and coimmunoprecipitation. Results Transforming growth factor-β (TGFβ) upregulates the expression of ATF3 in SSc fibroblasts. ATF3-deficient fibroblasts were less sensitive to TGFβ, whereas ectopic expression of ATF3 enhanced the profibrotic effects of TGFβ. Mechanistically, ATF3 interacts with Smad3 directly on stimulation with TGFβ and regulates Smad activity in a c-Jun-dependent manner. Knockout of ATF3 protected mice from bleomycin-induced fibrosis and fibrosis induced by overexpression of a constitutively active TGFβ receptor I. Reporter assays and analyses of the expression of Smad target genes demonstrated that binding of ATF3 regulates the transcriptional activity of Smad3. Conclusions We demonstrate for the first time a key role for ATF3 in fibrosis. Knockout of the ATF3 gene reduced the stimulatory effect of TGFβ on fibroblasts by interfering with canonical Smad signalling and protected the mice from experimental fibrosis in two different models. ATF3 might thus be a candidate for molecular targeted therapies for SSc.
INTRODUCTION
To cite: Mallano T, Palumbo-Zerr K, Zerr P, et al. Ann Rheum Dis Published Online First: [please include Day Month Year] doi:10.1136/ annrheumdis-2014-206214
Systemic sclerosis (SSc) is a chronic fibrotic connective tissue disease of unknown aetiology that affects the skin and internal organs. It is characterised by an aberrant activation of fibroblasts, resulting in excessive release of extracellular matrix.1 Accumulation of extracellular matrix proteins disturbs the physiological tissue function and causes high morbidity and mortality. Transforming growth factor-β (TGFβ) has been characterised as a key mediator of fibroblast activation in SSc and other fibrotic diseases.2 However, the intracellular signalling cascades that control TGFβ signalling
and the TGFβ-induced activation of fibroblasts are still incompletely understood. The family of activating transcription factor (ATF) consists of a large group of transcription factor members such as cAMP-responsive element binding protein (CREB), cAMP-response element modulator (CREM), B-ATF and ATF1–7.3 All family members can regulate gene expression via binding to consensus ATF/CREB cis-regulatory element via a basic-region leucine zipper domain (bZIP). The leucine zipper region is responsible for forming homodimers or heterodimers with other bZIP-containing proteins, while the basic region in this domain is responsible for specific DNA binding.4 Initially, ATF3 was suggested to regulate gene expression on the basis of the size of its isoforms— for example, the longer isoform is known to repress designated genes rather than to activate them.5 However, later investigations revealed that ATF3 can also form dimers with other ATF/CREB protein members such as ATF2,6 c-Jun, JunB7–9 and JunD.10–12 Depending on the promoter context, these heterodimers can act as either repressors or activators of transcription.13 This suggests a diverse repertoire of ATF3 functions depending on the cellular context and organs involved. Indeed, the expression of ATF3 is kept low in quiescent cells, while it can be rapidly induced by a wide variety of signals of cellular stress such as DNA damage, oxidative stress and cell injury,2 3 14 as well as by several cytokines such as interleukin-1β, tumour necrosis factor-α and TGFβ.13 15 Given the key role of oxidative stress in the pathogenesis of SSc,16 we thus aimed to study the role of ATF3 in fibroblast activation in SSc. Here, we demonstrate that TGFβ stimulates the expression of ATF3 in dermal fibroblasts and that ATF3 contributes to the profibrotic effects of TGFβ and to the formation of fibrosis in two mouse models. Significantly, ATF3 is elevated in the skin of patients with SSc, suggesting its relevance to human fibrosis.
MATERIALS AND METHODS Patients Dermal fibroblasts were isolated from skin biopsy specimens from 23 patients with SSc and 21 ageand sex-matched healthy volunteers. All patients fulfilled the American College of Rheumatology/ European League Against Rheumatism criteria from 2013.17 Sixteen patients were female, seven were
Mallano T, et al. Ann Rheum Dis 2015;0:1–7. doi:10.1136/annrheumdis-2014-206214
1
Copyright Article author (or their employer) 2015. Produced by BMJ Publishing Group Ltd (& EULAR) under licence.
Downloaded from http://ard.bmj.com/ on September 11, 2015 - Published by group.bmj.com
Basic and translational research male. The median age of patients with SSc was 45 years (range 19–65), and their median disease duration was 5 years (range 0.5–10). All patients and controls signed a consent form approved by the local institutional review board. Patients did not receive any potentially disease-modifying agents at the time of biopsy.
Reverse transcription coupled with quantitative PCR Gene expression was quantified by reverse transcription coupled with SYBR Green quantitative PCR (RT-qPCR) using the MX3005P Detection System (Agilent Technologies, Böblingen, Germany). Samples without enzyme in the reverse transcription reaction were used as negative controls. Unspecific signals caused by primer dimers were excluded by non-template controls and by dissociation curve analysis as described18–20 β-Actin was used to normalise for the amounts of cDNA within each sample.
efficiency was determined by cotransfection with vectors encoding β-galactosidase (Promega, Mannheim, Germany).
Animal studies The role of ATF3 in fibrosis was investigated in two different mouse models. (i) In the model of bleomycin-induced dermal fibrosis, fibrosis was induced by local injections of bleomycin for 4 weeks. Subcutaneous injections of 0.9% NaCl served as control. (ii) In the TGFβ receptor I (TBRI) model, injections of replication-deficient type 5 adenoviruses encoding a constitutively active TBRI construct induced localised skin fibrosis. Mice injected with type 5 adenoviruses encoding LacZ served as controls; 6.67×107 infectious units were injected intracutaneously. Fibrotic changes were analysed by quantification of the dermal thickness, myofibroblast counts and the hydroxyproline content as described previously.26 27 For direct visualisation of collagen, trichrome staining was performed.
Western blot analysis The protein concentration of cell lysates was determined by amido black assays. Proteins were separated by sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a poly(vinylidene difluoride) membrane. The membrane was incubated with antibodies against Smad3 (Santa Cruz Technologies, Heidelberg, Germany) or ATF3 (Santa Cruz Technologies) and horseradish peroxidase (HRP)-conjugated secondary antibodies (Dako, Hamburg, Germany). Blots were visualised by enhanced chemiluminescence and quantified by density analysis using ImageJ. β-Actin served as loading control.21 22
Quantification of collagen protein The amount of soluble collagen in cell culture supernatants was quantified using the SirCol collagen assay (Biocolor, Belfast, Northern Ireland).23 24 The total collagen content of tissue samples was determined by hydroxyproline assays.
Immunohistochemistry and immunofluorescence staining Formalin-fixed, paraffin-embedded skin sections or fibroblasts fixed in 4% paraformaldehyde and permeabilised with 0.25% Triton X-100 were stained with antibodies against prolyl-4hydroxylase β (Life Technologies), ATF3, vimentin (both Abcam) and c-Jun (Santa Cruz). HRP-conjugated or Alexa Fluor antibodies (Life Technologies) were used as secondary antibodies. Irrelevant isotype matched antibodies served as controls. Nuclei were counterstained using 4’,6-diamidino-2-phenylindole (Santa Cruz Biotechnology). Staining was analysed using a Nikon Eclipse 80i microscope (Nikon, Badhoevedorp, The Netherlands).
Coimmunoprecipitation (CoIP) Fibroblasts were collected in 150 mM NaCl lysis buffer. Twenty per cent of the lysates was used as loading control (input). Cell extracts were incubated with 20 μL protein A/G Sepharose and antibodies against Smad3, ATF3, c-Jun or IgG control immunoglobulins (Santa Cruz Biotechnology). The amounts of the respective proteins of interest in the immunoprecipitate were analysed by western blotting.
Nucleofection Fibroblasts were transfected with 10 μg pCMV-ATF3 or pCMV plasmid constructs in the absence or presence of the luciferase reporter driven by the CAGA Smad element using the Amaxa 4D-Nucleofector (Amaxa, Cologne, Germany).2 25 Transfection 2
Statistical analysis All data are presented as mean with SEM, and differences between the groups were tested for their statistical significance by the non-parametric Mann–Whitney U test. p Values
ARD Online First, published on January 14, 2015 as 10.1136/annrheumdis-2014-206214 Basic and translational research
EXTENDED REPORT
Activating transcription factor 3 regulates canonical TGFβ signalling in systemic sclerosis Tatjana Mallano,1 Katrin Palumbo-Zerr,1 Pawel Zerr,1 Andreas Ramming,1 Barbara Zeller,1 Christian Beyer,1 Clara Dees,1 Jingang Huang,1 Tsonwin Hai,2 Oliver Distler,3 Georg Schett,1 Jörg H W Distler1 Handling editor Tore K Kvien 1
Department of Internal Medicine 3, Institute for Clinical Immunology, University of Erlangen-Nuremberg, Erlangen, Germany 2 Department of Molecular and Cellular Biochemistry, The Ohio State University, Columbus, Ohio, USA 3 Rheumaklinik, University Hospital Zurich, Zurich, Switzerland Correspondence to Dr Jörg H W Distler, Department of Medicine 3, Institute for Clinical Immunology, University of Erlangen-Nuremberg, Ulmenweg 18, D-91054 Erlangen, Germany; [email protected] Received 4 July 2014 Revised 4 November 2014 Accepted 20 December 2014
ABSTRACT Background Activating transcription factor 3 (ATF3), a member of the ATF/cAMP-responsive element binding (CREB) family of transcription factors, regulates cellular response to stress including oxidative stress. The aim of this study was to analyse the role of ATF3 in fibroblast activation in systemic sclerosis (SSc). Methods ATF3 was analysed by reverse transcription quantitative PCR, western blot and immunohistochemistry. ATF3 knockout fibroblasts and mice were used to study the functional role of ATF3. Knockdown experiments, reporter assays and coimmunoprecipitation were performed to study the effects of ATF3 on Smad and activation protein 1 (AP-1) signalling. The role of c-Jun was analysed by costaining, specific inactivation and coimmunoprecipitation. Results Transforming growth factor-β (TGFβ) upregulates the expression of ATF3 in SSc fibroblasts. ATF3-deficient fibroblasts were less sensitive to TGFβ, whereas ectopic expression of ATF3 enhanced the profibrotic effects of TGFβ. Mechanistically, ATF3 interacts with Smad3 directly on stimulation with TGFβ and regulates Smad activity in a c-Jun-dependent manner. Knockout of ATF3 protected mice from bleomycin-induced fibrosis and fibrosis induced by overexpression of a constitutively active TGFβ receptor I. Reporter assays and analyses of the expression of Smad target genes demonstrated that binding of ATF3 regulates the transcriptional activity of Smad3. Conclusions We demonstrate for the first time a key role for ATF3 in fibrosis. Knockout of the ATF3 gene reduced the stimulatory effect of TGFβ on fibroblasts by interfering with canonical Smad signalling and protected the mice from experimental fibrosis in two different models. ATF3 might thus be a candidate for molecular targeted therapies for SSc.
INTRODUCTION
To cite: Mallano T, Palumbo-Zerr K, Zerr P, et al. Ann Rheum Dis Published Online First: [please include Day Month Year] doi:10.1136/ annrheumdis-2014-206214
Systemic sclerosis (SSc) is a chronic fibrotic connective tissue disease of unknown aetiology that affects the skin and internal organs. It is characterised by an aberrant activation of fibroblasts, resulting in excessive release of extracellular matrix.1 Accumulation of extracellular matrix proteins disturbs the physiological tissue function and causes high morbidity and mortality. Transforming growth factor-β (TGFβ) has been characterised as a key mediator of fibroblast activation in SSc and other fibrotic diseases.2 However, the intracellular signalling cascades that control TGFβ signalling
and the TGFβ-induced activation of fibroblasts are still incompletely understood. The family of activating transcription factor (ATF) consists of a large group of transcription factor members such as cAMP-responsive element binding protein (CREB), cAMP-response element modulator (CREM), B-ATF and ATF1–7.3 All family members can regulate gene expression via binding to consensus ATF/CREB cis-regulatory element via a basic-region leucine zipper domain (bZIP). The leucine zipper region is responsible for forming homodimers or heterodimers with other bZIP-containing proteins, while the basic region in this domain is responsible for specific DNA binding.4 Initially, ATF3 was suggested to regulate gene expression on the basis of the size of its isoforms— for example, the longer isoform is known to repress designated genes rather than to activate them.5 However, later investigations revealed that ATF3 can also form dimers with other ATF/CREB protein members such as ATF2,6 c-Jun, JunB7–9 and JunD.10–12 Depending on the promoter context, these heterodimers can act as either repressors or activators of transcription.13 This suggests a diverse repertoire of ATF3 functions depending on the cellular context and organs involved. Indeed, the expression of ATF3 is kept low in quiescent cells, while it can be rapidly induced by a wide variety of signals of cellular stress such as DNA damage, oxidative stress and cell injury,2 3 14 as well as by several cytokines such as interleukin-1β, tumour necrosis factor-α and TGFβ.13 15 Given the key role of oxidative stress in the pathogenesis of SSc,16 we thus aimed to study the role of ATF3 in fibroblast activation in SSc. Here, we demonstrate that TGFβ stimulates the expression of ATF3 in dermal fibroblasts and that ATF3 contributes to the profibrotic effects of TGFβ and to the formation of fibrosis in two mouse models. Significantly, ATF3 is elevated in the skin of patients with SSc, suggesting its relevance to human fibrosis.
MATERIALS AND METHODS Patients Dermal fibroblasts were isolated from skin biopsy specimens from 23 patients with SSc and 21 ageand sex-matched healthy volunteers. All patients fulfilled the American College of Rheumatology/ European League Against Rheumatism criteria from 2013.17 Sixteen patients were female, seven were
Mallano T, et al. Ann Rheum Dis 2015;0:1–7. doi:10.1136/annrheumdis-2014-206214
1
Copyright Article author (or their employer) 2015. Produced by BMJ Publishing Group Ltd (& EULAR) under licence.
Downloaded from http://ard.bmj.com/ on September 11, 2015 - Published by group.bmj.com
Basic and translational research male. The median age of patients with SSc was 45 years (range 19–65), and their median disease duration was 5 years (range 0.5–10). All patients and controls signed a consent form approved by the local institutional review board. Patients did not receive any potentially disease-modifying agents at the time of biopsy.
Reverse transcription coupled with quantitative PCR Gene expression was quantified by reverse transcription coupled with SYBR Green quantitative PCR (RT-qPCR) using the MX3005P Detection System (Agilent Technologies, Böblingen, Germany). Samples without enzyme in the reverse transcription reaction were used as negative controls. Unspecific signals caused by primer dimers were excluded by non-template controls and by dissociation curve analysis as described18–20 β-Actin was used to normalise for the amounts of cDNA within each sample.
efficiency was determined by cotransfection with vectors encoding β-galactosidase (Promega, Mannheim, Germany).
Animal studies The role of ATF3 in fibrosis was investigated in two different mouse models. (i) In the model of bleomycin-induced dermal fibrosis, fibrosis was induced by local injections of bleomycin for 4 weeks. Subcutaneous injections of 0.9% NaCl served as control. (ii) In the TGFβ receptor I (TBRI) model, injections of replication-deficient type 5 adenoviruses encoding a constitutively active TBRI construct induced localised skin fibrosis. Mice injected with type 5 adenoviruses encoding LacZ served as controls; 6.67×107 infectious units were injected intracutaneously. Fibrotic changes were analysed by quantification of the dermal thickness, myofibroblast counts and the hydroxyproline content as described previously.26 27 For direct visualisation of collagen, trichrome staining was performed.
Western blot analysis The protein concentration of cell lysates was determined by amido black assays. Proteins were separated by sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a poly(vinylidene difluoride) membrane. The membrane was incubated with antibodies against Smad3 (Santa Cruz Technologies, Heidelberg, Germany) or ATF3 (Santa Cruz Technologies) and horseradish peroxidase (HRP)-conjugated secondary antibodies (Dako, Hamburg, Germany). Blots were visualised by enhanced chemiluminescence and quantified by density analysis using ImageJ. β-Actin served as loading control.21 22
Quantification of collagen protein The amount of soluble collagen in cell culture supernatants was quantified using the SirCol collagen assay (Biocolor, Belfast, Northern Ireland).23 24 The total collagen content of tissue samples was determined by hydroxyproline assays.
Immunohistochemistry and immunofluorescence staining Formalin-fixed, paraffin-embedded skin sections or fibroblasts fixed in 4% paraformaldehyde and permeabilised with 0.25% Triton X-100 were stained with antibodies against prolyl-4hydroxylase β (Life Technologies), ATF3, vimentin (both Abcam) and c-Jun (Santa Cruz). HRP-conjugated or Alexa Fluor antibodies (Life Technologies) were used as secondary antibodies. Irrelevant isotype matched antibodies served as controls. Nuclei were counterstained using 4’,6-diamidino-2-phenylindole (Santa Cruz Biotechnology). Staining was analysed using a Nikon Eclipse 80i microscope (Nikon, Badhoevedorp, The Netherlands).
Coimmunoprecipitation (CoIP) Fibroblasts were collected in 150 mM NaCl lysis buffer. Twenty per cent of the lysates was used as loading control (input). Cell extracts were incubated with 20 μL protein A/G Sepharose and antibodies against Smad3, ATF3, c-Jun or IgG control immunoglobulins (Santa Cruz Biotechnology). The amounts of the respective proteins of interest in the immunoprecipitate were analysed by western blotting.
Nucleofection Fibroblasts were transfected with 10 μg pCMV-ATF3 or pCMV plasmid constructs in the absence or presence of the luciferase reporter driven by the CAGA Smad element using the Amaxa 4D-Nucleofector (Amaxa, Cologne, Germany).2 25 Transfection 2
Statistical analysis All data are presented as mean with SEM, and differences between the groups were tested for their statistical significance by the non-parametric Mann–Whitney U test. p Values
