Neue Regulatorische Effekte Von GLI2 Und HHAT Auf Die Aktivierung Der Hedgehog Signalkaskade in Der Systemischen Sklerose Und Ch

Neue regulatorische Effekte von GLI2 und HHAT auf die Aktivierung der Hedgehog Signalkaskade in

der systemischen Sklerose und Charakterisierung

der Rolle des Orphan Nuclear Receptors RORα in

der Pathogenese fibrotischer Erkrankungen

Der Naturwissenschaftlichen Fakultät

der Friedrich-Alexander-Universität Erlangen-Nürnberg

zur

Erlangung des Doktorgrades Dr. rer. nat.

vorgelegt von

Ruifang Liang

2019

Als Dissertation genehmigt von der Naturwissenschaftlichen Fakultät der

Friedrich-Alexander-Universität Erlangen-Nürnberg

Tag der mündlichen Prüfung: 13th Dec 2019

Vorsitzender des Promotionsorgans: Prof. Dr. Georg Kreimer

Gutachter/in: Prof. Dr. rer. nat. Michael Stürzl

Prof. Dr. Steffen Backert

Novel Insights into Hedgehog Signaling in

Systemic Sclerosis at the level of GLI2 and HHAT

and

Characterization of the Role of the Orphan Nuclear

Receptor RORα in Fibroblast Activation and

Tissue Fibrosis

To the Faculty of Natural Sciences of

Friedrich-Alexander-University Erlangen-Nuremberg

for

the obtainment of the academic degree

doctor rerum naturalium (Dr. rer. nat.)

submitted by

Ruifang Liang

2019

Approved by the Faculty of Natural Sciences of

Friedrich-Alexander-University Erlangen-Nuremberg

Date of oral examination: 13th Dec 2019

Chairman of examination board: Prof. Dr. Georg Kreimer

Referee: Prof. Dr. rer. nat. Michael Stürzl

Prof. Dr. Steffen Backert

Index of contents

Zusammenfassung ...... 1

Summary ...... 3

1 Introduction ...... 5

1.1 Systemic sclerosis ...... 5

1.1.1 Clinical overview (Epidemiology and classification)...... 5

1.1.2 Pathogenesis ...... 6

1.2 TGF signaling – key mediator of fibrosis ...... 14

1.2.1 TGF superfamily ...... 15

1.2.2 TGF intracellular signaling ...... 15

1.2.3 TGF signaling in fibrotic responses ...... 16

1.3 Hedgehog pathway ...... 17

1.3.1 Canonical Hedgehog signaling ...... 18

1.3.2 Non-Canonical Hedgehog signaling ...... 19

1.3.3 Hedgehog in fibrosis and cross talk with TGFβ signaling ...... 20

1.3.4 GLI proteins ...... 20

1.3.5 Skinny hedgehog ...... 21

1.4 Nuclear receptors ...... 22

1.4.1 ROR family ...... 25

1.4.2 Physiological Functions of RORα ...... 27

1.5 Animal models of Systemic sclerosis ...... 30

1.5.1 Bleomycin-induced skin fibrosis ...... 30

1.5.2 Fibrosis induced by overexpression of a constitutively active TGFβ receptor type 1 (TBRIact) ...... 31

1.5.3 DNA Topoisomerase I-induced fibrosis ...... 31

1.5.4 Bleomycin-induced pulmonary fibrosis ...... 32

1.6 Aims of the study ...... 33

2 Materials and Methods ...... 35

2.1 Material ...... 35

2.1.1 Chemicals ...... 35

2.1.2 Auxiliary materials ...... 39

2.1.3 Instruments ...... 40

2.1.4 Commercially available systems (kits) ...... 42

2.1.5 Antibodies ...... 43

2.1.6 Primers ...... 44

2.1.7 Media, buffers and solutions ...... 47

2.2 Methods ...... 51

2.2.1 Human skin biopsies...... 51

2.2.2 Cell culture ...... 51

2.2.3 Nucleofection ...... 53

2.2.4 FuGENE HD Plasmids Transfection ...... 53

2.2.5 Plasmids ...... 54

2.2.6 Plasmid isolation (Maxi Prep) ...... 54

2.2.7 Side-directed mutagenesis ...... 55

2.2.8 Chromatin immunoprecipitation ...... 55

2.2.9 Luciferase-repoter assay ...... 56

2.2.10 Coculture systems ...... 56

2.2.11 Mice ...... 57

2.2.12 RNA Analysis ...... 60

2.2.13 Histological Analysis...... 60

2.2.14 Protein analysis ...... 61

2.2.15 Amplification and purification of adenoviral vectors ...... 64

2.2.16 Statistical Analysis ...... 65

3 Results ...... 66

3.1 Evaluation of the HH transcription factor GLI2 in SSc ...... 66

3.1.1 Upregulated expression of GLI2 in fibrotic conditions ...... 66

3.1.2 GLI2 is upregulated by TGFβ in a Smad-dependent manner ...... 67

3.1.3 GLI2 regulates TGFβ induced fibroblast activation ...... 73

3.1.4 Fibroblast-specific knockout of Gli2 prevents TBRact-induced fibrosis ...... 75

3.1.5 Pharmacological inhibition of GLI2 inhibits TGF-β-dependent fibroblast activation .. 77

3.1.6 Pharmacological inhibition of Gli-2 ameliorates TBRact -induced fibrosis ...... 78

3.1.7 Inactivation of Gli2 induces regression of pre-established bleomycin-induced pulmonary fibrosis 79

3.2 The Hedgehog aceyltransferase Hhat regulates canonical TGFβ-dependent fibroblast activation in SSc ...... 80

3.2.1 HHAT is upregulated in activated fibroblasts in fibrotic skin ...... 80

3.2.2 HHAT expression is induced by canonical TGF signaling ...... 84

3.2.3 HHAT promotes TGF-dependent fibroblast-to-myofibroblast-differentiation ...... 86

3.2.4 Knockdown of Hhat ameliorates experimental fibrosis ...... 89

3.3 The orphan Nuclear Receptor Rorα is a Key Regulator of TGF- and WNT- Signaling in Fibrosis ...... 91

3.3.1 RORα expression pattern in fibrotic conditions ...... 91

3.3.2 Canonical Wnt signaling induces RORα expression in fibroblasts ...... 92

3.3.3 Inactivation of RORα reduces the stimulatory effects of TGFinduces fibroblast activation ...... 95

3.3.4 Fibroblast specific knockout Rorα mice are protected from experimental fibrosis ...... 98

3.3.5 Pharmacological inhibition of Rorα ameliorates experimental fibrosis ...... 99

4 Discussion ...... 104

4.1 Evaluation of the HH transcription factor GLI2 in SSc ...... 104

4.2 The Hedgehog aceyltransferase Hhat regulates canonical TGFβ-dependent fibroblast activation in SSc ...... 106

4.3 Orphan Nuclear Receptor Rorα Is a Key Regulator of TGF- and WNT- Signaling in Fibrosis ...... 107

5 Conclusion ...... 110

6 References...... 111

7 Abbreviations ...... 124

8 Acknowledgement ...... Fehler! Textmarke nicht definiert.

9 Curriculum vitae ...... Fehler! Textmarke nicht definiert.

Zusammenfassung

Die Systemische Sklerose (SSc) ist eine prototypische systemische fibrosierende Erkrankung mit unklarer Ätiologie, welche durch eine persistierende Aktivierung von Fibroblasten charakterisiert ist. Die Krankheit ist durch eine ausgedehnte Fibrose der Haut und der inneren Organe, durch Gefäßanomalien und Immundysfunktion gekennzeichnet. Obwohl einzelne Schlüsselfaktoren der Fibroblastenaktivierung, wie beispielsweise Transforming Growth Factor β (TGFβ) oder die kanonische Hedgehog Signalkaskade, identifiziert werden konnten, sind die Auswirkungen einer gleichzeitigen Aktivierung mehrerer pro-fibrotischer Signalwege unbekannt. Ebenso sind die Verbindungen zwischen den individuellen Signalwegen in fibrotischen Erkrankungen bisher kaum charakterisiert. Die gegenseitige Verstärkung pro- fibrotischer Signale könnte jedoch für die persistierende Aktivierung von zentraler Bedeutung sein. Darüber hinaus könnte die Identifizierung von Amplifikationsschleifen zwischen pro- fibrotischen Signalwegen neue Zielmoleküle für gerichtete Therapien hervorbringen, welche die kombinierte Hemmung verschiedener fibrotischer Signalwege ermöglicht. Im ersten Teil dieser Studie wurde die Rolle des nicht-kanonischen Hedgehog-Signalweges in der SSc evaluiert und die Effektivität einer direkten Inhibition von GLI getestet, welche gleichzeitig die kanonische und die nicht-kanonische Hedgehog Signalkaskade hemmt. Unsere Daten zeigen, dass GLI2 Smad3-abhängig durch TGFβ hochreguliert wird und die nukleäre Akkumulation und die Bindung von GLI2 an DNA induziert. Ein Knockout von GLI2 in Fibroblasten schützte Mäuse vor Fibrose, welche durch adenovirale Expression eines konstitutiv-aktiven TGFβ-Rezeptors I (TBRIact) induziert wurde. In experimenteller dermaler Fibrose zeigte eine kombinierte Blockade des kanonischen und des nicht-kanonischen Hedgehog Signalweges mit GLI Inhibitoren stärkere anti-fibrotische Effekte als eine selektive Blockade des kanonischen Hedgehog Signalweges mit SMO Inhibitoren. Darüber hinaus war die Inhibition von GLI auch bei vorbestehender Bleomycin-induzierter Lungenfibrose wirksam. Im zweiten Teil der Arbeit untersuchten wir die Rolle der Hedgehog Acyltransferase „Skinny Hedgehog“ (HHAT) in der Pathogenese der SSc. Wir konnten zeigen, dass HHAT in der SSc durch TGFβ reguliert wird und wiederum den TGFβ-induzierten endokrinen Hedgehog Signalweg stimuliert. Hierdurch fördert es die Aktivierung von Fibroblasten und die Fibroseentstehung. Eine Inaktivierung von HHAT reduzierte die durch TGFβ mediierte Aktivierung von Fibroblasten, die Produktion von Kollagen und die Expression von TGFβ-

Zusammenfassung Zielgenen. Außerdem hemmte der Knockdown von HHAT mittels siRNA die Fibrosierung der Haut in verschiedenen Mausmodellen. Der dritte Teil der Arbeit charakterisiert eine mögliche Beteiligung von Retinoic-acid related Orphan Receptor-alpha (RORα) an der pathologischen Aktivierung von Fibroblasten in SSc Patienten und evaluiert das anti-fibrotische Potential einer Inhibition von RORα.Wir konnten zeigen, dass RORα sowohl in humaner als auch muriner fibrotischer Lunge und Leber hochreguliert ist. Eine Aktivierung des kanonischen WNT/β-catenin Signalweges ahmte diesen Anstieg von RORα in Fibrose nach und induzierte dessen Expression. Eine Inhibition von RORα durch Inkubation mit SR3335 blockierte sowohl die WNT- als auch die TGFβ-abhängige Myofibroblastendifferenzierung und reduzierte effektiv die Abgabe von Kollagen. Reporter Assays zeigten, dass SR3335 die Aktivität des TGFβ/SMAD Signalweges inhibiert. Ein Knockout von RORα reduzierte die stimulatorischen Effekte von TGFβ und WNT auf die Fibroblastenaktivierung und die Freisetzung von Kollagen. Außerdem schützte ein Fibroblasten-spezifischer Knockout von RORα vor experimenteller Fibrose. Darüber hinaus reduzierte eine Behandlung mit SR3335 effektiv Fibrose in Wnt10b transgenen Mäusen. Des Weiteren verbesserte die pharmakologische Blockade von RORα das Ausmaß der Fibrose in verschiedenen murinen Modellen der Haut-, Lungen- und Leberfibrose. Diese Erkenntnisse könnten translationale Implikationen haben. Nicht-selektive Inhibitoren von GLI2 sind bereits im klinischen Gebrauch und selektive Substanzen sind aktuell in der Entwicklung. Eine zielgerichtete Therapie gegen HHAT könnte ein neuer Ansatz sein, selektiv die pro-fibrotischen Effekte des endokrinen Hedgehog Signalweges anzugreifen. Eine Intervention gegen RORα hemmt simultan TGFβ- und WNT-Signalwege an, welche zwei zentralen Signalwege in der Pathogenese fibrotischer Erkrankungen darstellen. Die Inhibition dieser zentralen Signalwege resultiert in starken anti-fibrotischen Effekten in verschiedenen Modellen und Organsystemen.

Summary Summary

Systemic sclerosis (SSc) is a prototypical systemic fibrotic disease of unknown etiology, which is characterized by aberrant activation of resident fibroblasts that display a persistently activated phenotype. The disease is characterized by widespread fibrosis of the skin and internal organs, vascular abnormalities and immune disturbances. Although individual key mediators of fibroblast activation such as transforming growth factor-β (TGF-β) or canonical hedgehog signaling have been identified, the consequences of the concomitant upregulation of multiple profibrotic pathways are unknown and crosstalk between individual pathways in fibrotic diseases is currently poorly characterized. However, mutual activation and amplification of profibrotic signals might be central for the persistent activation of fibroblasts. Moreover, identification of amplification loops between profibrotic pathways may yield novel candidate molecules for targeted therapies that allow for combined inhibition of different fibrotic pathways. The purpose of the first part of the study was to evaluate the role of non-canonical hedgehog signaling in SSc and to test the efficacy of direct GLI inhibitors that target simultaneously canonical and non-canonical hedgehog pathways. GLI2 is upregulated by TGFβ in a Smad3- dependent manner and induced nuclear accumulation and DNA binding of GLI2. Fibroblast- specific knockout of GLI2 protected mice from fibrosis induced by adenoviral expression of constitutively active TGFβ receptor type I (TBRIact). Combined targeting of canonical and non- canonical hedgehog signaling with direct GLI inhibitors exerted more potent ant-fibrotic effects than selective targeting of canonical hedgehog signaling with SMO inhibitors in experimental dermal fibrosis. Furthermore, targeting of GLI was also effective when initiated after the onset of bleomycin-induced pulmonary fibrosis. In the second part of this thesis, we investigated the role of the hedgehog acyltransferase skinny hedgehog (HHAT) in the pathogenesis of SSc. HHAT is regulated in SSc in a TGFβ-dependent manner and in turn stimulates TGFβ-induced endocrine hedgehog signaling to promote fibroblast activation and tissue fibrosis. Inactivation of HHAT reduced the TGFβ mediated activation of fibroblasts and production of collagens and target genes. Moreover, siRNA knockdown of HHAT ameliorated fibrosis in murine dermal fibrosis models. The third part of the study characterizes whether Retinoic-acid related Orphan Receptor-alpha (RORα) contributes to the pathologic activation of fibroblasts in patients with SSc and evaluates the anti-fibrotic potential of RORα inhibition. We observed that the expression of RORα was upregulated in fibroblasts in both human and murine fibrotic lung and liver. Activation of 3

Summary canonical WNT/β-catenin signaling mimicked the increase of RORα in fibrosis and potently induced RORα expression. Inhibition of RORα signaling by incubation with SR3335 inhibited WNT and TGFβ-dependent myofibroblast differentiation and effectively reduced the release of collagen. Reporter assay demonstrated that SR3335 dramatically inhibited TGFβ/SMAD signaling pathway activity. Knockout of RORα reduced the stimulatory effects of TGFβ and WNT on fibroblast activation and collagen release. In addition, fibroblast specific knockout of RORα in vivo protected from experimental fibrosis. Furthermore, treatment with SR3335 effectively reduced fibrosis in Wnt10b transgenic mice. In addition, pharmacological inhibition of RORα ameliorated fibrosis in several murine models of dermal, pulmonary and hepatic fibrosis. These findings may have translational implications as non-selective inhibitors of GLI2 are in clinical use and selective molecules are currently in development. Targeting of HHAT might be a novel approach to more selectively interfere with the profibrotic effects of endocrine hedgehog signaling. Targeting of RORα simultaneously interferes with TGFβ- and WNT signaling as two core pathways in the pathogenesis of fibrotic diseases. The inhibition of those core pathways translates into potent antifibrotic effects across different models and organ systems.

Introduction

1 Introduction

1.1 Systemic sclerosis

1.1.1 Clinical overview (Epidemiology and classification)

Systemic sclerosis (SSc), also known as scleroderma, is a heterogeneous autoimmune and connective tissue disease of unknown etiology. The term is from the Greek words "sklerosis" meaning "hardness" and "derma" meaning "skin and refers to the most obvious sign of the disease, the fibrosing and hardening of the skin. However, fibrosis of the skin is not the only hallmark of the systemic sclerosis as it includes also systemic manifestations such as autoimmunity and organ fibrosis. Systemic sclerosis is characterized by vascular impairment followed by defective neovascularization and remodeling, aberrant immune activation, and extensive tissue fibrosis of the skin and various internal organs such as lung, kidney, etc. (Armando Gabrielli 2009, van den Hoogen et al. 2013). The prevalence rates of scleroderma-like conditions range from 7 to 489 cases per million individuals. Incidence figures for SSc are 0.6 to 122 per million persons per year; the actual prevalence is probably at the high end of the range noted above. There are regional differences in incidence. Incidence rates and prevalence estimates are fairly similar for Europe, the United States, Australia, and Argentina suggesting a prevalence of 150–300 cases per million with a lower prevalence noted in Scandinavia, Japan, the UK, Taiwan, and India (Barnes and Mayes 2012).Age, sex, ethnicity, and genetic factors all influence the development of the disease. For example, higher rates are seen in the United States and Australia than in Japan or Europe, and in blacks than whites (Chifflot et al. 2008, Gelber et al. 2013). Studies revealed a higher susceptibility to scleroderma 3 to 5 times more frequent for women than men and the age at diagnosis ranges from 30 to 50 years, though the disease could also occur in children and elderly people (Mayes et al. 2003). The incidence of scleroderma in China is the third of connective tissue diseases, after rheumatoid arthritis and systemic lupus erythematosus (China Public Health Network 2016). Depending on the extent of skin involvement, SSc can be grouped into limited cutaneous systemic sclerosis (lcSSc) and diffuse cutaneous systemic sclerosis (dcSSc) subsets. There are several differences between these two main subsets (Varga and Abraham 2007). In limited cutaneous SSc, skin fibrosis is mainly restricted to the hands (sclerodactyly), face and the 5

Introduction extremities distal to the elbows and knees. Raynaud’s phenomenon is present for several months to years before fibrosis appears, pulmonary hypertension is frequent, and anticentromere antibodies occur in 50 to 90% of patients. In contrast, in diffuse cutaneous sclerosis, skin thickening is confined to face, the proximal extremities including the trunk and associated with more severe internal organ complications (Armando Gabrielli 2009, Asano 2018). dcSSc is more often connect with either topoisomerase I- or RNA polymerase III-specific antibodies (Steen 2005).

1.1.2 Pathogenesis

Although the etiology of SSc still remains ambiguous, there is consensus that conjugation of genetic factors and environmental influences will affect the development risk of SSc (Angiolilli et al. 2018, Salazar and Mayes 2015). There are three main characteristics of the pathogenesis of SSc, which are microangiopathy, inflammation and autoimmunity and extensive fibrosis of the skin and visceral organ fibrosis (Varga and Abraham 2007, Asano 2018).

Vasculopathy

Clinical symptoms and histological data suggest that endothelial damage and vascular injury are the earliest, and possibly primary, pathogenic events in systemic sclerosis (Trojanowska 2010, Varga and Abraham 2007). The structural and functional abnormalities of SSc vasculature are caused by complex interaction of various pathological processes. It is generally accepted that initial vascular changes are caused by aberrant activation of the immune system (Wick et al. 2013, Kahaleh 2004, Asano 2018). γδT cells have been reported as one potential mediator of immune-mediated damage of the endothelium (Giacomelli et al. 1998). In SSc patients, the majority of γδT cells in the peripheral circulation, skin, and bronchoalveolar lavage fluid are positive for Vδ1+ (Giacomelli et al. 1998). These peripheral Vδ1+ γδT cells crosstalk with endothelial cells by expression of activation marker CD49d, which interact with vascular cell adhesion molecule-1 (VCAM-1) to further promote the adhesion of these cells to endothelial cells (Giacomelli et al. 1998, Kahaleh, Fan Ps Fau - Otsuka and Otsuka 1999). Another mechanism that maybe involved in initial endothelial cell damage is anti-endothelial cell antibodies (AECAs). AECAs induce apoptosis of endothelial cells through antibody-dependent natural killer cell mediated cytotoxicity (ADCC) via the Fas pathway (Ahmed et al. 2006, Sgonc et al. 2000).

Introduction The autoimmune vascular attacks on endothelial cells result in structural abnormalities characteristic of SSc. The histological features of SSc structural abnormality include the dilation of capillaries, decrease in the number of small vessels, and stenosis of arterioles and small arteries. Structural abnormalities are classified into destructive vasculopathy and proliferative obliterative vasculopathy, which are due to impaired neovascularization and hypertrophic vascular remodelling, respectively. The processes of vascular remodeling and neovascularization consist of vasculogenesis and angiogenesis. Accumulating evidence strongly implicated that both of vasculogenesis and angiogenesis are remarkably impaired in SSc, leading to the development of vascular structural changes, such as loss of small vessels, dilation of capillaries, and stenosis of arterioles and small arteries (Distler, Gay and Distler 2006, Rabquer and Koch 2012). Vascular activation and tissue hypoxia caused by vascular structural changes promote tissue fibrosis. The term vasculogenesis represents the de novo formation of a blood vessel via differentiation of endothelial cells and pericytes/vascular smooth muscle cells, through the recruitment and differentiation of endothelial progenitor cells (EPCs). Embryonic vasculogenesis and postnatal vasculogenesis have been classified as two distinct vasculogenesis categories. The latter one is the vascular remodeling mediated by bone marrow derived EPCs to ischemic tissues in order to initiate and restore vascular supply which occurs throughout adult life (Fischer, Schneider M Fau - Carmeliet and Carmeliet 2006). Avouac et al. (Avouac et al. 2011) have reported dramatically reduced numbers of circulating EPCs in SSc, particularly in the late stages of the disease. Moreover, aberrant activation of monocytic pro-angiogenic hematopoietic cells (PHCs), one type of Circulating EPC, has been observed in SSc patients and these cells are preferentially differentiated into fibroblast-like cells (Yamaguchi et al. 2010, Yamaguchi and Kuwana 2013). Furthermore, the markedly decreased expression of CCN1, which regulates the recruitment of EPCs in dermal small vessels, is also reported in SSc (Saigusa et al. 2015b). Therefore, the decreased number, dysfunction and/or impaired recruitment of circulating EPC are the potential factors underlying inadequate vascular repair. The vascular density in the skin of SSc patients is strongly reduced as compared to healthy individuals (Konttinen et al. 2003). Surprisingly, one of the most striking features of the disease is the well characterized both locally and systemically upregulation of potent angiogenic factors such as vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), PDGF, placental growth factor (PIGF) and IL-8/CXCL8 in the context of diminished angiogenesis (Manetti et al. 2010, Distler et al. 2002). Interestingly, the upregulation of many antiangiogenic mediators (e.g., angiostatin, endostatin, angiopoietin 2, thrombospondin) has

Introduction also been observed in SSc patients. Presumably, the balance in the ratio of these mediators may simply be tipped in favor of inhibition of angiogenesis and progressive vascular disease (Matucci-Cerinic, Kahaleh and Wigley 2013). The paradox is that many of these proangiogenic cytokines are also potent activators of vascular smooth muscle cells, which indicates that they correlate with progressive microvascular loss, proliferative vasculopathy and disease progression. Moreover, switch in SSc from proangiogenic to antiangiogenic VEGF165b isoforms may contribute to insufficient angiogenic response to chronic ischemia (Guiducci et al. 2011). Impaired compensatory vasculogenesis and angiogenesis leads to the loss of neovascularization (destructive vasculopathy) and the development of fibroproliferative vascular change (proliferative obliterative vasculopathy) (Asano 2018). Vascular activation and tissue hypoxia caused by vascular structural changes promote tissue fibrosis. Endothelial cell damage by autoimmune-mediated mechanisms alters the expression profiles of cell adhesion molecules, chemokines, cytokines and growth factors activate T and B lymphocytes and monocytes and promote their infiltration into the perivascular areas. The expression and function of cell adhesion molecules expressing on endothelial cells and inflammatory cells are the key regulator of the intensity and property of inflammation. Several studies have shown that cell adhesion molecules expressed on endothelial cells, including E- selectin, P-selectin, intercellular adhesion molecule (ICAM), junctional adhesion molecule (JAM), platelet endothelial cell adhesion molecule (PECAM), and vascular adhesion molecule (VCAM) are elevated in the serum of SSc patients in general or at least in certain SSc subtypes (Mostmans et al. 2017). High levels of adhesion molecules, especially ICAM-1 and GlyCAM- 1, to a less extent E-selectin and P-selectin, are expressed in skin of patients with early diffuse cutaneous SSc (Matsushita et al. 2006). The expression of a cluster of gene-encoding molecules, including key leukocyte adhesion molecules L-selectin, integrin α6 and selectin-P, targeting leukocytes to the endothelium is upregulated in the peripheral blood mononuclear cells from SSc patients (Tan et al. 2006). Therefore, the altered phenotype of endothelial cells in SSc fosters evasion of immune cells from the blood into the skin and other affected organs. The association between overexpression of adhesion molecules on endothelial cells with the pathological tissue fibrosis has been also studied in mouse model. Yoshizaki et al. (Yoshizaki et al. 2010) have shown that loss of L-selectin and/or ICAM-1 reduced Th2 and Th17 cell numbers in bronchoalveolar lavage fluid, whereas loss of P-selectin, E-selectin reduced Th1 cell infiltration (Figure 1) (Asano 2018).

Introduction Moreover, endothelial-to-mesenchymal transition (EndoMT) with transdifferentiation of endothelial cells into myofibroblasts has been implicated in the pathogenesis of SSc with emerging data from preclinical models (Hashimoto et al. 2010, Jimenez 2013, Jimenez and Piera-Velazquez 2016), but also on ex vivo samples from skin and lungs and SSc patients (Mendoza et al. 2016, Manetti et al. 2017). Moreover, damaged endothelial cells also promote the activation of platelets. Platelets can release several profibrotic mediators including PDGF, TGFβ and serotonin into the circulation that fibroblast activation. Moreover, clotting with formation of intravascular fibrin deposits can aggravate luminal narrowing and vessel obstruction (Lee P Fau - Norman et al. 1985, Marvi and Chung 2010, Truchetet et al. 2016).

Figure 1: Vascular structural changes in systemic sclerosis. Autoimmune attacks to endothelial cells result in vascular activation and/or vascular injury. Vascular activation promotes tissue fibrosis through the induction of T- helper (Th)2/Th17 cell infiltration, endothelial to mesenchymal transition and impaired coagulation/fibrinolysis system. Vascular injury results in vascular structural abnormalities, such as capillary loss and arteriolar stenosis, due to abnormally activated angiogenesis and defective vasculogenesis. Capillary loss (destructive vasculopathy) and arteriolar stenosis (proliferative obliterative vasculopathy) cause tissue hypoxia, contributing to the development of tissue fibrosis. Arteriolar stenosis also directly leads to pulmonary arterial hypertension, scleroderma renal crisis and digital ulcers. Figure adapted and modified from (Asano 2018).

Introduction

Inflammation and autoimmunity

Both the innate and adaptive immune systems play a role in the pathogenesis of SSc and impact on dysregulated fibroblast extracellular matrix deposition, hallmark of the disease in conjunction with fibroproliferative vasculopathy (Chizzolini et al. 2011). Inflammatory infiltrates from innate immunity in early during the pathogenesis of fibrosis mainly consist of monocytes/macrophages, mast cells and lymphoid cells including natural killer/natural killer T (NK/NKT) (Wick et al. 2013). Different macrophage subpopulations regulate the fibrogenesis, by inducing release of MMPs from stromal cells to indirectly mediate degradation of ECM (inflammatory M1) or stimulating production and stabilization of already deposited ECM components and supporting synthesis as well as secretion of new ECM molecules (patrolling M2) (Sica and Mantovani 2012, Wick et al. 2013). Mice deficient in mast cells are partially protected from to bleomycin-induced lung fibrosis(O'Brien-Ladner, Wesselius and Stechschulte 1993). Moreover, human mast cells increase human skin fibroblast proliferation, collagen synthesis and collagen gel contraction (Garbuzenko et al. 2002). NK and NKT cells display mainly antifibrotic characteristics. NKT cell–deficient mice develop more severe lung fibrosis upon bleomycin challenge (Kim et al. 2005). Several cellular players of the adaptive immune system are implicated in the pathogenesis of SSc. Studies on skin section from patients with early SSc showed that inflammatory cell infiltrates, i.p. T cells, is predominantly localized in a perivascular distribution (Kalogerou et al. 2005). An altered balance between Th1/Th2/Th17 cell subsets in response to tissue injury is a potential hypothesis driving inflammation in the early stages of disease (Th1 and Th17 predominant) and fibrosis in the later stages (Th2 predominant). A predominantly Th2 profile (IL-4, IL-5, and IL-13) has been revealed in skin-infiltrating T cells and peripheral blood T cells in SSc patients (Sakkas and Platsoucas 2004, Abraham and Varga 2005, Chizzolini et al. 2003). IL-13 may contribute to fibrosis via the induction of TGFβ production by macrophages, and it also induces fibrosis through TGF-β-independent mechanisms (Abraham and Varga 2005). In contrast, reduced levels of the Th1 cytokine IFN- that inhibits collagen production by fibroblasts was observed in SSc patients. Studies of peripheral-blood cells and bronchoalveolar lavage cells from SSc patients also revealed the presence of numerous IL-17-producing CD4+ T cells, and higher levels of IL-17 may be observed in earlier disease stages (Meloni et al. 2009). There are contradictory results for the role of T-regulatory cells (Tregs) in altering immune homeostasis in SSc (Fuschiotti 2016).

Introduction Autoimmunity is best exemplified by the presence of multiple autoantibodies, namely the anti– topoisomerase 1 (Scl-70), anticentromere, and anti–RNA polymerase III antibodies described above, which identify distinct clinical subsets (Arnett 2006). The presence of these autoantibodies in SSc implicates a significant role for B cells, the precursors of plasma cells that produce antibodies. An activated B-cell signature has been found in lesional skin and affected the lung tissues of patients with SSc, with upregulation of cell-surface expression of CD19 and CD21 (Lafyatis et al. 2007, Whitfield et al. 2003). Furthermore, recent studies have shown that B cells from SSc patients can induce contact-dependent human dermal fibroblasts activation and upregulation of type I collagen and that depletion of B cells in a mouse model of SSc led to reduced fibrosis (Francois et al. 2013, Hasegawa et al. 2006).

Fibrosis

Vascular damage and tissue fibrosis are widespread in SSc and largely account for the protean clinical manifestations and substantial morbidity and mortality. The most discussed feature in the pathology of SSc is cutaneous fibrosis; the fibrosis of visceral organs in SSc manifests as pulmonary fibrosis, gastrointestinal dysmotility and malabsorption, and impaired cardiac function. Functional impairment of lung is the most common cause of death in patients with SSc (Bhattacharyya, Wei and Varga 2011). The fundamental mechanisms regulating excessive fibrosis in SSc are unknown. It is believed that vascular instability and immune perturbations precede and cause persistent fibroblast activation and progressive injury through ‘vicious’ cycles of profibrotic events (Varga and Abraham 2007). Fibrogenesis in SSc is a multistage process, now increasingly seen as a sequela of deregulated tissue repair responses and that the many different causes of fibrosis all tilt tissue homeostasis towards interstitial hyperplasia and excessive accumulation of extracellular matrix (ECM) (Ho et al. 2014). Under normal circumstances, the fibroblast repair program is self-limited during wound healing and tissue repair. In SSc, however, inappropriate fibroblast activation leads to permanent scarring and replacement of normal tissue architecture with a largely acellular, collagen-rich, rigid connective tissue (Allanore et al. 2015). Fibroblasts from different tissues, and even those within the same tissue, are heterogeneous, displaying distinct properties and functional activities. This heterogeneity appears to be even more profound during wound repair and fibrogenesis (Figure 2 ) (Tomasek et al. 2002).

Introduction

Figure 2: Fibrogenesis in SSc. Fibrosis is initiated by tissue injury induced by genetic mutation and/or defects or environmental triggers, which induces autoimmunity and autoantibody production. Tissue injury also activates innate immune cells. The adaptive immune response is also activated and TH1 cells secret proinflammatory cytokines, whereas TH2 cells predominantly secret factors that stimulate tissue remodeling. This results in the transdifferentiation of resting fibroblasts to myofibroblasts. The activation of resting fibroblasts with differentiation into myofibroblasts is a general hallmark of fibrotic diseases. Activated myofibroblasts synthesize excessive amounts of ECM, leading to ECM accumulation, increased collagen crosslinking, contraction and fibrosis. Figure adapted and modified from (Ho et al. 2014).

1.1.2.3.1 Origin of Myofibroblasts

Myofibroblasts are key effector cells of fibrogenesis. A significant increase in myofibroblasts number especially within the deeper dermis has been observed in skin biopsies from SSc patients, compared with healthy controls. Myofibroblasts are contractile cells expressing α- SMA and myosin bundles. They synthesize excessive amounts of ECM and are more resistant to apoptosis, which facilities their persistence in fibrotic diseases. The origins of the myofibroblasts in SSc continue to be a subject of intense research. Multiple cellular precursors may contribute to pool of Myofibroblasts in fibrotic tissues. Potential sources include (a) resident fibroblasts, (b) epithelial cells undergoing epithelial-to- mesenchymal transition (EMT) and endothelial cells undergoing endothelial-to-mesenchymal transition (EndoMT), (c) pericytes, and (d) circulating fibroblast-like cells termed fibrocytes as well as tissue resident mesenchymal progenitor cells (Bhattacharyya et al. 2011). TGFβ and 12

Introduction coexpression of the extra domain A (ED-A) variant form of fibronectin are central effectors mediating myofibroblast transdifferentiation from normal fibroblasts. Both EMT and EndoMT have been characterized in animal models of fibrosis and accumulating evidence suggests that these processes also occur in humans; however, the functional relevance of these processes to tissue fibrosis in human diseases is less well established. Pericytes are mesenchymal cells with smooth-muscle-like structural cells features that are normally localized to the walls of small blood vessels and capillaries. Upregulation of pericytes in the dermis in SSc has been reported showing features of myofibroblast differentiation (Rajkumar et al. 2005). Transdifferentiation of pericytes into myofibroblast-like cells also promotes the expansion and differentiation of resident ‘immature’ fibroblasts into myofibroblasts. The roles of pericytes, fibrocytes, and other monocyte-derived circulating fibroblast progenitors in the pathogenesis of fibrosis in SSc remain to be fully elucidated (Wynn and Ramalingam 2012). In addition, John Varga group indicates that intradermal adipocyte progenitors are the major contribution of myofibroblasts in the fibrotic dermis in mice (Figure 3) (Marangoni et al. 2015).

Figure 3: Origins of fibroblasts and fibrogenesis in SSc. Mesenchymal fibroblasts that contribute to excessive tissue repair or fibrogenesis (the production of ECM, notably collagen type I, and the development of contractile myofibroblasts) in SSc can be derived from different progenitor populations: (a) circulating precursors; (b) microvascular pericytes; (c) resident connective tissue fibroblasts can also become activated directly along the myofibroblast lineage. Characteristic features of activated mesenchymal cells in SSc include altered signaling pathways and a dysregulated TGFβ–SMAD pathway, which promotes the activation of transcription of collagen 13

Introduction and other ECM genes and ECM accumulation. Repeated injury promotes enhanced repair and scar formation, which progresses into a fibrotic lesion (replacement fibrosis) with excessive deposition of ECM. Figure adapted and modified from (Abraham and Varga 2005).

1.1.2.3.2 ECM

The ECM consists of a cellular compartment of resident fibroblasts and recruited cells and a connective tissue compartment comprises collagens, proteoglycans, elastin, glycosaminoglyc- an, fibrillins, fibronectin, and adhesion molecules (Allanore et al. 2015). The studies of global transcriptome analyses performed on normal and SSc fibroblasts show that many ECM genes are differentially expressed in SSc fibroblasts, including collagens, fibronectin and fibrillins. Collagens are the most abundant ECM components, which are a family of proteins with critical roles in organ development, growth, and differentiation. The ECM also functions as a reservoir for TGFβ, CTGF, and other growth factors and matrix cellular proteins that control mesenchymal cell differentiation, function, and survival. Under normal circumstances, fibroblasts only produce little ECM. The local release of cytokines and growth factors from infiltrating inflammatory cells, activated platelets, endothelial cells, and epithelial cells induced by tissue injury incites the differentiation of fibroblasts into myofibroblasts.

1.2 TGF signaling –key mediator of fibrosis Transforming growth factor  (TGFβ) is a pleiotropic cytokine with vital homeostatic functions that regulates diverse biological activities including cell proliferation, cell apoptosis, differentiation, morphogenesis, tissue homeostasis and regeneration. A fundamental role for TGFβ has been identified among of many elucidated mediators for pathological fibrogenesis (Varga and Pasche 2009). TGFβ promotes fibroblast proliferation, differentiation, migration, adhesion, survival, and myofibroblasts differentiation, and most importantly, upregulates the synthesis of collagen and extracellular matrix (Blobe, Schiemann and Lodish 2000). In addition, TGFβ stimulates the production of protease inhibitors that prevent enzymatic breakdown of the ECM. A subset of patients with the diffuse cutaneous SSc displays a ‘TGFβ responsive gene signature’ on gene expression profiling of the lesional skin (Whitfield et al. 2003, Milano et al. 2008). These patients have more severe disease (higher Rodnan skin scores and greater risk of lung involvement) than patients without this pattern of gene expression (Varga and Pasche 2009).

Introduction 1.2.1 TGF superfamily

The TGFβ superfamily includes a diverse range of proteins in mammals including the prototypic factor TGFβs, bone morphogenic proteins (BMPs), growth/differentiation factors (GDFs), activins and inhibins, nodal, myostatin and anti-Müllerian hormone. The family has a shared structure, similar signaling pathways and regulates wide-ranging and diverse roles in development, differentiation, and homeostasis (Massague 1998). TGFβ is a 25 kDa homodimeric polypeptide with many conserved structural motifs. It has been known that there is TGF-β1, -β2, -β3 three mammalian isoforms, all of them stimulate mesenchymal cells to proliferate and produce extracellular matrix and induce a fibrotic response in various tissues in vivo.

1.2.2 TGF intracellular signaling

TGFβ is secreted by many cell types including fibroblasts, myofibroblasts, T cells, monocytes and macrophages, and platelets, in a latent precursor complexed with two other polypeptides, latent TGFβ binding proteins (LTBP) and latency-associated peptide (LAP). Activation of latent TGFβ to its biologically active form capable of inducing cellular responses can be mediated by integrins (e.g., αvβ6), thrombospondin 1, proteases (matrix metalloproteinases 2 and 9), THY-1, or plasmin (Leask 2006). Once activated, TGFβ binds to a heteromeric receptor complex consisting of two transmembrane serine/threonine kinase receptors, types I and II, which are coexpressed by most cells, to exert its multiple biological actions (IHN 2002). Binding of TGFβ to type II receptor resulting in the recruitment and phosphorylation of type I receptor triggers an intracellular signal transduction cascade that leads to the induction of target genes. TGF- signal transduction is a complex process involving multiple primary and accessory receptors, signal intermediates, transcriptional factors, co-activators, repressors and posttranslational modifications (Abraham and Varga 2005). A major downstream pathway involves Smad signaling. Smad signaling is activated by phosphorylation of cytoplasmic proteins of receptor-associated Smad proteins. TGFβ receptor type I specifically recognizes and phosphorylates the ligand-specific receptor activated Smads (R-Smad), Smad2 and 3 that form heteromeric complexes with common mediator Smad, Smad4. The R-Smad/Smad4 complex is then translocated into the nucleus, where it binds to a consensus sequence “CAGAC” that defines the consensus SMAD-binding element (SBE) in the regulatory regions of target genes. The inhibitory Smad (I-Smad) proteins, such as Smad6 or Smad7, bind to TGFβ receptor type 15

Introduction I and prevent R-Smad phosphorylation and subsequent nuclear translocation (Leask and Abraham 2004). Except phosphorylation of the SMAD family of proteins by canonical TGFβ signaling, it has been also reported that non-SMAD molecules could be activated by TGF-β. All of these other intracellular pathways are summarized as non-canonical TGFβ pathways. They include several protein kinases (p38/MAPK, JNK, FAK, c-Abl), the lipid kinase phosphoinositide 3-kinase (PI3K and its downstream target Akt), and the phosphatase calcineurin (Varga and Abraham 2007).

1.2.3 TGF signaling in fibrotic responses

TGFβ is considered to be the master regulator of both physiological and pathological fibrogenesis of the multiple cytokines implicated in SSc. The expression of TGFβ1 and TGFβ2 is most prominent around dermal vessels and is associated with perivascular infiltrating mononuclear cells (Kulozik et al. 1990, Querfeld et al. 1999). The expression levels of TGFβ receptors are markedly upregulated on SSc fibroblasts, in particular the TGFβ type 1 receptor. In the fibrotic cellular milieu, latent complex TGFβ is sequestered in the ECM. Activated SSc dermal fibroblasts may be constitutively activated by establishing a self-activation system at least partially via autocrine TGFβ signaling (Asano 2018). TGFβ upregulates the expression of components of the ECM, including collagens, fibronectin, tenascins, proteoglycans and their transmembrane receptor integrins and suppresses the activity of ECM degradation genes such as matrix metalloproteinases (Wynn and Ramalingam 2012). CTGF is also induced by TGFβ and coordinates the actions of TGFβ by stimulating fibroblast proliferation and upregulating collagen, fibronectin, and integrin production. In addition, evidence supports the existence of autocrine loops that involve TGFβ, CTGF, and ET-1 as contributors to the persistent activation of fibroblasts in SSc (Eckes et al. 2007).

Introduction 1.3 Hedgehog pathway

The hedgehog (HH) pathway is considered as a major morphogen pathway which was first identified by genetic screens in Drosophila melanogaster (Nüsslein-Volhard and Wieschaus 1980). It earned its name from the appearance of embryos with null alleles of hh, which display a lawn of disorganized, hair-like bristles reminiscent of hedgehog spines (Briscoe and Therond 2013). It plays pivotal roles in many fundamental processes including self-renewal, growth, cell survival, differentiation, migration, and tissue polarity, during embryonic development. Furthermore, it also participates in vertebrate body patterning and morphogenesis of several organs. HH is also involved in progenitor and stem cells regulation in organ repair and in homeostatic mechanisms. Consequently, HH signaling is of major importance from early embryo development to adult tissue maintenance, requiring a very tight spatial and temporal regulation to ensure its correct function (Fernandes-Silva, Correia-Pinto and Moura 2017). HH signaling is highly conserved in mammals and other vertebrate species. Mammalian HH proteins can be classified into three subgroups including Sonic hedgehog (SHH), Indian hedgehog (IHH) and Desert hedgehog (DHH), and they are located in different tissues. DHH and IHH have best effect on developing tissues, IHH regulates bone and cartilage development, whereas DHH is essential for germ cell development in the testis and peripheral nerve sheath formation, but SHH has key role effect on developed and adult tissues and is the best studied one (Rimkus et al. 2016). The Hedgehog pathway consists of a chain of molecular events through which a signal produced by a cell (termed the sending cell) controls the behavior of another cell (referred to as the receiving cell). HH interacts with its co-receptor(s) and receptor Patched (PTCH), triggering an intracellular signal transduction cascade, which ultimately results in specific changes in gene expression (Petrov, Wierbowski and Salic 2017). Secreted HH ligands control developmental outcomes in a concentration and duration-dependent manner. The ability of Hedgehog signaling to initiate distinct developmental outcomes in cells exposed to an HH ligand at different concentrations or for different lengths of time is critical for HH-dependent establishment of the dorsal ventral axis during early neural development and formation of the anterior-posterior axis in developing limbs (Pak and Segal 2016).

Introduction 1.3.1 Canonical Hedgehog signaling

The HH pathway is a complex signaling network comprising both canonical and non-canonical signaling. The HH signaling cascade is initiated when one of the three HH secreted ligands binds to its receptor Patched (PTCH), a conserved 12-pass transmembrane domain receptor. In humans there are seven patched related genes named PTCH1, PTCH2, PTCHD1, PTCHD2, PTCHD3, NPC1, and PTCHD4. PTCH1 is a canonical receptor for HH ligands. PTCH2 can respond to SHH in the absence of PTCH (Alfaro et al. 2014). In contrast to most other ligand– receptor pairs, HH represses rather than activates PTCH upon binding. Peculiarly, this repression of PTCH results in activation of HH signaling. In the unstimulated state, PTCH1 is located in the primary cilium membrane. The primary cilium (PC) is a microtubule-based organelle, which emerges from the cell surface of most vertebrate cells. It is important to process several cellular signals and/or extracellular environmental changes necessary for animal development, as Wingless (WNT), PDGF, Notch and mTOR (Goetz and Anderson 2010). PTCH1 sits in the PC membrane and represses and excludes G protein–coupled receptor (GPCR)-like protein Smoothened (SMO) from the cilium through a physical interaction. Meanwhile, Zn-finger family of transcription factors GLI are sequestered and suppressed by Suppressor of Fused (SuFu) at the tip of the primary cilium. Binding of HH ligands to PTCH1 relieves the repression of SMO by PTCH1, allowing SMO to enter the cilium and to initiate Hh signal transduction to the cytoplasm. Active SMO relieves the repression of GLI transcription factors by SuFu, resulting in translocation of GLI transcription factors from the cilium to the nucleus, where they activate transcription of target genes (Rimkus et al. 2016, Taipale and Beachy 2001). In summary, in canonical hedgehog signaling, ligands such as sonic hedgehog (SHH) bind to Patched and stimulate the activation of GLI transcription factors to regulate the expression of target genes in a smoothened (SMO)-dependent manner (Figure 4).

Introduction

Figure 4: Hedgehog (Hh) signaling in vertebrates. (a) In the unstimulated state, Ptc1 sits in the cilium membrane and represses and excludes Smoothened (Smo) from the cilium. Gli transcription factors are sequestered and suppressed by Suppressor of Fused (SuFu) at the tip of the primary cilium. (b) In the stimulated state, upon binding of Shh to Ptc1, the repression of Smo by Ptc1 is relieved, allowing Smo to enter the cilium and Ptc1 to leave the cilium. This then allows Smo to repress SuFu, relieving repression of Gli at the tip of the cilium. Gli is thus freed to be post-translationally modified to form Gli activator form (GliA), which is transported out of the cilium to the nucleus to activate expression of downstream target genes. Figure adapted and modified from (Hui and Angers 2011).

1.3.2 Non-Canonical Hedgehog signaling

The transcription of hedgehog target genes can also be induced by non-canonical hedgehog signaling. Non-canonical HH activation can occur by two different mechanims: ligand- independent HH activation originating from PTCH and/or SMO, but independent of GLI- mediated transcription (Jenkins 2009, Brennan et al. 2012) or through direct stimulation of the GLI transcription factors, independent of PTCH and SMO (Stecca and Ruiz 2010, Robbins, Fei and Riobo 2012). PTCH can trigger apoptosis, or participates in cell cycle regulation through Cyclin B1 in an apparently SMO- and GLI-independent manner (Brennan et al. 2012). SMO as a G-protein-coupled receptor can start signal transmission by using small GTPases molecules to modulate intracellular cyclic AMP levels (Ogden et al. 2008). The expression and activation of GLI transcription factor can also be modulated independent of HH and SMO, by molecules such as RAS, TGF-β, phosphoinositide 3-kinase (PI3K), MAPK , PKC, FGF, and EGF (Lauth and Toftgard 2007).

Introduction 1.3.3 Hedgehog in fibrosis and cross talk with TGFβ signaling

Aberrant activation of Hedgehog signaling pathway is associated with different types of disease. Deactivation of this signaling may result in hereditary developmental disorders such as holoprosencephaly, whereas overactivation of this signaling by mutations may lead to cancer in brain, lung, pancreas, breast, and prostate (Fattahi, Pilehchian Langroudi and Akhavan-Niaki 2018, Xie 2005). Furthermore, we and others previously demonstrated that the activation of canonical SHH hedgehog signaling plays a crucial role in the pathogenesis of fibrotic diseases, such as pulmonary fibrosis, liver fibrosis and kidney fibrosis (Zhou, Tan and Liu 2016, Bolanos et al. 2012, Kramann 2016, Horn et al. 2012a, Horn et al. 2012b). SHH stimulates the release of collagen from fibroblasts, and its overexpression in murine skin is sufficient to induce fibrosis (Horn et al. 2012b). In contrast, inhibition of canonical hedgehog signaling by inactivation of SMO exerts potent antifibrotic effects in various preclinical models of SSc (Distler et al. 2014, Zahreddine et al. 2014) and in other fibrotic diseases (Bolanos et al. 2012, Fabian et al. 2012, Michelotti et al. 2013, Pereira et al. 2013). Emerging evidence suggests that SHH signaling pathway can cooperate with other signaling components, such as TGFβ, epidermal growth factor receptor (EGFR), platelet-derived growth factor receptor α (PDGFRα), KRAS, PKA, Notch, and Wnt/β-catenin (Xie et al. 2001, Bigelow et al. 2005, Kasper et al. 2006, Maeda et al. 2006, Riobo et al. 2006, Schnidar et al. 2009). However, the transcription of hedgehog target genes can also be induced by non-canonical hedgehog pathways. TGFβ has been shown to induce the transcription of GLI2 independent of hedgehog proteins such as SHH and of the cell surface receptors patched homolog (PTCH) and SMO in epithelial cells (Fan et al. 2010, Dennler et al. 2007, Javelaud, Pierrat and Mauviel 2012, Javelaud et al. 2011).

1.3.4 GLI proteins

GLI (glioma-associated oncogene family member’s proteins) belong to the Kruppel-like family of transcription factors with highly conserved zinc finger DNA-binding domains. In mammalian cells, there are three GLI proteins, GLI1, GLI2, and GLI3, and each is encoded by distinct genes. These transcription factors proteins are among the first human transcription factors for which the DNA-binding site was determined (Kinzler and Vogelstein 1990) and the 3D structure of their DNA-binding domain was resolved (Pavletich and Pabo 1993). Moreover, the GLI genes were also among the first developmental control genes implicated in human diseases including cancer (Kinzler et al. 1988, Ruppert, Vogelstein and Kinzler 1991, Vortkamp, Gessler and Grzeschik 1991). GLI binds to a consensus sequence of 5’-

Introduction GACCACCCA-3’ through the DNA-binding domain. GLI1 undergoes several posttranslational modifications to serve as a signal amplifier downstream of GLI2. GLI2 and GLI3 are the primary mediators of SHH signaling and are essential for embryogenesis (Hui and Angers 2011). GLI2 is the most important pathway activator, while GLI3 functions mainly repressor function. GLI2 is directly and transcriptionally induced by the TGFβ pathway in a wide range of cell types and in various pathological conditions (Dennler et al. 2007, Edson et al. 2010, Hu et al. 2008). Mauviel et al. identified GLI2 as an early gene target of the TGFβ /SMAD cascade independent of Hedgehog signaling (Dennler et al. 2007). The study suggested that GLI2 is a direct target of SMADs in response to TGFβ mediated by the rapid recruitment of both SMAD3 and β -catenin to distinct elements of the GLI2 promoter (Dennler et al. 2009). Howei et al. showed simultaneous expression of TGF-β1 and SHH in epithelial cells from both human lung fibrosis (cryptogenic fibrosing alveolitis and bronchiectasis), and allergen-induced lung fibrosis in mice (Stewart et al. 2003).

1.3.5 Skinny hedgehog

In mammals, secreted SHH transcripts are translated into precursor proteins of ~45 kDa, which undergoes a series of processing events. It starts with the removal of the N-terminal signal sequence as the protein enters the secretory pathway. And the C-terminal region of the SHH precursor facilitates an autocleavage reaction that produces the two distinct amino-terminals (a ~25 kDa C-terminal (ShhC) and a ~19 kDa N-terminal (ShhN)) that retains all signaling activity in a non–cell autonomous manner and can act directly on the cell in which it was made or on cells neighboring or far from its source (Mann and Beachy 2004). ShhN is heavily modified with two distinct lipids prior to transport and release. Cholesterol is covalently attached to the C-terminal during the autoprocessing reaction (Porter, Young and Beachy 1996). The N- terminus of ShhN is modified by covalent attachment of the 16-carbon fatty acid palmitate to the N-terminal cysteine via an amide linkage (Pepinsky et al. 1998, Buglino and Resh 2008). Both cholesterol and palmitoylation are important for effective Shh signaling (Chen et al. 2004). Transmembrane enzyme Hedgehog acyltransferase (HHAT, also known as skinny hedgehog) catalyzes the attachment of the fatty acid palmitate onto Sonic Hedgehog, which is an ER- resident, membrane-bound O-acyltransferase (MBOAT) family (Zeina Chamoun 2001, Buglino and Resh 2008). Multiple studies have established that palmitoylation of Shh by Hhat is critical for SHH signaling activity both in vitro and in vivo (Dawber et al. 2005, Micchelli et al. 2002, Chen et al. 2004, Lee and Treisman 2001). When palmitoylation of Shh is absent in 21

Introduction transgenic mice, Shh distribution in the developing embryos is disrupted, resulting in defects in neural tube and limb patterning. Palmitoylation of SHH by HHAT is essentially required for multimerisation of SHH proteins to large signaling complexes. The formation of those large hedgehog signaling complexes enables long-range signaling of SHH in an exocrine manner (Chen et al. 2004). Inhibition of HHAT thus interferes predominantly with exocrine long range hedgehog signaling, with minor effects on autocrine and paracrine functions. In contrast to other targets in the hedgehog pathway such as SMOOTHENED (SMO) or GLI-transcription factors that equally interfere with short- and long-range signaling, the predominant inhibition of long- range signaling upon inhibition of HHAT may allow for residual homeostatic functions executed by auto- and paracrine short-range signaling. This may be particularly relevant in SSc, as the serum levels of SHH are upregulated in SSc patients and correlate with fibrotic manifestations (Beyer et al. 2018) .

1.4 Nuclear receptors The nuclear receptor superfamily, a group of structurally related, transcription factors, contains many ligand-dependent receptors but also a large number of ‘orphan’receptors for which no ligand has yet been identified(or commonly agreed on) (Sever and Glass 2013). These proteins regulate gene expression in response to exocrine, endocrine, paracrine and intracranial signals, typically in the form of hormones inc1uding sex (estrogens, progesterons and androgens) and adrenal steroids (glucocorticoids and mineralocorticoids), thyroid hormones and retinoids, vitamin D and dietary ligands (fatty acids)(Levin and Hammes 2016). They function as key regulators of numerous physiological processes like cell growth, differentiation, proliferation, metabolism, homeostasis, reproduction and apoptosis that occur during embryonic development and in the adult (R.Tata 2002, Jetten, Kurebayashi and Ueda 2001). The nuc1ear receptor superfamily comprises 48 family members in humans that can be grouped into six subfamilies on the basis of nuclear receptor sequences using molecular phylogeny (Gronemeyer, Gustafsson and Laudet 2004, Germain et al. 2006). Subfamily 1: Thyroid Hormone Receptor-like Group A: Thyroid hormone receptor (Thyroid hormone) Group B: Retinoic acid receptor (Vitamin A and related compounds) Group C: Peroxisome proliferator-activated receptor Group D: Rev-erb Group F: Retinoid-related orphan receptor Group H: Liver X receptor-like

Introduction Group I: Vitamin D receptor-like Subfamily 2: Retinoid X Receptor-like Group A: Hepatocyte nuclear factor-4 (HNF4) Group B: Retinoid X receptor (RXR) Group C: Testicular receptor Group E: TLX/PNR Group F: COUP/EAR Subfamily 3: Estrogen Receptor-like (Steroid hormone receptor) Group A: Estrogen receptor (Sex hormone receptors) Group B: Estrogen related receptor Group C: 3-Ketosteroid receptors Subfamily 4: Nerve Growth Factor IB-like Group A: NGFIB/NURR1/NOR1 Subfamily 5: Steroidogenic Factor-like Group A: SF1/LRH1 Subfamily 6: Germ Cell Nuclear Factor-like Group A: GCN1 Subfamily 0: Miscellaneous Group B: DAX/SHP

Each receptor is described by the letters ‘NR’ (for ‘nuclear receptor’) and a three-digit identifier: this denotes the subfamily to which a given receptor belongs(indicated by the first digit, an Arabic numeral), the group (denoted by capital letters) and the individual gene(again denoted by Arabic numerals). For example, human thyroid hormone receptor-α, which belongs to subfamily I, group A and which is the first described gene in that group, is called NR1A1. This new nomenclature has been endorsed by the major groups in the field and by the International Union of Basic and Clinical Pharmacology Committee on Receptor Nomenclature and Drug Classification (NC-IUPHAR) (Table1).

Introduction

Table 1:Human nuclear receptors. Classification of nuclear receptors with their trivial name, abbreviations, the nomenclature and known ligands. From Hinrich Gronemeyer, Jan-Åke Gustafsson and Vincent Laudet. NATURE REVIEWS DRUG DISCOVERY 2004.3:950-964.

Nuclear receptors share a common structure, comprising four domains, a highly variable amino- terminal domain that includes several distinct transactivation regions (the region A/B; also referred to as AF1 for activation function 1), a central conserved DNA-binding domain (DBD or region C) that includes two Zn fingers, a linking hinge domain (region D) responsible for nuclear localization, and a large fairly well-conserved carboxy-terminal AF-2 ligand-binding domain (LBD or region E) that also contributes to interactions of the subset of nuclear receptors that form heterodimers(Figure 5) (Castrillo and Tontonoz 2004).

Figure 5: Schematic representation of functional domains of nuclear receptors. AF-1: activation function-1; DBD: DNA binding domain; LBD: ligand binding domain; AF-2: activation function-2; A/B: activation domain of ligand-independent transcription; C: DNA binding domain; D: hinge region; E: ligand binding domain. Nuclear receptors modulate gene transcription by binding to specific DNA sequences, a sequence of 6 base pairs as. The responsive core recognition motif consists in the nucleotide hexamer (A/G) GGTCA. Figure adapted and modified from (Berrabah et al. 2011). 1.4.1 ROR family

The retinoic acid-related orphan receptors (RORs) (NRIF) subfamily represented by the three subtypes RORα, RORβ and RORγ, which are encoded by three different genes and display constitutive transcriptional activity in the absence of an exogenous ligand. Thus they are so- called “orphan” receptors. The RORα gene maps to human chromosome 15q22.2 and spans a 25

Introduction relatively large 730 kb genomic region comprised of 15 exons. The RORβ and RORγ genes map to 9q21.13, and 1q21.3 and cover approximately 188 and 24 kb, respectively (Jetten 2009). RORs exhibit a modular structure that is characteristic for nuclear receptors; the DNA-binding domain (DBD) is highly conserved and the ligand-binding domain (LBD) is moderately conserved among RORs. These transcription factors regulate gene expression by binding to ROR response elements (ROREs) consisting of the half-site consensus core motif (A/G) GGTCA proceeded by a 6-bp long A/T-rich sequence in the regulatory regions of target genes (Jetten et al. 2001). RORE-dependent transcriptional activation by RORs is highly cell type- specific and modulated by interactions with nuclear cofactors. RORα is ubiquitously expressed. The highest level of expression is found in the Purkinje cells in the cerebellum and peripheral blood leukocytes. Staggerer mice, which carry a spontaneous inactivating mutation of RORα with in the exon encoding part of the ligand binding domain, thereby generating a truncated protein (RORαsg), as well as non-conditional RORα-/- mice exhibit severe cerebellar ataxia due to a defect in Purkinje cell development. Expression of ROR is very restricted. ROR is expressed in three principal components of the mammalian biological clock, the retina, the pineal gland, and suprachiasmatic nucleus. ROR-/- mice were shown to have alterations in circadian behavior. ROR plays an important role in thymopoiesis as it is most highly expressed in the thymus. Thymocytes from these mice undergo accelerated apoptosis. ROR-/- mice also lack lymph nodes, suggesting that ROR is essential for lymph node development. Overexpression of ROR has been shown to inhibit T cell receptor-mediated apoptosis in T cell hybridomas and to repress the induction of Fas-ligand and interleukin 2 (He et al. 1998). Each ROR gene generates several isoforms that only diverge from their amino terminus A/B regions by a combination of different promoter usage and alternative exon splicing. Most isoforms exhibit a distinct tissue-specific pattern of expression and regulate different biological processes and target genes.

RORα signaling RORs exert transcriptional control from their ROR response elements (RORE) located in the regulatory regions of target genes through recruitment of coregulators comprising of coactivators and corepressors which potentiate or attenuate gene transcription, respectively. In contrast to most nuclear receptors that bind as dimers to the DNA, RORα binds as monomer to the half-core site motif. The activation function (AF-2) of ROR proteins is localized in the Ligand-binding domain (LBD) within the C-terminus of RORs and is involved in recruitment of co-activators or co-repressors.

Introduction In ligand-bound nuclear receptors, conformational changes in the receptor LBD through the repositioning of a short, amphipathic helix in the LBD, referred to as activation function-2 (AF- 2) helix induces activation of the receptor (Renaud and Moras 2000, Greschik and Moras 2003). Upon ligand binding (holo: ligand-bound), the AF-2 folds against the LBD core, leading to dissociation of corepressors and formation of an interface more suitable for coactivator recruitment (Nolte et al. 1998). Ligand-dependent activation highly relies on the integrity of the AF-2 helix. In the absence of ligand (apo:-ligand-free), the AF-2 helix extends away from the LBD and is commonly associated with corepressors. In contrast, RORα displays constitutive activity independent of ligand binding. A homology model of RORα study has shown that there is a non-conserved amino acid in helix 11 of RORα (Phe-491) and a short-length helix at the N terminus of AF-2. These conditions may ensure that AF-2 is locked permanently in the holo-conformation thus enabled ligand-independent recruitment of coactivators. Moreover, RORα activity was downregulated by mutation of Phe- 491 eliminating recruitment of coactivators and transcriptional activation (Harris et al. 2002). Direct interaction of coactivator such as NCOA1 (SRC1), NCOA2 (TIF2 or GRIP1), PGC-1α, p300, and CBP with the RORα LBD have been shown. RORα can also interact with nuclear receptor corepressors (N-CoR) such as NCOR1, NCOR2, RIP140, and the neuronal interacting factor X (NIX1) (Jetten 2009). A conformational change in the LBD induced by interaction with pharmaceutical agonists such as the selective RORα agonist SR1078 allows release of the corepressor complex and promotes assembly of a coactivator complex resulting in the transcriptional activation by RORs, while the inverse agonist like SR3335 induces a conformational change within the receptor that decreases the affinity of the receptor for a cofactor protein. These observations not only suggested that RORs function as ligand-dependent transcription factors, but also indicated that RORs might be potential therapeutic targets to treat disease (Kumar et al. 2011, Solt and Burris 2012).

1.4.2 Physiological Functions of RORα

RORs play critical roles in the regulation of a variety of physiological processes (Figure 6). The physiological functions of RORα can be characterized in detail by taking advantage of the natural RORα-deficient Staggerer (RORαsg/sg) mice, which were first described in 1962 (Sidman, Lane and Dickie 1962). These mice develop severe ataxia (hence the name “staggerer”) and severe cerebellar atrophy, due to significantly fewer in Purkinje cell and a loss of cerebellar granule cells that impairs mobility and normal feeding behavior. The RORα null 27

Introduction mice display essentially an identical phenotype recapitulating the severe ataxia (Dussault et al. 1998). In addition to the cerebellar phenotype, RORαsg/sg exhibit abnormally thin and long, mass and density of bones, suggesting a pivotal role for RORα in the formation and maintenance of the bone tissue. RORα gene expression is increased during the osteogenic differentiation of mesenchymal stem cells derived from bone marrow (Meyer et al. 2000). RORα mRNA is highly expressed in skeletal muscle, suggesting a regulatory in myogenesis or muscle function. The observations showing that RORαsg/sg mice develop muscular atrophy provide evidence that RORα play a critical role in homeostasis of skeletal muscles. Lau et al. reported that lack of functional RORα delays the morphological differentiation of myotubes and induction of helix-loop-helix transcription factors MyoD and myogenin, transcription factors that are critical for myotubule differentiation (Lau et al. 1999). Furthermore, the RORαsg/sg mutant mice display a pronounced hypo-α-lipoproteinemia and have lower levels of total plasma cholesterol, ApoA1, the major constituent of HDL, ApoC3, ApoA2, triglycerides, and high density lipoprotein (HDL) on standard diet compared to WT mice. Accumulating evidence strongly suggests that RORα play an important role in metabolic pathways, particularly lipid and steroid metabolism (Lau et al. 2008, Wada et al. 2008a, Wada et al. 2008b, Boukhtouche, Mariani and Tedgui 2004). The expression of sterol regulatory element-binding protein 1 isoform c (SREBP1c), which is a critical regulator of lipogenesis and several lipogenic genes, was significantly reduced in liver and skeletal muscle of RORαsg/sg (Lau et al. 2008, Wada et al. 2008a). Functional ROREs have been identified in the promoter regulatory region of APOC2 and APOA1 (Vu-Dac et al. 1997, Raspe et al. 2001). The reduced susceptibility of RORα-deficient mice to hepatic steatosis and obesity suggests a role for RORα in energy homeostasis. Abnormalities in immune response of Staggerer mutant mice characterized by delayed thymic development and a defect in terminating T-cell responses (Trenkner and Hoffmann 1986). Mature T and B lymphocytes are significantly reduced in RORα-/- spleen, suggesting that RORα plays a critical role in lymphocyte development. Yang et al. have shown that Th17 differentiation was completely impaired in Thp cells deficient in both RORα and RORγ (Yang et al. 2008a, Yang et al. 2008b). Moreover, Staggerer mice are less susceptible to ovalbumin (OVA)-induced lung inflammation with lower level of infiltration of inflammatory cells as the induction of Th2 cytokines IL-4, IL-5, and IL-13 is greatly compromised (Tilley et al. 2007, Jaradat et al. 2006). RORα also plays a key role in the regulation of circadian rhythmicity. Shorter free-running activity period lengths is observed in entrained light-dark cycles Staggerer

Introduction mice under constant darkness conditions (Sato et al. 2004). Moreover, heterozygous Staggerer mutant mice show a faster adaptation to altered light cycles (Akashi and Takumi 2005).

Cerebellar development and

aging

Anti- Bone inflammatory metabolism ROR

Smooth and skeletal muscle cell Lipid differentiation metabolism

Central circadian rhythm

Figure 6: Physiological functions of RORα. The schema describes the main target tissue of the ROR action. Figure adapted and modified from (Boukhtouche, Mariani J Fau - Tedgui and Tedgui 2004, Gutstein et al. 2012)

Introduction 1.5 Animal models of Systemic sclerosis

Several murine and also one avian models have been developed to study mechanistic links between inflammation, vascular damage, and fibrosis in SSc. They are invaluable for discovering and testing of targeted therapies and characterization of their mechanism of action. To better mimic the complex pathogenesis of the human disease, it is critical to carefully select animal models as all of these models can only reproduce partial features of human SSc (Figure 7) (Beyer et al. 2010, Marangoni, Varga and Tourtellotte 2016).

Figure 7: Overview of common mouse models of SSc. Key pathophysiologic features (vasculopathy, fibrosis, inflammation, and autoimmunity) in systemic sclerosis (SSc) and their representation by different animal models. Adapted and modified from Beyer et al. and Asano et al. (Beyer et al. 2010, Asano and Sato 2013)

1.5.1 Bleomycin-induced skin fibrosis

Bleomycin is a chemotherapeutic agent widely used for cancer treatment, which was originally isolated from the fungus Streptomyces verticillus (Umezawa et al. 1966). The most serious complication of bleomycin is pulmonary fibrosis. Fibrosis can be induced in mice by subcutaneous injection of bleomycin. Moreover, cutaneous fibrosis has been described (Remlinger 2003). This clinical observation was translated into a mouse model of SSc. Dermal fibrosis was induced by means of subcutaneous injections of bleomycin into the shaved back skins (Yamamoto et al. 1999). The fibrotic response study comparing sex, age, and strain of the mice 30

Introduction showed that male mice responded with increased fibrosis compared with females, and older mice with increased fibrosis compared with younger animals. Moreover, Balb/C mice developed more severe skin fibrosis than C57BL/6 mice (Ruzehaji et al. 2015). Dermal sclerosis can be reproducibly be induced within relatively short periods. In this mouse model, the sequence of histopathological changes resembles that seen in SSc - a number of mononuclear cells including T cells, macrophages and mast cells infiltrate in the lesional skin and upregulated TGFβ and chemokine expression followed by dermal fibrosis with accumulation of α-SMA–expressing myofibroblasts (Yamamoto and Nishioka 2005). The release of reactive oxygen species in bleomycin-treated skin causes endothelial cell damage and platelet activation, leading to the upregulation of expression of adhesion molecules or secretion of inflammatory or fibrogenic cytokines. Bleomycin can also upregulate collagen mRNA expression in human dermal fibroblasts in vitro. Taken together, the bleomycin-induced dermal fibrosis model is a suitable model to study early inflammatory stages of fibrosis in SSc

1.5.2 Adenoviral overexpression of a constitutively active TGFβ receptor type 1 (TBRIact) model

TGFβ is the major cytokine implicated in the pathogenesis of fibrosis. TGFβ has potent profibrotic activities by promoting collagen synthesis, secretion, processing, and cross-linking, as well as inducing myofibroblast differentiation and apoptosis resistance (Marangoni et al. 2016). Intradermal injection of adenovirus encoding for a constitutively active mutant of the type 1 TGFβ receptor induces local fibrosis (Beyer et al. 2015).

1.5.3 DNA Topoisomerase I -induced fibrosis

The presence of autoantibodies is a central feature of SSc, and antibodies against topoisomerase I (anti–topo I) are associated with dcSSc and thus more extensive skin fibrosis, higher risk for internal organ involvement and SSc-related death (Kuwana et al. 2000). Yoshizaki et al. established a new animal model of SSc by biweekly injection of human topoisomerase I antigen with complete Freund’s adjuvant (CFA) in the back skin of C57BL/6mice. Upregulation of IL-6, TGF-β1, and IL-17 production and reduced IL-10 production are observed in mice treated with topo I and CFA. This immunization with topoisomerase I induce dermal fibrosis with Th2/Th17 immune polarization (Yoshizaki et al. 2011). This animal model mimics the fibrotic, inflammation and autoimmunity aspects of SSc

Introduction (Asano and Sato 2013).

1.5.4 Bleomycin-induced pulmonary fibrosis

Pulmonary fibrosis is associated with high rates of mortality and morbidity. Bleomycin is well known to induce lung injury/fibrosis in experimental animal models. Bleomycin-induced pulmonary fibrosis is an established murine model and is the most common approach to induce experimental pulmonary fibrosis. Intratracheal administration of a single dose of bleomycin causes epithelial apoptosis and provokes an transient inflammatory response in the first 7-10 days after instillation, resulting in maximal deposition of collagen after 4 weeks (Mouratis and Aidinis 2011).

Introduction 1.6 Aims of the study

In the first part of the thesis, we aimed to investigate crosstalk between TGFβ and hedgehog signaling at the level of GLI2 in fibrosis, characterize GLI2 is a downstream mediator of the pro-fibrotic effects of TGFβ and test, whether inhibition of GLI2 might be a target for the treatment of fibrotic diseases.

The first part of the thesis is comprised of:

1: Assessment of the effects of GLI2 deletion and amplification on the release of extracellular matrix of cultured dermal fibroblasts stimulated with TGFβ.

2: Interaction study of GLI2 with members of TGFβ signaling.

3: Effects of deletion of GLI2 on inflammation-driven models of early inflammatory stages of

SSc mimicked by the mouse models of bleomycin induced dermal fibrosis.

4: Evaluation of the same effects in the TBRIact model of SSc as a model of late, inflammation- independent stages of SSc.

In the second part of the thesis, we aimed to investigate potential contributions of disturbed

HHAT expression to increased hedgehog signaling in SSc, to test the role of HHAT in fibroblast activation, and to analyze the therapeutic effects of targeted inactivation of HHAT in preclinical models of SSc.

This includes the following readouts:

1: Expression pattern analysis of HHAT in human SSc skin and experimental fibrotic skin.

2: Assessment of the regulation of HHAT by TGFβ, a central profibrotic cytokine.

3: Analysis of the regulatory effects of HHAT on fibroblasts and assessment of the intracellular targets of TGFβ.

4: Fibroblast-specific inactivation of HHAT in murine models of SSc.

At the last part, our goal is to evaluate the role of RORα in the pathogenesis of fibrotic diseases.

This part consists of following parts:

Introduction 1: Expression pattern analysis of ROR in human lung and liver and experimental fibrotic lung and liver.

2: Assessment of the regulation of ROR by Wnt signaling.

3: Analysis of the regulatory effects of ROR on fibroblasts and assessment of the intracellular targets of TGFβ.

4: Targeting RORα ameliorates fibrosis in different murine models.

Materials and Methods 2 Materials and Methods

2.1 Material

2.1.1 Chemicals

Chemical Source 1,4-Dithiothreit (DTT) Roth, Germany 2-Propanol Roth, Germany 3,3’-Diaminobenzidine-tetrahydrochloride dihydrate Sigma Aldrich, USA (DAB) 30% Polyacrylamide Sigma, Germany 4',6-diamidino-2-phenylindole (DAPI) Santa Cruz, Germany 4-(Dimethylamino)benzaldehyde Fluka, Germany 10x PCR Buffer Applied Biosystems, Germany Acetic acid Merck, Germany

Acetone AppliChem, Germany Acid chloride Merck, Germany

Agarose Standard Carl Roth, Germany

Aniline Blue Sigma, Germany

Ascorbic acid Sigma, Germany Ammonium chloride Carl Roth, Germany Ammonium peroxide sulfate (APS) Sigma, Germany Amphotericin B Gibco® Life technologies, Germany Ampicillin Roth, Germany Bleomycin dissolved in 0.9 % sodium chloride University Hospital

Erlangen, Germany Bio-Rad Protein Assay Dye Reagent Concentrate (5X Bio-Rad, Germany Bradford dye) Biebrich scarlet-acid fuchsin solution Sigma, Germany Bovine serum albumin (BSA) Roth, Germany Bouin solution Sigma, Germany

Materials and Methods Chloramine T Sigma, Germany Citric acid monohydrate Roth, Germany Collagenase D Sigma, Germany Deoxyribonucleosidetriphosphates (dNTPs) Roche Diagnostics, Germany Dimethyl sulfoxide (DMSO) Roth, Germany Direct Red 80 Sigma, Germany Disodium ethylenediaminetetraacetate Roth, Germany dihydrate(Na2EDTA) Dispase II Sigma, Germany di-Sodium hydrogen phosphate dehydrate Merck, Germany DMEM/F12 (1:1) Gibco® Life technologies, Germany DNase I Sigma, Germany Ethanol Roth, Germany Ethanol Roth, Germany Ethylenediaminetetraacetic acid (EDTA) Roth, Germany Fetal bovine serum (FBS, FCS) Gibco® Life technologies, Germany FuGENE® HD Promega, Germany G418 (Geneticin) InvivoGen, Germany GANT 61 TOCRIS, Germany GenLadder 100bp plus 1.5kbp Genaxxon bioscience, Germany Gibco® Phosphate buffered saline (PBS) Life technologies, Germany Glacial acetic acid Merck, Germany Glycin Roth, Germany Goat serum Life technologies, Germany HEPES Sigma, Germany Haematoxylin Merck, Germany Horse serum Life technologies, Germany Histofix 4% Roti® (PFA) Roth, Germany Histokitt Roti® Roth, Germany Hydrochloric acid Roth, Germany Hydrogen peroxide 30 % Roth, Germany Hydroxyproline (cis-L) Sigma, Germany Isoflurane Abbott, Canada Kanamycin Roth, Germany

Materials and Methods Ketavet Zoetis Belgium S.A LB Broth (Luria/Miller), granuliert Roth, Germany LB Agar (Luria/Miller), granuliert Roth, Germany L-glutamine Life technologies, Germany Magnesium chloride 25mM Applied Biosystems, Germany Methanol Roth, Germany Methoxyethanol Sigma, Germany

Milk powder, blotting grade Roth, Germany Methyl red, Indicator pH 4.2-6.2 Roth, Germany Microscopy aquatex Merck, Germany MultiScribe Reverse Transcriptase Applied Biosystems, Germany N’ ,N’ ,N’ ,N’-Tetraethylmethylenediamine (TEMED) Sigma, Germany 1-methyl-2 pyrrolidone Sigma, Germany Penicillin Life technologies, Germany Picric Acid Sigma, Germany Phosphotungstic Acid Sigma, Germany Phosphomolybdic Acid Solution Sigma, Germany Perchloric acid 70% Roth, Germany PEQGREEN VWR, Germany Phenylmethlsulfonyl fluoride Roth, Germany Protease Inhibitor Cocktail (DMSO solution) Sigma, Germany Proteinase K Roth, Germany Protein A/G PLUS-Agarose Santa Cruz, Germany Potassium chloride Merck, Germany potassium hydrogen carbonate Roth, Germany

Potassium sulfate dodecahydrate Roth, Germany Random Hexamers Applied Biosystems, Germany RedMastermix (2X) Taq PCR Mastermix Genaxxon bioscience,Germany Rhodamine phalloidin Life technologies, Germany RNase Inhibitor Applied Biosystems, Germany Rompun® Bayer, Germany Sodium acetate Roth, Germany Sodium chloride Roth, Germany

Materials and Methods Sodium chloride 0.9 % Berlin-Chemic, Germany Sodium citrate dihydrate Roth, Germany Sodium dihydrogen phosphate dihydrate Merck, Germany Sodium dodecyl sulfate (SDS) Sigma, Germany Sodium fluoride Roth, Germany Sodium hydroxide Roth, Germany Sodium iodate Merck, Germany Streptomycin Life technologies, Germany SYBR Select Master Mix Applied Biosystems, Germany TopoisomeraseI Abcam, Germany Transforming Growth Factor β (TGF-β) R&D Systems, Germany Tris-(hydroxymethyl)-aminomethane (Tris) Roth, Germany Tri-Sodium citrate dihydrate Merck, Germany Triton X-100 Sigma, Germany Trypan blue solution 0.5% Sigma, Germany Trypsin/EDTA Gibco® Life technologies, Germany Tween 20 Roth, Germany Tween 80 Roth, Germany Wiegert’s Iron-Hematoxylin-Set Sigma, Germany Xylene Roth, Germany Zeocin™ InvivoGen, Germany β-Glycerophosphate Roth, Germany β-Mercaptoethanol Sigma, Germany Table 2: Chemicals

Materials and Methods 2.1.2 Auxiliary materials

Material Source

6,12,24,48,96-well plate Nalge Nunc International, Denmark Amersham™ ECL™ Prime Sigma, Germany Biopsy Punch 3 mm pfm medical AG, Germany Cell strainer 70 μm Nylon BD falcon, Germany Cellstar® polystyrene conical tubes (15, 50 ml) BD falcon, Germany Cellstar® serological pipette (2, 5,10, 25,50 ml) Greiner, Germany Cellstar® tissue culture flask (T25, T75, T175) Greiner, Germany Cell Culture Insert Companion Plates 24-well plate BD falcon, Germany Chamber slides, 8 well Nalge Nunc International, Denmark Cover glass 24x500 mm Menzel-Gläser, Germany Cryo tube vials(1, 2ml) Nalge Nunc International, Denmark Dako Faramount Aqueous Mounting medium Dako, USA Dako Faramount Fluorescent mounting medium Dako, USA Developer and fixer for X-ray films Tetenal Photowerk, Germany Disposable Sterile filter (0.22 μm pore size) Schleicher & Schüll, Germany Discardit™II syringe(1,10,20 ml) BD falcon, Germany Dual Gel Caster (SE 245) Hoefer, USA Eppendorf reaction tubes (0.5, 1.5, 2, 5 ml) Eppendorf AG, Germany Eppendorf research pipettes (2.5, 10, 100, 1000 μl) Eppendorf AG, Germany Eppendorf tips Eppendorf AG, Germany

Feather disposable scalpel (No.11, No.21) pfm medical AG, Germany Filter CA-membrane 0.45 μm; 0.20 μm BD falcon, Germany Freeze box for cryo vials Nalge Nunc International, Denmark

Incidin OxyWipe S Tissue Ecolab, USA

Inserts for 24-well plates BD falcon, Germany Microlance™ 3 needle (0.4x19 mm, 0.5x16 mm) BD falcon, Germany Microlance™ 3 needle (0.9x40 mm,0.8x40 mm) BD falcon, Germany Marker (PageRuler/plus Prestained Protein Ladder) Thermo -Fisher, Germany Neubauer counting chamber Paul Marienfeld GmbH, Germany 39

Materials and Methods Optical 8 cap strip, MicroAmp® Applied Biosystems, Germany Optical 96-well reaction plate, Micro-Amp® Applied Biosystems, Germany Optical 384-well reaction plate, Micro-Amp® Applied Biosystems, Germany PCR reaction tubes, single cap Biozym Scientific GmbH, Germany Petri dish Greiner bio-one GmbH, Germany pH indicator paper Carl Roth, Germany Precellys Lysing Kit CK14_2ml Precellys Bertin Technologies,Germany PVDF membrane Hybond™-P Amersham Biosciences, UK Roti Histokitt Carl Roth, Germany RNase Zap Applied Biosystems, Germany Safe Seal-Tips professional Biozym, Germany Steritop® and Stericup® Millipore GmbH, Germany X-Ray cassettes Amersham Biosciences, UK X-Ray film GE Healthcare, Germany

Table 3: Auxiliary materials

2.1.3 Instruments

Instrument Source

4D-Nucleofector Core Unit Lonza Group, Switzerland -20 Freezer comfort Liebherr, Germany -80 Freezer Heraeus, Germany

Agilent Stratagene MX3005P qPCR System Agilent, Germany

ABI StepOne™ Real-Time PCR System Applied Biosystems, Germany Biofuge fresco Heraeus, Germany Biological safety cabinet Class II ESCO, UK Camera DXC-390P Sony, Germany Centrifuge function line Heraeus, Germany DarkHood DH-20 Biostep, Germany Digital block heater HX-2 Peqlab, Germany Eppendorf Centrifuge 5417R Eppendorf, Germany Eppendorf Centrifuge 5804 Eppendorf, Germany

Materials and Methods GraphPad Prism V. 5.03 software GraphPad Prism, USA Heraeus Megafuge 1G Thermo Scientific, Germany ImageJ V. 142q software National Institutes of Health, USA Incubator Heraeus, Germany Incubator Memmert, Germany Incubator 1000 Heidolph Instruments, Germany Incubating orbital Shaker VWR, USA LaminAir HB 2448 Heraeus, Germany Meditome A550 Medite. Germany Megafuge 16R Heraeus, Germany Microscope Primo Vert Zeiss, Germany MP-3AP power supply Talron Biotech. L.T.D., Israel MRX ELISA reader Dynex Technologies, USA neoBlock – Heizer Duo 2-2504 NeoLab, Germany Nikon Eclipse 80i microscope Nikon, Badhoevedorp, Netherlands PeqSTAR 96 universal Peqlab, Germany pH-Meter pH340 WTW, Germany Pipetboy Integra Biosciences, USA PowerPac™ HC High-Current Power Supply BIO-RAD, Germany PowerPack P25T Biometra GmbH, Germany Precellys 24 tissue homogenizer PrecellysBertinTechnologies,Germany Promax 1020 Heidolph Instruments, Germany QuantStudio™ 6 Flex Real-Time PCR System Thermo Scientific, Germany RCT classic, magnetic heater IKA®, Germany Rocker 2D basic IKA®, Germany Roller10 basic IKA®, Germany Serva Blue Power Serva, Germany Sonicator SpectraMax 190 GMI, inc, USA Thermocycler T48 personal Biometra GmbH, Germany Thermomixer compact Eppendorf, Germany Tissue flotation bath TFB55 Medite, Germany TKA Micropure water purification system Thermo Scientific, Germany

Materials and Methods Transilluminator Biostep, Germany Water bath Memmert, Germany

Table 4: Instruments

2.1.4 Commercially available systems (kits)

System Source

Adeno-X Rapid Titer Kit Takara, Germany AL10 hand-held pH meter, AquaLytic Roth, Germany Amaxa P2 Primary Cell 4D-Nucleofactor X Kit Lonza Group, Switzerland Cell Counting Kit - 8 Sigma, Germany Clarity™ Western blot ECL Blotting substrate Bio Rad GmbH, Germany ChIP-IT® Express Chromatin Immunoprecipitation Kits Active Motif, Germany ChIP DNA Purification Kit Active Motif, Germany Dual-Luciferase® Reporter Assay System Promega, Germany ECL prime kit GE Healthcare, Germany LightSwitchTM Transfection Optimization kit Cell Signaling SimpleChIP® Human CTGF Promoter Primers Eurogentec GmbH, Germany MESA FAST qPCR MasterMix Plus for SYBR® ROX Eurogentec GmbH, Germany NucleoSpin® RNA II Machery-Nagel, Germany

SirCol™ Soluble Collagen Assay Biocolor, UK SBE Reporter kit BPS Bioscience Ringer lactate-free solution B. Braun, Germany PureYield™ Plasmid Miniprep System Promega, USA QIAGEN Plasmid Maxi Kit Qiagen, Germany

Table 5: Commercially available systems (kits)

Materials and Methods 2.1.5 Antibodies

Antibody Source Dilution/ Application

Alexa Fluor 488 goat anti-mouse IgG Invitrogen™, Germany 1:200/ IF Alexa Fluor 488 goat anti-Rabbit IgG Invitrogen™, Germany 1:200/ IF Alexa Fluor 594 goat anti-rabbit IgG Invitrogen™, Germany 1:200/ IF Alexa Fluor 594 donkey anti-goat IgG Invitrogen™, Germany 1:200/ IF Alexa Fluor 594 Chicken antimouse Invitrogen™, Germany 1:200/ IF IgG Invitrogen™, Germany 1:200/ IF Alexa Fluor 647 donkey anti-goat IgG Invitrogen™, Germany 1:200/ IF Alexa Fluor 647 donkey anti-mouse IgG Invitrogen™, Germany 1:200/ IF Alexa Fluor 647 donkey anti-Rabbit Santa Cruz, Germany 1:1000/ IF IgG DAPI (SC 3598) Monoclonal mouse IgG antibodies Calbiochem, USA 1:1000/ IHC Mouse monoclonal anti- Collagen I Abcam, UK 1:1000/WB Mouse monoclonal anti- Collagen I Abcam, UK 1:1000/WB Polyclonal rabbit anti- Collagen I Abcam, UK 1:1000/WB Polyclonal rabbit anti-Gli2 Santa Cruz, Germany 1:50/IHC Polyclonal rabbit Anti-Gli2 ChIP Grade Abcam, UK 1:200/ ChIP Polyclonal Goat anti-Gli2 Santa Cruz, Germany 1:100/ IF Mouse monoclonal anti-Gli2 Santa Cruz, Germany 1:50/ IF Polyclonal rabbit anti-p4Hβ Acris Antibodies, 1:50/IF Polyclonal mouse anti-p4Hβ Germany 1:50/IF Thermo-Fisher, Germany Monoclonal mouse anti-Vimentin Abcam, UK 1:500/IF

Polyclonal mouse anti- Vimentin CST, Germany 1:50/IF Polyclonal Goat anti- Vimentin Abcam, UK 1:50/IF Monoclonal mouse anti-α - Sigma, Germany 1:1000/ IHC 1:500/ IF smooth muscle actin (α-SMA) 1:10000/ WB Monoclonal mouse anti--actin Sigma, Germany Rabbit monoclonal anti-Phospho- Cell signaling, Germany 1:200/IF Smad2/Smad3 Rabbit monoclonal Anti-pSmad3 Abcam, UK 1:200/IF 43

Materials and Methods Rabbit monoclonal Anti-Smad3 Cell signaling, Germany 1:100/IF

Polyclonal rabbit anti-mouse IgG/HRP Dako 1:200/ IHC Cytomation,Denmark 1:5000/ WB Polyclonal rabbit anti-mouse CD45 1:150/IF Rhodamine phalloidin (R415) Abcam, UK 1:200/IF Recombinant Human Sonic Thermo Fisher, Germany 1:100/ WB Hedgehog/Shh R&D, Germany Polyclonal rabbit anti-human HHAT 1:50/ IHC/IF Thermo Fisher, Germany 1:500/ WB Polyclonal rabbit anti-mouse HHAT 1:50/ IHC/IF 1:250/ WB Sigma-Aldrich, Germany

Table 6: Antibodies

2.1.6 Primers

Primers Sequence human β-actin forward 5’-AGA AAA TCT GGC ACC ACA CC-3’ human β-actin reverse 5’-TAG CAC AGC CTG GAT AGC AA-3’ human col1a1 forward 5’-ACG AAG ACA TCC CAC CAA TC-3’ human col1a1 reverse 5’-ATG GTA CCT GAG GCC GTT C-3’ human col1a2 forward 5’- GGT CAG CAC CAC CGA TGT C-3’ human col1a2 reverse 5’- CAC GCC TGC CCT TCC TT-3’ human Cyclin D1 forward 5’- AAC CTG AGG AGC CCC AAC-3’ human Cyclin D1 forward 5’- AAG CGT GTG AGG CGG TAG-3’ human PAI-1 forward 5’- TCA TTG CTG CCC CTT ATG A-3’ human PAI-1 reverse 5’- GTT GGT GAG GGC AGA GAG AG-3’ human CTGF forward 5’- AAC TCA CAC AAC AAC TCT TCC CCG C -3’

Materials and Methods human CTGF reverse 5’- GAG TCG CAC TGG CTG TCT CCT CT -3’ human α-SMA forward 5’-AAG AGG AAT CCT GAC CCT GAA-3’ human α-SMA reverse 5’-TGG TGA TGA TGC CAT GTT CT-3’ human Gli-2 forward 5’- CTG CTC GAA GGC CTA CTC C -3’ human Gli-2 reverse 5’- ACC GCA GGT GTG TCT TCA G-3’ human smad3 forward 5’- TGG GCT GAA GCG CAC TGA CC -3’ human smad3 reverse 5’- CCG CGG CTC TTG CCC ACA T -3’ human Smad7 forward 5’-TAC TCC AGA TAC CCG ATG GAT T-3’ human Smad7 reverse 5’-TCT GGA CAG TCT GCA GTT GG-3’ human HHAT forward 5’- AAC TCT CAG CGT AGG CAT CG -3’ human HHAT reverse 5’- CAC TGC TTC CCC CAT TCC AT-3’ human Ptch-1 forward 5’- ACA AAC TCC TGG TGC AAA CC -3’ human Ptch-1 reverse 5’- GCT GAT GTC GAT GGG CTT AT -3’ human Ptch-2 forward 5’- TGT GGT GGG AGG CTA TCT G -3’ human Ptch-2 reverse 5’- GCA TGG TCA CAC AGG CAT AG -3’ murine β-actin forward 5’-TCT TTG ATG TCA CGC ACG AT-3’ murine β-actin reverse 5’-TAC AGC TTC ACC ACC ACA-3’ murine Gli-2 forward 5’- GCT GCA CCA AGA GGT ACA-3’ murine Gli-2 reverse 5’- CTT CAC ATG CTT GCG GAG T-3’ murine col1a1 forward 5’-GAA GCA CGT CTG GTT TGG A - 3’ murine col1a1 reverse 5’- ACT CGA ACG GGA ATC CAT C-3’ murine col1a2 forward 5’-CCA ACA AGC ATG TCT GGT TAG GA- 3’ murine col1a2 reverse 5’- TCA AAC TGG CTG CCA CCA T-3’ 45

Materials and Methods murine Cyclin D1 forward 5’-CAG AGG CGG ATG AGA ACA AG- 3’ murine Cyclin D1 reverse 5’- GTT GTG CGG TAG CAG GAG AG-3’ murine α-SMA forward 5’-ATG CCT CTG GAC GTA CAA CTG-3’ murine α-SMA reverse 5’-CAC ACC ATC TCC AGA GTC CA-3’ murine PAI-1 forward 5’-ACG TTG TGG AAC TGC CCT AC-3’ murine PAI-1 reverse 5’-AGC GAT GAA CAT GCT GAG G-3’ murine smad3 forward 5’- GTG ACC CTT CGG TGC CAG CC -3’ murine smad3 reverse 5’- CGT GAG GGA GCC CCT TCC GA -3’ murine Smad7 forward 5’-GCT CAA TTC GGA CAA CAA GAG-3’ murine Smad7 reverse 5’-TCT TGC TCC GCA CTT TCT G-3’ murine Hhat forward 5’- AGT TGG CCA CGT TAC TCA CA -3’ murine Hhat reverse 5’- GAC CAT GAC AAT CCA GGG TCT-3’ murine siRNA Hhat forward 5’-GUUAAGAGAAGGUGGUACAUU-3’ murine siRNA Hhat reverse 5’-PUGUACCACCUUCUCUUAACUU-3’ murine Ctgf forward 5’- CTG CCT ACC GAC TGG AAG AC -3’ murine Ctgf reverse 5’- TCG CAT CAT AGT TGG GTC TG s-3’ human CTGF ChIP_2 forward 5’- TCC ATT CAG CTC ATT GGC GAG-3’ human CTGF ChIP_2 reverse 5’- TCA GCG GGG AAG AGT TGT TG -3’ human RORa forward 5’- AGC GCC ACA CAC TGC ACA-3’ human RORa reverse 5’- CCC TCC TTT GCC TGA CCC-3’ murine Rora forward 5’-ACC TAC TCC TGT CCT CGT CA-3’ murine Rora reverse 5’-TCT GCT GGT CCG ATC AAT CA-3’ human ARNTL forward 5’- AGC GGA TTG GTC GGA AAG TA -3’ 46

Materials and Methods human ARNTL reverse 5’- AGT CAG TGG AGC TGC CTT TC -3’

Table 7: Primers

2.1.7 Media, buffers and solutions

Fibroblast growth media

10 % DMEM/F-12 0.1 % DMEM/F-12

10 % (v/v) FBS 10 % (v/v) FBS 100 units/ml Penicillin 100 units/ml Penicillin 100 units/ml Streptomycin 100 units/ml Streptomycin 2 mM L-Glutamine 2 mM L-Glutamine 2.5 µg/ml Amphotericin B 2.5 µg/ml Amphotericin B

SHH Light II cell growth media

10 % DMEM/F-12-Glutamax 0.1 % DMEM/F-12Glutamax

10 % (v/v) FBS 10 % (v/v) FBS 0.4mg/ml G418 0.4mg/ml G418 0.15mg/ml Zeocin 0.15mg/ml Zeocin

HEK293 culture media

10 % DMEM culture media Serum free DMEM wash media

10 % (v/v) FBS 50 units/ml Penicillin 50 units/ml Penicillin 50 µg Streptomycin 50 µg Streptomycin 1 mM L-Glutamine 1 mM L-Glutamine 0.5 µg/ml Amphotericin B

Amido black assay

Materials and Methods Amido black solution Washing solution Elution solution

1.3 % Amido black 10B 9: 1 MeOH / HOAc 0.1 M NaOH in 9: 1 MeOH / HOAc

Hematoxylin-Eosin Staining

Hematoxylin Solution Eosin Solution

3.3 mM Haematoxylin 4.8 mM Eosin 1 mM sodium iodate 0.1 % (v/v) glacial acetic acid 105.5 mM potash alum 303 mM chloral hydrat 5.2 mM citric acid 1 % HCl (37 %) in 70 % 2-Propanol

Hydroxyproline assay

HP buffer (pH 6.0) p-Dimethylaminobenzamide solution

260 mM citric acid monohydrate 15 % w/v) p-DMBA 1.46 M sodium acetate trihydrate 60 % (v/v) Methoxyethanol 850 mM sodium hydroxide 26 % (v/v) Perchloric acid 303 mM chloral hydrate 12 % (v/v) glacial acetic acid

Immunohistochemistry

10x PBS (pH 7.2) Sodium citrate buffer (pH 6.0) Tris/ EDTA buffer (pH 9.0)

1.37 M NaCl 10 mM sodium citrate 10 mM Tris 27 mM KCl 0.05 % Tween 20 1 mM EDTA

80 mM Na2HPO4 ad H2O ad H2O

20 mM KH2PO4 ad H2O

Materials and Methods BSA blocking solution Horse serum blocking solution Peroxidase blocking solution

2-10 % (v/v) BSA 5 % (v/v) horse serum 3 % (v/v) 30 % H2O2 in 1 x PBS in 1 x PBS in 1 x PBS

DAB stock solution DAB peroxidase substrate solution

1 % (w/v) DAB 5 % (v/v) DAB ad H2O 5 % (v/v) 0.3 % H2O2 in 1 x PBS

SDS-PAGE and Western Blot

NP-40 lysis buffer 5x loading buffer

150 mM NaCl 250 mM Tris-HCl pH 6.8 1% NP-40 10% (v/v) SDS 50mM pH 8.0 Tris 25mM β-glycerophosphate 50% (v/v) Glycerine 1mM DTT 0.5% (v/v) Bromphenol blue

50mM NaF 5 % (v/v) β-Mercaptoethanol 1μg/ml Proteinase inhibitor 1mM PMSF

4x Stacking buffer (pH 8.8) 4x Resolving buffer (pH 6.8)

1.88 mM Tris/ HCl 1.88 mM Tris/ HCl

10 % Resolving gel 5% Stacking gel

33 % (v/v) 30 % Polyacrylamid 13.3 % (v/v) 30 % Polyacrylamid 25 % (v/v) 4x resolving buffer 25 % (v/v) 4x stacking buffer 1.4 % SDS 0.1 % (v/v) SDS (v/v)

41 % (v/v) ddH2O 57 % (v/v) ddH2O 0.08 % (v/v) APS 1.2 % (v/v) APS 0.05 % (v/v) TEMED 0.08 % (v/v) TEMED

Materials and Methods

10x Running buffer (pH 8.8) 10x Transfer buffer (pH 8.0) 1x Transfer buffer (pH 8.0)

2.5 M Glycine 1.05 M Glycine 10 % (v/v) 10x Transfer buffer 0.25 M Tris 0.5 M Tris 20 % (v/v) Methanol

1 % (w/v) SDS 70 % (v/v) ddH2O

10x TBS (pH 7.6) 1x TBST (pH 7.6) 1x PBST (pH 7.6)

200 mM Tris 200m Tris 10 mM Tris M 1.54M NaCl 1.54M NaCl 1 mM EDTA

0.1% Tween 20 ad H2O

Genotyping digestion buffer 50x TAE buffer pH 8.5 50 mM Tris-HCl pH 8.0 2M Tris-HCl Buffer

100 mM EDTA pH8.0 100mM EDTA

100 mM NaCl

1% SDS

Materials and Methods 2.2 Methods

2.2.1 Human skin biopsies

Skin biopsies were obtained from 45 SSc patients and 50 age- and sex-matched healthy volunteers. Seventeen patients were female, six were male. The median age of SSc patients was 44 years (range: 19 - 61 years), and their median disease duration was 4 years (range: 0.5 - 8 years). Scar tissues from normal skin wounds were obtained from seven volunteers by excisional biopsies. Five of these volunteers were female, two were male. The median age was 41 years (range: 18 – 65 years). Lung tissue was obtained from seven patients with IPF and seven matched non-fibrotic controls. Of the patients with IPF, five were females and two were males. The median age was 51 years (range 40 - 68 years). All patients and control individuals gave informed consent as approved by the local institutional ethical board. The respective number of samples used in each experiment is given in the respective figure legends.

2.2.2 Cell culture

Human fibroblast culture Human dermal fibroblast cultures were prepared as outgrowth cultures from thirteen SSc patients and 14 volunteers. All SSc patients presented with diffuse-cutaneous SSc, and 3 mm punches were taken from lesional skin at the volar side of the forearm. Skin biopsies were digested with 0.1% type I collagenase at 37°C for 2 h. After inactivation with normal medium and centrifugation (1400 r.p.m, 5 min), the cell pellets were washed with sterile PBS. At the end resuspended cell pellets in Dulbecco’s modified Eagle’s medium Ham’s F-12 (DMEM/F- 12 )medium containing 10% heat inactivated fetal calf serum (FCS), 25 mM HEPES, 100 U/ml penicillin, 100 g/ml streptomycin, 2 mM L-glutamine, 2.5 g/ml amphotericin B. Fibroblasts from passages 4-9 were used for all experiments.

Human fibroblast stimulation and treatment with GANT-61

Fibroblasts were seeded in 6-well plates and grown in DMEM/F-12 supplemented with 10 % FBS 100.0 U/mL penicillin, 100.0 g/ml streptomycin, 2.0 mM L-glutamine, 2.5 g/ml amphotericin B (all Gibco BRL, Basel, Switzerland) until cells reached 80-90% confluence. FBS was reduced to 0.1 % and supplemented with vitamin C (conc.1000ng/ml).

Materials and Methods Cells were stimulated with 10.0 ng/ml human recombinant TGFβ (Preprotech, Rocky Hill, New Jersey, USA) for twenty-four hours. Another 24 hours after stimulation, cells were lysed in RA1 buffer (Macherey-Nagel™ RNA Isolation) for RNA analysis or in NP-40 lysis buffer for Western Blot. Supernatants were collected for collagen measurements. For specific experiments, cells were pretreated with GANT-61 (Tocris, Germany), in a concentration of 10.0 µM 1 hour before TGFβ stimulation.

Murine fibroblast culture

Murine fibroblasts were isolated from skin biopsies of GLI2fl/fl mice. Mice dorsal skin was shaved and cleaned with 70% Ethanol and all subcutaneous fat was removed. Incubate it in 5mg/ml Dispase solution for 45 min at 37°C on a shaker (80 rpm) then separate dermis from epidermis. Dermis was cut the in small pieces and digested in 0.4mg/ml Collagenase D plus 0.2mg/ml DNase I in RPMI medium for 30 min at 37°C on a shaker (1000 rpm). Enzymatic digestion was stopped by 10% FCS RPMI plus 10mM EDTA, the samples were filtered with 100 µl cellstrainer, centrifuged, resuspended and cultured with DMEM/F12 medium containing 10 % heat inactivated fetal calf serum (FCS), 25 mM HEPES, 100 U/ml penicillin, 100 g/ml streptomycin, 2 mM L-glutamine, 2.5 g/ml amphotericin B (all Gibco BRL, Basel, Switzerland). Fibroblasts from passages 4-8 were used for all experiments. Cre mediated recombination in murine fibroblasts isolated from Gli2fl/fl mice were induced by infection with adeno-associated viruses encoding for Cre recombinase at an ifu /fibroblast ratio of 100. Adeno-associated virus encoding for LacZ served as controls. To stimulate cells recombinant TGFβ (10 ng/ml) was added into medium. In selective experiments, cells were pretreated with GANT-61 (Tocris, Germany), in a concentration of 10.0 µM 1 hour before TGFβ stimulation.

Chamber slides for stress fibers staining and quantification

To investigate the potential of GANT-61 to impair or induce stress fiber development, fibroblasts were incubated with GANT-61 for 1 h in 0.1 % DMEM/F12 and stimulated with TGFβ (10.0 mg/ml, Peprotech, Rocky Hill, New Jersey, USA). After 24 hours, the cultured fibroblasts were first washed with 1x PBS and then fixed with 4% methanol free paraformaldehyde at room temperature for 10 minutes. Fixed cells were permeabilized for five minutes at room temperature with 0.25 % Triton X-100. Cells were incubated with 2 % BSA blocking solution for one hour. α-SMA was detected with mouse monoclonal anti-α-SMA 52

Materials and Methods (clone 1A4, Sigma-Aldrich, Steinheim, Germany) overnight at 4°C. Polyclonal goat anti- mouse Alexa Fluor 488 (Invitrogen, Karlsruhe, Germany) was used as secondary antibody. For visualizing stress fiber formation, rhodamine phalloidin (1:40 dilution in 2 % BSA, #R415, Sigma-Aldrich) which labels cytoskeletal F-actin was stained at RT for 20 min . In addition, cell nuclei were stained with DAPI. Images were captured at a 200-fold magnification. Fluorescence intensity was quantified in a blinded manner using Fiji software.

Cell Viability and Cytotoxicity Assays

Cell viability of cultured cells was quantified using the Cell Counting Kit 8 (Dojindo Molecular Technologies, Maryland, USA) and an MRX ELISA reader (Dynex Technologies, Chantilly, USA). In Brief, cells were seeded in a 96-well plate at numbers of 3000 cells/well. The next day, cells were treated with GANT-61 in concentrations of 1 to 1000 µM. After an incubation of 20 hours, 10 μl of CCK8 were added into the culture medium of each well. The plates were then incubated another 4 hours. The OD at 450 nm was quantified using a MRX ELISA reader (Dynex Technologies, Chantilly, USA).

2.2.3 Nucleofection

Human Smad3 siRNA, human skinny hedgehog siRNA (skn, s5438) were obtained from Eurogentec (Cologne, Germany). Non-targeting siRNAs (Life Technologies, Darmstadt, Germany) served as controls. Fibroblasts were transfected with 3 μg of siRNA by using the Amaxa 4D-Nucleofector (Amaxa, Cologne, Germany). 0.5-1.0 x 106 fibroblasts were resuspended in Nucleofection solution (Amaxa P2 Primary Cell 4D-Nucleofactor X Kit) containing 3 μg of either targeting siRNA or non-target siRNA then transferred into a Nucleo- cuvette. A predefined Nucleofector program for neonatal human dermal fibroblasts (program number DT-130 which provided by the manufacturer with unspecified assay conditions) was used to perform the transfection. Fibroblasts were transferred from the cuvette with plastic pipettes into 6-well plates or chamber slide with pre-warmed DMEM/ F12 medium immediately. The medium was changed to DMEM/ F12 with 0.1% FCS post 12hrs and followed by 24hrs starvation. In selective experiments, cells were incubated with recombinant TGFβ (10 ng/ml) (PeproTech, Hamburg, Germany), inhibitor GANT-61 (Tocris, Germany).

2.2.4 FuGENE HD Plasmids Transfection

Materials and Methods Plasmid transfection into cell line by FuGENE HD reagent (Promega, Mannheim, Germany) followed manufacture´s protocol. Briefly, cells were plated one or two days before the transfection experiment and grown to approximately 80% confluency. On the day of transfection, DNA was diluted to 2µg per 100µl of serum-free medium. FuGENE HD Transfection Reagent was added to achieve 3:1 ratio of reagent to DNA and incubated for 15 minutes. 5µl (96-well plate), 13µl (48-well plate) and 25µl (24-well plate) of FuGENE HD Transfection reagent:DNA mixture was added to each well of cells. There is no need to remove serum or change culture conditions. Cells were incubated for 24–48 hours.

2.2.5 Plasmids

The LightSwitch-Promoter-GoClone CTGF promoter reporter plasmid and the control vector were purchased from Active Motif (Active Motif, Belgium). Mutations of the GLI binding sites on CTGF promoter region with substitution of CC to GG were introduced into the CTGF promoter at –623 (CTGF_Mut1_-623) and –9 (CTGF_Mut1_-9) using QuickChange-Multisite Mutagenesis Kit (Agilent Technologies) of the CTGF core promoter sequence.

2.2.6 Plasmid isolation (Maxi Prep)

Plasmid preparation of the LightSwitch-Promoter-GoClone CTGF plasmid; the control vector and two mutant CTGF promoter reporter plasmids was perfomed according to the manufacture’s protocol (Qiagen, Düsseldorf, Germany). Single colonies were picked from selective plates and inoculated into a starter culture of 5 ml LB medium containing specific antibiotics. LB medium was incubated for 8 h at 37 °C with vigorous shaking at 200 rpm. Then the starting culture was added into 250 ml LB medium also with antibiotics and kept under growing conditions for 15 h at 37 °C and 200 rpm. Bacteria were harvested by centrifugation and the pellet was re-suspended in 10 ml Buffer P1 containing RNase A. After lysed the bacteria by using 10 ml Buffer P2 at RT for 5 min, 10 ml of ice-cold Buffer P3 was added to the cells and incubated on ice for 20 min. The lysate was applied to the equilibrated column (Qiagen-tip 500). After two times washing, DNA was eluted by using 15 ml Buffer QF. The DNA was precipitated by adding 10.5 ml isopropanol and subsequent centrifugation. The supernatant was carefully discarded and the pellet was resuspended in 5 ml of 70 % ethanol. After the last centrifugation, ethanol was removed carefully and the pellet was dried at RT for 5-10 min and

Materials and Methods the DNA was dissolved in 0.5 ml nuclease-free water. The concentration and the quality of DNA were determined by using NanoDrop™ 2000/2000c device.

2.2.7 Side-directed mutagenesis

For mutated GLI binding sites on CTGF promoter region (Bs1, Bs2), two essential C nucleotides (underlined) were changed to two G nucleotides. In vitro site-directed mutagenesis was performed according to the manufacture’s procedure (QuikChange Multi Site-Directed Mutagenesis Kit, Agilent Technologies, Germany). In brief, first the mutagenic primer (5'- gtctttgttctctttcttgtggcaaaaccgttacctcaagtg-3' for GLI Bs 1 at -623 CTGF promoter region and 5'- tcgggccgcccgcggcaaactcacacaac-3' for Bs 2 at -9 CTGF promoter region) was annealed to denatured template DNA. PfuTurbo DNA polymerase then extended the mutagenic primer with high fidelityll and without primer displacement, generating ds-DNA molecules with one strand bearing multiple mutations and containing nicks. The nicks were sealed by components in the enzyme blend. Afterwards, the thermal cycling reaction (1 cycle 95°C 1min, 30 cycles of (95°C 1min, 55°C 1min, 65°C 2 minutes/kb of plasmid length) products were treated at 37°C for 1 hour with the restriction endonuclease Dpn I , which is specific for methylated and hemimethylated DNA6 and was used to digest the parental DNA template. Finally, the reaction mixture, enriched for multiply mutated single stranded DNA, was transformed into XL10-Gold ultracompetent cells, where the mutant closed circle ss-DNA was converted into duplex form in vivo. The transformation plates were incubated at 37°C for >16 hours. Single colonies were picked from selective plates and inoculated into a starter culture of 5 ml LB medium containing specific antibiotics for mini preparation then sent for sequencing to identify correct mutated plasmids. Widetype CTGF promoter reporter plasmid and two mutant reporter plasmids were transfected into human fibroblast, and then luciferase activity was measured upon TGF challenge. .

2.2.8 Chromatin immunoprecipitation

Chromatin immunoprecipitation (ChIP) assays were performed using the ChIP-IT® Express Chromatin Immunoprecipitation Kits (Active Motif, Germany) according to manufacturer’s instructions. Briefly, 20 μg of sonicated chromatin extracts were incubated with anti-Gli2 antibodies (abcam 26056, Germany) or normal rabbit IgG antibodies (Santa Cruz Biotechnology, Heidelberg, Germany) and complexes were precipitated by magnetic beads.

Materials and Methods After reversal of cross-linking, the immunoprecipitated chromatin DNA was purified using the ChromatinIP-DNA-Purification Kit (Active Motif). ChIP and input DNA was measured by quantitative real-time PCR and normalized to input values.

2.2.9 Luciferase-repoter assay

Fibroblast cells were transfected with wild type or mutated CTGF promoter luciferase reporter plasmids or pSv-β-galactosidase (β-gal) using FuGENE HD Transfection Reagent (Promega, Mannheim, Germany). Experiments were conducted 24–48 h after transfection. Luciferase activity was normalized for transfection efficiency to internal pSv-β-galactosidase or Renila luciferase activity. Luciferase activities were determined using a microplate luminometer (Berthold Technologies, Bad Herrenalb, Germany). NIH3T3-Light2 cells were kindly provided by Prof. Dr. Suzanne Eaton (Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany). Cells were transfected with siRNA against Hhat or non-targeting control siRNA (n.t.siRNA) by nucleofection as described. 24h after transfection, cells were serum starved and stimulated with recombinant TGF. The fluorescence intensity was measured at a Luminoskan Ascent Microplate-Luminometer (ThermoFisher, Bonn, Germany).

2.2.10 Coculture systems

In transwell co-culture assays, direct cell-cell contact was prevented by using cell culture insert with a 0.4 µm pore size polycarbonate membrane (Falcon) in a 24-well plate. 3 x 104 of SHH- light II reporter cells were cultured in the bottom of the well (100% confluence), whereas 3 x 104 NIH3T3 fibroblasts transfected with n.t.siRNA or Hhat siRNA were placed in the insert of 24-well plate (100% confluence). NIH3T3 cells were serum starved for 24 h and infected with adenoviruses encoding for LacZ or TBR. 24 hours after infection, the insert was removed, SHH- light II reporter cells were lysed and subsequent analyzed by Dual-Luciferase assays. In direct co-culture system, 2 x 104 of NIH3T3 cells transfected with Hhat siRNA or n.t. siRNA and 2 x 104 of SHH-light II (together with the NIH3T3 cells 100% confluency) cells were placed in a single well of 24-well plate. Cells were serum starved for 24 h and infected with adenoviruses LacZ or TBR. 24 h after infection, cells were lysed and Dual-luciferase were performed.

Materials and Methods 2.2.11 Mice

C57Bl/6 mice were purchased from Janvier (Le Genest Saint Isle, France). GANT-61 (Tocris, Germany) was dissolved in DMSO and further diluted in PBS (1:10 and 1:20) for intraperitoneal application (2.5 mg/kg and 5.0 mg/kg) twice daily during bleomycin challenge. Vismodegib (Celgene, Summit, New Jersey, USA) was suspended in 1 % methylcellulose and applied orally twice daily during bleomycin challenge (5.0 mg/kg and 25.0 mg/kg). The RORα specific inhibitor SR3335 and ROR RORα/γ inhibitor SR1001 were dissolved in 10% tween- 80 and 10% DMSO in PBS and injected intraperitoneally at a dose of 15mg/kg and 25mg/kg, respectively. Negative control groups were injected with the vehicle 10% tween and 10% DMSO in PBS. Mice were treated with inhibitors every 12 hours.

Conditional knockout of GLI2 in murine models

Mice carrying two conditional alleles of Gli2 (Gli2fl/fl) (provided by Walter Birchmeier, Max Delbrück Center for Molecular Medicine, Berlin, Germany) were crossbred with Col1a2- CreER*TBRI mice (Feil et al. 1997, Zheng et al. 2002). Cre-mediated recombination was induced by repeated intraperitoneal injections of approximately 75 mg tamoxifen/kg body weight. Dissolve tamoxifen (Sigma-Aldrich) in corn oil at a concentration of 20 mg/ml, and was applied once every 24 hours for a total of 5 consecutive days. Control groups were injected with corn oil. The role of Gli2 mediated signaling in fibrosis was investigated. The following four groups were analyzed: 1) Gli2fl/fl X Col1a2-CreER*TBRwt/wt mice administered with oil. 2) Gli2fl/fl X Col1a2-CreER*TBRact mice administered with oil. 3) Gli2fl/fl X Col1a2- CreER*TBRwt/wt mice administered with tamoxifen. 4) Gli2fl/fl X Col1a2-CreER*TBRact mice administered with tamoxifen.

TBRact -induced dermal fibrosis treated with GANT-61

TBRact-induced skin fibrosis was induced in 4-week-old mice (either wildtype mice on a C57BL/6 wildtype purchased from Janvier( Illkirch, France) by receiving of 6.67 × 107 pfu/mouse of replication-deficient type 5 adenoviruses encoding for TBRIact into defined areas of 1 cm2 at the upper back four times per 2 months. Mice injected with 0.9 % sodium chloride served as controls. Mice were treated with intraperitoneal injection of GLI inhibihiotr GANT- 61(2.5 mg/kg and 5.0 mg/kg) twice daily or an oral gavage of Smo inhibitor Vismodegib (5.0 mg/kg and 25.0 mg/kg) twice daily. 57

Materials and Methods

Bleomycin-induced lung fibrosis treated with GANT-61

Pulmonary fibrosis model was induced by single intratracheal instillation of 50µl Bleomycin (0.5 mg/ml) in 8 weeks old C57BL/6 mice (Janvier, France) by using a high-pressure syringe (Penn-Century, Wyndmoor, PA, USA). The mice were sacrificed for further analysis at day 10 or 4 weeks later. Mice were treated with intraperitoneal injection of GLI inhibihiotr GANT- 61(2.5 mg/kg and 5.0 mg/kg) twice daily or an oral gavage of Smo inhibitor Vismodegib (5.0 mg/kg and 25.0 mg/kg) twice daily. Instillation of equal volumes of 0.9 % NaCl served as a control.

Hhat siRNA knockdown in Bleomycin-or Topo-induced dermal fibrosis

Bleomycin-induced skin fibrosis was induced by local injections of bleomycin at a concentration of 0.5 mg/ml in defined areas of 1 cm2 at the upper back every other day for 4 weeks (6 weeks of age, mixed genders). Topo-induced fibrosis was induced in 6-week-old female C57Bl/6 mice by subcutaneous injections of 150 µl of 500 U/ml of recombinant TopoisomeraseI mix with 100 µl of Complete Freund’s Adjuvant (CFA) in a 1cm2 defined areas (5 points) on the upper back of the mice every other week for eight weeks. Controls were injected with 150µl NaCl with 100µl of CFA. After 8-week Topoisomerase I challenge, mice were sacrificed and the injected skin processed for further analysis. Hhat siRNA was prepared as 10 μM with RNAse-free siRNA buffer (Atelocollagen Kit; KOKEN, Japan). Mix Hhat siRNA with atelocollagen as 1:1 in a syringe (with 27G needle) and inject 20μl into each of the five positions of defined area. Inject mice once a week and knockdown efficacy is determined via Real-Time PCR and Western Blot.

Conditional knockout of ROR in murine fibrosis models

Mice carrying conditional alleles of Ror (Rorfl/fl) (Purchased from Institut Clinique de la Souris (ICS), France) were crossbred with Col1a2-CreER mice. Cre-mediated recombination was induced by repeated intraperitoneal injections of 100µl of 20 mg/ml tamoxifen for once every 24 hours for a total of 5 consecutive days. Control groups were injected with corn oil. The role of Ror mediated signaling in fibrosis was investigated in Bleomycin-induced skin fibrosis and lung firbosis. The following four groups were analyzed: 1) Rorfl/fl X Col1a2- CreER mice administered with corn oil and NaCl. 2) Rorfl/fl X Col1a2-CreER mice 58

Materials and Methods administered with corn oil and Bleomycin. 3) Rorfl/fl X Col1a2-CreER mice administered with tamoxifen and NaCl. 4) Rorfl/fl X Col1a2-CreER mice administered with tamoxifen and Bleomycin.

Wnt10b transgenic mice

In these studies, we used transgenic mice overexpressing murine Wnt10b under the control of a FABP4 promoter (Wnt10b tg mice). (Longo et al. 2004)Wnt10b tg mice resulted in a massive generalized dermal fibrosis. fibrotic changes were already observed at an age of 3 weeks and steadily progressed over time. At the age of 12 weeks, the dermis was almost sixfold thicker in Wnt-10b tg mice than in control mice. The hydroxyproline content of the skin progressively increased in Wnt-10b tg mice and was tenfold higher in 12-week-old mice. Myofibroblast counts were significantly increased in Wnt-10b tg mice. (Akhmetshina et al. 2012) The RORα specific inhibitor SR3335 injected intraperitoneally at a dose of 15mg/kg twice daily.

ROR inhibitor SR3335 and SR1001 treatment in Bleomycin-or TBRact- induced dermal fibrosis

Bleomycin-induced and TBRact-induced skin fibrosis model were applied as described above. The RORα specific inhibitor SR3335 and ROR RORα/ inhibitor SR1001 were dissolved in 10% tween-80 and 10% DMSO in PBS and injected intraperitoneally at a dose of 15mg/kg and 25mg/kg, respectively. Negative control groups were injected with the vehicle 10% tween and 10% DMSO in PBS. Mice were treated with inhibitors every 12 hours.

ROR inhibitor SR3335 treatment in Bleomycin-induced lung fibrosis

Bleomycin-induced lung fibrosis was induced as described above. The RORα specific inhibitor SR3335 was dissolved in 10% tween-80 and 10% DMSO in PBS and injected intraperitoneally at a dose of 15mg/kg. Mice were treated with inhibitors every 12 hours.

ROR inhibitor SR3335 treatment in CCL4-indeuced liver fibrosis model

CCL4-induced hepatic fibrosis was initiated by intraperitoneal (i.p.) injections of CCL4 diluted in sunflower oil (week 1:1:31 dilution; week 2: 1:15 dilution; week 3: 1:7 dilution; week 4–6: 1:3 dilution) in mice (8 weeks of age, mixed genders) three times per week. Sunflower oil was

Materials and Methods used in the control group.(Hernandez-Gea et al. 2012). The mice were sacrificed for further analysis at 3-week old or 6 weeks later. The RORα specific inhibitor SR3335 was dissolved in 10% tween-80 and 10% DMSO in PBS and injected intraperitoneally at a dose of 15mg/kg. Mice were treated with inhibitors every 12 hours.

2.2.12 RNA Analysis

RNA isolation

Total RNA samples were isolated with the NucleoSpin RNA II extraction system (Macherey- Nagel, Düren, Germany) by following the instructions of the manufacturer.

Reverse transcription

Reverse transcription of RNA into cDNA was performed using random hexamers and the TaqPolymerase (Applied Biosystems, Foster City, California, USA) on a Thermocycler T personal (Biometra GmbH, Germany). The cDNA samples from the group without added reverse transcriptase in the reverse transcription reaction served as Non-RT-controls (Negative controls) were used to exclude genomic contamination.

Quantitative real-time PCR

Gene expression was quantified by SYBR Green real-time PCR with the StepOne™ System qPCR System (Thermo Fischer Scientific, Waltham, Massachusetts, USA) or Stratagene Mx3005 System (Agilent Technologies, Santa Clara, CA, USA). β-actin was used to normalize for the amounts of loaded cDNA within each sample. The gene expression level was calculated with the threshold cycle (Ct) and the comparative Ct method (referred as the 2−ΔΔCT method) for relative quantification.

2.2.13 Histological Analysis

After sacrifice, skin and lung samples were fixed in formalin overnight and then embedded in paraffin. Skin sections were stained with hematoxylin and eosin and lung samples were stained with Sirius Red. Dermal thickness was measured at four different sites in each mouse in a

Materials and Methods blinded manner. For visualization of collagen proteins, Trichrome and Sirius Red staining were performed (Sigma-Aldrich, Steinheim, Germany)(Liang et al. 2017).

Hematoxylin/Eosine staining (HE)

For determination of skin thickness and scoring of inflammatory infiltrates, murine skin specimen were fixed in 4 % paraformaldehyde, embedded in paraffin and cut into 5 µm sections. The sections were then stained with hematoxylin and eosin. Dermal thickness and were analyzed at 100-fold magnification (Nikon Eclipse 80i) by measuring the distance between epidermal-dermal junction and the dermal-subcutaneous fat junction. Dermal infiltration by leukocytes was assessed by a semi-quantitative scoring with 0 = no / 1 = slight / 2 = moderate / 3 = severe inflammatory infiltrate / 4 = very severe inflammatory infiltrate.

Trichrome staining

After deparaffinization and rehydration, the sections were incubated in Bouin’s Solution for 15 min at 56°C and then stained with Weigert’s iron hematoxylin and Biebrich Scarlet-Acid Fuchsin solution. Incubation of the slides with phosphotungstic and phosphomolybdic acid were followed by two minutes’ incubation with aniline blue. Under the microscope, collagen proteins were stained in light blue (Liang et al. 2017).

Sirius red staining

To show collagen bundles in murine lung and skin, specimen were fixed in 4 % paraformaldehyde, embedded in paraffin and cut into 5 µm sections. The sections were then stained with Sirius red reagents for one hour followed by hematoxylin staining. For lung sections, images from sections stained with Sirius Red were captured using a Nikon Eclipse 80i microscope at 40-fold or 100-fold magnification and the fibrotic area in each image was calculated using Fiji with ThresholdColor plugin (Varga and Hinchcliff 2014). Briefly, the relative of fibrotic area was calculated by dividing the area, in which the intensity is above a threshold set by the threshold color menu divided by the total area. The same thresholds and system settings were used for all mice within the same experiment.

2.2.14 Protein analysis

Materials and Methods Hydroxyproline assay for tissue samples

To measure the amount of collagen protein in tissue samples from skin was determined via hydroxyproline assay. The skin biopsies (Ø 3 mm) were digested in 6 M HCl for three hours at 120 °C, and adjusted the samples to pH 7 with 6 M NaOH. Subsequently, 0.06 M chloramine T was added to each sample and incubated for 20 min at RT. Next, 3.15 M perchloric acid and 20 % p-dimethylaminobenzaldehyde were added and incubated for another 20 min at 60 °C. The absorbance of 557 nm was measured with a Spectra MAX 190 microplate spectrophotometer (Avouac et al. 2012). The amount of collagen protein in lung tissue samples were also measured using hydroxyproline assays. The special procedure for the lung samples was to remove extra water from the tissue by heated the sample at 80 °C until the weight did not decrease anymore. The net weight of lung was measured and the dry lungs were digested in 6 M HCl at 120 °C for overnight. After digestion, the supernatant was collected. The following steps were the same as hydroxyproline assay for skin.

Quantification of soluble collagen protein

The amount of collagen which secreted by fibroblast into cell culture supernatants was quantified via using the SirCol collagen assay (Biocolor, Belfast, Northern Ireland). According to the instruction of the manufacturer, 200 μl of collected supernatant mixed with 400 μl of Sircol Dye Reagent, an anionic dye that reacts specifically with basic side chain groups of collagens then incubated in a gentle mechanical shaker for 30 min at RT to precipitate the collagen-dye complex. The precipitate was collected and dissolved in 250 μl Alkali Reagent. The absorbance was measured at 555 nm in a microwell plate MRX ELISA reader (Dynex Technologies, Chantilly, VA, USA).

Immunohistochemistry staining (IHC)

For IHC, paraffin-embedded skin sections from human or mice were first deparaffinized by xylene and ethanol. Heat-induced epitope/antigen retrieval was performed by boiling the deparaffinized sections in citrate buffer (pH6) and Tris-EDTA (pH9) and followed by blocking with 5% BSA/PBS or 5% horse serum in 2% BSA/PBS. Afterwards, the slides were incubated with primary antibodies Hhat (1:50, Thermo fisher, Germany) or SMA (1:250, Life Technologies, Germany) at 4°C overnight. Species-specific HRP-conjugated antibodies served 62

Materials and Methods as secondary antibody. Slides incubated with isotype control antibodies and proceeded as described for used as negative controls. The staining of the slides was visualized using the Nikon Eclipse 80i microscope. In addition, myofibroblasts were identified as single double- positive cells for αSMA and vimentin stained.

Immunofluorescence staining (IF)

Formalin-fixed, paraffin-embedded skin sections were deparaffinized by xylene and ethanol. After heat-induced epitope/antigen retrieval and blocking, sections stained with primary antibodies against αSMA (1:1000,Life Technologies, Germany), rhodamine phalloidin (1: 1000, Life Technologies, Darmstadt, Germany), Gli2 (1:100, sc-280, Santa Cruz, Heidelberg, Germany), p4Hβ (1:50, AP08767PU-N, Acris Antibodies, Herford, Germany),Vimentin (1:1000, ab20346, Abcam), Smad3 (1:100, ab52903, Abcam ), Hha t(1:100, SAB1303011, Sigma-Aldrich ), RORα (1:200, sc-28612, Santa Cruz), -catenin (1:100, ab2365, abcam) at 4 °C overnight. After washing with 1X PBS, Alexa Fluor antibodies (Life Technologies) were used as secondary antibodies at RT for 1 h. irrelevant isotype matched antibodies served as controls. Nuclei were counterstained using DAPI (1:800, sc-3598, Santa Cruz, Heidelberg, Germany). Concentration-matched species-specific immunoglobulins (Vector Laboratories) served as control antibodies. The staining was analyzed using a Nikon Eclipse 80i microscope (Nikon, Badhoevedorp, Netherlands).

Co-immunoprecipitation (CoIP)

Protein samples were collected in NP-40 lysis buffer. 20 μg of lysate served as input. Before the CoIP, using 500 μg of Cell lysates incubated with 30 μl Protein A/G Sepharose beads for 30 mins in 4°C to remove the unspecific binding as a pre-cleaning step. Afterward, we collected the supernatant and incubated it with target antibodies in 4°C overnight and then added 30 μl Protein A/G Sepharose beads to bind with target antibodies for another 2 to 4 hours. Samples were then washed with 0.1% NP-40 lysis buffer to remove unbound proteins. Protein A/G Sepharose beads bound protein complexes were separated via SDS-PAGE and further analyzed by Western blotting.

Western blot analysis

Materials and Methods Protein samples were separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel and electrotransferred onto polyvinylidene fluoride membranes (Millipore, Billerica, Massachusetts, USA). After blocking, membranes were incubated with polyclonal antibodies against GLI2 (Santa Cruz, Heidelberg, Germany), polyclonal antibodies against Smad3 (Santa Cruz), polyclonal antibodies against HHAT (Santa Cruz, Heidelberg, Germany), polyclonal antibodies against SHH (Santa Cruz) were used overnight at 4°C. Membranes were incubated with horseradish-peroxidase-conjugated secondary antibodies (Dako, Glostrup, Denmark). Blots were visualized by ECL. Beta-actin antibody was used as loading control. Western Blots were quantified using the ImageJ Software (version 1.41). Conditioned cell supernatants from fibroblasts transfected with Hhat siRNA or non-targeting siRNA were collected and centrifuged at 500 g for 15 minutes to remove cellular debris. The supernatants were concentrated by using Amicon Ultra-0.5 mL Centrifugal Filters 50 kDa to enrich for oligomeric SHH (75 kDa, 120 kDa, 180 kDa)(Hernandez-Gea et al. 2012).The flow- through was concentrated by trichloroacetic acid precipitation to enrich for monomeric SHH (19 kDa). After addition of reducing SDS sample buffer, samples were heated at 95 °C for 10 minutes, briefly centrifuged at 14,000 x g for 5 minutes, and loaded onto the 12% (for oligomeric SHH) and 6% (for monomeric SHH) SDS-PAGE. We confirmed these findings by an additional approach. In this approach, proteins in the supernatants were cross-linked with 1% PFA for 10min. The supernatants were then applied to Amicon Ultra-0.5 mL Centrifugal Filters and the flow-through was precipitated by TCA and further preceded as described above.

2.2.15 Amplification and purification of adenoviral vectors

HEK293A cells were grown to 70-80% confluency for virus infection. Several rounds of infection were performed to increase the yield. Two to three days after infection, more than 70% of cells were floating, indicating that a sufficient number of viral particles were available for collection and further purification. After spinning down of the cell debris and three cycles of freezing and thawing with liquid nitrogen and 37°C incubation in the water bath, a CsCl2 gradient solution was prepared and the virus supernatant was loaded on top. The gradient solution was centrifuged at 35000rpm for 2 h at 12°C with SW41 rotor by Optima LE-80 ultracentrifuge (Beckman Coulter, Pasadena, USA). The viral particles appeared as a bluish- white rim in the centrifuge tube. The viral particles were collected and further purified using Bio-Rad EconoPac columns (Bio-Rad, Hercules, USA). Afterwards, the titer of viral particles was determined by Adeno-X Rapid titer kits (Takara Bio USA, CA, USA).

Materials and Methods

Adenovirus Rapid titer kits

HEK293 cells were seeded at a density of 5 x 105 cells/ml in 12 well plates. Right after seeding, the virus was added with 10-fold serial dilutions ranging from 10-2 to 10-6ml. The cells were incubated at 37°C in 5% CO2 for 48 h. Then, the medium was aspirated and the cell culture plates were dried in the hood. The cells were fixed with 1ml ice-cold 100% methanol and incubated at -20°C for 10 mins. After removal of methanol, the cells were incubated with mouse anti-Hexon antibodies for 1 h at 37°C. After washing with PBS with 1% BSA, the cells were incubated with HRP conjugated rat anti-mouse antibodies for 1 h at 37°C. DAB solution was added for 10 mins and the staining cells were counted at 20-fold magnification.

2.2.16 Statistical Analysis

All data are presented as median with interquartile range (IQR). Differences between the groups were tested for their statistical significance by Mann-Whitney U test for non-related samples and by the Wilcoxon signed rank tests for related samples. In a subset of experiments, the mean values of the control groups were set to 1. All other values were expressed as fold changes compared with the respective controls used as ‘comparison mean values’. P-values less than 0.05 were considered significant. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001.

Results 3 Results

3.1 Evaluation of the HH transcription factor GLI2 in SSc

3.1.1 Upregulated expression of GLI2 in fibrotic conditions

We and others previously demonstrated that canonical HH signaling components, such as SHH, PTCH, and SMO are upregulated in patients with SSc and in other fibrotic diseases (Bolanos et al. 2012, Kramann 2016, Horn et al. 2012a, Horn et al. 2012b, Distler et al. 2014). In this part of the thesis, we aimed to characterize the role of GLI2, which has recently been shown to be upregulated in idiopathic pulmonary fibrosis (Bolanos et al. 2012). To investigate the role of GLI2 in the pathogenesis of SSc, we then analyzed the expression of GLI2 in different fibrotic conditions. The mRNA level of GLI2 was significantly increased in fibrotic skin of SSc patients compared to matched healthy individuals (Figures 8A). A significant increase in protein levels was also detected in cultured SSc fibroblasts as compared to normal controls (Figure 8B). Quantification of the western blots with Image J reflects the relative amounts of the proteins versus the loading control β-actin.

Figure 8: The expression of GLI2 is increases in fibroblasts in SSc skin. (a) Real-time PCR analysis showing the absolute mRNA expression levels of GLI2 in SSc skin and in skin of matched healthy individuals (n ≥ 5 for both groups). (b) Fold changes in the protein levels of GLI2 in SSc skin vs. healthy skin (n ≥ 6 for both groups). Representative Western blot results (left) and quantification (bar graph, right) of the results are shown (n ≥ 6 for both groups). Results are shown as median ± IQR. The statistical significance was determined by Mann-Whitney testing. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001.

Skin sections derived from patients with SSc were triple stained for GLI2, the fibroblast marker prolyl-4-hydroxylase-β and 4, 6-diamidino-2-phenylindole (DAPI).Semi-quantitative analysis

Results further highlighted the prominent induction of GLI2 in SSc fibroblasts compared to those in healthy skin (Figure 9).

Figure 9: GLI2 is upregulated in the skin of SSc patients Immunofluorescence staining for GLI2 in skin sections of SSc patients and matched controls co-stained with the fibroblast marker P4H. Representative images and a quantification with Image J are shown (200- and 600-fold magnification, scale bars, 10 μm; n = 5 per group). *: 0.01 ≤ p < 0.05, **: 0.001 ≤ p < 0.01, ***: p < 0.001

3.1.2 GLI2 is upregulated by TGFβ in a Smad-dependent manner

Aberrant TGFβ signaling is a major hallmark of SSc and other fibrotic diseases. The upregulation of GLI2 persisted in cultured SSc fibroblasts, which maintained high mRNA and protein levels of GLI2 compared with control fibroblasts even after several passages in culture. To analyze whether TGFβ contributes to the upregulation of GLI2 in fibrotic skin, human dermal fibroblasts were stimulated with recombinant TGFβ.Indeed, short-term stimulation with TGFβ upregulated the mRNA and protein levels of GLI2 in cultured human dermal fibroblasts in time-dependent manner with maximal effects after 3 h for mRNA and 12-24 h for protein (Figure 10A, B). In addition, immunofluorescent staining GLI2 and stress fiber on healthy human dermal fibroblasts with TGFβ also showed the same pattern (Figure 10C).

Results

Figure 10: TGFβ induces the expression of GLI2 in cultured fibroblasts. (A–C) TGFβ signaling upregulates GLI2 expression and in human healthy dermal cultured fibroblasts. mRNA (A) and protein levels (B and C) of GLI2 as rt-qPCR, western blot and immunofluorescence staininig, respectively, in TGFβ stimulated fibroblasts (n=4 for both experimental settings). Representative images are shown at 200-fold magnification.). Results are shown as median ± IQR. The statistical significance was determined by Mann Whitney testing. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001.

Furthermore, increased levels of GLI2 were also observed in the skin of mice overexpressing a constitutively active TGFβ receptor type I (TBRact) in vivo (Figure 11). Furthermore, when TGFβ signaling was blocked by the selective TBRI inhibitor SD-208 upregulation of GLI2 was strongly abrogated in TBRact-induced and bleomycin-induced fibrosis as well as in Tsk-1 mice on mRNA and protein level respectively. Taken together, these results demonstrate that TGFβ could directly lead to induction of GLI2 expression in fibroblasts.

Results

Figure 11: Gli2 expression in experimental models of skin fibrosis models with and without SD-208 treatment. (A) Gli2 expression in TBRact-induced dermal fibrosis as analysed by western blotting. Quantification (bar graph, right) of the results is shown. (B-F) Effects of the selective transforming growth factor receptor (TBR) inhibitor SD-208 in on the mRNA levels of Gli2 and protein level (costaining for Gli2, vimentin and DAPI) in three experimental models of skin fibrosis: (B, C) TBRact-induced fibrosis and (D, E) bleomycin-induced skin fibrosis as well as in (F, G) Tsk-1 mice. Representative images are shown by at a 1000-fold magnification. (n=4 for all groups and models).Results are shown as median ± IQR. The statistical significance was determined by Mann Whitney testing. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001.

Results Triple staining with the fibroblast marker prolyl-4-hydroxylase-β (P4Hβ) and TGFβ sinalling mediator SMAD3 showed that GLI2-positive cells also stained positive for pSMAD3 in SSc fibroblasts, which is demonstrating active canonical TGFβ signaling in GLI2-positive fibroblasts (Figure 12). As in human SSc, GLI2-positive cells uniformly stained positive for pSMAD3 in the skin of TBRact-mice.

Figure 12: Co-localization of Gli2 and pSmad2/3 in SSc and in TBR-induced fibrosis. Representative images of costainings for Gli2, pSmad2/3 and DAPI in the skin of SSc patients and healthy volunteers (n = 6 for healthy and 5 for SSc) (A) and in vehicle-treated LacZ- and TBRact mice and

TBRact-mice treated with SD208 mice (n = 6 mice in each group) (B). Representative images are shown by at a 1000-fold magnification. All data are presented as median with IQR. The statistical significance was determined by Mann Whitney testing. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001.

We next aimed to analyze the role of canonical TGFβ signaling by siRNA-mediated inactivation of Smad3. Silencing of Smad3 abrogated the stimulatory effects of TGFβ on GLI2 both at mRNA and protein levels in cultured fibroblasts, implying that TGFβ regulates GLI2 via canonical TGFβ/Smad signaling (Figure 13 A, B). The transfection efficiency of siRNA of Smad3 was confirmed by significantly decreased mRNA levels and protein levels (Figure 13C, D).

Results

Figure 13: Knockdown of Smad3 prevents the induction of GLI2 by TGFβ. (A) Effects of the siRNA knockdown of Smad3 on GLI2 mRNA levels in TGFβ-stimulated human fibroblasts (n = 6). (B) Representative images of costainings for GLI2, Smad3 and DAPI in fibroblasts transfected with siRNA against Smad3 and stimulated with TGFβ shown at a 1000-fold magnification. (C-D) siRNA against Smad3 decreases the mRNA and protein levels of Smad3 in cultured dermal fibroblasts (n = 4). All data are presented as median with IQR. The statistical significance was determined by Mann Whitney testing. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001.

Moreover, knockdown of Smad3 in the skin of mice overexpressing TBRact also largely prevented the upregulation of GLI2 (Figure 14 A-D). These data demonstrate that TGFβ activates hedgehog signaling in a canonical manner via SMAD3-dependent pathways.

Results

Figure 14: Knockdown of Smad3 decreases the induction of Gli2 in TBRact-induced dermal fibrosis. siRNA knockdown of Smad3 inhibits the upregulation of Gli2 mRNA (A) and protein (B) in TBRact-driven fibrosis (n = 5 mice per group). Representative images are shown at 1000-fold magnification. siRNA mediated knockdown reduces the mRNA (C) and protein(D) levels of Smad3 in murine skin (n = 5). All data are presented as median with IQR. The statistical significance was determined by Mann Whitney testing. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001.

We had previously shown that TGFβ upregulates the mRNA levels of SHH (Horn et al. 2012b). In extension of those findings, we now demonstrate that SHH mRNA and protein are increased in the skin of mice upon challenge with bleomycin. However, treatment with SD-208 only partially blocked this upregulation (Figure 15), suggesting that the upregulation of SHH in experimental fibrosis is only partially dependent on TGFβ and that other factors contribute to its induction to a significant extent.

Results

Figure 15: The upregulation of SHH in bleomycin-induced skin fibrosis is only partially dependent on TGF- β. mRNA (A) and protein levels (B) of SHH as analyzed by real-time qPCR and Western blot, respectively, in bleomycin-challenged mice with and without co-treatment with the TGFβ receptor I kinase inhibitor SD-208 and in non-fibrotic control mice (n = 5 mice in each group). Data are presented as median with IQR. The statistical significance was determined by Mann Whitney testing. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001.

3.1.3 GLI2 regulates TGFβ induced fibroblast activation

To investigate whether GLI2 in turn regulate TGF-β-induced fibroblast activation, we first knocked out GLI2 by infecting dermal fibroblasts from Gli2fl/fl mice with AdCre. Fibroblasts lacking GLI2 demonstrated impaired responsiveness to TGF-β. The induction of classical TGFβ target genes such as Pai-1 and Ctgf was significantly reduced in Gli2 knockout fibroblasts compared with control cells (Figure 16A). Moreover, myofibroblast differentiation was impaired in Gli2 knockout fibroblasts and TGFβ did not upregulate the mRNA or protein levels of α-SMA or induces the formation of stress fibers (Figure 16B, C). Consistently, the stimulatory effects of TGFβ on Col1a2 mRNA levels and on collagen release were inhibited in Gli2 knockout fibroblasts (Figure 16D, E).

Results

Figure 16: Fibroblasts deficient in Gli2 are less sensitive to transforming growth factor β (TGFβ)-induced activation. (A–C) Inactivation of Gli2 by infection with adeno viruses encoding Cre recombinase in murine fibroblasts reduces the induction of TGFβ target genes. (A) mRNA levels of Pai-1 and Ctgf (n=6 in all experiments). (B and C) Myofibroblast differentiation is impaired in Gli2 knockout fibroblasts. (B) mRNA level of α-Sma and (C) immunofluorescence staining of α-Sma and stress fibres upon stimulation with TGF-β. Representative images are shown at 200-fold magnification. n=6 in all experiments. (D and E) The stimulatory effects of TGFβ on Col1a2 mRNA levels and on collagen release are reduced in Gli2 knockout fibroblasts. (D) mRNA levels of Col1a2 and (E) collagen in the supernatant (n=6 in all experiments). α-SMA, α-smooth muscle actin; AdCre, type 5 adeno-associated viruses encoding for Cre; AdLacZ, type 5 adeno-associated viruses encoding for LacZ. All data are presented as median with IQR. *p<0.05; **p<0.01; ***p<0.001.

We next aimed to analyse whether GLI2 as a transcription factor can directly upregulate the expression of TGFβ target genes. In silico analyses predicted two GLI-binding sites in the promoter of the Ctgf gene (Figure 17 A). Potential GLI binding sites were also identified in the promoter of Pai-1. ChIP assays demonstrated that TGFβ induces binding of GLI2 to both predicted binding sites (Figure 17 B). The activity of the Ctgf promoter is induced by TGFβ in reporter assays, and this induction is reduced upon mutation of either binding site (Figure 17 C).

Results

Figure 17: GLI2 directly binds to and stimulates the activity of the CTGF promoter. (A) Potential binding sites for GLI2 in the promoter of the CTGF gene predicted by in silico analysis. (B) ChIP assays demonstrating enrichment of GLI2 protein on both predicted DNA binding sites upon stimulation with TGFβ (n = 3 fibroblast lines with two technical replicates each). (C) Reporter assays using a non-mutated CTGF promoter and two constructs with selective mutation of the predicted GLI2 binding sites with and without recombinant TGFβ (n = 6).

3.1.4 Fibroblast-specific knockout of Gli2 prevents TBRact-induced fibrosis

The increase of GLI2 in fibrotic diseases prompted us to investigate the outcome of mice with fibroblast-specific knockout of Gli2 in experimental fibrosis. TGFβ-dependent skin fibrosis was induced by fibroblast-specific overexpression of TBRact. Col1a2-CreER mice, expressing a tamoxifen-sensitive CreERT recombinase under the control of a 6 kbp, fibroblast-specific col1a2 promoter fragment (Col1a2-CreER) (Sonnylal et al. 2007), were cross-bred with TBRact mice that harbour a Cre-inducible TBRact mutation, which has been targeted to the ROSA locus to generate TBRact; Col1a2-CreER double transgenic mice. To generate Gli2fl/fl TBRact; Col1a2-CreER triple mutant mice, we crossed mice harboring Gli2 alleles flanked by loxP sites Gli2fl/fl with TBRact; Col1a2-CreER mice. Upon induction of recombination by 75

Results tamoxifen, Gli2 expression was specifically depleted in fibroblast that express TBRact. Gli2fl/fl TBRact; Col1a2-CreER mice exhibited mitigated transcription of the classical TGFβ target genes Ctgf and plasminogen activator inhibitor-1 (Pai-1) as well as lower level of the hedgehog target genes Ptch-1, Ptch-2, Cyclin D1 and Gli-2 with reduced dermal collagen accumulation as compared to Gli2fl/fl TBRwt; Col1a2-CreER mice. Histological evaluation demonstrated more pronounced reduction of dermal thickening with less deposition of collagen and decreased myofibroblast counts in Gli2fl/fl TBRact; Col1a2-CreER mice compared to Gli2fl/fl TBRwt; Col1a2-CreER mice (Figure 18A-D).

Figure 18: Fibroblast-specific knockout of Gli2 ameliorates TBRact-induced fibrosis. Knockout of Gli2 ameliorates the histological features of TBRact-induced fibrosis (A), inhibited dermal thickening, decreased the number of myofibroblasts and reduced the hydroxyproline content (B) in the skin of TBRact-mice. Representative images of H&E-stained and trichrome-stained sections are shown at 100-fold magnification. Mice with fibroblast- specific knockout of Gli2 were also protected from TBRact-induced upregulation of the transforming growth factor-β (TGFβ) target genes Pai-1 and Ctgf (C) as well as from induction of the hedgehog target genes Ptch-1, Ptch-2, Cyclin D1 and Gli-2 (D). N ≥ 6 per group. All data are presented as dot blots in which the median is represented by a red bar. *p<0.05; **p<0.01; ***p<0.001.

Results 3.1.5 Pharmacological inhibition of GLI2 inhibits TGFβ-dependent fibroblast activation

Drugs that inhibit GLI2 such as arsenic trioxide (AsO) are already in clinical use, and selective inhibitors of GLI2 are currently in development. We therefore aimed to evaluate the translational potential of targeting GLI2 in fibrosis using GANT-61, which serves as a lead compound for the development of selective GLI2 inhibitors(Lauth and Toftgard 2007). GANT- 61 reduced the stimulatory effects of TGFβ on human dermal fibroblasts. Incubation with GANT-61 decreased the expression of prototypical TGFβ target genes (Figure 19A), inhibited myofibroblast differentiation (Figure 19B, C) and decreased collagen release (Figure 19D, E) as compared to vehicle-treated fibroblasts cells.

Figure 19: The selective GLI2 inhibitor GANT-61 blocks transforming growth factor-β (TGFβ)-induced fibroblast activation. Incubation of TGFβ-stimulated human dermal fibroblasts with GANT-61 reduces the upregulation of the TGFβ target genes Pai-1 and Ctgf (A) and inhibits α-Sma mRNA (B) and protein as well as stress fibre formation (C) (n=6 for all outcomes). Representative images are shown at 200-fold magnification. GANT-61 also inhibits the upregulation of Col1a2 mRNA (D) and the secretion of collagen protein by TGFβ (E) (n=6). All data are presented as median with IQR. *p<0.05; **p<0.01; ***p<0.001.

Results 3.1.6 Pharmacological inhibition of Gli-2 ameliorates TBRact -induced fibrosis

We next analysed the antifibrotic effects of the selective Gli2 inhibitor GANT-61 and of the SMO inhibitor vismodegib in TBRact-induced skin fibrosis. GANT-61, which inhibits canonical as well as non-canonical hedgehog signaling, significantly ameliorated TBRact- induced fibrosis and reduced dermal thickening, collagen deposition and myofibroblast counts compared with vehicle-treated mice overexpressing TBRact (Figure 20A, B). In line with a TGFβ-dependent, non-canonical activation of Gli2, treatment with the SMO inhibitor vismodegib did not affect TBRact-induced fibrosis (Figure 20A, B). Consistent with the in vitro findings, the levels of prototypical TGFβ/SMAD target genes such as Pai-1 and Ctgf were decreased in TBRact mice treated with GANT-61, but were unaffected by treatment with vismodegib (Figure 20C). GANT-61 also reduced the TBRact-mediated induction of hedgehog target genes such as Ptch-1, Ptch-2, CyclinD1 and GLI2 (Figure 20D).

Results Figure 20: Pharmacological inhibition of Gli-2, but not of Smo, ameliorates TBRact-induced skin fibrosis. Treatment with GANT-61 reduced the histological features of TBRact-induced fibrosis (A), inhibited dermal thickening, decreased the number of myofibroblasts and reduced the hydroxyproline content (B) in the skin of TBRact-mice. In contrast, the Smo inhibitor vismodegib did not ameliorate TBRact-induced fibrosis (A and B). Representative images of H&E-stained and Sirius Red-stained sections are shown at 100-fold magnification. Treatment with GANT-61, but not with vismodegib, also reduced the mRNA levels of Pai-1 and Ctgf (C) and of Ptch-1, Ptch-2, Cyclin D1 and Gli-2 (D) in mice overexpressing TBRact. N=6–8 per group. All data are presented as dot blots in which the median is represented by a red bar. *p<0.05; **p<0.01; ***p<0.001.

3.1.7 Inactivation of Gli2 induces regression of pre-established bleomycin- induced pulmonary fibrosis

We also evaluated the efficacy of GANT-61 in bleomycin-induced pulmonary fibrosis. To better resemble the clinical situation, treatment with GANT-61 was initiated 10 days after challenge with bleomycin, at a time point when fibrotic changes were already evident.18 Treatment with GANT-61 prevented progression of bleomycin-induced pulmonary fibrosis with reduced fibrotic area, decreased hydroxyproline content and lowered myofibroblast counts compared with vehicle-treated mice (Figure 21A, B). Treatment with GANT-61 also reduced the levels of TGFβ as well as of hedgehog target genes more effectively than vismodegib (Figure 21C, D).

Results Figure 21: Therapeutic dosing of GANT-61 ameliorates bleomycin-induced pulmonary fibrosis. Treatment with GANT-61 initiated after the onset of fibrosis prevents progression of bleomycin-induced pulmonary fibrosis (A) with reduced fibrotic area, lower myofibroblast counts and decreased hydroxyproline content (B). Representative images of Sirius Red-stained sections are shown at 200-fold magnification. Pharmacological inhibition of Gli2 also reduced the mRNA levels of Pai-1 and Ctgf (C) and of Ptch-1, Ptch-2, Cyclin D1 and Gli- 2 (D) in mice challenged with bleomycin. N=6–8 per group. All data are presented as dot blots in which the median is represented by a red bar. *p<0.05; **p<0.01; ***p<0.001.

3.2 The Hedgehog aceyltransferase Hhat regulates canonical TGFβ-dependent fibroblast activation in SSc

3.2.1 HHAT is upregulated in activated fibroblasts in fibrotic skin

To investigate potential contributions of disturbed HHAT expression to increased hedgehog signaling in SSc, we first analyzed the expression of HHAT in SSc skin. Expression of HHAT was upregulated as compared to matched skin samples from healthy individuals (Figure 22A). HHAT expression tended to be higher in patients with higher modified Rodnan skin score (mRSS) scores and active disease, but these findings did not reach statistical significance. Costaining with the fibroblast-marker prolyl-4-hydroxylase-β demonstrated that fibroblasts prominently express HHAT and that the number of HHAT-positive fibroblasts is increased in SSc (Figure 22A). Quantification of the fluorescence intensity revealed an increased expression in fibrotic skin of patients with diffuse-cutaneous SSc as compared to non-fibrotic SSc skin and to skin samples from matched healthy individuals. The upregulation of HHAT in SSc fibroblasts persisted under culture conditions with increased mRNA and protein levels of HHAT even after several passages in vitro (Figure 22 B).

Results

Figure 22: HHAT is upregulated in fibroblasts in SSc. (A) Costainings of HHAT with the fibroblast marker prolyl-4-hydroxylase-β (P4H) at 200-fold and 600-fold magnification. Semi-quantitative analysis of HHAT staining in fibroblasts in the skin of SSc patients and healthy volunteers (n=5 SSc patients and 5 matched controls). (B): mRNA and protein levels of HHAT in fibroblasts of SSc patients and healthy individuals (n=9). Results are shown as median ± IQR. The statistical significance was determined by Mann-Whitney testing. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001.

Furthermore, HHAT-positive cells also co-stained intensely for the hedgehog transcription factor GLI2 with higher numbers of HHAT and GLI2 double positive fibroblasts in SSc than in controls (Figure 23).

Results

Figure 23: Hedgehog signaling is active in fibroblasts expressing HHAT. (A) Co-stainings of HHAT with the hedgehog transcription factor GLI2 and the fibroblast marker prolyl-4-hydroxylase-β (P4H) at 200-fold magnification. Semi-quantitative analysis of HHAT staining in fibroblasts in the skin of SSc patients and healthy volunteers (n=5 for SSc patients and 5 for matched healthy controls). (B) Immunofluorescence staining (400-fold and 1000-fold magnification) with quantification in healthy and SSc patients skins (n=6). Results are shown as median ± IQR. The statistical significance was determined by Mann-Whitney testing. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001.

In addition to human SSc, the expression of Hhat was also upregulated in murine models of skin fibrosis. The mRNA and protein levels of Hhat were increased in the skin of mice challenged with bleomycin-induced skin fibrosis compared to non-fibrotic control mice injected with sodium chloride (Figure 24 A-C).

Results

Figure 24: Increased expression of Hhat in bleomycin-induced skin fibrosis. Expression of Hhat in the skin of bleomycin-induced skin fibrosis: (A) mRNA levels, (B) protein levels analyzed by Western blot and (C) IHC staining (400.-fold magnification) with quantification (n=6.). Results are shown as median ± IQR. The statistical significance was determined by Mann-Whitney testing. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001.

Results 3.2.2 HHAT expression is induced by canonical TGF signaling

Persistent activation of TGF signaling is a common denominator of fibrotic conditions and of cultured SSc fibroblasts (Ramming, Dees and Distler 2015). We therefore hypothesized that TGF signaling might drive the upregulation of HHAT in fibrotic SSc skin, in SSc fibroblasts and in experimental fibrosis. Stimulation of cultured fibroblasts with TGF increased the mRNA and protein levels of HHAT in a time-dependent manner (Figure. 25A, B).

Figure 25: TGFβ induces the expression of HHAT in human fibroblasts. (A) mRNA and protein (B) levels of HHAT in human fibroblasts stimulated with TGFβ for different time periods (n=6. biological replicates with ≥ 2 technical replicates). Results are shown as median ± IQR. The statistical significance was determined by Mann- Whitney testing. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001.

Consistent with those in vitro data, activation of TGF signaling by overexpression of a constitutively active TGF-receptor-type I (TBRIact) in the skin of mice upregulated Hhat expression (Figure 26A, B). The central regulatory effects of TGF on Hhat expression in fibrosis were further highlighted by selective inhibition of the TGF receptor type I kinase by SD208 in murine models of fibrosis. Treatment with SD208 prevented the increases in Hhat mRNA and protein in mice challenged with bleomycin (Figure 26C, D).

Results

Figure 26: Hhat is upregulated in experimental dermal fibrosis. (A) mRNA and (B) protein levels of Hhat in the skin of mice overexpressing TBRIact (n=3 biological replicates with ≥ 2 technical replicates). (C) mRNA and (D) protein levels of Hhat in the skin of control mice, mice challenged with bleomycin, and bleomycin challenged mice treated with the TGFβ receptor type I kinase inhibitor SD208 (n=5 biological replicates with ≥ 2 technical replicates). Data are shown as median ± IQR. The statistical significance was determined by Mann-Whitney testing. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001.

To determine whether TGF regulates HHAT via canonical SMAD signaling, we first knocked down SMAD3 in human dermal fibroblasts by siRNA. Knockdown of SMAD3 reduced the stimulatory effects of TGF on HHAT expression in mRNA and protein level (Figure 27A, B). To confirm the relevance of a Smad3-dependent induction of Hhat in vivo, we costained skin sections of mice challenged with bleomycin or overexpressing TBRIact for Hhat and phosphorylated Smad3 (pSmad3). Hhat staining colocalized with pSmad3 and all Hhat-positive cells were positive for pSmad3 (Figure 27 C). These results further highlighted the central role of TGFβ in regulating Hhat expression in fibrosis.

Results

Figure 27: TGFβ induces HHAT expression in a SMAD3-dependent manner. (A, B) Effects of siRNA- mediated knockdown of SMAD3 on the mRNA and protein levels of HHAT in human dermal fibroblasts (n=4 biological replicates with ≥2 technical replicates). (C) Costaining for Hhat, Smad3 and DAPI in murine models of skin fibrosis (n=5). Data are shown as median ± IQR. The statistical significance was determined by Mann- Whitney testing. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001.

3.2.3 HHAT promotes TGF-dependent fibroblast-to-myofibroblast- differentiation

Given the TGF-dependent induction of HHAT, the requirement of HHAT for the secretion of active hedgehog ligands and the increased levels of SHH in SSc, we hypothesized that the upregulation of HHAT may contribute to the profibrotic effects of TGF. SiRNA-mediated knockdown of HHAT inhibited the stimulatory effects of TGF on hedgehog activity in NIH3T3-Light2 reporter cells (Figure 28A) and also reduced the upregulation of the hedgehog target genes PTCH1, PTCH2, CYCLIN D1 and GLI2 by TGFin human dermal fibroblasts (Figure 28B). Moreover, knockdown of HHAT inhibited the stimulatory effects of TGF on collagen production in SSc fibroblasts with reduced mRNA levels of Col1A1 and decreased release of collagen protein (Figure 28C). Knockdown of HHAT also partially blocked TGF-

Results induced fibroblast-to-myofibroblast transition with reduced expression of αSMA and formation of stress fibers (Figure 28C).

Figure 28: HHAT promotes TGF-dependent fibroblast-to-myofibroblast-differentiation. (A) Hedgehog reporter activity in TGFβ stimulated 3T3 fibroblasts with or without Hhat knockdown (n=.3. biological replicates with ≥ 2 technical replicates). (B) mRNA levels of the hedgehog target genes PTCH-1, PTCH-2, CYCLIN D1 and GLI2 in human dermal fibroblasts. (C) Levels of COL1A1 mRNA and collagen protein as well as expression of α-

SMA and stress fiber formation (representative images shown at 200 fold magnification and quantification). (n≥

4 biological replicates with ≥ 2 technical replicates for all readouts in B,C). Data are shown as median ± IQR. The statistical significance was determined by Mann-Whitney testing. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001.

To confirm that knockdown of HHAT blocks long-range hedgehog signaling, we cocultured hedgehog reporter cells and fibroblasts with modulated HHAT expression in different conditions; either as mixed culture with direct physical contact or with physical separation in transwell assays. In transwell assays, the reporter can only be activated by long-range signaling, whereas it is activated by both, short- and long-range signaling, in direct cocultures. Consistent

Results with a potent inhibition of long-range hedgehog signaling upon Hhat inactivation, knockdown of Hhat completely abrogated TBR-induced hedgehog-driven reporter activity in transwell assays. In mixed cultures, knockdown of Hhat reduced the reporter activity by 65 ± 9%, but allowed for some residual induction by TBR(35 %) due to maintained short-range signaling

(Figure 29 A,B). Consistent with these findings, knockdown of HHhat reduced the release of oligomeric SHH complexes at 180, 120 and 75 kDa, but not of monomeric SHH at 19 kDa in both settings (Figure 29C).

Figure 29: Knockdown of Hhat abrogates TBR-induced long-range hedgehog signaling. (A) Transwell assays with physical separation of fibroblasts and reporter cells and activation of the reporter exclusively by long-range hedgehog signaling; schematic presentation of the experiment (left) and quantification of the reporter activity

(right). (B) Direct coculture of reporter cells and fibroblasts. Reporter activity is mediated by short- and long-range hedgehog signaling. Schematic presentation of the experiment (left) and quantification of the reporter activity

Results (right). Fibroblasts were transfected with a constitutively active TGFβ receptor type I (TBR) (or LacZ) and Hhat siRNA (or scrambled siRNA) (n=10). (C) Western blot analysis of oligomeric and monomeric forms of SHH in the supernatant of fibroblasts transfected with a constitutively active TGFβ receptor type I (TBR) (or LacZ) and

Hhat siRNA (or scrambled siRNA) in direct coculture and transwell settings. One representative WB (coculture) and quantification (n=4 independent experiments for each setting).

3.2.4 Knockdown of Hhat ameliorates experimental fibrosis

To further investigate the role of HHAT in experimental fibrosis, we targeted Hhat expression in the skin of mice challenged with bleomycin using siRNA / atelocollagen complexes. Injection of siRNA against Hhat decreased the expression of hedgehog target genes such as cyclin D1 and Gli2 as compared to bleomycin-challenged mice injected with non-targeting siRNA (Figure 30 A). The inhibition of hedgehog signaling translated into potent antifibrotic effects. Knockdown of Hhat reduced bleomycin-induced dermal thickening, myofibroblast counts and hydroxyproline levels (Figure 30 B, C). Meanwhile, the knockdwon efficiency of siRNA of Hhat was confirmed by significantly decreased protein levels from mouse skin. (Figure 30 D)

Results Figure 30: Knockdown of Hhat ameliorates Bleomycin-induced skin fibrosis. (A) mRNA levels of the hedgehog target genes Ptch-1, Ptch-2, Cyclin D1 and Gli2. (B) Effects of Hhat-knockdown on bleomycin- induced dermal thickening, myofibroblast counts and the hydroxyproline content. (C) Representative trichrome-stained and hematoxylin and eosin–stained skin sections at 200.-fold magnification. n = 6 mice for all groups and outcomes. (D) Efficiency of siRNA-mediated knockdown of Hhat on the protein levels in mouse skin. n = 6 mice for all groups and outcomes. Data are shown as median ± IQR. The statistical significance was determined by Mann-Whitney testing. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001.

SiRNA against Hhat also prevented the upregulation of Hhat and decreased the expression of hedgehog target genes in topoisomerase-challenged mice (Figure 31A). Knockdown of Hhat ameliorated topoisomerase-induced dermal thickening, inhibited myofibroblast differentiation and reduced the deposition of hydroxyproline content as compared to CFA challenged mice (Figure 31 B-D).

Figure 31: Knockdown of Hhat ameliorates Topo-induced skin fibrosis. (A) mRNA levels of the hedgehog target genes Ptch-1, Ptch-2, Cyclin D1 and Gli2. (B) Dermal thickness, myofibroblast counts and hydroxyproline content. (C) Representative trichrome-stained and hematoxylin and eosin–stained skin sections at 200-fold magnification. (D) Efficiency of siRNA-mediated knockdown of Hhat on the protein levels in mouse skin. n = 6 mice for all groups and outcomes. Data are shown as median ± IQR. The statistical significance was determined by Mann-Whitney testing. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001.

Results 3.3 The Orphan Nuclear Receptor RORα is a Key Regulator of TGF- and WNT-Signaling in Fibrosis

3.3.1 RORα expression pattern in fibrotic conditions

Several members of the superfamily of nuclear receptors have recently been shown to play important roles tissue responses to injury and in fibroblast activation (Beyer et al. 2013, Wu et al. 2009, Zerr et al. 2015) Here, we aimed to characterize the role of RORin the pathogenesis of fibrotic diseases. We first analyzed the expression of ROR in idiopathic pulmonary fibrosis and non-viral liver fibrosis. Semi-quantitative analysis results of co-staining of ROR and the fibroblast marker prolyl-4- hydroxylase-β (P4Hβ) showed increased RORlevels in fibroblasts (Figure 32 A-B).

Figure 32: ROR accumulates in human lung and liver fibrosis. Immunofluorescence showing staining for ROR (green) in 4-PH (red) positive cells in pulmonary (A), and hepatic fibrosis (B) (n = 6 for IPF and nonalcoholic hepatic fibrosis and n ≥ 7 for non-fibrotic controls). All sections were counterstained with DAPI (blue). Results are shown as median ± IQR. Significance was determined by Mann-Whitney test. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001.

Western blot and immunofluorescence staining results demonstrated that RORexpression was also upregulated in the murine models of bleomycin-induced pulmonary fibrosis (Figure 33 A, B) and carbon tetrachloride (CCL4)-induced liver fibrosis (Figure 33 C-D).

Results

Figure 33: Expression of ROR is increased in murine models of lung and liver fibrotic diseases. Levels of ROR protein in mouse models of pulmonary and hepatic-fibrosis analyzed by immunofluorescence (A, C) and western blot. (B, D) (n = 5 per group). Results are shown as median ± IQR. Significance was determined by Mann- Whitney test. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001.

3.3.2 Canonical Wnt signaling induces RORα expression in fibroblasts We and others have recently demonstrated that canonical WNT ligands, such as WNT1 and WNT10B, are overexpressed in patients with SSc and in other fibrotic diseases (Wei et al. 2011; Akhmetshina et al. 2012). In light of the connection between canonical WNT and ROR ((Lee et al. 2010)) in colon cancer, we wondered whether canonical WNT may drive the overexpression of RORin fibrotic diseases. Indeed, the mRNA level of ROR showed a time- dependent increase of upon WNT1stimulation with highest level after 6h in cultured human fibroblasts (Figure 34 A). Moreover, we observed a time-dependent upregulation of ROR protein level by WNT1 by Western blot with a peak after 12 h (Figure 34 B).

Results

Figure 34: WNT dependent upregulation of ROR. (A) ROR mRNA (n = 5) and (B) ROR protein (n = 4) in human dermal fibroblasts upon short-term incubation with WNT1 as determined by qPCR and Western blot. Results are shown as median ± IQR. Significance was determined by Mann-Whitney test. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001.

After demonstrating that canonical Wnt signaling upregulates the expression of ROR, we next analyzed the expression of Ror in Wnt10b transgenic (tg) mice. These mice overexpress Wnt10b under a FABP4 promoter in adipocytes and are characterized by massive generalized dermal fibrosis. Indeed, upregulation of RORm and protein levels were found in the skin of Wnt10b tg mice as compared to control littermates (Figure 35A-C).

Figure 35: RORis upregulated in Wnt-10b transgenic mice. Increased mRNA (A) and protein (B) levels in skin samples of Wnt-10b mice and control Bl/6WT mice by qPCR and Western Blot, respectively (n=5 for both). (C) Immunofluorescence staining demonstrating co-localization of ROR and vimentin in Wnt-10b and Bl/6WT mice skin samples (n=5 each). Representative images are shown in 200- and 600-fold magnification. Results are shown as median ± IQR. Significance was determined by Mann-Whitney test. *: 0.01 ≤ p < 0.05, **: 0.001 ≤ p < 0.01, ***: p < 0.001. Conversely, inhibition of canonical WNT signaling by recombinant Dickkopf-1 (DKK1) decreased the mRNA levels of ROR (Figure 36A). The downregulation of ROR on protein level was also confirmed by immunofluorescence staining (Figure 36 B). Consistently, DKK1 93

Results also blocked the WNT-induced upregulation of the RORtarget gene ARNTL (Figure 35A). Moreover, DKK1 blocked the WNT-induced transcriptional activation of the ARNTL promoter in reporter assays (Figure 36C).

Figure 36: Canonical WNT signaling promotes upregulation of ROR effect can be blocked by DKK. (A) ROR and mRNA level in human dermal fibroblasts upon stimulation with WNT with or without DKK. (B) Analysis of the expression level of ROR (green) by immunofluorescence staining. Counterstaining with -catenin (red and DAPI (blue). (C) ARNTL promoter activity in response to WNT1 and DKK (n = 4 per group). Results are shown as median ± IQR. Significance was determined by Mann-Whitney test. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001. Our group reported previously that canonical Wnt signaling play a key role for fibroblast activation and collagen release in fibrotic diseases. To investigate the role of RORα on WNT signaling in fibrosis, we used the specific small molecule inverse agonist SR3335. The induction of stress fiber formation and aSMA expression were downregulated by SR3335 (Figure 37A). Moreover, the mRNA levels of COL1A1, COL1A2 and ACTA2 were decreased as well with SR3335 in Wnt3a stimulated fibroblasts to a similar extend as with the Wnt antagonist DKK (Figure 37B).

Results

Figure 37: SR3335 prevented the stimulatory effects of canonical Wnt signaling on human fibroblasts. (A) SR3335 decreased the formation of stress fibres and SMA expression in Wnt1 stimulated cultured fibroblasts (n = 6). (B) Incubation with SR3335 reduced the mRNA level of COL1A1; COL1A2 and ACTA2 (n = 6 per group). Of note, the inhibitory effects of SR3335on fibroblasts were comparable with those of DKK. Results are shown as median ± IQR. Significance was determined by Mann-Whitney test. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001.

3.3.3 Inactivation of RORα reduces the stimulatory effects of TGFinduces fibroblast activation

We next analyzed whether the upregulation of RORmay regulate TGF signaling. As a first approach, we inhibited the activity of RORα using SR3335 and also the combined RORα and RORγ inhibitor SR1001. Inactivation of RORprevented the stimulatory effect of TGF on fibroblasts, with decreased mRNA levels of COL1A1, COL1A2 and ACTA-2 mRNA (Figure

Results 38A) as well as reduced protein levels of αSMA (Figure 38B). In addition, inhibition of RORmitigated the expression of myofibroblast markers in resting fibroblasts with decreased expression of α-SMA and reduction of stress fibers (Figure 38C). To further investigate the molecular mechanisms underlying the modulatory effects of ROR on TGF signaling, we analyzed whether targeting of RORaffects TGFSmad3 signaling. Inactivation of RORby SR3335 reduced the levels of phosphorylated Smad3 in TGFstimulated human fibroblast (Figure 38 D). Moreover, inhibition of ROR also repressed TGFβ-induced SBE reporter activity (Figure 38E).

Figure 38: Inhibition of ROR decreases the stimulatory effects of TGFβ on fibroblasts. (A) mRNA levels of COL1A1 and COL1A2 analyzed by qPCR and collagen release as analyzed by SirCol assays (n =5). (B) Protein level of -SMA analyzed by Western blot (n =5). (C) Stress fiber formation in fibroblasts with or without TGF, SR3335 and SR1001. Representative images are shown at 200-fold magnification. (D) Levels of pSmad3 in fibroblasts analyzed by Western Blotting. (E) Transcriptional activity of Smad in Smad-binding elements (SBEs) reporter assays in fibroblasts stimulated with TGFβ and co-incubated with SR3335 or SR1001. Results are shown as median ± IQR. Significance was determined by Mann-Whitney test. *: 0.01 ≤ p < 0.05, **: 0.001 ≤ p < 0.01, ***: p < 0.001.

Results Based on the stimulatory effects of ROR on fibroblasts, we next investigated whether knocked out of ROR is sufficient to suppress the stimulatory effects of TGF in murine fibroblasts. Effective knockout of ROR was confirmed by quantitative polymerase chain reaction (Figure 39A). Knockout of ROR attenuated TGF-β-induced differentiation of resting fibroblasts into myofibroblasts with decreased expression of α-smooth muscle actin (αSMA) and reduced formation of stress fibers (Figure 39B). The stimulatory effect of TGFβ on collagen synthesis was also reduced in RORknockout fibroblasts with decreased levels of Col1a1 and Acta2 mRNA and decreased release of collagen protein (Figure 39C, D). Deficiency of ROR in mouse fibroblast attenuated the levels of phosphorylated Smad3 upon TGF stimulation (Figure 39E).

Figure 39: Fibroblasts deficient in Ror are less sensitive to TGFβ-induced activation. (A) mRNA protein levels of Ror upon infection with adenoviruses encoding Cre recombinaseor LacZ. (B) Immunofluorescence staining for α-Sma and stress fibres upon stimulation with TGFβ. Representative images are shown at 200-fold magnification. n=6 in all experiments. mRNA levels of Col1A1 and Col1A2 (C) and release of collagen protein (D). (E) Levels of pSmad3 in fibroblasts analyzed by Western Blotting. (n=6 in all experiments). AdCre, type 5 97

Results adeno-associated viruses encoding for Cre; AdLacZ, type 5 adeno-associated viruses encoding for LacZ. All data are presented as median with IQR. *p<0.05; **p<0.01; ***p<0.001.

3.3.4 Mice with fibroblast-specific knockout of Rorα are protected from experimental fibrosis

To investigate the specific role of ROR in fibroblasts in fibrosis in vivo, we generated a mouse strain with inducible deletion of Ror (Rorfl/fl) in fibroblasts. Col1a2-CreER mice express a tamoxifen responsive Cre-ER fusion protein under the control of a fibroblast-specific 6 kBP Col1a2 promoter sequence. Rorfl/fl x Col1a2-CreER enable fibroblast-specific deletion of Ror upon challenge with tamoxifen. In the absence of bleomycin, fibroblast-specific deletion of Ror that the expression of Ror in fibroblasts is not required for maintenance of skin homeostasis. However, bleomycin-induced skin fibrosis was significantly ameliorated in Rorfl/fl x Col1a2-CreER mice treated with tamoxifen compared to Rorfl/fl x Col1a2-CreER mice injected with corn oil as shown by reduced dermal thickening, inhibited myofibroblast- differentiation and decreased hydroxyproline content in lesional skin (Figures 40A-D).

Figure 40: Fibroblast-specific deletion of Rorα ameliorates bleomycin-induced skin fibrosis. (A) Representative hematoxylin and eosin–stained skin sections at 100-fold magnification. (B-D) Effects of Ror- deficiency on bleomycin induced dermal thickening (B), (C) the myofibroblast counts and the hydroxyproline content (D) (n ≥ 6 mice for all groups and outcomes). All data are presented as median with IQR. *p<0.05; **p<0.01; ***p<0.001.

Mice with fibroblast-specific deletion of Rorα were also protected from bleomycin-induced pulmonary fibrosis as an inflammation-driven model of lung fibrosis with significantly decreased deposition of collagen, lower Ashcroft scores (a histological score of pulmonary fibrosis that is also used for quantification of fibrotic damage in human lungs) and reduced

Results hydroxyproline content as compared to control littermates (Figures 41A-D). As for skin, lung histology was not altered by deletion of Rorα in fibroblasts in the absence of bleomycin.

Figure 41: Mice with fibroblast-specific deletion of Rorameliorates bleomycin-induced pulmonary fibrosis. (A) Representative Sirius red lung sections at 200-fold magnification. (B-D) Effects of Ror-deficiency on bleomycin induced collagen accumulation in the lung (B), on Ashcroft scores (C) and on hydroxyproline content (D) (n ≥ 6 mice for all groups and outcomes). All data are presented as median with IQR. *p<0.05; **p<0.01; ***p<0.001.

3.3.5 Pharmacological inhibition of Rorα ameliorates experimental fibrosis

We next aimed to investigate the therapeutic potential of pharmacological inhibition of ROR. Based on our in vitro findings, we first evaluated, whether SR3335 can inhibit WNT-induced fibrosis. Treatment with SR3335 ameliorates skin fibrosis in Wnt-10b mice at well-tolerated doses with reduced dermal thickening, decreased myofibroblast counts and reduced hydroxyproline content as compared to vehicle-treated WNT10b mice. SR3335 also inhibited the upregulation of Col1a1, Col1a2 and Acta2 mRNA (Figure 42A-E).

Results

Figure 42: Pharmacologic inhibition of RorSR3335 ameliorates dermal fibrosis in Wnt-10b transgenic mice skin. (A) Representative hematoxylin and eosin–stained skin sections are shown at 100-fold magnification. (B-D) Inhibition of Rorreduces dermal thickening (B), myofibroblast differentiation and hydroxyproline content (D) and Col1a1, Col1a2 and Acta2 mRNA levels (E) in Wnt-10b transgenic mice (n≥ 6 for each group in all readouts). All data are presented as median with IQR. *p<0.05; **p<0.01; ***p<0.001.

In mice overexpressing TBRI, preventive dosing of both SR3335, but also of the combined RORα/γ inhibitor SR1001, ameliorated dermal thickening. myofibroblast differentiation and collagen deposition (as measured by hydroxyproline content) (Figure 43A-D). Apart from TGFβ-driven fibrosis, inhibition of RORsignaling by SR3335 or by SR1001 showed potent antifibrotic effects in bleomycin-induced skin fibrosis (Figure 43E-H).

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Results

Figure 43: Pharmacologic inhibition of Rorameliorates bleomycin- and TBRI-induced skin fibrosis. (A- D) Bleomycin-induced fibrosis: (A) Representative hematoxylin and eosin–stained skin sections in bleomycin- induced dermal fibrosis are shown at 100-fold magnification. (B-D) Inhibition of Rordecreases dermal thickening (B), myofibroblast differentiation (C) and hydroxyproline content (D). (E-H) TBRI-induced fibrosis: (E) Representative trichrom-stained skin sections in TBRact-induced dermal fibrosis are shown at 100-fold magnification. (F-H) Inhibition of Rordecreases dermal thickening (F), myofibroblast differentiation (G) and hydroxyproline content (H). N≥ 5 for each group in all readouts. All data are presented as median with IQR. *p<0.05; **p<0.01; ***p<0.001.

The anti-fibrotic effects of SR3335 were not restricted to skin fibrosis, but also occurred in other organs. Treatment with SR3335 attenuated bleomycin-induced pulmonary fibrosis with decreased collagen accumulation, impaired myofibroblasts differentiation and reduced hydroxyproline content (Figure 44).

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Results

Figure 44: Inactivation of Rorameliorates bleomycin-induced pulmonary fibrosis. (A) Representative Sirius red stained lung sections at 200-fold magnification. (B-D) Effects on collagen staining (B), hydroxyproline content (C) and Ashcroft scores (D) (n ≥ 7 mice for all groups and outcomes). All data are presented as median with IQR. *p<0.05; **p<0.01; ***p<0.001.

We used preventive dosing in all models so far with treatment initiated together with the first profibrotic insult. However, clinically more relevant is a situation with pre-established fibrosis, in which treatment is started once fibrosis has already manifested with the aim reduce further progression of fibrosis. Optimally, treatment would not only halt progression, but even induce regression of pre-established fibrotic changes. We thus tested the effect of RORα inhibition in the setting of pre-established fibrosis. Treatment with SR3335 was also effective, when initiated after fibrosis had already manifested. Therapeutic application of SR3335 in mice with pre-established fibrosis prevented progression of fibrosis in carbon tetrachloride (CCL4)-induced hepatic fibrosis despite further challenges with CCl4 and with decreased hydroxyproline content and lower Scheuer´s scores (a standard histological readout of liver fibrosis) as compared to vehicle-treated mice (Figure 45).

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Results

Figure 45: SR3335 is effective for the treatment of pre-established experimental hepatic fibrosis. (A) Representative Sirius Red stained liver sections at 200-fold magnification. (B) Scheuer´s score and (C) hydroxyproline content. N≥ 6 per group. Results are shown as median ± IQR. Significance was determined by Mann-Whitney test. *p<0.05; **p<0.01; ***p<0.001.

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Discussion 4 Discussion

4.1 Evaluation of the HH transcription factor GLI2 in the pathogenesis of SSc

We demonstrate in the present study that GLI2 is increased in SSc fibroblasts in vitro, in SSc skin ex vivo and in fibroblasts of fibrotic murine skin. We further presented that this upregulation of GLI2 is mediated by TGFβ. Incubation with recombinant TGFβ increased the mRNA and protein levels of GLI2 in cultured fibroblasts. Thus, TGFβ may contribute directly to the increased accumulation of GLI2 in fibrotic diseases such as SSc. The relevance of this non canonical pathway for the activation of hedgehog signaling in fibrosis is highlighted by the potent suppressive effects of selective inactivation of TGFβ signaling on GLI2 expression. Inhibition of TGFβ signaling by SD-208, a selective inhibitor of TGFβ receptor type 1 (Uhl et al. 2004) strongly reduced the expression of GLI2 in bleomycin-induced fibrosis and in Tsk-1 mice, demonstrating that TGFβ-dependent, non-canonical hedgehog signaling significantly contributes to the increased transcription of hedgehog target genes in fibrosis.

We showed a crosstalk between TGFβ and hedgehog signaling in SSc. However, as TGFβ also contributes to the upregulation of SHH in experimental fibrosis, further studies in other models are required to determine the relative contribution of canonical and non-canonical mechanisms to the aberrant activation of hedgehog signaling.

We also show that the activation of non-canonical hedgehog signaling is required for the profibrotic effects of TGFβ. GLI2 binds to the promoters of TGFβ target genes such as Ctgf, and knockdown of GLI2 in cultured fibroblasts prevented the differentiation of resting fibroblasts into myofibroblasts and reduced the release of collagen. In addition to inhibition of the differentiation of resting fibroblasts into myofibroblasts, targeting of GLI2 may also inhibit myofibroblast proliferation by induction of cell cycle arrest (Kramann et al. 2015).We observed a clear trend towards higher levels of Pai-1 and Ctgf in Gli2fl/fl x Col1a2-Cre-ER at baseline. The underlying mechanisms require further studies. However, the induction of Pai-1 and Ctgf by TBRact was strongly reduced in Gli2fl/fl x Col1a2-Cre-ER mice compared with control mice with normal expression of GLI2. Moreover, knockdown of GLI2 also significantly ameliorated fibrosis induced by overexpression of a constitutively active TGFβ receptor type I

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Discussion with decreased skin thickening, impaired myofibroblast differentiation and reduced the hydroxyproline content. Considering the potent stimulatory effects of TGFβ on hedgehog signaling, GLI2 inhibitors may theoretically offer benefits over SMO inhibitors for the treatment of fibrosis as they simultaneously interfere with canonical and non-canonical hedgehog signaling and also reduce the pro-fibrotic effects of TGFβ, whereas SMO inhibitors only interfere with canonical hedgehog signaling(Riobo and Manning 2007). Indeed, our study provides first evidence that targeted therapies against GLI2 may be effective for the treatment of fibrosis. The direct GLI inhibitor GANT-61 prevented the accumulation of GLI2 and the induction of hedgehog target genes induced by overexpression of a constitutively active TGFβ receptor type I, whereas the SMO inhibitor vismodegib showed no significant effects in readouts of fibrosis this setting. In addition to TBRact-induced fibrosis, GLI inhibitors also suppressed the expression of hedgehog target genes in bleomycin-induced fibrosis more potently than vismodegib, thereby further highlighting the crucial role of non-canonical, TGFβ- dependent activation of hedgehog signaling in experimental fibrosis. Treatment with GLI inhibitors did not only block hedgehog signaling, but also interfered with TGFβ signaling. GLI2 inhibitors effectively decreased the expression of TGFβ target genes, while inhibition of SMO had only mild, statistically not significant effects. We demonstrated previously that TGFβ can induce that expression of SHH in fibroblasts (Horn et al. 2012b), implying the possibility that TGFβ may also promote canonical hedgehog signaling to stimulate the transcription of target genes. However, based on our current mechanistic findings, the strong induction of GLI2 by TGFβ, the almost complete dependency of this upregulation on TGFβ signaling and the modest regulatory effects of TGFβ on SHH expression in experimental fibrosis, we believe that the TGFβ activates the transcription of hedgehog target genes predominantly by inducing GLI2, whereas the modest upregulation of SHH is of minor importance. This conclusion is further supported by the finding that treatment with an SMO inhibitor does not significantly reduce TBRact-induced fibrosis. The inhibitory effects of GLI2 inhibitors on canonical and non-canonical hedgehog pathways and on the expression of TGFβ target genes directly translate into potent antifibrotic effects in experimental models of SSc. Preventive treatment with GANT-61 effectively reduced TBRact- induced skin thickening, accumulation of collagen and differentiation of resting fibroblasts into myofibroblasts. Treatment with GANT-61 was also effective when initiated after the onset of fibrosis. Of note, treatment with GANT-61 was well tolerated without evidence for adverse events in clinical monitoring or on necropsy, indicating that the use of GLI2 inhibitors in adults may not be limited by toxicity. These findings have translational implications because drugs

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Discussion that inhibit GLI2 such as arsenic trioxide, darinaparsin and itraconazole are already approved for cancer therapy. Moreover, more specific compounds are currently in clinical development (Gonnissen, Isebaert and Haustermans 2015).Considering the prominent accumulation of GLI2 in fibrotic diseases (Horn et al. 2012b, Dennler et al. 2007, Lauth and Toftgard 2007, Hui and Angers 2011) , the potent antifibrotic effects of GLI2 inhibitors in preventive and therapeutic dosing regimens, the current use of GLI2-targeting agents in the clinic and the ongoing development of selective GLI2 inhibitors, GLI2 may be an interesting candidate for targeted antifibrotic therapies.

4.2 The Hedgehog aceyltransferase Hhat regulates canonical TGFβ-dependent fibroblast activation in SSc

Long-range hedgehog signaling seems to play a central role in the pathogenesis of SSc as evidenced by the recent demonstration of elevated serum levels of SHH in patients with SSc and their correlation with fibrotic manifestations.(Beyer et al. 2018) Selective targeting of long- range, hedgehog signaling via HHAT offers theoretical advantages over broad-spectrum inhibition of autocrine, paracrine and endocrine hedgehog signaling. Although the broad inhibition of hedgehog signaling seems better tolerated than interference with other stem cell pathways,(Bergmann and Distler 2016) safety concerns with impaired differentiation of stem cells remain with long-term treatment. Selective inhibition of long-range hedgehog signaling would ameliorate those concerns. Targeted inactivation of HHAT interferes with the formation of oligomeric SHH and thus with long-range hedgehog signaling, but allows to maintain the homeostatic autocrine and paracrine functions of monomeric SHH required e.g. for differentiation and maturation of stem cells (Buglino and Resh 2008, Chen et al. 2004). Targeting long-range hedgehog signaling may thus be an approach to reduce the adverse events of broad spectrum hedgehog inhibition, but maintain its antifibrotic effects. Indeed, siRNA- mediated knockdown of Hhat did not induce obvious alterations of the skin histology and potently inhibited fibroblast-to-myofibroblast transition and collagen release in vitro and in two mouse models in vivo. We and others provided evidence for a crosstalk of hedgehog- and TGFβ-signaling and demonstrated that TGFβ promotes hedgehog signaling by inducing the ligand SHH and in particular the transcription factor GLI2. (Horn et al. 2012a, Horn et al. 2012b, Liang et al. 2017, Bolanos et al. 2012, Kramann et al. 2015, Distler et al. 2014, Omenetti et al. 2007, Michelotti

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Discussion et al. 2013, Syn et al. 2009, Dennler et al. 2007) We now extend those findings and provide evidence that TGFβ may particularly promote long-range hedgehog signals. The upregulation of HHAT by TGFβ may shift the balance from rather homeostatic autocrine and paracrine signals to endocrine signaling to promote tissue remodeling. Indeed, the upregulation of HHAT is central for TGFβ-induced hedgehog activity and directly contributes to the profibrotic effects of TGFβ. TGFβ is a core pathway of fibrosis and is thus an obvious candidate for antifibrotic therapies. Targeting long-range hedgehog signaling as an important profibrotic downstream mediator of TGFβ may offer advantages over other approaches: (1) upstream targeting of TGFβ signaling at the levels of TGFβ isoforms or TGFβ receptors is associated with the formation of keratoacanthomas and increased risk of autoimmunity. (2) In contrast to many other downstream mediators of TGFβ signaling, we already have gathered considerable clinical experience with inhibition of hedgehog signaling, although not with direct inhibition of HHAT. In summary, we demonstrate that TGFβ induces HHAT expression to promote a shift from homeostatic towards profibrotic long-range hedgehog signaling and that inhibition of long- range signaling by inactivation of HHAT maintains potent antifibrotic effects. These findings may have translational implications as HHAT inhibitors are in development.

4.3 The Orphan Nuclear Receptor Rorα is a Key Regulator of TGF- and WNT-Signaling in Fibrosis

As part of my thesis, we characterized the role of orphan nuclear receptor RORNr1f1) in experimental fibrosis. We demonstrated that RORexpression is upregulated in the fibrotic lungs of patients with idiopathic pulmonary fibrosis and non-viral liver fibrosis as well as fibrotic mouse lung and liver. We further showed that this induction of ROR is mediated by canonical Wnt signaling. Incubation with recombinant Wnt1 increased the mRNA and protein levels of ROR in cultured human dermal fibroblasts with time dependent manner. In line with these in vitro results, both mRNA and protein expression level of ROR were highly upregulated in Wnt10b transgenic mice, which develop massive generalized dermal fibrosis (Akhmetshina et al. 2012). Furthermore, treatment with recombinant DKK protein, an antagonist of canonical Wnt signaling, prevented the upregulation of ROR in cultured human dermal fibroblasts. Apart from Wnt, other factors may also contribute to the expression of ROR given that it is suggested as a target gene of hypoxia-inducible factor (HIF) (Caroline

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Discussion CHAUVET 2004). The implicated function of HIF in fibrosis include stimulation of excessive ECM, vascular remodeling, and futile angiogenesis (Xiong and Liu 2017). The stimulatory effects of canonical Wnt signaling on human fibroblasts were inhibited by SR3335. Moreover, SR3335 exerted potent anti-fibrotic effects in transgenic overexpression of Wnt10b mice. In addition, we showed that the inhibition of RORactivity downregulates canonical TGFβ/Smad signaling in human fibroblasts. SR3335 inhibited the stimulatory effects of TGFβ on myofibroblast differentiation and on the release of collagen in vitro. Inactivation of ROR also reduced Smad-dependent transcription with decreased Smad reporter activity and downregulation of the levels of pSmad3. Additionally, inactivation of ROR also ameliorated skin fibrosis in mice with TBRact-induced dermal fibrosis and in bleomycin-challenged mice at well-tolerated doses. R3335 also exerted ant-fibrotic effect in preclinical mouse models with fibrosis in internal organs such bleomycin-induced pulmonary fibrosis and CCL4-induced liver cirrhosis. The exact mechanisms by which RORregulates TGFβ/Smad signaling remains to be further elucidated. As a nuclear transcription factor, ROR훼 could bind directly on the promoter region of RORE in target genes. However, in silico algorithms did not predict classical RORE on the putative SMAD3 promoter region. Several studies suggested that ROR훼 can also regulate gene expression by transactivation/transrepression. In this scenario, ROR훼 associates with other transcription factors to modulate their transcriptional activity. In colon carcinoma cells, ROR훼 was shown to bind 훽-catenin directly and to regulate 훽-catenin-mediated transcriptional activation of the target genes such as cyclin D1 and c-myc (Lee et al. 2010). In epithelial cells, RORα binds to E2F1 and inhibits its acetylation and DNA-binding activity by recruiting histone deacetylase 1 (HDAC1) (Xiong and Xu 2014). Whether similar mechanisms are involved in the regulation of TGFβ/Smad remains unknown and requires further studies.

Our findings suggested that pharmacological inhibition of RORα, either selectively by SR3335 are in combination with RORγ by SR1001exerts antifibrotic effects without causing the obvious gross toxicity. This is consistent with the lack of adverse effects observed in other studies in mice (Sun et al. 2015, Solt et al. 2011). Together, these findings indicate that RORα might be a potential target for the treatment of fibrosis. However, considering the complex pathogenesis and the heterogeneity of SSc, further in vivo studies are required to confirm these findings. Particular attention should be appointed to the effects of RORα inhibition on macrophage polarization. Several studies demonstrated that RORα activation induced M2 macrophage 108

Discussion polarization (Han et al. 2017, Xiao et al. 2016), which is thought to play an important role in fibrosis by the release of pro-fibrotic mediators such as IL-4 and IL-13 (Wynn and Vannella 2016, Cook, Kang and Jetten 2015). In summary, we demonstrate that RORα as a key checkpoint of TGFβ- and WNT-induced fibroblast activation. RORα is induced in fibrotic diseases in a WNT-dependent manner to promote TGFβ- and WNT-induced fibroblast activation. Targeting of RORα simultaneously interferes with TGFβ- and WNT-signaling as two core pathways in the pathogenesis of fibrotic diseases. The inhibition of those core pathways translates into potent antifibrotic effects across different models and organ systems.

109

Conclusion 5 Conclusion

In summary, the first part of my thesis demonstrate a direct interaction between TGFβ and hedgehog signaling in fibroblasts and highlight that both pathways are integrated by GLI2 in fibrotic conditions. Moreover, we identify GLI2 as a crucial downstream mediator of the profibrotic effects of TGFβ. The second part of my thesis demonstrates that Hhat is upregulated in a TGFβ-dependent manner in SSc. Hhat facilitates long-range hedgehog signaling downstream of TGFβ. Inactivation of Hhat reduces TGFβ-dependent fibroblast activation and ameliorates experimental fibrosis. The last part of my thesis characterizes RORα as a key checkpoint of TGFβ- and WNT- induced fibroblast activation. RORα is induced in fibrotic diseases in a WNT-dependent manner to promote TGFβ- and WNT-induced fibroblast activation and tissue fibrosis. Targeting of RORα simultaneously interferes with TGFβ- and WNT-signaling as two core pathways in the pathogenesis of fibrotic diseases. The inhibition of those core pathways translates into potent antifibrotic effects across different models and organ systems. We thus provide novel examples for a highly complex signaling network with crosstalk of TGFβ signaling to multiple other growth factor pathways that promotes the aberrant activation of fibroblasts and the excessive deposition of collagen in SSc and other fibrotic disorders.

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References

6 References

Abraham, D. J. & J. Varga (2005) Scleroderma: from cell and molecular mechanisms to disease models. Trends Immunol, 26, 587-95. Ahmed, S. S., F. C. Tan Fk Fau - Arnett, L. Arnett Fc Fau - Jin, Y.-J. Jin L Fau - Geng & Y. J. Geng (2006) Induction of apoptosis and fibrillin 1 expression in human dermal endothelial cells by scleroderma sera containing anti-endothelial cell antibodies. Arthritis Rheum, 54, 2250-62. Akashi, M. & T. Takumi (2005) The orphan nuclear receptor RORalpha regulates circadian transcription of the mammalian core-clock Bmal1. Nat Struct Mol Biol, 12, 441-8. Akhmetshina, A., K. Palumbo, C. Dees, C. Bergmann, P. Venalis, P. Zerr, A. Horn, T. Kireva, C. Beyer, J. Zwerina, H. Schneider, A. Sadowski, M. O. Riener, O. A. MacDougald, O. Distler, G. Schett & J. H. Distler (2012) Activation of canonical Wnt signalling is required for TGF-beta-mediated fibrosis. Nat Commun, 3, 735. Alfaro, A. C., B. Roberts, L. Kwong, M. F. Bijlsma & H. Roelink (2014) Ptch2 mediates the Shh response in Ptch1-/- cells. Development, 141, 3331-9. Allanore, Y., R. Simms, O. Distler, M. Trojanowska, J. Pope, C. P. Denton & J. Varga (2015) Systemic sclerosis. Nat Rev Dis Primers, 1, 15002. Angiolilli, C., W. Marut, M. van der Kroef, E. Chouri, K. A. Reedquist & T. Radstake (2018) New insights into the genetics and epigenetics of systemic sclerosis. Nat Rev Rheumatol, 14, 657-673. Armando Gabrielli, M. D., Enrico V. Avvedimento, M.D., and Thomas Krieg, M.D. (2009) Scleroderma. The new england journal of medicine, 360, 1989-2003. Arnett, F. C. (2006) Is scleroderma an autoantibody mediated disease? Curr Opin Rheumatol, 18, 579-81. Asano, Y. (2018) Systemic sclerosis. J Dermatol, 45, 128-138. Asano, Y. & S. Sato (2013) Animal models of scleroderma: current state and recent development. Curr Rheumatol Rep, 15, 382. Avouac, J., J. H. Cagnard N Fau - Distler, Y. Distler Jh Fau - Schoindre, B. Schoindre Y Fau - Ruiz, P. O. Ruiz B Fau - Couraud, G. Couraud Po Fau - Uzan, C. Uzan G Fau - Boileau, G. Boileau C Fau - Chiocchia, Y. Chiocchia G Fau - Allanore & Y. Allanore (2011) Insights into the pathogenesis of systemic sclerosis based on the gene expression profile of progenitor-derived endothelial cells. Arthritis Rheum, 63, 3552-62. Avouac, J., K. Palumbo, M. Tomcik, P. Zerr, C. Dees, A. Horn, B. Maurer, A. Akhmetshina, C. Beyer, A. Sadowski, H. Schneider, S. Shiozawa, O. Distler, G. Schett, Y. Allanore & J. H. Distler (2012) Inhibition of activator protein 1 signaling abrogates transforming growth factor beta-mediated activation of fibroblasts and prevents experimental fibrosis. Arthritis Rheum, 64, 1642-52. Barnes, J. & M. D. Mayes (2012) Epidemiology of systemic sclerosis: incidence, prevalence, survival, risk factors, malignancy, and environmental triggers. Curr Opin Rheumatol, 24, 165-70. Bergmann, C. & J. H. Distler (2016) Canonical Wnt signaling in systemic sclerosis. Lab Invest, 96, 151-5. Berrabah, W., P. Aumercier, P. Lefebvre & B. Staels (2011) Control of nuclear receptor activities in metabolism by post-translational modifications. FEBS Lett, 585, 1640-50. Beyer, C., J. Huang, J. Beer, Y. Zhang, K. Palumbo-Zerr, P. Zerr, A. Distler, C. Dees, C. Maier, L. Munoz, G. Kronke, S. Uderhardt, O. Distler, S. Jones, S. Rose-John, T. Oravecz, G.

111

References Schett & J. H. Distler (2015) Activation of liver X receptors inhibits experimental fibrosis by interfering with interleukin-6 release from macrophages. Ann Rheum Dis, 74, 1317- 24. Beyer, C., D. Huscher, A. Ramming, C. Bergmann, J. Avouac, S. Guiducci, F. Meier, S. Vettori, E. Siegert, V. K. Jaeger, B. Maurer, G. Riemekasten, U. Walker, U. Muller-Ladner, G. Valentini, M. Matucci-Cerinic, Y. Allanore, O. Distler, G. Schett & J. H. W. Distler (2018) Elevated serum levels of sonic hedgehog are associated with fibrotic and vascular manifestations in systemic sclerosis. Ann Rheum Dis, 77, 626-628. Beyer, C., G. Schett, O. Distler & J. H. Distler (2010) Animal models of systemic sclerosis: prospects and limitations. Arthritis Rheum, 62, 2831-44. Beyer, C., A. Skapenko A Fau - Distler, C. Distler A Fau - Dees, H. Dees C Fau - Reichert, L. Reichert H Fau - Munoz, J. Munoz L Fau - Leipe, H. Leipe J Fau - Schulze-Koops, O. Schulze-Koops H Fau - Distler, G. Distler O Fau - Schett, J. H. W. Schett G Fau - Distler & J. H. Distler (2013) Activation of pregnane X receptor inhibits experimental dermal fibrosis. Ann Rheum Dis. , 72, 621–625. Bhattacharyya, S., J. Wei & J. Varga (2011) Understanding fibrosis in systemic sclerosis: shifting paradigms, emerging opportunities. Nat Rev Rheumatol, 8, 42-54. Bigelow, R. L., E. Y. Jen, M. Delehedde, N. S. Chari & T. J. McDonnell (2005) Sonic hedgehog induces epidermal growth factor dependent matrix infiltration in HaCaT keratinocytes. J Invest Dermatol, 124, 457-65. Blobe, G. C., W. P. Schiemann & H. F. Lodish (2000) Role of transforming growth factor beta in human disease. N Engl J Med, 342, 1350-8. Bolanos, A. L., C. M. Milla, J. C. Lira, R. Ramirez, M. Checa, L. Barrera, J. Garcia-Alvarez, V. Carbajal, C. Becerril, M. Gaxiola, A. Pardo & M. Selman (2012) Role of Sonic Hedgehog in idiopathic pulmonary fibrosis. Am J Physiol Lung Cell Mol Physiol, 303, L978-90. Boukhtouche, F., A. Mariani J Fau - Tedgui & A. Tedgui (2004) The "CholesteROR" protective pathway in the vascular system. Arterioscler Thromb Vasc Biol. , 24, 637-43. Boukhtouche, F., J. Mariani & A. Tedgui (2004) The "CholesteROR" protective pathway in the vascular system. Arterioscler Thromb Vasc Biol, 24, 637-43. Brennan, D., X. Chen, L. Cheng, M. Mahoney & N. A. Riobo (2012) Noncanonical Hedgehog signaling. Vitam Horm, 88, 55-72. Briscoe, J. & P. P. Therond (2013) The mechanisms of Hedgehog signalling and its roles in development and disease. Nat Rev Mol Cell Biol, 14, 416-29. Broen, J. C., T. R. Radstake & M. Rossato (2014) The role of genetics and epigenetics in the pathogenesis of systemic sclerosis. Nat Rev Rheumatol, 10, 671-81. Buglino, J. A. & M. D. Resh (2008) Hhat is a palmitoylacyltransferase with specificity for N- palmitoylation of Sonic Hedgehog. J Biol Chem, 283, 22076-88. Caroline CHAUVET, B. B.-J., Edurne BERRA, Jacques POUYSSEGUR and Jean-Louis DANAN (2004) The gene encoding human retinoic acid-receptor-related orphan receptor α is a target for hypoxia-inducible factor 1. Biochem. J. , 384, 79–85. Castrillo, A. & P. Tontonoz (2004) Nuclear receptors in macrophage biology: at the crossroads of lipid metabolism and inflammation. Annu Rev Cell Dev Biol, 20, 455-80. Chen, M. H., Y. J. Li, T. Kawakami, S. M. Xu & P. T. Chuang (2004) Palmitoylation is required for the production of a soluble multimeric Hedgehog protein complex and long-range signaling in vertebrates. Genes Dev, 18, 641-59. Chifflot, H., B. Fautrel, C. Sordet, E. Chatelus & J. Sibilia (2008) Incidence and prevalence of systemic sclerosis: a systematic literature review. Semin Arthritis Rheum, 37, 223-35. China Public Health Network. 2016. Systemic sclerosis. Chizzolini, C., N. C. Brembilla, E. Montanari & M. E. Truchetet (2011) Fibrosis and immune dysregulation in systemic sclerosis. Autoimmun Rev, 10, 276-81. 112

References Chizzolini, C., Y. Parel, C. De Luca, A. Tyndall, A. Akesson, A. Scheja & J. M. Dayer (2003) Systemic sclerosis Th2 cells inhibit collagen production by dermal fibroblasts via membrane-associated tumor necrosis factor alpha. Arthritis Rheum, 48, 2593-604. Cook, D. N., H. S. Kang & A. M. Jetten (2015) Retinoic Acid-Related Orphan Receptors (RORs): Regulatory Functions in Immunity, Development, Circadian Rhythm, and Metabolism. Nucl Receptor Res, 2. Dawber, R. J., S. Hebbes, B. Herpers, F. Docquier & M. van den Heuvel (2005) Differential range and activity of various forms of the Hedgehog protein. BMC Dev Biol, 5, 21. Dennler, S., J. Andre, I. Alexaki, A. Li, T. Magnaldo, P. ten Dijke, X. J. Wang, F. Verrecchia & A. Mauviel (2007) Induction of sonic hedgehog mediators by transforming growth factor-beta: Smad3-dependent activation of Gli2 and Gli1 expression in vitro and in vivo. Cancer Res, 67, 6981-6. Dennler, S., J. Andre, F. Verrecchia & A. Mauviel (2009) Cloning of the human GLI2 Promoter: transcriptional activation by transforming growth factor-beta via SMAD3/beta-catenin cooperation. J Biol Chem, 284, 31523-31. Dieude, P., M. Dawidowicz K Fau - Guedj, Y. Guedj M Fau - Legrain, J. Legrain Y Fau - Wipff, E. Wipff J Fau - Hachulla, E. Hachulla E Fau - Diot, J. Diot E Fau - Sibilia, L. Sibilia J Fau - Mouthon, J. Mouthon L Fau - Cabane, Z. Cabane J Fau - Amoura, J.-L. Amoura Z Fau - Crakowski, P. Crakowski Jl Fau - Carpentier, J. Carpentier P Fau - Avouac, O. Avouac J Fau - Meyer, A. Meyer O Fau - Kahan, C. Kahan A Fau - Boileau, Y. Boileau C Fau - Allanore & Y. Allanore (2010) Phenotype-haplotype correlation of IRF5 in systemic sclerosis: role of 2 haplotypes in disease severity. J Rheumatol, 37, 987- 92. Distler, A., V. Lang, T. Del Vecchio, J. Huang, Y. Zhang, C. Beyer, N. Y. Lin, K. Palumbo- Zerr, O. Distler, G. Schett & J. H. Distler (2014) Combined inhibition of morphogen pathways demonstrates additive antifibrotic effects and improved tolerability. Ann Rheum Dis, 73, 1264-8. Distler, J. H., S. Gay & O. Distler (2006) Angiogenesis and vasculogenesis in systemic sclerosis. Rheumatology (Oxford), 45 Suppl 3, iii26-7. Distler, O., A. Del Rosso, R. Giacomelli, P. Cipriani, M. L. Conforti, S. Guiducci, R. E. Gay, B. A. Michel, P. Bruhlmann, U. Muller-Ladner, S. Gay & M. Matucci-Cerinic (2002) Angiogenic and angiostatic factors in systemic sclerosis: increased levels of vascular endothelial growth factor are a feature of the earliest disease stages and are associated with the absence of fingertip ulcers. Arthritis Res, 4, R11. Dussault, I., D. Fawcett, A. Matthyssen, J. A. Bader & V. Giguere (1998) Orphan nuclear receptor ROR alpha-deficient mice display the cerebellar defects of staggerer. Mech Dev, 70, 147-53. Eckes, B., N. Hunzelmann, P. Moinzadeh & T. Krieg (2007) Scleroderma -- news to tell. Arch Dermatol Res, 299, 139-44. Edson, M. A., R. L. Nalam, C. Clementi, H. L. Franco, F. J. Demayo, K. M. Lyons, S. A. Pangas & M. M. Matzuk (2010) Granulosa cell-expressed BMPR1A and BMPR1B have unique functions in regulating fertility but act redundantly to suppress ovarian tumor development. Mol Endocrinol, 24, 1251-66. Fabian, S. L., R. R. Penchev, B. St-Jacques, A. N. Rao, P. Sipila, K. A. West, A. P. McMahon & B. D. Humphreys (2012) Hedgehog-Gli pathway activation during kidney fibrosis. Am J Pathol, 180, 1441-53. Fan, Q., M. He, T. Sheng, X. Zhang, M. Sinha, B. Luxon, X. Zhao & J. Xie (2010) Requirement of TGFbeta signaling for SMO-mediated carcinogenesis. J Biol Chem, 285, 36570-6. Fattahi, S., M. Pilehchian Langroudi & H. Akhavan-Niaki (2018) Hedgehog signaling pathway: Epigenetic regulation and role in disease and cancer development. J Cell Physiol, 233, 5726-5735. 113

References Feil, R., D. Wagner J Fau - Metzger, P. Metzger D Fau - Chambon & P. Chambon (1997) Regulation of Cre recombinase activity by mutated estrogen receptor ligand-binding domains. Biochem Biophys Res Commun., 237, 752-757. Fernandes-Silva, H., J. Correia-Pinto & R. S. Moura (2017) Canonical Sonic Hedgehog Signaling in Early Lung Development. J Dev Biol, 5. Fischer, C., P. Schneider M Fau - Carmeliet & P. Carmeliet (2006) Principles and therapeutic implications of angiogenesis, vasculogenesis and arteriogenesis. Handb Exp Pharmacol, 176, 157-212. Francois, A., E. Chatelus, D. Wachsmann, J. Sibilia, S. Bahram, G. Alsaleh & J. E. Gottenberg (2013) B lymphocytes and B-cell activating factor promote collagen and profibrotic markers expression by dermal fibroblasts in systemic sclerosis. Arthritis Res Ther, 15, R168. Fuschiotti, P. (2016) Current perspectives on the immunopathogenesis of systemic sclerosis. Immunotargets Ther, 5, 21-35. Garbuzenko, E., A. Nagler, D. Pickholtz, P. Gillery, R. Reich, F. X. Maquart & F. Levi-Schaffer (2002) Human mast cells stimulate fibroblast proliferation, collagen synthesis and lattice contraction: a direct role for mast cells in skin fibrosis. Clin Exp Allergy, 32, 237-46. Gelber, A. C., R. L. Manno, A. A. Shah, A. Woods, E. N. Le, F. Boin, L. K. Hummers & F. M. Wigley (2013) Race and association with disease manifestations and mortality in scleroderma: a 20-year experience at the Johns Hopkins Scleroderma Center and review of the literature. Medicine (Baltimore), 92, 191-205. Germain, P., B. Staels, C. Dacquet, M. Spedding & V. Laudet (2006) Overview of nomenclature of nuclear receptors. Pharmacol Rev, 58, 685-704. Giacomelli, R., P. Matucci-Cerinic M Fau - Cipriani, I. Cipriani P Fau - Ghersetich, R. Ghersetich I Fau - Lattanzio, A. Lattanzio R Fau - Pavan, A. Pavan A Fau - Pignone, M. L. Pignone A Fau - Cagnoni, T. Cagnoni Ml Fau - Lotti, G. Lotti T Fau - Tonietti & G. Tonietti (1998) Circulating Vdelta1+ T cells are activated and accumulate in the skin of systemic sclerosis patients. Arthritis Rheum, 41, 327-34. Giguere, V. (1999) Orphan nuclear receptors: from gene to function. Endocr Rev, 20, 689-725. Goetz, S. C. & K. V. Anderson (2010) The primary cilium: a signalling centre during vertebrate development. Nat Rev Genet, 11, 331-44. Gonnissen, A., S. Isebaert & K. Haustermans (2015) Targeting the Hedgehog signaling pathway in cancer: beyond Smoothened. Oncotarget, 6, 13899-913. Greschik, H. & D. Moras (2003) Structure-activity relationship of nuclear receptor-ligand interactions. Curr Top Med Chem, 3, 1573-99. Gronemeyer, H., J. A. Gustafsson & V. Laudet (2004) Principles for modulation of the nuclear receptor superfamily. Nat Rev Drug Discov, 3, 950-64. Guiducci, S., M. Manetti, E. Romano, B. Mazzanti, C. Ceccarelli, S. Dal Pozzo, A. F. Milia, S. Bellando-Randone, G. Fiori, M. L. Conforti, R. Saccardi, L. Ibba-Manneschi & M. Matucci-Cerinic (2011) Bone marrow-derived mesenchymal stem cells from early diffuse systemic sclerosis exhibit a paracrine machinery and stimulate angiogenesis in vitro. Ann Rheum Dis, 70, 2011-21. Gutstein, D. E., D. Krishna R Fau - Johns, H. K. Johns D Fau - Surks, H. M. Surks Hk Fau - Dansky, S. Dansky Hm Fau - Shah, Y. B. Shah S Fau - Mitchel, J. Mitchel Yb Fau - Arena, J. A. Arena J Fau - Wagner & J. A. Wagner (2012) Anacetrapib, a novel CETP inhibitor: pursuing a new approach to cardiovascular risk reduction. Clin Pharmacol Ther., 91, 109-22. Han, Y. H., H. J. Kim, H. Na, M. W. Nam, J. Y. Kim, J. S. Kim, S. H. Koo & M. O. Lee (2017) RORalpha Induces KLF4-Mediated M2 Polarization in the Liver Macrophages that Protect against Nonalcoholic Steatohepatitis. Cell Rep, 20, 124-135.

114

References Harris, J. M., P. Lau, S. L. Chen & G. E. Muscat (2002) Characterization of the retinoid orphan- related receptor-alpha coactivator binding interface: a structural basis for ligand- independent transcription. Mol Endocrinol, 16, 998-1012. Hasegawa, M., Y. Hamaguchi, K. Yanaba, J.-D. Bouaziz, J. Uchida, M. Fujimoto, T. Matsushita, Y. Matsushita, M. Horikawa, K. Komura, K. Takehara, S. Sato & T. F. Tedder (2006) B-lymphocyte depletion reduces skin fibrosis and autoimmunity in the tight-skin mouse model for systemic sclerosis. The American journal of pathology, 169, 954-966. Hashimoto, N., S. H. Phan, K. Imaizumi, M. Matsuo, H. Nakashima, T. Kawabe, K. Shimokata & Y. Hasegawa (2010) Endothelial-mesenchymal transition in bleomycin-induced pulmonary fibrosis. Am J Respir Cell Mol Biol, 43, 161-72. He, Y. W., M. L. Deftos, E. W. Ojala & M. J. Bevan (1998) RORgamma t, a novel isoform of an orphan receptor, negatively regulates Fas ligand expression and IL-2 production in T cells. Immunity, 9, 797-806. Hernandez-Gea, V., Z. Ghiassi-Nejad, R. Rozenfeld, R. Gordon, M. I. Fiel, Z. Yue, M. J. Czaja & S. L. Friedman (2012) Autophagy releases lipid that promotes fibrogenesis by activated hepatic stellate cells in mice and in human tissues. Gastroenterology, 142, 938- 46. Hihi, A. K., L. Michalik & W. Wahli (2002) PPARs: transcriptional effectors of fatty acids and their derivatives. Cell Mol Life Sci, 59, 790-8. Ho, Y. Y., D. Lagares, A. M. Tager & M. Kapoor (2014) Fibrosis—a lethal component of systemic sclerosis. Nat Rev Rheumatology, 10, 390. Horn, A., T. Kireva, K. Palumbo-Zerr, C. Dees, M. Tomcik, C. Cordazzo, P. Zerr, A. Akhmetshina, M. Ruat, O. Distler, C. Beyer, G. Schett & J. H. Distler (2012a) Inhibition of hedgehog signalling prevents experimental fibrosis and induces regression of established fibrosis. Ann Rheum Dis, 71, 785-9. Horn, A., K. Palumbo, C. Cordazzo, C. Dees, A. Akhmetshina, M. Tomcik, P. Zerr, J. Avouac, J. Gusinde, J. Zwerina, H. Roudaut, E. Traiffort, M. Ruat, O. Distler, G. Schett & J. H. Distler (2012b) Hedgehog signaling controls fibroblast activation and tissue fibrosis in systemic sclerosis. Arthritis Rheum, 64, 2724-33. Hu, M., J. Yao, D. K. Carroll, S. Weremowicz, H. Chen, D. Carrasco, A. Richardson, S. Violette, T. Nikolskaya, Y. Nikolsky, E. L. Bauerlein, W. C. Hahn, R. S. Gelman, C. Allred, M. J. Bissell, S. Schnitt & K. Polyak (2008) Regulation of in situ to invasive breast carcinoma transition. Cancer Cell, 13, 394-406. Hui, C. C. & S. Angers (2011) Gli proteins in development and disease. Annu Rev Cell Dev Biol, 27, 513-37. IHN, H. I. (2002) The Role of TGF-b Signaling in the Pathogenesis of Fibrosis in Scleroderma.pdf. Jaradat, M., C. Stapleton, S. L. Tilley, D. Dixon, C. J. Erikson, J. G. McCaskill, H. S. Kang, M. Angers, G. Liao, J. Collins, S. Grissom & A. M. Jetten (2006) Modulatory role for retinoid-related orphan receptor alpha in allergen-induced lung inflammation. Am J Respir Crit Care Med, 174, 1299-309. Javelaud, D., V. I. Alexaki, S. Dennler, K. S. Mohammad, T. A. Guise & A. Mauviel (2011) TGF-beta/SMAD/GLI2 signaling axis in cancer progression and metastasis. Cancer Res, 71, 5606-10. Javelaud, D., M. J. Pierrat & A. Mauviel (2012) Crosstalk between TGF-beta and hedgehog signaling in cancer. FEBS Lett, 586, 2016-25. Jenkins, D. (2009) Hedgehog signalling: emerging evidence for non-canonical pathways. Cell Signal, 21, 1023-34. Jetten, A. M. (2009) Retinoid-related orphan receptors (RORs): critical roles in development, immunity, circadian rhythm, and cellular metabolism. Nucl Recept Signal, 7, e003. 115

References Jetten, A. M., S. Kurebayashi & E. Ueda (2001) The ROR nuclear orphan receptor subfamily: critical regulators of multiple biological processes. Prog Nucleic Acid Res Mol Biol, 69, 205-47. Jimenez, S. A. (2013) Role of endothelial to mesenchymal transition in the pathogenesis of the vascular alterations in systemic sclerosis. ISRN Rheumatol, 2013. Jimenez, S. A. & S. Piera-Velazquez (2016) Endothelial to mesenchymal transition (EndoMT) in the pathogenesis of Systemic Sclerosis-associated pulmonary fibrosis and pulmonary arterial hypertension. Myth or reality? Matrix Biol, 51. Kahaleh, M. B. (2004) Raynaud phenomenon and the vascular disease in scleroderma.pdf. Current Opinion in Rheumatology, 16, 718-722. Kahaleh, M. B., T. Fan Ps Fau - Otsuka & T. Otsuka (1999) Gammadelta receptor bearing T cells in scleroderma: enhanced interaction with vascular endothelial cells in vitro. Clin Immunol, 91, 188-95. Kalogerou, A., E. Gelou, S. Mountantonakis, L. Settas, E. Zafiriou & L. Sakkas (2005) Early T cell activation in the skin from patients with systemic sclerosis. Ann Rheum Dis, 64, 1233-5. Kasper, M., H. Schnidar, G. W. Neill, M. Hanneder, S. Klingler, L. Blaas, C. Schmid, C. Hauser-Kronberger, G. Regl, M. P. Philpott & F. Aberger (2006) Selective modulation of Hedgehog/GLI target gene expression by epidermal growth factor signaling in human keratinocytes. Mol Cell Biol, 26, 6283-98. Kim, J. H., H. Y. Kim, S. Kim, J. H. Chung, W. S. Park & D. H. Chung (2005) Natural killer T (NKT) cells attenuate bleomycin-induced pulmonary fibrosis by producing interferon- gamma. Am J Pathol, 167, 1231-41. Kinzler, K. W., J. M. Ruppert, S. H. Bigner & B. Vogelstein (1988) The GLI gene is a member of the Kruppel family of zinc finger proteins. Nature, 332, 371-4. Kinzler, K. W. & B. Vogelstein (1990) The GLI gene encodes a nuclear protein which binds specific sequences in the human genome. Mol Cell Biol, 10, 634-42. Konttinen, Y. T., Z. Mackiewicz, P. Ruuttila, A. Ceponis, A. Sukura, D. Povilenaite, M. Hukkanen & I. Virtanen (2003) Vascular damage and lack of angiogenesis in systemic sclerosis skin. Clin Rheumatol, 22, 196-202. Kramann, R. (2016) Hedgehog Gli signalling in kidney fibrosis. Nephrol Dial Transplant, 31, 1989-1995. Kramann, R., S. V. Fleig, R. K. Schneider, S. L. Fabian, D. P. DiRocco, O. Maarouf, J. Wongboonsin, Y. Ikeda, D. Heckl, S. L. Chang, H. G. Rennke, S. S. Waikar & B. D. Humphreys (2015) Pharmacological GLI2 inhibition prevents myofibroblast cell-cycle progression and reduces kidney fibrosis. J Clin Invest, 125, 2935-51. Kulozik, M., A. Hogg, B. Lankat-Buttgereit & T. Krieg (1990) Co-localization of transforming growth factor beta 2 with alpha 1(I) procollagen mRNA in tissue sections of patients with systemic sclerosis. J Clin Invest, 86, 917-22. Kumar, N., D. J. Kojetin, L. A. Solt, K. G. Kumar, P. Nuhant, D. R. Duckett, M. D. Cameron, A. A. Butler, W. R. Roush, P. R. Griffin & T. P. Burris (2011) Identification of SR3335 (ML-176): a synthetic RORalpha selective inverse agonist. ACS Chem Biol, 6, 218-22. Kuwana, M., J. Kaburaki, T. Mimori, Y. Kawakami & T. Tojo (2000) Longitudinal analysis of autoantibody response to topoisomerase I in systemic sclerosis. Arthritis Rheum, 43, 1074-84. Lafyatis, R., C. O'Hara, C. A. Feghali-Bostwick & E. Matteson (2007) B cell infiltration in systemic sclerosis-associated interstitial lung disease. Arthritis Rheum, 56, 3167-8. Lau, P., P. Bailey, D. H. Dowhan & G. E. Muscat (1999) Exogenous expression of a dominant negative RORalpha1 vector in muscle cells impairs differentiation: RORalpha1 directly interacts with p300 and myoD. Nucleic Acids Res, 27, 411-20.

116

References Lau, P., R. L. Fitzsimmons, S. Raichur, S. C. Wang, A. Lechtken & G. E. Muscat (2008) The orphan nuclear receptor, RORalpha, regulates gene expression that controls lipid metabolism: staggerer (SG/SG) mice are resistant to diet-induced obesity. J Biol Chem, 283, 18411-21. Lauth, M. & R. Toftgard (2007) Non-canonical activation of GLI transcription factors: implications for targeted anti-cancer therapy. Cell Cycle, 6, 2458-63. Leask, A. (2006) Scar wars: is TGFbeta the phantom menace in scleroderma? Arthritis Res Ther, 8, 213. Leask, A. & D. J. Abraham (2004) TGF-beta signaling and the fibrotic response. FASEB J, 18, 816-27. Lee, J. D. & J. E. Treisman (2001) Sightless has homology to transmembrane acyltransferases and is required to generate active Hedgehog protein. Curr Biol, 11, 1147-52. Lee, J. M., I. S. Kim, H. Kim, J. S. Lee, K. Kim, H. Y. Yim, J. Jeong, J. H. Kim, J. Y. Kim, H. Lee, S. B. Seo, H. Kim, M. G. Rosenfeld, K. I. Kim & S. H. Baek (2010) RORalpha attenuates Wnt/beta-catenin signaling by PKCalpha-dependent phosphorylation in colon cancer. Mol Cell, 37, 183-95. Lee P Fau - Norman, C. S., S. Norman Cs Fau - Sukenik, C. A. Sukenik S Fau - Alderdice & C. A. Alderdice (1985) The clinical significance of coagulation abnormalities in systemic sclerosis (scleroderma). J Rheumatol, 12, 514-7. Levin, E. R. & S. R. Hammes (2016) Nuclear receptors outside the nucleus: extranuclear signalling by steroid receptors. Nat Rev Mol Cell Biol, 17, 783-797. Liang, R., B. Sumova, C. Cordazzo, T. Mallano, Y. Zhang, T. Wohlfahrt, C. Dees, A. Ramming, D. Krasowska, M. Michalska-Jakubus, O. Distler, G. Schett, L. Senolt & J. H. Distler (2017) The transcription factor GLI2 as a downstream mediator of transforming growth factor-beta-induced fibroblast activation in SSc. Ann Rheum Dis, 76, 756-764. Longo, K. A., W. S. Wright, S. Kang, I. Gerin, S. H. Chiang, P. C. Lucas, M. R. Opp & O. A. MacDougald (2004) Wnt10b inhibits development of white and brown adipose tissues. J Biol Chem, 279, 35503-9. Maeda, O., M. Kondo, T. Fujita, N. Usami, T. Fukui, K. Shimokata, T. Ando, H. Goto & Y. Sekido (2006) Enhancement of GLI1-transcriptional activity by beta-catenin in human cancer cells. Oncol Rep, 16, 91-6. Manetti, M., S. Guiducci, L. Ibba-Manneschi & M. Matucci-Cerinic (2010) Mechanisms in the loss of capillaries in systemic sclerosis: angiogenesis versus vasculogenesis. J Cell Mol Med, 14, 1241-54. Manetti, M. A.-O. h. o. o., E. Romano, I. Rosa, S. Guiducci, S. Bellando-Randone, A. De Paulis, L. Ibba-Manneschi & M. Matucci-Cerinic (2017) Endothelial-to-mesenchymal transition contributes to endothelial dysfunction and dermal fibrosis in systemic sclerosis. Ann Rheum Dis, 76, 924-934. Mann, R. K. & P. A. Beachy (2004) Novel lipid modifications of secreted protein signals. Annu Rev Biochem, 73, 891-923. Marangoni, R. G., B. D. Korman, J. Wei, T. A. Wood, L. V. Graham, M. L. Whitfield, P. E. Scherer, W. G. Tourtellotte & J. Varga (2015) Myofibroblasts in murine cutaneous fibrosis originate from adiponectin-positive intradermal progenitors. Arthritis Rheumatol, 67, 1062-73. Marangoni, R. G., J. Varga & W. G. Tourtellotte (2016) Animal models of scleroderma: recent progress. Curr Opin Rheumatol, 28, 561-70. Marvi, U. & L. Chung (2010) Digital ischemic loss in systemic sclerosis. LID - 10.1155/2010/130717 [doi] LID - 130717 [pii]. Int J Rheumatol, 2010. Massague, J. (1998) TGF-beta signal transduction. Annu Rev Biochem, 67, 753-91.

117

References Matsushita, T., M. Hasegawa, Y. Hamaguchi, K. Takehara & S. Sato (2006) Longitudinal analysis of serum cytokine concentrations in systemic sclerosis: association of interleukin 12 elevation with spontaneous regression of skin sclerosis. J Rheumatol, 33, 275-84. Matucci-Cerinic, M., B. Kahaleh & F. M. Wigley (2013) Review: evidence that systemic sclerosis is a vascular disease. Arthritis Rheum, 65, 1953-62. Mayes, M. D., J. V. Lacey, Jr., J. Beebe-Dimmer, B. W. Gillespie, B. Cooper, T. J. Laing & D. Schottenfeld (2003) Prevalence, incidence, survival, and disease characteristics of systemic sclerosis in a large US population. Arthritis Rheum, 48, 2246-55. Meloni, F., N. Solari, L. Cavagna, M. Morosini, C. M. Montecucco & A. M. Fietta (2009) Frequency of Th1, Th2 and Th17 producing T lymphocytes in bronchoalveolar lavage of patients with systemic sclerosis. Clin Exp Rheumatol, 27, 765-72. Mendoza, F. A., S. Piera-Velazquez, J. L. Farber, C. Feghali-Bostwick & S. A. Jimenez (2016) Endothelial Cells Expressing Endothelial and Mesenchymal Cell Gene Products in Lung Tissue From Patients With Systemic Sclerosis-Associated Interstitial Lung Disease. Arthritis Rheumatol, 68. Meyer, T., M. Kneissel, J. Mariani & B. Fournier (2000) In vitro and in vivo evidence for orphan nuclear receptor RORalpha function in bone metabolism. Proc Natl Acad Sci U S A, 97, 9197-202. Micchelli, C. A., I. The, E. Selva, V. Mogila & N. Perrimon (2002) Rasp, a putative transmembrane acyltransferase, is required for Hedgehog signaling. Development, 129, 843-51. Michelotti, G. A., G. Xie, M. Swiderska, S. S. Choi, G. Karaca, L. Kruger, R. Premont, L. Yang, W. K. Syn, D. Metzger & A. M. Diehl (2013) Smoothened is a master regulator of adult liver repair. J Clin Invest, 123, 2380-94. Milano, A., S. A. Pendergrass, J. L. Sargent, L. K. George, T. H. McCalmont, M. K. Connolly & M. L. Whitfield (2008) Molecular subsets in the gene expression signatures of scleroderma skin. PLoS One, 3, e2696. Mostmans, Y., M. Cutolo, C. Giddelo, S. Decuman, K. Melsens, H. Declercq, E. Vandecasteele, F. De Keyser, O. Distler, J. Gutermuth & V. Smith (2017) The role of endothelial cells in the vasculopathy of systemic sclerosis: A systematic review. Autoimmunity Reviews, 16, 774-786. Mouratis, M. A. & V. Aidinis (2011) Modeling pulmonary fibrosis with bleomycin. Curr Opin Pulm Med, 17, 355-61. Nolte, R. T., G. B. Wisely, S. Westin, J. E. Cobb, M. H. Lambert, R. Kurokawa, M. G. Rosenfeld, T. M. Willson, C. K. Glass & M. V. Milburn (1998) Ligand binding and coactivator assembly of the peroxisome proliferator-activated receptor-gamma. Nature, 395, 137-43. Nüsslein-Volhard, C. & E. Wieschaus (1980) Mutations affecting segment number and polarity in Drosophila. Nature, 287, 795. O'Brien-Ladner, A. R., L. J. Wesselius & D. J. Stechschulte (1993) Bleomycin injury of the lung in a mast-cell-deficient model. Agents Actions, 39, 20-4. Ogden, S. K., D. L. Fei, N. S. Schilling, Y. F. Ahmed, J. Hwa & D. J. Robbins (2008) G protein Galphai functions immediately downstream of Smoothened in Hedgehog signalling. Nature, 456, 967-70. Omenetti, A., L. Yang, Y. X. Li, S. J. McCall, Y. Jung, J. K. Sicklick, J. Huang, S. Choi, A. Suzuki & A. M. Diehl (2007) Hedgehog-mediated mesenchymal-epithelial interactions modulate hepatic response to bile duct ligation. Lab Invest, 87, 499-514. Pak, E. & R. A. Segal (2016) Hedgehog Signal Transduction: Key Players, Oncogenic Drivers, and Cancer Therapy. Dev Cell, 38, 333-44. Pavletich, N. P. & C. O. Pabo (1993) Crystal structure of a five-finger GLI-DNA complex: new perspectives on zinc fingers. Science, 261, 1701-7. 118

References Pepinsky, R. B., C. Zeng, D. Wen, P. Rayhorn, D. P. Baker, K. P. Williams, S. A. Bixler, C. M. Ambrose, E. A. Garber, K. Miatkowski, F. R. Taylor, E. A. Wang & A. Galdes (1998) Identification of a palmitic acid-modified form of human Sonic hedgehog. J Biol Chem, 273, 14037-45. Pereira, T. A., G. Xie, S. S. Choi, W. K. Syn, I. Voieta, J. Lu, I. S. Chan, M. Swiderska, K. B. Amaral, C. M. Antunes, W. E. Secor, R. P. Witek, J. R. Lambertucci, F. L. Pereira & A. M. Diehl (2013) Macrophage-derived Hedgehog ligands promotes fibrogenic and angiogenic responses in human schistosomiasis mansoni. Liver Int, 33, 149-61. Petrov, K., B. M. Wierbowski & A. Salic (2017) Sending and Receiving Hedgehog Signals. Annu Rev Cell Dev Biol, 33, 145-168. Porter, J. A., K. E. Young & P. A. Beachy (1996) Cholesterol modification of hedgehog signaling proteins in animal development. Science, 274, 255-9. Querfeld, C., B. Eckes, C. Huerkamp, T. Krieg & S. Sollberg (1999) Expression of TGF-beta 1, -beta 2 and -beta 3 in localized and systemic scleroderma. J Dermatol Sci, 21, 13-22. R.Tata, J. (2002) Signalling through nuclear receptors.pdf. NATURE REVIEWS | MOLECULAR CELL BIOLOGY, 3, 702-710. Rabquer, B. J. & A. E. Koch (2012) Angiogenesis and vasculopathy in systemic sclerosis: evolving concepts. Curr Rheumatol Rep, 14, 56-63. Rajkumar, V. S., K. Howell, K. Csiszar, C. P. Denton, C. M. Black & D. J. Abraham (2005) Shared expression of phenotypic markers in systemic sclerosis indicates a convergence of pericytes and fibroblasts to a myofibroblast lineage in fibrosis. Arthritis Res Ther, 7, R1113-23. Ramming, A., C. Dees & J. H. Distler (2015) From pathogenesis to therapy--Perspective on treatment strategies in fibrotic diseases. Pharmacol Res, 100, 93-100. Raspe, E., H. Duez, P. Gervois, C. Fievet, J. C. Fruchart, S. Besnard, J. Mariani, A. Tedgui & B. Staels (2001) Transcriptional regulation of apolipoprotein C-III gene expression by the orphan nuclear receptor RORalpha. J Biol Chem, 276, 2865-71. Remlinger, K. A. (2003) Cutaneous reactions to chemotherapy drugs: the art of consultation. Arch Dermatol, 139, 77-81. Renaud, J. P. & D. Moras (2000) Structural studies on nuclear receptors. Cell Mol Life Sci, 57, 1748-69. Repa, J. J. & D. J. Mangelsdorf (1999) Nuclear receptor regulation of cholesterol and bile acid metabolism. Curr Opin Biotechnol, 10, 557-63. Rimkus, T. K., R. L. Carpenter, S. Qasem, M. Chan & H. W. Lo (2016) Targeting the Sonic Hedgehog Signaling Pathway: Review of Smoothened and GLI Inhibitors. Cancers (Basel), 8. Riobo, N. A., K. Lu, X. Ai, G. M. Haines & C. P. Emerson, Jr. (2006) Phosphoinositide 3- kinase and Akt are essential for Sonic Hedgehog signaling. Proc Natl Acad Sci U S A, 103, 4505-10. Riobo, N. A. & D. R. Manning (2007) Pathways of signal transduction employed by vertebrate Hedgehogs. Biochem J, 403, 369-79. Robbins, D. J., D. L. Fei & N. A. Riobo (2012) The Hedgehog signal transduction network. Sci Signal, 5, re6. Ruppert, J. M., B. Vogelstein & K. W. Kinzler (1991) The zinc finger protein GLI transforms primary cells in cooperation with adenovirus E1A. Mol Cell Biol, 11, 1724-8. Ruzehaji, N., J. Avouac, M. Elhai, M. Frechet, C. Frantz, B. Ruiz, J. H. Distler & Y. Allanore (2015) Combined effect of genetic background and gender in a mouse model of bleomycin-induced skin fibrosis. Arthritis Res Ther, 17, 145. Saigusa, R., Y. Asano, T. Taniguchi, T. Yamashita, Y. Ichimura, T. Takahashi, T. Toyama, A. Yoshizaki, K. Sugawara, D. Tsuruta, T. Taniguchi & S. Sato (2015a) Multifaceted

119

References contribution of the TLR4-activated IRF5 transcription factor in systemic sclerosis. Proc Natl Acad Sci, 112, 15136-41. Saigusa, R., Y. Asano, T. Taniguchi, T. Yamashita, T. Takahashi, Y. Ichimura, T. Toyama, Z. Tamaki, Y. Tada, M. Sugaya, T. Kadono & S. Sato (2015b) A possible contribution of endothelial CCN1 downregulation due to Fli1 deficiency to the development of digital ulcers in systemic sclerosis. Exp Dermatol, 24, 127-32. Sakkas, L. I. & C. D. Platsoucas (2004) Is systemic sclerosis an antigen-driven T cell disease? Arthritis Rheum, 50, 1721-33. Salazar, G. & M. D. Mayes (2015) Genetics, Epigenetics, and Genomics of Systemic Sclerosis. Rheum Dis Clin North Am, 41, 345-66. Sato, T. K., S. Panda, L. J. Miraglia, T. M. Reyes, R. D. Rudic, P. McNamara, K. A. Naik, G. A. FitzGerald, S. A. Kay & J. B. Hogenesch (2004) A functional genomics strategy reveals Rora as a component of the mammalian circadian clock. Neuron, 43, 527-37. Schnidar, H., M. Eberl, S. Klingler, D. Mangelberger, M. Kasper, C. Hauser-Kronberger, G. Regl, R. Kroismayr, R. Moriggl, M. Sibilia & F. Aberger (2009) Epidermal growth factor receptor signaling synergizes with Hedgehog/GLI in oncogenic transformation via activation of the MEK/ERK/JUN pathway. Cancer Res, 69, 1284-92. Sever, R. & C. K. Glass (2013) Signaling by nuclear receptors. Cold Spring Harb Perspect Biol, 5, a016709. Sgonc, R., G. Gruschwitz Ms Fau - Boeck, N. Boeck G Fau - Sepp, J. Sepp N Fau - Gruber, G. Gruber J Fau - Wick & G. Wick (2000) Endothelial cell apoptosis in systemic sclerosis is induced by antibody-dependent cell-mediated cytotoxicity via CD95. Arthritis Rheum, 43, 2550-62. Sica, A. & A. Mantovani (2012) Macrophage plasticity and polarization: in vivo veritas. J Clin Invest, 122, 787-95. Sidman, R. L., P. W. Lane & M. M. Dickie (1962) Staggerer, a new mutation in the mouse affecting the cerebellum. Science, 137, 610-2. Solt, L. A. & T. P. Burris (2012) Action of RORs and their ligands in (patho)physiology. Trends Endocrinol Metab, 23, 619-27. Solt, L. A., N. Kumar, P. Nuhant, Y. Wang, J. L. Lauer, J. Liu, M. A. Istrate, T. M. Kamenecka, W. R. Roush, D. Vidovic, S. C. Schurer, J. Xu, G. Wagoner, P. D. Drew, P. R. Griffin & T. P. Burris (2011) Suppression of TH17 differentiation and autoimmunity by a synthetic ROR ligand. Nature, 472, 491-4. Sonnylal, S., C. P. Denton, B. Zheng, D. R. Keene, R. He, H. P. Adams, C. S. Vanpelt, Y. J. Geng, J. M. Deng, R. R. Behringer & B. de Crombrugghe (2007) Postnatal induction of transforming growth factor beta signaling in fibroblasts of mice recapitulates clinical, histologic, and biochemical features of scleroderma. Arthritis Rheum, 56, 334-44. Stecca, B. & I. A. A. Ruiz (2010) Context-dependent regulation of the GLI code in cancer by HEDGEHOG and non-HEDGEHOG signals. J Mol Cell Biol, 2, 84-95. Steen, V. D. (2005) Autoantibodies in Systemic Sclerosis. Seminars in Arthritis and Rheumatism, 35, 35-42. Stewart, G. A., G. F. Hoyne, S. A. Ahmad, E. Jarman, W. A. Wallace, D. J. Harrison, C. Haslett, J. R. Lamb & S. E. Howie (2003) Expression of the developmental Sonic hedgehog (Shh) signalling pathway is up-regulated in chronic lung fibrosis and the Shh receptor patched 1 is present in circulating T lymphocytes. J Pathol, 199, 488-95. Sun, Y., C. H. Liu, J. P. SanGiovanni, L. P. Evans, K. T. Tian, B. Zhang, A. Stahl, W. T. Pu, T. M. Kamenecka, L. A. Solt & J. Chen (2015) Nuclear receptor RORalpha regulates pathologic retinal angiogenesis by modulating SOCS3-dependent inflammation. Proc Natl Acad Sci U S A, 112, 10401-6. Syn, W. K., Y. Jung, A. Omenetti, M. Abdelmalek, C. D. Guy, L. Yang, J. Wang, R. P. Witek, C. M. Fearing, T. A. Pereira, V. Teaberry, S. S. Choi, J. Conde-Vancells, G. F. Karaca & 120

References A. M. Diehl (2009) Hedgehog-mediated epithelial-to-mesenchymal transition and fibrogenic repair in nonalcoholic fatty liver disease. Gastroenterology, 137, 1478-1488 e8. Taipale, J. & P. A. Beachy (2001) The Hedgehog and Wnt signalling pathways in cancer. Nature, 411, 349-54. Tan, F. K., X. Zhou, M. D. Mayes, P. Gourh, X. Guo, C. Marcum, L. Jin & F. C. Arnett, Jr. (2006) Signatures of differentially regulated interferon gene expression and vasculotrophism in the peripheral blood cells of systemic sclerosis patients. Rheumatology (Oxford), 45, 694-702. Tilley, S. L., M. Jaradat, C. Stapleton, D. Dixon, X. Hua, C. J. Erikson, J. G. McCaskill, K. D. Chason, G. Liao, L. Jania, B. H. Koller & A. M. Jetten (2007) Retinoid-related orphan receptor gamma controls immunoglobulin production and Th1/Th2 cytokine balance in the adaptive immune response to allergen. J Immunol, 178, 3208-18. Tomasek, J. J., G. Gabbiani, B. Hinz, C. Chaponnier & R. A. Brown (2002) Myofibroblasts and mechano-regulation of connective tissue remodelling. Nat Rev Mol Cell Biol, 3, 349-63. Trenkner, E. & M. K. Hoffmann (1986) Defective development of the thymus and immunological abnormalities in the neurological mouse mutation "staggerer". J Neurosci, 6, 1733-7. Trojanowska, M. (2010) Cellular and molecular aspects of vascular dysfunction in systemic sclerosis. Nat Rev Rheumatol, 6, 453-60. Truchetet, M. E., B. Demoures, J. Eduardo Guimaraes, A. Bertrand, P. Laurent, V. Jolivel, I. Douchet, C. Jacquemin, L. Khoryati, P. Duffau, E. Lazaro, C. Richez, J. Seneschal, M. S. Doutre, J. L. Pellegrin, J. Constans, T. Schaeverbeke, P. Blanco & C. Contin-Bordes (2016) Platelets Induce Thymic Stromal Lymphopoietin Production by Endothelial Cells: Contribution to Fibrosis in Human Systemic Sclerosis. Arthritis Rheumatol, 68, 2784- 2794. Uhl, M., S. Aulwurm, J. Wischhusen, M. Weiler, J. Y. Ma, R. Almirez, R. Mangadu, Y. W. Liu, M. Platten, U. Herrlinger, A. Murphy, D. H. Wong, W. Wick, L. S. Higgins & M. Weller (2004) SD-208, a novel transforming growth factor beta receptor I kinase inhibitor, inhibits growth and invasiveness and enhances immunogenicity of murine and human glioma cells in vitro and in vivo. Cancer Res, 64, 7954-61. Umezawa, H., K. Maeda, T. Takeuchi & Y. Okami (1966) New antibiotics, bleomycin A and B. J Antibiot (Tokyo), 19, 200-9. Van den Hoogen, F., D. Khanna, J. Fransen, S. R. Johnson, M. Baron, A. Tyndall, M. Matucci- Cerinic, R. P. Naden, T. A. Medsger, Jr., P. E. Carreira, G. Riemekasten, P. J. Clements, C. P. Denton, O. Distler, Y. Allanore, D. E. Furst, A. Gabrielli, M. D. Mayes, J. M. van Laar, J. R. Seibold, L. Czirjak, V. D. Steen, M. Inanc, O. Kowal-Bielecka, U. Muller- Ladner, G. Valentini, D. J. Veale, M. C. Vonk, U. A. Walker, L. Chung, D. H. Collier, M. E. Csuka, B. J. Fessler, S. Guiducci, A. Herrick, V. M. Hsu, S. Jimenez, B. Kahaleh, P. A. Merkel, S. Sierakowski, R. M. Silver, R. W. Simms, J. Varga & J. E. Pope (2013) 2013 classification criteria for systemic sclerosis: an American College of Rheumatology/European League against Rheumatism collaborative initiative. Arthritis Rheum, 65, 2737-47. Varga, J. & D. Abraham (2007) Systemic sclerosis: a prototypic multisystem fibrotic disorder. J Clin Invest, 117, 557-67. Varga, J. & M. Hinchcliff (2014) Connective tissue diseases: systemic sclerosis: beyond limited and diffuse subsets? Nat Rev Rheumatol, 10, 200-2. Varga, J. & B. Pasche (2009) Transforming growth factor beta as a therapeutic target in systemic sclerosis. Nat Rev Rheumatol, 5, 200-6. Vortkamp, A., M. Gessler & K. H. Grzeschik (1991) GLI3 zinc-finger gene interrupted by translocations in Greig syndrome families. Nature, 352, 539-40. 121

References Vu-Dac, N., P. Gervois, T. Grotzinger, P. De Vos, K. Schoonjans, J. C. Fruchart, J. Auwerx, J. Mariani, A. Tedgui & B. Staels (1997) Transcriptional regulation of apolipoprotein A-I gene expression by the nuclear receptor RORalpha. J Biol Chem, 272, 22401-4. Wada, T., H. S. Kang, M. Angers, H. Gong, S. Bhatia, S. Khadem, S. Ren, E. Ellis, S. C. Strom, A. M. Jetten & W. Xie (2008a) Identification of oxysterol 7alpha-hydroxylase (Cyp7b1) as a novel retinoid-related orphan receptor alpha (RORalpha) (NR1F1) target gene and a functional cross-talk between RORalpha and liver X receptor (NR1H3). Mol Pharmacol, 73, 891-9. Wada, T., H. S. Kang, A. M. Jetten & W. Xie (2008b) The emerging role of nuclear receptor RORalpha and its crosstalk with LXR in xeno- and endobiotic gene regulation. Exp Biol Med (Maywood), 233, 1191-201. Whitfield, M. L., D. R. Finlay, J. I. Murray, O. G. Troyanskaya, J. T. Chi, A. Pergamenschikov, T. H. McCalmont, P. O. Brown, D. Botstein & M. K. Connolly (2003) Systemic and cell type-specific gene expression patterns in scleroderma skin. Proc Natl Acad Sci U S A, 100, 12319-24. Wick, G., C. Grundtman, C. Mayerl, T. F. Wimpissinger, J. Feichtinger, B. Zelger, R. Sgonc & D. Wolfram (2013) The immunology of fibrosis. Annu Rev Immunol, 31, 107-35. Wu, M., E. Melichian Ds Fau - Chang, M. Chang E Fau - Warner-Blankenship, A. K. Warner- Blankenship M Fau - Ghosh, J. Ghosh Ak Fau - Varga & J. Varga (2009) Rosiglitazone abrogates bleomycin-induced scleroderma and blocks profibrotic responses through peroxisome proliferator-activated receptor-gamma. Am J Pathol., 174, 519–533. Wynn, T. A. & T. R. Ramalingam (2012) Mechanisms of fibrosis: therapeutic translation for fibrotic disease. Nat Med, 18, 1028-40. Wynn, T. A. & K. M. Vannella (2016) Macrophages in Tissue Repair, Regeneration, and Fibrosis. Immunity, 44, 450-462. Xiao, L., Z. Zhang, X. Luo, H. Yang, F. Li & N. Wang (2016) Retinoid acid receptor-related orphan receptor alpha (RORalpha) regulates macrophage M2 polarization via activation of AMPKalpha. Mol Immunol, 80, 17-23. Xie, J. (2005) Hedgehog signaling in prostate cancer. Future Oncol, 1, 331-8. Xie, J., M. Aszterbaum, X. Zhang, J. M. Bonifas, C. Zachary, E. Epstein & F. McCormick (2001) A role of PDGFRalpha in basal cell carcinoma proliferation. Proc Natl Acad Sci U S A, 98, 9255-9. Xiong, A. & Y. Liu (2017) Targeting Hypoxia Inducible Factors-1alpha As a Novel Therapy in Fibrosis. Front Pharmacol, 8, 326. Xiong, G. & R. Xu (2014) RORalpha binds to E2F1 to inhibit cell proliferation and regulate mammary gland branching morphogenesis. Mol Cell Biol, 34, 3066-75. Yamaguchi, Y. & M. Kuwana (2013) Proangiogenic hematopoietic cells of monocytic origin: roles in vascular regeneration and pathogenic processes of systemic sclerosis. Histol Histopathol, 28, 175-83. Yamaguchi, Y., Y. Okazaki, N. Seta, T. Satoh, K. Takahashi, Z. Ikezawa & M. Kuwana (2010) Enhanced angiogenic potency of monocytic endothelial progenitor cells in patients with systemic sclerosis. Arthritis Res Ther, 12, R205. Yamamoto, T. & K. Nishioka (2005) Cellular and molecular mechanisms of bleomycin- induced murine scleroderma: current update and future perspective. Exp Dermatol, 14, 81-95. Yamamoto, T., S. Takagawa, I. Katayama, K. Yamazaki, Y. Hamazaki, H. Shinkai & K. Nishioka (1999) Animal model of sclerotic skin. I: Local injections of bleomycin induce sclerotic skin mimicking scleroderma. J Invest Dermatol, 112, 456-62. Yang, L., D. E. Anderson, C. Baecher-Allan, W. D. Hastings, E. Bettelli, M. Oukka, V. K. Kuchroo & D. A. Hafler (2008a) IL-21 and TGF-beta are required for differentiation of human T(H)17 cells. Nature, 454, 350-2. 122

References Yang, X. O., S. H. Chang, H. Park, R. Nurieva, B. Shah, L. Acero, Y. H. Wang, K. S. Schluns, R. R. Broaddus, Z. Zhu & C. Dong (2008b) Regulation of inflammatory responses by IL- 17F. J Exp Med, 205, 1063-75. Yoshizaki, A., Y. Yanaba K Fau - Iwata, K. Iwata Y Fau - Komura, A. Komura K Fau - Ogawa, Y. Ogawa A Fau - Akiyama, E. Akiyama Y Fau - Muroi, T. Muroi E Fau - Hara, F. Hara T Fau - Ogawa, M. Ogawa F Fau - Takenaka, K. Takenaka M Fau - Shimizu, M. Shimizu K Fau - Hasegawa, M. Hasegawa M Fau - Fujimoto, T. F. Fujimoto M Fau - Tedder, S. Tedder Tf Fau - Sato & S. Sato (2010) Cell adhesion molecules regulate fibrotic process via Th1/Th2/Th17 cell balance in a bleomycin-induced scleroderma model. J Immunol, 185, 2502-15. Yoshizaki, A., K. Yanaba, A. Ogawa, Y. Asano, T. Kadono & S. Sato (2011) Immunization with DNA topoisomerase I and Freund's complete adjuvant induces skin and lung fibrosis and autoimmunity via interleukin-6 signaling. Arthritis Rheum, 63, 3575-85. Zahreddine, H. A., B. Culjkovic-Kraljacic, S. Assouline, P. Gendron, A. A. Romeo, S. J. Morris, G. Cormack, J. B. Jaquith, L. Cerchietti, E. Cocolakis, A. Amri, J. Bergeron, B. Leber, M. W. Becker, S. Pei, C. T. Jordan, W. H. Miller & K. L. Borden (2014) The sonic hedgehog factor GLI1 imparts drug resistance through inducible glucuronidation. Nature, 511, 90-3. Zeina Chamoun, R. K. M., Denise Nellen, Doris P. von Kessler, Manolo Bellotto,Philip A. Beachy,Konrad Basler (2001) Skinny Hedgehog, an Acyltransferase Required for Palmitoylation and Activity of the Hedgehog Signal. Science, 293, 2080-2084. Zerr, P., S. Vollath, K. Palumbo-Zerr, M. Tomcik, J. Huang, A. Distler, C. Beyer, C. Dees, K. Gela, O. Distler, G. Schett & J. H. Distler (2015) Vitamin D receptor regulates TGF-beta signalling in systemic sclerosis. Ann Rheum Dis, 74, e20. Zheng, B., C. M. Zhang Z Fau - Black, B. Black Cm Fau - de Crombrugghe, C. P. de Crombrugghe B Fau - Denton & C. P. Denton (2002) Ligand-dependent genetic recombination in fibroblasts : a potentially powerful technique for investigating gene function in fibrosis. Am J Pathol., 160, 1609-1617. Zhou, D., R. J. Tan & Y. Liu (2016) Sonic hedgehog signaling in kidney fibrosis: a master communicator. Sci China Life Sci, 59, 920-9.

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Abbreviations 7 Abbreviations

Ad adeno viral

AF-2 activation function-2

ACR American College of Rheumatology

α-SMA α-smooth muscle actin

ADCC antibody dependent cell mediated cytotoxicity

AECAs anti-endothelial cell antibodies

AsO arsenic trioxide

Bleo Bleomycin

BMPs Bone Morphogenic Proteins

BSA bovine serum albumin

bFGF Basic Fibroblast Growth Factor

cDNA complementary DNA

cGvHD chronic graft-versus-host disease

CFA Complete Freund’s Adjuvant

CoIP Co-immunoprecipitation

Ct threshold cycle

DAB 3,3-diaminobenzidine tetrahydrochloride

DBD DNA-binding domain

DAPI 4’-6-Diamindino-2-phenylindole

dcSSc diffuse cutaneous SSc

ddH20 double-distilled water

DKK Dickkopf

DMEM dulbecco's modified eagle's medium

124

Abbreviations DMSO dimethylsulfoxid

DNA deoxyribonucleic acid

DHH Desert hedgehog dNTPs desoxyribonukleosidtriphosphate

ECL enhanced chemiluminescence

ECM extracellular matrix

EDTA ethylenediaminetetraacetic acid

EGFR epidermal growth factor receptor

EMT epithelial-to-mesenchymal transition

EndoMT endothelial to mesenchymal transition

EPCs endothelial progenitor cells

ER estradiol

EtOH ethanol

ED-A extra domain A

FACS fluorescence activated cell sorting

FBS fetal bovine serum

FCS fetal calf serum

FAK focal adhesion kinase

Fn Fibronectin

GDFs growth/differentiation factors

HDL high density lipoprotein

HE Hematoxylin-eosin staining

HH Hedgehog

HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid

125

Abbreviations HRP Horseradish peroxidase

HLA Human Leukocyte Antigen ifu infection unites

IHH Indian hedgehog

IL-6 interleukin-6 i.p. intraperitoneally

Irf5 interferon regulatory factor 5

IPF idiopathic pulmonary fibrosis

ICAM intercellular adhesion molecule

JAM junctional adhesion molecule

JNK c-Jun N-terminal kinases

LAP Latency Associated Peptide lcSSc Limited cutaneous SSc

LBD ligand-binding domain

LTBP latent TGFβ binding proteins

MAPK mitogen-activated protein kinase

MBOAT membrane-bound O-acyltransferase

MeOH methanol

MMP Matrix metalloproteinases mRNA Messenger ribonucleic acid

NK/NKT natural killer/natural killer T cell

OVA ovalbumin

P4H Prolyl-4-hydroxylase-β

PAI-1 Plasminogen activator inhibitor 1

126

Abbreviations PACAM platelet endothelial cell adhesion molecule

PAH pulmonary arterial hypertension

PBS phosphate buffered saline

PCR polymerase chain reaction

PDGF Platelet-derived growth factor

PDGFRα Platelet-derived growth factor receptor α

PFA paraformaldehyde

PI3K Phosphoinositide 3 kinase

PlGF Placental growth factor pH Pondus Hydrogenii

PLC phospholipase C

PC primary cilium

PKC protein kinase C

PPAR peroxisome proliferator-activated receptor

PR progesterone

Ptch-1 Patched homologue 1

PHCs pro-angiogenic hematopoietic cells

PVDF polyvinylidenfluorid

RORs Retinoic Acid-Related Orphan Receptors

ROR Retinoic Acid Receptor-Related Orphan Receptor Alpha

ROREs ROR response elements

RBC red blood cell lysis buffer

RNA ribonucleic acid

RT Room temperature

127

Abbreviations RTK receptor tyrosine kinase

RYK receptor-like tyrosine kinase

SBE Smad binding element scl cGvHD sclerodermatous chronic graft-versus-host disease

SDS sodium dodecyl sulfate s.c. Subcutaneous siRNA small interfering RNA

SHH Sonic Hedgehog

SMO Smoothened

SREBP1c sterol regulatory element-binding protein 1 isoform c

SSc systemic sclerosis

SuFu Suppressor of Fused

TCR T-cell receptor

TEMED N,N,N',N'-Tetramethylethylendiamin

TGFβ transforming growth factor β

TR thyroid hormones

Tregs T-regulatory cells

TNF tumour-necrosis factor

Topo Topoisomerase

Tsk-1 tight-skin 1

TSP-1 thrombospondin-1

Tris tris(hydroxymethyl)aminomethane

(v/v) volume per volume

VCAM vascular adhesion molecule

128

Abbreviations VEGF vascular endothelial growth factor

VDR vitamin D receptor

(w/v) weight per volume

WT Wildtype

129