Supplementary material Ann Rheum Dis

Online Supplement for the manuscript:

The acyltransferase skinny hedgehog regulates TGF-dependent fibroblast activation in

SSc

by

Ruifang Liang1, Rosebeth Kagwiria1, Ariella Zehender1, Clara Dees1, Christina Bergmann1,

Andreas Ramming1, Dorota Krasowska2, Małgorzata Michalska-Jakubus2, Alexander

Kreuter3, Georg Schett1, Jörg H. W. Distler1

Supplementary Material and Methods

Patient and Public Involvement

Patients were not involved in the design of the study or the interpretation of the results, but

donated biopsies for this study. The results of the study will be presented on congress to

enable distribution also by patients.

Patients and fibroblasts

Dermal fibroblasts were isolated from skin biopsies of 23 SSc patients and 21 matched

healthy volunteers. Biopsies were taken at the forearm, 15 ± 2 cm away from the styloid

processus. All patients fulfilled the 2013 ACR/EULAR criteria for SSc.[1] Sixteen patients

were female, seven were male. The median age of SSc patients was 45 years (range: 19-65

years), and their median disease duration was 5 years (range: 0.5-10 years). All patients and

healthy volunteers signed a consent form approved by the local institutional review board.

Liang R, et al. Ann Rheum Dis 2019; 0:1269–1273. doi: 10.1136/annrheumdis-2019-215066 Supplementary material Ann Rheum Dis

Murine models of SSc

Three different murine models of SSc were employed: Bleomycin-induced, TBRact-induced

and Topoisomerase 1 (topo)-induced dermal fibrosis. Bleomycin was injected every other day

at concentrations of 0.5 mg/ml as described [2, 3]. Injections with the vehicle, 0.9% NaCl

served as controls. For TBRact-induced fibrosis, 4-week-old mice received of 6.67 × 107

pfu/mouse of replication-deficient type 5 adenoviruses encoding for constitutively active

TBRI construct into defined areas of at the upper back four times every other week [4].

Injections of 6.67 × 107 pfu/mouse of replication-deficient type 5 adenoviruses encoding for

LacZ served as controls. In the Topo model, fibrosis was induced by four local injections of

topo every other week. Recombinant human topo was diluted to 500 units/ml and mixed with

Complete Freund’s Adjuvant (CFA) (Sigma-Aldrich, Taufkirchen, Germany) 3:2

(volume/volume). 250µl were injected subcutaneously into C57BL/6 mice (female, 6 weeks

old) in defined areas of the upper back [5]. Injections of the vehicle, including CFA served as

controls.

Reporter assay

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 [6]. 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).

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

Liang R, et al. Ann Rheum Dis 2019; 0:1269–1273. doi: 10.1136/annrheumdis-2019-215066 Supplementary material Ann Rheum Dis

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 (Supplementary Fig. 3 A).

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 (Supplementary Fig. 3B).

siRNA-mediated knockdown of HHAT and Smad3

Complexes of siRNA and atelocollagen (Koken, Tokyo, Japan) were prepared as described

previously [7, 8]. The following siRNA duplexes (Thermo Fisher) were used: mHhat: 5´-

GUUAAGAGAAGGUGGUACAUU-3´, antisense 5´-PUGUACCACCUUCUCUUAACUU-

3´. Non-targeting siRNA duplexes served as controls. Atelocollagen/siRNA complexes were

injected intracutaneously once weekly.

Analysis of dermal fibrosis and adverse effects

Dermal thickness, -smooth muscle actin and hydroxyproline content were analyzed as

described previously [2, 3, 9-14]. Mice were monitored daily for activity, weight loss, texture

of the fur and diarrhea.

Quantitative real time-PCR

Liang R, et al. Ann Rheum Dis 2019; 0:1269–1273. doi: 10.1136/annrheumdis-2019-215066 Supplementary material Ann Rheum Dis

Gene expression was quantified by SYBR-Green real-time-PCR using the MX3005P

Detection System (Agilent Technologies, Santa Clara CA, US).[15, 16] Samples without

enzyme in the reverse transcription reaction, without template and dissociation curve analysis

served as controls. All primers are summarized in supplementary table 1.

Western blotting

Protein samples were separated by SDS-polyacrylamide gel and electrotransferred onto

polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). After blocking,

membranes were incubated with polyclonal antibodies against HHAT (Santa Cruz,

Heidelberg, Germany) overnight at 4°C. For the detection of SHH, polyclonal antibodies

against SHH (Santa Cruz) were used. Membranes were incubated with horseradish-

peroxidase-conjugated secondary antibodies (Dako, Glostrup, Denmark).

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) [17]. 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.

Immunofluorescence staining

Liang R, et al. Ann Rheum Dis 2019; 0:1269–1273. doi: 10.1136/annrheumdis-2019-215066 Supplementary material Ann Rheum Dis

Paraffin-embedded skin sections or cultured fibroblasts were stained with antibodies against

prolyl-4-hydroxylase- (P4H), -smooth muscle actin (SMA) (Sigma-Aldrich), HHAT

(Sigma-Aldrich), GLI2 and DAPI. Myofibroblasts were identified as single, spindle shaped

cells in the dermis positive for -smooth muscle actin. Concentration-matched species-

specific immunoglobulins (Vector Laboratories) served as control antibodies. The staining

was analyzed using a Nikon Eclipse 80i microscope (Nikon, Tokyo, Japan).

Statistics

All in vitro data are presented as median with interquartile range (IQR), and all in vivo data as

dot blots. Differences between the groups were tested by non-parametric Mann-Whitney U

test. P-values less than 0.05 were considered as significant.

Liang R, et al. Ann Rheum Dis 2019; 0:1269–1273. doi: 10.1136/annrheumdis-2019-215066 Supplementary material Ann Rheum Dis

Supplementary Figures

Supplementary Figure 1: Hedgehog signaling is active in fibroblasts expressing HHAT.

A, Co-stainings of HHAT with the hedgehog GLI2 and the fibroblast

marker prolyl-4-hydroxylase- (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 by two

independent human observers (n=6).

Supplementary Figure 2: TGF induces HHAT expression in a SMAD3-dependent

manner.

A-B, Effects of siRNA-mediated knockdown of SMAD3 on the mRNA (A) and protein (B)

levels of HHAT in human dermal fibroblasts (n=4 biological replicates with ≥2 technical

replicates).C-D, Costaining for Hhat, Smad3, Vimentin and DAPI in murine models of skin

fibrosis with quantification (C) and representative images (D) (200-fold and 600-fold

magnification n=5).

Supplementary Figure 3: 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

Liang R, et al. Ann Rheum Dis 2019; 0:1269–1273. doi: 10.1136/annrheumdis-2019-215066 Supplementary material Ann Rheum Dis

signaling. Schematic presentation of the experiment (left) and quantification of the reporter

activity (right). Fibroblasts were transfected with a constitutively active TGF 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 TGF 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).

Supplementary Figure 4: Knockdown efficacy of Hhat in murine skin.

Protein levels of Hhat in the skin of bleomycin- (A) or Topo-challenged (B) mice with or

without atellocollagen/siRNA complexes (n=6).

Liang R, et al. Ann Rheum Dis 2019; 0:1269–1273. doi: 10.1136/annrheumdis-2019-215066 Supplementary material Ann Rheum Dis

References for supplementary Material and Methods

1. van den Hoogen F, Khanna D, Fransen J, Johnson SR, Baron M, Tyndall A, et al. 2013 classification criteria for systemic sclerosis: an American College of Rheumatology/European League against Rheumatism collaborative initiative. Arthritis and rheumatism. 2013 Nov; 65(11):2737-2747. 2. Dees C, Akhmetshina A, Zerr P, Reich N, Palumbo K, Horn A, et al. Platelet-derived serotonin links vascular disease and tissue fibrosis. The Journal of experimental medicine. 2011 May; 208(5):961-972. 3. Dees C, Zerr P, Tomcik M, Beyer C, Horn A, Akhmetshina A, et al. Inhibition of Notch signaling prevents experimental fibrosis and induces regression of established fibrosis. Arthritis and rheumatism. 2011 May; 63(5):1396-1404. 4. Chakraborty D, Sumova B, Mallano T, Chen CW, Distler A, Bergmann C, et al. Activation of STAT3 integrates common profibrotic pathways to promote fibroblast activation and tissue fibrosis. Nature communications. 2017 Oct; 8(1):1130. 5. Bergmann C, Brandt A, Merlevede B, Hallenberger L, Dees C, Wohlfahrt T, et al. The histone demethylase Jumonji domain-containing protein 3 (JMJD3) regulates fibroblast activation in systemic sclerosis. Annals of the rheumatic diseases. 2018 Jan; 77(1):150-158. 6. Palumbo-Zerr K, Zerr P, Distler A, Fliehr J, Mancuso R, Huang J, et al. Orphan nuclear receptor NR4A1 regulates transforming growth factor-beta signaling and fibrosis. Nature medicine. 2015 Feb; 21(2):150-158. 7. Minakuchi Y, Takeshita F, Kosaka N, Sasaki H, Yamamoto Y, Kouno M, et al. Atelocollagen-mediated synthetic small interfering RNA delivery for effective silencing in vitro and in vivo. Nucleic acids research. 2004 Jul; 32(13):e109. 8. Horn A, Kireva T, Palumbo-Zerr K, Dees C, Tomcik M, Cordazzo C, et al. Inhibition of hedgehog signalling prevents experimental fibrosis and induces regression of established fibrosis. Annals of the rheumatic diseases. 2012 May; 71(5):785-789. 9. Akhmetshina A, Palumbo K, Dees C, Bergmann C, Venalis P, Zerr P, et al. Activation of canonical Wnt signalling is required for TGF-beta-mediated fibrosis. Nature communications. 2012 Mar; 3:735. 10. Avouac J, Palumbo K, Tomcik M, Zerr P, Dees C, Horn A, et al. Inhibition of activator protein 1 signaling abrogates transforming growth factor beta-mediated activation of fibroblasts and prevents experimental fibrosis. Arthritis and rheumatism. 2012 May; 64(5):1642-1652. 11. Bergmann C, Akhmetshina A, Dees C, Palumbo K, Zerr P, Beyer C, et al. Inhibition of glycogen synthase kinase 3beta induces dermal fibrosis by activation of the canonical Wnt pathway. Annals of the rheumatic diseases. 2011 Dec; 70(12):2191-2198. 12. Beyer C, Reich N, Schindler SC, Akhmetshina A, Dees C, Tomcik M, et al. Stimulation of soluble guanylate cyclase reduces experimental dermal fibrosis. Annals of the rheumatic diseases. 2012 Jun; 71(6):1019-1026. 13. Beyer C, Schramm A, Akhmetshina A, Dees C, Kireva T, Gelse K, et al. beta-catenin is a central mediator of pro-fibrotic Wnt signaling in systemic sclerosis. Annals of the rheumatic diseases. 2012 May; 71(5):761-767. 14. Zerr P, Palumbo-Zerr K, Distler A, Tomcik M, Vollath S, Munoz LE, et al. Inhibition of hedgehog signaling for the treatment of murine sclerodermatous chronic graft-versus-host disease. Blood. 2012 Oct; 120(14):2909-2917. 15. Beyer C, Zenzmaier C, Palumbo-Zerr K, Mancuso R, Distler A, Dees C, et al. Stimulation of the soluble guanylate cyclase (sGC) inhibits fibrosis by blocking non-canonical TGFbeta signalling. Annals of the rheumatic diseases. 2015 Jul ; 74(7):1408-1416.

Liang R, et al. Ann Rheum Dis 2019; 0:1269–1273. doi: 10.1136/annrheumdis-2019-215066 Supplementary material Ann Rheum Dis

16. Zerr P, Vollath S, Palumbo-Zerr K, Tomcik M, Huang J, Distler A, et al. Vitamin D receptor regulates TGF-beta signalling in systemic sclerosis. Annals of the rheumatic diseases. 2015 Mar ;74(3) :e20. 17. Dierker T, Dreier R, Migone M, Hamer S, Grobe K. Heparan sulfate and transglutaminase activity are required for the formation of covalently cross-linked hedgehog oligomers. The Journal of biological chemistry. 2009 Nov; 284(47):32562-32571.

Liang R, et al. Ann Rheum Dis 2019; 0:1269–1273. doi: 10.1136/annrheumdis-2019-215066