ARTHRITIS & RHEUMATISMVol. 62, No. 6, June 2010, pp 1733–1743DOI 10.1002/art.27443© 2010, American College of Rheumatology

MicroRNA-29, a Key Regulator ofCollagen Expression in Systemic Sclerosis

Britta Maurer,1 Joanna Stanczyk,1 Astrid Jungel,1 Alfiya Akhmetshina,2 Michelle Trenkmann,1

Matthias Brock,1 Otylia Kowal-Bielecka,3 Renate E. Gay,1 Beat A. Michel,1

Jorg H. W. Distler,2 Steffen Gay,1 and Oliver Distler1

Objective. To investigate the role of microRNA(miRNA) as posttranscriptional regulators of profi-brotic genes in systemic sclerosis (SSc).

Methods. MicroRNA, which target collagens, wereidentified by in silico analysis. Expression of miRNA-29(miR-29) was determined by TaqMan real-time poly-merase chain reaction analysis of skin biopsy andfibroblast samples from SSc patients and healthy con-trols as well as in the mouse model of bleomycin-induced skin fibrosis. Cells were transfected with pre-cursor miRNA (pre-miRNA)/anti-miRNA of miR-29

using Lipofectamine. Collagen gene expression was alsostudied in luciferase reporter gene assays. For stimula-tion, recombinant transforming growth factor �(TGF�), platelet-derived growth factor B (PDGF-B), orinterleukin-4 (IL-4) was used. The effects of inhibitingPDGF-B and TGF� signaling on the levels of miR-29were studied in vitro and in the bleomycin model.

Results. We found that miR-29a was stronglydown-regulated in SSc fibroblasts and skin sections ascompared with the healthy controls. Overexpression inSSc fibroblasts significantly decreased, and accordingly,knockdown in normal fibroblasts increased, the levels ofmessenger RNA and protein for type I and type IIIcollagen. In the reporter gene assay, cotransfection withpre-miR-29a significantly decreased the relative lucif-erase activity, which suggests a direct regulation ofcollagen by miR-29a. TGF�, PDGF-B, or IL-4 reducedthe levels of miR-29a in normal fibroblasts to those seenin SSc fibroblasts. Similar to human SSc, the expressionof miR-29a was reduced in the bleomycin model of skinfibrosis. Inhibition of PDGF-B and TGF� pathways bytreatment with imatinib restored the levels of miR-29ain vitro and in the bleomycin model in vivo.

Conclusion. These data add the posttranscrip-tional regulation of collagens by miR-29a as a novelaspect to the fibrogenesis of SSc and suggest miR-29a asa potential therapeutic target.

Systemic sclerosis (SSc) is a multisystemic fibroticdisorder with high morbidity and mortality rates (1). Theprogressive replacement of normal tissue by collagen-rich extracellular matrix leads to impairment and, ulti-mately, to failure of affected organs. Fibroblasts, thecellular key players of this process, are activated byprofibrotic cytokines and growth factors, such asinterleukin-4 (IL-4), transforming growth factor �(TGF�), and platelet-derived growth factor B (PDGF-

Dr. Maurer’s work was supported by the Olga MayenfischFoundation. Dr. Stanczyk’s work was supported by the Foundation forthe Medical Research University of Zurich and the Swiss NationalScience Foundation. Dr. Jungel’s work was supported by the EuropeanUnion Sixth Framework Programme (project AutoCure) and SeventhFramework Programme (project Masterswitch). Mr. Brock’s work wassupported by the Zurich Center of Integrative Human Physiology. Dr.J. H. W. Distler’s work was supported by the DFG (grant DI 1537/2-1),the Interdisciplinary Center of Clinical Research in Erlangen (grantA20), and a Career Support Award of Medicine from the Ernst JungFoundation. Dr. O. Distler’s work was supported by a EULAR OrphanDisease Programme grant.

1Britta Maurer, MD, Joanna Stanczyk, MD, PhD, AstridJungel, PhD, Michelle Trenkmann, MSc, Matthias Brock, MSc, Re-nate E. Gay, MD, Beat A. Michel, MD, Steffen Gay, MD, OliverDistler, MD: University Hospital Zurich and Zurich Center of Inte-grative Human Physiology, Zurich, Switzerland; 2Alfiya Akhmetshina,PhD, Jorg H. W. Distler, MD: University of Erlangen–Nuremberg,Erlangen, Germany; 3Otylia Kowal-Bielecka, MD: Medical Universityof Bialystok, Bialystok, Poland.

Drs. Maurer, S. Gay, and O. Distler have filed an applicationfor a patent for the use of miR-29 as a novel pharmacologic treatmentof scleroderma. Dr. J. H. W. Distler has received consulting fees,speaking fees, and/or honoraria from Actelion, Pfizer, and Glaxo-SmithKline (less than $10,000 each). Dr. O. Distler has receivedconsulting fees, speaking fees, and/or honoraria from Actelion, Pfizer,Encysive, FibroGen, ErgoNex, NicOx, Biovitrum, and Bristol-MyersSquibb (less than $10,000 each).

Address correspondence and reprint requests to Oliver Dis-tler, MD, Center of Experimental Rheumatology, University HospitalZurich, Gloriastrasse 25, 8091 Zurich, Switzerland. E-mail: oliver.distler@usz.ch.

Submitted for publication March 30, 2009; accepted in revisedform February 23, 2010.

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B). Despite the ongoing research in identifying stimulithat contribute to the continuous activation of fibro-blasts in SSc, the intracellular molecular events thatinduce and sustain the status of activation have yet to beelucidated.

MicroRNA (miRNA) have recently been discov-ered to be a new class of posttranscriptional repressorsof gene expression (2). Up to date, more than 700 ofthese small, noncoding, evolutionarily conserved RNAshave been experimentally validated in humans. In silicoanalyses estimate that miRNA account for 2–3% of thehuman genome (3). The term miRNA refers to themature molecule of 19–25 nucleotides in length, which isgenerated in a multistep process in the nucleus and inthe cytoplasm. The miRNA mediate the posttranscrip-tional regulation of gene expression by binding to par-tially complementary sites in the 3�-untranslated region(3�-UTR) of target messenger RNA (mRNA). Theformation of miRNA–mRNA duplexes leads to mRNAdegradation or translational repression (4).

Since miRNA are predicted to regulate �30% ofthe genes in humans (5), dysregulation of miRNA seemsa likely scenario for the development of diseases such ascancer (6) or neurologic (7), cardiovascular (8), orautoimmune (9) disorders.

There is rapidly growing evidence from in vitrostudies and studies of animal models that the modifica-tion of altered miRNA levels with chemically modifiedanti-miR or miRNA mimics may provide a novel, spe-cific, molecular-based therapy (10–13).

The role of miRNA in the pathogenesis of SSchas not been addressed so far. Thus, the objectives ofour present study were to identify differentially ex-pressed miRNA in patients with SSc, to investigate theregulation of selected miRNA by profibrotic mediators,and to characterize the therapeutic effects of miRNAmodulation on collagen production.

PATIENTS AND METHODS

Patients, biopsy specimens, and cell culture. Skinbiopsy specimens were obtained by punch biopsy from 12 SScpatients and 11 healthy donors. Skin fibroblasts were expandedby outgrowth culture in Dulbecco’s modified Eagle’s medium(DMEM; Gibco Invitrogen) as described previously (14). Cellsfrom passages 3–8 were used for the experiments. All patientsfulfilled the criteria for SSc as described by LeRoy et al (15),and the study was approved by the local ethics committee.(Detailed characteristics of the 12 SSc patients, as well asadditional information concerning the methodology used inthese studies, are available upon request from the authors.)

Reagents and stimulation assays. After 24 hours inserum-reduced DMEM (0.5% fetal calf serum), cells werestimulated for 24 and 48 hours with TGF� (10 ng/ml),PDGF-B (40 ng/ml), or IL-4 (10 ng/ml) (R&D Systems). Insubsets of experiments, dermal fibroblasts were incubated for24 hours with imatinib mesylate that had been dissolved in0.9% NaCl to a final concentration of 1.0 �g/ml (stockconcentration 10 mg/ml; Novartis) (14). Myofibroblast differ-entiation was induced by stimulating SSc fibroblasts withTGF� (2 ng/ml) for 4 days (16).

Transfection experiments. Cells were transfected withsynthetic precursor miRNA (pre-miR), with inhibitors ofmiR-29 (anti-miR), or with negative controls (Pre-miR/Anti-miR Negative Control #1; Ambion/Applied Biosystems) at afinal concentration of 100 nM with the use of Lipofectamine2000 reagent (Invitrogen). At 72 hours after transfection,cellular lysates were collected to analyze the expression oftypes I and III collagen. Transfection efficiency was controlledby TaqMan-based real-time polymerase chain reaction (PCR).Additionally, normal skin fibroblasts were treated with TGF�,PDGF-B, or IL-4 for 48 hours after overnight serum starva-tion, then transfected with pre-miR-29a or negative control,and lysed after 24 hours to measure COL1A1 and COL3A1mRNA.

In another subset of experiments, SSc skin fibroblastswere transfected with small interfering RNA (siRNA) againstc-abl, PDGF receptor � (PDGFR�), and c-kit (Qiagen) at afinal concentration of 50 nM and lysed after 24 hours toexamine the expression of miR-29.

Computational prediction analysis of miRNA target-ing collagen genes. Screening for miRNA targeting collagengenes as major components of the secreted extracellularproteins in SSc was performed by merging the results of severalcomputational prediction algorithms provided at the miRGendatabase (http://www.diana.pcbi.upenn.edu/miRGen.html) (17).

RNA isolation and quantitative real-time PCR analy-sis of miRNA. Skin biopsy specimens (0.5 cm2) were homoge-nized with TissueLyser (Qiagen). A mirVana miRNA Isolationkit and a RecoverAll Total Nucleic Acid Isolation kit wereused for isolation of total RNA (Ambion/Applied Biosystems).Specific single TaqMan miRNA assays (Ambion/Applied Bio-systems) were used to measure the expression levels of selectedmiRNA in a model 7500 real-time PCR system analyzer(Applied Biosystems). Expression of the U6B small nuclearRNA (RNU6B) or snoRNA-202 was used as endogenouscontrol to normalize the data for human and murine samples,respectively. For relative quantification, the comparativethreshold cycle (Ct) method was used (18). To measure theexpression of collagen genes, total RNA was isolated using anRNeasy Mini kit (Qiagen). Gene expression was quantifiedusing SYBR Green real-time PCR, as previously described (14).

Western blot analysis for type I�2 and type III colla-gen. Equivalent amounts of protein from each cell lysate wereseparated by sodium dodecyl sulfate–10% polyacrylamide gelelectrophoresis and electrotransferred onto nitrocellulose mem-branes, as described previously (14). Polyclonal antibodiesagainst type I�2 and type III collagen (Santa Cruz Biotechnol-ogy) and horseradish peroxidase–conjugated goat anti-rabbitsecondary antibodies (Jackson ImmunoResearch) were used.Semiquantitative analysis based on densitometry was per-formed using AlphaImager software.

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Luciferase reporter assay for targeting the 3�-UTR ofCOL3A1. To validate the idea that miR-29a/29b/29c directlyregulate the expression of type III collagen by binding to thecomplementary seed matches within the 3�-UTR of collagengenes, we performed a luciferase reporter gene assay. Thehuman embryonic kidney cell line HEK 293 was used in theseexperiments, and the results were confirmed with primarydermal fibroblasts. (Data available upon request from theauthors.) For the experiment, a COL3A1 3�-UTR segment of973 bp that contained 2 conserved binding sites for miR-29 wasamplified by PCR from human genomic DNA and insertedinto the pGL3 control vector (Promega) by using the Xba I siteimmediately downstream of the stop codon of the luciferase.HEK 293 cells were transfected with the pGL3 control 3�-UTRof the COL3A1 construct (0.15 �g/well) using Lipofectamine(Invitrogen). For normalization, a control vector containingRenilla luciferase (pRL-SV40 vector, 0.8 �g/well; Promega)was added. For each well, a 100 nM concentration of pre-miR-

29a, pre-miR-29b, pre-miR-29c, or scrambled control(Ambion/Applied Biosystems) was used.

Firefly and Renilla luciferase activities were measuredin the cell lysates by the Dual-Luciferase Reporter Assaysystem (Promega) 24 hours after transfection. For the 3�-UTRof COL1A1 and COL1A2, conserved seed matches for miR-29were likewise predicted (19).

Bleomycin-induced skin fibrosis in mice. Skin fibrosiswas induced in 6-week-old female DBA mice by local intracu-taneous injection of bleomycin as described previously(14,20,21). The animal protocol was approved by the localanimal care and use committee. Briefly, the mice were chal-lenged every other day for 6 weeks with intracutaneousinjections of bleomycin (100 �l dissolved in 0.9% NaCl of a 0.5mg/ml concentration) in defined areas of 1.5 cm2 on the upperback. One group of mice was treated with intraperitonealinjections of imatinib 150 mg/kg/day for the last 3 weeks toassess the impact of imatinib on miR-29 levels in established

Figure 1. Expression of microRNA-29 (miR-29) in systemic sclerosis (SSc) patients and healthycontrols, as determined by TaqMan-based real-time polymerase chain reaction analysis. Expressionof miR-29 family member genes in SSc patient samples was determined relative to the healthycontrols, which was defined as 100%. A, All members of the miR-29 family (miR-29a, miR-29b, andmiR-29c) were expressed in SSc dermal fibroblasts. Lower �Ct values indicate higher levels ofexpression. MicroRNA-29a showed the highest baseline expression (n � 8), whereas miR-29bexpression was near the detection limit. Horizontal lines show the mean. B, Levels of all miR-29family members, in particular, miR-29a, were markedly reduced in SSc skin fibroblasts (n � 6) ascompared with healthy controls (n � 6). C, Similar to the in vitro findings, the expression ofmiR-29a in paraffin-embedded skin sections from SSc patients (n � 12) was down-regulated ascompared with healthy controls (n � 11). D, Expression of miR-29a in fresh skin biopsy samplesfrom SSc patients (n � 5) was down-regulated as compared with healthy donors (n � 5). Valuesin B–D are the mean and SEM. � � P � 0.05 versus healthy controls.

miR-29 REGULATION OF COLLAGEN EXPRESSION IN SSc 1735

fibrosis. Another group was treated with 0.9% NaCl instead ofimatinib for the last 3 weeks. The respective control group forbleomycin exposure received intracutaneous injections of 100�l of 0.9% NaCl for 6 weeks instead of bleomycin andadditionally received 0.9% NaCl for the last 3 weeks. After 6weeks, animals were asphyxiated with CO2. The skin wasremoved and processed for further analysis.

Statistical analysis. GraphPad Prism software wasused for statistical analyses. Normal distribution of the datawas confirmed using the Kolmogorov-Smirnov test. Student’sunpaired t-test was used for unrelated parametric data. Dataare expressed as the mean � SEM. P values less than 0.05 wereconsidered statistically significant.

RESULTS

Identifying miRNA-targeting collagen genes.Biocomputational prediction algorithms are well-established screening tools for miRNA profiling (22).Among other miRNA, such as the let-7 family, miR-26,and miR-155, the miR-29 family members (miR-29a/29b/29c) were identified as potential posttranscriptionalregulators of collagen genes. The miR-29 family wasselected for further analysis because it was predicted tohave the best seed matches with the collagens (http://www.targetscan.org) and because it is broadly conservedamong most vertebrates (23). MicroRNA-29a and miR-29b1 are clustered together and are located in chromo-some 7 in humans, whereas the miR-29b2/miR-29ccluster is found on chromosome 1 (http://www.mirbase.org). Other predicted profibrotic targets ofmiR-29a included, for example, PDGF-B, PDGFR�,Sp-1, thrombospondin, and secreted protein, acidic andrich in cysteine (SPARC).

Down-regulation of baseline miR-29 expressionin SSc. Given that most miRNA act as posttranscrip-tional repressors and that in SSc, the expression ofcollagen genes is remarkably up-regulated, we expecteda down-regulation of the miR-29 family in SSc patientsas compared with healthy controls. Therefore, we ana-lyzed the expression of the miR-29 family and of type Iand type III collagen in skin fibroblasts and biopsysamples from SSc patients and healthy controls. Wefound that all members of the miR-29 family wereexpressed in skin fibroblasts from SSc patients, with nodifferences in disease subsets (limited versus diffuseSSc) or disease stages (early [�2 years] versus late [�2years]). (Data on the expression of miR-29 in differentdisease subsets and stages of SSc are available uponrequest from the authors.)

Within the miR-29 family, miR-29a showed thehighest level of expression (mean � SEM �Ct 17.4 �0.2), followed by miR-29c (�Ct 22.8 � 0.1), and miR-29b

(�Ct 27.1 � 0.3) or miR-29c (�Ct 22.8 � 0.1) (Figure1A). However, compared with normal skin fibroblasts,the baseline levels of miR-29 were consistently down-regulated in SSc fibroblasts: by a mean � SEM of 60 �15% for miR-29a (P � 0.004), 45 � 15% for miR-29b(P � 0.001), and 58 � 7% for miR-29c (P � 0.0001)(Figure 1B). In the same SSc fibroblasts, this wasparalleled by increased levels of COL1A1 and COL3A1mRNA (mean � SEM 1.5 � 0.2–fold and 1.6 �0.4–fold, respectively; P � 0.06) in SSc fibroblasts ascompared with healthy control fibroblasts (Figure 2A).

Next, we investigated the expression of themiR-29 family in skin samples from SSc patients. First,the expression of miR-29 in tissue extracts isolated fromparaffin-embedded skin sections was analyzed, and sim-ilar to the findings in SSc fibroblasts, decreased levels ofthe miR-29 family members were observed. In particu-lar, miR-29a was down-regulated in the dermis of SScpatients as compared with healthy controls (by 79 � 9%;P � 0.0001) (Figure 1C).

To exclude the possibility that paraffin processinginfluenced the content of miRNA in the tissue, weadditionally investigated the expression of the miR-29family in fresh skin biopsy samples. Consistent with the

Figure 2. Expression of type I and type III collagen in systemicsclerosis (SSc) patients and healthy controls, as determined by SYBRGreen real-time polymerase chain reaction analysis. Whereas expres-sion of the microRNA-29 family members was found to be up-regulated in SSc patients, the expression of type I and type III collagenin these same patients was increased as compared with healthycontrols. Expression of collagen genes in SSc patient samples wasdetermined relative to the healthy controls, which was defined as 1.A, Levels of mRNA for COL1A1 and COL3A1 were increased in SScfibroblasts (n � 6) as compared with normal skin fibroblasts (n � 6).B, Levels of mRNA for COL1A1 and COL3A1 were also increased infresh skin biopsy samples from SSc patients (n � 5) as compared withhealthy controls (n � 5). Values are the mean and SEM. � � P � 0.05versus healthy controls.

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previous results, miR-29a was found to be down-regulated by 42 � 8% (P � 0.0011) in fresh skin biopsysamples from SSc patients as compared with healthycontrols (Figure 1D). In the same SSc skin biopsysamples, this was again paralleled by increased levels ofCOL1A1 and COL3A1 mRNA (8.0 � 2.5–fold [P �0.09] and 2.7 � 0.6–fold [P � 0.03], respectively) ascompared with healthy control skin in the same samples(Figure 2B).

Taken together, the miR-29 family members and

miR-29a in particular were consistently expressed at aremarkable down-regulation in skin fibroblasts and skintissue sections from SSc patients as compared withhealthy controls.

Altered collagen expression following overex-pression and knockdown of miR-29. We next aimed toanalyze the functional effects of altered miR-29 expres-sion on fibrogenesis. According to our hypothesis, therestoration of miR-29 levels would decrease collagenlevels in SSc fibroblasts, whereas down-regulation of

Figure 3. Influence of overexpression and knockdown of microRNA-29a (miR-29a) on the expression ofcollagen. Expression of type I and type III collagen in systemic sclerosis (SSc) patient samples wasdetermined relative to the controls transfected with scrambled RNA, which was defined as 100% or as 1.A, At the mRNA level, transfection of SSc fibroblasts (n � 5) with precursor miR-29a (pre-miR-29a) ascompared with scrambled RNA–transfected controls (n � 5) decreased the levels of type I and type IIIcollagen, as determined by SYBR Green real-time polymerase chain reaction (PCR) analysis. B, At theprotein level, overexpression of miR-29a in SSc fibroblasts (n � 6) also decreased the expression of typeI�2 and type III collagen as compared with scrambled RNA–transfected controls (n � 6), as determinedby Western blotting (top). Representative duplicate results are shown. The semiquantitative analysis wasperformed by densitometry (bottom). C, At the mRNA level, knockdown of miR-29a in fibroblasts fromhealthy donors (n � 5) as compared with scrambled RNA–transfected controls (n � 5) increased the levelsof type I and type III collagen, as determined by SYBR Green real-time PCR. D, At the protein level,knockdown of miR-29a in normal skin fibroblasts (n � 3) similarly increased the expression of type I�2and type III collagen as compared with scrambled RNA–transfected controls (n � 5), as determined byWestern blotting (top). Representative duplicate results are shown. The semiquantitative analysis wasperformed by densitometry (bottom). Values are the mean and SEM. In the Western blots, �-tubulin wasused as a loading control. � � P � 0.05 versus scrambled RNA–transfected controls.

miR-29 REGULATION OF COLLAGEN EXPRESSION IN SSc 1737

miR-29 in normal skin fibroblasts would induce anSSc-like phenotype, with an increase in the expression ofcollagen.

In SSc fibroblasts, transfection with pre-miR-29a/29b/29c increased the levels of the respective miRNA bya mean � SEM of 147.4 � 26.9–fold, 1,625 � 1,271–fold, and 57.5 � 0.4–fold. Transfection with the respec-tive pre-miR did not substantially alter the levels of theother miR-29 family members. Knockdown with anti-miR reduced the expression of the respective miRNA toa mean � SEM of 0.08 � 0.05–fold for miR-29a, 0.1 �0.04–fold for miR-29b, and 0.5 � 0.04–fold for miR-29c.Except for knockdown of miR-29a, which also affectedthe levels of miR-29b, the effects were specific.

In all gain-and-loss of function experiments, miR-29a, as compared with miR-29b/29c, showed the stron-gest effects. The expression of types I and III collagendecreased by a mean � SEM of 66 � 10% (P � 0.0003)and 65 � 8% (P � 0.0001), respectively, on the mRNAlevel, as analyzed by TaqMan-based real-time PCR

(Figure 3A), and by 37 � 5% (P � 0.0001) and 45 � 9%(P � 0.0006), respectively, on the protein level, asanalyzed by Western blotting (Figure 3B). Knockdownof miR-29a in normal skin fibroblasts increased theexpression of types I and III collagen by 1.8 � 0.5–foldand 2.0 � 0.6–fold, respectively, on the mRNA level(Figure 3C) and by 2.1 � 0.3–fold (P � 0.03) and 1.8 �0.4–fold, respectively, on the protein level (Figure 3D).

Overexpression of miR-29b in SSc fibroblastsdown-regulated the expression of mRNA for type I andtype III collagen by 35 � 9% (P � 0.01) and 40 � 15%(P � 0.03), respectively, and reduced the level of typeI�2 collagen protein by 44 � 22% (P � 0.05). Theexpression of type III collagen protein did not changesignificantly. Similar to the results with miR-29a, knock-down of miR-29b in healthy dermal fibroblasts increasedthe expression of types I and III collagen mRNA, but theeffects at the protein level were less distinct. (Datashowing the effects of miR-29b on the expression ofcollagen are available upon request from the authors.)

Figure 4. Direct regulation of collagen expression by members of the microRNA-29 (miR-29)family. Luciferase activity in HEK 293 cells transfected with precursor miR-29 (pre-miR-29) orwith anti-miR-29 was determined relative to controls transfected with scrambled RNA, which wasdefined as 100%. A, HEK 293 cells (n � 8) were transfected with the pGL3 luciferase reportercontaining a segment of the 3�-untranslated region (3�-UTR) sequence of COL3A1 that waspredicted to have 2 harbor binding sites for the miR-29 family: 242–248 and 682–688. B,Cotransfection of HEK 293 cells with pre-miR-29a, pre-miR-29b, or pre-miR-29c, which mimicsup-regulation of the respective miRNA, significantly reduced the relative luciferase activity ascompared with the scrambled RNA–transfected controls (n � 8). C, Cotransfection of HEK 293cells with the pGL3 luciferase vector and inhibitory molecules of miR-29a, miR-29b, or miR-29c(n � 6) significantly increased the relative luciferase activity as compared with the scrambledRNA–transfected controls (n � 8). All experiments were performed at least in duplicate. Valuesare the mean and SEM. � � P � 0.05 versus scrambled RNA–transfected controls.

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Enforced expression of miR-29c in SSc fibro-blasts decreased type I collagen mRNA by 62 � 14%(P � 0.001) and type III collagen mRNA by 61 � 14%(P � 0.002), whereas on the protein level, expression oftypes I�2 and III collagen was reduced by 14 � 7% (P �0.05) and 30 � 5% (P � 0.0002), respectively. Theeffects of miR-29c knockdown in healthy control fibro-blasts on the expression of types I and III collagen weresimilar to the effects of the other miR-29 family mem-bers examined. (Data showing the effects of overexpres-sion and knockdown of miR-29c on collagen expressionare available upon request from the authors.)

COL3A1, a direct target of miR-29. The observedreduction in collagen expression by miR-29 family mem-

bers could be caused indirectly, for example, by amodification of profibrotic cytokines and growth factors,or directly, by direct posttranscriptional regulation ofcollagen gene expression. To assess whether there wasdirect regulation of collagen genes, we created a lucif-erase reporter gene system by cloning a part of the3�-UTR of COL3A1 with respective binding sites formiR-29a, miR-29b, and miR-29c into the pGL3 controlvector (Figure 4A).

All miR-29 family members were expressed inHEK 293 cells, but at lower baseline levels as comparedwith the expression in skin fibroblasts (mean � SEM�Ct 22.87 � 0.25 for miR-29a, 28.09 � 1.9 for miR-29b,and 24.32 � 0.44 for miR-29c). Transfection of HEK 293

Figure 5. MicroRNA-29a (miR-29a), a downstream mediator of profibrotic molecules. Expression of miR-29a,miR-29b, or miR-29c in normal or systemic sclerosis (SSc) skin fibroblasts was determined relative to the respectivecontrols, which was defined as 100% or 1. A and B, Normal skin fibroblasts obtained from healthy donors were treatedwith recombinant transforming growth factor � (TGF�), platelet-derived growth factor B (PDGF-B), or interleukin-4(IL-4) to explore the effects on miR-29 expression, as measured by TaqMan-based real-time polymerase chain reaction.Stimulation of normal skin fibroblasts (n � 6) reduced the expression of miR-29a as compared with untreated controls(n � 6) (A). Compared with unstimulated normal skin fibroblasts, treatment with TGF�, PDGF-B, or IL-4 increased theexpression of mRNA for COL1A1 and COL3A1. However, overexpression of miR-29a by using precursor miR-29a(pre-miR-29a) in cytokine-stimulated normal skin fibroblasts decreased the expression of mRNA for COL1A1 andCOL3A1 as compared with stimulated and scrambled small interfering RNA (siRNA)–transfected cells (n � 5 for eachcondition) (B). C and D, In skin fibroblasts from SSc patients, PDGF-B and TGF� signaling pathways were inhibited,and the effects on the expression of miR-29 were analyzed. Treatment of SSc fibroblasts (n � 6) with imatinib increasedthe expression of miR-29 family members as compared with untreated controls (n � 6) (C). SSc skin fibroblasts were trans-fected with siRNA against c-abl, PDGF receptor � (PDGFR�), or c-kit to explore potential mechanisms of how imatinibrestores the levels of miR-29a. Inhibition of c-abl and PDGFR�, but not c-kit signaling, increased the expression ofmiR-29a in SSc skin fibroblasts (n � 4) as compared with scrambled siRNA-transfected controls (n � 4) (D).

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cells with pre-miR was specific and had no substantialeffects on the other family members. In HEK 293 cells,transfection with pre-miR-29a/29b/29c increased the lev-els of the respective miRNA by a mean � SEM of 510 �189–fold, 5,517 � 1,597–fold, and 420 � 152–fold.Knockdown of the miR-29 decreased their expression to0.36 � 0.07–fold for miR-29a, 0.13 � 0.09–fold formiR-29b, and 0.06 � 0.02–fold for miR-29c. Except forknockdown of miR-29c, which also affected the levels ofmiR-29b and, to a lesser extent, miR-29a, the effectswere specific.

In the reporter gene assay, cotransfection ofHEK 293 cells with pre-miR-29a and the luciferasereporter plasmid containing the 3�-UTR of COL3A1(Figure 4A) decreased the relative luciferase activity bya mean � SEM of 47 � 10% (P � 0.007) compared withcells transfected with the plasmid and scrambled con-trols (Figure 4B). Accordingly, cotransfection with anti-

miR-29a increased the relative enzyme activity by 20 �6% (P � 0.01) (Figure 4C). Similar effects were ob-served for miR-29b and miR-29c. Cotransfection withpre-miR-29b reduced the relative luciferase activity by67 � 10% (P � 0.0001) (Figure 4B), and cotransfectionwith anti-miR-29b increased the relative enzyme activityby 30 � 7% (P � 0.008) (Figure 4C). Cotransfectionwith pre-miR-29c decreased the relative luciferase ac-tivity by 70 � 6% (P � 0.0001) (Figure 4B), whereascotransfection with anti-miR-29c did not show signifi-cant effects (Figure 4C). In summary, these findings provethat miR-29 directly regulates collagen production.

MicroRNA-29, a downstream mediator of profi-brotic molecules. To explore factors that potentiallydown-regulate miR-29 levels in SSc, we analyzed theeffects of major profibrotic cytokines and growth factors.Treatment of normal skin fibroblasts with profibroticcytokines induced an SSc-like phenotype, with down-regulation of miR-29a to an extent similar to that seen inSSc fibroblasts. The most pronounced effects were ob-served for miR-29a after 48 hours of treatment, with areduction in the levels of mRNA for TGF� by 80 � 8%(P � 0.0001), for PDGF-B by 70 � 20% (P � 0.003), andfor IL-4 by 74 � 11% (P � 0.0001) (Figure 5A). Thelevels of miR-29b and miR-29c were also reduced, but toa lesser extent. (Data showing miR-29b and miR-29c asdownstream mediators of profibrotic molecules areavailable upon request from the authors.)

To confirm miR-29a as a downstream mediatorof the profibrotic effects of these cytokines, we investi-gated whether rescuing cytokine-treated normal fibro-blasts by overexpression of miR-29a could decrease thelevels of collagens induced by these cytokines. Stimula-tion with TGF�, PDGF-B, or IL-4 increased the levels ofCOL1A1 and COL3A1 mRNA in nonrescued scram-bled controls, but this effect was abolished by overex-pression of miR-29a (Figure 5B). Thus, it seems likelythat miR-29 is indeed a downstream mediator of theprofibrotic effects of the cytokines we examined.

We next analyzed whether treatment with TGF�,PDGF-B, or IL-4 could further decrease the reducedlevels of miR-29 in SSc fibroblasts. In contrast to healthyfibroblasts, no additional decrease in miR-29a/29b/29clevels was observed. Accordingly, transdifferentiation ofSSc fibroblasts into myofibroblasts by long-term stimu-lation with TGF� for 4 days did not further reduce thelevels of miR-29 (data not shown). The recombinantproteins used in these experiments were biologicallyactive, as proven by the strong decrease in miR-29 inhealthy fibroblasts and by an induction in downstreammolecules, such as type I collagen, in both healthy and

Figure 6. MicroRNA-29 (miR-29) in the murine model of bleomycin-induced skin fibrosis and in vivo effects of imatinib treatment, asdetermined by TaqMan-based real-time polymerase chain reactionanalysis. Expression of miR-29 family members in mice treated withbleomycin (bleo) only or with bleomycin plus imatinib (ima) wasdetermined relative to the saline-treated controls, which was definedas 100% or 1. A, Mice were challenged with bleomycin or saline for 6weeks (n � 10 mice per group). Similar to the findings in systemicsclerosis patients, the expression of miR-29a, miR-29b, and miR-29cwas significantly down-regulated in bleomycin-treated mice as com-pared with saline-treated controls. B, All mice were challenged withbleomycin for 6 weeks. Then, one group received imatinib 150mg/kg/day during the last 3 weeks, and the control group receivedNaCl during the last 3 weeks (n � 8 mice per group). This is abiologically relevant concentration that corresponds to mean plasmapeak and trough concentrations in humans. The expression of miR-29family members was increased in imatinib-treated mice as comparedwith control mice (only challenged with bleomycin). Values are themean and SEM. � � P � 0.05 versus saline-treated controls.

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SSc fibroblasts. A potential explanation for this findingwas that miR-29 levels in SSc fibroblasts were alreadymaximally suppressed and therefore could not be furtherdecreased by profibrotic cytokines. To further analyzethis hypothesis, SSc fibroblasts were incubated with thetyrosine-kinase inhibitor imatinib, which abrogatesTGF� and PDGF-B signaling (14,21). Indeed, treatmentof SSc fibroblasts with imatinib increased the levels ofmiR-29a by a mean � SEM of 40 � 7% (P � 0.002),miR-29b by 60 � 3% (P � 0.05), and miR-29c by 30 �6% (Figure 5C).

To further investigate which of the imatinib tar-gets mediates the observed effects, we performed selec-tive knockdown experiments for c-abl, PDGFR�, andc-kit in SSc fibroblasts and analyzed the levels of miR-29.Inhibition of c-abl and PDGFR�, but not c-kit, increasedthe levels of miR-29a by a mean � SEM of 9.1 �4.6–fold and 5.6 � 3.7–fold (P � 0.03), respectively, ascompared with the scrambled controls (Figure 5D).Similar effects were observed for miR-29c and formiR-29b (data available upon request from the authors).These results suggest abrogation of PDGFR� and c-ablsignaling as the main mechanism of how imatinib re-stores the levels of miR-29 in SSc fibroblasts.

MicroRNA-29 in the murine model of bleomycin-induced skin fibrosis. Finally, we wanted to investigatewhether the reduced levels of miR-29 in human SSc aremimicked in the SSc model of bleomycin-induced skinfibrosis in mice. Similar to the findings in human SSc(Figure 1B), the expression of all miR-29 family mem-bers in the skin of mice with bleomycin-induced fibrosiswas down-regulated after 6 weeks of bleomycin chal-lenge: miR-29a was reduced by a mean � SEM of 34 �19% (P � 0.02), miR-29b by 50 � 27% (P � 0.01), andmiR-29c by 69 � 11% (P � 0.0001) (Figure 6A).

We next used imatinib to analyze the effects ofimpaired PDGF-B and TGF� signaling on the levels ofmiR-29 in vivo. To mimic the clinical situation, we useda modified bleomycin model in which fibrosis is estab-lished before treatment with imatinib is started (24).Remarkably, imatinib treatment of mice with bleomycin-induced fibrosis increased the expression of miR-29a bya mean � SEM of 2.0 � 1.4–fold (P � 0.003) ascompared with NaCl control treatment. The levels of theother miR-29 family members showed a similar, but notsignificant, up-regulation (Figure 6B). These resultssuggest that even in preestablished fibrosis, abrogationof PDGFR� and c-abl/TGF� signaling could increasethe levels of miR-29a. As compared with normal con-trols (i.e., mice treated with saline instead of bleomycinfor 6 weeks and then treated with NaCl instead of

imatinib during the last 3 weeks), the levels of miR-29a,but not miR-29b or miR-29c, were normalized (data notshown).

DISCUSSION

The direct posttranscriptional regulation of col-lagens by miR-29 adds a novel aspect to the complexnetwork of factors that regulate the expression of colla-gen genes in SSc at the level of transcription. Thisincludes deregulated stimulatory (e.g., Sp-1, Smad3,p300/CREB binding protein) and inhibitory transcrip-tion factors and cofactors (e.g., Smad7, Fli-1, peroxi-some proliferator–activated receptor �), as well as sev-eral stimulatory cytokines and growth factors (25). Therelative importance of these factors in the pathogenesisof SSc is unclear. Similarly, there is limited knowledgeabout the extent to which down-regulated repressors andup-regulated stimulators of collagen transcription inter-play. Of note, miR-29a as a posttranscriptional repres-sor, which is induced by the stimulators mentionedabove, acts downstream of most of the previously iden-tified profibrotic molecules.

Thus, the main hypothesis derived from thepresent study is that targeting miR-29 family members asposttranscriptional regulators could be a potent antifi-brotic approach. This needs to be tested in futurestudies. Specifically, the effects of the modification ofmiR-29 levels on the synthesis of extracellular matrixshould be analyzed in several animal models of SSccovering different aspects of the disease. Optimally,miR-29–knockout mice could be created and potentiallycrossed with genetic fibrotic animal models or chal-lenged with profibrotic stimuli. If the findings of theseexperiments turned out to be promising, modification ofmiR-29 levels could be adapted to humans and the firstclinical proof-of-concept studies and studies on toxicitycould be performed.

So far, miRNA-based therapeutic approaches inanimal studies of other diseases have been promising,since toxic effects were not observed (10–13). Accord-ingly, our experiments with the restoration of miR-29 inskin fibroblasts did not have overt toxic effects on cellsurvival and proliferation (data not shown). Our resultsalso demonstrated that the bleomycin model of skinfibrosis, although it does not cover all of the features ofhuman SSc, represents one of the potential in vivomodels in which to test the different strategies of miR-29restoration in SSc because it displayed the reduced levelsof miR-29 that are seen in humans with SSc. Abrogationof PDGFR� and, eventually, TGF� signaling by admin-

miR-29 REGULATION OF COLLAGEN EXPRESSION IN SSc 1741

istration of imatinib increased the levels of miR-29 invivo. The direct therapeutic modulation of miR-29 mighthave even more pronounced effects. Whether imatinibalso restores miR-29 levels in human SSc should beanalyzed in the ongoing imatinib trials.

Interestingly, miR-29 might additionally influ-ence the production of other profibrotic molecules, suchas PDGF-B, PDGFR�, thrombospondin, and SPARC,since in silico analyses have shown these molecules to bepredicted targets of miR-29. The ability of miR-29 tocoordinate such a broad spectrum of profibrotic mole-cules illustrates the potential of miRNA to controlcomplex molecular interactions. Further research shouldinclude the analysis of miRNA expression profiles in SScbiologic samples, as well as the identification of miRNAthat regulate other key molecules, such as growth fac-tors, profibrotic cytokines, and matrix metalloprotein-ases.

The precise mechanisms that lead to the down-regulation of miR-29, especially miR-29a, in SSc remainto be determined, but our study showed that TGF�,PDGF-B, and IL-4, all of which are major profibroticmediators in SSc, decreased the expression of miR-29ain normal skin fibroblasts. Moreover, the stimulatoryeffects of these cytokines on collagen synthesis could bereduced by rescuing cells with miR-29a, suggesting thatthe reduced levels of miR-29a occur downstream ofthese cytokines and that miR-29 at least partially medi-ates their profibrotic effects. Accordingly, inhibition ofTGF� and PDGF-B signaling by imatinib or, morespecifically, by knockdown of c-abl or PDGFR� restoredthe decreased levels of miR-29 in SSc fibroblasts. Sincethese major profibrotic factors are not specific for SSc,the miR-29 family could also play a role in other fibroticdiseases. Indeed, there is evidence that miR-29b plays arole in cardiac fibrosis (26), and miR-29c was recentlyfound to be involved in the mechanisms of fibrosisduring the development of nasopharyngeal carcinomas(24).

Additional factors potentially involved in thedown-regulation of miR-29 in SSc fibroblasts may in-clude intrinsic mechanisms, because the low expressionof miR-29 family members was maintained from early tolate cell culture (passages 3–8) without additional pro-fibrotic stimuli and because stimulation with the profi-brotic cytokines did not further decrease the levels ofmiR-29 in SSc fibroblasts.

Epigenetic dysregulation of gene expression mayalso be part of this scenario. Fli-1, a negative regulator ofcollagen synthesis, has been proven to be epigeneticallysilenced in SSc fibroblasts by DNA methylation and

histone deacetylation (27). Changes in the expression ofhistone deacetylases have also been found to play a rolein the profibrotic mechanisms of SSc (28,29). Thus,epigenetic silencing of miR-29 might also contribute tothe activated state of SSc fibroblasts. Recently, a study ofskeletal myogenesis and the pathogenesis of rhabdomyo-sarcoma demonstrated that in rhabdomyosarcoma cellsand primary tumors with impaired differentiation,miR-29 was epigenetically silenced by an activated NF-�B/YY-1 pathway (30). On the other hand, DNA methyltransferases are experimentally validated targets ofmiR-29 (31), and thus, it might also be hypothesized thatmiR-29 plays a role in the epigenetic regulation ofprofibrotic genes in SSc.

So far, the available data on miR-29 indicate ageneral role in the development of fibrosis not limited toSSc. To evaluate in SSc whether low miR-29 levelscorrelate with individual profibrotic activity, as assessedby the modified Rodnan skin thickness score (MRSS) orby disease stage (early versus late), longitudinal, ratherthan cross-sectional, studies with larger cohorts of pa-tients will be needed, since in our study, patients withhigh MRSS scores and/or early disease were underrep-resented and, in general, the patients were only assessedat a single time point. In addition, it seems that there issome organ specificity in terms of which member of themiR-29 family has a predominant action. This fits verywell with the findings of previous studies demonstratingthat fibroblasts from different anatomic sites showedremarkable differences in their patterns of gene expres-sion (32).

In conclusion, our study shows that in SSc miR-29a directly regulates collagen expression at the post-transcriptional level, which is a novel aspect of thecomplex regulatory network of fibrosis in SSc. Further-more, our data indicate that the dysregulation of miR-29a is mediated, at least in part, by profibrotic cytokinesand growth factors. Based on the encouraging resultsfrom miRNA-based therapeutic approaches in animalstudies (10–12,33) and given the fact that the first humantrial targeting a liver-specific miRNA involved in thereplication of the hepatitis C virus has been launched(34), the development of strategies to maintain theexpression or to prevent the repression of miR-29a inSSc appears to be a promising future therapeutic strat-egy.

ACKNOWLEDGMENTS

We thank Ferenc Pataky and Peter Kunzler for excel-lent technical support.

1742 MAURER ET AL

AUTHOR CONTRIBUTIONS

All authors were involved in drafting the article or revising itcritically for important intellectual content, and all authors approvedthe final version to be published. Dr. O. Distler had full access to all ofthe data in the study and takes responsibility for the integrity of thedata and the accuracy of the data analysis.Study conception and design. Maurer, Stanczyk, J. H. W. Distler, S.Gay, O. Distler.Acquisition of data. Maurer, Akhmetshina, Trenkmann, Brock.Analysis and interpretation of data. Maurer, Stanczyk, Jungel, Akh-metshina, Kowal-Bielecka, R. E. Gay, Michel, S. Gay, O. Distler.

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