Journal of Dermatological Science 68 (2012) 179–186

Knockdown of electron transfer flavoprotein b subunit reduced TGF-b-induceda-SMA mRNA expression but not COL1A1 in fibroblast-populatedthree-dimensional collagen gel cultures

Shigenari Hirokawa a,b,1,*, Tomomasa Shimanuki a, Hiroyuki Kitajima a, Yasutomo Nishimori a,Makoto Shimosaka b

a R&D Laboratories, POLA PHARMA INC., Yokohama, Japanb Division of Applied Biology, Faculty of Textile Science and Technology, Shinshu University, Nagano, Japan

A R T I C L E I N F O

Article history:

Received 18 October 2011

Received in revised form 22 August 2012

Accepted 16 September 2012

Keywords:

ETFB

Collagen

TGF-beta

Alpha smooth muscle actin

A B S T R A C T

Background: The inhibition of transforming growth factor b (TGF-b)-induced myofibroblast differentia-

tion is a key objective for the treatment of hypertrophic scarring. We previously reported that

knockdown of the electron transfer flavoprotein b subunit (ETFB) reduced mechanoregulated cell

number in fibroblast-populated collagen gel cultures [1].

Objective: To characterize the effects of ETFB knockdown, we investigated gel contraction, TGF-b-

induced collagen, a-SMA mRNA expression and stress fiber formation.

Methods: Fibroblasts were transfected with negative control or ETFB-specific siRNAs and embedded in

collagen gels in an attached or detached condition. The gel contraction assay was performed in three

different concentrations of collagen (0.5, 1.0 or 1.5 mg/mL) and was analyzed by measuring the changes

in the gel area throughout the culture period. The attached collagen gel culture was performed in the

presence of rTGF-b and the mRNA levels of a-SMA and COL1A1 were measured by qRT-PCR. The effect of

ETFB knockdown on proliferation and stress fiber organization in monolayer cultures was investigated

by conducting AlamarBlue assays and phalloidin staining.

Results: The transfection of ETFB siRNA did not alter gel contraction compared to the negative control in

all collagen concentrations. When the cells were treated with TGF-b under mechanical stress conditions,

ETFB knockdown attenuated a-SMA mRNA expression to a level comparable to that observed in the

absence of TGF-b. However, no inhibitory effect on COL1A1 mRNA levels was observed. The AlamarBlue

assay indicated that the knockdown had no effect on the proliferation of cells cultured on plastic.

Phalloidin staining of a monolayer culture showed that ETFB knockdown weakened the stress fiber

organization induced by rTGF-b.

Conclusion: ETFB knockdown can affect TGF-b-induced tissue remodeling and/or fibrotic processes in

vitro.

� 2012 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights

reserved.

Contents lists available at SciVerse ScienceDirect

Journal of Dermatological Science

jou r nal h o mep ag e: w ww .e lsev ier . co m / jds

Abbreviations: a-SMA, alpha smooth muscle actin; bFGF, basic fibroblast growth

factor; COL1A1, collagen type I a I; CTGF, connective tissue growth factor; ETFB,

electron transfer flavoprotein b subunit; ECM, extracellular matrix; IFN-g,

interferon gamma; PDGF, platelet-derived growth factor; PPAR, peroxisome

proliferator-activated receptor; rTGF-b, recombinant transforming growth factor

b; 2DE, two-dimensional gel electrophoresis; 2D, two-dimensional; 3D, three-

dimensional.

* Corresponding author at: R&D Laboratories, POLA PHARMA INC., 560 Kashio-

cho, Totsuka-ku, Yokohama 244-0812, Japan. Tel.: +81 45 826 7251;

fax: +81 45 826 7259.

E-mail address: s-hirokawa@pola-pharma.co.jp (S. Hirokawa).1 Present address: Business Development Department, POLA PHARMA INC., 8-9-5

Nishi gotanda, Shinagawa-ku, Tokyo 141-0031, Japan. Tel.: +81 3 5436 2461;

fax: +81 3 5436 5671.

0923-1811/$36.00 � 2012 Japanese Society for Investigative Dermatology. Published b

http://dx.doi.org/10.1016/j.jdermsci.2012.09.012

1. Introduction

Granulation tissue is formed as part of the initial response towounding during the healing and repair process in the dermis andis composed of small vessels, abundant fibroblasts and variablenumbers of inflammatory cells [2]. When the dermis is injured, thefibroblasts acquire a migratory phenotype and generate compara-bly small traction forces to promote wound closure in response tochanges in the composition, organization and mechanical proper-ties of the ECM. The fibroblasts also respond to cytokines releasedlocally by inflammatory and other resident cells by proliferatingand producing ECM components such as fibronectin and collagen[3]. In response to the increasing ECM stress resulting from these

y Elsevier Ireland Ltd. All rights reserved.

S. Hirokawa et al. / Journal of Dermatological Science 68 (2012) 179–186180

remodeling activities, some fibroblasts differentiate into myofi-broblasts and upregulate a-SMA, the most widely used myofi-broblast marker [3]. As myofibroblasts exhibit higher contractileforces and ECM production, it is generally accepted that thefibroblast-to-myofibroblast differentiation represents a key eventduring wound healing and tissue repair. The high contractile forcegenerated by myofibroblasts is beneficial for physiological tissueremodeling but detrimental for tissue function when it becomesexcessive, as in hypertrophic scars, which are histologicallycharacterized by an overabundance of dermal collagen andmyofibroblasts [4].

Many studies have evaluated candidate anti-fibrosis factors orreagents by assessing their impact on TGF-b-induced collagen anda-SMA expression in fibroblasts. For example, a Rac1 inhibitor [5],IFN-g [6], a p38 inhibitor [7], Vitamin D [8], SB431542 (TGF-b1inhibitor) [9], a PPAR-g agonist [10] and CTGF antisense oligonu-cleotides [11] can inhibit the increase in collagen and a-SMAexpression induced by TGF-b. PPAR-g activation and CTGFinhibition were also effective in in vivo models of fibrosis [12,13].Recently, bFGF was shown to reduce excessive fibrosis by inhibitingthe differentiation of fibroblasts into myofibroblasts [14] and mayserve as a therapeutic tool for hypertrophic scarring [15].

Fibroblast-populated three-dimensional (3D) collagen gelculture has been widely accepted as an in vitro model of woundclosure or fibrosis [16–19]. In the detached-free-floating culturecondition, tractional forces generated by fibroblasts cause thecollagen matrix to contract, making this culture method a useful in

vitro wound closure model [16,18,20]. Taken together, in theattached-tethering culture condition, the cells are proliferative,morphologically elongated and bipolar, and some populationsdifferentiate into a-SMA-expressing myofibroblasts. This condi-tion has therefore been recognized to mimic fibrosis [17,19].

To find new pharmaceutical targets for the treatment ofhypertrophic scars, we performed a 2DE differential displaycomparison of 3D collagen gel cultures in the attached anddetached conditions to identify factors that reduce mechanor-egulated fibroblast cell proliferation. The electron transferflavoprotein b subunit (ETFB) was one of the candidates identified[1]. In the present report, we verified the inhibitory effect of ETFBon fibroblast cell number in our 3D collagen culture system anddetermined the ETFB mRNA expression pattern. To further validateETFB as an anti-fibrosis factor, we examined the effects of ETFBknockdown on the induction of collagen and a-SMA mRNAexpression by rTGF-b in attached collagen gel cultures.

2. Materials and methods

2.1. Cell culture and reagents

CCD-1113sk cells (ATCC No. CRL2439), which are normalhuman skin fibroblasts isolated from a 46-year-old female, werepurchased from the American Type Culture Collection at the fourthpassage. The cells were cultured in DMEM (1000 mg/mL glucose,0.584 g/L L-glutamine and 3.7 g/L NaHCO3; D-6046, Sigma, MO)supplemented with 10% FBS and no antibiotics in a tissue cultureflask (353024, BD Falcon, NJ). The experiments were performedbefore passage 13 because senescence was observed after 16passages.

The collagen gel culture was performed as previously described[1]. Briefly, for one 24-well plate, a collagen matrix solution wasprepared by mixing 7 mL of 5 mg/mL bovine dermis-derived, acid-solubilized type I collagen (Cellgen, Koken, Tokyo), 3.5 mL FBS,7 mL of 5� DMEM (4500 mg/mL glucose, final concentration,HEPES(�), NaHCO3(�); 12800-017, Invitrogen, CA), 3.5 mL of200 mM HEPES, 3.5 mL of 2.2% NaHCO3, 1.75 mL of 0.1 N NaOH and5.25 mL of H2O. The final concentrations of collagen and FBS were

1 mg/mL and 10% (v/v), respectively. For the gel contraction assay,the volume of collagen was adjusted with the volume of H2O. Thesolution was stored at 12 8C before use. Before culturing, 250 mL ofthe solution was seeded into each well of a 24-well plate as a basallayer and incubated for 15 min. After mixing with 2.5 mL ofsuspended fibroblasts (1 � 106 cells/mL) and 22.5 mL of thecollagen matrix solution, 1 mL of the mixture was seeded ontothe basal layer. The final calculated cell number was 1 � 105 cells/well. For the groups cultured in the detached condition, the gel andbasal layer were released from the well with a spatula after 2 h ofincubation. For TGF-b treatments, 300 ng/mL rTGF-b (100-B-010,R&D Systems, MN) was mixed into the collagen solution.

To measure cell numbers, the matrices were incubated with1000 U/mL collagenase (C-1889, Sigma, MO) in DMEM (2 mLcollagenase solution/matrix) for 30 min with agitation at 37 8C.The cells were then harvested by centrifugation at 3000 rpm for5 min, and the pellet was suspended in DMEM. The cell numberwas automatically counted by trypan blue staining using Vi-CellXR (Beckman Coulter, CA). The statistical differences between theETFB siRNA- and negative control siRNA-transfected cultures wereevaluated by Student’s t-test (p < 0.05) at each time point.

2.2. Transfection

The day before transfection, 7.25 � 105 cells were plated onto a10-cm dish. Transfection was performed according to themanufacturer’s instructions with Lipofectamine 2000 (Invitrogen,CA). Briefly, the siRNA and Opti-MEM (Invitrogen, Carlsbad, CA)were mixed to a final concentration of 33 nM siRNA. Lipofectamine2000 and Opti-MEM were mixed to achieve a final concentration of50 mL Lipofectamine 2000 per dish and then incubated for 5 min atroom temperature. Equal volumes of the two mixtures werecombined and incubated for 20 min at room temperature. Duringthe incubation, the culture medium of the 10-cm dishes wasreplaced with 12.5 mL of fresh medium (DMEM supplementedwith 10% (v/v) FBS). Next, 2.5 mL of the siRNA–Lipofectaminemixture was added to each dish and incubated for 24 h. At the endof the experiment, the transfected cells were trypsinized andsubjected to collagen gel culture and monolayer culture.

The siRNA for ETFB was purchased from GE Healthcare (UK; D-010494-04). The AllStars Negative Control siRNA was used fornegative controls (Qiagen, CA).

2.3. qRT-PCR

Total RNA was extracted from collagenase-dissolved cells (3gels/extraction) using the RNeasy RNA extraction kit (Qiagen,CA) according to the manufacturer’s instructions. The RNAconcentration of each sample was measured with a NanoDrop1000 (ThermoFisher Scientific, MA) and adjusted to 50 ng/mLwith RNase-free H2O. The concentration-adjusted RNA sampleswere visibly checked with agarose gel electrophoresis (E-gelSystem, Invitrogen, CA). The cDNA synthesis was performed withthe QuantiTect cDNA synthesis kit (Qiagen, CA). The concentra-tion of the RNA template was 250 ng per 20 mL reaction. Next,qRT-PCR was conducted using the SYBR green method (Quanti-Tect SYBR Green PCR Kit, Qiagen, CA) and an ABI7500 instrument(Applied Biosystems, CA). A total of 5 mL cDNA was used in afinal volume of 25 mL. The PCR reaction consisted of 55 cycles ofdenaturation at 94 8C for 15 s, annealing at 60 8C for 30 s, andextension at 72 8C for 34 s.

The data were analyzed with 7500 SDS System Software(Applied Biosystems, CA). The relative mRNA expression of thesamples compared to that of GAPDH was analyzed with the ddCtmethod. For statistical analysis, Student’s t-test (p < 0.05) wasperformed using dCt values [21].

S. Hirokawa et al. / Journal of Dermatological Science 68 (2012) 179–186 181

The following primers were used for qRT-PCR. COL1A1 primerswere designed with the PRIMER3 software: forward, 50-GTGGCCATC-CAGCTGACC-30 and reverse, 50-AGTGGTAGGTGATGTTCTGGGAG-30.ETFB primers were also designed with the PRIMER3 software:forward, 50-GGGGACAAGTTGAAAGTGGA-30 and reverse, 50-CAGA-GAGCTTGGAGGTCAGG-30. The primers for a-SMA were purchasedfrom Qiagen (QuantiTect primer assay, QT00088102). The primersfor GAPDH were custom-made by Invitrogen using a sequence from aprevious report [22].

2.4. Gel contraction assay

For this assay, fibroblasts were embedded in collagen at threedifferent concentrations (0.5, 1.0 or 1.5 mg/mL). The gels weredetached and cultured for 4 days. Each 24-well plate collagen gelculture was photographed with a digital camera. The image wasanalyzed with ImageJ 1.45s software to determine the area ofthe well and the gel and then calculate the relative percentage ofthe gel area. The area of the well was set as 100%. The differencesin the areas of the negative control siRNA- and ETFB siRNA-transfected cultures were evaluated by Aspin–Welch’s t-tests(p < 0.05) at each time point.

2.5. AlamarBlue assay

The siRNA-transfected cells were seeded onto 96-well plates(353072, BD Falcon, NJ) at 3 � 103 cells/well in 100 mL of medium.At days 1 and 3 of incubation, 10 mL of AlamarBlue (BUF012A, AbDSerotec, UK) was added and incubated for 2 h. Absorbances at 600and 570 nm were measured with a microplate reader (Spectra Max190, Molecular Devices, CA). The percentage reduction wascalculated according to the manufacturer’s instruction as follows:

Percentage reduction ¼ ðO2 � A1Þ � ðO1 � A2ÞðR1 � N2Þ � ðR2 � N1Þ � 100%

where O1 = 80,586 (molar extinction coefficient (E) of oxidizedAlamarBlue (Blue) at 570 nm), O2 = 117,216 (E of oxidizedAlamarBlue at 600 nm), R1 = 155,677 (E of reduced AlamarBlue

Fig. 1. Effect of ETFB knockdown on cell number and expression of ETFB mRNA in fibroblas

and embedded in collagen gel. The viable cell number was counted every day over 4 day

respectively. The diamond and square symbols represent negative control siRNA- and

triplicate cultures at each time point of at least three independent experiments. The asterisk

ETFB siRNA-transfected cultures as evaluated by Student’s t-test (p < 0.05). Messenger RNA

the ddCt method, and the relative expression level of the negative control siRNA-transfected

(p < 0.05). The graph represents the mean � SE of triplicate samples. The asterisks indicat

differences were observed between the negative control siRNA-transfected cultures. The

representative of two independent experiments.

(Red) at 570 nm), R2 = 14,652 (E of reduced AlamarBlue at 600 nm),A1 = absorbance of test wells at 570 nm, A2 = absorbance of testwells at 600 nm, N1 = absorbance of negative control well (mediaplus AlamarBlue but no cells) at 570 nm, N2 = absorbance of negativecontrol well (media plus AlamarBlue but no cells) at 600 nm.

For statistical analyses, the percentage reductions wereevaluated by Student’s t-test (p < 0.05) or a simple linearregression was fitted to the percentage reduction at days 1 and3, and a slope was calculated for each transfection treatment. Thestatistical significance of the slope between the negative controlsiRNA- and ETFB siRNA-transfected cultures was evaluated usingthe analysis of covariance (ANCOVA, p < 0.05).

2.6. Phalloidin staining

To visualize actin stress fibers, 5 � 104 cells of siRNA-transfected cells were plated onto coverslips in 6-well cultureplates in the presence or absence of TGF-b (11 ng/mL) for 3 days.After washing twice with PBS, the cells were fixed with formalin for20 min at room temperature and again washed twice with PBS. Thecells were then treated with 0.2% Triton-X-100-PBS for 15 minfollowed by 3 washes in Tris-buffered saline (TBS). Next, 5 units/mL of Phalloidin Alexa 488 (A-12379, Invitrogen, CA) was added tothe sample and incubated for 20 min. After 3 more washes withTBS, the coverslips were enclosed with Fluoromount G (K024,Diagnostics BioSystems, CA). Microscopic observation was per-formed with a fluorescent microscope (ECLIPSE E600, Nikon,Tokyo), and the samples were photographed with a digital camera(D100, Nikon, Tokyo) with a shutter speed of 2 s.

3. Results

We previously reported that knockdown of ETFB inhibitedmechanoregulated cell number in fibroblast-populated 3D colla-gen gel cultures [1]. To confirm these findings, we transfectedfibroblasts with ETFB or negative control siRNA and then countedcell numbers every day for 4 days of attached or detached collagengel culture (Fig. 1A). In the cells transfected with negative control

t-populated 3D collagen gel cultures. CCD-1113sk cells were transfected with siRNA

s of culture. The filled and open symbols represent attached and detached cultures,

ETFB siRNA-transfected cells, respectively. The data represent the mean � SD of

s indicate statistically significant differences between the negative control siRNA- and

levels of ETFB were measured by qRT-PCR at days 1 and 4. The data were analyzed with

culture at day 1 was set to 1. Student’s t-test was performed to evaluate the dCt values

e statistical significance. For ETFB siRNA-transfected cultures, statistically significant

open and filled bars represent days 1 and 4 of culture, respectively. The data are

Table 1Effect of ETFB siRNA transfection on gel contraction.

Collagen (mg/mL) siRNA % of area

Day 1 Day 2 Day 3 Day 4

0.5 Negative control 13.1 � 1.4 12.3 � 1.1 12.3 � 0.9 11.6 � 0.9

ETFB 13.6 � 1.2 13.5 � 1.7 13.5 � 1.9 13.1 � 1.2*

1.0 Negative control 28.0 � 1.8 23.9 � 1.0 25.3 � 1.1 23.6 � 1.4

ETFB 32.1 � 2.8* 26.8 � 3.1 28.1 � 1.7* 27.4 � 2.2*

1.5 Negative control 35.4 � 3.1 32.5 � 3.9 32.1 � 3.8 31.8 � 3.6

ETFB 41.4 � 2.1* 35.5 � 2.9 34.4 � 3.8 35.4 � 4.6

The cells were transfected with control or ETFB siRNA and cultured in detached collagen gels (0.5, 1.0 or 1.5 mg/mL).

The wells were photographed every day for 4 days, and the relative area of the gel was calculated.

The data represent the mean � SD of 6 wells at each time point.*ETFB siRNA-transfected cultures were significantly different from the negative control siRNA-transfected cultures according to Aspin–Welch’s t-test (p < 0.05).

Fig. 2. Photograph of detached collagen gel culture at day 4. The cells were

transfected with control or ETFB siRNA and cultured in detached collagen gels at

three different collagen concentrations (0.5, 1.0 or 1.5 mg/mL). The photograph

represents the gels cultured at day 4. The upper panel represents the negative

control siRNA-transfected cultures, and the lower panel represents ETFB siRNA-

transfected cultures.

S. Hirokawa et al. / Journal of Dermatological Science 68 (2012) 179–186182

siRNA and cultured in the attached condition, we found a three-fold increase in cell number after 4 days of culture; however, cellnumber decreased in the detached culture condition, as previouslydescribed [23]. In the cells transfected with ETFB siRNA andcultured in the attached condition, cell number declined to almostthe same level as the negative control-transfected detachedcultures. Statistically significant reductions in cell numbercompared with the negative control siRNA-transfected attachedcultures were observed at days 3 and 4 (p < 0.05). No statisticallysignificant change in cell number was observed in the ETFB siRNA-transfected cells compared to the negative control siRNA-transfected cells in the detached condition at all time points. Thisfinding indicates that cell toxicity is not a significant consequenceof ETFB knockdown. Expression of ETFB mRNA at days 1 and 4 wasmeasured by qRT-PCR (Fig. 1B). On day 1 in the negative controlsiRNA-transfected cells, ETFB expression in detached cultures wasapproximately 4% of the level observed in the attached cultures(p < 0.05). However, ETFB levels increased in the detachedcultures to a level comparable to that of the attached culturesby day 4. The effect of ETFB siRNA knockdown was maintainedthroughout the culture period (p < 0.05).

ETFB knockdown was initially shown to reduce mechanor-egulated cell number [1]. Because ETFB is involved in electrontransfer during the b-oxidation cascade [24,25], it is possible thatthe inhibitory effect of ETFB knockdown affects multiple cellularprocesses in treated fibroblasts. For ETFB knockdown to beconsidered a candidate treatment for hypertrophic scarring, it isimportant that the knockdown have no harmful effects on normalwound healing processes, especially wound closure. The gelcontraction assay has been an excellent in vitro model of woundcontraction and matrix remodeling [16]. We thereforedetermined the effect of ETFB knockdown on gel contraction.We investigated three different concentrations of collagen (0.5, 1.0or 1.5 mg/mL) because it has been reported that the initialconcentration of collagen affected the outcomes of the gelcontraction assay [26]. For this analysis, we measured the areaof the detached gels containing fibroblasts transfected with ETFBor negative control siRNA over 4 days of culture (Table 1). Thephotographs of the gels at day 4 are shown in Fig. 2. The collagengels of both groups contracted similarly when the collagenconcentration was at 0.5 mg/mL, while statistical significancebetween the negative control siRNA- and ETFB siRNA-transfectedculture was observed at day 4. Statistically significant differencesbetween the treatments were observed at all time points except atday 2 in the 1.0 mg/mL collagen gel; however, the differences were3–6% through the culturing. When the concentration was 1.5 mg/mL, a significant difference was not observed unless the data fromday 1 was included (approximately 6% of difference). Thedifferences between the transfections during day 2 to 4 wereapproximately 2–4%. From these results, we conclude thatthere was no overall difference between the negative control

siRNA-transfected and ETFB siRNA-transfected cultures on gelcontraction because of the following reasons: (a) the differencewas small and was not dependent on collagen concentration. (b)We did not observe statistical significance in the 1.5 mg/mLculture. (c) ETFB siRNA was completely effective during theculture, as indicated in Fig. 1. These findings indicate that ETFBknockdown did not inhibit overall mechanical activation ormajorly impact matrix contraction in vitro.

Inhibiting the differentiation of fibroblasts into myofibroblastshas been recognized as a therapeutic target for hypertrophicscarring. Many candidate factors have been evaluated in vitro usingassays that measure the expression of a-SMA, a marker ofmyofibroblast differentiation. TGF-b is a potent inducer of a-SMAexpression in fibroblasts. Determining the effects of candidateantifibrosis factors on TGF-b-induced a-SMA expression incollagen gel culture is a useful method for assessing theirantifibrotic and antihypertrophic scarring potencies [27–30]. Wetherefore investigated the effect of ETFB knockdown on a-SMAmRNA expression in the presence of rTGF-b. COL1A1 mRNA levelswere also measured in this experiment. Fibroblasts weretransfected with ETFB siRNA and embedded in collagen gel inthe presence of 300 ng/mL rTGF-b1. After 1 day of culture, a-SMAand COL1A1 mRNA levels were measured. In the negative controlsiRNA-transfected cells, exposure to TGF-b induced significantincreases in COL1A1 (approximately 2.5-fold; p < 0.05; Fig. 3A)and a-SMA expression (approximately 1.7-fold; p < 0.05; Fig. 3B).The addition of TGF-b tended to slightly increase ETFB expression(approximately 1.5-fold; Fig. 3C), but no statistically significantdifference was detected. In contrast, ETFB knockdown significantly

Fig. 3. Effect of ETFB knockdown on TGF-b-induced COL1A1 and a-SMA mRNA expression. ETFB siRNA- or negative control siRNA-transfected fibroblasts were embedded in

attached collagen gels in the presence or absence of 300 ng/mL rTGF-b. After 24 h of culture, the cells were harvested and subjected to qRT-PCR analysis. The relative

expression levels (ddCt values) were normalized by setting the expression value of the negative control siRNA-transfected cultures in the absence of rTGF-b to 1. The

statistical significance of the dCt values was evaluated by Student’s t-test. The graph represents the mean � SE of triplicate samples. The asterisks indicate significant differences

(p < 0.05). The data represent two independent experiments.

S. Hirokawa et al. / Journal of Dermatological Science 68 (2012) 179–186 183

attenuated the increase in a-SMA expression to approximatelycontrol levels (p < 0.05); COL1A1 expression was not affected.

Interestingly, fibroblasts transfected with ETFB siRNA andcultured in monolayers showed no morphological changes by lightmicroscopy relative to the negative controls (data not shown). Wetherefore determined the effect of ETFB knockdown on theproliferation of fibroblasts cultured on plastic tissue culture-treated plates. We used the AlamarBlue assay, which is a rapid andsensitive measure of proliferation and cytotoxicity that serves asan alternative to the [3H]thymidine incorporation assay [31]. ThesiRNA-transfected cells were seeded onto 96-well plates, and theabsorbance was measured at days 1 and 3 following the addition ofAlamarBlue dye. The absorbance values were reduced by36.8 � 0.3% and 38.4 � 2.2% on day 1 and by 47.5 � 1.4% and52.8 � 4.2% on day 3 in the negative control siRNA- and ETFB siRNA-transfected cells, respectively (Fig. 4A). Statistically significantdifferences of the percent reduction at day 3 compared to day 1were observed in both cultures. However, no significant differenceswere observed between the ETFB siRNA- and negative control siRNA-transfected cells at days 1 and 3. Moreover, the slopes of simple linearregressions for the negative control- and ETFB siRNA-transfected cellswere 5.37 and 7.19, respectively, and no statistically significantdifference between the cultures was observed by ANCOVA (p > 0.05).These data strongly indicate that ETFB siRNA-transfected cells wereas proliferative as negative control cells when cultured as monolayerson plastic tissue culture-treated plates.

Because ETFB knockdown inhibits TGF-b-induced a-SMAexpression, stress fiber formation may also be affected. Wecultured siRNA-treated fibroblasts for 3 days on coverslips in thepresence or absence of rTGF-b1 and then visualized stress fiberformation by phalloidin staining. Both groups of siRNA-transfected

cells were widely spread on the coverslips (Fig. 4B). Obvious stressfibers were observed in negative control-transfected cells in boththe presence and absence of TGF-b (Fig. 4Ba, left). However, in theETFB siRNA-transfected TGF-b-treated cells, the overall fluores-cence was weaker than in the negative control siRNA-transfectedcells, and obvious stress fibers were rarely observed (Fig. 4Ba,right). The difference was clearer in the presence of TGF-b than inthe absence of TGF-b. The experiments were conducted at thesame time and photographed using the same shutter speed (2 s). Ata higher magnification, fragile bundles of stress fibers appeared inthe ETFB siRNA-transfected TGF-b-treated cells (Fig. 4Bb).

4. Discussion

We identified ETFB in a previous loss-of-function screen forfactors that affect mechanoregulated cell proliferation in collagengel culture [1]. As we previously described [1], it has been reportedthat the 3D collagen culture system can mimic the proliferative orquiescent state of fibroblasts by changing mechanical stress bytethering the well (attached culture) or floating freely in the medium(detached culture). In the present study of the time courseexperiment through cell number or mRNA analysis, we found thatETFB knockdown attenuates the increase in cell number bymechanical stress without decreasing the cell number when freefrom mechanical stress. We also found that ETFB expression at day 1in detached culture showed a significantly reduced mRNA level andprotein level [1]. From the results, it is conceivable that ETFB wouldbe involved in a proliferative signal that is initially transmittedthrough mechanoregulation, presumably due to the contact fromthe ECM–cell interaction. Interestingly, knockdown of ETFB in 2Dmonolayer culture did not affect cell proliferation or viability as

Fig. 4. AlamarBlue assay of fibroblasts transfected with ETFB siRNA and phalloidin staining of TGF-b-treated ETFB siRNA-transfected fibroblasts in a monolayer culture. The

siRNA-transfected cells were seeded onto 96-well plates, and the absorbance of the AlamarBlue dye was measured at days 1 and 3. The experiment was performed in triplicate

over two independent experiments. The percent reduction in absorbance was calculated, and the difference was evaluated by Student’s t-test (p < 0.05). The asterisks indicate

the statistically significant difference between days 1 and 3 for each transfection. ‘‘NS’’ indicates that no significance was observed between the ETFB siRNA- and negative

control siRNA-transfected cells. The siRNA-transfected cells were cultured on coverslips for 3 days in the presence or absence of rTGF-b (11 ng/mL). The cells were fixed and

stained with phalloidin Alexa 488. (a) Images at 40� magnification and (b) overlay with phase-contrast image of phalloidin (green) and DAPI (blue) at 200� magnification,

indicating the transfected cells in the presence of rTGF-b. The left and right panels are the negative control siRNA- and ETFB siRNA-transfected cells, respectively. The

arrowheads indicate fragile stress fibers. The bars indicate 20 mm.

S. Hirokawa et al. / Journal of Dermatological Science 68 (2012) 179–186184

determined by the AlamarBlue assay. The cells grown in 3D cultureshow different morphological and biochemical properties whencompared with cells grown in monolayers and are similar to cellsfrom an in vivo environment [17,20,32]. Therefore, 3D culture-basedscreening is a promising method to identify new targets overlookedin monolayer culture-based screens. Mechanoregulation was alsoimplicated by its interaction with collagen as an important factor forexerting the effect of ETFB knockdown.

It was unclear why, at day4, the expression of ETFB mRNA in thedetached culture was similar to the levels observed in the negativecontrol attached culture. As described in our previous report, wescreened the target proteins from the cultures at day 1 because wewanted to omit secondary signals from mechanical force [1]. Therestoration of the mRNA level may reflect such secondary signals inthe detached culture.

We concluded that ETFB knockdown did not display a majorinhibitory effect on gel contraction. We previously observed thatthe a-SMA mRNA level was low in detached cultures in theabsence of TGF-b (data not shown). Therefore, our observationconcerning the gel contraction could be due to fibroblast tractionalforces or proto-myofibroblasts, which express stress fibers but donot express a-SMA [2]. The forces and proto-myofibroblasts havean integral role in early granulation tissue formation or woundclosure [2].

The b-oxidation cascade takes place in peroxisomes andmitochondria. Peroxisome proliferators, such as ciprofibrate,activate the b-oxidation cascade. The ciprofibrate-induced upre-gulation of fatty acyl-CoA oxidase (FAO), which is the first enzymein the b-oxidation pathway, was reported to be more robust inhepatocytes cultured on collagen gels compared with cellscultured on plastic plates [33]. ETFB reportedly transfers electronsto FAO [24,25]. Given these results and the report that peroxisomeproliferators increase cell proliferation [34], it is conceivable thatthe ECM can influence the b-oxidation pathway and that

mechanoregulation-specific cascades for proliferation may existin dermal fibroblasts. We observed a fragile stress fiber formationafter ETFB knockdown. This effect suggests a hypothesis that ETFBknockdown could affect focal adhesion complex formation such asFAK or paxillin. The adaptor protein paxillin participates in celladhesion-mediated signal transduction and belongs to a LIM-containing protein family [35,36]. Dong et al. demonstrated thatphosphorylation of Ser272 within the LD4 motif, which contains abinding domain of GIT1, and FAK1 regulated nuclear export andthat nuclear-localization paxillin LIM domains stimulated DNAsynthesis and cell proliferation [36]. Therefore, if ETFB knockdowninhibited paxillin phosphorylation or nuclear localization in the 3Dculture, there may be a relationship between paxillin regulationand the present experiment on the inhibition of cell number.Additionally, a signaling cascade of antifibrosis involving paxillinphosphorylation is PGE2 stimulation. PGE2 has been reported to bean antifibrosis factor inhibiting TGF-b-induced a-SMA andcollagen expression and also to inhibit the phosphorylation ofpaxillin [37]. In this report, the authors speculate that the signalingpathways of collagen and a-SMA genes are different but over-lapping because the PGE2 inhibition of TGF-b-induced collagensecretion appears to occur earlier than the PGE2 inhibition of a-SMA expression [37]. Our observation that ETFB knockdownaffected only a-SMA mRNA expression induced by TGF-b canpartially account for the resemblance to the PGE2 signalingcascade. However, a discrepancy exists between PGE2 stimulationand our observation because PGE2 stimulation reduced prolifera-tion both in 3D and 2D cultures [38], whereas ETFB knockdownattenuated proliferation only in 3D culture. Further investigationsare needed to clarify the relationship between focal adhesionsignals, PGE2 stimulation and the involvement of ETFB.

Alternatively, the b-oxidation cascade also acts as an energysupplier for the TCA cycle by oxidizing fatty acids. With respect toenergy metabolism, ETFB is an essential component of the

ECM (Collagen)

Mechanical stress

ETFB

β-oxidation

Proliferation α-SMA

expression Collagen

production

Fibroblast

Stres s fiber

formation

Energy (ATP ?)

activation ?

activation

signal TGF-β

TGF-βsignal

Fig. 5. Schematic diagram of the involvement of ETFB on mechanoregulation. The

current hypothesis of the involvement of ETFB on mechanical stress transmitted by

ECM–cell communication.

S. Hirokawa et al. / Journal of Dermatological Science 68 (2012) 179–186 185

oxidative phosphorylation pathway [24,25] and regulates at leastnine mitochondrial matrix dehydrogenases [39]. There are anumber of studies that link b-oxidation to cancer. Changes in lipidmetabolism can affect numerous cellular processes including cellgrowth, proliferation, differentiation and motility [40]. Forexample, fatty acid oxidation is reported to be a dominantpathway for energy generation in prostate cancer [41] andenhanced mitochondrial b-oxidation of fatty acids has been linkedto tumor promotion in pancreatic cancer [42]. Moreover, energymetabolism reportedly increased in the burned animal model [43]and in keloid patients [44]. Based on these reports, we speculatethat b-oxidation may represent an energy source used by activatedfibroblasts to respond to mechanical stress or during theirdifferentiation into myofibroblasts. We hypothesize that ETFBknockdown may deprive a cell of the excess energy needed for cellactivation without perturbing basic energy metabolism. Theincrease in ETFB expression following TGF-b treatment and thefragile stress fiber organization resulting from ETFB knockdownwithout affecting the proliferation on monolayer culture maysupport our hypothesis. Although more studies are needed toconfirm this hypothesis, a schematic diagram is shown in Fig. 5based on previous reports and the present study. Mechanical stressis transmitted through contact with ECM–cell communicationsuch as integrins. The signal upregulates the expression of TGF-b[23] and FAO [33], which lead to the enhancement of the b-oxidation cascade. The signal from ECM and/or TGF-b signalingelucidates proliferation, stress fiber formation and a-SMA mRNAexpression in fibroblasts. The enhancement of b-oxidation canspecifically provide energy for these responses. ETFB knockdowninhibits the first step of b-oxidation, and thus these responses, byreducing the energy supply. The cascade of collagen production issupposed to be independent from b-oxidation. On the other hand,in an environment free of mechanical stress, cells can survive withenergy supplied by cascades other than b-oxidation such as theTCA cycle.

We observed that ETFB knockdown inhibited not only themechanoregulated increase in cell number but also the TGF-b-induced increase in a-SMA mRNA expression. The results of our gelcontraction assay and the cell number experiment in the detachedculture condition show that these activities cannot be attributed totoxic effects. Many studies of factors that regulate these activities in

vitro have been reported in addition to those described in theIntroduction. For example, Sugiura et al. reported that N-acetyl-L-cysteine (NAC) inhibited TGF-b-induced a-SMA expression in

detached collagen gel cultures [27], and Genever et al. reportedthat phenytoin stimulated cell proliferation under nonretractingconditions [45]. To our knowledge, this is the first report showingthat ETFB knockdown induces similar effects. Hypertrophic scar orfibrosis is characterized by the hypercellularity of fibroblasts and theabundance of myofibroblasts [16,46,47]. Our findings support theidea that ETFB knockdown affects TGF-b-induced tissue remodelingand/or fibrotic processes in vitro by inhibiting mechanoregulation-induced cell proliferation and a-SMA mRNA expression. Althoughthe knockdown did not inhibit collagen mRNA expression, totalexcessive collagen in the fibrotic tissue could be reduced, attenuat-ing excessive cellularity and a-SMA mRNA expression, which willlead to myofibroblast differentiation. ETFB knockdown had noobvious detrimental effects on gel contraction or the viability ofdetached cultured fibroblasts. Collectively, these results suggest thatETFB knockdown has the potential to provide a new therapeuticmechanism, which could specifically inhibit an excessive activationof fibroblasts induced by mechanoregulation. Future studies shouldinvestigate the in vivo relevance of this effect such as using knockoutmice or staining of human hypertrophic scar tissue and itsrelationship with signaling molecules.

Acknowledgements

We thank Dr. Nobuo Kubota and Akiko Miyamae for kindlysupporting the gel contraction assay.

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