Wound healing-related properties detected in an experimental model with a collagen gel contraction assay are affected in the absence of tenascin-X

Author’s Accepted Manuscript Wound healing-related properties detected in an experimental model with a collagen gel contraction assay are affected in the absence of tenascin-X Kei Hashimoto, Naoyo Kajitani, Miyamoto, Ken-ichi Matsumoto

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PII: DOI: Reference:

S0014-4827(17)30679-1 https://doi.org/10.1016/j.yexcr.2017.12.025 YEXCR10864

To appear in: Experimental Cell Research Received date: 11 May 2017 Revised date: 19 December 2017 Accepted date: 27 December 2017 Cite this article as: Kei Hashimoto, Naoyo Kajitani, Yasunori Miyamoto and Ken-ichi Matsumoto, Wound healing-related properties detected in an experimental model with a collagen gel contraction assay are affected in the absence of tenascin-X, Experimental Cell Research, https://doi.org/10.1016/j.yexcr.2017.12.025 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Wound healing-related properties detected in an experimental model with a collagen gel contraction assay are affected in the absence of tenascin-X Kei Hashimotoa,b,c,d,e, Naoyo Kajitanie,f, Yasunori Miyamotoa,c and Ken-ichi Matsumotoe,*

a

Graduate School of Humanities and Sciences, Ochanomizu University, Tokyo, Japan

b

Program for Leading Graduate Schools, Ochanomizu University, Tokyo, Japan

c

Institute for Human Life Innovation, Ochanomizu University, Tokyo, Japan

d

Research Fellow of Japan Society for the Promotion of Science, Tokyo, Japan

e

Department of Biosignaling and Radioisotope Experiment, Interdisciplinary Center for Science

Research, Organization for Research and Academic Information, Shimane University, Izumo, Japan f

Department of Experimental Animals, Interdisciplinary Center for Science Research, Organization for

Research and Academic Information, Shimane University, Izumo, Japan

* Correspondence to: Department of Biosignaling and Radioisotope Experiment, Interdisciplinary Center for Science Research, Organization for Research and Academic Information, Shimane University, Enya-cho, Izumo 693-8501, Japan. Email address: [email protected] (K. Matsumoto)

Abbreviations: BrdU, bromodeoxyuridine; BSA, bovine serum albumin; 2D, 2-dimensional; DAPI, 4’, 6-diamidino-2-phenylindole dihydrochloride; E, embryonic day; ECM, extracellular matrix; EDS, Ehlers-Danlos syndrome; ELISA, enzyme-linked immunosolvent assay; FBG, fibrinogen-like domain; FBS, fetal bovine serum; MEF, mouse embryonic fibroblast; MMP, matrix metalloproteinase; PBS, phosphate buffered saline; PS, penicillin-streptomycin; RT-PCR, reverse transcription-polymerase chain reaction; siRNA, small interfering RNA; TGF-1, transforming growth factor-1; TNX, tenascin-X; Tnxb, mouse tenascin-X gene; TBS, Tris-buffered saline; WT, wild type.

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ABSTRACT

Patients with tenascin-X (TNX)-deficient type Ehlers-Danlos syndrome (EDS) do not exhibit delayed wound healing, unlike classic type EDS patients, who exhibit mutations in collagen genes. Similarly, in TNX-knockout (KO) mice, wound closure of the skin is normal even though these mice exhibit a reduced breaking strength. Therefore, we speculated that the wound healing process may be affected in the absence of TNX. In this study, to investigate the effects of TNX absence on wound healing-related properties, we performed collagen gel contraction assays with wild-type (WT) and TNX-KO mouse embryonic fibroblasts (MEFs). Collagen gels with embedded TNX-KO MEFs showed significantly greater contraction than those containing WT MEFs. Subsequently, we assessed collagen gel contraction-related properties, such as the activities of matrix metalloproteinase (MMP)-2 and MMP-9 and the protein and mRNA expression levels of transforming growth factor 1 (TGF-1) in the collagen gels. The activities of MMP-2 and MMP-9 and the expression level of TGF-1 were elevated in the absence of TNX. Furthermore, filopodia-like protrusion formation, cell proliferation, migration, and collagen expression in MEFs were promoted in the absence of TNX. These results indicate that these wound healing-related properties are affected in a TNX-deficient extracellular environment.

Keywords: Tenascin-X Wound healing Matrix metalloproteinase 2

TGF-1 Ehlers-Danlos syndrome

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1. Introduction

Tenascin-X (TNX), a member of the tenascin family, is an extracellular matrix (ECM) protein that is ubiquitously expressed in a wide variety of tissues [1]. TNX plays essential roles in the ECM architecture and tissue integrity. Several in vitro and in vivo observations have suggested that TNX interacts with types I, III, and V fibrillar collagens [2, 3]; types XII and XIV fibril-associated collagens [3, 4]; and the proteoglycan decorin [5]. Furthermore, TNX is involved in collagen deposition [6], fibrillogenesis [2, 7], and stiffness [8], as well as elastic fiber and microfibril formation [9]. Complete deficiency of TNX in humans causes an autosomal recessive form of the classic type of Ehlers-Danlos syndrome (EDS), a heritable connective tissue disorder [10, 11]. In contrast, several lines of evidence have suggested that mutations in the genes encoding the types I and V collagens lead to the development of the classic type of EDS with autosomal dominant inheritance [12, 13]. Thus, TNX deficiency is an example of a causative mutation for the classic type of EDS caused by factors other than collagens or collagen-processing enzymes. In general, classic EDS caused by mutations in the types I and V collagen genes is a disorder characterized by skin hyperextensibility, fragile and soft skin, easy bruising, joint hypermobility, and delayed wound healing with atrophic scars (cigarette paper-like skin) [13, 14]. Interestingly, unlike classic EDS, patients with TNX-deficient type EDS exhibit macroscopically normal wound healing with no atrophic scars [11, 15]. In a previous study, TNX-knockout (KO) mice with wounds showed that there was no significant difference in the period of wound healing between wild-type (WT) mice and TNX-KO 4

mice. However, the tensile strength of healing wounds in TNX-KO mice was significantly weaker than that in WT mice, suggesting that TNX is responsible for increasing the biomechanical strength of the skin [16]. In general, it is known that abnormal collagen deposition and fibrillogenesis results in aberrant wound healing [17]. However, even though aberrant collagen deposition and fibrillogenesis are induced in the absence of TNX [2, 6], abnormal wound healing is not detected in TNX-deficient EDS patients and mice, suggesting that wound healing process are altered in the absence of TNX. Wound healing is a highly orchestrated biological process that involves interactions among platelets and a wide variety of cell types, such as neutrophils, macrophages, fibroblasts, endothelial cells, and keratinocytes, as well as ECM components [18, 19]. The wound healing process includes the stages of hemostasis, inflammation, granulation tissue formation, and scar tissue formation. In the granulation tissue formation stage, newly synthesized ECM components are deposited, the surrounding tissues are pulled via cell migration and contraction, and the wound is closed. In the scar tissue formation stage, the production and decomposition of ECM components occur, and granulation tissues are remodeled. The mechanism through which the wound healing process is altered in the absence of TNX is not well understood. It has been shown that matrix metalloproteinase (MMP)-2 and MMP-9 expression is induced in the absence of TNX in vivo [20] and in vitro [21]. MMP-9 regulates wound healing through cell recruitment and migration around the lesion and the synthesis and degradation of the ECM [22]. In addition, MMP-2 is activated during the wound healing process in the skin and regulates healing while interacting with plasminogen [23]. Therefore, it is possible that the wound healing process is affected via the activation of MMP-2 and MMP-9 in the

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absence of TNX. In the present study, to assess wound healing-related properties in TNX-KO mice, we investigated the matrix contraction process using collagen gels with embedded WT or TNX-KO mouse embryonic fibroblasts (MEFs), providing an in vitro model for studying wound contraction. We found that the contraction of collagen gels through the up-regulation of MMP-2 and MMP-9 activities, and the expression level of transforming growth factor-1 (TGF-1) is promoted in the absence of TNX. Moreover, these factors may facilitate the formation of filopodia-like protrusions, cell proliferation, and migration of MEFs in collagen gels. These results suggest that the promotion of matrix contraction in the absence of TNX may explain the macroscopically normal wound healing observed in patients with TNX-deficient type EDS.

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2. Materials and methods

2.1. Mice

C57BL/6J mice were obtained from CLEA Japan (Tokyo, Japan), and TNX-KO mice were originally generated by homologous recombination using embryonic stem cells [20]. Thereafter, TNX-KO mice were backcrossed into a congenic line, C57BL/6 [24]. This study was approved by the Ethical Committee for Animal Research of Shimane University, and all of the experimental procedures were performed according to the institutional guidelines.

2.2. Cell culture

MEFs were isolated from embryonic day 13.5 (E13.5) or E17.5 embryos of WT and TNX-KO

mice

in

phosphate-buffered

saline

(PBS)

supplemented

with

penicillin-streptomycin (PS) (Thermo Fisher Scientific). A clump of cells from whole body apart from blood forming tissues such as liver was minced. The isolated cells were allowed to attach to plastic dishes. MEFs were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Nissui, Tokyo, Japan) supplemented with 10% fetal bovine serum (FBS) (Thermo Fisher Scientific, Waltham, MA) and PS (Thermo Fisher Scientific) at 37oC in 5% CO2. MEFs from the 5th to 15th passages were used for experiments.

2.3. Reverse transcripts-polymerase chain reaction (RT-PCR) and real-time RT-PCR 7

RNAs were extracted from cultured MEFs. The RNAs were reverse-transcribed using a PrimeScript 1st strand cDNA Synthesis Kit (Takara Bio Inc., Shiga, Japan). RT-PCR analysis was performed to confirm the expression of Tnxb, Actb, and Gapdh mRNAs in MEFs. PCR was performed as previously reported [21]. Real-time RT-PCR analysis was performed to quantify Gapdh, Tgfb1, Mmp9, Mmp2 and Col1a2 mRNA levels. The mRNA levels were quantified with an ABI 7300 real-time PCR machine (Thermo Fisher Scientific) using KOD SYBR qPCR Mix (Toyobo Co., Ltd., Osaka, Japan). The primer sequences used for real-time RT-PCR analysis are listed in Table.1 [25-28].

2.4. Collagen gel contraction assay

Contraction of collagen gels was performed in 6-well plates. The plates were pre-coated with 2% bovine serum albumin (BSA) (Wako Pure Chemical Industries, Ltd., Osaka, Japan) at 37oC overnight and then washed three times with phosphate buffered saline (PBS). After the density of MEFs had been adjusted to 7.5 x 105 cells/ml with DMEM supplemented with 12% FBS and PS, the same volumes of the cell suspensions, Cellmatrix type I-A (Nitta Gelatin, Inc., Osaka, Japan) and 2 x DMEM were mixed. The final concentration of the mixed collagen solution was 2.5 x 105 MEFs/ml in 1 x DMEM, 4% FBS, and 1.0 mg/ml collagen. Then 1.5 ml of the mixture was added to each well in the BSA pre-coated 6-well culture plates and gelated at 37oC over a period of 30 min. After gelation, the gels were detached from the plastic surface by forceful ejection of a medium of DMEM supplemented with 4% FBS and PS. The floating collagen gels were

8

cultured at 37oC in 5% CO2. After the contraction of the collagen gels, the gel areas were analyzed using ImageJ. For inhibitory experiments, both of gels and media were incubated with MMP inhibitors, 50 M batimastat (AnaSpec, Fremont, CA) and 50 M GM6001 (Merck Millipore, Birellica, CA), a TGF-1 receptor I (ALK5) inhibitor, 2.0 g/ml SB525334 (Wako), and an actin polymerization inhibitor, 1.0 g/ml cytochalasin B (Sigma Aldrich, St. Louis, MO). To label the MEFs with bromodeoxyuridine (BrdU), BrdU at a final concentration of 20 M (Nacalai Tesque, Inc., Kyoto. Japan) was added to the culture medium 24 h before fixation. In addition, to measure the number of cells in the gels, the collagen gels were digested with DMEM containing 450 g/mL collagenase type II (Life technologies), 0.025% trypsin (Life technologies), and 0.05 mg/mL deoxyribonuclease I (Sigma-Aldrich), and the number of isolated MEFs were counted.

2.5. Transfection with DNA plasmid or small interfering RNA (siRNA)

Transfection with DNA plasmids or siRNA was performed as described by Kobayashi et al. [29]. Briefly, 7.5 x 105 MEFs were seeded on 6-well plates with DMEM and 10% FBS and cultured overnight at 37oC in 5% CO2. Transfection with 2.5 g plasmid DNA, pSec-FTNX2 [30] or pSec-Tag2/Hygro B (pSec-Tag2B) (Thermo Fisher Scientific) as an empty vector or with 200 pmol MMP-9 siRNA (MSS275783; Thermo Fisher Scientific), MMP-2 siRNA (MSS206700; Thermo Fisher Scientific) or control siRNA (Thermo Fisher Scientific) was performed using Lipofectamine 2000 (Thermo Fisher Scientific) for 6 h at 37oC in 5% CO2. After the transfection, media were changed to DMEM with 10% FBS and PS. The transfected cells were cultured

9

overnight and then subjected to a collagen gel contraction assay.

2.6. Gelatin zymography

Gelatin zymography was performed as described by Matsumoto et al. using conditioned media from collagen gel culture containing MEFs with DMEM supplemented with 4% FBS [21]. The media were subjected to electrophoresis under non-reducing conditions on a sodium dodecyl sulfate–polyacrylamide (10%) gel containing 0.1% gelatin. The gelatinolytic activities were quantified with a gel image analyzer, Odyssey infrared imaging system (LI-COR Biosciences, Lincoln, NE).

2.7. Enzyme-linked immunosolvent assay (ELISA) for TGF-1

ELISA was performed to measure concentrations of TGF-1 in conditioned media of collagen gel culture containing MEFs using a Mouse/Rat/Porcine/Canine TGF-1 Quantikine ELISA Kit (R&D Systems, Minneapolis, MN) according to the manufacturer’s instructions. To measure total amount of active and latent TGF-1, samples were incubated with HCl for 10 min.

2.8. Immunostaining

Cultured collagen gels containing MEFs were fixed with 10% formalin overnight and cryoprotected in 15% to 30% sucrose solution. Sections (10 m thick) were obtained by using a cryostat (CM3050S; Leica, Wetzlar, Germany). For the detection of

10

BrdU, the sections were boiled in 10 mM citrate buffer (pH 6.0) for 10 min and were blocked by a blocking buffer (10% FBS, 3% BSA, 130 mM glycine, 0.4% Triton-X100; in Tris-buffered saline (TBS) [25 mM Trizma base, 137 mM NaCl, 2.68 mM KCl, (pH 7.4)]. The sections were incubated overnight with anti-BrdU (1:500; MAB3222; Merck Millipore) as a primary antibody, washed three times with TBS, and further incubated for 1 h with Alexa Fluor 555-conjugated anti-muose IgG (1:300; Thermo Fisher Scientific) as a secondary antibody. For the detection of F-actin, after blocking, the sections were incubated with Acti-stainTM 555 Fluorescent Phalloidin (1:150; PHDH1; Cytoskeleton, Denver, CO) for 30 min. After fluorescent staining, the sections were mounted with ProLong® Gold Antifade Mountant with 4′, 6-diamidino-2-phenylindole dihydrochloride (DAPI) (Thermo Fisher Scientific) to stain the nuclei of MEFs. Fluorescent images were captured with a fluorescent microscope (ECLIPSE 80i; Nikon, Tokyo, Japan). As for analysis of filopodia-like protrusion formation, MEFs with filopoida-like protrusions longer than 2 m were regarded as the cells exhibiting protrusions. According to this criterion, the ratio of cells exhibiting filopodia-like protrusions to total cells was analyzed. In addition, as for quantification of the lengths of filopodia-like protrusions, the lengths were measured with ImageJ.

2.9. Migration assay

Collagen gels with 100 l of gel volume containing MEFs (2.5 x 104 cells) were gelated in a 96-well culture plate pre-coated with 2% BSA at 37oC for 30 min as described in the section “Collagen gel contraction assay”. The gel was removed from the 96-well plate and placed in the wells of a non-coated 6-well culture plate. Next,

11

cell-free collagen solution in DMEM with 4% FBS and PS was poured into the 6-well plate to embed the gel from the 96-well plate and then the collagen solution was gelated. After 1-, 2-, and 3-day culture of the 6-well plates, images of cells migrating beyond the boundary line between the embedded gels and cell-free gels were captured using a microscope (CKX53, Olympus, Tokyo, Japan). The cells that had migrated beyond the boundary line were counted after 3-day culture.

2.10. Quantification of collagen protein

The total amount of collagen protein in the collagen gel containing MEFs was measured with a total collagen assay kit (Quick Zyme Biosciences, Leiden, Netherlands) and a total protein assay kit (Quick Zyme Biosciences) according to the manufacturer’s instructions.

2.11. Fibrin gel contraction assay

Contraction assay of fibrin gels was performed with 6-well plates according to a previous report [31]. Each well of a 6-well plate was coated with -1.5 ml of SYLGARD (Dow-Chemicals, Midland, Michigan, USA) and left at 55oC for 48 h. E13.5 or E17.5 MEFs were suspended in 10 mg bovine fibrinogen (Sigma Aldrich) and 0.88 units of human thrombin (Sigma Aldrich) to a final concentration of 7.5 x 105 cells per 1.5 ml of DMEM supplemented with 10% FBS and spread over the surface of the coated wells. The MEFs-embedded fibrin gels were left for 30 min at 30oC. Then 2 ml of DMEM supplemented with 10% FBS and 0.2 mM L-ascorbic acid 2-phosphate (Sigma Aldrich)

12

was added. The medium was changed every day. Contraction of fibrin gels was analyzed every day. Gelatin zymography using conditioned media of MEFs-embedded fibrin gels with DMEM supplemented with 4% FBS and 0.2 mM L-ascorbic acid 2-phosphate was performed as described above.

2.12. Statistical analysis

We used pairs of the MEFs derived from TNX-KO (n=2) and WT (n=2) mice at E13.5 and E17.5. We performed at least two independent experiments about each pair of MEFs. The data were analyzed by a one-tailed Student’s t-test. The values were expressed as means ± standard error of the mean (SEM). Changes were considered significant if the p value from Student’s t-test was less than 0.05, *: p < 0.05, **: p < 0.01, and ***: p < 0.001.

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3. Results

3.1. Contraction of collagen gels is stimulated in the absence of TNX

To determine the effect of TNX absence on the wound healing-related properties of MEFs, we examined the contraction of collagen gels embedded with MEFs derived from WT (TNX+/+) or TNX-KO (TNX-/-) mice. First, to confirm whether TNX is expressed in MEFs, the mRNA expression of Tnxb was analyzed in E13.5 MEFs by RT-PCR. As shown in Fig. 1A, Tnxb mRNA was detected in WT MEFs but not TNX-KO MEFs. Next, WT and TNX-KO MEFs were cultured in floating collagen gels. After 5 days of culture, the contraction of TNX-KO gels was significantly greater than that in WT gels (Fig. 1B, C). Similar to the gels containing E13.5 TNX-KO MEFs, gel contraction in the absence of TNX was observed in collagen gels containing E17.5 TNX-KO MEFs (Supplementary Fig. 1A-C). The contraction of MEF-embedded fibrin gels was also examined as an alternative to collagen gels, and contraction was also promoted in fibrin gels containing E13.5 or 17.5 TNX-KO MEFs (Supplementary Fig. 1D-F). In addition, to determine whether the promotion of gel contraction in the absence of TNX could be suppressed by the expression of TNX, a TNX expression plasmid (pSec-FTNX2) and control vector (pSec-Tag2B) were introduced into TNX-KO MEFs, with transfection with the pSec-FTNX2 plasmid resulting in increased expression of Tnxb mRNA (Fig. 1D). Cells were then subjected to the gel contraction assay. After 3-day culture of TNX-overexpressed TNX-KO MEFs in collagen gels, TNX overexpression suppressed TNX-KO-induced gel contraction, while the contraction of WT collagen gels was not affected by TNX-overexpression (Fig. 1E). These results 14

indicate that the contraction of collagen gels is promoted in the absence of TNX.

3.2. The activities of MMP-2 and MMP-9 in conditioned media from collagen gel culture are elevated in the absence of TNX

It has been reported that the activities of MMP-2 and -9 are increased in melanoma-inoculated foot pads of TNX-KO mice [20]. In addition, MMP-9 stimulates the contraction of collagen gel containing lung fibroblasts [29]. These reports led us to speculate that the activities of MMP-2 and -9 are involved in the promotion of MEF-embedded collagen gel contraction in the absence of TNX. To evaluate this possibility, the activities of MMP-2 and -9 in the conditioned media from collagen gels containing E13.5 TNX-KO or WT MEFs after 5-day culture were examined by gelatin zymography. The activities of MMP-2 and -9 in TNX-KO MEFs in collagen gels were higher than those in WT gels (Fig. 2A-C). In addition, MMP activities in the media from collagen gels containing E17.5 MEFs and fibrin gels containing E13.5 or E17.5 MEFs were also increased in the absence of TNX (Supplementary Fig. 2). To examine whether the up-regulations of MMP activity in TNX-KO gels are dependent on regulation at the transcriptional level, we quantified Mmp2 and Mmp9 mRNA expression levels in E13.5 TNX-KO and WT MEFs in collagen gels. The loss of TNX increased the Mmp9 mRNA level (Fig. 2E) but decreased the Mmp2 mRNA level (Fig. 2D). These results indicate that the activities of MMP-2 and -9 are regulated in the absence of TNX not only at the active protein level, but also at the transcriptional level in collagen gels containing MEFs. Next, to confirm whether MMP activities were regulated by TNX, the TNX expression vector was transfected into MEFs, which were then subjected to the gel

15

contraction assay. After 3-day culture of TNX-overexpressed TNX-KO MEFs in collagen gels, MMP-2 and -9 activities, which were increased in the absence of TNX, were suppressed by TNX-overexpression (Fig. 2F, G).

3.3. Promotion of collagen gel contraction in the absence of TNX is suppressed by inhibition of MMP activities

To determine the involvement of MMP-2 and -9 in the promotion of collagen gel contraction in the absence of TNX, MMP activity was inhibited in collagen gel assays. First, E13.5 TNX-KO MEFs were cultured in collagen gels with the MMP inhibitors, batimastat and GM6001. We checked the inhibition of MMP-2 and 9 activities in the gels cultured with batimastat and GM6001. While MMP-2 activity was significantly reduced by batimastat and GM6001 treatment, MMP-9 activity was not inhibited by batimastat and was only slightly reduced by GM6001 (Fig. 3E, F), indicating that, after 3 days of culture, the activity of MMP-2 was significantly inhibited in the media of the collagen gels containing WT or TNX-KO MEFs by batimastat or GM6001. In addition, the inhibition of MMP activity significantly suppressed the contraction of both WT and TNX-KO collagen gels (Fig. 3A-D). Next, MMP-2, and -9 were knocked down with MMP-2-siRNA and -9-siRNA in floating collagen gels containing E13.5 TNX-KO and WT MEFs. The knock-down efficiencies of MMP-2 activity were 77.4% in WT and 59.7% in TNX-KO gels, while those of MMP-9 activity were 93.7% and 75.3%, respectively. MMP-2 and -9 knock-down significantly suppressed the contraction of TNX-KO gels (Fig. 3G). Furthermore, MMP-2 and -9 double knock-down suppressed

16

gel contraction even more than either of the single gene knock-downs. These results indicate that an increase in MMP activity contributes to the promotion of collagen gel contraction in the absence of TNX.

3.4. Increase in TGF-1 induced in the absence of TNX promotes collagen gel contraction and MMP activity

It has been reported that MMP-9 activates TGF-1, thereby promotes collagen gel contraction [29, 32]. Therefore, we speculated that TGF-1 is involved in the promotion of gel contraction in the absence of TNX. To evaluate this possibility, we measured the levels of total (active and latent) TGF-1 in media collected from the 3-day culture of collagen gels containing E13.5 WT or TNX-KO MEFs using an ELISA. As shown in Fig. 4A, the production of total TGF-1 by TNX-KO MEFs in collagen gels was significantly higher than that in WT MEFs. The mRNA expression level of Tgfb1 by TNX-KO MEFs in collagen gels was also elevated (Fig. 4B). Next, to determine whether TGF-1 affects collagen gel contraction induced by TNX-KO MEFs, SB525334, a selective inhibitor of the TGF- receptor I (ALK5), was added to the gels. The addition of SB525334 suppressed the contraction of both TNX-KO and WT gels, with this effect being stronger in the TNX-KO gels (Fig. 4C, D). Furthermore, to examine the effects of TGF-1 on Mmp9 mRNA expression, SB525334 was added to collagen gel culture. The inhibition of TGF-1 suppressed the up-regulation of the Mmp9 mRNA level in the absence of TNX (Fig. 4E). These results indicate that the up-regulation of TGF-1 induced by the absence of TNX contributes to the promotion of collagen gel contraction and the up-regulation of Mmp9 mRNA expression level.

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3.5. Formation of filopodia-like protrusions in MEFs is promoted in the absence of TNX

The appearance of filopodia-like protrusions in MEFs was assessed after 3-day culture of collagen gels in order to clarify involvement of actin polymerization in the promotion of gel contraction in the absence of TNX. Consequently, both the ratio of MEFs with filopodia-like protrusions and protrusion length were increased in TNX-KO MEFs compared with those in WT MEFs (Fig. 5A-C), suggesting that actin polymerization in MEFs is promoted in the absence of TNX. To confirm that actin polymerization in MEFs is required for collagen gel contraction, TNX-KO MEFs in collagen gels were exposed to cytochalasin B, an actin polymerization inhibitor, and gel contraction was suppressed by cytochalasin B (Fig. 5D, E). In addition, to clarify whether MMPs and TGF-1 affect the formation of filopodia-like protrusions in TNX-KO MEFs, the effects of batimastat and SB525334 on the filopodia-like protrusions were observed in the gels. The ratio of TNX-KO MEFs with filopodia-like protrusions and the protrusion length were reduced by the inhibition of MMPs and TGF-1 (Fig. 5F-J). These results indicate that MMPs and TGF-1 contribute to the promotion of filopodia-like protrusions in the absence of TNX.

3.6. Proliferation of MEFs in collagen gels is promoted in the absence of TNX

The proliferation of fibroblasts is a critical process during wound healing [33, 34]. We therefore examined the effect of TNX absence on the proliferation rate of MEFs. First, we counted the number of cells in collagen gels cultured for 3 days. The number

18

of TNX-KO MEF was significantly higher than that of WT MEFs (Fig. 6A). Next, to determine whether the promotion of MEF proliferation in the absence of TNX occurs in the 3D collagen gels, we counted the number of 24 h-labeled BrdU-positive MEFs in the gels. The ratio of 24 h-labeled BrdU-positive MEFs to total cells was elevated in TNX-KO gels compared with that in WT gels (Fig. 6B, C). These results indicate that the proliferation of MEFs in collagen gels is promoted in the absence of TNX.

3.7. Migration of MEFs in collagen gels is promoted in the absence of TNX

The migration of fibroblasts to injured regions is an important process for effective wound repair [33, 34]. Therefore, to analyze the migration of MEFs in the collagen gels, a piece of collagen gel containing MEFs was embedded in a cell-free collagen gel, and the number of MEFs that had migrated beyond the boundary line between the embedded gel and the cell-free gel after 3-day culture was counted. The number of migrated MEFs was greater in TNX-KO gels than in WT gels (Fig. 7A, B). In addition, the distance over which the MEFs had migrated from the boundary line was greater in TNX-KO gels than in WT gels (Fig. 7B).

3.8. Expression of type I collagen 2 is increased in TNX-deficient MEFs cultured in collagen gels

In order to repair a wound, it is necessary for fibroblasts to produce collagen around the lesion [34]. Accordingly, we measured the amount of collagen in collagen gels containing TNX-KO or WT MEFs. First, we analyzed the mRNA expression of Col1a2

19

in MEFs cultured in the collagen gels. The results showed that the expression of Col1a2 mRNA was higher in TNX-KO MEFs than in WT MEFs (Fig. 8A). We also measured the amount of collagen protein in collagen gels containing TNX-KO or WT MEFs. The amount of collagen protein in TNX-KO gels was greater than that in WT gels (Fig. 8B). These results indicate that the production of collagen protein is promoted in the absence of TNX.

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4. Discussion

In this study, we sought to determine how wound healing-related properties, such as collagen gel contraction, the migration and proliferation of fibroblasts, and collagen production, are affected in the absence of TNX. To determine how collagen gel contraction, which mimics scar formation in the wound healing process, is affected in the absence of TNX, a MEF-embedded collagen gel contraction assay was performed as an in vitro wound-healing model. The up-regulation of MMP-2 and MMP-9 activities and TGF-1 expression in the absence of TNX resulted in the promotion of collagen gel contraction. Furthermore, in the collagen gel, the formation of filopodia-like protrusions and the proliferation and migration of MEFs were facilitated in the absence of TNX. In addition, the expression of Col1a2 mRNA in TNX-KO MEFs and the total amount of collagen protein in collagen gels containing TNX-KO MEFs were higher than those in WT MEFs. In the wound-healing process, fibroblasts migrate to wound regions, proliferate, produce ECM proteins such as collagen, and form a scar [33, 34]. The present study showed that several cellular properties related to the wound-healing process are promoted in the absence of TNX. Regarding MMP activities, we found that the promotion of gel contraction by TNX-KO MEFs is dependent on the up-regulation of MMP activities. We previously reported that MMP activities are increased in the absence of TNX. We showed that the activities of MMP-2 and -9 in TNX-KO mouse foot-pads were increased compared with those in WT mice [20], and we also reported that MMP-2 activity was induced in mouse fibroblasts in the absence of TNX [21]. These previous findings are consistent with our 21

present results. MMP-2 and -9 activities are known to induce collagen gel contraction. For example, the contraction of collagen gels containing lung fibroblasts derived from MMP-9-KO mice is suppressed compared with that in gels derived from WT mice [29]. It has also been reported that the inhibition of MMP-2 activity in human periodontal ligament cells by MMP inhibitors reduces the contraction of collagen gels [35]. We revealed that the activities of MMP-2 and -9 in collagen gels are increased in the absence of TNX and that the up-regulation of MMP-2 and -9 activities contributes to the promotion of gel contraction. However, as shown in Fig. 2D, the expression of Mmp2 mRNA was decreased in TNX-KO MEFs compared with that in WT MEFs. This result is not consistent with the finding that MMP-2 activity is elevated in TNX-KO MEFs compared with that in WT MEFs (Fig. 2B). At present, the reason for this discrepancy is not clear. However, it is possible that despite the lower expression of Mmp2 at the transcriptional level, the stability of the MMP-2 protein is higher in TNX-KO MEFs than WT MEFs due to a lower degradation rate in the floating collagen gels containing TNX-KO MEFs than in those containing WT-MEFs. In addition to MMPs, TGF-1 is also known to contribute to the contraction of collagen gels. In the culture media of MMP-9-KO mouse-derived fibroblasts, TGF-1 levels are decreased and thus collagen gel contraction is suppressed [29], indicating that TGF-1 is located downstream of MMP-9. In contrast, the results of the present study showed that the inhibition of TGF-1 activity suppressed the up-regulation of Mmp9 mRNA in the absence of TNX. Another study showed that TGF-1 induces MMP-9 expression in rat vascular smooth muscle cells [36], indicating that TGF-1 is located upstream of MMP-9. These findings suggest that TGF-1 regulates MMP-9 activity in a positive feedback manner. The mechanisms by which the loss of TNX up-regulates the

22

quantity of total (latent and active) TGF-1 at the transcriptional and translational levels remain to be understood. Alcaraz et al. showed that the FBG domain of TNX interacts with the small latent TGF- complex in vitro and in vivo and converts the latent form of TGF- into an active molecule in epithelial cells [37]. There are various known ways to activate the latent form of TGF- in a TNX-independent manner, such as through the involvement of thrombospondin-1 and plasmin [38]. However, there are no reports that the amount of TGF-1 is promoted in the absence of TNX. However, we speculate that the activities of MMPs are increased in the absence of TNX as shown in previous studies [20, 21] and in the present study; therefore, the increased activities of MMPs may augment the expression of TGF-1 [29]. The mechanisms by which MMPs and TGF-1 induce collagen gel contraction have not been fully elucidated. A previous study showed that during the contraction of collagen gel, fibroblasts in collagen gels exhibit protruded processes, migrate, and accumulate collagen fibrils in the vicinity of fibroblasts, and collagen fibrils are condensed in their vicinity [39, 40]. Consequently, fibril condensation on the cells causes overall gel contraction [40]. Another study showed that the network of collagen fibrils is perturbed by heparin, which thereby suppresses the contraction of collagen gels [41]. Therefore, filopodia-like protrusions of TNX-KO MEFs may help to accumulate collagen fibrils to their vicinity, thereby promoting gel contraction. The filopodia-like protrusions are also involved in cell migration. The increase in the formation of filopodia-like protrusions in the absence of TNX may also cause the up-regulation of migration activity in TNX-KO MEFs. In contrast, filopodia-like protrusion formation in the absence of TNX was suppressed by inhibitors of MMPs and TGF-1 in collagen gels, indicating that the up-regulation of MMPs and TGF-1 contributes to the increase

23

in filopodia-like protrusions in TNX-KO MEFs. Our results coincide with those of a previous study, in which filopodia formation in some tumor cells was induced by TGF-1 [42]. It was reported that the contraction ability of collagen gels is dependent on the number of cells embedded in the gels [43]. The results of the present study showed that the proliferation of MEFs in collagen gels was increased in the absence of TNX. However, since the proliferation rate of fibroblasts is suppressed in floating collagen gels [44], the increase in proliferation in the absence of TNX may have little impact on the contraction of floating collagen gels. We examined the contraction of fibrin gels induced by MEFs as an alternative in vitro wound healing model. Collagen gels are known to be weak and secrete limited additional collagen to the ECM. In contrast, it has been reported that fibrin gels are also initially weak but that rapid degradation of fibrin gels by embedded cells occurs, and the cells produce an abundance of ECM proteins including type I collagen, leading to a strong extracellular environment [45, 46]. In the present study, we also demonstrated the promotion of fibrin gel contraction in the absence of TNX via accelerated activation of MMP-2 and MMP-9. These results indicate that the promotion of gel contraction in the absence of TNX can also be induced in extracellular environments other than collagen gels. Wound healing impairments, such as the delayed healing of skin wounds, can be explained as the consequence of mutations affecting different genes, not only those encoding collagens and collagen-modified enzymes but also those encoding organizers of the ECM [14]. However, even though collagen fibrils from TNX-deficient patients are less densely packed and not aligned to neighboring fibrils [47], these patients exhibit

24

no macroscopic impairment of the wound healing process [11, 15]. Our study suggests that the nearly normal wound healing observed in TNX-deficient EDS patients may be caused by increased activation of MMPs and up-regulation of TGF-1, followed by up-regulation of collagen synthesis, which leads to accelerated matrix contraction and tissue remodeling in scar tissues locally.

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5. Conclusions

Our results indicate that increased activation of MMPs and up-regulation of TGF-1 in MEFs derived from TNX-KO mice, followed by the promotion of cell proliferation and migration, might explain the almost normal wound healing detected in the skin of TNX-KO mice. In the future, it will be necessary to investigate whether the increased activation of MMPs and up-regulation of TGF-1 and the accumulation of collagens actually occur during the wound healing process in TNX-KO mice in vivo. Finally, the activation of MMP and up-regulation of TGF-1 pathways may represent a novel clinical intervention to accelerate the healing of wounds in patients with classical EDS.

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Acknowledgements

The authors thank Dr. Kohei Kawakami (Shimane University) for providing the mutant mice and Dr. Hiromichi Sakai (Shimane University), Mr. Naoki Fukunaga (Shimane University) and Ms. Kazumi Satoh (Shimane University) for the analyses of data. The authors gratefully acknowledge the work of past and present members of the Matsumoto laboratory, the Miyamoto laboratory, and the Leading Promotion Center. This work was funded by Program for Leading Graduate Schools, Ochanomizu University and by the Japan Society for the Promotion of Science (JSPS) KAKENHI Grant Number 26462296 from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

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Figure Legends

Fig. 1. Effect of TNX absence on collagen gel contraction by MEFs. (A) Expression of Tnxb mRNAs in 2D-cultured E13.5 WT (TNX+/+) and TNX-KO (TNX-/-) MEFs. The amplified band of Tnxb mRNA by RT-PCR was detected in 2% agarose gel. Actb mRNA was used as an internal control. (B, C) Effect of TNX deficiency on collagen gel contraction. Collagen gel contractions by E13.5 TNX+/+ and TNX-/- MEFs were observed after 5-day culture. (D, E) Effect of TNX overexpression in TNX+/+ and TNX-/- MEFs on collagen gel contraction. The level of Tnxb mRNA expression (D) and gel contractions (E) by E13.5 MEFs transfected with a DNA plasmid encoding mouse TNX (pSec-FTNX-2) and a control plasmid (pSec-Tag2B). (C, E) The area of gels was analyzed using ImageJ. The areas of the contracted TNX+/+ gels without transfection (Control) were regarded as 1, and the areas of contracted TNX-/- and transfected gels were normalized by the areas of TNX+/+ gels (Control). * p < 0.05, ** p < 0.01, *** p < 0.001. Data are the means ± SEM of five independent experiments for TNX overexpression, and other data are means ± SEM of three independent experiments.

Fig. 2. Effects of TNX absence on MMP-2 and MMP-9 activities in collagen gel culture. (A-C) Gelatin zymography for analysis of MMP-2 and MMP-9 activities in conditioned media from WT (TNX+/+) and TNX-KO (TNX-/-) collagen gel cultures. Five-day cultured media from collagen gel containing E13.5 TNX+/+ or TNX-/- MEFs were subjected to gelatin zymography (A). Most of gelatinolytic bands other than those for MMP-2 and -9 indicated were MMPs derived from FBS in the media. MMP-2 (B) and MMP-9 (C) activities in the media were quantified according to the density of 35

gelatinolytic bands. The value of MMP activities in the media of TNX+/+ gels was regarded as 1, and the values of MMP activities in the media of TNX-/- gels were normalized by the TNX+/+ value. (D, E) Expression levels of Mmp2 and Mmp9 mRNA in MEFs. Total RNAs were extracted from 3-day cultured collagen gels containing E13.5 TNX+/+ or -/- MEFs, and mRNA levels were examined by real-time RT-PCR. The mRNA level by TNX+/+ MEF was regarded as 1, and the value of TNX-/- MEF was normalized by the value of TNX+/+ MEF. (F, G) Analysis of MMP-2 and MMP-9 activities in conditioned media from TNX over-expressed TNX+/+ and TNX-/- collagen gel cultures using gelatin zymography. The value of MMP activities in the media of pSec-Tag2B vector-transfected TNX+/+ gels were regarded as 1, and the values of MMP activities in the media of other gels were normalized by the control TNX+/+ value. * p < 0.05, ** p < 0.01. Data of MMPs activities are means ± SEM of three independent experiments. Other data are means ± SEM of seven independent experiments.

Fig. 3. Collagen gel contraction containing TNX-WT or TNX-KO MEFs with inhibitors of MMPs. (A-F) Effects of inhibitors of MMPs on collagen gel contraction containing WT (TNX+/+) or TNX-KO (TNX-/-) MEFs. Collagen gels containing E13.5 TNX+/+ or TNX-/- MEFs were cultured with MMP inhibitors, batimastat (A, B) and GM6001 (C, D). Dimethyl sulfoxide (DMSO) was added as a vehicle. After 3-day culture, the contracted gels were observed (A, C), the areas of contracted gels were quantified (B, D), and the MMP-2 and -9 activities were measured using gelatin zymography (E, F). The area of contracted gel by vehicle-treated TNX+/+ MEF was regarded as 1, and areas of other gels were normalized by the area with vehicle TNX+/+ MEF. (G) Effects

36

of MMP-2 and -9 knockdowns on collagen gel contraction. The contractions of collagen gels by E13.5 TNX+/+ and -/- MEFs were suppressed by siRNAs for MMP-2 (siMMP-2) and MMP-9 (siMMP-9) after 3-day culture. siMMP-2, 9 indicates double knock-down of the MMP-2 and -9 expressions. For negative control experiments, control siRNA (siControl) was used. The area of contracted gel by control siRNA-treated TNX+/+ and -/- MEF was regarded as 1, and areas of contracted gels were normalized by the area with control siRNA-treated gels. * p < 0.05, ** p < 0.01. Data are means ± SEM of three independent experiments.

Fig. 4. Effects of TGF-1 on collagen gels containing TNX-WT or TNX-KO MEFs. (A) The level of total (active and latent) TGF-1 protein in conditioned media from contracted collagen gel. The amount of total TGF-1 in 3-day cultured conditioned media from gels containing E13.5 WT (TNX+/+) or TNX-KO (TNX-/-) MEFs was measured by ELISA. (B) Expression levels of Tgfb1 mRNA in MEFs. Total RNAs were extracted from 3-day cultured collagen gels containing E13.5 TNX+/+ or -/- MEFs, and Tgfb1 mRNA levels in the gels were examined by real-time RT-PCR. The Tgfb1 mRNA level by TNX+/+ MEF was regarded as 1, and the value of TNX-/- MEF was normalized by the value of TNX+/+ MEF. (C, D) Effect of TGF-1 inhibition on collagen gel contraction by MEFs. Collagen gels containing E13.5 TNX +/+ or -/MEFs were cultured with a TGF- receptor I (ALK5) inhibitor, SB525334, for 3 days. Methanol was added as a vehicle. Areas of the contracted collagen gels were quantified with ImageJ. The areas of contracted gels by vehicle-treated TNX+/+ and TNX -/- MEF were regarded as 1, and areas of other contracted gels were normalized by the area of vehicle-treated TNX+/+ and TNX-/- MEF, respectively. (E) Effects of TGF-1

37

inhibition on Mmp9 mRNA expression level in collagen gels containing MEFs. Gels containing E13.5 TNX+/+ or -/- MEFs were cultured in collagen gels with SB525334 for 3 days. mRNA expression levels were detected using real-time RT-PCR. * p < 0.05, ** p < 0.01, *** p < 0.001. ELISA data are from two independent experiments, and triplicate measurements were performed in each experiment. Data for collagen gel experiments are means ± SEM of triplicate experiments. Real-time RT-PCR data are means ± SEM of six experiments.

Fig. 5. Effect of TNX absence on the formation of filopodia-like protrusions of MEFs cultured in contracted collagen gels. (A) Immunostaining of collagen gels containing E13.5 WT (TNX+/+) or TNX-KO (TNX-/-) MEFs. After 3-day culture, the gels were stained with F-actin (red) and DAPI (blue). (B) Analysis of filopodia-like protrusion formation. The number of cells exhibiting filopodia-like protrusions longer than 2 m to total cells was analyzed. (C) Quantification of the lengths of filopodia-like protrusions. The lengths were measured with ImageJ. (D, E) Effects of cytochalasin B on collagen gel contraction by MEFs. Collagen gels containing E13.5 TNX+/+ or -/- MEFs were cultured with an actin polymerization inhibitor, cytochalasin B (CytoB), for 3 days. DMSO was added as a vehicle. Areas of the contracted gels were determined by ImageJ. The area of contracted gel by vehicle-treated TNX+/+ MEF was regarded as 1, and areas of contracted gels were normalized by the area of vehicle-treated TNX+/+ MEF. (F) Immunostaining of collagen gels containing E13.5 TNX-/- MEFs cultured with batimastat or SB525334 after 3-day culture. The sections of gels were stained for F-actin (red) and DAPI (blue). (G, H) Effect of inhibition of the activities of MMPs and TGF-1 on filopodia-like protrusion formation of TNX-/- MEFs. The ratio of cells with

38

protrusions to total cells was determined after 3-day culture by ImageJ. (I, J) Effect of inhibition of the activities of MMPs and TGF-1 on the length of filopodia-like protrusions of TNX-/- MEFs. The length of filopodia-like protrusions was measured after 3-day culture by ImageJ. Scale bar: 30 m. * p < 0.05, ** p < 0.01, *** p < 0.001. Ratios of cells exhibiting filopodia-like protrusions are from two independent experiments with 20 fields observed in each experiment. Length of filopodia-like protrusions is from two independent experiments with 25 protrusions measured in each experiment. Gel area data are from four independent experiments.

Fig. 6. Effects of TNX absence on proliferation of MEFs in collagen gels. (A) The numbers of E13.5 WT (TNX+/+) and TNX-KO (TNX-/-) MEFs in collagen gels determined after 3-day culture. (B, C) Immunostaining of collagen gels containing E13.5 TNX +/+ or -/- MEFs after 24-h culture with BrdU. The MEFs were stained with anti-BrdU (red) and DAPI (blue). (C) The ratio of 24 h-labeled BrdU-positive MEFs to DAPI-positive MEFs in collagen gels was determined. For the analysis of the number of MEFs, three independent experiments were performed, and two independent experiments were performed with 20 fields observed in each experiment in collagen gels. Scale bar: 50 m. * p < 0.05.

Fig. 7. Effects of TNX absence on migration of MEFs in collagen gels. (A) Phase-contrast images of migrating E13.5 WT (TNX+/+) and TNX-KO (TNX-/-) MEFs after 1-, 2-, and 3-day-culture in collagen gels. Black lines indicate the boundary lines between the embedded collagen gels with MEFs and cell-free collagen gels. The number of cells of TNX-KO MEFs that had migrated beyond the boundary line was

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greater than that in WT gels. (B) Effect of TNX absence on migration of MEFs. The number of cells that had migrated beyond the boundary line was counted after 3-day culture. Five rectangular regions adjacent to the boundary line (length of 1749 m in the vertical direction against the boundary line and width of 1 mm along the boundary line) were arbitrarily chosen from the surrounding area of three embedded TNX+/+ and -/gels. Then the cells that had migrated beyond the boundary line were classified at 250-m intervals in the distance range of 0 - 1749 m, and the number of cells in each interval was counted. Scale bar: 500 m. * p < 0.05, ** p < 0.01. Data are means of the numbers of cells that had migrated from an embedded gel ± SEM in at least three independent experiments. In each experiment, 5 fields of view in collagen gels were observed.

Fig. 8. Effects of TNX absence on expression of collagen type I in collagen gels. (A) Quantification of mRNA levels of Col1a2 in E13.5 WT (TNX+/+) and TNX-KO (TNX-/-) MEFs cultured in collagen gels after 3-day culture. (B) Analyses of collagen protein levels in collagen gels containing E13.5 TNX+/+ or -/- MEFs cultured after 3-day culture. The amount of collagen protein was determined with a total collagen assay kit. * p < 0.05. Data are from two independent experiments, and duplicate measurements were performed in each experiment.

Supplementary Fig. 1. Effects of TNX deficiency on collagen and fibrin gel contractions by MEFs. (A) Expression of Tnxb mRNAs in 2D-cultured MEFs derived from E17.5 WT (TNX+/+) and TNX-KO (TNX-/-) mice. Actb mRNA was used as an internal control. (B, C)

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Contraction of collagen gels by E17.5 TNX+/+ or -/- MEFs. The contracted gels were observed after 5-day culture (B). The areas were measured with ImageJ (C). The area of contracted WT (TNX+/+) gel was regarded as 1, and the areas of contracted KO (TNX-/-) gel were normalized by the WT area. (D-F) Contraction of fibrin gels by E13.5 or 17.5 TNX+/+ or -/- MEFs. The contracted gels were observed (D) and the areas were measured (E, F) at each day after 0-6 day culture (D). The ratio of TNX-/gel area to TNX+/+ gel area was identified each day. Data for collagen gel contraction by E17.5 TNX+/+ or -/- MEFs represents the means ± SEM of triplicate independent experiments. Data of fibrin contraction by E13.5 or 17.5 TNX+/+ or -/- MEFs represents the means ± SEM of four independent experiments. Other data represents the means ± SEM of two independent experiments. ** p < 0.01.

Supplementary Fig. 2. Effects of TNX deficiency on MMP-2 and MMP-9 activities in collagen and fibrin gel cultures. (A-C) Gelatin zymography for analyses of MMP-2 and MMP-9 activities in the conditioned media of collagen gel cultures containing E17.5 WT (TNX+/+) or TNX-KO (TNX-/-) MEFs after 5-day culture. MMP-2 (B) and MMP-9 (C) activities were quantified according to the density of gelatinolytic bands. (D-H) Gelatin zymography for analysis of MMP-2 and MMP-9 activities in conditioned media of fibrin gel cultures containing E13.5 or 17.5 TNX+/+ or -/- MEFs after 3-day or 4-day culture. The value of WT gel was regarded as 1, and the value of TNX-KO gel was normalized by the WT value. Data of fibrin and collagen contraction by E13.5 or E17.5 TNX+/+ or -/- MEFs represents the means ± SEM of triplicate experiments. Other data represents the means ± SEM of two independent experiments. * p < 0.05.

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Table.1 Primer sequences used for real-time RT-PCR Gene Tgf1 Col1a2 Mmp9 Mmp2 Gapdh

Primer Sequence Forward

5'-CCCTATATTTGGAGCCTGGA-3'

Reverse

5'-CTTGCGACCCACGTAGTAGA-3'

Forward

5'-CCGTGCTTCTCAGAACATCA-3'

Reverse

5'-CTTGCCCCATTCATTTGTCT-3'

Forward

5'-GCGGACATTGTCATCCAGTTTG-3'

Reverse

5'-CGTCGTCGAAATGGGCATC-3'

Forward

5'-ACCCAGATGTGGCCAACTAC-3'

Reverse

5'-TACTTTTAAGGCCCGAGCAA-3'

Forward

5'-CGTGTTCTACCCCCAATGT-3'

Reverse

5'-TGTCATCATACTTGGCAGGTTTCT-3'

Highlight  

Contraction of collagen gels containing MEFs is promoted in the absence of TNX. Activities of MMPs in collagen gels with MEFs are increased in the absence of TNX.

  

TGF-1 level in collagen gels including MEFs is increased in the absence of TNX. Actin polymerization of MEFs in collagen gels is activated in the absence of TNX. Cell migration of MEFs in collagen gels is promoted in the absence of TNX.

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