The FASEB Journal article fj.13-248476. Published online May 27, 2014.

The FASEB Journal • Research Communication

A potential wound-healing-promoting peptide from salamander skin Lixian Mu,*,†,1 Jing Tang,*,†,1 Han Liu,*,†,1 Chuanbin Shen,*,† Mingqiang Rong,* Zhiye Zhang,*,† and Ren Lai*,2 *Key Laboratory of Animal Models and Human Disease Mechanisms, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, Yunnan, China; and †Kunming College of Life Science, University of Chinese Academy of Sciences, Beijing, China Although it is well known that wound healing proceeds incredibly quickly in urodele amphibians, such as newts and salamanders, little is known about skin-wound healing, and no bioactive/effector substance that contributes to wound healing has been identified from these animals. As a step toward understanding salamander wound healing and skin regeneration, a potential wound-healing-promoting peptide (tylotoin; KCVRQNNKRVCK) was identified from salamander skin of Tylototriton verrucosus. It shows comparable wound-healing-promoting ability (EC50ⴝ11.14 ␮g/ml) with epidermal growth factor (EGF; NSDSECPLSHDGYCLHDGVCMYIEALDKYACNCVVGYIGERCQYRDLKWWELR) in a murine model of full-thickness dermal wound. Tylotoin directly enhances the motility and proliferation of keratinocytes, vascular endothelial cells, and fibroblasts, resulting in accelerated reepithelialization and granulation tissue formation in the wound site. Tylotoin also promotes the release of transforming growth factor ␤1 (TGF-␤1) and interleukin 6 (IL-6), which are essential in the wound healing response. Gene-encoded tylotoin secreted in salamander skin is possibly an effector molecule for skin wound healing. This study may facilitate understanding of the cellular and molecular events that underlie quick wound healing in salamanders.—Mu, L., Tang, J., Liu, H., Shen, C., Rong, M., Zhang, Z., Lai, R. A potential wound-healing-promoting peptide from salamander skin.

ABSTRACT

Abbreviations: ␣-SMA, ␣ smooth muscle actin; DMEM, Dulbecco’s modified Eagle medium; ECM, extracellular matrix; EGF, epidermal growth factor; ELISA, enzyme-linked immunosorbent assay; ERK, extracellular regulated protein kinase; FBS, fetal bovine serum; H&E, hematoxylin and eosin; HSF, human skin fibroblast; HUVEC, human umbilical vein endothelial cell; IL-1, interleukin1; IL-6, interleukin 6; JNK, c-Jun NH2-terminal kinase; LSGS, low serum growth supplement; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; MAPK, mitogen-activated protein kinase; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide; PBS, phosphate-buffered saline; RP-HPLC, reversed-phase high-performance liquid chromatography; TGF-␤1, transforming growth factor ␤1; TNF-␣, tumor necrosis factor ␣ 0892-6638/14/0028-0001 © FASEB

Key Words: TGF-␤ 䡠 urodele amphibians 䡠 full-thickness incision Adult skin consists of two tissue layers: a keratinized stratified epidermis and an underlying thick layer of collagen-rich dermal connective tissue providing support and nourishment (1–2). The skin serves as a protective barrier against the outside world, and any break in it must be rapidly and efficiently mended. Wound healing is essential for organisms to survive. Cutaneous wound healing is divided into 3 continuous and overlapping processes: inflammation, proliferation, and remodeling phase (1, 3–5). In the inflammatory phase, inflammatory cells, composed mainly of neutrophils and macrophages, infiltrate to the wound site and trigger the process of wound healing (2). In the proliferative phase, the major events are reepithelialization and granulation tissue formation, both of which require cell proliferation and migration of keratinocytes, fibroblasts, and endothelial cells (3). In the contraction and remodeling phase, myofibroblasts differentiated from fibroblasts play a key role in wound contraction and control synthesis and degradation of ECM proteins. This complex process is executed and regulated by an equally complex signaling network involving numerous growth factors, cytokines, and chemokines, such as transforming growth factor ␤ (TGF-␤), interleukin 1 (IL-1), and interleukin 6 (IL-6) (6). Salamanders are unique among adult vertebrates in their ability to regenerate structurally complete and fully functional limbs (7). Wounding is believed to initiate limb regeneration. One of the earliest steps for wound healing is reepithelialization of the wounded surface. Wound healing proceeds incredibly quickly in salamanders. Compared with the process of reepithelialization in mammalian wounds, which normally takes 2–3 d, the same process in salamanders takes ⬍10 h. Little is known about skin wound healing in amphibi1

These authors contributed equally to this work. Correspondence: Key Laboratory of Animal Models and Human Disease Mechanisms, 32 Jiaochangdonglu, Kunming 650223, Yunnan, China. E-mail: [email protected] doi: 10.1096/fj.13-248476 This article includes supplemental data. Please visit http:// www.fasebj.org to obtain this information. 2

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ans. Some genes, including prx1, Tbx5, Fgf8, Fgf10, and Msx1, have been found to take part in amphibian skin wound healing and limb regeneration (8), but no effector substance that exerts a skin wound-healing function has been identified from amphibians. The current work aims to identify bioactive substance with wound-healing-promoting activity from salamander skin.

MATERIALS AND METHODS Tylototriton verrucosus sample Adult T. verrucosus (either sex, 20⫾5 g) were collected from the Yunnan province of China. Animals were anesthetized using 2.5% vaporized inhaled isofluorane, and the dorsal skin was cut off after cleansing with distilled water. The skin was homogenized by tissue homogenizer with 0.1 M phosphatebuffered saline (PBS; pH 6.0) containing 1% (v/v) protease inhibitor cocktail (P8340-5; Sigma, St. Louis, MO, USA). The skin homogenate solutions were quickly centrifuged (10,000 g for 10 min), and the supernatants were lyophilized. All experiments were approved by Kunming Institute of Zoology, Chinese Academy of Sciences. Peptide purification An aliquot (1 g) of lyophilized homogenate supernatant of skin was dissolved in 10 ml PBS and centrifuged at 5000 g for 10 min. The supernatant was applied to a Sephadex G-50 (Superfine, 2.6 cm diameter, 100 cm length; Amersham Biosciences, Castle Hill, NSW, Australia) gel filtration column equilibrated with 0.1 M PBS for preliminary separation. Elution was performed with the same buffer, collecting fractions of 3.0 ml. The eluted fractions were monitored at 280 nm and subjected to cell proliferation assays. The fraction containing cell proliferation activity was further purified by a C18 reversed-phase high-performance liquid chromatography (RP-HPLC) column (5 ␮m particle size, 110 Å pore size, 250 mm length, 4.6 mm diameter; Gemini C18, Phenomenex, Torrance, CA, USA). The elution was performed using a linear gradient of 0 – 80% acetonitrile containing 0.1% (v/v) trifluoroacetic acid in 0.1% (v/v) trifluoroacetic acid/water over 60 min as illustrated in Supplemental Fig. S1A. UVabsorbing peaks were collected, lyophilized, and assayed for cell proliferation activity.

according to the manufacturer’s instructions. The Smart cDNA Library Construction Kit (Clontech, Mountain View, CA, USA) was used to synthesize cDNA. The synthesized cDNA was used as template for PCR to screen the cDNAs encoding the peptide (tylotoin). Two pairs of oligonucleotide primers [S1: 5=-AC(A/C/G/T)C(G/T)(C/T)TT(A/G)TT(A/G)TT(C/T)TG(A/C/G/T)C-3=, according to the sequence determined by Edman degradation, in the antisense direction, and primer II A: 5=-AAGCAGTGGTATCAACGCAGAGT-3; S2: 5=-ATGGAGCTATGCCTCATACTCAC-3= and primer II A] were used in PCR reactions. The PCR conditions were 2 min at 95°C and 30 cycles of 10 s at 92°C, 30 s at 50°C, and 40 s at 72°C, followed by 10 min extension at 72°C. The PCR products were cloned into pGEM-T Easy vector (Promega, Madison, WI, USA). DNA sequencing was performed on an ABI Prism 377 DNA sequencer (Applied Biosystems, Foster City, CA, USA). Synthetic peptide Tylotoin (KCVRQNNKRVCK) was synthesized by GL Biochem Ltd. (Shanghai, China) and analyzed by HPLC and mass spectrometry to confirm its purity ⬎98%. Cell proliferation assay The proliferation of immortalized human HaCaT keratinocyte cells, human skin fibroblasts (HSFs), and human umbilical vein endothelial cells (HUVECs) was measured using a colorimetric assay. Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS) and penicillin (100 U/ml)-streptomycin (100 mg/ml) at 37°C in a humidified 5% CO2 atmosphere. HaCaT cells, HSFs, and HUVECs (2⫻104 cells/well, 180 ␮l) were plated into 96-well plates. After adhering to the plate, cells were incubated with vehicle (DMEM) or tylotoin at different concentrations (2, 5, 10, 20 ␮g/ml) for 24 h. Then 20 ␮l of 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) solution (R&D Systems Inc. Minneapolis, MN, USA) was added to each well for a further 4 h incubation at 37°C. After the cells were washed 3 times with PBS (pH 7.4), the insoluble formazan product was dissolved by incubation with 150 ␮l DMSO. The absorbance of each well was measured on an enzyme-linked immunosorbent assay (ELISA) microplate reader at 570 nm. The optical density reflects the level of cell metabolic activity. Each experiment was performed in quintuplicate. Regulation of cytokine production.

Primary structure analysis Purified peptides were subjected to amino acid sequencing by automated Edman degradation analysis on a pulsed liquidphase Shimadzu protein sequencer (PPSQ-31A; Shimadzu, Kyoto, Japan) according to the manufacturer’s instructions. Purified peptide (0.5 ␮l) in 0.1% (v/v) trifluoroacetic acid/ water was spotted onto a matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) plate with 0.5 ␮l ␣-cyano-4-hydroxycinnamic acid matrix (10 mg/ml in 60% acetonitrile) and analyzed by an UltraFlex I mass spectrometer (Bruker Daltonics, Billerica, MA, USA) in positive ion mode. Construction and screening of a cDNA library Total RNA was extracted from the skin of T. verrucosus using the RNeasy Protect Mini Kit (Qiagen, Hilden, Germany) 2

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Raw 264.7 murine macrophage cells (1⫻106) were seeded and adhered to a 96-well culture plate. The cells were treated with tylotoin (2, 5, 10, and 20 ␮g/ml) or vehicle for 16 h, then supernatants were collected for EGF, IL-1, IL-6, TGF-␤1, and tumor necrosis factor ␣ (TNF-␣) analysis using ELISA kits (Dakawe, Beijing, China). ELISA was performed according to the manufacturer’s instructions. Endothelial cell tube formation assay HUVECs were cultured in M200 medium (Invitrogen) supplemented with 20% FBS and 1⫻ low serum growth supplement (LSGS; Invitrogen) at 37°C in a humidified 5% CO2 atmosphere. Matrigel matrix (50 ␮l) was added to each well of a 96-well plate and incubated at 37°C for 30 min to allow for gel formation. After detaching by trypsinization, HUVECs were washed and resuspended in serum-free medium con-

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taining 1⫻ LSGS. The cells (1⫻106) were seeded into the 96-well plate pretreated with Matrigel matrix. Cells were incubated with vehicle (M200), tylotoin, or stylotoin at 20 ␮g/ml concentration for 18 h. Tubes were photographed using a microscope (Olympus, Tokyo, Japan) and quantified using the OpenLab program (Improvision, Coventry, UK). Western blot analysis Raw 264.7 murine macrophage cells (1⫻106/well) were plated into a 6-well culture plate and transferred to serumfree DMEM for a 16 h incubation. After incubation with tylotoin (2, 5, 10, and 20 ␮g/ml) or vehicle, 1 h incubation for mitogen-activated protein kinase (MAPK) pathway and 18 h incubation for Smad pathway, the cells were collected by centrifugation (1000 g for 5 min) and washed twice with ice-cold PBS. The washed cell pellets were lysed in 250 ␮l RIPA lysis buffer (50 mM Tris-HCl, pH 7.4; 1% Nonidet P-40; 0.25% sodium deoxycholate; 150 mM NaCl; 1 mM EDTA; 1 mM phenylmethylsulfonyl fluoride; 1 ␮g/ml each of aprotinin, leupeptin, and pepstatin; 1 mM sodium orthovanadate; and 1 mM NaF) and incubated for 30 min on ice. The concentration of protein was determined by the Bradford protein assay. Next, 30 ␮g of cellular proteins was separated on a 12% SDS-PAGE gel and electroblotted onto a polyvinylidene difluoride membrane. Primary antibodies against ␤-actin (1:2000; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and extracellular regulated protein kinase 1/2 (Erk1/ 2), SAPK/c-Jun NH2-terminal kinase (JNK), p38 MAPK, Smad2, Smad3, and Smad7 (1:2000; Cell Signaling Technology, Beverly, MA, USA) were used in Western blot analysis. Wound healing scratched assay HaCaT cells (1⫻106) were seeded into a 6-well plate and grown to monolayer confluency. After 24 h of serum starvation (DMEM supplemented with 1% FBS), the cell monolayer was subjected to a mechanical scratch wound using a sterile pipette tip. After washing twice with PBS to remove floating cells, cells were then cultured for additional periods (from 0 to 48 h) in a serum-free basal medium in the continued presence of vehicle, tylotoin, or stylotoin (20 ␮g/ml). Images of the wounded cell monolayers were obtained using a microscope (Olympus) at 0, 24, and 48 h after scratch wounding. Cell migration activity was expressed as the percentage of the gap relative to the total area of the cell-free region immediately after scratch wounding, named the repair rate of scarification, using ImageJ software (U.S. National Institutes of Health, Bethesda, MD, USA). For each plate, 6 randomly selected images were acquired. All experiments were carried out in quintuplicate. Full-thickness wounds and quantification of healing Male Kunming mice (age 6 – 8 wk) were anesthetized using 1% pentobarbital sodium (0.1 ml/20 g body weight). Dorsal hairs were removed by an electric clipper, and the dorsal skin was cleansed with Betadine. Two full-thickness wounds were created in the skin on the back of each mouse using a 9-mm-diameter biopsy punch. After wounding, mice were caged individually until termination of the experiment. Mice were treated with vehicle, tylotoin (20 ␮l, 20 ␮g/ml) or EGF (20 ␮l, 100 ␮g/ml) applied directly to the wound site twice daily. Wound healing was macroscopically monitored by taking digital photographs at the indicated time points. The wound areas were calculated from the photographs using PhotoShop (Adobe Photoshop Element 2.0; Adobe Systems, San Jose, CA, USA; n⫽10/group). The experimental protoWOUND-HEALING-PROMOTING PEPTIDE

cols were approved by the Animal Care and Use Committee at Kunming Institute of Zoology, Chinese Academy of Sciences. Histology and immunohistochemistry The biopsy specimens involving the central part of the wounds (different days after wounding) were obtained from mice for light microscopy. Skin specimens were fixed in 10% formalin, dehydrated through a graded series of ethanol, cleared in xylene, and embedded in paraffin wax. Thick sections (5 ␮m) were prepared and stained with hematoxylin and eosin (H&E) for histological analysis. IPLab imaging software (BD Biosciences, Bedford, MA, USA) was used to measure changes in the wound. Width of the wound and distance of the neoepithelium were measured on H&Estained sections, and percentage reepithelialization was calculated as (distance covered by neoepithelium)/(distance between wound bed) ⫻ 100 (n⫽6/group). All slices were used to evaluate epidermal regeneration and granulation, by using a semiquantitative score system (9). In this system, a 3-point scale was used to evaluate dermal and epidermal regeneration (1, little regeneration; 2, moderate regeneration; 3, complete regeneration) and 4-point scales were used to evaluate granulation tissue formation (1, thin granulation layer; 2, moderate granulation layer; 3, thick granulation layer; 4, very thick granulation layer). For immunohistochemistry, 3 ␮m sections were reacted with anti-F4/80 primary antibody (1:200 dilution, ab111101; Abcam, Cambridge, MA, USA), anti-TGF-␤1 primary antibody (1:500, ab92486; Abcam), and anti-␣ SMA primary antibody (1:1000 dilution, ab32575; Abcam) after blocking endogenous peroxidase and nonspecific binding. Then, they were incubated with biotinylated goat anti-rabbit IgG (1:200 dilution, ab150077; Abcam) for 1 h at room temperature. As a control, sections were treated with the same dilution buffer without the primary antibodies. Infiltration of macrophages was evaluated by counting the cells immunostained with anti-F4/80 antibody (n⫽6/group). Skin tissue ELISA The biopsy specimens involving the central part of the wounds (d 0, 1, 2, 3, 4, 6, 8, and 10) were obtained from mice for tissue ELISA. Skin specimens were homogenized in 1 ml 0.1 M PBS/g tissue using a glass homogenizer. The homogenates were transferred to 1.5 ml Eppendorf tubes and centrifuged at 13,000 g for 30 min at 4°C, and the supernatant was stored at ⫺80°C until analyzed. TGF-␤1 protein levels were determined using ELISA kits (Dakawe). Statistical analysis Statistical differences were determined using the 1-way analysis of variance test or Student’s t test. Values of P ⬍ 0.05 were considered significant. Results are shown as means ⫾ sem.

RESULTS Purification of tylotoin As illustrated in Supplemental Fig. S1A, the skin homogenate supernatant of T. verrucosus was divided into 6 fractions after Sephadex G-50 gel filtration. The fraction containing activity to promote cell proliferation was pooled and subjected to a C18 RP-HPLC 3

column for further purification (Supplemental Fig. S1B). The purified peptide with cell proliferation activity was named tylotoin (indicated arrow in Supplemental Fig. S1B).

␮g/ml) for 18 h increased tube formation by 112%. Stylotoin resulted in not such good cell motility and proliferation effects as tylotoin, indicating that the sequence specificity was critical for cell motility and proliferation ability of tylotoin.

Structural characterization Effects on cytokine secretion The complete amino acid sequence of purified tylotoin was determined as KCVRQNNKRVCK by Edman degradation. Tylotoin is composed of 12 aa residues including 2 cysteines, which are able to form an intramolecular disulfide bridge. MALDI-TOF-MS gave an observed mass of 1473.80 Da, which is matched well with the theoretical molecular weight (1473.79) of tylotoin containing an intramolecular disulfide bridge. The presence of an intramolecular disulfide bridge in native tylotoin was further confirmed by synthesized peptide, which had the same observed mass and RP-HPLC elution manner with the native tylotoin. cDNA cloning The complete nucleotide sequence encoding tylotoin precursor (GenBank accession number KF031133) and the encoded amino acid sequence are shown in Supplemental Fig. S2. The sequence contains a coding region of 438 nt, and the encoded amino acid sequence corresponds to a polypeptide of 146 aa including mature tylotoin. BLAST search indicated that the precursor is a member of cathelicidin family containing a conserved cathelin domain and a variable C terminus (Supplemental Fig. S3). Tylotoin enhances proliferation and motility of keratinocytes, fibroblasts, and vascular endothelial cells Effects of tylotoin on proliferation of HaCaT cells, HSFs, and HUVECs are illustrated in Fig. 1A–C. Tylotoin enhanced the proliferation of HaCaT cells, HSFs, and HUVECs in a concentration-dependent manner. At concentrations of 10 and 20 ␮g/ml, the rates of proliferation were increased by 73 and 86% (HaCaT cells), 55 and 70% (HSFs), and 63 and 87% (HUVECs), respectively. The scrambled version of tylotoin, called stylotoin, did not show any activities on the proliferation of keratinocytes, fibroblasts, and vascular endothelial cells (data not shown). The wound-healing scratch assay in which monolayers of cultured HaCaT keratinocyte cells were mechanically wounded with a pipette tip indicated that tylotoin also promoted keratinocyte migration, as illustrated in Fig. 1D, E. The migration of HaCaT cells across the wound chasm was significantly enhanced in cells treated with tylotoin compared to those treated with vehicle. The repair rate of scarification of tylotoin was reached to 60 or 80% at 24 or 48 h, respectively. As illustrated in Fig. 1F, G, endothelial cell tube formation assay indicated that tylotoin significantly promoted tube formation of HUVECs. Tylotoin treatment (20 4

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Except for phagocytosis and antigen presentation, macrophages at the site of injury can produce many of the specific proteins, including chemoattractants that recruit and activate additional macrophages at the site of injury and growth factors that are relevant to wound healing, such as TGF-␤1, IL-1␤, IL-6, and TNF-␣ (10). Effects of tylotoin on cytokine secretion in murine macrophage cell line RAW264.7 were tested using ELISA. As illustrated in Fig. 2A, B, tylotoin significantly increased TGF-␤1 and IL-6 secretion in a dose-dependent manner in the culture supernatants of RAW264.7. Compared with the control 328 pg/ml, the concentration of TGF-␤1 in the cells treated with tylotoin at 2, 5, 10, and 20 ␮g/ml for 16 h were 439 pg/ml (33.5%), 613 pg/ml (86.4%), 685.5 pg/ml (108.4%), and 763.2 pg/ml (132.1%), respectively. Compared with the control of 43 pg/ml, tylotoin treatment for 16 h at 2, 5, 10, and 20 ␮g/ml induced 53, 61, 75, and 94 pg/ml IL-6 secretion, respectively. Tylotoin had no significant effect on IL-1␤, EGF, and TNF-␣ secretion (Supplemental Fig. S4). Effects of tylotoin on Smad2/Smad3 activation Smad family proteins are key regulators in TGF-␤ signaling pathways. Smad proteins, including Smad2, Smad3, and Smad7, are essential components of downstream TGF-␤ signaling, which either positively (via activation of Smad2/3) or negatively (through the negative feedback mechanism of Smad7) regulates biological activities of TGF-␤ (11). As illustrated in Fig. 3, tylotoin increased phosphorylation of Smad2 and Smad3, but there was no obvious effect on Smad7 expression. Especially, phosphorylation of Smad2 was markedly increased by tylotoin. After 18 h tylotoin treatment at concentrations of 2, 5, 10, and 20 ␮g/ml, Smad2 phosphorylation was up-regulated by 1.9, 4.6, 5.8, and 7.7 times, respectively. Effects of tylotoin on MAPKs As mentioned above, tylotoin induced TGF-␤1 secretion in vitro (Fig. 2A) and in vivo (Fig. 2D–F). To examine whether MAPK kinases are involved in the process of TGF-␤1 secretion induced by tylotoin, specific MAPK kinase inhibitor was cocultured with tylotoin in the macrophage cells, and the level of TGF-␤1 was tested. As illustrated in Fig. 2C, the level of TGF-␤1 was significantly reduced in the supernatants of the cells treated with ERK- and JNK-specific inhibitors, but there was no obvious change for p38-specific inhibitors. This indicated that ERK and JNK signaling pathways

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Figure 1. Tylotoin enhanced the motility and proliferation of keratinocytes, fibroblasts, and vascular endothelial cells. A–C) Cultured keratinocytes (A), fibroblasts (B), and vascular endothelial cells (C) were treated with vehicle (control) or tylotoin with indicated concentration, and relative cell numbers were estimated by O.D readings. Values are the means ⫾ sem (n⫽5). D, E) Effects of tylotoin or stylotoin on scratch wounding closure in cultured keratinocytes. D) Representative images. E) Quantification of repair rate of scarification. F, G) Effects of tylotoin or stylotoin on tube formation of HUVECs seeded on Matrigel-precoated wells. Tube formation, designated as the number of branch points was evaluated 18 h after cell plating. F) Representative images. G) Quantitative data. Scale bars ⫽ 200 ␮m. Values are means ⫾ sem (n⫽5 cultures). *P ⬍ 0.05, **P ⬍ 0.01 vs. control.

have been involved in TGF-␤1 release induced by tylotoin. Western immunoblot analysis was performed to further explore the effect of tylotoin on MAPK signaling pathway in RAW 264.7 macrophage cells. As illustrated in Fig. 4, tylotoin significantly increased ERK and JNK WOUND-HEALING-PROMOTING PEPTIDE

phosphorylation in a concentration-dependent manner, but showed little effect on p38 phosphorylation. After 1 h treatment with 2, 5, 10, and 20 ␮g/ml tylotoin, ERK phosphorylation was increased by 1.7, 3.1, 5.3, and 7.5 times, while the corresponding JNK up-regulation was 1.5, 7.7, 17, and 23 times, respectively. 5

Figure 2. Tylotoin induced TGF-␤1 and IL-6 secretion in murine macrophage cell line RAW264.7 and induced macrophage recruitment, TGF-␤1 secretion, and fibroblast-to-myofibroblast transition in skin wounds. A, B) Tylotoin significantly increased TGF-␤1 (A) and IL-6 (B) secretion in a dose-dependent manner in the culture supernatants of RAW264.7 cells. C) Inhibition of tylotoin-induced TGF-␤1 expression by MAPK inhibitors. Cells were untreated or treated with the specific ERK inhibitor PD98059 (20 ␮M), the specific JNK inhibitor SP600125 (20 ␮M), or the specific p38 inhibitor SB203580 (20 ␮M). Data are means ⫾ sem. **P ⬍ 0.01 vs. control; ##P ⬍ (continued on next page)

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Figure 3. Effects of tylotoin on TGF-␤/Smad signaling pathways. A) Western blot shows effects of tylotoin on Smad2/3 phosphorylation and Smad7 expression in a dose-dependent manner. B–D) Relative activation of Smad2 (B), Smad3 (C), and Smad7 (D) expression by tylotoin. Values for tylotoin treatment are significantly different from control values. *P ⬍ 0.05, **P ⬍ 0.01 (n⫽5).

Tylotoin accelerates the healing of full-thickness wounds in mice Considering that tylotoin has strong ability to promote proliferation, migration, and tube formation of HUVECs, we employed a mouse model to determine whether topical application of tylotoin would modify the healing of full-thickness dermal wounds. On postinjury d 2, wound area in tylotoin- and EGF-treated mice was ⬃10 and 15% smaller than that in control mice (Fig. 5A, B). Subsequently, there was a rapid acceleration of wound healing in tylotoin- and EGF-treated mice. On d 8, the wound was ⬃65 and 70% smaller than controls, respectively. On d 10, the wounds of tylotoin- and EGF-treated mice were almost completely closed, whereas the wounds of control mice were ⬃26% open (Fig. 5A, B). EC50 calculation revealed values of 11.14 ␮g/ml for tylotoin on d 10 (Fig. 5C). No adverse effects on body weight, general health, or behavior of the mice were observed for the topical tylotoin treatment. In a parallel experiment, we euthanized mice in tylotoin-treated and control groups at postinjury d 3, 6, and 10, and then performed a histological evaluation of skin tissue sections stained with H&E. Tylotoin-treated

mice exhibited enhanced restoration of dermal and epidermal and formation of granulation tissue in the wound (Fig. 5D–F). Tylotoin induced macrophage recruitment, TGF-␤1 secretion, and fibroblast-to-myofibroblast transition in skin wounds Through orchestrating the complex processes of cellular proliferation and functional tissue regeneration within wounds, macrophages play a critical role in the wound-healing process (12). Tylotoin has been demonstrated to significantly increase TGF-␤1 and IL-6 secretion in vitro (Fig. 2A, B). Immunohistochemical analysis was performed to delineate the effects of tylotoin on macrophages in the wound site. As illustrated in Fig. 2G, H, macrophages were obviously recruited in and around the wound site treated with tylotoin. Both on postoperative d 2 and 3, numbers of F4/80-positive stained macrophages were significantly higher (almost 3- to 4-fold) in tylotoin-treated skin wounds than in the controls. At the same time, macrophage recruitment showed a dynamic increase from d 2 to 3.

0.01 vs. tylotoin-induced group without MAPK inhibitors (n⫽3). D) Tylotoin significantly increased the expression of TGF-␤1 in the wound site at the indicated day after injury. E) Expression of TGF-␤1 in wounded skin treated with tylotoin or vehicle at d 4 after wounding. F) Quantification of TGF-␤1-positive area. G) Recruitment of macrophage cells in wound healing in mice treated with tylotoin or vehicle at d 2 or 3 after wounding. Arrowheads indicate macrophage cells. H) Number of macrophages per microscopic field. I) Expression of ␣-SMA in wounded skin treated with tylotoin or vehicle at d 6 or 8 after wounding. J) Quantification of ␣-SMA-positive area. Scale bars ⫽ 100 ␮m. All values represent means ⫾ sem (n⫽6/group). **P ⬍ 0.01 vs. control. WOUND-HEALING-PROMOTING PEPTIDE

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Figure 4. Effects of tylotoin on MAPK signaling pathways. A) Western blot shows effects of tylotoin on ERK, JNK, and p38 protein kinase phosphorylation. B–D) Relative activation analysis. Results were quantified by ImageJ. Densitometry of phosphorylated ERK (B), p38 (C), and JNK (D) were normalized to total ERK, p38, and JNK and graphed as means ⫾ sem (n⫽5). Values for tylotoin treatment are significantly different from control values. **P ⬍ 0.01.

The expression of TGF-␤1 in wound sites treated with tylotoin was significantly higher than that in the controls, as illustrated in Fig. 2D. After tylotoin treatment for 1, 2, 4, 6, 8, and 10 d, TGF-␤1 concentration at the wound skin was ⬃79, 141, 218, 182, 144, and 33 pg/mg, while that in the control was ⬃33, 46, 69, 78, 99, and 60 pg/mg, respectively. TGF-␤1 in vivo expression was also detected by immunohistochemical analysis, as illustrated in Fig. 2E, F. Compared with the vehicle-treated group, TGF-␤1-positive areas were much more extensive in the tylotoin-treated group (Fig. 2E). There were ⬃15 and 31 TGF-␤1positive areas in the vehicle- and tylotoin-treated groups on postoperative d 4, respectively (Fig. 2F). Tylotoin’s effect on TGF-␤1 expression level in vivo was identical to that in vitro (Fig. 2A). Fibroblast-to-myofibroblast transition plays important role in cutaneous wound healing (13). Wound tissues at postinjury d 6 or 8 were stained with an antibody against ␣ smooth muscle actin (␣-SMA), a differentiation marker of smooth muscle cells. Figure 2I, J shows strong expression of ␣-SMA in myofibroblasts of the granulation tissue below the wound surface at d 6 or d 8 in tylotoin-treated mice, but only very weak signals in vehicle-treated mice. There was ⬃15 and 3.5% ␣-SMA-positive area in wound tissue of mice treated with tylotoin and vehicle, respectively, on postoperative d 6, while the proportion of ␣-SMApositive area reached 28 and 13%, respectively, postoperative d 8. 8

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DISCUSSION Urodele amphibians (salamanders) are the only adult vertebrates that are able to regenerate lost or damaged body parts, including their limbs, perfectly. They have excellent wound-healing ability (7). Much of our current understanding of salamander wound healing is related to the pathways and signal events. No salamander bioactive/effector substance that directly promotes wound healing or initiates the pathways and signal events was reported. As a step toward understanding salamander wound healing and skin regeneration, a wound-healing-promoting peptide (tylotoin) was identified from salamander skin in this work. Tylotoin is a short peptide (KCVRQNNKRVCK) composed of 12 amino acid residues. The precursor of tylotoin was found to belong to the protein family of cathelicidin. Cathelicidins are a conserved family among vertebrates. After endogenous enzymatic processing, most of them release bioactive peptides with direct antimicrobial activity (14 –17). These bioactive peptides are located at the C terminus of cathelicidins. Similarly, mature tylotoin is located at the C terminus of the cathelicidin. Different from other bioactive peptides released from other cathelicidins, tylotoin did not show any antimicrobial activity (data not shown), but it showed strong wound-healing ability (Fig. 5). Wound healing is an evolutionarily conserved, complex, multicellular process. This process involves the coordinated efforts of several cell types, including

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MU ET AL.

Figure 5. Topical application of tylotoin accelerated the healing of full-thickness wounds in mice. A) Images of a representative mouse from each group taken on postinjury d 0, 2, 4, 6, 8, and 10. B) Wound area on different days postinjury. Values represent means ⫾ sem (n⫽10/group). *P ⬍ 0.05, **P ⬍ 0.01 vs. control. C) Wound area and dose response curves for tylotoin on postinjury d 10. D) Images of skin tissue sections stained with H&E at d 3, 6, and 10 postwounding. Yellow dotted line indicates the neoepithelium. Scale bars ⫽ 100 ␮m. E, F) Histological scores of epidermis and dermis regeneration (E) and granulation thickness (F) at d 3, 6, and 10 postwounding. Values represent means ⫾ sem (n⫽6/group). *P ⬍ 0.05, **P ⬍ 0.01 vs. control.

keratinocytes, fibroblasts, endothelial cells, macrophages, and platelets. The migration, infiltration, proliferation, and differentiation of these cells resulted in the formation of new tissue and ultimately wound closure (3, 6, 18). Tylotoin treatment markedly enWOUND-HEALING-PROMOTING PEPTIDE

hanced the migration and proliferation of keratinocytes, fibroblasts, and vascular endothelial cells in vitro (Fig. 1), suggesting that it has the ability to promote wound healing. In vivo experimental results further demonstrated tylotoin’s ability for wound healing. Al9

though wound healing in mice is a little different from that in humans, as it partly occurs via contraction, the repair process is also in part dependent on reepithelialization and new tissue formation. Thus, the murine model of full thickness dermal wounding is widely used for the study of wound healing. Topical application of tylotoin greatly accelerated full-thickness skin wound healing in a mouse model of full-thickness skin wounding (Fig. 5). Accelerated wound closure was observed in the mice treated with tylotoin. Some fibroblasts differentiate into myofibroblasts, and these contractile cells will help bridge the gap between the wound edges (13). As illustrated in Fig. 2I, J, tylotoin significantly promoted the expression of ␣-SMA, a differentiation marker of smooth muscle cells, in vivo, suggesting that tylotoin accelerated fibroblast-to-myofibroblast transition to accelerate wound closure. In addition, tylotoin was found to induce endothelial cell tube formation, as illustrated in Fig. 1F, G, suggesting that it has ability to promote angiogenesis, which plays a critical role in wound healing (19), because it provide nutrients and oxygen to the wound. Macrophages play a critical role in the wound-healing process, taking part in almost every stage. They can recruit other inflammatory and fibroblastic cells and influence cell proliferation and tissue remodeling (12). Immunohistochemical analysis using the monocyte/ macrophage-specific monoclonal antibodyF4/80 indicated that tylotoin markedly promoted recruitment of macrophages to excisional wounds in mice (Fig. 2G, H). In addition, macrophages can produce many cytokines associated with wound healing, such as TNF-␣, IL-1␤, IL-6, and TGF-␤1 (20). TGF-␤1 plays a fundamental role in wound-healing processes. It is a multifunctional cytokine that plays important roles in cell proliferation, differentiation, and formation of extracellular matrix (ECM). In wound healing, TGF-␤1 is important in inflammation, angiogenesis, reepithelialization, and connective tissue regeneration (21, 22). Furthermore, TGF-␤1 helps monocytes convert to macrophages, then macrophages initiate the development of granulation tissue and release a variety of proinflammatory cytokines and growth factors that contributing to wound healing. As illustrated in Fig. 2A, B, tylotoin significantly increased TGF-␤1 and IL-6 secretion in a dose-dependent manner in the culture supernatants of RAW264.7. It also significantly enhanced the expression of TGF-␤1 at the wound site (Fig. 2D–F). These results indicated that tylotoin not only induced macrophage recruitment but also stimulated macrophages to secrete TGF-␤1 and IL-6 in vitro and in vivo. To further demonstrate that tylotoin up-regulates TGF-␤1, effects of tylotoin on Smad2, Smad3, and Smad7, which are essential components of downstream TGF-␤ signaling, were investigated. As illustrated in Fig. 3, Smad2 and Smad3 were significantly activated by tylotoin. The MAPK signaling system has been implicated in regulating TGF-␤ production and release from skin cells and has been demonstrated to play an important role in wound healing (6, 23–25). Western blot analysis 10

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showed that tylotoin significantly increased the activation of ERK and JNK subgroups of the MAPK signaling pathway in a concentration-dependent manner (Fig. 4). Specific inhibitors for ERK and JNK blocked the up-regulation of TGF-␤1 induced by tylotoin (Fig. 2C), suggesting that these kinases are involved in the process of tylotoin-induced TGF-␤1 secretion. In summary, tylotoin, identified from salamander skin, is a bioactive/effector compound with potential wound-healing ability through promotion of migration and proliferation of keratinocytes and fibroblasts, macrophage recruitment, TGF-␤1 expression, and fibroblast-to-myofibroblast differentiation. It may facilitate understanding of salamander wound healing and skin regeneration. In addition, tylotoin contains only 12 amino acid residues and might be a potential biomaterial or template for development of novel woundhealing agent. The authors thank all staff of the Animal Center at Kunming Institute of Zoology for taking good care of the animals; and members of the Cell Banks at Kunming Institute of Zoology for providing cells and culture instruction. This work was supported by the Ministry of Science and Technology (2010CB529800, 2013CB911300), the National Natural Science Foundation (31025025, U1132601, U1302221, 31200590, 31260208), and Jiangsu Province (BK2012365, BE2012748). The authors declare no conflicts of interest.

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