Skip to main content
NCBI home page
Wiley Open Access Collection logoLink to Wiley Open Access Collection
. 2017 Nov 28;106(6):2403–2413. doi: 10.1002/jbm.b.34034

Shotgun label‐free proteomic analysis for identification of proteins in HaCaT human skin keratinocytes regulated by the administration of collagen from soft‐shelled turtle

Tetsushi Yamamoto 1, Saori Nakanishi 1, Kuniko Mitamura 1, Atsushi Taga 1,✉
PMCID: PMC6175320  PMID: 29193735

Abstract

Soft‐shelled turtles (Pelodiscus sinensis) are widely distributed in some Asian countries, and we previously reported that soft‐shelled turtle tissue could be a useful material for collagen. In the present study, we performed shotgun liquid chromatography (LC)/mass spectrometry (MS)‐based global proteomic analysis of collagen‐administered human keratinocytes to examine the functional effects of collagen from soft‐shelled turtle on human skin. Using a semiquantitative method based on spectral counting, we were able to successfully identify 187 proteins with expression levels that were changed more than twofold by the administration of collagen from soft‐shelled turtle. Based on Gene Ontology analysis, the functions of these proteins closely correlated with cell–cell adhesion. In addition, epithelial–mesenchymal transition was induced by the administration of collagen from soft‐shelled turtle through the down‐regulation of E‐cadherin expression. Moreover, collagen‐administered keratinocytes significantly facilitated wound healing compared with nontreated cells in an in vitro scratch wound healing assay. These findings suggest that collagen from soft‐shelled turtle provides significant benefits for skin wound healing and may be a useful material for pharmaceuticals and medical care products. © 2017 The Authors Journal of Biomedical Materials Research Part B: Applied Biomaterials Published by Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 2403–2413, 2018.

Keywords: collagen, soft‐shelled turtle, E‐cadherin, epithelial–mesenchymal transition, wound healing

INTRODUCTION

Collagen is a ubiquitous structural protein in both invertebrates and vertebrates, comprising >20 different types based on the function in each tissue.1, 2 These proteins are involved in the formation of fibrillar and microfibrillar networks of extracellular matrix and basement membranes to maintain the extracellular matrix environment.3, 4, 5, 6, 7 Recent reports have demonstrated that collagen is able to interact with several cell surface receptors and regulate cell proliferation or apoptosis.8, 9 In addition, collagen is used for skin substitutes and drug delivery.10, 11, 12, 13, 14, 15 Therefore, collagen is an important material for cosmetics, pharmaceuticals, and medical care products.

Most of the collagen presently in use is derived from bovine and porcine skin. However, allergic reactions and connective tissue disorders, such as arthritis and lupus, have been reported with the use of collagen from these animals.16 Moreover, these materials can potentially carry animal diseases, such as bovine spongiform encephalopathy and foot and mouth disease. Thus, these animals have been reconsidered as the main source for collagen products. In addition, many Muslims and Jews do not consume pig‐derived food products, and many Hindus do not consume cow‐derived products.17 Therefore, collagen of marine origin, such as fish, sponges, and mollusks, was recently considered as a useful alternative to mammalian sources because of its high availability.18, 19, 20, 21, 22, 23, 24, 25 In addition, we previously reported that soft‐shelled turtle (Pelodiscus sinensis) tissue could be a useful alternative for collagen.26 Recently, several reports demonstrated its usefulness,27, 28 making collagen from soft‐shelled turtle a useful material for cosmetics, pharmaceuticals, and medical care products.

However, collagen from soft‐shelled turtle may differ greatly from that of mammalian resources in regards to physicochemical properties, amino acid compositions, and physiological functions due to the difference in the habitat environment. Therefore, further research is needed before using collagen from soft‐shelled turtle as a source for collagen products. In the present study, we performed shotgun liquid chromatography (LC)/mass spectrometry (MS)‐based global proteomic analysis of collagen‐administered human keratinocytes to examine the functional effects of collagen from soft‐shelled turtle on human skin. We found that 187 proteins were differentially expressed in the collagen‐administered keratinocytes compared with nontreated cells, and these proteins may be involved in wound healing in human skin.

MATERIALS AND METHODS

Chemicals

The chemicals used in this study were of the highest grade available and purchased from Wako Pure Chemical Industries (Osaka, Japan).

Turtles

Emperor tissue, a soft tissue in the region around the shell of soft‐shelled turtles (P. sinensis), was provided by Shin‐uoei (Osaka, Japan).

Collagen extraction

Collagen extraction was performed in accordance with the our previous study.26 Briefly, emperor tissue was treated with 0.1M formic acid at a ratio of 1:10 (w/v) for 24 h for demineralization. The sample was then treated with 0.1M sodium hydroxide (NaOH) at a ratio of 1:10 (w/v) for 3 days to remove noncollagenous proteins, including endogenous proteases. The NaOH solution was changed every day. Finally, the sample was incubated with 0.03M citric acid for 24 h. After incubation, the solution was centrifuged at 6500g for 20 min at 4°C and the supernatant collected as the collagen solution.

Cell culture

HaCaT immortalized human keratinocytes were purchased from CLS Cell Lines Service GmbH (Eppelheim, Germany). The cells were cultured in RPMI1640 medium supplemented with 10% fetal bovine serum (FBS) (Gibco, Carlsbad, CA, USA) in an atmosphere containing 5% CO2.

Cell growth assay

Cells were plated at a density of 5 × 103 cells per well in a 96‐well plate and grown in culture medium. The next day, the medium was changed and cells grown in collagen‐containing culture medium. After 72 h, the cells were incubated with WST‐8 cell counting reagent (Wako) and the optical density of the culture solution in the plate measured using an ELISA plate reader.

Protein preparation

HaCaT cells were plated in a 60‐mm dish at a density of 2 × 105 cells per dish and grown in culture medium. The next day, the medium was changed and the cells grown in collagen‐containing culture medium. After 72 h, the cells were solubilized in urea lysis buffer (7M urea, 2M thiourea, 5% CHAPS, 1% Triton X‐100). The protein concentration was measured using the Bradford method.

In‐solution trypsin digestion

A gel‐free digestion approach was performed in accordance a previously described protocol.29 Briefly, 10 μg of protein extract from each sample was reduced by the addition of 45 mM dithiothreitol and 20 mM tris(2‐carboxyethyl)phosphine, and then alkylated using 100 mM iodoacetic acid. After alkylation, the samples were digested with trypsin gold, mass spectrometry grade (Promega Corp., Madison, WI, USA) at 37°C for 24 h. Next, the digests were purified using PepClean C‐18 Spin Columns (Thermo, Rockford, IL, USA) according to the manufacturer's protocol.

LC–MS/MS analysis for protein identification

Peptide samples (∼2 μg) were injected into a peptide L‐trap column (Chemicals Evaluation and Research Institute, Tokyo, Japan) using an HTC PAL autosampler (CTC Analytics, Zwingen, Switzerland) and further separated through a Paradigm MS4 (AMR, Tokyo, Japan) using a reverse‐phase C18‐column (L‐column, 3 μm diameter gel particles and 120 Å pore size, 0.2 × 150 mm, Chemicals Evaluation and Research Institute). The mobile phase consisted of 0.1% formic acid in water as solution A and acetonitrile as solution B. The column flow rate was 1 μL/min with a concentration gradient of 5% B to 40% B over 120 min. Gradient‐eluted peptides were analyzed using an LTQ ion‐trap mass spectrometer (Thermo). The results were acquired in a data‐dependent manner in which MS/MS fragmentation was performed on the two most intense peaks of every full MS scan.

All MS/MS spectral data were searched against the SwissProt Homo Sapiens database using Mascot (version 2.4.01, Matrix Science, London, UK). The search criteria were set as follows: enzyme, trypsin; allowance of up to two missed cleavage peptides; mass tolerance ±2.0 Da and MS/MS tolerance ±0.8 Da; and modifications of cysteine carbamidomethylation and methionine oxidation.

Semiquantitative analysis of identified proteins

The fold changes in expressed proteins on a base 2 logarithmic scale were calculated using the Rsc based on spectral counting.30 Relative amounts of identified proteins were calculated using the normalized spectral abundance factor (NSAF).31 Differentially expressed proteins were chosen so that their Rsc was >1 or ≤1, which correspond to fold changes of >2 or <0.5.

Bioinformatics

Functional annotations for proteins identified to be regulated by collagen administration were processed using the Database for Annotation, Visualization, and Integrated Discovery (DAVID) version 6.8 (http://david.abcc.ncifcrf.gov/home.jsp).32, 33, 34

Western blot analysis

A total of 5 µg of cell extract was added to each well and subjected to SDS‐PAGE under reducing conditions. The separated proteins were transferred to polyvinylidene fluoride transfer membranes. Following blocking in TBS–Tween‐20 (0.1%) buffer with 5% skim milk for 2 h at room temperature, the membranes were incubated at 4°C overnight with an anti‐E‐cadherin antibody (1:5,000; Cell Signaling Technology, Beverly, MA), antivimentin antibody (1:1000; Cell Signaling Technology), or antisnail antibody (1:1000; Cell Signaling Technology). Next, the membranes were washed and incubated with HRP‐conjugated antirabbit IgG antibody (American Qualex, San Clemente, CA). Following washing, the blots were visualized using SuperSignal West Dura Extended Duration substrate (Thermo Fisher Scientific) and bands detected using the myECL Imager system (version 2.0; Thermo Fisher Scientific). Next, the same membranes were reprobed with an anti‐β‐actin antibody (Santa Cruz Biotechnology, Dallas, TX) to confirm equal loading of the proteins. All Western blot analyses were performed in triplicate.

Scratch assay

Cells were plated in 35 mm dishes (5 × 105 cells/dish) and incubated for 24 h at 37°C in a humidified 5% CO2 atmosphere to assure confluency. The center of the monolayer was scratched by scraping the cells with a sterile 200‐μL pipette tip.35 After scratching, the dish was gently washed with PBS to remove the detached cells and the medium changed in collagen‐containing culture medium. A microscope system was used to take photographs from the scratch area 0 and 8 h after scratching (Olympus, Tokyo, Japan).

Statistical analysis

All data are presented as the mean ± standard error of the mean. The data were analyzed using one‐way analysis of variance followed by Dunnett's test or the unpaired t test. P < 0.01 was considered significant in all analyses. Computations were performed in GraphPad Prism version 5.1 (GraphPad Software, La Jolla, CA, USA).

RESULTS

Cytotoxicity of collagen against HaCaT cells

To examine the cytotoxic effect of collagen on HaCaT cells, we assessed the cell growth rate when cells were grown in culture medium containing the collagen solution at a concentration of 0.1–100 μg/mL. The growth rate of HaCaT cells cultured in the medium containing collagen was not inhibited at 72 h compared with nontreated cells (Fig. 1). Therefore, we used 100 μg/mL collagen in the following experiments.

Figure 1.

Figure 1

Open in a new tab

Cytotoxic effect of collagen administration in HaCaT cells. Suitable concentrations of collagen that are not cytotoxic to HaCaT cells were determined. No effect was observed on cell proliferation of HaCaT cells with collagen administration.

Protein identification and semiquantitative comparison of identified proteins in collagen‐administered HaCaT cells

To investigate the effect of collagen on the cells in the basal layer of the skin, we determined the molecular profile of proteins in HaCaT cells whose expression levels were regulated by collagen using shotgun proteomics. We performed a label‐free semiquantitative method based on spectral counting to determine the proteins whose expression levels were regulated by collagen. In Figure 2, each R sc value is plotted against the corresponding protein (X‐axis) in increasing order from left to right for proteins identified in collagen‐administered HaCaT cells (collagen) and nontreated cells (nontreatment). A positive value indicates increased expression in the collagen‐treated cells and a negative value decreased expression in the collagen‐treated cells. The NSAF value (Fig. 2, bar) was also plotted on the X‐axis for each corresponding protein with collagen treatment above the X‐axis and control below. Proteins with a high positive or negative R sc value would be candidates for proteins regulated by collagen.

Figure 2.

Figure 2

Open in a new tab

Semiquantitative comparison of identified proteins in collagen‐administered and nontreated HaCaT cells. R sc and normalized spectral abundance factor (NSAF) values calculated for identified proteins are on the X‐axis. Protein expression is compared for collagen versus control. Proteins highly expressed in either collagen‐administered cells or nontreated cells are near the right or left side of the X‐axis. Housekeeping proteins are located around the center of the X‐axis.

As a result of semiquantification, a total of 187 differentially expressed proteins were identified (Table 1). The expression levels of housekeeping proteins β‐actin, GAPDH, and histone H4 were not changed by collagen administration.

Table 1.

Differentially Expressed Proteins (>2‐fold) Upon Administration of Collagen

No. ID Accession Number and Description Number of Amino Acids Fold Change
(R sc)
1 H2B1K_HUMAN O60814 Histone H2B type 1‐K 126 −3.690
2 EF1A3_HUMAN Q5VTE0 Putative elongation factor 1‐alpha‐like 3 462 −3.080
3 H2B1M_HUMAN Q99879 Histone H2B type 1‐M 126 −2.698
4 K2C3_HUMAN P12035 Keratin, type II cytoskeletal 3 628 −2.178
5 H2A1H_HUMAN Q96KK5 Histone H2A type 1‐H 128 −1.611
6 RL10_HUMAN P27635 60S ribosomal protein L10 214 −1.611
7 ARF3_HUMAN P61204 ADP‐ribosylation factor 3 181 −1.611
8 DYHC1_HUMAN Q14204 Cytoplasmic dynein 1 heavy chain 1 4646 −1.477
9 TBAL3_HUMAN A6NHL2 Tubulin alpha chain‐like 3 446 −1.359
10 ENOB_HUMAN P13929 Beta‐enolase 434 −1.359
11 FLNB_HUMAN O75369 Filamin‐B 2602 −1.359
12 PDLI1_HUMAN O00151 PDZ and LIM domain protein 1 329 −1.359
13 FLNA_HUMAN P21333 Filamin‐A 2647 −1.359
14 MYH14_HUMAN Q7Z406 Myosin‐14 1995 −1.359
15 K2C80_HUMAN Q6KB66 Keratin, type II cytoskeletal 80 452 −1.359
16 K2C72_HUMAN Q14CN4 Keratin, type II cytoskeletal 72 511 −1.053
17 POTEF_HUMAN A5A3E0 POTE ankyrin domain family member F 1075 −1.053
18 GDIA_HUMAN P31150 Rab GDP dissociation inhibitor alpha 447 −1.053
19 RS27A_HUMAN P62979 Ubiquitin‐40S ribosomal protein S27a 156 −1.053
20 CAH2_HUMAN P00918 Carbonic anhydrase 2 260 −1.053
21 SEPT9_HUMAN Q9UHD8 Septin‐9 586 −1.053
22 PRP8_HUMAN Q6P2Q9 Pre‐mRNA‐processing‐splicing factor 8 2335 −1.053
23 IMB1_HUMAN Q14974 Importin subunit beta‐1 876 −1.053
24 HS105_HUMAN Q92598 Heat shock protein 105 kDa 858 −1.053
25 PLST_HUMAN P13797 Plastin‐3 630 −1.053
26 H2A1D_HUMAN P20671 Histone H2A type 1‐D 130 −1.036
27 AL1A3_HUMAN P47895 Aldehyde dehydrogenase family 1 member A3 512 1.020
28 HNRH1_HUMAN P31943 Heterogeneous nuclear ribonucleoprotein H 449 1.102
29 PEPL_HUMAN O60437 Periplakin 1756 1.102
30 LDHB_HUMAN P07195 l‐lactate dehydrogenase B chain 334 1.102
31 TPM4_HUMAN P67936 Tropomyosin alpha‐4 chain 248 1.102
32 2AAA_HUMAN P30153 Serine/threonine‐protein phosphatase 2A 65 kDa regulatory subunit A alpha isoform 589 1.102
33 EZRI_HUMAN P15311 Ezrin 586 1.102
34 COR1C_HUMAN Q9ULV4 Coronin‐1C 474 1.102
35 SPTN1_HUMAN Q13813 Spectrin alpha chain, nonerythrocytic 1 2472 1.182
36 H2AV_HUMAN Q71UI9 Histone H2A.V 128 1.245
37 ARP3_HUMAN P61158 Actin‐related protein 3 418 1.245
38 TCPB_HUMAN P78371 T‐complex protein 1 subunit beta 535 1.245
39 AHSA1_HUMAN O95433 Activator of 90 kDa heat shock protein ATPase homolog 1 338 1.245
40 KMT2A_HUMAN Q03164 Histone‐lysine N‐methyltransferase 2A 3969 1.245
41 SYLC_HUMAN Q9P2J5 Leucine‐tRNA ligase, cytoplasmic 1176 1.245
42 PGAM1_HUMAN P18669 Phosphoglycerate mutase 1 254 1.245
43 ICAL_HUMAN P20810 Calpastatin 708 1.245
44 CISY_HUMAN O75390 Citrate synthase, mitochondrial 466 1.245
45 LIMA1_HUMAN Q9UHB6 LIM domain and actin‐binding protein 1 759 1.245
46 CAPR1_HUMAN Q14444 Caprin‐1 709 1.245
47 MYADM_HUMAN Q96S97 Myeloid‐associated differentiation marker 322 1.245
48 PDCD4_HUMAN Q53EL6 Programmed cell death protein 4 469 1.245
49 APEX1_HUMAN P27695 DNA‐(apurinic or apyrimidinic site) lyase 318 1.245
50 MARE1_HUMAN Q15691 Microtubule‐associated protein RP/EB family member 1 268 1.408
51 NACAM_HUMAN E9PAV3 Nascent polypeptide‐associated complex subunit alpha, muscle‐specific form 2078 1.562
52 SHLB2_HUMAN Q9NR46 Endophilin‐B2 395 1.562
53 LIMS1_HUMAN P48059 LIM and senescent cell antigen‐like‐containing domain protein 1 325 1.562
54 EHD1_HUMAN Q9H4M9 EH domain‐containing protein 1 534 1.562
55 TNPO1_HUMAN Q92973 Transportin‐1 898 1.562
56 PYGB_HUMAN P11216 Glycogen phosphorylase, brain form 843 1.562
57 BZW1_HUMAN Q7L1Q6 Basic leucine zipper and W2 domain‐containing protein 1 419 1.562
58 AP2B1_HUMAN P63010 AP‐2 complex subunit beta 937 1.562
59 CMC1_HUMAN O75746 Calcium‐binding mitochondrial carrier protein Aralar1 678 1.562
60 SKAP_HUMAN Q9Y448 Small kinetochore‐associated protein 316 1.562
61 CD9_HUMAN P21926 CD9 antigen 228 1.562
62 P4HA1_HUMAN P13674 Prolyl 4‐hydroxylase subunit alpha‐1 534 1.562
63 PPAC_HUMAN P24666 Low molecular weight phosphotyrosine protein phosphatase 158 1.562
64 FUBP2_HUMAN Q92945 Far upstream element‐binding protein 2 711 1.562
65 RGPD2_HUMAN P0DJD1 RANBP2‐like and GRIP domain‐containing protein 2 1756 1.562
66 RAB1C_HUMAN Q92928 Putative Ras‐related protein Rab‐1C 201 1.562
67 HUWE1_HUMAN Q7Z6Z7 E3 ubiquitin‐protein ligase HUWE1 4374 1.562
68 IPYR2_HUMAN Q9H2U2 Inorganic pyrophosphatase 2, mitochondrial 334 1.562
69 CERS2_HUMAN Q96G23 Ceramide synthase 2 380 1.562
70 IRS4_HUMAN O14654 Insulin receptor substrate 4 1257 1.562
71 DDX3X_HUMAN O00571 ATP‐dependent RNA helicase DDX3X 662 1.562
72 PARP1_HUMAN P09874 Poly[ADP‐ribose] polymerase 1 1014 1.562
73 MAP4_HUMAN P27816 Microtubule‐associated protein 4 1152 1.562
74 LAT1_HUMAN Q01650 Large neutral amino acids transporter small subunit 1 507 1.562
75 CARD6_HUMAN Q9BX69 Caspase recruitment domain‐containing protein 6 1037 1.562
76 PCD16_HUMAN Q96JQ0 Protocadherin‐16 3298 1.562
77 CP250_HUMAN Q9BV73 Centrosome‐associated protein CEP250 2442 1.562
78 MCM3_HUMAN P25205 DNA replication licensing factor MCM3 808 1.562
79 SYSC_HUMAN P49591 Serine‐tRNA ligase, cytoplasmic 514 1.562
80 EPHA4_HUMAN P54764 Ephrin type‐A receptor 4 986 1.562
81 NT5D1_HUMAN Q5TFE4 5‐nucleotidase domain‐containing protein 1 455 1.562
82 GIPC3_HUMAN Q8TF64 PDZ domain‐containing protein GIPC3 312 1.562
83 MXRA5_HUMAN Q9NR99 Matrix‐remodeling‐associated protein 5 2828 1.562
84 CO4A4_HUMAN P53420 Collagen alpha‐4 (IV) chain 1690 1.562
85 POTEB_HUMAN Q6S5H4 POTE ankyrin domain family member B 581 1.562
86 MYH1_HUMAN P12882 Myosin‐1 1939 1.562
87 NFRKB_HUMAN Q6P4R8 Nuclear factor related to kappa‐B‐binding protein 1299 1.562
88 NAC2_HUMAN Q9UPR5 Sodium/calcium exchanger 2 921 1.562
89 NRK2_HUMAN Q9NPI5 Nicotinamide riboside kinase 2 230 1.562
90 BRM1L_HUMAN Q5PSV4 Breast cancer metastasis‐suppressor 1‐like protein 323 1.562
91 SAP3_HUMAN P17900 Ganglioside GM2 activator 193 1.562
92 APBA1_HUMAN Q02410 Amyloid beta A4 precursor protein‐binding family A member 1 837 1.562
93 RS14_HUMAN P62263 40S ribosomal protein S14 151 1.562
94 ENDOV_HUMAN Q8N8Q3 Endonuclease V 282 1.562
95 UBE4B_HUMAN O95155 Ubiquitin conjugation factor E4 B 1302 1.562
96 F134C_HUMAN Q86VR2 Protein FAM134C 466 1.562
97 ACSM5_HUMAN Q6NUN0 Acyl‐coenzyme A synthetase ACSM5, mitochondrial 579 1.562
98 DPOE1_HUMAN Q07864 DNA polymerase epsilon catalytic subunit A 2286 1.562
99 SRRT_HUMAN Q9BXP5 Serrate RNA effector molecule homolog 876 1.562
100 EXOC1_HUMAN Q9NV70 Exocyst complex component 1 894 1.562
101 GDE1_HUMAN Q9NZC3 Glycerophosphodiester phosphodiesterase 1 331 1.562
102 CAMP3_HUMAN Q9P1Y5 Calmodulin‐regulated spectrin‐associated protein 3 1249 1.562
103 BCAS3_HUMAN Q9H6U6 Breast carcinoma‐amplified sequence 3 928 1.562
104 NXF2_HUMAN Q9GZY0 Nuclear RNA export factor 2 626 1.562
105 HIC1_HUMAN Q14526 Hypermethylated in cancer 1 protein 733 1.562
106 VP13C_HUMAN Q709C8 Vacuolar protein sorting‐associated protein 13C 3753 1.562
107 DCE1_HUMAN Q99259 Glutamate decarboxylase 1 594 1.562
108 RUVB2_HUMAN Q9Y230 RuvB‐like 2 463 1.562
109 UBA1_HUMAN P22314 Ubiquitin‐like modifier‐activating enzyme 1 1058 1.562
110 ANX11_HUMAN P50995 Annexin A11 505 1.562
111 2AAB_HUMAN P30154 Serine/threonine‐protein phosphatase 2A 65 kDa regulatory subunit A beta isoform 601 1.562
112 TFG_HUMAN Q92734 Protein TFG 400 1.562
113 1433Z_HUMAN P63104 14‐3‐3 protein zeta/delta 245 1.562
114 C1TC_HUMAN P11586 C‐1‐tetrahydrofolate synthase, cytoplasmic 935 1.562
115 PRDX4_HUMAN Q13162 Peroxiredoxin‐4 271 1.562
116 TENA_HUMAN P24821 Tenascin 2201 1.562
117 MIF_HUMAN P14174 Macrophage migration inhibitory factor 115 1.562
118 NIPS2_HUMAN O75323 Protein NipSnap homolog 2 286 1.562
119 CTNB1_HUMAN P35222 Catenin beta‐1 781 1.562
120 ADIRF_HUMAN Q15847 Adipogenesis regulatory factor 76 1.562
121 COASY_HUMAN Q13057 Bifunctional coenzyme A synthase 564 1.562
122 TF_HUMAN P13726 Tissue factor 295 1.562
123 MATR3_HUMAN P43243 Matrin‐3 847 1.562
124 RAB4A_HUMAN P20338 Ras‐related protein Rab‐4A 218 1.562
125 IF4H_HUMAN Q15056 Eukaryotic translation initiation factor 4H 248 1.562
126 ERP29_HUMAN P30040 Endoplasmic reticulum resident protein 29 261 1.562
127 RL30_HUMAN P62888 60S ribosomal protein L30 115 1.562
128 PPCE_HUMAN P48147 Prolyl endopeptidase 710 1.562
129 UBFL1_HUMAN P0CB47 Putative upstream‐binding factor 1‐like protein 1 393 1.562
130 HGB1A_HUMAN B2RPK0 Putative high mobility group protein B1‐like 1 211 1.562
131 TM163_HUMAN Q8TC26 Transmembrane protein 163 289 1.562
132 DCK_HUMAN P27707 Deoxycytidine kinase 260 1.562
133 PSB6_HUMAN P28072 Proteasome subunit beta type‐6 239 1.562
134 GLYC_HUMAN P34896 Serine hydroxymethyltransferase, cytosolic 483 1.562
135 ETFB_HUMAN P38117 Electron transfer flavoprotein subunit beta 255 1.562
136 SEPT2_HUMAN Q15019 Septin‐2 361 1.562
137 IG2AS_HUMAN Q6U949 Putative insulin‐like growth factor 2 antisense gene protein 168 1.562
138 SYEP_HUMAN P07814 Bifunctional glutamate/proline‐tRNA ligase 1512 1.562
139 GGH_HUMAN Q92820 Gamma‐glutamyl hydrolase 318 1.562
140 SMC5_HUMAN Q8IY18 Structural maintenance of chromosomes protein 5 1101 1.562
141 3BHS2_HUMAN P26439 3 beta‐hydroxysteroid dehydrogenase/Delta 5–>4‐isomerase type 2 372 1.562
142 SIAS_HUMAN Q9NR45 Sialic acid synthase 359 1.562
143 DYH7_HUMAN Q8WXX0 Dynein heavy chain 7, axonemal 4024 1.562
144 GRM2_HUMAN Q14416 Metabotropic glutamate receptor 2 872 1.562
145 PLCB_HUMAN O15120 1‐acyl‐sn‐glycerol‐3‐phosphate acyltransferase beta 278 1.562
146 PNPO_HUMAN Q9NVS9 Pyridoxine‐5‐phosphate oxidase 261 1.562
147 GFPT1_HUMAN Q06210 Glutamine‐fructose‐6‐phosphate aminotransferase [isomerizing] 1 699 1.562
148 INADL_HUMAN Q8NI35 InaD‐like protein 1801 1.562
149 CPMD8_HUMAN Q8IZJ3 C3 and PZP‐like alpha‐2‐macroglobulin domain‐containing protein 8 1885 1.562
150 CO9A1_HUMAN P20849 Collagen alpha‐1(IX) chain 921 1.562
151 DNJA2_HUMAN O60884 DnaJ homolog subfamily A member 2 412 1.562
152 GASP1_HUMAN Q5JY77 G‐protein coupled receptor‐associated sorting protein 1 1395 1.562
153 BIRC3_HUMAN Q13489 Baculoviral IAP repeat‐containing protein 3 604 1.562
154 IL2RG_HUMAN P31785 Cytokine receptor common subunit gamma 369 1.562
155 FUCM_HUMAN A2VDF0 Fucose mutarotase 154 1.562
156 KAD3_HUMAN Q9UIJ7 GTP:AMP phosphotransferase AK3, mitochondrial 227 1.562
157 GSX2_HUMAN Q9BZM3 GS homeobox 2 304 1.562
158 MIMIT_HUMAN Q8N183 Mimitin, mitochondrial 169 1.562
159 CYC_HUMAN P99999 Cytochrome c 105 1.562
160 CC141_HUMAN Q6ZP82 Coiled‐coil domain‐containing protein 141 1450 1.562
161 ZN503_HUMAN Q96F45 Zinc finger protein 503 646 1.562
162 CHD7_HUMAN Q9P2D1 Chromodomain helicase DNA binding protein 7 2997 1.562
163 RADI_HUMAN P35241 Radixin 583 1.633
164 CAN1_HUMAN P07384 Calpain‐1 catalytic subunit 714 1.633
165 CATB_HUMAN P07858 Cathepsin B 339 1.660
166 EF1G_HUMAN P26641 Elongation factor 1‐gamma 437 1.875
167 CNN2_HUMAN Q99439 Calponin‐2 309 1.938
168 GELS_HUMAN P06396 Gelsolin 782 1.938
169 KRT81_HUMAN Q14533 Keratin, type II cuticular Hb1 505 2.094
170 EIF3E_HUMAN P60228 Eukaryotic translation initiation factor 3 subunit E 445 2.094
171 DAZP1_HUMAN Q96EP5 DAZ‐associated protein 1 407 2.094
172 SURF4_HUMAN O15260 Surfeit locus protein 4 269 2.094
173 GGCT_HUMAN O75223 Gamma‐glutamylcyclotransferase 188 2.094
174 HNRH2_HUMAN P55795 Heterogeneous nuclear ribonucleoprotein H2 449 2.094
175 AT1A1_HUMAN P05023 Sodium/potassium‐transporting ATPase subunit alpha‐1 1023 2.094
176 OLA1_HUMAN Q9NTK5 Obg‐like ATPase 1 396 2.094
177 RL1D1_HUMAN O76021 Ribosomal L1 domain‐containing protein 1 490 2.094
178 IF4A3_HUMAN P38919 Eukaryotic initiation factor 4A‐III 411 2.094
179 MESD_HUMAN Q14696 LDLR chaperone MESD 234 2.094
180 K1C27_HUMAN Q7Z3Y8 Keratin, type I cytoskeletal 27 459 2.094
181 CNDP2_HUMAN Q96KP4 Cytosolic nonspecific dipeptidase 475 2.191
182 H2A2A_HUMAN Q6FI13 Histone H2A type 2‐A 130 2.481
183 PYGL_HUMAN P06737 Glycogen phosphorylase, liver form 847 2.481
184 H2A1C_HUMAN Q93077 Histone H2A type 1‐C 130 2.481
185 ADT1_HUMAN P12235 ADP/ATP translocase 1 298 3.039
186 VPP4_HUMAN Q9HBG4 V‐type proton ATPase 116 kDa subunit a isoform 4 840 3.049
187 H2B1H_HUMAN Q93079 Histone H2B type 1‐H 126 4.672
Open in a new tab

Functional annotation of proteins regulated by collagen

Gene ontology (GO) analysis was performed with the candidate proteins for each biological process [Fig. 3(A)], cellular component [Fig. 3(B)], and molecular function [Fig. 3(C)] using DAVID. Some of the differentially expressed proteins were related to cell adhesion, and we focused on the function of proteins classified as cadherin binding involved in cell–cell adhesion (Table 2).

Figure 3.

Figure 3

Open in a new tab

Gene ontology (GO) analysis for identified proteins. (A) Proteins assigned to biological process, (B) cellular component, and (C) molecular function GO term categories. Only significant categories (p < 0.05) are shown.

Table 2.

Differentially Expressed Proteins Categorized as Cadherin Binding Involved in Cell–Cell Adhesion Proteins in Gene Ontology

No. Accession Number and Description Fold Change
(R sc)
1 O00151 PDZ and LIM domain protein 1 −1.359
2 P21333 Filamin‐A −1.359
3 O75369 Filamin‐B −1.359
4 Q9UHD8 Septin‐9 −1.053
5 P15311 Ezrin 1.102
6 O60437 Periplakin 1.102
7 Q13813 Spectrin alpha chain, nonerythrocytic 1 1.182
8 Q9UHB6 LIM domain and actin‐binding protein 1 1.245
9 O95433 Activator of 90 kDa heat shock protein ATPase homolog 1 1.245
10 P20810 Calpastatin 1.245
11 Q15691 Microtubule‐associated protein RP/EB family member 1 1.408
12 Q7L1Q6 Basic leucine zipper and W2 domain‐containing protein 1 1.562
13 Q9NR46 Endophilin‐B2 1.562
14 O00571 ATP‐dependent RNA helicase DDX3X 1.562
15 P35222 Catenin beta‐1 1.562
16 Q9H4M9 EH domain‐containing protein 1 1.562
17 Q15056 Eukaryotic translation initiation factor 4H 1.562
18 P63104 14‐3‐3 protein zeta/delta 1.562
19 Q15019 Septin‐2 1.562
20 P28072 Proteasome subunit beta type‐6 1.562
21 P35241 Radixin 1.633
22 P26641 Elongation factor 1‐gamma 1.875
23 Q99439 Calponin‐2 1.938
24 P60228 Eukaryotic translation initiation factor 3 subunit E 2.094
25 Q9NTK5 Obg‐like ATPase 1 2.094
26 O76021 Ribosomal L1 domain‐containing protein 1 2.094
Open in a new tab

Effect of collagen administration on the expression level of E‐cadherin and EMT marker proteins in HaCaT cells

To investigate whether collagen administration affected the level of cadherin expression, we examined the expression of E‐cadherin in collagen‐administered HaCaT cells. The expression of E‐cadherin clearly decreased with collagen administration compared with nontreated cells (Fig. 4). Next, we examined the expression levels of vimentin and snail to investigate whether epithelial–mesenchymal transition (EMT) was induced in correlation with the downregulation of E‐cadherin. The expression of vimentin and snail clearly increased with collagen administration compared with nontreated cells (Fig. 4).

Figure 4.

Figure 4

Open in a new tab

Expression levels of E‐cadherin and EMT markers in HaCaT cells. E‐cadherin expression was decreased with the administration of collagen, whereas the expression levels of vimentin and snail were increased by the administration of collagen compared with nontreated cells.

Effect of collagen administration on keratinocyte migration in a scratch‐wound healing process

To investigate whether EMT affected the migration capability of HaCaT cells, we performed an in vitro wound healing study using the HaCaT scratch model. Photographs were taken before treatment and after 8 h of incubation at 37°C in 5% CO2 [Fig. 5(A)]. Collagen‐administered cells significantly facilitated wound healing compared with nontreated cells [Fig. 5(B)].

Figure 5.

Figure 5

Open in a new tab

Wound healing assay. (A) Microscopic images of wound healing over 8 h. (B) The percentage of wounded area in collagen‐administered HaCaT cells was significantly larger than in nontreated cells. *p < 0.01.

DISCUSSION

In this study, we used a gel‐free LC–MS‐based proteomics approach to examine the functional effects of collagen from soft‐shelled turtle on human skin. Although spectral counting may not accurately reflect the quantity information,36 it is useful and has been used in many studies, including those searching for novel diagnostic biomarkers.37, 38, 39, 40, 41, 42 We were able to successfully identify several proteins whose expression levels were changed >2‐fold in HaCaT cells by the administration of collagen using a semiquantitative method based on spectral counting.

To examine the role of these identified proteins, we performed GO analysis. The functional category that directly relates to cell–cell adhesion was obtained from among the GO terms on molecular function, biological process, and cellular component. We focused on the functions of proteins classified as cadherin binding involved in cell–cell adhesion because they play important roles in cadherin‐mediated cell adhesion; thus, changes in the expression levels of these proteins with the administration of collagen from soft‐shelled turtle may affect the expression of cadherin. To evaluate this hypothesis, we examined the expression of a major cadherin protein in epithelial cells, E‐cadherin; its expression level was decreased with the administration of collagen from soft‐shelled turtle. As down‐regulation of E‐cadherin is an important factor in EMT induction, we examined the expression of EMT markers in HaCaT cells to investigate whether EMT was induced in keratinocytes by the administration of collagen. The increase in expression of vimentin, a mesenchymal marker,43 and snail, a major inducer of EMT via suppression of E‐cadherin expression,43, 44 in collagen‐administered HaCaT cells compared with nontreated cells suggests that the administration of collagen from soft‐shelled turtle induces EMT in human keratinocytes. Recent studies reported that human collagen type I can induce EMT in some cell types,45, 46, 47, 48 and collagen from soft‐shelled turtle as used in this study may have a similar effect.

EMT was originally described as a phenomenon observed during gastrulation in the early embryo.49 Recently, EMT was considered to be associates with tissue repair responses to injuries in parenchymal organs, including skin.43, 50 Therefore, we performed an in vitro wound healing assay using a cell scratch model to clarify the effect of EMT of HaCaT cells induced by the administration of collagen from soft‐shelled turtle on the wound healing process. The significant promotion of wound healing in HaCaT cells administered collagen suggests that administration of collagen from soft‐shelled turtle enhances the wound healing ability of keratinocytes through the induction of EMT. However, the mechanism of the induction of EMT of keratinocytes upon administration of collagen from soft‐shelled turtle is unclear. In this study, we focused on the function of proteins listed in Table 2, in which the expression level of β‐catenin was increased with collagen administration. A previous report demonstrated that overexpression of β‐catenin induced cell migration and invasion through the induction of EMT via up‐regulation of mesenchymal markers, including vimentin, and down‐regulation of epithelial markers, including E‐cadherin.51 Therefore, increased expression of β‐catenin may be one of the mechanisms underlying the induction of EMT after the administration of collagen from soft‐shelled turtle. Further studies are necessary to clarify the mechanism of increased β‐catenin expression and the other mechanisms for EMT induction.

In conclusion, we measured the changes in protein expression in HaCaT cells administered collagen from soft‐shelled turtle using a shotgun LC/MS‐based global proteomic analysis and found that the administration of collagen induced the EMT of keratinocytes and facilitated wound healing. Therefore, collagen from soft‐shelled turtle may provide significant benefits for skin wound healing and be a useful material for pharmaceuticals and medical care products.

ACKNOWLEDGMENTS

We are grateful to Mr. T. Aboshi for providing the soft‐shelled turtles used in the study (Shin‐uoei, Inc.).

How to cite this article: Yamamoto T, Nakanishi S, Mitamura K, Taga A 2018. Shotgun label‐free proteomic analysis for identification of proteins in HaCaT human skin keratinocytes regulated by the administration of collagen from soft‐shelled turtle. J Biomed Mater Res Part B 2018:106B:2403–2413.

REFERENCES

  • 1. Gelse K, Poschl E, Aigner T. Collagens–structure, function, and biosynthesis. Adv Drug Deliv Rev 2003;55(12):1531–1546. [DOI] [PubMed] [Google Scholar]
  • 2. Myllyharju J, Kivirikko KI. Collagens, modifying enzymes and their mutations in humans, flies and worms. Trends Genet 2004;20(1):33–43. [DOI] [PubMed] [Google Scholar]
  • 3. Birk DE, Trelstad RL. Extracellular compartments in tendon morphogenesis: Collagen fibril, bundle, and macroaggregate formation. J Cell Biol 1986;103(1):231–240. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4. Adachi E, Hayashi T. Anchoring of epithelia to underlying connective tissue: Evidence of frayed ends of collagen fibrils directly merging with meshwork of lamina densa. J Electron Microsc (Tokyo) 1994;43(5):264–271. [PubMed] [Google Scholar]
  • 5. Park KH, Bae YH. Phenotype of hepatocyte spheroids in Arg‐GLY‐Asp (RGD) containing a thermo‐reversible extracellular matrix. Biosci Biotechnol Biochem 2002;66(7):1473–1478. [DOI] [PubMed] [Google Scholar]
  • 6. Liu B, Weinzimer SA, Gibson TB, Mascarenhas D, Cohen P. Type I alpha collagen is an IGFBP‐3 binding protein. Growth Horm IGF Res 2003;13(2–3):89–97. [DOI] [PubMed] [Google Scholar]
  • 7. Di Lullo GA, Sweeney SM, Korkko J, Ala‐Kokko L, San Antonio JD. Mapping the ligand‐binding sites and disease‐associated mutations on the most abundant protein in the human, type I collagen. J Biol Chem 2002;277(6):4223–4231. [DOI] [PubMed] [Google Scholar]
  • 8. Saby C, Buache E, Brassart‐Pasco S, El Btaouri H, Courageot MP, Van Gulick L, Garnotel R, Jeannesson P, Morjani H. Type I collagen aging impairs discoidin domain receptor 2‐mediated tumor cell growth suppression. Oncotarget 2016;7(18):24908–24927. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9. Maquoi E, Assent D, Detilleux J, Pequeux C, Foidart JM, Noel A. MT1‐MMP protects breast carcinoma cells against type I collagen‐induced apoptosis. Oncogene 2012;31(4):480–493. [DOI] [PubMed] [Google Scholar]
  • 10. Gorell ES, Leung TH, Khuu P, Lane AT. Purified type I collagen wound matrix improves chronic wound healing in patients with recessive dystrophic epidermolysis bullosa. Pediatr Dermatol 2015;32(2):220–225. [DOI] [PubMed] [Google Scholar]
  • 11. Shevchenko RV, Sibbons PD, Sharpe JR, James SE. Use of a novel porcine collagen paste as a dermal substitute in full‐thickness wounds. Wound Repair Regen 2008;16(2):198–207. [DOI] [PubMed] [Google Scholar]
  • 12. Wollina U, Meseg A, Weber A. Use of a collagen‐elastin matrix for hard to treat soft tissue defects. Int Wound J 2011;8(3):291–296. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13. Barhoumi A, Salvador‐Culla B, Kohane DS. NIR‐triggered drug delivery by collagen‐mediated second harmonic generation. Adv Healthc Mater 2015. [DOI] [PubMed] [Google Scholar]
  • 14. Wallace DG, Rosenblatt J. Collagen gel systems for sustained delivery and tissue engineering. Adv Drug Deliv Rev 2003;55(12):1631–1649. [DOI] [PubMed] [Google Scholar]
  • 15. Friess W. Collagen–biomaterial for drug delivery. Eur J Pharm Biopharm 1998;45(2):113–136. [DOI] [PubMed] [Google Scholar]
  • 16. Lynn AK, Yannas IV, Bonfield W. Antigenicity and immunogenicity of collagen. J Biomed Mater Res B Appl Biomater 2004;71(2):343–354. [DOI] [PubMed] [Google Scholar]
  • 17. Pranoto Y, Lee CM, Park HJ. Characterizations of fish gelatin films added with gellan and kappa‐carrageenan. LWT Food Sci Technol 2007;40(5):766–774. [Google Scholar]
  • 18. Muralidharan N, Jeya Shakila R, Sukumar D, Jeyasekaran G. Skin, bone and muscle collagen extraction from the trash fish, leather jacket (Odonus niger) and their characterization. J Food Sci Technol 2013;50(6):1106–1113. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19. Wang Y, Regenstein JM. Effect of EDTA, HCl, and citric acid on Ca salt removal from Asian (silver) carp scales prior to gelatin extraction. J Food Sci 2009;74(6):C426–C431. [DOI] [PubMed] [Google Scholar]
  • 20. Wang C, Zhan CL, Cai QF, Du CH, Liu GM, Su WJ, Cao MJ. Expression and characterization of common carp (Cyprinus carpio) matrix metalloproteinase‐2 and its activity against type I collagen. J Biotechnol 2014;177:45–52. [DOI] [PubMed] [Google Scholar]
  • 21. Benjakul S, Thiansilakul Y, Visessanguan W, Roytrakul S, Kishimura H, Prodpran T, Meesane J. Extraction and characterisation of pepsin‐solubilised collagens from the skin of bigeye snapper (Priacanthus tayenus and Priacanthus macracanthus). J Sci Food Agric 2010;90(1):132–138. [DOI] [PubMed] [Google Scholar]
  • 22. Nalinanon S, Benjakul S, Kishimura H. Collagens from the skin of arabesque greenling (Pleurogrammus azonus) solubilized with the aid of acetic acid and pepsin from albacore tuna (Thunnus alalunga) stomach. J Sci Food Agric 2010;90(9):1492–1500. [DOI] [PubMed] [Google Scholar]
  • 23. Tziveleka LA, Ioannou E, Tsiourvas D, Berillis P, Foufa E, Roussis V. Collagen from the Marine sponges Axinella cannabina and Suberites carnosus: Isolation and morphological, biochemical, and biophysical characterization. Mar Drugs 2017;15(6). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24. Pallela R, Venkatesan J, Janapala VR, Kim SK. Biophysicochemical evaluation of chitosan‐hydroxyapatite‐marine sponge collagen composite for bone tissue engineering. J Biomed Mater Res A 2012;100(2):486–495. [DOI] [PubMed] [Google Scholar]
  • 25. Coelho RCG, Marques ALP, Oliveira SM, Diogo GS, Pirraco RP, Moreira‐Silva J, Xavier JC, Reis RL, Silva TH, Mano JF. Extraction and characterization of collagen from Antarctic and Sub‐Antarctic squid and its potential application in hybrid scaffolds for tissue engineering. Mater Sci Eng C Mater Biol Appl 2017;78:787–795. [DOI] [PubMed] [Google Scholar]
  • 26. Yamamoto T, Uemura K, Sawashi Y, Mitamura K, Taga A. Optimization of method to extract collagen from “emperor” tissue of soft‐shelled turtles. J Oleo Sci 2016;65(2):169–175. [DOI] [PubMed] [Google Scholar]
  • 27. Zou Y, Wang L, Cai P, Li P, Zhang M, Sun Z, Sun C, Xu W, Wang D. Effect of ultrasound assisted extraction on the physicochemical and functional properties of collagen from soft‐shelled turtle calipash. Int J Biol Macromol 2017. [DOI] [PubMed] [Google Scholar]
  • 28. Yang Y, Li C, Song W, Wang W, Qian G. Purification, optimization and physicochemical properties of collagen from soft‐shelled turtle calipash. Int J Biol Macromol 2016;89:344–352. [DOI] [PubMed] [Google Scholar]
  • 29. Bluemlein K, Ralser M. Monitoring protein expression in whole‐cell extracts by targeted label‐ and standard‐free LC–MS/MS. Nat Protoc 2011;6(6):859–869. [DOI] [PubMed] [Google Scholar]
  • 30. Old WM, Meyer‐Arendt K, Aveline‐Wolf L, Pierce KG, Mendoza A, Sevinsky JR, Resing KA, Ahn NG. Comparison of label‐free methods for quantifying human proteins by shotgun proteomics. Mol Cell Proteomics 2005;4(10):1487–1502. [DOI] [PubMed] [Google Scholar]
  • 31. Zybailov B, Coleman MK, Florens L, Washburn MP. Correlation of relative abundance ratios derived from peptide ion chromatograms and spectrum counting for quantitative proteomic analysis using stable isotope labeling. Anal Chem 2005;77(19):6218–6224. [DOI] [PubMed] [Google Scholar]
  • 32. Dennis G, Jr. , Sherman BT, Hosack DA, Yang J, Gao W, Lane HC, Lempicki RA. DAVID: Database for annotation, visualization, and integrated discovery. Genome Biol 2003;4(5):P3. [PubMed] [Google Scholar]
  • 33. Huang da W, Sherman BT, Lempicki RA. Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat Protoc 2009;4(1):44–57. [DOI] [PubMed] [Google Scholar]
  • 34. Huang da W, Sherman BT, Lempicki RA. Bioinformatics enrichment tools: Paths toward the comprehensive functional analysis of large gene lists. Nucleic Acids Res 2009;37(1):1–13. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35. Liang CC, Park AY, Guan JL. In vitro scratch assay: A convenient and inexpensive method for analysis of cell migration in vitro . Nat Protoc 2007;2(2):329–333. [DOI] [PubMed] [Google Scholar]
  • 36. Lundgren DH, Hwang SI, Wu L, Han DK. Role of spectral counting in quantitative proteomics. Expert Rev Proteomics 2010;7(1):39–53. [DOI] [PubMed] [Google Scholar]
  • 37. Yamamoto T, Kudo M, Peng WX, Naito Z. Analysis of protein expression regulated by lumican in PANC1 cells using shotgun proteomics. Oncol Rep 2013;30(4):1609–1621. [DOI] [PubMed] [Google Scholar]
  • 38. Takaya A, Peng WX, Ishino K, Kudo M, Yamamoto T, Wada R, Takeshita T, Naito Z. Cystatin B as a potential diagnostic biomarker in ovarian clear cell carcinoma. Int J Oncol 2015;46(4):1573–1581. [DOI] [PubMed] [Google Scholar]
  • 39. Kanzaki A, Kudo M, Ansai S‐I, Peng W‐X, Ishino K, Yamamoto T, Wada R, Fujii T, Teduka K, Kawahara K, Kawamoto Y, Kitamura T, Kawana S, Saeki H, Naito Z, et al. Insulin‐like growth factor 2 mRNA‐binding protein‐3 as a marker for distinguishing between cutaneous squamous cell carcinoma and keratoacanthoma. Int J Oncol 2016;48(3):1007–1015. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40. Yamamoto T, Kudo M, Peng W‐X, Takata H, Takakura H, Teduka K, Fujii T, Mitamura K, Taga A, Uchida E, Naito Z, et al. Identification of aldolase A as a potential diagnostic biomarker for colorectal cancer based on proteomic analysis using formalin‐fixed paraffin‐embedded tissue. Tumour Biol 2016;37(10):13595–13606. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41. Takata H, Kudo M, Yamamoto T, Ueda J, Ishino K, Peng WX, Wada R, Taniai N, Yoshida H, Uchida E, et al. Increased expression of PDIA3 and its association with cancer cell proliferation and poor prognosis in hepatocellular carcinoma. Oncol Lett 2016;12(6):4896–4904. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42. Kawamura T, Nomura M, Tojo H, Fujii K, Hamasaki H, Mikami S, Bando Y, Kato H, Nishimura T. Proteomic analysis of laser‐microdissected paraffin‐embedded tissues: (1) stage‐related protein candidates upon non‐metastatic lung adenocarcinoma. J Proteomics 2010;73(6):1089–1099. [DOI] [PubMed] [Google Scholar]
  • 43. Zeisberg M, Neilson EG. Biomarkers for epithelial–mesenchymal transitions. J Clin Invest 2009;119(6):1429–1437. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44. Barrallo‐Gimeno A, Nieto MA. The snail genes as inducers of cell movement and survival: Implications in development and cancer. Development 2005;132(14):3151–3161. [DOI] [PubMed] [Google Scholar]
  • 45. Shintani Y, Hollingsworth MA, Wheelock MJ, Johnson KR. Collagen I promotes metastasis in pancreatic cancer by activating c‐Jun NH(2)‐terminal kinase 1 and up‐regulating N‐cadherin expression. Cancer Res 2006;66(24):11745–11753. [DOI] [PubMed] [Google Scholar]
  • 46. Shintani Y, Wheelock MJ, Johnson KR. Phosphoinositide‐3 kinase‐Rac1‐c‐Jun NH2‐terminal kinase signaling mediates collagen I‐induced cell scattering and up‐regulation of N‐cadherin expression in mouse mammary epithelial cells. Mol Biol Cell 2006;17(7):2963–2975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 47. Koenig A, Mueller C, Hasel C, Adler G, Menke A. Collagen type I induces disruption of E‐cadherin‐mediated cell–cell contacts and promotes proliferation of pancreatic carcinoma cells. Cancer Res 2006;66(9):4662–4671. [DOI] [PubMed] [Google Scholar]
  • 48. Shintani Y, Maeda M, Chaika N, Johnson KR, Wheelock MJ. Collagen I promotes epithelial‐to‐mesenchymal transition in lung cancer cells via transforming growth factor‐beta signaling. Am J Respir Cell Mol Biol 2008;38(1):95–104. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 49. Hay ED. An overview of epithelio‐mesenchymal transformation. Acta Anat (Basel) 1995;154(1):8–20. [DOI] [PubMed] [Google Scholar]
  • 50. Haensel D, Dai X. Epithelial‐to‐mesenchymal transition in cutaneous wound healing: Where we are and where we are heading. Dev Dyn 2017. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 51. Chen L, Mai W, Chen M, Hu J, Zhuo Z, Lei X, Deng L, Liu J, Yao N, Huang M, Peng Y, Ye W, Zhang D, et al. Arenobufagin inhibits prostate cancer epithelial–mesenchymal transition and metastasis by down‐regulating beta‐catenin. Pharmacol Res 2017;123:130 – 142. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Biomedical Materials Research. Part B, Applied Biomaterials are provided here courtesy of Wiley

RESOURCES