Collagen peptides from soft‑shelled turtle induce calpain‑1 expression and regulate inflammatory cytokine expression in HaCaT human skin keratinocytes

  • Authors:
    • Tetsushi Yamamoto
    • Saori Nakanishi
    • Kuniko Mitamura
    • Atsushi Taga
  • View Affiliations

  • Published online on: May 8, 2018     https://doi.org/10.3892/ijmm.2018.3659
  • Pages: 1168-1180
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Abstract

Collagen peptides (CPs), derived by hydrolyzing collagen with chemicals or enzymes, are often used as functional materials, due to their various bioactivities and high bioavailability. A previous study by our group reported that collagen from soft‑shelled turtle, Pelodiscus sinensis, induces keratinocytes to undergo epithelial‑mesenchymal transition and facilitates wound healing. Therefore, CPs derived from soft‑shelled turtle collagen may have useful effects on the skin. In the present study, the functional effects of CPs on human skin were examined by analyzing CP‑treated human keratinocytes with a shotgun liquid chromatography/mass spectrometry‑based global proteomic approach. A semi‑quantitative method based on spectral counting was applied and 211 proteins that exhibited >2‑fold changes in expression after CP treatment were successfully identified. Based on a Gene Ontology analysis, the functions of these proteins were indicated to be closely linked with protein processing. In addition, CP treatment significantly increased the expression of calpain‑1, a calcium‑dependent intracellular cysteine protease. Furthermore, CP‑treated keratinocytes exhibited elevated interleukin (IL)‑1α and IL‑8 expression and reduced IL‑6 expression. CPs also induced the expression of proteins implicated in cell‑cell adhesion and the skin barrier. Therefore, CPs from soft‑shelled turtle may provide significant benefits for maintaining the biological environment of the skin, and may be useful as components of pharmaceuticals and medical products.

Introduction

Collagen is a ubiquitous structural protein. There are more than 20 different types of collagen, with specific functions in each tissue (1,2). These proteins have important roles in the maintenance of the extracellular matrix environment (3-7). Certain studies have demonstrated that collagen regulates cell proliferation or apoptosis (8,9). In this decade, collagens of marine origin (e.g., fish, sponges and mollusks) have been considered a useful resource due to their high availability (10-17). These collagens have been widely used as functional foods or dietary supplements. Collagen has also been used for skin substitutes and drug delivery vehicles (18-23).

Recently, collagen peptides (CPs), derived from chemical and enzymatic collagen hydrolysis (24,25), have been increasingly used as functional materials, due to their various bioactivities and high bioavailability (26,27). Several studies have demonstrated the beneficial effects of CPs. For instance, CPs derived from fish skin were demonstrated to have several protective effects on skin photo-aging and wound healing, as they improved moisture retention and repaired endogenous collagen and elastin protein fibers (28-32). Therefore, CPs are considered a useful material for the development of cosmetics, pharmaceuticals and medical products.

Previous studies by our group reported that tissue from soft-shelled turtle, Pelodiscus sinensis, may be a useful alternative source of collagen (33). Due its ability to induce keratinocytes to enter the epithelial-mesenchymal transition (EMT), which facilitates wound healing, this collagen may be a useful component of pharmaceuticals and medical products (34). Furthermore, CPs from soft-shelled turtle may have beneficial effects on skin. However, due to differences in habitat environments, collagen from soft-shelled turtle may differ greatly from collagen from mammalian or marine sources, in terms of physicochemical properties, amino acid composition and physiological functions. Therefore, further research is required prior to the use of CPs derived from soft-shelled turtle tissue in commercial products.

In the present study, a shotgun liquid chromatography/mass spectrometry (LC/MS)-based global proteomic analysis of human keratinocytes treated with CPs was performed to examine the functional effects of CPs on human skin. A total of 211 differentially expressed proteins was identified in keratinocytes treated with CPs compared with untreated keratinocytes. It was investigated whether any of these proteins may be involved in the induction of inflammatory factors in human skin.

Materials and methods

Chemicals and reagents

The highest-grade chemicals and reagents available were purchased from Wako Pure Chemical Industries (Osaka, Japan). Emperor tissue, a soft tissue in the region around the shell of soft-shelled turtles (P. sinensis), was provided by Shin-uoei, Inc. (Osaka, Japan).

Collagen extraction

Collagen extraction was performed as described in a previous study (33). In brief, emperor tissue was treated with 0.1 M formic acid at a ratio of 1:10 (w/v) for 24 h for demineralization. The sample was then treated with 0.1 M NaOH at a ratio of 1:10 (w/v) for 3 days to remove non-collagenous proteins, including endogenous proteases. The NaOH solution was changed every day. Finally, the sample was incubated with 0.03 M citric acid for 24 h. After the incubation, the solution was centrifuged at 6,500 x g for 20 min at 4°C, and the supernatant was collected. This collagen solution was used in the subsequent experiments.

Tryptic digestion of collagen

The extracted collagen solution was subjected to proteolytic activation with bovine pancreatic trypsin (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) in 100 mM ammonium bicarbonate buffer (pH 8.0). The collagen was incubated with trypsin at a trypsin/collagen ratio of 1:100 (w:w) at 37°C. At each indicated time-point, reaction solutions were quickly removed and heated to 100°C to terminate trypsin digestion.

Tricine-SDS-PAGE

The molecular weights of the tryptic digestion products were determined with tricine-SDS-PAGE, as described previously (35). For comparison, molecular weight markers ranging from 3.5 to 42 kDa (Wako Pure Chemical Industries) were used. The electrophoresed gel was stained with Coomassie brilliant blue at room temperature for 1 h.

Matrix-assisted laser desorption-time of flight/mass spec- trometry (MALDI-TOF/MS)

CPs were applied onto the MALDI target plate (Shimadzu, Kyoto, Japan) with 10 mg/ml of α-cyano-4-hydroxy cinnamic acid (Sigma-Aldrich; Merck KGaA) in 50% acetonitrile and 0.05% trifluoroacetic acid. The mass spectra of the CPs were determined with an AXIMA Confidence (Shimadzu) in reflector mode. Prior to acquiring the peptide mass spectrum of the sample, the system was calibrated with a ProteoMass Peptide & Protein MALDI-MS Calibration Kit (cat. no. MSCAL1-1KT; Bradykinin fragment 1-7, 757.3997; P14R, 1,533.8582; insulin oxidized B-chain, 3,494.6513; Sigma-Aldrich; Merck KGaA).

Cell culture

HaCaT immortalized human keratinocytes were purchased from CLS Cell Lines Service GmbH (Eppelheim, Germany). The cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) in an atmosphere containing 5% CO2 at 37°C.

Cell growth assay

Cells were plated at a density of 5×103 cells/well in a 96-well plate and grown in culture medium. The medium was changed the next day, and different concentrations of CPs were added. After 72-h treatments, the cells were incubated with the WST-8 cell counting reagent (Wako Pure Chemical Industries), and the optical density of the culture solution was measured at 450 nm with 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 medium was changed the next day and CPs were added. After 72-h treatments, the cells were solubilized in urea lysis buffer (7 M urea, 2 M thiourea, 5% CHAPS, 1% Triton X-100). The protein concentration was measured with the Bio-Rad Protein Assay (cat. no. 5000006JA; Bio-Rad Laboratories, Hercules, CA, USA).

In-solution trypsin digestion

The gel-free digestion method was applied as described previously (36). In brief, 10 μg protein extract from each sample was chemically reduced by adding 45 mM dithiothreitol and 20 mM tris(2-carboxyethyl)phos-phine. Subsequently, the protein was alkylated with 100 mM iodoacetamide. After the alkylation, the samples were digested with mass spectrometry grade trypsin gold (Promega Corp., Madison, WI, USA) at 37°C for 24 h. Next, the digests were purified with PepClean C-18 Spin Columns (Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol.

Liquid chromatography tandem MS (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) with an HTC PAL autosampler (CTC Analytics, Zwingen, Switzerland). The peptides were separated further in a Paradigm MS4 (AMR Inc., Tokyo, Japan) with 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 (solution A) and acetonitrile (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 with an LTQ ion-trap mass spectrometer (Thermo Fisher Scientific, Inc.). The results were acquired in a data-dependent manner, where MS/MS fragmentation was performed on the two most intense peaks of each full MS scan.

All MS/MS spectral data were entered into a search for comparisons against the SwissProt Homo sapiens database with the Mascot tool (version 2.4.01; Matrix Science, London, UK). The search criteria were as follows: Enzyme, trypsin; with the following allowances: Up to two missed cleavage peptides; mass tolerance, ±2.0 kDa; MS/MS tolerance, ±0.8 kDa; and cysteine carbamidomethylation and methionine oxidation modifications.

Semiquantitative analysis of identified proteins

The fold-change in expression was calculated as the log2 of the ratio of protein abundances (Rsc), evaluated by spectral counting (37). For comparison, the relative amounts of identified proteins were calculated using the normalized spectral abundance factor (NSAF) (38). Differential expression of proteins were considered significant when the Rsc was >1 or <-1, which corresponded to fold-changes of >2 or <0.5, respectively.

Bioinformatics

The function of proteins that exhibited a significant change in expression with CP treatment was investigated. These sequences were processed by examining their functional annotations in the Database for Annotation, Visualization, and Integrated Discovery (DAVID) version 6.8 (http://david.abcc.ncifcrf.gov/home.jsp) (39-41).

Western blot analysis

Total protein (5 μg) that had been extracted from CP-treated cells was added to each well of an SDS-PAGE gel and electrophoresis was performed under reducing conditions. The separated proteins were transferred to polyvinylidene fluoride membranes (Merck KGaA) for 30 min at 15 V. After blocking in TBS-Tween-20 (0.1%) buffer with 5% skimmed milk for 2 h at room temperature, the membranes were incubated with an anti-calpain 1 antibody (1:1,000 dilution; cat. no. 2556; Cell Signaling Technology, Inc., Beverly, MA, USA) at 4°C overnight. The membranes were then washed and incubated with horseradish peroxidase-conjugated anti-rabbit immunoglobulin (Ig)G antibody (cat. no. A106PU; American Qualex, San Clemente, CA, USA) at room temperature for 1 h. The blots were washed and visualized with SuperSignal West Dura Extended Duration substrate (Thermo Fisher Scientific, Inc.). The bands were analyzed with the myECL Imager system (version 2.0; Thermo Fisher Scientific, Inc.). Next, the membranes were stripped by Restore Western Blot Stripping buffer (Thermo Fisher Scientific, Inc.), and the same membranes were re-probed with an anti-β-actin antibody (1:5,000 dilution; cat. no. sc-47778; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) at 4°C overnight, which served as the protein loading control. The intensities of calpain-1 and β-actin were quantified with myImageAnalysis software (version 2.0; Thermo Fisher Scientific, Inc.). The relative quantities of calpain-1 over β-actin were used to evaluate calpain-1 expression under different conditions. All western blot analyses were performed as three independent experiments.

Reverse transcription-quantitative polymerase chain reaction (RT-qPCR)

Total RNA was extracted from HaCaT cells with the GenElute Mammalian Total RNA Miniprep kit (cat. no. RTN70-1KT; Sigma-Aldrich; Merck KGaA). Complementary (c)DNA was synthesized with the High Capacity cDNA Reverse Transcription kit (cat. no. 4368814; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocols. To measure the expression levels of interleukin (IL)-1α, IL-6, IL-8 and tumor necrosis factor (TNF)-α, PCR amplification was performed in the 7500 system (Applied Biosystems; Thermo Fisher Scientific, Inc.). Primers and TaqMan probes for detecting IL-1α (assay ID, Hs00174092_m1), IL-6 (assay ID, Hs00985639_m1), IL-8 (assay ID, Hs00174103_m1), TNF-α (assay ID, Hs01113624_g1) and 18S ribosomal (r)RNA (assay ID, Hs03928990_g1) were supplied with the TaqMan Gene Expression Assay (Applied Biosystems; Thermo Fisher Scientific, Inc.). The relative gene expression was calculated via the ΔΔCq method (42-46). The ΔΔCq method uses the normalized ΔCq value of each sample, which was calculated with 18S rRNA as the endogenous control gene. The ΔΔCq value is the difference between treated and control samples. Finally, the fold-change was determined as 2−ΔΔCq. Gene expression was evaluated in triplicate.

Statistical analysis

All data are presented as the mean ± standard error of the mean. The data were analyzed by one-way analysis of variance followed by Dunnett's test or the unpaired Student's t-test for two groups. P<0.05 was considered to indicate a signifi-cant difference. Computations were performed with GraphPad Prism version 5.1 (GraphPad Software Inc., La Jolla, CA, USA).

Results

Tryptic digestion of collagen from soft-shelled turtle

Collagen extracted from soft-shelled turtle was digested with trypsin to obtain CPs with molecular weights of <3.5 kDa. The collagen digestion was monitored by extracting samples at different time-points. The samples were separated on a 15% tricine-SDS-PAGE gel (Fig. 1A). After 1 h of trypsin digestion, bands that corresponded to collagen or CPs at around 42 kDa were observed (Fig. 1A; black square). After 96 h, these bands completely disappeared (Fig. 1A). The molecular weight distribution of the digested CPs was evaluated using MALDI-TOF/MS. The results indicated that the collagen was digested to CPs with a molecular weight of <4.0 kDa, and most CPs had mass-to-charge (m/z) ratios of 800-2,500 (Fig. 1B).

Cytotoxicity of CPs to HaCaT cells

To examine the possible cytotoxic effects of CPs on HaCaT cells, it was assessed whether the cell growth rate was affected when the cells were grown in culture medium containing CPs at concentrations of 0.1-100 μg/ml. The results indicated that CPs did not inhibit the HaCaT cell growth rate at any of the tested concentrations (Fig. 2). Therefore, the CPs were used at a concentration of 100 μg/ml in the subsequent experiments.

Identification and semi-quantitative comparison of differen- tially expressed proteins in CP-treated HaCaT cells

Next, the potential effect of CPs on cells in the basal layer of the skin was investigated by treating HaCaT cells with CPs. To determine the molecular profile of proteins that are regulated by CPs, a shotgun proteomics approach was used. A label-free semi-quantitative method based on spectral counting was utilized to evaluate the proteins expressed in HaCaT cells. The Rsc values were calculated for proteins that had been identified in CP-treated HaCaT cells and untreated cells. A positive value indicated increased expression by CP-treatment and a negative value indicated reduced expression by CP-treatment (Fig. 3; light grey area). For each protein that had been identified in CP-treated HaCaT cells and untreated cells, the NSAF value was also calculated (Fig. 3; black bars; peptides, grey bar; control). Proteins with a >1 and <-1 Rsc value were considered candidate CP-regulated proteins.

Based on this semi-quantitative procedure, a total of 211 proteins that were differentially expressed with CP treatment were identified (Table I). The expression of housekeeping proteins, including β-actin and GAPDH, was not altered by CP treatment.

Table I

Proteins differentially expressed (≥2-fold) after treatment with collagen peptides.

Table I

Proteins differentially expressed (≥2-fold) after treatment with collagen peptides.

IDAccession numberDefinitionNumber of amino acidsFold change (Rsc)
TBA1B_HUMANP68363Tubulin α-1B chain451−2.937
H2B1L_HUMANQ99880Histone H2B type 1-L126−2.890
H2B1B_HUMANP33778Histone H2B type 1-B126−2.683
H2B1K_HUMANO60814Histone H2B type 1-K126−2.683
K2C75_HUMANO95678Keratin, type II cytoskeletal 75551−2.507
K2C4_HUMANP19013Keratin, type II cytoskeletal 4534−2.443
K2C79_HUMANQ5XKE5Keratin, type II cytoskeletal 79535−2.073
ATPA_HUMANP25705ATP synthase subunit α, mitochondrial553−2.073
HS902_HUMANQ14568Putative heat shock protein HSP 90-α A2343−1.691
HSP7C_HUMANP11142Heat shock cognate 71 kDa protein646−1.577
H2A1B_HUMANP04908Histone H2A type 1-B/E130−1.577
K2C6C_HUMANP48668Keratin, type II cytoskeletal 6C564−1.409
K2C5_HUMANP13647Keratin, type II cytoskeletal 5590−1.384
TBA3C_HUMANQ13748Tubulin α-3C/D chain450−1.383
HS90B_HUMANP08238Heat shock protein HSP 90-β724−1.343
K1C19_HUMANP08727Keratin, type I cytoskeletal 19400−1.245
TBA3E_HUMANQ6PEY2Tubulin α-3E chain450−1.171
TPIS_HUMANP60174Triosephosphate isomerase286−1.171
LMNA_HUMANP02545Prelamin-A/C664−1.171
K2C3_HUMANP12035Keratin, type II cytoskeletal 3628−1.171
PPIA_HUMANP62937Peptidyl-prolyl cis-trans isomerase A165−1.125
K2C73_HUMANQ86Y46Keratin, type II cytoskeletal 73540−1.005
HS904_HUMANQ58FG1Putative heat shock protein HSP 90-α A4418−1.005
RS12_HUMANP2539840S ribosomal protein S12132−1.005
H2A1A_HUMANQ96QV6Histone H2A type 1-A131−1.005
FAS_HUMANP49327Fatty acid synthase2,511−1.005
K1C18_HUMANP05783Keratin, type I cytoskeletal 184301.009
KRT84_HUMANQ9NSB2Keratin, type II cuticular Hb46001.029
H2B1A_HUMANQ96A08Histone H2B type 1-A1271.029
PSA2_HUMANP25787Proteasome subunit α type-22341.029
PHB2_HUMANQ99623Prohibitin-22991.029
ALBU_HUMANP02768Serum albumin6091.029
SPTN1_HUMANQ13813Spectrin α chain, non-erythrocytic 12,4721.029
S10AE_HUMANQ9HCY8Protein S100-A141041.029
HSPB1_HUMANP04792Heat shock protein β-12051.029
PDIA3_HUMANP30101Protein disulfide-isomerase A35051.071
1433S_HUMANP3194714-3-3 protein σ2481.071
ACTG_HUMANP63261Actin, cytoplasmic 23751.076
ACTBM_HUMANQ9BYX7Putative β-actin-like protein 33751.165
RSSA_HUMANP0886540S ribosomal protein SA2951.165
IASPP_HUMANQ8WUF5RelA-associated inhibitor8281.190
FUMH_HUMANP07954Fumarate hydratase, mitochondrial5101.190
HMGB1_HUMANP09429High mobility group protein B12151.190
COR1B_HUMANQ9BR76Coronin-1B4891.190
COTL1_HUMANQ14019Coactosin-like protein1421.190
LA_HUMANP05455Lupus La protein4081.190
NCBP1_HUMANQ09161Nuclear cap-binding protein subunit 17901.190
SQRD_HUMANQ9Y6N5Sulfide:Quinone oxidoreductase, mitochondrial4501.190
TRAP1_HUMANQ12931Heat shock protein 75 kDa, mitochondrial7041.190
EZRI_HUMANP15311Ezrin5861.190
RL22_HUMANP3526860S ribosomal protein L221281.190
RS3_HUMANP2339640S ribosomal protein S32431.190
ARF4_HUMANP18085ADP-ribosylation factor 41801.190
COR1C_HUMANQ9ULV4Coronin-1C4741.190
MARE1_HUMANQ15691 Microtubule-associated protein RP/EB family member 12681.190
MYO1B_HUMANO43795Unconventional myosin-Ib1,1361.190
FREM1_HUMANQ5H8C1FRAS1-related extracellular matrix protein 12,1791.190
VPS35_HUMANQ96QK1Vacuolar protein sorting-associated protein 357961.190
TBA1C_HUMANQ9BQE3Tubulin α-1C chain4491.242
PRDX6_HUMANP30041 Peroxiredoxin-62241.257
SERPH_HUMANP50454Serpin H14181.257
AN32B_HUMANQ92688Acidic leucine-rich nuclear phosphoprotein 32 family member B2511.334
CX6B1_HUMANP14854Cytochrome c oxidase subunit 6B1861.334
ROA1_HUMANP09651Heterogeneous nuclear ribonucleoprotein A13721.334
COX5A_HUMANP20674Cytochrome c oxidase subunit 5A, mitochondrial1501.417
PSME1_HUMANQ06323Proteasome activator complex subunit 12491.417
4F2_HUMANP081954F2 cell-surface antigen heavy chain6301.417
K1C9_HUMANP35527Keratin, type I cytoskeletal 96231.528
LEG7_HUMANP47929Galectin-71361.721
EIF3M_HUMANQ7L2H7Eukaryotic translation initiation factor 3 subunit M3741.721
HNRDL_HUMANO14979Heterogeneous nuclear ribonucleoprotein D-like4201.721
TCPB_HUMANP78371T-complex protein 1 subunit β5351.721
CPNE3_HUMANO75131Copine-35371.721
DEST_HUMANP60981Destrin1651.721
TFR1_HUMANP02786Transferrin receptor protein 17601.721
CNDP2_HUMANQ96KP4Cytosolic non-specific dipeptidase4751.721
AHSA1_HUMANO95433Activator of 90 kDa heat shock protein ATPase homolog 13381.721
DDB1_HUMANQ16531DNA damage-binding protein 11,1401.721
ICAL_HUMANP20810Calpastatin7081.721
ASNA_HUMANO43681ATPase ASNA13481.721
CISY_HUMANO75390Citrate synthase, mitochondrial4661.721
CSK23_HUMANQ8NEV1Casein kinase II subunit α 33911.721
CNN2_HUMANQ99439Calponin-23091.721
SYYC_HUMANP54577Tyrosine-tRNA ligase, cytoplasmic5281.721
SODM_HUMANP04179Superoxide dismutase [Mn], mitochondrial2221.721
ACTY_HUMANP42025B-centractin3761.721
ML12A_HUMANP19105Myosin regulatory light chain 12A1711.721
RL32_HUMANP6291060S ribosomal protein L321351.721
MYO1C_HUMANO00159Unconventional myosin-Ic1,0631.721
FSCN1_HUMANQ16658Fascin4931.722
RL3_HUMANP3902360S ribosomal protein L34031.722
RPN1_HUMANP04843 Dolichyl-diphosphooligosaccharide-protein glycosyltransferase subunit 16071.722
B2MG_HUMANP61769 B-2-microglobulin1191.722
RBP56_HUMANQ92804TATA-binding protein-associated factor 2N5921.722
2AAA_HUMANP30153 Serine/threonine-protein phosphatase 2A 65 kDa regulatory subunit A α isoform5891.722
ENOG_HUMANP09104Γ-enolase4341.722
RL27A_HUMANP4677660S ribosomal protein L27a1481.722
DCD_HUMANP81605Dermcidin1101.722
RS7_HUMANP6208140S ribosomal protein S71941.722
IQGA1_HUMANP46940Ras GTPase-activating-like protein IQGAP11,6571.723
TERA_HUMANP55072Transitional endoplasmic reticulum ATPase8061.723
SPTB2_HUMANQ01082Spectrin β chain, non-erythrocytic 12,3641.723
RL18A_HUMANQ0254360S ribosomal protein L18a1761.723
CALX_HUMANP27824Calnexin5921.723
TBA4B_HUMANQ9H853Putative tubulin-like protein α-4B2411.724
TCPE_HUMANP48643T-complex protein 1 subunit ε5411.724
K2C80_HUMANQ6KB66Keratin, type II cytoskeletal 804521.724
HNRPK_HUMANP61978Heterogeneous nuclear ribonucleoprotein K4631.865
VPP4_HUMANQ9HBG4V-type proton ATPase 116 kDa subunit a isoform 48402.029
EF1G_HUMANP26641Elongation factor 1-γ4372.110
G6PI_HUMANP06744Glucose-6-phosphate isomerase5582.110
H2B1M_HUMANQ99879Histone H2B type 1-M1262.119
RALY_HUMANQ9UKM9RNA-binding protein Raly3062.253
TXTP_HUMANP53007Tricarboxylate transport protein, mitochondrial3112.253
CAN1_HUMANP07384Calpain-1 catalytic subunit7142.253
DHB12_HUMANQ53GQ0Estradiol 17-β-dehydrogenase 123122.253
DHX9_HUMANQ08211ATP-dependent RNA helicase A1,2702.253
MYADM_HUMANQ96S97Myeloid-associated differentiation marker3222.253
CUTA_HUMANO60888Protein CutA1792.253
CECR2_HUMANQ9BXF3Cat eye syndrome critical region protein 21,4842.253
KRT81_HUMANQ14533Keratin, type II cuticular Hb15052.569
GLOD4_HUMANQ9HC38Glyoxalase domain-containing protein 43132.569
SURF4_HUMANO15260Surfeit locus protein 42692.569
P4HA1_HUMANP13674Prolyl 4-hydroxylase subunit α-15342.569
ANX11_HUMANP50995Annexin A115052.569
CALU_HUMANO43852Calumenin3152.569
TFG_HUMANQ92734Protein TFG4002.569
ECHA_HUMANP40939Trifunctional enzyme subunit α, mitochondrial7632.569
PA2G4_HUMANQ9UQ80 Proliferation-associated protein 2G43942.569
SF3A2_HUMANQ15428Splicing factor 3A subunit 24642.569
SHLB2_HUMANQ9NR46Endophilin-B23952.569
MBOA7_HUMANQ96N66Lysophospholipid acyltransferase 74722.569
AT1A1_HUMANP05023 Sodium/potassium-transporting ATPase subunit α-11,0232.569
PPME1_HUMANQ9Y570Protein phosphatase methylesterase 13862.569
IF4G2_HUMANP78344Eukaryotic translation initiation factor 4 γ 29072.569
IPYR2_HUMANQ9H2U2Inorganic pyrophosphatase 2, mitochondrial3342.569
CKAP4_HUMANQ07065 Cytoskeleton-associated protein 46022.569
COPB2_HUMANP35606Coatomer subunit β9062.569
DLG1_HUMANQ12959Disks large homolog 19042.569
ATX2L_HUMANQ8WWM7Ataxin-2-like protein1,0752.569
RMXL1_HUMANQ96E39RNA binding motif protein, X-linked-like-13902.569
CERS2_HUMANQ96G23Ceramide synthase 23802.569
RM46_HUMANQ9H2W639S ribosomal protein L46, mitochondrial2792.569
FDFT_HUMANP37268Squalene synthase4172.569
CP26A_HUMANO43174Cytochrome P450 26A14972.569
EIF3A_HUMANQ14152Eukaryotic translation initiation factor 3 subunit A1,3822.569
GLYM_HUMANP34897Serine hydroxymethyltransferase, mitochondrial5042.569
ARHGJ_HUMANQ8IW93Rho guanine nucleotide exchange factor 198022.569
GFPT1_HUMANQ06210 Glutamine-fructose-6-phosphate aminotransferase[isomerizing] 16992.569
NOD2_HUMANQ9HC29Nucleotide-binding oligomerization domain-containing protein 21,0402.569
NAGK_HUMANQ9UJ70 N-acetyl-D-glucosamine kinase3442.569
SYAC_HUMANP49588Alanine-tRNA ligase, cytoplasmic9682.569
CLAP1_HUMANQ7Z460CLIP-associating protein 11,5382.569
SPTN4_HUMANQ9H254Spectrin β chain, non-erythrocytic 42,5642.569
MTU1_HUMANO75648Mitochondrial tRNA-specific 2-thiouridylase 14212.569
PAOX_HUMANQ6QHF9Peroxisomal N(1)-acetyl-spermine/spermidine oxidase6492.569
MCM5_HUMANP33992DNA replication licensing factor MCM57342.569
WNK3_HUMANQ9BYP7 Serine/threonine-protein kinase WNK31,8002.569
LIMK2_HUMANP53671LIM domain kinase 26382.569
VIPR2_HUMANP41587Vasoactive intestinal polypeptide receptor 24382.569
DOS_HUMANQ8N350Protein Dos7252.569
TRDN_HUMANQ13061Triadin O7292.569
ZN318_HUMANQ5VUA4Zinc finger protein 3182,2792.569
SUCO_HUMANQ9UBS9SUN domain-containing ossification factor1,2542.569
2AAB_HUMANP30154 Serine/threonine-protein phosphatase 2A 65 kDa regulatory subunit A β isoform6012.569
PCBP2_HUMANQ15366Poly(rC)-binding protein 23652.569
1433Z_HUMANP6310414-3-3 protein ζ/δ2452.569
MDHC_HUMANP40925Malate dehydrogenase, cytoplasmic3342.569
DNM1L_HUMANO00429Dynamin-1-like protein7362.569
ARL8A_HUMANQ96BM9ADP-ribosylation factor-like protein 8A1862.569
DIAP1_HUMANO60610Protein diaphanous homolog 11,2722.569
IF4H_HUMANQ15056Eukaryotic translation initiation factor 4H2482.569
TEX35_HUMANQ5T0J7Testis-expressed sequence 35 protein2332.569
GGCT_HUMANO75223 Γ-glutamylcyclotransferase1882.569
SF3A1_HUMANQ15459Splicing factor 3A subunit 17932.569
ARPC2_HUMANO15144Actin-related protein 2/3 complex subunit 23002.569
PP14B_HUMANQ96C90Protein phosphatase 1 regulatory subunit 14B1472.569
ARHGH_HUMANQ96PE2Rho guanine nucleotide exchange factor 172,0632.569
SYSC_HUMANP49591Serine-tRNA ligase, cytoplasmic5142.569
ALPK2_HUMANQ86TB3A-protein kinase 22,1702.569
SUSD3_HUMANQ96L08Sushi domain-containing protein 32552.569
DP13B_HUMANQ8NEU8DCC-interacting protein 13-β6642.569
XRCC6_HUMANP12956X-ray repair cross-complementing protein 66092.569
HBS1L_HUMANQ9Y450HBS1-like protein6842.569
RK_HUMANQ15835Rhodopsin kinase5632.569
SPRY7_HUMANQ5W111SPRY domain-containing protein 71962.569
BRSK2_HUMANQ8IWQ3 Serine/threonine-protein kinase BRSK27362.569
STAT1_HUMANP42224Signal transducer and activator of transcription 1-α/β7502.569
ZEP1_HUMANP15822Zinc finger protein 402,7182.569
SOX4_HUMANQ06945Transcription factor SOX-44742.569
REXO1_HUMANQ8N1G1RNA exonuclease 1 homolog1,2212.569
ZN521_HUMANQ96K83Zinc finger protein 5211,3112.569
DLG5_HUMANQ8TDM6Disks large homolog 51,9192.569
TM155_HUMANQ4W5P6Protein TMEM1551302.569
ZYX_HUMANQ15942Zyxin5722.569
UBAC2_HUMANQ8NBM4 Ubiquitin-associated domain-containing protein 23442.569
STPG2_HUMANQ8N412Sperm-tail PG-rich repeat-containing protein 24592.569
K0556_HUMANO60303Uncharacterized protein KIAA05561,6182.569
KLH11_HUMANQ9NVR0Kelch-like protein 117082.569
TTLL6_HUMANQ8N841Tubulin polyglutamylase TTLL68432.569
CFA36_HUMANQ96G28Cilia- and flagella-associated protein 363422.569
MMP14_HUMANP50281Matrix metalloproteinase-145823.101
ADT1_HUMANP12235ADP/ATP translocase 12983.101
GPNMB_HUMANQ14956Transmembrane glycoprotein NMB5723.101
PYGL_HUMANP06737Glycogen phosphorylase, liver form8473.101
NIPS2_HUMANO75323Protein NipSnap homolog 22863.101
ERP29_HUMANP30040Endoplasmic reticulum resident protein 292613.101
ADT3_HUMANP12236ADP/ATP translocase 32983.101
LBN_HUMANQ86UK5Limbin1,3083.101
RAB1B_HUMANQ9H0U4Ras-related protein Rab-1B2013.201
TF_HUMANP13726Tissue factor2953.490

[i] Rsc, log2 of the ratio of protein abundance; ATP, adenosine triphosphate; ADP, adenosine diphosphate; FRAS-1, fraser syndrome 1; MCM, mini-chromosome maintenance; WNK, with no K (lysine); DCC, deleted in colorectal cancer.

Functional annotation of proteins regulated by CPs

A gene ontology (GO) analysis of the candidate CP-regulated proteins was then performed. GO terms associated with 'pathway' (Fig. 4A) and 'molecular function' (Fig. 4B) were searched for in DAVID. The search focused on proteins classified as 'protein processing in endoplasmic reticulum' (Table II).

Table II

Differentially expressed proteins categorized as 'protein processing in endoplasmic reticulum' in a Gene Ontology analysis in human skin keratinocytes.

Table II

Differentially expressed proteins categorized as 'protein processing in endoplasmic reticulum' in a Gene Ontology analysis in human skin keratinocytes.

Accession numberDescriptionFold change (Rsc)
P11142Heat shock cognate 71 kDa protein−1.577
P08238Heat shock protein HSP 90-β−1.343
P30101Protein disulfide-isomerase A31.071
P04843 Dolichyl-diphosphooligosaccharide-protein glycosyltransferase subunit 11.722
P27824 Calnexin1.723
P55072Transitional endoplasmic reticulum ATPase1.723
P07384Calpain-1 catalytic subunit2.253
Q07065 Cytoskeleton-associated protein 42.569
P30040Endoplasmic reticulum resident protein 293.101

[i] Rsc, log2 of the ratio of protein abundance.

Effect of CP on calpain-1 expression in HaCaT cells

To confirm that CP treatment altered caipain-1 expression, caipain-1 protein levels were examined in CP-treated HaCaT cells. It was identified that caipain-1 expression was signifi-cantly increased with CP treatment compared with that in control cells (Fig. 5).

Effect of CP treatment on the expression of inflammatory factors in HaCaT cells

To investigate whether the CP-induced increase in calpain-1 expression was associated with the expression of inflammatory cytokines in HaCaT cells, expression levels of IL-1α, IL-6, IL-8 and TNF-α were examined (Fig. 6). Although CP-treated HaCaT cells tended to exhibit increases in IL-1α expression (Fig. 6A), the change was not significant (P=0.0781). However, IL-6 expression was significantly decreased in HaCaT cells treated with CP, and by contrast, IL-8 expression was significantly increased with CP treatment (Fig. 6B and C). Finally, CPs had no effect on TNF-α expression (Fig. 6D).

Discussion

In the present study, a gel-free LC-MS-based proteomics approach was used to examine the functional effects of soft-shelled turtle CPs on human skin cells. A total of 211 proteins that exhibited >2-fold changes in expression after CP treatment were successfully identified in HaCaT cells, based on a semi-quantitative method of spectral counting. To examine the roles of these identified proteins, a GO analysis was performed. The study focused on the functions of proteins classified as 'protein processing in endoplasmic reticulum', as they have important roles in the synthesis of correctly folded proteins and in the degradation of misfolded proteins. The function of calpain-1, which is a member of this pathway, was also examined.

To validate the spectral counting results, a western blot analysis was performed to examine whether calpain-1 expression is increased in HaCaT cells with CP treatment. Calpain-1 is a calcium-dependent intracellular cysteine protease (47,48). It has an important role in various biological processes, including cell proliferation, cell migration, apoptosis and cytoskeletal remodeling (49,50). Therefore, calpain is considered a therapeutic target for disorders involving inflammation, wound healing and tumor progression. A previous study reported that downregulation of calpain-1 expression in IgE-activated mast cells led to a reduced expression of cytokines, including IL-6 and TNF-α. Thus, they concluded that calpain-1 may regulate IgE-mediated allergic inflammation (51). Another study indicated that downregulation of calpain-1 expression in lung fibroblast cells also reduced the expression of cytokines, including IL-6, IL-8, and TNF-α (52). Furthermore, calpain-1 knockout mice exhibited impaired bactericidal activity in an acute bacterial peritonitis model, due to a reduction in IL-1α production (53). Therefore, calpain-1 is considered a key factor in the immune response through its regulation of inflammation. Accordingly, it was hypothesized that CP-induced increases in calpain-1 expression in HaCaT cells may affect the expression of inflammatory cytokines in keratinocytes. The present results support this hypothesis. Therefore, CP treatment may regulate the immune system in the setting of skin wounds.

In the present GO analysis in the category molecular function, several proteins classified as 'cadherin binding involved in cell-cell adhesion' exhibited altered expression with CP treatment. A previous study by our group reported that in HaCaT cells, these proteins were changed by treatment with collagen derived from soft-shelled turtle. Furthermore, it was observed that changing the expression of these proteins enhanced the wound healing properties of HaCaT cells by inducing EMT (34). In the present study, it was revealed that CP treatment was associated with >2-fold increases in ceramide synthase 2 expression in HaCaT cells compared with that in control cells (Table I). Ceramide is an epidermal sphingolipid that has important roles in maintaining the skin barrier and supporting wound healing processes (54-56). The present result is similar to previous ones reported for collagen derived from soft-shelled turtles and for CPs derived from other origins (25,31,32,57). It is therefore suggested that CPs from soft-shelled turtles may facilitate wound healing.

Although the present study suggested that CPs from soft-shelled turtles may be a useful material for pharmaceuticals and medical products, it remains elusive whether these CPs affect the wound healing processes in keratinocytes in vivo. Of note, certain CP-regulated inflammatory cytokines including IL-6 and IL-8 were not been investigated to determine their potential effects on the wound healing process in keratinocytes. Further in vitro and in vivo studies are necessary to clarify the effect of CPs from soft-shelled turtle on wound healing and the role of CP-regulated inflammatory cytokines in keratinocytes. In addition, it is also necessary to investigate the correlation between the expression of calpain-1 and inflammatory cytokines in CP-treated keratinocytes.

In conclusion, the present shotgun LC/MS-based global proteomic analysis revealed that CP treatment regulated the expression of inflammatory cytokines in HaCaT cells and induced the expression of proteins associated with cell-cell adhesion and skin barrier maintenance. Therefore, CPs from soft-shelled turtles may provide significant benefits in maintaining the biological environment of the skin. These peptides may be a useful material for pharmaceuticals and medical products.

Funding

This study was supported in part by a Grant-in-Aid for Scientific Research (C) from the Japanese Society for the Promotion of Science to T.Y. (grant no. 15K09054).

Availability of data and materials

All data generated or analyzed during this study are included in this published article.

Authors' contributions

TY and AT designed the study and analyzed the data. TY and SN performed the experimental work. TY drafted the manuscript. KM contributed conduct the literature review. AT critically evaluated the study and final version of the manuscript. All authors participated in discussion of the study and gave final approval.

Ethical approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Abbreviations:

MALDI-TOF/MS

matrix-assisted laser desorptiontime of flight/mass spectrometry

LC-MS/MS

liquid chromatography tandem mass spectrometry

NSAF

normalized spectral abundance factor

RT-PCR

reverse transcription-polymerase chain reaction

IL

interleukin

TNF

tumor necrosis factor

Acknowledgments

The authors are grateful to Mr. Takashi Aboshi (Shin-uoei, Inc.) for providing the soft-shelled turtle tissue used in the present study.

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August-2018
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Yamamoto T, Nakanishi S, Mitamura K and Taga A: Collagen peptides from soft‑shelled turtle induce calpain‑1 expression and regulate inflammatory cytokine expression in HaCaT human skin keratinocytes. Int J Mol Med 42: 1168-1180, 2018.
APA
Yamamoto, T., Nakanishi, S., Mitamura, K., & Taga, A. (2018). Collagen peptides from soft‑shelled turtle induce calpain‑1 expression and regulate inflammatory cytokine expression in HaCaT human skin keratinocytes. International Journal of Molecular Medicine, 42, 1168-1180. https://doi.org/10.3892/ijmm.2018.3659
MLA
Yamamoto, T., Nakanishi, S., Mitamura, K., Taga, A."Collagen peptides from soft‑shelled turtle induce calpain‑1 expression and regulate inflammatory cytokine expression in HaCaT human skin keratinocytes". International Journal of Molecular Medicine 42.2 (2018): 1168-1180.
Chicago
Yamamoto, T., Nakanishi, S., Mitamura, K., Taga, A."Collagen peptides from soft‑shelled turtle induce calpain‑1 expression and regulate inflammatory cytokine expression in HaCaT human skin keratinocytes". International Journal of Molecular Medicine 42, no. 2 (2018): 1168-1180. https://doi.org/10.3892/ijmm.2018.3659