Modulation of Cell Adhesion and Differentiation on Collagen Gels by

Subscriber access provided by UniSA Library

Article

Modulation of Cell Adhesion and Differentiation on Collagen Gels by the Addition of the Ovalbumin Secretory Signal Peptide Chie Kojima, Yuri Narita, Yusuke Nakajima, Naoya Morimoto, Takashi Yoshikawa, Nobuyuki Takahashi, Akihiro Handa, Tomonori Waku, and Naoki Tanaka ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.8b01505 • Publication Date (Web): 18 Apr 2019 Downloaded from http://pubs.acs.org on April 20, 2019

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 39 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Biomaterials Science & Engineering

Modulation of Cell Adhesion and Differentiation on Collagen Gels by the Addition of the Ovalbumin Secretory Signal Peptide Chie Kojima1*, Yuri Narita2, Yusuke Nakajima1, Naoya Morimoto2, Takashi Yoshikawa2, Nobuyuki Takahashi3, Akihiro Handa4, Tomonori Waku2*, Naoki Tanaka2$

1Department of Applied Chemistry, Graduate School of Engineering, Osaka Prefecture University,

1-2 Gakuen-cho, Naka-ku, Sakai, Osaka 599-8570, Japan 2Faculty

of Molecular Chemistry and Engineering, Kyoto Institute of Technology, Gosyokaido-

cho, Matsugasaki, Sakyo-ku, Kyoto 606-8585, Japan 3Laboratory

of Applied Structural Biology, Division of Applied Life Sciences, Graduate School

of Agriculture, Kyoto University, Gokasho, Uji, Kyoto, 611-0011, Japan 4R

& D Division, Kewpie Corporation, 2-5-7 Sengawa-cho, Chofu, Tokyo, 182-0002, Japan

$Deceased

13th November 2018.

*To whom correspondence should be addressed. CK: Department of Applied Chemistry, Graduate School of Engineering, Osaka Prefecture University, 1-2 Gakuen-cho, Naka-ku, Sakai, Osaka 599-8570, Japan. Tel.: +81 72 254 8190; Fax: +81 72 254 8190; E-mail: [email protected]

ACS Paragon Plus Environment

1

ACS Biomaterials Science & Engineering 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 39

TW: Faculty of Molecular Chemistry and Engineering, Kyoto Institute of Technology, Gosyokaido-cho, Matsugasaki, Sakyo-ku, Kyoto 606-8585, Japan. Tel.: +81 75 724 7811; Fax: +81 75 724 7800; E-mail: [email protected]

ABSTRACT

Ovalbumin (OVA) is the most abundant protein in egg whites, that is unnecessary in the egg yolkbased food industry. The development of OVA-based functional materials is of great interest. Collagen is a major component of the extracellular matrix. In this study, an OVA fragment, the OVA secretory signal peptide (OVA SP), was loaded in collagen gels, which were used as a cell scaffold for various types of cells. And, we examined the effect of OVA SP loaded in collagen gels to cell properties. The peptide was initially bound to the collagen fibers and then released from the gel. Our results indicate that the released OVA SP suppressed the integrin-mediated cell adhesion and focal adhesion formation. However, the adhesion of NIH3T3 cells was not suppressed by treatment with EDTA and an anti-1 antibody. These suggest that OVA SP nonspecifically interacts with cell surface proteins. The adhesion of various cell types on collagen gels were changed by the addition of OVA SP, depending on their integrin expression pattern. Additionally, the differentiation of MC3T3-E1 osteoblastic cells was promoted on the OVA SPloaded collagen gels. This suggests that OVA SP may modulate both the differentiation and the adhesion of cells cultured on the collagen gels.

KEYWORDS. collagen, hydrogel, ovalbumin, secretory peptide, integrin, cell scaffold


2

Page 3 of 39 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Introduction Ovalbumin (OVA) is the most abundant protein in egg whites.1 During the production of food products using egg yolks, such as sweets and mayonnaise, a large amount of the egg whites become unnecessary. Thus, developing OVA-based functional materials is of great interest. Although OVA belongs to the serpin superfamily categorized into a major secretory protein, its predominant role is unknown. The OVA secretory signal sequence (1–43) contains the hydrophilic α helix A (hA, 1–22) and the hydrophobic α helix B (hB, 32–43) regions. Previous reports indicate that the N-terminal region of OVA is important for secretion.2 The peptide fragment of the hA region (pN1– 22,

acetyl-GSIGAASMEFCFDVFKELKVHH) can be specifically separated from the native OVA

by treatment with pepsin at pH 4.3 This peptide is named OVA secretory signal peptide (OVA SP) in this study (Figure 1). Collagen, a major component of the extracellular matrix, is widely used as a cell scaffold. Cell scaffolds are used for in vivo experiments to improve the viability and functions of implant cells.4 And, controlling the properties of collagen gels in cell adhesion and differentiation processes is useful for tissue and cell engineering.4-6 There are 28 known isoforms of collagen in humans; type I collagen forms fibrils to provide mechanical strength for tissues.5 Thus, type I collagen is generally used for preparation of collagen gels. Integrins are heterodimeric transmembrane receptors, which are involved in cell adhesion. They transmit divergent signals into the cell, such as those for proliferation, differentiation, and survival. It has been reported that proliferation and differentiation are regulated by the interaction between integrins and collagen.5,6 Thus, the modulation of this interaction may regulate the cell properties on a collagen gel.


3


Page 4 of 39

We previously reported that OVA SP formed amyloid-like fibrillar aggregates, promoted the formation of OVA aggregates, and enhanced the stiffness of OVA hydrogels.7 Interestingly, the peptide also promoted the formation of aggregates of collagen fibers, and enhanced the stiffness of collagen hydrogels.7 Because the substrate stiffness affects both cell differentiation and proliferation, controlling the physicochemical properties of collagen gels is important to develop suitable cell culture systems.8 In a previous work, we prepared OVA SP-loaded collagen gels as a cell scaffold and examined the physicochemical properties.9 In this study, we elucidated the involvement of OVA SP in cell adhesion and differentiation, and demonstrated that the collagen gels loaded with OVA SP may be useful for tissue engineering. First, adhesive properties of NIH3T3 cells (mouse embryonic fibroblasts) to the OVA SP-loaded collagen gels were examined to elucidate a possible mechanism for the interaction of OVA SP. Various cells with different integrin expression patterns were cultured on OVA SP-loaded collagen gels to investigate the effect of OVA SP on the cell adhesion and differentiation.

Figure 1. 3D Structure of OVA. The region of OVA SP is colored in blue.

2. Experimental Section


4

Page 5 of 39 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


2.1. Materials pN1–22

(OVA

SP)

was

obtained

from

GenScript

(Piscataway,

NJ,

USA).

5-

Iodoacetamidofluorescein (5-IAF), mouse monoclonal anti-vinculin-FITC antibody, bovine serum albumin (BSA) and normal goat serum were purchased from Sigma-Aldrich (St. Louis, MO, USA). Collagen type I-A was acquired from Nitta Gelatin, Inc. (Osaka, Japan). Fetal bovine serum (FBS) was purchased from Biowest (Nuaillé, France). Purified anti-mouse/rat CD29 antibody (HMβ1-1) was obtained from BioLegend (San Diego, CA, USA). Cell Counting Kit-8 was purchased from Dojindo Molecular Technologies (Kumamoto, Japan). NIH3T3 cells were obtained from the Japanese Collection of Research Bioresources Cell Bank (Osaka, Japan). RAW264, MC3T3-E1, and MDCK cells were obtained from the RIKEN Bioresource Research Center (BRC) through the National Bio-Resource Project of MEXT (Japan).

2.2. Preparation of OVA SP-loaded collagen gels OVA SP-loaded collagen gels were prepared, according to our previous report.9 Briefly, 1.4 mL of type I-A collagen solution (3.0 mg/mL), 0.4 mL of 5 Dulbecco's Modified Eagle Medium (DMEM) without NaHCO3, 0.2 mL of the reconstruction solution (50 mM NaOH, 200 mM HEPES, and 260 mM NaHCO3) with or without OVA SP were mixed on ice in that order. The solution (0.5 mL) was then added to a 24-well plate and as incubated at 37 °C for 1 h to form the hydrogel.

2.3. Preparation of fluorescent OVA SP and its observation in collagen gels


5


Page 6 of 39

Four equivalents of 5-IAF were mixed with OVA SP. Then, the solution pH was adjusted to 9.86 and the reaction was performed at 37 °C for 1 h. The mixture was purified with ultrafiltration using a CENTRI SPIN-10 Column (Princeton Separations, Inc., NJ, USA), followed by freezedrying. The obtained sample was characterized by MALDI TOF-mass spectrometry using an autoflex speed-kF spectrometer (Bruker Daltonics K. K., Japan). The observed mass was 2,823 ([M+H] +, calculated 2,823). A solution of 3% fluorescent dye-labeled OVA SP was prepared, and 100 M OVA SP of the solution were loaded into a collagen gel. The prepared hydrogel was observed on a cover glass by total internal reflection fluorescence microscopy (TIRFM, TE2000-TIRF2, Nikon, Tokyo, Japan). An Ar+ laser (IMA1010 40ALS; CVI Melles Griot, Albuquerque, NM, USA) was used for excitation (488 nm), and the fluorescence emission was collected using an oil-immersion microscope objective lens (100x). Fluorescence images were obtained using a band-pass filter for GFP (Nikon) and an electron multiplier CCD camera (ImagEM C9100-13, Hamamatsu Photonics, Shizuoka, Japan).

2.4. Release of fluorescent OVA SP from the collagen gel Fluorescent dye-labeled OVA SP and the unlabeled peptide were mixed at a 1:2 ratio to control the fluorescent dye concentration. The mixed OVA SP-loaded collagen gels (500 L, OVA SP 100 M, dye 33 M) were prepared in a 24-well-plate by mixing type I-A collagen solution and 100 mM phosphate buffer (pH 7.5), followed by the incubation at 25 °C for 1 h.9 Phosphate buffer (1 mL, 100 mM, pH 7.5) was added to the gel, and the absorbance at 491 nm was measured with


6

Page 7 of 39 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


a UV-1650 PC spectrophotometer (Shimadzu Corp., Kyoto, Japan). The 100% release was defined as the point where the dye concentration in the solution was 11 M.

2.5. Cell culture HeLa and NIH3T3 cells were cultured at 37 °C under a 5% CO2 atmosphere, using 10% and 5% FBS-containing DMEM, respectively. RAW264.7 and MDCK cells were cultured using 10% FBS-containing minimal essential medium (MEM) with nonessential amino acids. MC3T3-E1 cells were cultured using 10% FBS-containing MEM alpha. The differentiation medium was prepared by adding β-glycerophosphate (10 mM) and L-ascorbic acid (50 μg/mL) to the growth medium. MC3T3-E1 cells were seeded on the OVA SP-loaded collagen gels and cultured in the growth medium for 24 h. Differentiation was induced by replacing the growth medium with the differentiation one. And, the differentiation medium was changed every 72 h. After 2 weeks, the differentiated cells were fixed with 4% paraformaldehyde for 15 min and then treated with von Kossa stain or alkaline phosphatase (ALP) assay to obtain their images.10 For the von Kossa stain procedure, cells were treated with 5% silver nitrate solutions prior to the observation. An ALP staining kit (Muto Pure Chemicals Co., Ltd.) was used for the ALP assay, according to the manufacturer’s instructions with a longer staining time (5 h).

2.6. Cell adhesion to OVA SP-loaded collagen gels


7


Page 8 of 39

HeLa and RAW264 cells (1.2  104 cells) were cultured on the collagen gels loaded with OVA SP at different concentrations for 48 h. Then, the gels were washed with phosphate-buffered saline (PBS) solution, and the remaining cells were observed using an inverted microscope (ECLIPSE Ti-U, Nikon, Tokyo, Japan). MC3T3-E1 and MDCK cells (3.0  103 cells) were cultured on a culture dish and on the collagen gels loaded with OVA SP at different concentrations to perform the same experiment. For the quantitative analysis, NIH3T3, HeLa, RAW264.7, and MC3T3 cells (2.4 x 103 cells/well) were cultured on the collagen gels loaded with OVA SP at different concentrations in a 96-well-plate for 48 h. Then, the number of cells in each well was evaluated using a Cell Counting Kit-8, according to the manufacturer’s instructions. The cell adhesion inhibition assay was performed according to the previous reports.11,12 For the inhibition assay using EDTA, NIH3T3 cells (4  104 cells) were cultured on the collagen gels with and without OVA SP (100 M). After 3 h of incubation in the absence or the presence of 5 mM EDTA, the gels were washed with PBS, and the remaining cells were observed and counted using an inverted microscope (IMT-2; Olympus Corp., Japan). This experiment was also carried out using the Cell Counting Kit-8. In this case, NIH3T3 cells (1.5 x 105 cells) were cultured on the collagen gels loaded with OVA SP (100 M) in a 24-well-plate. For the inhibition assay using the anti-1 integrin antibody, NIH3T3 cells (2  105 cells) were pre-incubated with the anti-CD29 antibody (10 g/mL) for 20 min in serum-free DMEM. The pre-treated cells (2.0 x 104 cells) were then seeded on the collagen gels with and without OVA SP (100 M) in a 96-well-plate and cultured for 3 h in serum-free medium. After washing with PBS, the number of adhered cells was evaluated using the Cell Counting Kit-8.


8

Page 9 of 39 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


2.7. Subcellular localization of OVA SP NIH3T3 cells (8  104 cells) were cultured overnight on 35-mm poly-D-lysine-coated glassbottomed dishes (Matsunami Glass Ind. Ltd.). The cells were washed thrice with PBS and then treated with PBS containing fluorescent OVA SP (50 M) for 1 h at 37 °C. Once washed, the cells were observed using a laser confocal microscope (LSM510 ver. 3.2 (Zeiss) or FV10i (Olympus Corp.)).

2.8. Observation of focal adhesion Collagen gels with or without OVA SP were prepared in glass-bottomed dishes with a 14-mm hole (non-coated 35-mm dishes, Matsunami Glass Ind. Ltd.). NIH3T3 cells (1  105 cells) were cultured on the collagen gels for 1 day. The cells were then washed twice with PBS and treated with cold methanol for 10 min at −20 °C. After drying, they were treated with cold acetone for 1 min at −20 °C. Again, after drying, they were washed with PBS. The cells were permealized in 0.1% Triton X-100 for 5 min, and then treated with 3% BSA in PBS for 1 h for blocking. A PBS solution with mouse monoclonal anti-vinculin-FITC antibody (1:100, 22 ng/mL) was added. After 1 h of incubation at 37 °C and a subsequent wash with PBS, the cells were observed with the laser confocal microscope.

3. Results 3.1. Behavior of OVA SP in the collagen gel


9


Page 10 of 39

In our previous work, we found that OVA SP changed the physical properties of collagen gels: the storage modulus increased and the contact angle decreased.9 In this work, the localization of OVA SP in the collagen gel was examined. OVA SP was labeled with a green fluorescent dye (5IAF) and used for preparation of the OVA SP-loaded collagen gel. The TIRFM observations revealed a fibrous structure (Figure 2), suggesting that OVA SP was adsorbed to the collagen fibers in the gel. The fluorescent dye-labeled OVA SP-loaded collagen gel was immersed in PBS, to examine the retention of OVA SP in the collagen gel. The fluorescent signal of the solution gradually increased, and after 5 h, almost all of the OVA SP was completely released from the gel (Figure 3). Thus, these suggest that the OVA SP adsorption to the collagen fibers is weak, and the OVA SP is released from the collagen gel.

Figure 2. Localization of OVA SP in the collagen gel. Scale bar: 20 m.


10

Page 11 of 39 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 3. Release of OVA SP from the collagen gel.

3.2. Influence of OVA SP on the adhesion of NIH3T3 cells to collagen gels In our previous work, the number and morphology of the adhered NIH3T3cells changed when adding the peptide to the collagen gels, suggesting that OVA SP affects the cell adhesion.9 In this work, we examined the subcellular localization of OVA SP in NIH3T3 cells using fluorescent dyelabeled OVA SP. Large aggregates of OVA SP were observed predominantly at the plasma membrane (Figure 4). Integrins are heterodimeric transmembrane receptors that are involved in cell adhesion. Because the α and β chains of integrins form a heterodimer by chelating calcium ions, the addition of chelate molecules, such as EDTA, suppresses the integrin-mediated cell adhesion.11 To


11


Page 12 of 39

determine whether OVA SP affects the integrin-mediated cell adhesion, the adhesion of NIH3T3 cells to collagen gels was compared in the absence and presence of EDTA by the cell counting method and the WST-8 assay. In the cell counting method, the number of adhered cells was lower in the presence of EDTA on the intact collagen gel (i.e., without OVA SP), suggesting that integrins mediate this process. Conversely, the number of adhered cells in OVA SP-loaded gels remained almost unchanged in the absence and presence of EDTA (Figure 5A). This suggests that the cells can adhere to the OVA SP-loaded collagen gel via an integrin-independent mechanism. In the WST-8 assay, the cell amount decreased in the presence of EDTA, both with and without OVA SP (Figure 5B). Because only living cells are detected by the WST-8 assay, the cells adhered to the OVA SP-loaded collagen gel were dead in the presence of EDTA. This suggests that the cell viability possibly depended on the integrin-mediated cell adhesion. It has been reported that α1β1 and α2β1 integrins bind to collagen.6 Hence, an inhibition assay for the adhesion of NIH3T3 cells was performed using an anti-β1 integrin (CD29) antibody. The antibody suppressed cell adhesion to the intact collagen but not to the OVA SP-loaded gel (Figure 6). This indicates that NIH3T3 cells can adhere to the OVA SP-loaded collagen gel in a β1 integrin-independent manner. It is known that focal adhesions form during the integrin-mediated cell process. And, vinculin, a well-known component of focal adhesions, has been used as a marker.13 The immunofluorostaining of vinculin was performed to visualize focal adhesions in our system. Bright dots corresponding to the focal adhesions were observed in the NIH3T3 cells cultured on the intact collagen gels, but were not detected on the OVA SP-loaded gels (Figure 7). This suggests that OVA SP suppresses the integrin-mediated cell adhesion and the formation of focal adhesion.


12

Page 13 of 39 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 4. Subcellular localization of OVA SP in NIH3T3 cells. (B) is an enlargement of the cell marked with an arrow in (A). Scale bar: 20 m.


13


Page 14 of 39

Figure 5. Effect of EDTA on the NIH3T3 cell adhesion to collagen gels. Cells were cultured on collagen gels with and without OVA SP (100 M) in the absence (black bars) or presence (white bars) of EDTA. (A) Adhered cells were counted by microscopic observation. (B) Relative absorbance normalized to that in the absence of EDTA, which was obtained from the WST-8 assay. *p < 0.01, n = 3.


14

Page 15 of 39 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 6. Inhibition assay of the adhesion of NIH3T3 cells to collagen gels with and without OVA SP (100 M) by anti-1 integrin antibody. Relative absorbance normalized to that in the absence of the antibody, which was obtained from the WST-8 assay. Black and white bars correspond to the non-pretreated and anti-1 integrin antibody-treated cells, respectively. *p < 0.01, n = 5.


15


Page 16 of 39

Figure 7. Focal adhesion formation in NIH3T3 cells on collagen gels. Representative images of vinculin immunostaining in NIH3T3 cells cultured on the intact (A) or the OVA SP (100 M)loaded collagen gel. Scale bar: 20 m.

3.3. Cell adhesion of various cell types to OVA SP-loaded collagen gels In addition to NIH3T3 cells, a variety of cell types, such as RAW264.7 (mouse macrophage-like cells), HeLa (human cervix adenocarcinoma), and MC3T3-E1 (mouse osteoblasts), were also


16

Page 17 of 39 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


cultured on collagen gels loaded with OVA SP at different concentrations. The number of adhered living cells was quantitatively evaluated from the WST-8 assay (Figure 8). The adhered HeLa cells slightly increased with increasing OVA SP concentrations. Contrastingly, the adhesion of NIH3T3 and MC3T3-E1 cells was suppressed on the OVA SP-loaded gels. Typical phase contrast images of these cells are shown in Figure 9. The number and morphology of the adhered cells in the intact collagen gel were different for each cell type. Less RAW264.7 cells were adhered to the intact collagen gel, compared to other cells. And, RAW264.7 cells formed cell aggregates unlike other cells. MDCK cells are commonly used as a model of epithelial cells because they form a monolayer.14,15 Single MDCK cells were observed on the intact collagen gel. As the OVA SP concentration increased, they aggregated and formed an epithelial monolayer-like structure (Figure 9).

Figure 8. Adhesion of different cells on OVA SP-loaded collagen gels after 48 h. Relative absorbance normalized to that in the absence of OVA SP, which was obtained from the WST-8 assay. n=3-5.


17


Page 18 of 39

Figure 9. Cultures of different cell types on collagen gels loaded with increasing concentrations of OVA SP (0, 30, 50, or 100 M) after 48 h. Scale bar: 100 m.

3.4. Differentiation of osteoblastic cells on OVA SP-loaded collagen gels The differentiation of pre-osteoblasts MC3T3-E1 cells is known to be induced by the presence of β-glycerophosphate and ascorbic acid. Mineralized nodules are known to be produced by differentiated MC3T3-E1 cells.10 Here, we carried out the osteoblastic differentiation on the collagen gels with and without OVA SP. Figure 10 shows that more mineralized nodules were produced from the cells cultured on the OVA SP-loaded collagen gel, compared to the intact


18

Page 19 of 39 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


collagen gel. Mineralized nodules were stained by the von Kossa method. Furthermore, higher ALP activities were observed in the MC3T3-E1 cells cultured on the loaded gels. These suggest that OVA SP promotes the differentiation of MC3T3-E1 cells.

Figure 10. Differentiation of MC3T3-E1 cells on the intact or the OVA SP (100 M)-loaded collagen gel. (A) Mineralization, (B) von Kossa staining, and (C) alkaline phosphatase assay. Scale bar: 1 mm.

4. Discussion In this work, we examined the detailed adhesion mechanism of NIH3T3 cells on the OVA SPloaded collagen gels. Figure 7 shows that NIH3T3 cells did not form focal adhesions on the OVA


19


Page 20 of 39

SP-loaded collagen gel, suggesting that OVA SP suppressed the integrin-mediated cell adhesion. On the other hand, Figures 5 and 6 show that the adhesion of NIH3T3 cells to OVA SP-loaded collagen gels was not affected by the presence of EDTA and the pre-treatment of anti-1 integrin antibody, indicating that the adhesion was independent in the integrin. Our previous report indicated that OVA SP promoted the aggregation of various kinds of proteins with different pI.7 Thus, it can be assumed that OVA SP nonspecifically interact with some adhesion molecules at the cell surface, including 1 integrin. This is consistent with our finding that the peptide was localized at the surface of adhered NIH3T3 cells (Figure 4). Although our previous works revealed that OVA SP changed some physical properties of collagen gels, such as the stiffness and surface hydrophobicity,7,9 Figure 3 shows that the loaded OVA SP was easily released from the collagen gel. This suggests that the physical effects of OVA SP to the collagen gel was limited in the cell culture. When NIH3T3 cells were seeded after washing out the released peptide, the number and morphology of adhered cells on the OVA SP-loaded collagen gels were similar to those on the intact collagen gel (data not shown). Conversely, when OVA SP was added to the medium on the collagen gel while the NIH3T3 cells were seeded, the peptide is expected to interact with the cells, prior to adhesion. These cells formed large aggregates and showed the cytotoxicity (data not shown), unlike those cultured on the OVA SP-loaded gels. This suggests the nonspecific interaction of OVA SP with cell surface proteins to induce the cell-cell interaction and the cytotoxicity. It is also suggested that the OVA SP released from the gel modulates cell adhesion and that the gradual release of OVA SP is important to suppress any undesirable effects. Based on the obtained results, the mechanism of OVA SP effect to cell adhesion is illustrated in Figure 11. It is likely that OVA SP is released from the collagen gel and nonspecifically associates with cell surface proteins, including integrins. It is also likely that the interaction of OVA SP suppresses the


20

Page 21 of 39 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


integrin-mediated cell adhesion and simultaneously induces the integrin-independent cell adhesion, because OVA SP possibly binds to various proteins with different pI.7 In this study, we also investigated the effect of OVA SP to adhesion and differentiation of various cells, which are known to express different integrin heterodimers (Table 1). It has been reported that α1β1 and α2β1 integrins bind to collagen, but the binding mode differs. α2β1 binds to type I collagen fibrils, while α1β1 binds to monomers of type I collagen. α1β1 can also associate with collagen fibrils via COLINBRI molecules (COLlagen INtegrin BRIdging).6 The functions of these integrins are also different: α1β1 stimulates cell proliferation and acts as a negative regulator for collagen synthesis, whereas α2β1 inhibits the growth of some cell types and increases matrix synthesis.5,6 NIH3T3 cells have been reported to express both α1β1 and α2β1 integrins,16 while HeLa cells express only α1β1, and MC3T3-E1 only α2β1.17,18 The number of NIH3T3 and MC3T3-E1 cells on the collagen gel tended to decrease in the presence of OVA SP, contrary to the effect observed in HeLa cells (Figure 8). Therefore, OVA SP likely suppressed the direct adhesion of α2β1 integrin by interacting with β1, but not the indirect adhesion of α1β1 integrin. OVA SP may promote and/or stabilize the association of COLINBRI, because of its properties as promoter of protein aggregation.7 In contrast, RAW264.7 cells do not express either α1β1 or α2β1 integrins.19 Thus, the morphology of adhered RAW264.7 cells was different than those of all the other cells. The adhesion properties of MDCK to the collagen gel drastically changed by the addition of OVA SP. The morphology of these cells cultured on the intact collagen gel was fibroblastic, which was different from an epithelial monolayer-like structure on a plastic dish.14 It is because the cell–cell interaction decreases and MDCK cells spread on intact collagen gels.15 Interestingly, MDCK cells cultured on the gels loaded with OVA SP at high concentration became epitheliallike (Figure 9). The interaction between collagen and α2β1 integrins has been reported to affect


21


Page 22 of 39

the cell polarity of epithelial MDCK cells.20 It is possible that OVA SP may interfere with the function of the α2β1 integrin, thus leading to the suppression of integrin signaling. Our results imply that OVA SP is a candidate to inhibit the change from an epithelial phenotype to a mesenchymal one (i.e., EMT). This should be further explored in future investigations. The differentiation of osteoblastic MC3T3-E1 cells was promoted on the OVA SP-loaded collagen gel (Figure 10), suggesting that this peptide also functions as a promotor of osteoblastic differentiation. OVA SP likely interacts with the α2β1 integrin at the surface of MC3T3-E1 cells, similar to NIH3T3 cells, which might induce the differentiation. It has been reported that the binding of α2β1 to collagen induced the differentiation of MC3T3-E1 cells, but other types of integrins, such as αvβ3 and/or αvβ5, also induced it.21-23 Thus, OVA SP possibly simulates some integrin pathways to induce the differentiation. Since bone mainly contains collagen and hydroxyapatite, collagen matrixes are often used as a scaffold of osteoblastic cells.24 Thus, the OVA SP-loaded collagen gel is useful for bone regenerative medicine.

Figure 11. Possible cell adhesion mechanism to collagen gels with and without OVA SP.


22

Page 23 of 39 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Table 1. Integrin expression patterns for the cells used in this study. Cell NIH3T3 HeLa MC3T3-E1 RAW264.7 MDCK

Origin mouse embryonic fibroblasts human cervix adenocarcinoma cell line mouse osteoblastic cell line mouse macrophage cell line dog kidney epithelial cell line

Integrin heterodimer with 1 11, 21 11, 51 21, 51, v1 41, 51 21, 31

Reference 16 17 18 19 20

5. Conclusions In this study, we examined the adhesion of NIH3T3 cells on collagen gels loaded with OVA SP and proposed a mechanism of the effect of OVA SP to cell adhesion. We also demonstrated that the adhesion of various cells types to the collagen gel and the differentiation of osteoblastic cells can be modulated by OVA SP. Our results indicate that this peptide is released from the collagen gel and nonspecifically associates with cell surface proteins to affect the adhesion and the differentiation processes. The findings of this study suggest that OVA SP is a potent modulator of cell scaffolds. Moreover, the OVA SP-loaded collagen, which may be useful for bone regenerative medicine, is a good example of the effective use of egg white in the egg-yolk-based food production.

Conflicts of interest There are no conflicts of interest to declare.


23


Page 24 of 39

REFERENCES [1] Huntington, J.A.; Stein, P.E.; Structure and Properties of Ovalbumin. J. Chromatogr. B 2001, 756, 189-198. [2] a) Arii, Y.; Takahashi, N.; Hirose, M.; Periplasmic Secretion of Native Ovalbumin Without Signal Cleavage in Escherichia Coli. Biosci. Biotech. Biochem. 2003, 67, 368-371; b) Belin, D.; Guzman, L.M.; Bost, S.; Konakova, M.; Silva, F.; Beckwith, J.; Functional Activity of Eukaryotic Signal Sequences in Escherichia Coli: The Ovalbumin Family of Serine Protease Inhibitors. J. Mol. Biol. 2004, 335, 437-453. [3] Kitabatake, N.; Indo, K.; Doi, E.; Limited Proteolysis of Ovalbumin by Pepsin. J. Agric. Food Chem. 1998, 36, 417-420. [4] Gelse, K.; Poschl, E.; Aigner. T.; Collagens—Structure, Function, and Biosynthesis. Adv. Drug Deliv. Rev. 2003, 55, 1531-1546. [5] Jokinen, J.; Dadu, E.; Nykvist, P.; Käpylä, J.; White, D.J.; Ivaska, J.; Vehviläinen, P.; Reunanen, H.; Larjava, H.; Häkkinen, L.; Heino, J.; Integrin-Mediated Cell Adhesion to Type I Collagen Fibrils. J. Biol. Chem. 2004, 279, 31956-31963. [6] a) Zeltz, C.; Orgel, J.; Gullberg, D.; Molecular Composition and Function of Integrin-Based Collagen Glues-Introducing COLINBRIs. Biochim. Biophys. Acta 2014, 1840, 2533-2548; b) Zeltz, C.; Gullberg, D.; The Integrin-Collagen Connection--A Glue for Tissue Repair? J. Cell Sci. 2016, 129, 653-664. [7] Kawachi, Y.; Kameyama, R.; Handa, A.; Takahashi, N.; Tanaka, N.; Role of the N-Terminal Amphiphilic Region of Ovalbumin During Heat-Induced Aggregation and Gelation. J. Agric. Food Chem. 2013, 61, 8668-5675. [8] a) Engler, A.J.; Sen, S.; Sweeney, H.L.; Discher, D.E.; Matrix Elasticity Directs Stem Cell Lineage Specification. Cell 2006, 126, 677-689; b) Hadjipanayi, E.; Mudera, V.; Brown, R. A.; Close Dependence of Fibroblast Proliferation on Collagen Scaffold Matrix Stiffness. J. Tissue Eng. Regen. Med. 2009, 3, 77-84.


24

Page 25 of 39 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


[9] Kojima, C.; Narita, Y.; Waku, T.; Morimoto, N.; Togawa, D.; Takahashi, N.; Handa, A.; Tanaka, N.; Ovalbumin Secretory Signal Peptide-Containing Collagen Gel as a Cell Scaffold. Chem. Lett. 2017, 46, 395-397. [10] Kim, S.W.; Her, S.J.; Park, S.J.; Kim, D.; Park, K.S.; Lee, H.K.; Han, B.H.; Kim, M.S.; Shin, C.S.; Kim, S.Y.; Ghrelin Stimulates Proliferation and Differentiation and Inhibits Apoptosis in Osteoblastic MC3T3-E1 Cells. Bone 2005, 37, 359-369. [11] Elices, M.J.; Hemler, M.E.; The Human Integrin VLA-2 is a Collagen Receptor on Some Cells and a Collagen/Laminin Receptor on Others. Proc. Natl. Acad. Sci. USA 1989, 86, 99069910. [12] Senger, D.R.; Claffey, K.P.; Benes, J.E.; Perruzzi, C.A.; Sergiou, A.P.; Detmar, M.; Angiogenesis Promoted by Vascular Endothelial Growth Factor: Regulation Through α1β1 and α2β1 Integrins. Proc. Natl. Acad. Sci. USA 1997, 94, 13612-13617. [13] Turner, C.E.; Burridge, K.; Transmembrane Molecular Assemblies in Cell-Extracellular Matrix Interactions. Curr. Opin. Cell Biol. 1991, 3, 849-853. [14] Zuk, A.; Matlin, K.S.; Hay, E.D.; Type I Collagen Gel Induces Madin-Darby Canine Kidney Cells to Become Fusiform in Shape and Lose Apical-Basal Polarity. J. Cell Biol. 1989, 108, 903-919. [15] Hay, E.D.; Zuk, A.; Transformations Between Epithelium and Mesenchyme: Normal, Pathological, and Experimentally Induced. Am. J. Kidney Dis. 1995, 26, 678-690. [16] Defilippi, P.; Olivo, C.; Tarone, G.; Mancini, P.; Torrisi, M.R.; Eva, A.; Actin Cytoskeleton Polymerization in Dbl-Transformed NIH3T3 Fibroblasts is Dependent on Cell Adhesion to Specific Extracellular Matrix Proteins. Oncogene 1997, 14, 1933-1943. [17] Albiges-Rizo, C.; Frachet, P.; Block, M.R.; Down Regulation of Talin Alters Cell Adhesion and the Processing of the Alpha 5 Beta 1 Integrin. J. Cell Sci. 1995, 108, 3317-3329.


25


Page 26 of 39

[18] Hendesi, H.; Barbe, M.F.; Safadi, F.F.; Monroy, M.A.; Popoff, S.N.; Integrin Mediated Adhesion of Osteoblasts to Connective Tissue Growth Factor (CTGF/CCN2) induces Cytoskeleton Reorganization and Cell Differentiation. PLoS One 2015, 10, e0115325. [19] Martchenko, M.; Jeong, S.Y.; Cohe, S.N.; Heterodimeric Integrin Complexes Containing Beta1-Integrin Promote Internalization and Lethality of Anthrax Toxin. Proc. Natl. Acad. Sci. USA 2010, 107, 15583-15588. [20] Schoenenberger, C.A.; Zuk, A.; Zinkl, G.M.; Kendall, D.; Matilin, K.S.; Integrin Expression and Localization in Normal MDCK Cells and Transformed MDCK Cells Lacking Apical Polarity. J. Cell Sci. 1994, 107, 527-541. [21] Zeng, Q.; Guo, Y.; Liu, Y.; Li, R.; Zhang, X.; Liu, L.; Wang, Y.; Zhang, X.; Zou, X.; Integrin-β1, Not Integrin-β5, Mediates Osteoblastic Differentiation and ECM Formation Promoted by Mechanical Tensile Strain. Biol. Res. 2015, 48, 25. [22] Su, J.-L.; Chiou, J.; Tang, C.-H.; Zhao, M.; Tsai, C.-H.; Chen, P.-S.; Chang, Y.-W.; Chien, M.-H.; Peng, C.-Y.; Hsiao, M.; Kuo, M.-L.; Yenk, M.-L.; CYR61 Regulates BMP-2-Dependent Osteoblast Differentiation through the αvβ3 Integrin/Integrin-linked Kinase/ERK Pathway. J. Biol. Chem. 2010, 285, 31325-31336. [23] Taubenberger, A.V.; Woodruff, M.A.; Bai, H.; Muller, D.J.; Hutmacher, D.W.; The Effect of Unlocking RGD-motifs in Collagen I on Pre-Osteoblast Adhesion and Differentiation. Biomaterials. 2010, 31, 2827-2835. [24] Wahl, D.A.; Czernuszka, J.T.; Collagen-Hydroxyapatite Composites for Hard Tissue Repair. Eur. Cell. Mater. 2006, 11, 43-56.


26

Page 27 of 39 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


For Table of Contents Only Modulation of Cell Adhesion and Differentiation on Collagen Gels by the Addition of the Ovalbumin Secretory Signal Peptide Chie Kojima1*, Yuri Narita2, Yusuke Nakajima1, Naoya Morimoto2, Takashi Yoshikawa2, Nobuyuki Takahashi3, Akihiro Handa4, Tomonori Waku2*, Naoki Tanaka2$


27


Figure 1. 3D Structure of OVA. The region of OVA SP is colored in blue. 49x59mm (300 x 300 DPI)


Page 28 of 39

Page 29 of 39 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 2. Localization of OVA SP in the collagen gel. Scale bar: 20 μm. 82x88mm (300 x 300 DPI)



Figure 3. Release of OVA SP from the collagen gel. 94x94mm (300 x 300 DPI)


Page 30 of 39

Page 31 of 39 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 4. Subcellular localization of OVA SP in NIH3T3 cells. (B) is an enlargement of the cell marked with an arrow in (A). Scale bar: 20 μm. 87x169mm (300 x 300 DPI)



Figure 5. Effect of EDTA on the NIH3T3 cell adhesion to collagen gels. Cells were cultured on collagen gels with and without OVA SP (100 μM) in the absence (black bars) or presence (white bars) of EDTA. (A) Adhered cells were counted by microscopic observation. (B) Relative absorbance normalized to that in the absence of EDTA, which was obtained from the WST-8 assay. *p < 0.01, n = 3. 105x167mm (300 x 300 DPI)


Page 32 of 39

Page 33 of 39 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 6. Inhibition assay of the adhesion of NIH3T3 cells to collagen gels with and without OVA SP (100 μM) by anti-β1 integrin antibody. Relative absorbance normalized to that in the absence of the antibody, which was obtained from the WST-8 assay. Black and white bars correspond to the non-pretreated and anti-β1 integrin antibody-treated cells, respectively. *p < 0.01, n = 5. 97x87mm (300 x 300 DPI)



Figure 7. Focal adhesion formation in NIH3T3 cells on collagen gels. Representative images of vinculin immunostaining in NIH3T3 cells cultured on the intact (A) or the OVA SP (100 μM)-loaded collagen gel. Scale bar: 20 μm. 46x87mm (300 x 300 DPI)


Page 34 of 39

Page 35 of 39 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 8. Adhesion of different cells on OVA SP-loaded collagen gels after 48 h. Relative absorbance normalized to that in the absence of OVA SP, which was obtained from the WST-8 assay. n=3. 121x115mm (300 x 300 DPI)



Figure 9. Cultures of different cell types on collagen gels loaded with increasing concentrations of OVA SP (0, 30, 50, or 100 μM) after 48 h. Scale bar: 100 μm. 138x117mm (300 x 300 DPI)


Page 36 of 39

Page 37 of 39 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 10. Differentiation of MC3T3-E1 cells on an intact or an OVA SP (100 μM)-loaded collagen gel. (A) Mineralization, (B) von Kossa staining, and (C) alkaline phosphatase assay. Scale bar: 1 mm. 79x101mm (300 x 300 DPI)



Figure 11. Possible cell adhesion mechanism to collagen gels with and without OVA SP. 115x60mm (300 x 300 DPI)


Page 38 of 39

Page 39 of 39 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Table of content 167x64mm (300 x 300 DPI)


Modulation of Cell Adhesion and Differentiation on Collagen Gels by

Recommend Documents