Accepted Manuscript Periostin promotes secretion of fibronectin from the endoplasmic reticulum Isao Kii, PhD., Takashi Nishiyama, Akira Kudo, PhD. PII:
S0006-291X(16)30140-1
DOI:
10.1016/j.bbrc.2016.01.139
Reference:
YBBRC 35256
To appear in:
Biochemical and Biophysical Research Communications
Received Date: 16 January 2016 Accepted Date: 22 January 2016
Please cite this article as: I. Kii, T. Nishiyama, A. Kudo, Periostin promotes secretion of fibronectin from the endoplasmic reticulum, Biochemical and Biophysical Research Communications (2016), doi: 10.1016/j.bbrc.2016.01.139. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Periostin promotes secretion of fibronectin from the
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endoplasmic reticulum
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3 Isao Kii,1,2,** Takashi Nishiyama,1 and Akira Kudo1*
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cho, Midori-ku, Yokohama 226-8501, Japan.
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Pathophysiological and Health Science Team, Imaging Application Group, Division of
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Department of Biological Information, Tokyo Institute of Technology, 4259 Nagatsuta-
Bio-Function Dynamics Imaging, RIKEN Center for Life Science Technologies, 6-7-3 Minatojima-minamimachi, Chuo-ku, Kobe, Hyogo 650-0047, Japan.
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Runnning head: Periostin in the secretory pathway
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*Corresponding author:
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Akira Kudo, PhD., 4259-B-33 Nagatsuta, Midori-ku, Yokohama 226–8501, Japan.
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Tel and Fax: +81-45-924-5718, E-mail: [email protected]
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**Corresponding author:
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Isao Kii, PhD., 6-7-3 Minatojima-minamimachi, Chuo-ku, Kobe, Hyogo 650-0047, Japan.
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Tel: +81-78-304-7192 (Ext. 95-8380), Fax: +81-78-304-7191, E-mail: [email protected]
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ABSTRACT
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Extracellular matrix (ECM) proteins are synthesized in the endoplasmic reticulum (ER),
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transported to the extracellular milieu through the secretory pathway, and assembled into
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an extracellular architecture. Previous studies of ours showed that periostin, a secretory
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protein, interacts with fibronectin and is involved in ECM remodeling. Here we show that
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periostin played a role in fibronectin secretion from the ER. Co-immunoprecipitation and
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in situ proximity ligation assays revealed an interaction between periostin and fibronectin
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in the ER. Although accumulation of fibronectin was detected in the ER of fibroblastic
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C3H10T1/2 cells, forced expression of periostin in those cells decreased the
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accumulation of fibronectin in the ER, suggesting that periostin promoted the secretion of
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fibronectin. A substitution mutant of tryptophan at the position 65 to alanine in the EMI
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domain of periostin, which caused periostin to lose its ability to interact with fibronectin,
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did not decrease the accumulation. Furthermore, targeted disruption of periostin in mice
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caused the non-fibrillar and ectopic deposition of fibronectin in the periodontal ligament.
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Thus, these results demonstrate a subcellular role of periostin in promotion of fibronectin
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secretion from the ER.
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Keywords
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Periostin; Fibronectin; Endoplasmic reticulum; Periodontal ligament
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1. Introduction Mechanical properties such as tensile strength and elasticity of the connective tissues depend on the extracellular matrix (ECM) architecture. The ECM architecture is
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constructed from a large number of ECM proteins and remodeled by dissociation and
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subsequent reconnection of the ECM proteins under the dynamic changes occurring in
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mechanical environments [1]. The ECM architecture is based on a framework of
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fibronectin matrix, which is required for the assembly of multiple ECM proteins [2]. This
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matrix, which consists of highly elastic fibronectin fibrils, is remodeled under dynamic
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movement [3-6]. Thus, fibronectin plays an important role in regulating ECM remodeling.
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The ECM proteins are synthesized in the endoplasmic reticulum (ER), transported into the extracellular milieu through the secretory pathway, and assembled into an
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extracellular architecture. The ECM proteins are folded into their native conformations
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with the aid of ER molecular chaperones before they are transported to post-ER
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compartments. General ER chaperones (e.g., calreticulin, calnexin, protein disulfide
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isomerase, and BiP) play a role in the folding of the ECM proteins [7-11]. Specific
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molecular chaperones for the ECM proteins have also been demonstrated. For example,
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heat shock protein 47 (Hsp47) is a chaperone for collagen in the ER [12, 13]. Type I
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collagen molecules in the ER of hsp47-null cells become insoluble aggregates [14].
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Chaperone-like activity for ECM proteins was also reported in the case of a matricellular
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protein, SPARC, otherwise known as osteonectin [15-17]. Thus, the general and specific
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molecular chaperones for the ECM proteins in the ER maintain ECM homeostasis.
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Periostin is a secretory protein involved in ECM remodeling in the periodontal ligament, myocardial infarction, and heart valve [18, 19]. Targeted disruption of the
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periostin gene in mice causes a defect in the ECM remodeling in the periodontal
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ligament, which is a dense collagenous tissue connecting tooth to alveolar bone, resulting
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in disturbance of incisor eruption [20]. We and others also demonstrated that the rate of
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heart ruptures and death caused by acute myocardial infarction is higher in periostin-/-
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mice than in wild-type counterparts [21, 22]. It has been reported that periostin directly
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binds to several ECM proteins, such as fibronectin, collagens, and tenascin-C [23, 24].
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Furthermore, we demonstrated that periostin functions as a scaffold between tenascin-C
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and the ECM proteins, including fibronectin and type I collagen [23]. As described above,
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periostin has been studied as one of the ECM proteins. On the other hand, several reports
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showed a subcellular localization of periostin [19, 23, 25-27], and the role of subcellular
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periostin is not yet well understood.
In this present study, we demonstrated an interaction between periostin and
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fibronectin in the ER. This interaction may enhance the solubility of fibronectin to
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prevent its aggregation in the ER. Furthermore, we found that periostin deficiency in
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mice caused the ectopic deposition of fibronectin in the periodontal ligament. Thus, the
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present study suggests that periostin interacted with fibronectin to promote the secretion
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of the latter from the ER.
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2. Materials and methods
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Cell culture and transfection C3H10T1/2 and 293T cells were maintained in low-glucose Dulbecco's Modified
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Eagle Medium (DMEM; Nacalai Tesque, Inc., Kyoto, Japan) supplemented with 10%
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fetal bovine serum (FBS; JRH Biosciences, Inc., Lenexa, KS), in which serum fibronectin
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had been depleted [23]. Cells were transfected with expression vectors with
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polyethylenimine (Sigma-Aldrich, Inc., St. Louis, MO), as described previously [23]. For
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immunocytochemistry, cells were cultured on coverslips (No. 1-S; Matsunami Glass Ind.,
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LTD, Osaka, Japan) in a sparse condition.
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2.2.
Antibodies
Rat monoclonal anti-mouse fibronectin antibody (A15-1) was generated
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previously in our laboratory (unpublished). Rabbit polyclonal anti-fibronectin antibody
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Ab-10 (NeoMarkers, Lab Vision Corporation, Thermo Fisher Scientific Inc.), monoclonal
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mouse anti-HA antibody (Nacalai Tesque), rabbit polyclonal anti-HA antibody (Santa
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Cruz Biotechnology Inc., CA), and rabbit polyclonal anti-calreticulin antibody (Affinity
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BioReagents Inc., Thermo Fisher Scientific Inc.) were purchased from the sources
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indicated. Alexa Fluor-labeled goat anti-rabbit, anti-mouse, anti-rat antibodies, Alexa
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Fluor 568-labeled phalloidin, and TO-PRO-3 were obtained from Molecular Probes Inc.
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(Thermo Fisher Scientific Inc.).
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2.3.
Immunofluorescence microscopy
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Immunohistochemistry was performed as described previously [23], and
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fluorescent images were collected with a laser-scanning confocal microscope (FV1000-
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BX61; Olympus Corporation, Japan).
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2.4.
Preparation of microsomal fraction
Cells (1 x 108) were detached with trypsin, washed with ice-cold PBS, and
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homogenized on ice with Dounce disruption in 20 mM Tris-HCl (pH 7.5), 5 mM EDTA
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with protease inhibitors. Before centrifugation at 700 × g, the solution was brought up to
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440 mM sucrose. The postnuclear supernatants were centrifuged further, first at 12,000 ×
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g for 15 min at 4 °C to pellet the mitochondria fraction; and the resulting supernatants
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were then ultracentrifuged at 120,000 × g for 1 hr at 4 °C. The pellets (microsomal
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fraction) were resuspended in 500 µL of the above buffer containing sucrose.
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2.5.
Animals
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Care and experiments with animals were in accordance with the guidelines of the
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Animal Care and Use Committee at Tokyo Institute of Technology. Generation of
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periostin-/- mice was described previously [20, 22].
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2.6.
Immunohistochemistry Tissue paraffin sections were obtained and immunostained, as described earlier
[20, 23].
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2.7.
Immunoprecipitation
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Immunoprecipitation were performed with anti-HA antibody-conjugated agarose
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(Sigma-Aldrich) or protein G Sepharose (GE Healthcare), as described previously [23].
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Conjugation of purified fibronectin protein and its 70-kDa fragment with Alexa
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Fluor 555
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Fibronectin and its 70-kDa fragment (Sigma-Aldrich) were diluted in 100 mM
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HEPES buffer (pH 8.0), and conjugated with Alexa Fluor 555 succinimidyl ester
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(Molecular Probes, Thermo Fisher Scientific Inc.). The unreacted ester was removed by
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dialysis against PBS.
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2.9.
EMI-hFc pull-down assay
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The fusion protein of the amino-terminal domain of periostin (EMI domain) and
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the Fc region of human IgG (EMI-hFc) [23] was purified from 293T cells stably
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expressing EMI-hFc by use of protein G Sepharose (GE Healthcare). Protein bound on
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protein G Sepharose was incubated with the Alexa-555-labeled fibronectin or its 70-kDa
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fragment in 25 mM HEPES (pH 7.2), 150 mM NaCl, 0.1% NP-40 for 1 hr, and then
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pulled down by centrifugation. The bound proteins were sequentially eluted with SDS
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sample buffer containing 50 mM DTT, subjected to SDS-PAGE, and then detected with
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Typhoon 8600 (GE Healthcare). The gels were also stained with a CBB rapid stain kit
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(Nacalai Tesque).
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2.10.
In situ proximity ligation assay (PLA)
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In situ PLA was carried out according to the manufacturer’s protocol using mouse
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anti-HA, rabbit anti-fibronectin (Ab-10) antibodies, and a Duolink Detection Kit (Olink
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Bioscience, Sweden), as described previously [23].
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2.11.
Treatment with Brefeldin A (BFA)
Cells were incubated with 10 µg/mL BFA (Sigma-Aldrich) for 12 hr prior to lysis for the immunoprecipitation assay.
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3. Results
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3.1.
Periostin is localized in the ER To examine the subcellular localization of periostin, we performed
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immunofluorescence analysis of C3H10T1/2 cells stably expressing periostin-HA
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(10T1/2-periostin-HA cells), in which an HA peptide tag was conjugated at the carboxyl-
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terminal end of mouse periostin [23]. Periostin-HA was detected inside the cells (Fig. 1A).
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These fluorescence signals coincided with those of calreticulin, an ER marker, indicating
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that periostin-HA was localized in the ER. Slightly strong fluorescence signals for the
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HA tag were detected around the nuclei, which corresponds to the Golgi [23, 26].
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3.2.
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the ER
Forced expression of periostin-HA decreases accumulation of fibronectin in
The periostin-HA localization in the ER prompted us to envisage a role of
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periostin in the ER. Periostin is known to interact with fibronectin [23, 24]. We compared
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the localization of fibronectin in the 10T1/2-periostin-HA cells with that in C3H10T1/2
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cells harboring the empty vector (10T1/2-control cells). In the 10T1/2-control cells,
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immunofluorescent signals of fibronectin were detected not only in the fibrillar ECM, but
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also inside the cells (Fig. 1B). The strong signals of fibronectin inside the cells appeared
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as a granular accumulation (Fig. 1B), which was similar to that in chicken embryonic
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fibroblasts reported previously [28]. In the 10T1/2-periostin-HA cells, the fibrillar
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fibronectin in the ECM was detected the same as in the 10T1/2-control ones, whereas the
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intracellular granular signals were rarely detected (Fig. 1C).
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To investigate the localization of fibronectin, we performed immunofluorescence
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analysis using anti-fibronectin and anti-calreticulin antibodies. The fluorescent signals of
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fibronectin were co-localized with those of calreticulin in the 10T1/2-control cells, but
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not in the 10T1/2-periostin-HA cells (Fig. 1D), indicating that periostin decreased the
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fibronectin accumulation in the ER. To quantify the fibronectin accumulation in the ER,
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we prepared the microsomal fraction, which is rich in ER. The amount of fibronectin was
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decreased in the microsomal fraction of the 10T1/2-periostin-HA cells compared with
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that in that of the 10T1/2-control cells (Fig. 1E), which is consistent with the results of
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the immunofluorescence analysis (Fig. 1B to 1D). Thus, these results suggest that
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periostin promoted the secretion of fibronectin from the ER.
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periostin-/- mice
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Non-fibrillar deposition of fibronectin in the periodontal ligament of
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To confirm the fibronectin accumulation observed in the in vitro analyses, we performed immunohistochemical staining using anti-fibronectin antibody to examine the
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expression of fibronectin in the periodontal ligament of incisors from wild-type and
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periostin-/- mice. As reported previously [20], sections stained with hematoxylin-eosin
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showed the absence of a region of the ECM remodeling, the so-called shear zone (Fig. 2A
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and arrows in 2B), in the periostin-/- periodontal ligament (Fig. 2C and 2D). In the
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periodontal ligament of the wild-type incisors, fibrillar immuno-reactivity indicating
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fibronectin was detected around the cells (Fig. 2F). In contrast, non-fibrillar and dappled
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immuno-signals for fibronectin were observed especially around the nuclei in the
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periodontal ligament of the periostin-/- incisors (Fig. 2H). Thus, these results indicate that
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the periostin deficiency caused ectopic deposition of fibronectin in the periodontal
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ligament of the incisors.
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3.4.
Periostin interacts with fibronectin in the ER
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An earlier study showed direct interaction of periostin with fibronectin coated on
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the surface of a microtiter plate [24], an interaction achieved through the amino-terminal
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EMI domain of periostin [23]. To examine whether periostin interacted with fibronectin
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in cells or culture supernatant, we performed a co-immunoprecipitation assay with 293T
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cells transiently co-transfected with the expression vectors for periostin-HA and
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fibronectin. Fibronectin was co-immunoprecipitated with periostin-HA in the total cell
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lysate, but not in the cell culture supernatant, even though the cell culture supernatant
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contained an abundance of fibronectin and periostin-HA proteins (Fig. 3A). To clarify the reason why the interaction between periostin and fibronectin was
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not detected in the cell culture supernatant, we examined whether periostin would interact
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with purified fibronectin in solution. We prepared the EMI-hFc fusion protein (see
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Materials & Methods) bound on protein G Sepharose resin, and mixed it with either the
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purified fibronectin or its 70-kDa fragment in solution. We detected a small amount of
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purified fibronectin pulled down with the EMI-hFc resin, compared with the amount of
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the 70-kDa fragment pulled down with it (Fig. 3B), indicating that the structure of
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fibronectin in solution may have interfered with the interaction between periostin and the
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70-kDa region of fibronectin.
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To confirm that periostin indeed interacted with fibronectin inside the cells, we looked for close proximity between periostin and fibronectin by performing in situ PLA,
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which depicts the close proximity (≤ 40 nm) of cellular molecules as fluorescent signals
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(PLA signals) [29]. Fibronectin and periostin-HA in the 10T1/2-control and -periostin-
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HA cells were labeled indirectly by using anti-rabbit PLUS probe and anti-mouse
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MINUS probe, respectively. If the PLUS and MINUS probes are proximate to each other,
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PLA signals emerge as bright fluorescent dots at the proximity site. The in situ PLA
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signals between periostin-HA and fibronectin were detected around the nuclei (Fig. 3C).
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These results indicate that periostin and fibronectin interacted only inside the cells,
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especially around the nuclei, probably at locations corresponding to the ER and the Golgi.
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If periostin interacted with fibronectin in the ER, blocking of the protein transport
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from the ER to the post-ER compartments should increase the amount of the complex of
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periostin and fibronectin. So we performed a co-immunoprecipitation assay using cells
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treated or not with Brefeldin A (BFA), which interferes with anterograde protein
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transport from the ER to the Golgi and causes the accumulation of secretory proteins
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inside the ER. The treatment with BFA increased the interaction between EMI-hFc and
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fibronectin in 293T cells transiently co-transfected with the expression vectors for EMI-
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hFc and fibronectin (Fig. 3D), indicating that the interaction between periostin and
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fibronectin had taken place in the ER.
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in secretion of fibronectin from the ER.
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Tryptophan residue at position 65 in the EMI domain of periostin is involved
To ask whether the interaction between periostin and fibronectin promoted the
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secretion of fibronectin from the ER, we examined a mutant periostin that loses its ability
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to interact with fibronectin. An earlier study of ours showed that the substitution mutant
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of tryptophan at position 65 to alanine in the EMI domain of periostin (Trp65Ala) fails to
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interact with fibronectin [23]. The granular accumulation of fibronectin was detected
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inside C3H10T1/2 cells stably expressing the mutant periostin (peri[Trp65Ala]-HA), just
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the same as in the 10T1/2-control cells (Fig. 4). Thus, this result suggests that the
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tryptophan residue was required for promotion of fibronectin secretion from the ER.
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DISCUSSION In this study, we found that periostin played a role in the secretion of fibronectin
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from the ER. Periostin consists of an amino-terminal EMI domain, a tandem repeat of 4
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fas1 domains (RD1, 2, 3, and 4), and a carboxyl-terminal region (CTR) [19]. The EMI
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domain directly binds to fibronectin [23]. Earlier we showed that recombinant RD4-CTR
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of periostin exhibits high solubility and monodispersity in solution [30]. These features
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may enhance the solubility of fibronectin in the ER, resulting in decreased granular
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accumulation of fibronectin in the ER.
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Periostin has no ER-retention signals such as the KDEL sequence. Indeed,
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periostin is efficiently secreted into culture medium [31]. Distinct from other ER
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chaperones, periostin functions in efficient secretion of ECM proteins with low solubility.
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Fibronectin in the ECM forms a huge insoluble assembly of multiple ECM proteins [32],
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and this insolubility probably contributes to stability of the ECM architecture. To strike a
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balance between its insolubility in the ECM and solubility in the ER, periostin have
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evolved to interact with fibronectin in the ER, enhancing the solubility of the latter, and
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to dissociate from it in the extracellular milieu. It should be noted that periostin did not
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interact with fibronectin in solution. Fibronectin exists in a compact or extended
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conformation, depending upon the surrounding environment [6, 33, 34]. For example,
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soluble fibronectin is folded into a compact conformation, and a hydrophobic surface
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extends fibronectin conformation [35]. Taken together, the data suggest that the
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interaction between periostin and fibronectin may depend on the conformation of
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fibronectin. Further studies are necessary to clarify the conditions for the interaction
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between periostin and fibronectin, and the conformational change in fibronectin during its
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passage through the secretory pathway through the ER to the extracellular milieu.
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The present study also provides a mechanistic insight into the mechanism of the disturbance in incisor eruption seen in periostin-/- mice. In the periodontal ligament of
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incisors from periostin-/- mice, the disorganized fibronectin matrix or the accumulated
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fibronectin probably disturbs the cellular functions necessary to remodel the ECM
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architecture [20]. The extracellular fibronectin matrix functions as a framework for the
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ECM architecture, and thus disorganization of this framework must result in disruption of
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the ECM remodeling.
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Several groups including ours have reported abnormalities in periostin-/- mice,
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such as those in the periodontal ligament, periosteum, infarcted myocardium, tendon,
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heart valve, and so on [18, 19]. Previously we demonstrated that the confined tibial
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periostitis in periostin-/- mice, which is similar to that seen in human sports injuries, is
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due to the decreased incorporation of tenascin-C into the ECM architecture [23]. In the
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case of myocardial infarction, integrin signaling may be involved in the periostin-
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mediated recruitment of myofibroblasts to the infarcted region [22]. This periostin-
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mediated integrin signaling plays essential roles in other physiological and pathological
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systems as well [18, 19]. These varieties of reports on the molecular functions of
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periostin suggest that periostin is a multifunctional secretory protein.
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Acknowledgements The authors thank Prof. Norio Amizuka (Hokkaido University, Japan) and Dr. Minqi Li (Hokkaido University, Japan) for preparation of paraffin sections of wild-type
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and periostin-/- mandibles. This work was supported by grants-in-aid for scientific
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research from the Ministry of Education, Science, Culture, and Sports of Japan
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(19791362, 19370093).
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fibrosis of bronchial asthma downstream of IL-4 and IL-13 signals, J. Allergy Clin.
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cutaneous wound healing, PLoS One 6 (2011) e18410. [26] B.Y. Kim, J.A. Olzmann, S.I. Choi, S.Y. Ahn, T.I. Kim, H.S. Cho, H. Suh, E.K. Kim,
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periostin and causes mislocalization to the lysosome, J. Biol. Chem. 284 (2009) 19580-
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[27] N. Yoshioka, S. Fuji, M. Shimakage, K. Kodama, A. Hakura, M. Yutsudo, H. Inoue,
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the TRIF52/periostin/OSF-2 gene, Exp. Cell Res. 279 (2002) 91-99. [28] K.M. Yamada, Immunological characterization of a major transformation-sensitive fibroblast cell surface glycoprotein. Localization, redistribution, and role in cell shape, J.
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the in situ proximity ligation assay, Methods 45 (2008) 227-232.
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[31] K. Horiuchi, N. Amizuka, S. Takeshita, H. Takamatsu, M. Katsuura, H. Ozawa, Y.
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Toyama, L.F. Bonewald, A. Kudo, Identification and characterization of a novel protein,
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periostin, with restricted expression to periosteum and periodontal ligament and
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increased expression by transforming growth factor beta, J. Bone Miner. Res. 14 (1999)
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1239-1249. [32] R. Pankov, K.M. Yamada, Fibronectin at a glance, J. Cell Sci. 115 (2002) 3861-3863.
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[33] K.J. Johnson, H. Sage, G. Briscoe, H.P. Erickson, The compact conformation of
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fibronectin is determined by intramolecular ionic interactions, J. Biol. Chem. 274
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(1999) 15473-15479.
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[34] H.P. Erickson, N.A. Carrell, Fibronectin in extended and compact conformations.
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Electron microscopy and sedimentation analysis, J. Biol. Chem. 258 (1983) 14539-
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14544.
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[35] M. Bergkvist, J. Carlsson, S. Oscarsson, Surface-dependent conformations of human
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plasma fibronectin adsorbed to silica, mica, and hydrophobic surfaces, studied with use
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of atomic force microscopy, J. Biomed. Mater. Res. A 64 (2003) 349-356.
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FIGURE LEGENDS
2 Fig. 1. Periostin decreases the accumulation of fibronectin in the ER.
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(A) Periostin-HA is detected in the ER. C3H10T1/2 cells were stably transfected with the
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expression vector of periostin-HA, in which an HA peptide tag was conjugated at the
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carboxyl-terminal end of mouse periostin. The cells were fluorescently stained with anti-
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HA and anti-calreticulin antibodies.
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(B and C) C3H10T1/2 cells stably expressing periostin-HA (10T1/2-periostin-HA; C)
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and C3H10T1/2 cells harboring empty vector (10T1/2-control; B) were stained with anti-
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fibronectin and anti-HA antibodies (upper columns) or with anti-fibronectin and
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fluorescently labeled phalloidin (lower columns). Arrowheads indicate strong immuno-
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reactions for fibronectin proteins inside the cells; and arrows, obscure ones inside them.
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(D) 10T1/2-control and -periostin-HA cells were fluorescently stained with anti-
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fibronectin and anti-calreticulin antibodies. Arrows indicate co-localization of strong
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immunoreactivity for fibronectin and calreticulin.
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(E) Microsomal fractions of 10T1/2-control and -periostin-HA cells were prepared by the
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ultracentrifugation method (see Materials and methods). The microsomal fractions and
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the total cell lysates were analyzed by Western blotting using anti-fibronectin, anti-HA,
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and anti-calreticulin antibodies.
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Fig. 2. Ectopic deposition of fibronectin proteins in the periodontal ligament of the
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incisors from periostin-/- mice.
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Paraffin sections of mandibles from 12-week-old wild-type (A, B, E, F) and periostin-/-
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(C, D, G, H) mice were stained with hematoxylin-eosin (HE) solution (A-D) or anti-
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fibronectin antibody (E-H). The periodontal ligaments between the dentin and bone in
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“A,” “C,” “E,” and “G” are shown in “B,” “D,” “F,” and “H,” respectively. Typical
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immunoreactions in “F” and “H” are presented as high-magnification images in the white
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rectangular boxes. Arrows indicate shear zone, which is the region of ECM remodeling.
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Arrowheads indicate strong immunoreactivity for fibronectin proteins around the nuclei.
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Representative images are shown.
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Fig. 3. Interaction between periostin and fibronectin in the ER.
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(A) Total cell lysates and cell culture supernatants of 10T1/2-control and -periostin-HA
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were immunoprecipitated with anti-HA antibody-conjugated agarose beads. The
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precipitated proteins, total cell lysates, and culture supernatants were analyzed by
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Western blotting using anti-fibronectin and anti-HA antibodies.
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(B) EMI-hFc fusion proteins bound on protein G Sepharose resin were incubated with
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either fibronectin or its 70-kDa fragment. The bound proteins on the resin were eluted
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and then subjected to SDS-PAGE.
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(C) Close proximity between periostin-HA and fibronectin inside the cells
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10T1/2-control and -periostin-HA cells were labeled with anti-fibronectin and anti-HA
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antibodies, and then detected with PLA probes. Nuclei were stained with TO-PRO-3.
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Close proximity between periostin-HA and fibronectin was visualized as bright
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fluorescent dot-like signals (PLA signals) around the nuclei. Representative images are
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shown.
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(D) 293T cells were transfected with either the empty vector (control) or the expression
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vector for EMI-hFc, and then incubated with or without Brefeldin A, which blocks
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vesicular transport from the ER to the Golgi. The total cell lysates were incubated with
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protein G Sepharose, and the proteins bound on the Sepharose and the total cell lysates
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(Input) were analyzed by Western blotting using anti-fibronectin and anti-human IgG
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(hFc) antibodies.
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Fig. 4. Tryptophan residue at position 65 in the EMI domain of periostin is necessary to
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decrease the amount of accumulated fibronectin.
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C3H10T1/2 cells were stably transfected with either the empty vector (control), the
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expression vector for periostin-HA (periostin-HA) or the substitution mutant of the
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tryptophan residue to alanine at position 65 in the EMI domain of periostin
13
(peri[Trp65Ala]-HA). The cells were fluorescently stained with anti-fibronectin and anti-
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HA peptide tag antibodies. Arrowheads indicate the granular accumulation of fibronectin
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inside the cells. Representative images are shown.
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Highlights Periostin is localized in the ER.
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Periostin interacts with fibronectin in the ER.
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Expression of periostin decreases accumulation of fibronectin in the ER.
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Fibronectin is accumulated around the nuclei in the periostin-/- connective tissue.
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S0006-291X(16)30140-1
DOI:
10.1016/j.bbrc.2016.01.139
Reference:
YBBRC 35256
To appear in:
Biochemical and Biophysical Research Communications
Received Date: 16 January 2016 Accepted Date: 22 January 2016
Please cite this article as: I. Kii, T. Nishiyama, A. Kudo, Periostin promotes secretion of fibronectin from the endoplasmic reticulum, Biochemical and Biophysical Research Communications (2016), doi: 10.1016/j.bbrc.2016.01.139. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Periostin promotes secretion of fibronectin from the
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endoplasmic reticulum
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3 Isao Kii,1,2,** Takashi Nishiyama,1 and Akira Kudo1*
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cho, Midori-ku, Yokohama 226-8501, Japan.
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Pathophysiological and Health Science Team, Imaging Application Group, Division of
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Department of Biological Information, Tokyo Institute of Technology, 4259 Nagatsuta-
Bio-Function Dynamics Imaging, RIKEN Center for Life Science Technologies, 6-7-3 Minatojima-minamimachi, Chuo-ku, Kobe, Hyogo 650-0047, Japan.
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Runnning head: Periostin in the secretory pathway
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*Corresponding author:
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Akira Kudo, PhD., 4259-B-33 Nagatsuta, Midori-ku, Yokohama 226–8501, Japan.
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Tel and Fax: +81-45-924-5718, E-mail: [email protected]
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**Corresponding author:
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Isao Kii, PhD., 6-7-3 Minatojima-minamimachi, Chuo-ku, Kobe, Hyogo 650-0047, Japan.
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Tel: +81-78-304-7192 (Ext. 95-8380), Fax: +81-78-304-7191, E-mail: [email protected]
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ABSTRACT
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Extracellular matrix (ECM) proteins are synthesized in the endoplasmic reticulum (ER),
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transported to the extracellular milieu through the secretory pathway, and assembled into
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an extracellular architecture. Previous studies of ours showed that periostin, a secretory
5
protein, interacts with fibronectin and is involved in ECM remodeling. Here we show that
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periostin played a role in fibronectin secretion from the ER. Co-immunoprecipitation and
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in situ proximity ligation assays revealed an interaction between periostin and fibronectin
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in the ER. Although accumulation of fibronectin was detected in the ER of fibroblastic
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C3H10T1/2 cells, forced expression of periostin in those cells decreased the
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accumulation of fibronectin in the ER, suggesting that periostin promoted the secretion of
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fibronectin. A substitution mutant of tryptophan at the position 65 to alanine in the EMI
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domain of periostin, which caused periostin to lose its ability to interact with fibronectin,
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did not decrease the accumulation. Furthermore, targeted disruption of periostin in mice
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caused the non-fibrillar and ectopic deposition of fibronectin in the periodontal ligament.
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Thus, these results demonstrate a subcellular role of periostin in promotion of fibronectin
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secretion from the ER.
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Keywords
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Periostin; Fibronectin; Endoplasmic reticulum; Periodontal ligament
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1. Introduction Mechanical properties such as tensile strength and elasticity of the connective tissues depend on the extracellular matrix (ECM) architecture. The ECM architecture is
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constructed from a large number of ECM proteins and remodeled by dissociation and
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subsequent reconnection of the ECM proteins under the dynamic changes occurring in
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mechanical environments [1]. The ECM architecture is based on a framework of
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fibronectin matrix, which is required for the assembly of multiple ECM proteins [2]. This
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matrix, which consists of highly elastic fibronectin fibrils, is remodeled under dynamic
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movement [3-6]. Thus, fibronectin plays an important role in regulating ECM remodeling.
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The ECM proteins are synthesized in the endoplasmic reticulum (ER), transported into the extracellular milieu through the secretory pathway, and assembled into an
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extracellular architecture. The ECM proteins are folded into their native conformations
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with the aid of ER molecular chaperones before they are transported to post-ER
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compartments. General ER chaperones (e.g., calreticulin, calnexin, protein disulfide
15
isomerase, and BiP) play a role in the folding of the ECM proteins [7-11]. Specific
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molecular chaperones for the ECM proteins have also been demonstrated. For example,
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heat shock protein 47 (Hsp47) is a chaperone for collagen in the ER [12, 13]. Type I
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collagen molecules in the ER of hsp47-null cells become insoluble aggregates [14].
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Chaperone-like activity for ECM proteins was also reported in the case of a matricellular
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protein, SPARC, otherwise known as osteonectin [15-17]. Thus, the general and specific
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molecular chaperones for the ECM proteins in the ER maintain ECM homeostasis.
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Periostin is a secretory protein involved in ECM remodeling in the periodontal ligament, myocardial infarction, and heart valve [18, 19]. Targeted disruption of the
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periostin gene in mice causes a defect in the ECM remodeling in the periodontal
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ligament, which is a dense collagenous tissue connecting tooth to alveolar bone, resulting
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in disturbance of incisor eruption [20]. We and others also demonstrated that the rate of
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heart ruptures and death caused by acute myocardial infarction is higher in periostin-/-
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mice than in wild-type counterparts [21, 22]. It has been reported that periostin directly
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binds to several ECM proteins, such as fibronectin, collagens, and tenascin-C [23, 24].
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Furthermore, we demonstrated that periostin functions as a scaffold between tenascin-C
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and the ECM proteins, including fibronectin and type I collagen [23]. As described above,
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periostin has been studied as one of the ECM proteins. On the other hand, several reports
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showed a subcellular localization of periostin [19, 23, 25-27], and the role of subcellular
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periostin is not yet well understood.
In this present study, we demonstrated an interaction between periostin and
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fibronectin in the ER. This interaction may enhance the solubility of fibronectin to
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prevent its aggregation in the ER. Furthermore, we found that periostin deficiency in
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mice caused the ectopic deposition of fibronectin in the periodontal ligament. Thus, the
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present study suggests that periostin interacted with fibronectin to promote the secretion
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of the latter from the ER.
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2. Materials and methods
2 2.1.
Cell culture and transfection C3H10T1/2 and 293T cells were maintained in low-glucose Dulbecco's Modified
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Eagle Medium (DMEM; Nacalai Tesque, Inc., Kyoto, Japan) supplemented with 10%
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fetal bovine serum (FBS; JRH Biosciences, Inc., Lenexa, KS), in which serum fibronectin
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had been depleted [23]. Cells were transfected with expression vectors with
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polyethylenimine (Sigma-Aldrich, Inc., St. Louis, MO), as described previously [23]. For
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immunocytochemistry, cells were cultured on coverslips (No. 1-S; Matsunami Glass Ind.,
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LTD, Osaka, Japan) in a sparse condition.
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2.2.
Antibodies
Rat monoclonal anti-mouse fibronectin antibody (A15-1) was generated
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previously in our laboratory (unpublished). Rabbit polyclonal anti-fibronectin antibody
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Ab-10 (NeoMarkers, Lab Vision Corporation, Thermo Fisher Scientific Inc.), monoclonal
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mouse anti-HA antibody (Nacalai Tesque), rabbit polyclonal anti-HA antibody (Santa
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Cruz Biotechnology Inc., CA), and rabbit polyclonal anti-calreticulin antibody (Affinity
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BioReagents Inc., Thermo Fisher Scientific Inc.) were purchased from the sources
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indicated. Alexa Fluor-labeled goat anti-rabbit, anti-mouse, anti-rat antibodies, Alexa
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Fluor 568-labeled phalloidin, and TO-PRO-3 were obtained from Molecular Probes Inc.
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(Thermo Fisher Scientific Inc.).
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2.3.
Immunofluorescence microscopy
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Immunohistochemistry was performed as described previously [23], and
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fluorescent images were collected with a laser-scanning confocal microscope (FV1000-
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BX61; Olympus Corporation, Japan).
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2.4.
Preparation of microsomal fraction
Cells (1 x 108) were detached with trypsin, washed with ice-cold PBS, and
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homogenized on ice with Dounce disruption in 20 mM Tris-HCl (pH 7.5), 5 mM EDTA
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with protease inhibitors. Before centrifugation at 700 × g, the solution was brought up to
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440 mM sucrose. The postnuclear supernatants were centrifuged further, first at 12,000 ×
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g for 15 min at 4 °C to pellet the mitochondria fraction; and the resulting supernatants
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were then ultracentrifuged at 120,000 × g for 1 hr at 4 °C. The pellets (microsomal
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fraction) were resuspended in 500 µL of the above buffer containing sucrose.
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2.5.
Animals
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Care and experiments with animals were in accordance with the guidelines of the
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Animal Care and Use Committee at Tokyo Institute of Technology. Generation of
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periostin-/- mice was described previously [20, 22].
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2.6.
Immunohistochemistry Tissue paraffin sections were obtained and immunostained, as described earlier
[20, 23].
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2.7.
Immunoprecipitation
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Immunoprecipitation were performed with anti-HA antibody-conjugated agarose
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(Sigma-Aldrich) or protein G Sepharose (GE Healthcare), as described previously [23].
3 2.8.
Conjugation of purified fibronectin protein and its 70-kDa fragment with Alexa
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Fluor 555
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Fibronectin and its 70-kDa fragment (Sigma-Aldrich) were diluted in 100 mM
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HEPES buffer (pH 8.0), and conjugated with Alexa Fluor 555 succinimidyl ester
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(Molecular Probes, Thermo Fisher Scientific Inc.). The unreacted ester was removed by
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dialysis against PBS.
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2.9.
EMI-hFc pull-down assay
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The fusion protein of the amino-terminal domain of periostin (EMI domain) and
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the Fc region of human IgG (EMI-hFc) [23] was purified from 293T cells stably
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expressing EMI-hFc by use of protein G Sepharose (GE Healthcare). Protein bound on
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protein G Sepharose was incubated with the Alexa-555-labeled fibronectin or its 70-kDa
16
fragment in 25 mM HEPES (pH 7.2), 150 mM NaCl, 0.1% NP-40 for 1 hr, and then
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pulled down by centrifugation. The bound proteins were sequentially eluted with SDS
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sample buffer containing 50 mM DTT, subjected to SDS-PAGE, and then detected with
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Typhoon 8600 (GE Healthcare). The gels were also stained with a CBB rapid stain kit
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(Nacalai Tesque).
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2.10.
In situ proximity ligation assay (PLA)
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In situ PLA was carried out according to the manufacturer’s protocol using mouse
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anti-HA, rabbit anti-fibronectin (Ab-10) antibodies, and a Duolink Detection Kit (Olink
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Bioscience, Sweden), as described previously [23].
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2.11.
Treatment with Brefeldin A (BFA)
Cells were incubated with 10 µg/mL BFA (Sigma-Aldrich) for 12 hr prior to lysis for the immunoprecipitation assay.
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3. Results
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3.1.
Periostin is localized in the ER To examine the subcellular localization of periostin, we performed
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immunofluorescence analysis of C3H10T1/2 cells stably expressing periostin-HA
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(10T1/2-periostin-HA cells), in which an HA peptide tag was conjugated at the carboxyl-
6
terminal end of mouse periostin [23]. Periostin-HA was detected inside the cells (Fig. 1A).
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These fluorescence signals coincided with those of calreticulin, an ER marker, indicating
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that periostin-HA was localized in the ER. Slightly strong fluorescence signals for the
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HA tag were detected around the nuclei, which corresponds to the Golgi [23, 26].
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3.2.
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the ER
Forced expression of periostin-HA decreases accumulation of fibronectin in
The periostin-HA localization in the ER prompted us to envisage a role of
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periostin in the ER. Periostin is known to interact with fibronectin [23, 24]. We compared
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the localization of fibronectin in the 10T1/2-periostin-HA cells with that in C3H10T1/2
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cells harboring the empty vector (10T1/2-control cells). In the 10T1/2-control cells,
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immunofluorescent signals of fibronectin were detected not only in the fibrillar ECM, but
18
also inside the cells (Fig. 1B). The strong signals of fibronectin inside the cells appeared
19
as a granular accumulation (Fig. 1B), which was similar to that in chicken embryonic
20
fibroblasts reported previously [28]. In the 10T1/2-periostin-HA cells, the fibrillar
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fibronectin in the ECM was detected the same as in the 10T1/2-control ones, whereas the
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intracellular granular signals were rarely detected (Fig. 1C).
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To investigate the localization of fibronectin, we performed immunofluorescence
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analysis using anti-fibronectin and anti-calreticulin antibodies. The fluorescent signals of
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fibronectin were co-localized with those of calreticulin in the 10T1/2-control cells, but
4
not in the 10T1/2-periostin-HA cells (Fig. 1D), indicating that periostin decreased the
5
fibronectin accumulation in the ER. To quantify the fibronectin accumulation in the ER,
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we prepared the microsomal fraction, which is rich in ER. The amount of fibronectin was
7
decreased in the microsomal fraction of the 10T1/2-periostin-HA cells compared with
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that in that of the 10T1/2-control cells (Fig. 1E), which is consistent with the results of
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the immunofluorescence analysis (Fig. 1B to 1D). Thus, these results suggest that
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periostin promoted the secretion of fibronectin from the ER.
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periostin-/- mice
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Non-fibrillar deposition of fibronectin in the periodontal ligament of
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To confirm the fibronectin accumulation observed in the in vitro analyses, we performed immunohistochemical staining using anti-fibronectin antibody to examine the
16
expression of fibronectin in the periodontal ligament of incisors from wild-type and
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periostin-/- mice. As reported previously [20], sections stained with hematoxylin-eosin
18
showed the absence of a region of the ECM remodeling, the so-called shear zone (Fig. 2A
19
and arrows in 2B), in the periostin-/- periodontal ligament (Fig. 2C and 2D). In the
20
periodontal ligament of the wild-type incisors, fibrillar immuno-reactivity indicating
21
fibronectin was detected around the cells (Fig. 2F). In contrast, non-fibrillar and dappled
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immuno-signals for fibronectin were observed especially around the nuclei in the
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periodontal ligament of the periostin-/- incisors (Fig. 2H). Thus, these results indicate that
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the periostin deficiency caused ectopic deposition of fibronectin in the periodontal
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ligament of the incisors.
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3.4.
Periostin interacts with fibronectin in the ER
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An earlier study showed direct interaction of periostin with fibronectin coated on
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the surface of a microtiter plate [24], an interaction achieved through the amino-terminal
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EMI domain of periostin [23]. To examine whether periostin interacted with fibronectin
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in cells or culture supernatant, we performed a co-immunoprecipitation assay with 293T
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cells transiently co-transfected with the expression vectors for periostin-HA and
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fibronectin. Fibronectin was co-immunoprecipitated with periostin-HA in the total cell
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lysate, but not in the cell culture supernatant, even though the cell culture supernatant
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contained an abundance of fibronectin and periostin-HA proteins (Fig. 3A). To clarify the reason why the interaction between periostin and fibronectin was
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not detected in the cell culture supernatant, we examined whether periostin would interact
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with purified fibronectin in solution. We prepared the EMI-hFc fusion protein (see
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Materials & Methods) bound on protein G Sepharose resin, and mixed it with either the
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purified fibronectin or its 70-kDa fragment in solution. We detected a small amount of
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purified fibronectin pulled down with the EMI-hFc resin, compared with the amount of
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the 70-kDa fragment pulled down with it (Fig. 3B), indicating that the structure of
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fibronectin in solution may have interfered with the interaction between periostin and the
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70-kDa region of fibronectin.
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To confirm that periostin indeed interacted with fibronectin inside the cells, we looked for close proximity between periostin and fibronectin by performing in situ PLA,
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which depicts the close proximity (≤ 40 nm) of cellular molecules as fluorescent signals
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(PLA signals) [29]. Fibronectin and periostin-HA in the 10T1/2-control and -periostin-
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HA cells were labeled indirectly by using anti-rabbit PLUS probe and anti-mouse
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MINUS probe, respectively. If the PLUS and MINUS probes are proximate to each other,
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PLA signals emerge as bright fluorescent dots at the proximity site. The in situ PLA
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signals between periostin-HA and fibronectin were detected around the nuclei (Fig. 3C).
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These results indicate that periostin and fibronectin interacted only inside the cells,
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especially around the nuclei, probably at locations corresponding to the ER and the Golgi.
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If periostin interacted with fibronectin in the ER, blocking of the protein transport
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from the ER to the post-ER compartments should increase the amount of the complex of
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periostin and fibronectin. So we performed a co-immunoprecipitation assay using cells
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treated or not with Brefeldin A (BFA), which interferes with anterograde protein
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transport from the ER to the Golgi and causes the accumulation of secretory proteins
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inside the ER. The treatment with BFA increased the interaction between EMI-hFc and
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fibronectin in 293T cells transiently co-transfected with the expression vectors for EMI-
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hFc and fibronectin (Fig. 3D), indicating that the interaction between periostin and
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fibronectin had taken place in the ER.
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3.5.
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in secretion of fibronectin from the ER.
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Tryptophan residue at position 65 in the EMI domain of periostin is involved
To ask whether the interaction between periostin and fibronectin promoted the
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secretion of fibronectin from the ER, we examined a mutant periostin that loses its ability
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to interact with fibronectin. An earlier study of ours showed that the substitution mutant
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of tryptophan at position 65 to alanine in the EMI domain of periostin (Trp65Ala) fails to
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interact with fibronectin [23]. The granular accumulation of fibronectin was detected
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inside C3H10T1/2 cells stably expressing the mutant periostin (peri[Trp65Ala]-HA), just
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the same as in the 10T1/2-control cells (Fig. 4). Thus, this result suggests that the
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tryptophan residue was required for promotion of fibronectin secretion from the ER.
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DISCUSSION In this study, we found that periostin played a role in the secretion of fibronectin
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from the ER. Periostin consists of an amino-terminal EMI domain, a tandem repeat of 4
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fas1 domains (RD1, 2, 3, and 4), and a carboxyl-terminal region (CTR) [19]. The EMI
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domain directly binds to fibronectin [23]. Earlier we showed that recombinant RD4-CTR
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of periostin exhibits high solubility and monodispersity in solution [30]. These features
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may enhance the solubility of fibronectin in the ER, resulting in decreased granular
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accumulation of fibronectin in the ER.
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Periostin has no ER-retention signals such as the KDEL sequence. Indeed,
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periostin is efficiently secreted into culture medium [31]. Distinct from other ER
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chaperones, periostin functions in efficient secretion of ECM proteins with low solubility.
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Fibronectin in the ECM forms a huge insoluble assembly of multiple ECM proteins [32],
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and this insolubility probably contributes to stability of the ECM architecture. To strike a
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balance between its insolubility in the ECM and solubility in the ER, periostin have
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evolved to interact with fibronectin in the ER, enhancing the solubility of the latter, and
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to dissociate from it in the extracellular milieu. It should be noted that periostin did not
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interact with fibronectin in solution. Fibronectin exists in a compact or extended
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conformation, depending upon the surrounding environment [6, 33, 34]. For example,
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soluble fibronectin is folded into a compact conformation, and a hydrophobic surface
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extends fibronectin conformation [35]. Taken together, the data suggest that the
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interaction between periostin and fibronectin may depend on the conformation of
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fibronectin. Further studies are necessary to clarify the conditions for the interaction
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between periostin and fibronectin, and the conformational change in fibronectin during its
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passage through the secretory pathway through the ER to the extracellular milieu.
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The present study also provides a mechanistic insight into the mechanism of the disturbance in incisor eruption seen in periostin-/- mice. In the periodontal ligament of
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incisors from periostin-/- mice, the disorganized fibronectin matrix or the accumulated
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fibronectin probably disturbs the cellular functions necessary to remodel the ECM
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architecture [20]. The extracellular fibronectin matrix functions as a framework for the
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ECM architecture, and thus disorganization of this framework must result in disruption of
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the ECM remodeling.
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Several groups including ours have reported abnormalities in periostin-/- mice,
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such as those in the periodontal ligament, periosteum, infarcted myocardium, tendon,
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heart valve, and so on [18, 19]. Previously we demonstrated that the confined tibial
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periostitis in periostin-/- mice, which is similar to that seen in human sports injuries, is
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due to the decreased incorporation of tenascin-C into the ECM architecture [23]. In the
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case of myocardial infarction, integrin signaling may be involved in the periostin-
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mediated recruitment of myofibroblasts to the infarcted region [22]. This periostin-
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mediated integrin signaling plays essential roles in other physiological and pathological
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systems as well [18, 19]. These varieties of reports on the molecular functions of
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periostin suggest that periostin is a multifunctional secretory protein.
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Acknowledgements The authors thank Prof. Norio Amizuka (Hokkaido University, Japan) and Dr. Minqi Li (Hokkaido University, Japan) for preparation of paraffin sections of wild-type
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and periostin-/- mandibles. This work was supported by grants-in-aid for scientific
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research from the Ministry of Education, Science, Culture, and Sports of Japan
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(19791362, 19370093).
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FIGURE LEGENDS
2 Fig. 1. Periostin decreases the accumulation of fibronectin in the ER.
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(A) Periostin-HA is detected in the ER. C3H10T1/2 cells were stably transfected with the
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expression vector of periostin-HA, in which an HA peptide tag was conjugated at the
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carboxyl-terminal end of mouse periostin. The cells were fluorescently stained with anti-
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HA and anti-calreticulin antibodies.
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(B and C) C3H10T1/2 cells stably expressing periostin-HA (10T1/2-periostin-HA; C)
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and C3H10T1/2 cells harboring empty vector (10T1/2-control; B) were stained with anti-
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fibronectin and anti-HA antibodies (upper columns) or with anti-fibronectin and
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fluorescently labeled phalloidin (lower columns). Arrowheads indicate strong immuno-
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reactions for fibronectin proteins inside the cells; and arrows, obscure ones inside them.
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(D) 10T1/2-control and -periostin-HA cells were fluorescently stained with anti-
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fibronectin and anti-calreticulin antibodies. Arrows indicate co-localization of strong
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immunoreactivity for fibronectin and calreticulin.
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(E) Microsomal fractions of 10T1/2-control and -periostin-HA cells were prepared by the
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ultracentrifugation method (see Materials and methods). The microsomal fractions and
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the total cell lysates were analyzed by Western blotting using anti-fibronectin, anti-HA,
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and anti-calreticulin antibodies.
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Fig. 2. Ectopic deposition of fibronectin proteins in the periodontal ligament of the
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incisors from periostin-/- mice.
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Paraffin sections of mandibles from 12-week-old wild-type (A, B, E, F) and periostin-/-
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(C, D, G, H) mice were stained with hematoxylin-eosin (HE) solution (A-D) or anti-
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fibronectin antibody (E-H). The periodontal ligaments between the dentin and bone in
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“A,” “C,” “E,” and “G” are shown in “B,” “D,” “F,” and “H,” respectively. Typical
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immunoreactions in “F” and “H” are presented as high-magnification images in the white
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rectangular boxes. Arrows indicate shear zone, which is the region of ECM remodeling.
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Arrowheads indicate strong immunoreactivity for fibronectin proteins around the nuclei.
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Representative images are shown.
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Fig. 3. Interaction between periostin and fibronectin in the ER.
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(A) Total cell lysates and cell culture supernatants of 10T1/2-control and -periostin-HA
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were immunoprecipitated with anti-HA antibody-conjugated agarose beads. The
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precipitated proteins, total cell lysates, and culture supernatants were analyzed by
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Western blotting using anti-fibronectin and anti-HA antibodies.
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(B) EMI-hFc fusion proteins bound on protein G Sepharose resin were incubated with
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either fibronectin or its 70-kDa fragment. The bound proteins on the resin were eluted
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and then subjected to SDS-PAGE.
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(C) Close proximity between periostin-HA and fibronectin inside the cells
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10T1/2-control and -periostin-HA cells were labeled with anti-fibronectin and anti-HA
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antibodies, and then detected with PLA probes. Nuclei were stained with TO-PRO-3.
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Close proximity between periostin-HA and fibronectin was visualized as bright
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fluorescent dot-like signals (PLA signals) around the nuclei. Representative images are
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shown.
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(D) 293T cells were transfected with either the empty vector (control) or the expression
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vector for EMI-hFc, and then incubated with or without Brefeldin A, which blocks
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vesicular transport from the ER to the Golgi. The total cell lysates were incubated with
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protein G Sepharose, and the proteins bound on the Sepharose and the total cell lysates
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(Input) were analyzed by Western blotting using anti-fibronectin and anti-human IgG
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(hFc) antibodies.
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Fig. 4. Tryptophan residue at position 65 in the EMI domain of periostin is necessary to
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decrease the amount of accumulated fibronectin.
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C3H10T1/2 cells were stably transfected with either the empty vector (control), the
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expression vector for periostin-HA (periostin-HA) or the substitution mutant of the
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tryptophan residue to alanine at position 65 in the EMI domain of periostin
13
(peri[Trp65Ala]-HA). The cells were fluorescently stained with anti-fibronectin and anti-
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HA peptide tag antibodies. Arrowheads indicate the granular accumulation of fibronectin
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inside the cells. Representative images are shown.
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Highlights Periostin is localized in the ER.
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Periostin interacts with fibronectin in the ER.
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Expression of periostin decreases accumulation of fibronectin in the ER.
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Fibronectin is accumulated around the nuclei in the periostin-/- connective tissue.
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