OPN and syndecan-4 preparations.
The three human OPN complementary DNAs (cDNAs) were inserted into pGEX-4T vector (GE Healthcare) in the same reading frame as the carrier gene GST and were transformed in E. coli
JM109 cells. Thus, three unglycosylated human OPN/GST fusion proteins were produced: OPN full/GST (M1-N314), OPN N half/GST (M1-R168), and OPN C half/GST (K170-N314). These proteins were purified as described previously (29
). To prepare the glycosylated forms of OPN (OPN/CHO and OPN N half/CHO), the full-length human and mouse OPN and OPN N half cDNAs were inserted into pcDNA3 (Invitrogen) and transfected to CHO-K1 cells with Lipofectamine 2000 (Invitrogen). The glycosylated form of human OPN was purified with a formyl-cellulofine column (Seikagaku Kogyo) coupled with anti-OPN (O-17) antibody (Immuno-Biological Laboratories Co., Ltd. [IBL]), as described previously (30
). Mouse OPN was purified with a formyl-cellulofine column coupled with rabbit anti–mouse OPN (M3) antibody raised against 155
(IBL). The cDNA for human syndecan-4 and the extracellular domain of human syndecan-4 (M1-E145) were PCR amplified from NRC-12 cells, and the cDNA for the extracellular domain of mouse syndecan-4 (M1-R143) was from B16-BL6 mouse melanoma cells using the following primers: human syndecan-4, 5′-TGCGGATCCGGTGCCATGGCCCCCGCCCGT-3′ (sense) and 5′-TGGCTCGAGTCACGCGTAGAACTCATTGGT-3′ (antisense); human syndecan-4 (M1-E145), 5′-TGCAAGCTTGGTGCCATGGCCCCCGCCCGT-3′ (sense) and 5′-TTGGGATCCTCCGTTCTCTCAAAGATGTT-3′ (antisense); and mouse syndecan-4 (M1-R143), 5′-AAAGAATTCGAAGCCATGGCGCCTGCCTGC-3′ (sense) and 5′-GTCGGATCCCTCTCAAAGATGTTGCTG-3′ (antisense). The amplified product of syndecan-4 was cloned into pcDNA3.1. The products of the extracellular domain of syndecan-4 were fused with a Fc portion of human IgG1 (Syn4Ig), cloned into pcDNA3.1, and transfected to CHO-K1 suspension culture cells with Lipofectamine 2000. Syn4Ig was purified by using protein A column chromatography.
Reagents and cell lines.
Thrombin and PMA were obtained from Sigma-Aldrich. Human renal cell carcinoma cell lines (NRC-12; IBL) and human B lymphocyte lines of Burkitt lymphoma origin (Namalwa; American Type Culture Collection) were cultured in TIL medium (IBL) supplemented with 10% FCS. The Namalwa cells were stably transfected with OPN cDNA and were referred to as OPN/Namalwa. CHO cells were also transfected with OPN alone, syndecan-4 alone, or both OPN and syndecan-4 cDNA. CHO-K1 (Cell Resource Center for Biomedical Research, Tohoku University) and CHO-K1 suspension culture (RIKEN Cell Bank) cells were cultured in DMEM/nutrient mixture F-12 (Wako) supplemented with 5% FCS. NIH3T3 and B16-BL6 (Cell Resource Center for Biomedical Research, Tohoku University) were cultured in DMEM supplemented with 5% FCS. Antibodies used to detect human OPN (1B20) and syndecan-4 in the Western blot analysis were purchased from IBL. Anti–α4 integrin (P1H4) antibody was obtained from Chemicon. Rabbit antibody (M5) specifically recognizing the cryptic epitope within mouse OPN molecules, which is exposed by thrombin cleavage, was obtained from IBL and used to neutralize the function of the thrombin-cleaved form of OPN (12
6–8-wk-old C57BL/6 mice were obtained from Japan SLC. Syndecan-4 KO mice (3
), backcrossed >10 times into the C57BL/6 background, were obtained from the Center for Animal Resources and Development. These animals were maintained in specific pathogen-free conditions in the animal facility of the Laboratory of Animal Experiment for Disease Model (Institute for Genetic Medicine, Hokkaido University). All animal experiments were in accordance with the guidelines of an institutional committee at Hokkaido University.
Enzyme treatment of syndecan-4.
100 μg Syn4Ig was dialyzed against a 1:1 mixture of 0.1 M sodium acetate buffer, pH 7, and 10 mM calcium acetate, and digested with 20 mU HSase (Seikagaku Kogyo), with 85 mU CSase (Sigma-Aldrich), or with both enzymes at 37°C for 15 h.
OPN binding to heparin and syndecan-4.
OPN proteins or synthetic peptides were coated onto a 96-well plate at various concentrations at 37°C for 1 h, then blocked with 0.1% BSA in Tris-buffered saline (TBS) containing 0.05% NaN3 at 37°C for at least 1 h The plates were washed two times with TBS and incubated with either 10 μg/ml of biotinylated heparin or 10 μg/ml Syn4Ig at 37°C for 1 h After a further three washes, a 1:5,000 dilution of peroxidase-conjugated streptavidin (Jackson ImmunoResearch Laboratories) for biotinylated heparin or peroxidase-conjugated anti–human IgG (Jackson ImmunoResearch Laboratories) for Syn4Ig were added to each well at room temperature for 30 min. Bound protein was quantified by a colorimetric assay using 3,3,5,5-tetramethylbenzidine solution (Thermo Fisher Scientific) as a substrate for 15 min at room temperature. Plates were read at a wavelength of 450 nm. To evaluate endogenous binding of OPN to syndecan-4, NRC-12 cells and OPN/Namalwa cells, stimulated with PMA for 30 min in serum-free medium, were cultured for an additional 48 h without PMA. The supernatant of NRC-12 cells was applied to an anti–syndecan-4 antibody–coupled formyl-cellulofine column and washed extensively. A rabbit IgG–coupled formyl-cellulofine column was used as a control column. Elute fraction with 0.2 M glycine-HCl, pH 2.5, was immediately neutralized and electrophoresed through 12% SDS-PAGE gel and probed with anti-OPN (1B20) or anti–syndecan-4 antibody. The supernatants of NRC-12 and OPN/Namalwa cells were also applied to an anti-OPN antibody (O-17; IBL)–coupled formyl-cellulofine column and Western blotted. The supernatants obtained from CHO cells, transiently transfected with OPN alone, syndecan-4 alone, or both OPN and syndecan-4 cDNA, were also tested for the presence of association between OPN and syndecan-4.
Thrombin treatment of OPN.
50 μl of 3 μg/ml hOPN/CHO protein was mixed with either 50 μl of human IgG1 (hIgG) or 6 μg/ml Syn4Ig and incubated for 1 h at 37°C, then digested with 2 U of thrombin for 30 min.
Cell adhesion test.
The 96-well plates were precoated with 10 μg/ml OPN/CHO protein in the presence or absence of 20 μg/ml Syn4Ig overnight at 4°C, followed by treatment with 0.5% BSA in TBS for 1 h at room temperature. Cells were suspended in DMEM containing 0.25% BSA, and 200 μl of cell suspension (at a cell density of 5 × 104 cells per well) was applied to 96-well plates and incubated for 1 h at 37°C. The medium was removed from the plates, and all wells were washed twice. The adherent cells were fixed and stained by 0.5% crystal violet in 20% methanol for 30 min. All wells were rinsed three times with water, and adherent cells were then lysed with 20% acetic acid. The resulting supernatants from each well were analyzed by an immunoreader (Immuno Mini NJ-2300; Nolge Nunc International), and the absorbance at 590 nm was measured to determine the relative number of cells adhered to wells. The binding of cells to OPN was expressed as 100%.
OPN, human syndecan-4 (IBL), and IFN-γ (BD Biosciences) concentrations were measured by using ELISA kits as specified by the manufacturers. The plasma level of mouse syndecan-4 was measured by using a ELISA system, which was established using 10 μg/ml of rabbit anti–mouse syndecan-4 antibody (IBL) for capture antibody and 3 μg/ml of biotinylated anti-HS antibody (Seikagaku Kogyo) for detection antibody. Purified mouse syndecan-4 Ig was used for standard. The thrombin-cleaved form of mouse OPN was detected by ELISA obtained from IBL. The detailed information on this ELISA for the thrombin-cleaved form of OPN is shown in Fig. S7.
Induction of ConA-induced liver injury in mice.
C57BL/6 mice were injected intravenously with 10 mg ConA (Sigma-Aldrich) per kilogram of body weight, dissolved in pyrogen-free PBS. In some experiments, 150 μg Syn4Ig or human IgG was administered to mice intraperitoneally 15 h before ConA challenge. Liver damage was evaluated by measuring the serum activity of ALT and AST by using a standard clinical autoanalyzer (DRI-CHEM 5500V; Fujifilm).
Livers were harvested at various times after ConA injection. All specimens were fixed in 10% buffered formalin and embedded in paraffin. Sections were cut and stained with hematoxylin and eosin (H-E). Light microscopy was performed to assess liver injury. Necrotic areas were measured in each section by using ImageJ (version 1.37; National Institutes of Health), followed by calculation of the necrotic area per section. For each tissue, data were obtained using at least three high power fields (×100).
Liver-infiltrating leukocytes were isolated as previously described (14
). In brief, livers were minced after a few minutes of perfusion, pressed through a stainless steel mesh, and suspended in PBS. After washing, cells were resuspended in 33% Percoll solution and centrifuged at 2,000 rpm for 15 min to remove liver parenchymal cells. The pellet was treated with red blood cell lysis solution, washed with PBS, and resuspended in 10% FCS-DMEM. The numbers of leukocytes and macrophages were determined by flow cytometry using monoclonal antibodies reacting to CD45 (BD Biosciences) and F4/80 (Serotec), respectively.
Analysis of messenger RNA (mRNA) expression.
Total RNA from livers was extracted by TRIzol (Invitrogen). The following primers were used: G3PDH, 5′-ACCACAGTCCATGCCATCAC-3′ (sense) and 5′-TCCACCACCCTGTTGCTGTA-3′ (antisense); and syndecan-4, 5′-ATTCGAGAGACAGAGGTCATC-3′ (sense) and 5′-CTCTGAGGGGACACGGATGCC-3′ (antisense). Quantitative real-time PCR analysis of mRNA expression was also performed with LightCycler Fast Start DNA Master SYBR Green I Systems (Roche). The expression of mRNA was calculated by LightCycler software (version 3; Roche). Data were standardized by G3PDH.
Data are presented as means ± SEM and are representative of at least three independent experiments. Significant differences between experimental groups in the adhesion test, thrombin-resistance test, and ConA-induced hepatitis model were analyzed using the Student's t test. Differences were considered to be significant when P < 0.05 (*) or 0.005 (**).
Online supplemental material.
Fig. S1 shows the structure of OPN. Fig. S2 shows the binding of heparin to the full-length form of OPN. Fig. S3 shows the structure of syndecan-4 and Syn4Ig. Fig. S4 shows the association between OPN and syndecan-4 in the supernatant of CHO transfectant cells. Fig. S5 shows the binding of Syn4Ig to the HBD of OPN. Fig. S6 shows the binding of OPN to plates in the presence or absence of Syn4Ig. Fig. S7 shows the specificity of the ELISA system for detection of the thrombin-cleaved form of mouse OPN. Online supplemental material is available at http://www.jem.org/cgi/content/full/jem.20071324/DC1