Syndecan-4 modulates OPN functions by masking functional domains of OPN
We thus examined whether the association of OPN and syndecan-4 modulates the availability of OPN to thrombin and/or its receptors. OPN proteins were mixed with either human IgG or Syn4Ig. Addition of thrombin resulted in the significant reduction of full-length OPN levels in OPN preparation when mixed with human IgG. However, when OPN was mixed with Syn4Ig, OPN was resistant to thrombin digestion (Fig. 2 A). Because the binding domain of syndecan-4 to OPN overlaps the thrombin cleavage site, the thrombin resistance may be achieved through the masking of the thrombin-cleaved site by syndecan-4. Syndecan-4 was originally detected in microvascular endothelial cells as an antithrombin binding molecule (19, 26). Antithrombin is a plasma serine protease inhibitor and, thus, inhibits thrombin activity by forming a covalent complex with thrombin in a 1:1 ratio. Importantly, after binding to syndecan-4 in vivo, antithrombin exhibits a 500-fold increase of its thrombin inhibitory activity (27). Therefore, it is likely that in vivo syndecan-4 regulates formation of thrombin-cleaved OPN by masking the thrombin cleavage site of OPN and/or inhibiting thrombin activity via association with the antithrombin molecule.

View larger version (9K): [in this window] [in a new window] [Download PPT slide]   Figure 2. The masking of functional domains of OPN by syndecan-4. (A) OPN becomes less sensitive to thrombin digestion when bound by syndecan-4. Recombinant OPN (50 µl of 3 µg/ml) was mixed with either 50 µl of human IgG1 (hIgG) or 6 µg/ml Syn4Ig. The mixture was subjected to thrombin digestion (2 U/ml) for 37°C for 30 min. The full-length form of OPN was measured using ELISA. Note that this ELISA is unable to detect thrombin-cleaved OPN. (B) OPN recognition by integrin receptors is inhibited by syndecan-4. The binding of CHO cells to OPN is RGD dependent. The binding of CHO cells to either precoated OPN alone or OPN together with Syn4Ig was examined. (C) The binding of CHO cells expressing 4 integrin (4/CHO) to OPN or OPN bound by syndecan-4 was examined in the presence or absence of 100 µg/ml GRGDS or 10 µg/ml anti–4 integrin antibody. Data are presented as means ± SEM. *, P < 0.05; **, P < 0.005.

Both OPN and syndecan-4 expression are up-regulated in a mouse model of ConA-induced hepatic injury
To investigate whether the described scenario operates in vivo, we examined the role of syndecan-4 in ConA-induced hepatic injury in mice. As expected, both plasma OPN and alanine aminotransferase (ALT) levels were significantly elevated after ConA injection (Fig. 3 A). Because antibodies against mouse syndecan-4 and HS are commercially available, we constructed a sandwich ELISA to measure mouse syndecan-4. Plasma syndecan-4 levels were elevated after ConA injection (Fig. 3 B). We also noted that syndecan-4 gene expression in the liver was significantly up-regulated at 2 h and persisted up to 12 h after ConA injection (Fig. 3 C).

View larger version (26K): [in this window] [in a new window] [Download PPT slide]   Figure 3. The augmented expression of OPN and syndecan-4 in a mouse model of fulminant hepatitis. C57BL/6 mice were intravenously injected with ConA (n = 3). (A) The plasma levels of ALT and OPN after ConA injection. (B) The plasma levels of syndecan-4 after ConA injection. (C) RT-PCR analysis and real-time PCR analysis of syndecan-4 expression in liver after ConA injection. Data are presented as means ± SEM.

View larger version (57K): [in this window] [in a new window] [Download PPT slide]   Figure 4. The high susceptibility of Syn4KO mice to ConA-induced hepatic injury. (A) The plasma levels of ALT and AST in wild-type (Cont) and Syn4KO mice at 12 h after ConA injection (n = 6 per each group). (B) Representative histology of liver sections stained by H-E 12 h after ConA injection, and the survival curve after ConA injection (n = 8 and 13 wild type and Syn4KO, respectively). Dotted lines indicate the area of liver degeneration. The degenerative area per liver section was quantified by using ImageJ (n = 3 per each group). Bar, 200 µm. (C) The plasma levels of full-length and thrombin-cleaved OPN in wild-type (Cont) and Syn4KO mice at 12 h after ConA injection (n = 4 and 6 wild type and Syn4KO, respectively). (D) The attenuation of ConA-induced hepatic injury in Syn4KO mice by anti-OPN (M5) antibody treatment (n = 5 per group). Syn4KO mice were intraperitoneally treated with M5 antibody and control normal rabbit IgG (Cont Ig; 400 µg/head) at 15 h before ConA injection. Plasma ALT levels and representative histology (HE) are shown. Degenerative area per liver section was quantified by using ImageJ (n = 5 per each group). Data are presented as means ± SEM. *, P < 0.05; **, P < 0.005. Bar, 500 µm.

Administration of exogenous recombinant syndecan-4 protects mice from ConA-induced hepatic injury
The final issue we addressed was whether the exogenous purified syndecan-4 can protect mice from ConA-induced hepatic injury. Syndecan-4–injected mice were protected from hepatic injury, as reflected by reduced levels of ALT and IFN- after ConA injection compared with those in control mice (Fig. 5 A). It has been previously shown that OPN is involved in cell adhesion, cell migration, and inflammation via integrin receptors (7, 10, 12, 14, 15, 17). Therefore, we tested whether administration of syndecan-4 would inhibit OPN-mediated inflammatory responses by flow cytometry. The infiltration of inflammatory cells into liver tissues in ConA-injected mice was significantly inhibited by the administration of exogenous syndecan-4 (Fig. 5 B). Infiltrated leukocytes and macrophages/Kupffer cells were defined by the expression of CD45 and F4/80, respectively. Nevertheless, hepatic necrosis in histology was significantly reduced by the administration of syndecan-4 (Fig. 5 C).

View larger version (30K): [in this window] [in a new window] [Download PPT slide]   Figure 5. Exogenous syndecan-4 protects mice from ConA-induced hepatic injury. (A) Exogenous syndecan-4 protects mice from ConA-induced hepatic injury, as judged by plasma ALT levels and plasma IFN- levels at 12 h after ConA injection. Normal human IgG (Cont Ig) was used as a control IgG (n = 10 per each group). (B) Liver-infiltrating leukocytes were prepared from Syn4Ig- or control normal human IgG (Cont Ig)–treated mice at 12 h after ConA injection (n = 5 per group). The number of CD45-positive leukocytes and F4/80-positive macrophages was determined by flow cytometry. (C) Representative histology of liver sections stained by H-E at 24 h after ConA injection. The degenerative area per liver section was quantified by using ImageJ (n = 4 per each group). Data are presented as means ± SEM. *, P < 0.05; **, P < 0.005. Bar, 500 µm. (D) Schematic illustration of the in vivo role of syndecan-4 in OPN-mediated inflammatory reactions.

	MATERIALS AND METHODS

Top ABSTRACT RESULTS AND DISCUSSION MATERIALS AND METHODS REFERENCES

 
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 ¹⁵⁵KSRSFQVSDEQYPDATDE¹⁷² (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–4 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, 14).

Animals.
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 x 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%.

ELISA.
OPN, human syndecan-4 (IBL), and IFN- (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).

Histology.
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 (x100).

Flow cytometry.
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.

Statistical analysis.
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.

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