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Thrombin-Cleaved COOH-Terminal Osteopontin Peptide Binds with Cyclophilin C to CD147 in Murine Breast Cancer Cells

Departments of ¹ Surgery and ² Cell Biology, Duke University Medical Center, Durham, North Carolina

Requests for reprints: Paul C. Kuo, 110 Bell Building, DUMC 3522, Durham, NC 27710. Phone: 919-668-1856; Fax: 919-684-8716; E-mail: kuo00004{at}mc.duke.edu.

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Osteopontin is a glycoprotein that has been linked to metastatic function in breast, lung, and prostate cancers. However, the mechanism by which osteopontin acts to induce metastatic properties is largely unknown. One intriguing feature of osteopontin is the presence of a conserved thrombin cleavage site that is COOH-terminal from a well-characterized RGD domain. Although the COOH-terminal fragment may bind to cell surface CD44 receptors, little is known about the COOH-terminal osteopontin fragment. In the current study, we use the murine mammary epithelial tumor cell lines 4T1 and 4T07; these cells are thioguanine-resistant sublines derived from the parental population of 410.4 cells from Balb/cfC3H mice. Using flow cytometry and Forster resonance energy transfer, we show that the COOH-terminal fragment of osteopontin binds with another marker of metastatic function (cyclophilin C or rotamase) to the CD147 cell surface glycoprotein (also known as extracellular matrix metalloproteinase inducer), to activate Akt1/2 and matrix metalloproteinase-2. In in vitro assays, thrombin cleavage of osteopontin to generate short COOH-terminal osteopontin in the presence of cyclophilin C increases migration and invasion of both 4T07 and 4T1 cells. This interaction between osteopontin peptide and cyclophilin C has not been previously described but assigns a heretofore unknown function for the thrombin-cleaved osteopontin COOH-terminal fragment. [Cancer Res 2007;67(9):4088–97]

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Osteopontin is a phosphorylated acidic glycoprotein that has recently been implicated as a regulator of metastatic function in breast, colon, and hepatocellular cancers (1–4). The underlying molecular mechanisms have not been fully characterized. One intriguing feature of osteopontin is the presence of a conserved thrombin cleavage site that is COOH-terminal from a well-characterized RGD domain (5). Senger et al. were among the first to show cleavage of osteopontin by catalytic naturally occurring concentrations of thrombin and the presence of thrombin-induced osteopontin cleavage products during blood coagulation (6, 7). These authors suggested that the presence of both osteopontin and thrombin were likely to occur in cancer, inflammation, and wound healing. Subsequently, multiple authors have shown activity of the thrombin-cleaved NH₂-terminal domain in mediating integrin binding and cell adhesion (5, 6, 8, 9). However, to date, very little is known of the activity and fate of the corresponding COOH-terminal domain. Although COOH-terminal fragment binds to cell surface CD44 receptors, many believe that osteopontin activity resides primarily in the RGD-containing NH₂-terminal fragment or the intact protein (6, 10).

Osteopontin is a secreted glycoprotein that seems to mediate cell-matrix interactions and cellular signaling through binding with integrin (primarily _vß₃) and CD44 receptors. Osteopontin functions to mediate cell adhesion, chemotaxis, macrophage-directed interleukin-10 suppression, stress-dependent angiogenesis, prevention of apoptosis, and anchorage-independent growth of tumor cells (10–13). Recently, a substantial body of data has linked osteopontin with the regulation of metastatic spread by tumor cells. However, the molecular mechanisms that define the role of osteopontin in tumor metastasis are incompletely understood. In the current study, we show that the COOH-terminal fragment of osteopontin binds with another marker of metastatic function (cyclophilin C or rotamase) to the CD147 cell surface glycoprotein (also known as extracellular matrix metalloproteinase inducer or EMMPRIN), to activate Akt1/2 and MMP-2. This interaction between osteopontin and cyclophilin C has not been previously described but shows a function for the thrombin-cleaved osteopontin COOH-terminal fragment.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Cell cultures. Mouse mammary tumor cell lines 4T1 and 4T07 were cultured in DMEM supplemented with 10% FCS, penicillin (100 units/mL), and streptomycin (100 µg/mL) and maintained at 37°C in a humidified atmosphere of 5% CO₂. For secreted-protein analysis, serum-free DMEM was centrifuged at 600 x g for 5 min to remove cellular material and concentrated 100-fold through Ultrafree Centrifugal filters (Millipore).

Yeast two-hybrid screening. Yeast Saccharomyces cerevisiae cells were maintained following the protocol of MATCHMAKER Gal4 two hybrid system (BD Biosciences Clontech). The mouse full-length osteopontin cDNA and its truncated mutants were separately fused in frame with the GAL4 DNA binding domain in pGBKT7 bait plasmid. Yeast strain AH109, Y187, or CG-1945 were separately cotransformed with the full-length or truncated osteopontin pGBKT7 bait plasmids and mouse 17-day embryo Yeast Two-Hybrid Library (BD Biosciences Clontech). Cotransformation of these yeast two-hybrid plasmids into yeast strain AH109, Y187, or CG-1945; screening of transformants; galactosidase activity of the lacZ reporter marker on Ade/His/Leu/Trp SD medium plates; yeast mating; and isolation of plasmid DNA from yeast were done according to Yeast Protocols Handbook (BD Biosciences Clontech).

Plasmids. The mouse full-length cyclophilin C cDNA, full-length osteopontin cDNA, and its truncated mutants were separately fused in frame into mammalian secretion expression vector pSecTag2/Hygro (Invitrogen), mammalian cell expression vector pcDNA3.1/His B (Invitrogen), and pECFP and pEYFP (BD Biosciences Clontech). The mouse full-length osteopontin cDNA was mutated (Mu-OPN) at its thrombin cleavage site, from aggtca (nucleotides –458 to –463) to agc ttt using PCR, and cloned into mammalian secretion expression vector pSecTag2/Hygro (Invitrogen); this mutates the thrombin cut site from R153-S154 to S153-F154 and renders Mu-OPN resistant to thrombin cleavage. The Mu-OPN plasmid was transfected into COS7 cells; the secreted Mu-OPN protein was purified from the culture medium using the MagneHis Protein Purification System (Promega) and quantified with Bio-Rad protein assay kit (Bio-Rad).

Transient transfection assay. Cells were transiently transfected with the above plasmids using LipofectAMINE 2000, according to the manufacturer's instruction (Invitrogen). Briefly, 4 x 10⁵ cells were seeded with antibiotic-free DMEM on each well of 12-well plates the day before transfection. Two micrograms of plasmid DNA and 4 µL LipofectAMINE 2000, diluted with Opti-MEM medium, were mixed gently and incubated with cells. Culture medium was changed after 6 h of transfection and incubated further at 37°C for 24 h. The control cells received LipofectAMINE 2000 alone.

Coimmunoprecipitation assay. 4T07 cells were cotransfected with pSecTag2/Hygro/CyPC combined with pSecTag2/Hygro/FL-OPN, pSecTag2/Hygro/LC-OPN, pSecTag2/Hygro/SC-OPN, pSecTag2/Hygro/SC-OPN-1, pSecTag2/Hygro/SC-OPN-2, pSecTag2/Hygro/N-OPN-1, or pSecTag2/Hygro/N-OPN-2 for 24 h. The clarified concentrated serum-free supernatant was incubated with osteopontin antibody (R&D Systems) and Protein G-agarose in Co-IP buffer [10 mmol/L Tris-HCl (pH 7.5), 3 mmol/L EGTA, 20 mmol/L NaCl, 0.02% Triton X-100, 1x protease inhibitors cocktail, 0.2 mmol/L DTT, 1 mmol/L phenylmethylsulfonyl fluoride (PMSF)] at 4°C for 4 h. Protein G-agarose beads were collected by centrifugation and washed thrice with the Co-IP buffer. The immune complexes were resuspended with SDS sample buffer and then loaded onto 4% to 20% SDS-PAGE gel for Western blot analysis.

Western blot analysis. Cells were lysed in buffer [0.8% NaCl, 0.02% KCl, 1% SDS, 10% Triton X-100, 0.5% sodium deoxycholic acid, 0.144% Na₂HPO₄, 0.024% KH₂PO₄, 2 mmol/L PMSF (pH 7.4)] and centrifuged at 12,000 x g for 10 min at 4°C. The protein concentration was determined by the Bio-Rad protein assay kit. The protein samples were separated by 4% to 20% SDS-PAGE and electrotransferred onto polyvinylidene difluoride membranes (Amersham Biosciences) by semi-dry transfer (Bio-Rad). The membranes were probed with the following primary antibodies for 1 h at room temperature: osteopontin polyclonal antibody (R&D Systems), CD147 antibody, cyclophilin C antibody, Akt1/2 antibody, MMP-2, phosphorylated Akt1/2 (P-Akt1/2) antibody (Santa Cruz Biotechnology). These antibodies were detected using the appropriate horseradish peroxidase–conjugated secondary antibody. The reactive proteins were visualized by means of the ECL kit (Amersham Bioscience).

Confocal microscopy and Forster resonance energy transfer acceptor bleaching assay. 4T07 cells were cultured on coverslips and cotransfected with pECFP-SC-OPN Forster resonance energy transfer (FRET) donor and pEYFP-CyC FRET acceptor plasmids. After 24 h post-transfection, the coverslips were rinsed thrice with ice-cold PBS followed by fixation for 15 min with 1% (w/v) paraformaldehyde. Coverslips were rinsed thrice with PBS and mounted onto a microscope slide using 50 µL mounting medium (Calbiochem). The coverslips were sealed by wax and kept at 4°C until analysis. Leica TCS SP2 confocal microscope was used for image acquisition. CFP and YFP emission spectra were first optimized at 458 and 514 nm, respectively. FRET was measured by acceptor photobleaching using the FRET-AB wizard in the Leica TS software. A pair of pre-bleach images of CFP and YFP images was collected for the cells of interest. Randomly chosen regions of interest were irradiated (bleached) with the 514-nm laser line set at 100% intensity to photobleach YFP only for the minimum number of iterations of bleaching required. Post-bleach CFP and YFP images were collected following photobleaching. FRET was indicated by an increase in CFP donor fluorescence intensity following YFP photobleaching. FRET efficiency was calculated as 100 x [(donor post-bleach – donor prebleach) / donor post-bleach], taking into account CFP and YFP background noise in each channel; FRET efficiency was measured and calculated automatically by Leica LAS AF software. The apparent K_d values were determined. Fluorescence intensity and apparent K_d values were measured by MetaMorph software (Molecular Devices) using the integrated fluorescence weighting measurement for 12-bit images.

Flow cytometric analysis. SC-OPN-CFP and CyC-YFP fusion proteins were purified from transfected COS7 cell line by MagneHis protein purification system (Promega) and quantified by Bio-Rad protein assay kit (Bio-Rad). 4T07 cells were detached with PBS containing 5 mmol/L EDTA. Following three washes with PBS, the cells were incubated at 37°C for 2 h in a humidified atmosphere of 5% CO₂ with SC-OPN-CFP (2 µg/mL), CyC-YFP (2 µg/mL), CD147 antibody (2 µg/mL; Santa Cruz Biotechnology), osteopontin antibody (2 µg/mL; R&D Systems), and cyclosporine A (100 nmol/L; Sigma-Aldrich). After three washes with PBS, the cells were suspended in PBS, and at least 10⁶ cells were analyzed on a FACSCalibur flow cytometer under standard flow for 120 s (Becton Dickinson). Mean fluorescence intensity was determined using Cell Quest software (Becton Dickinson). The results were obtained from four independent experiments.

Osteopontin small interfering RNA transfection. The sequences of osteopontin small interfering RNA (siRNA) and mismatch siRNA control were synthesized as previously described (14). 4T1 cells were harvested using trypsin 24 h before transfection and plated at a density of 5 x 10⁵ per well in six-well plates (Costar) in DMEM + 10% fetal bovine serum (FBS) without antibiotics. Annealed siRNA was diluted in Opti-MEM I (Invitrogen) to a final concentration of 50 nmol/L and transiently transfected into 4T1 using LipofectAMINE 2000 (Invitrogen). The medium was replaced with DMEM + 10% FBS after 4 h. Wild-type 4T1 cells and 4T1 incubated with mismatch siRNA were used as controls. Cells were harvested 48 h after transfection.

Gelatin zymography. Gelatin zymography was carried out as described by Novex Zymogram Gel manual (Invitrogen). Briefly, the concentrated serum-free DMEM medium was mixed with Novex Tris-Glycine SDS sample buffer and directly loaded on 10% Gelatin Novex Zymogram Gel. After running for 1.5 h, the gel was incubated in Zymogram renaturing buffer for 30 min at room temperature with gentle agitation. After incubating the gel with fresh Zymogram developing buffer at 37°C overnight and staining the gel with Colloidal Blue Staining kit (Invitrogen), images were acquired by AlphaImager 3400 (Alpha Innotech).

In vitro cell migration and invasion assay. The migration and invasiveness of cells were evaluated in 24-well Transwell chambers with upper and lower culture compartments separated by polycarbonate membranes with 8-µm-sized pores (Costar 3422, Corning). Cell culture medium DMEM was mixed with various combinations of full-length wild-type osteopontin (FL-OPN; 5 µmol/L), thrombin (0.02 NIH unit/mL, Sigma-Aldrich), thrombin inhibitor Thromstop (500 nmol/L, American Diagnostica, Inc.), short COOH-terminal osteopontin (SC-OPN; 5 µmol/L), NH₂-terminal osteopontin (N-OPN; 5 µmol/L), Mu-OPN (5 µmol/L), cyclophilin C antibody (2 µg/mL, Santa Cruz Biotechnology), or cyclophilin C (100 nmol/L, Sigma-Aldrich). Cells (5 x 10⁴) suspended in 100 µL of pretreated DMEM/0.1% bovine serum albumin were plated into the top chamber. DMEM/10% FBS was placed in the bottom chamber to act as a chemoattractant. After 24 h of incubation at 37°C in 5% CO₂ humidified air, the cells remaining at the upper surface of the membrane were removed with a cotton swab. The cells that migrated through the 8-µm-sized pores and adhered to the lower surface of the membrane were fixed with 3.7% paraformaldehyde, stained with 0.2% crystal violet, and washed with 1x PBS thrice. The dye was eluted using 30% acetic acid, and quantification of cell number was done using colorimetric analysis with a microplate reader (absorbance at 590 nm). In a similar fashion, the invasiveness of FL-OPN cells were evaluated in Matrigel (Collaborative Biomedical Products)–coated 24-well Transwell chambers. Cells, medium, experimental conditions, and analysis done were similar to those of migration assays. The absorbance obtained for control and experimental groups were each divided by the absorbance obtained for controls and expressed as a migration or invasion index. By definition, untreated 4T1 and 4T07 controls were assigned an index of 1. Triplicate assays were done for each group of cells.

Statistical analysis. All data are presented as mean ± SE. Analysis was done using the Student's t test. P < 0.05 was considered significant.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Yeast two-hybrid analysis of SC-OPN and cyclophilin C. Murine osteopontin contains 297 amino acid residues (ref. 12; see Fig. 1 ). It is acidic, hydrophilic, and highly negatively charged with features of a secreted protein. There a several potentially relevant structural domains: (a) an RGD domain at amino acids 145 to 147, (b) an NH₂-terminal hydrophobic leader sequence that ends at S16, (c) a thrombin cleavage site at R153-S154, and (d) two theoretical MMP-3/7 cleavage sites at amino acids 167 and 211. We therefore used a series of osteopontin constructs in our yeast two-hybrid assays: (a) FL-OPN (amino acids 1–297), (b) long COOH-terminal osteopontin (LC-OPN; amino acids 21–297), (c) SC-OPN (amino acids 155–297), and (d) N-OPN-1 (amino acids 21–160). SC-OPN corresponds to the thrombin-cleaved COOH-terminal fragment of osteopontin. A murine 17-day-old embryo cDNA library (BD Biosciences Clontech) was screened, and five distinct proteins that specifically interact with SC-OPN were isolated. The most frequently (n = 6) isolated clone corresponded to cyclophilin C. There was a single protein that was found to bind to LC-OPN; there were no target protein interactions found when FL-OPN or N-OPN was used as bait.

View larger version (11K): [in this window] [in a new window] [Download PPT slide]   Figure 1. Schematic representation of osteopontin (OPN) constructs used in yeast two-hybrid assay and coimmunoprecipitation studies.

View larger version (9K): [in this window] [in a new window] [Download PPT slide]   Figure 2. Osteopontin and cyclophilin C protein interactions. A, osteopontin and cyclophilin C (CyC) protein expression in culture media and cell lysates of 4T1 and 4T07 cells. Cells were lysed in buffer, and protein concentration was determined by the Bio-Rad protein assay kit. The protein samples were separated by 4% to 20% SDS-PAGE and electrotransferred onto polyvinylidene difluoride membranes by semi-dry transfer. The membranes were probed with the primary antibodies for osteopontin, cyclophilin C, and ß-actin. These antibodies were detected using the appropriate horseradish peroxidase–conjugated secondary antibody. The reactive proteins were visualized by means of chemiluminescence. Relative protein expression was analyzed by laser densitometry and normalized to a ß-actin standard. Blot is representative of four experiments. B, immunoprecipitation (IP) of osteopontin and cyclophilin C in culture medium of 4T07 cells. Cyclophilin C, osteopontin, and osteopontin-truncated mutant cDNA were separately fused in frame into mammalian secretion expression vectors. 4T07 cells were cotransfected, as described in Materials and Methods. The clarified concentrated serum-free supernatant was incubated with osteopontin antibody for immunoblot (IB) analysis (top), and separate aliquots were incubated with Protein G-agarose in Co-IP buffer. Protein G-agarose beads were collected by centrifugation and washed. The immune complexes were resuspended with SDS sample buffer and then loaded onto 4% to 20% SDS-PAGE gel for analysis using cyclophilin C antibody (bottom). Detection was done using the appropriate horseradish peroxidase–conjugated secondary antibody. The reactive proteins were visualized by means of the ECL kit (Amersham Bioscience). Blot is representative of four experiments. C, Western blot analysis of CD147 expression in 4T1 and 4T07 cells. Cells were lysed in buffer, and protein concentration was determined by the Bio-Rad protein assay kit. The protein samples were separated by 4% to 20% SDS-PAGE and electrotransferred onto polyvinylidene difluoride membranes by semi-dry transfer. The membranes were probed with the primary antibodies for CD147 and ß-actin. These antibodies were detected using the appropriate horseradish peroxidase–conjugated secondary antibody. The reactive proteins were visualized by means of chemiluminescence. Relative protein expression was analyzed by laser densitometry and normalized to a ß-actin standard. Blot is representative of four experiments.

View larger version (24K): [in this window] [in a new window] [Download PPT slide]   Figure 3. Binding of SC-OPN and cyclophilin C to CD147 receptor. Flow cytometric analysis of binding of SC-OPN and cyclophilin C proteins on 4T07 cells. SC-OPN-CFP and CyC-YFP fusion proteins were purified. 4T07 cells were incubated with SC-OPN-CFP (2 µg/mL), CyC-YFP (2 µg/mL), CD147 antibody (2 µg/mL), osteopontin antibody (2 µg/mL), and/or cyclosporine A (100 nmol/L), as indicated. At least 106 cells were analyzed on a FACSCalibur flow cytometer under standard flow for 120 s. Mean fluorescence intensity was determined using Cell Quest software. Percentage of cell gated in each quadrant. Representative results of three independent experiments. A, SC-OPN-CFP– and CyC-YFP–treated 4T07 cells. B, SC-OPN-CFP– and CyC-YFP–treated 4T07 cells with the cyclophilin inhibitor cyclosporine A. C, SC-OPN-CFP– and CyC-YFP–treated 4T07 cells with CD147 antibody. D, SC-OPN-CFP– and CyC-YFP–treated 4T07 cells with osteopontin antibody. E, 4T07 cells alone.

View larger version (40K): [in this window] [in a new window] [Download PPT slide]   Figure 4. Binding of cyclophilin C and SC-OPN to 4T07 cells. A, localization of cyclophilin C and SC-OPN to the 4T07 cell surface. 4T07 cells were cultured on coverslips and cotransfected with pECFP-SC-OPN and pEYFP-CyC plasmids. After 24 h post-transfection, the coverslips were rinsed, and a Leica TCS SP2 confocal microscope was used for image acquisition. Photo is representative of four experiments. B, FRET analysis. CFP and YFP emission spectra were collected following excitation at 458 nm and were used as reference spectra for linear unmixing of CFP and YFP emission spectra. FRET was measured by acceptor photobleaching. Confocal microscopy was used to visualize a 2-µm optical slice through 4T07 cells. Pre-bleach CFP and YFP images were collected simultaneously following excitation at 458 nm (38% laser intensity, detector gain = 740). A selected region of interest was irradiated with the 514-nm laser line (100% intensity, 60 iterations, using a 458-nm/514-nm dual dichroic mirror) for 5 to 10 s to photobleach YFP. Post-bleach CFP and YFP images were collected simultaneously (at 458 nm) immediately following photobleaching. FRET was measured as an increase in CFP fluorescence intensity following YFP photobleaching. FRET efficiency was calculated as 100 x [(CFP post-bleach – CFP prebleach) / CFP post-bleach], taking into account CFP and YFP background noise in each channel. FRET efficiency was measured and calculated by Leica LAS AF software. FRET was done on 50 cells per treatment group with three regions per cell. Photos are representative of five experiments. C, titration of FRET photobleaching. A photobleaching titration was done with determination of FRET efficiencies with a range of bleaching values of 20% to 100%. Points, mean of four independent experiments; bars, SD. D, binding of cyclophilin C to 4T07 cells. To directly measure the effect of CD147 receptor on recruitment of a SC-OPN + CyC complex, the dose response of cyclophilin C binding to CD147 was measured as a function of integrated fluorescence intensity in the presence and absence of saturating concentrations of CFP-SC-OPN (200 nmol/L). YFP-CyC concentrations varied from 5 to 80 nmol/L. CD147 dependence was assessed by addition of blocking CD147 antibody (2 µg). Points, mean of four independent experiments; bars, SD.

View larger version (12K): [in this window] [in a new window] [Download PPT slide]   Figure 5. MMP2 and Akt1/2 activation in 4T1 and 4T07 cells. A, total and P-Akt1/2 expression. Cells were lysed in buffer, and protein concentration was determined by the Bio-Rad protein assay kit. The protein samples were separated by 4% to 20% SDS-PAGE and electrotransferred onto polyvinylidene difluoride membranes by semi-dry transfer. The membranes were probed with the primary antibodies for osteopontin, total Akt1/2, P-Akt1/2, and ß-actin. These antibodies were detected using the appropriate horseradish peroxidase–conjugated secondary antibody. The reactive proteins were visualized by means of chemiluminescence. In selected instances, osteopontin expression in 4T1 cells was ablated using siRNA to osteopontin; mismatch siRNA (MM siRNA) served as a control. Blot is representative of four experiments. B, histogram representation of relative P-Akt1/2 expression. Relative P-Akt1/2 protein expression was analyzed by laser densitometry and normalized to total Akt1/2. Columns, mean of four independent experiments; bars, SD. *, P < 0.01, 4T1 versus 4T07, 4T1+ CD147 antibody, and 4T1 + OPN siRNA; **, P < 0.01, 4T07 + SC-OPN + CyC versus 4T07, 4T07 + SC-OPN, 4T07 + CyC, 4T1 + OPN siRNA, and 4T07+SC-OPN + CyC + CD147 antibody. C, MMP-2 activity. Gelatin zymography of MMP2 activity was determined in 4T1 and 4T07 cells. Western blot analysis of ß-actin and MMP-2 in cell lysate and concentrated culture medium was done as described in Materials and Methods. Blot is representative of four experiments.

View larger version (19K): [in this window] [in a new window] [Download PPT slide]   Figure 6. Osteopontin, thrombin cleavage, and cyclophilin C regulation of in vitro migration and invasion of 4T07 and 4T1 cells. A, Western blot analysis of osteopontin and Mu-OPN. The mouse full-length osteopontin cDNA was mutated (Mu-OPN) at its thrombin cleavage site, from agg tca (nucleotides –458 to –463) to agc ttt using PCR, and cloned into mammalian secretion expression vector pSecTag2/Hygro (Invitrogen). This mutates the thrombin cut site from R153-S154 to S153-F154 and renders it resistant to thrombin cleavage. The Mu-OPN plasmid was transfected into COS7 cells. The secreted Mu-OPN protein was purified from the culture medium using the MagneHis Protein Purification System (Promega) and quantified with Bio-Rad protein assay kit (Bio-Rad). Osteopontin and Mu-OPN were then exposed to thrombin (0.02 unit/mL) and/or a thrombin inhibitor (Thromstop, 500 nmol/L), and Western blot analysis was done. Thrombin cleavage of osteopontin would be expected to generate two fragments (33 and 30 kDa). Blot is representative of three experiments. B, migration and invasion characteristics of untreated 4T1 and 4T07 cells. In vitro migration and invasion assays were done as described in Materials and Methods. By definition, 4T1 cells were assigned an index of 1. Columns, mean of four experiments; bars, SE. *, P < 0.01, versus 4T1 for migration and invasion. C, effect of osteopontin, cyclophilin C, and thrombin on migration of 4T1 and 4T07 cells. Migration assays were done as described in Materials and Methods. By definition, untreated control 4T1 and 4T07 cells were assigned an index of 1. Culture medium was mixed with various combinations of FL-OPN (5 µmol/L), thrombin (0.02 NIH unit/mL; Sigma-Aldrich), thrombin inhibitor Thromstop (500 nmol/L; American Diagnostica), SC-OPN (5 µmol/L), N-OPN (5 µmol/L), Mu-OPN (5 µmol/L), cyclophilin C antibody (2 µg/mL; Santa Cruz Biotechnology), or cyclophilin C (100 nmol/L; Sigma-Aldrich). Columns, mean of four experiments; bars, SE. *, P < 0.01, versus control, FL-OPN, Mu-OPN, N-OPN, Mu-OPN + thrombin, and FL-OPN + thrombin + inhibitor; **, P < 0.02, versus 4T07 alone; @, P < 0.01, versus SC-OPN, FL-OPN, Mu-OPN, N-OPN, FL-OPN + thrombin, Mu-OPN + thrombin, and FL-OPN + thrombin + inhibitor; #, P < 0.01, versus 4T1 + cyclophilin C antibody. D, effect of osteopontin, cyclophilin C, and thrombin on invasion of 4T1 and 4T07 cells. Invasion assays were done as described in Materials and Methods. By definition, untreated control 4T1 and 4T07 cells were assigned an index of 1. Culture medium was mixed with various combinations of FL-OPN (5 µmol/L), thrombin (0.02 NIH unit/mL; Sigma-Aldrich), thrombin inhibitor Thromstop (500 nmol/L; American Diagnostica), SC-OPN (5 µmol/L), N-OPN (5 µmol/L), Mu-OPN (5 µmol/L), cyclophilin C antibody (2 µg/mL; Santa Cruz Biotechnology), or cyclophilin C (100 nmol/L; Sigma-Aldrich). Columns, mean of four experiments; bars, SE. *, P < 0.01, versus control, FL-OPN, Mu-OPN, N-OPN, Mu-OPN + thrombin, and FL-OPN + thrombin + inhibitor; **, P < 0.02, versus 4T07 alone; @, P < 0.01, versus SC-OPN, FL-OPN, Mu-OPN, N-OPN, FL-OPN + thrombin, Mu-OPN + thrombin, and FL-OPN + thrombin + inhibitor; #, P < 0.01, versus 4T1 + cyclophilin C antibody.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
In this study using 4T1 and 4T07 murine mammary epithelial tumor cell lines, we show that the thrombin cleaved COOH-terminal fragment of osteopontin interacts with cyclophilin C and binds to CD147 on the cell surface of 4T07 cells to phosphorylate Akt1/2 and activate MMP-2. In keeping with its nonmetastatic phenotype, 4T07 cells do not contain activated P-Akt1/2 or MMP-2. In in vitro assays, thrombin cleavage of osteopontin to generate SC-OPN in the presence of cyclophilin C increases migration and invasion of both 4T07 and 4T1 cells. This function for SC-OPN and its interaction with cyclophilin C has not been previously reported.

Posttranslational proteolytic cleavage of osteopontin by thrombin has been previously shown to mediate endothelial cell migration and integrin binding (5, 10). In rat, human, and mouse species, a single thrombin cleavage site generates an NH₂-terminal fragment that exposes a cryptic integrin-binding motif SVVYGLR on its COOH terminus, allowing the specific interaction with integrins. Although both resulting fragments have electrophoretic mobilities corresponding to a molecular weight of 35 kDa, further electrophoretic analysis under nondenaturing conditions indicate that the two fragments do not remain associated (5). Because the NH₂-terminal fragment contains the RGD-binding domain that is critical for integrin binding, this polypeptide has received a majority of the attention. It is felt that the NH₂-terminal fragment conveys the bulk of the function for osteopontin. In certain cell types, such as melanoma cells, cell binding only occurs with the thrombin-cleaved but not the intact osteopontin, suggesting that thrombin cleavage is critical for certain osteopontin-cell interactions. As for the COOH-terminal fragment of thrombin-cleaved osteopontin, it has been found to bind to CD44 to induce chemotaxis, and it may suppress RGD-dependent cell adhesion (18, 19). Otherwise, little is known of SC-OPN function.

CD147 (or EMMPRIN) is the principal signaling receptor for extracellular cyclophilins (20). Elevated CD147 levels are detected in numerous malignancies and correlate with tumor progression in experimental and clinical conditions. CD147 stimulates angiogenesis via vascular endothelial growth factor (VEGF) and invasion via stimulation of MMPs, anchorage-independent growth, and multidrug resistance. Tang et al. have shown that CD147 signaling occurs through Akt to mediate VEGF expression in human breast cancer cell line MDA-MB-231 (16). In a similar fashion, Tang et al. have also convincingly shown the role of CD147 in MMP expression in the setting of breast cancer (21–23). Thus, activation (phosphorylation) of Akt1/2 and MMP-2 were selected as functional correlates to the protein binding between SC-OPN and cyclophilin C in these studies. Structurally similar to cyclophilins A and B, cyclophilin C was originally characterized as a tissue specific cyclophilin with 212 amino acids (24). Overexpression of the cyclophilin family (A, B, C, and D) and its interactions with CD147 has been associated with increased proliferation and metastatic properties in pancreatic, breast, and non–small-cell lung cancers (25). In our studies, we have shown that SC-OPN and cyclophilin C protein-protein interactions activate Akt1/2 and MMP-2 via CD147. Exposure of 4T07 cells to SC-OPN and cyclophilin C induces a CD147-dependent metastatic profile similar to that of 4T1. In this regard, our findings are novel in that SC-OPN has not been previously associated with CD147 or cyclophilin C and indicate that SC-OPN regulates metastatic functions in a manner independent of previously described interactions between full-length osteopontin and integrin or CD44 cell surface receptors.

The interactions between SC-OPN and cyclophilin C were analyzed using FRET combined with confocal microscopy to determine the proximity of CFP- and YFP-tagged proteins on the plasma membrane of 4T07 cells. Resonance energy can be transferred from CFP to YFP if they are within 10 nm of each other, and if their dipoles are appropriately aligned. Energy transfer from CFP (donor) to YFP (acceptor) results in the quenching of CFP fluorescence and an increase in YFP fluorescence and can be measured as an increase in CFP fluorescence following selective irradiation of the YFP fluorophore. Laser scanning confocal microscopy allows the photobleaching to be confined to very discrete intracellular regions. The relationship of CD147 in the SC-OPN and cyclophilin C interactions was determined using FACS analysis and FRET. Our results indicate that the SC-OPN + CyC complex binds to the CD147 with an apparent K_d 05 nmol/L, suggesting a high degree of avidity. This binding can be inhibited in the presence of cyclosporine A, indicating its dependence upon the cyclosporine binding domain of cyclophilin C. However, we recognize the severe limitations of the estimates given the potential error in calculation of K_d. In addition, our tagged proteins CFP-SC-OPN and YFP-CyC were expressed and isolated in COS-1 cells. Although the osteopontin proteins correspond in molecular weight on SDS-PAGE with wild-type osteopontin, the question arises whether posttranslational modifications, which take place in COS-1 cells, are similar to those found in breast cancer cells. Because osteopontin may undergo a number of posttranslational modifications in different cells, functional consequences may vary as a result. The type and extent of osteopontin posttranslational modification is a current topic of ongoing investigation in our lab.

In conclusion, our studies show that the thrombin cleaved COOH-terminal fragment of osteopontin is a biologically relevant peptide. SC-OPN can form a complex with cyclophilin C, as shown by FRET, to bind to CD147 to activate Akt1/2 and MMP-2 in 4T07 murine breast cancer cells. Our findings indicate that this OPN-CyC complex regulates in vitro migration and invasion properties of 4T1 and 4T07 breast cancer cells.

	Acknowledgments

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 11/ 6/06. Revised 1/26/07. Accepted 2/22/07.

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