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Originally published In Press as doi:10.1074/jbc.M310276200 on January 21, 2004

J. Biol. Chem., Vol. 279, Issue 14, 13711-13720, April 2, 2004
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Resistance of B16 Melanoma Cells to CD47-induced Negative Regulation of Motility as a Result of Aberrant N-Glycosylation of SHPS-1*

Takeshi Ogura{ddagger}, Tetsuya Noguchi{ddagger}§, Reiko Murai-Takebe{ddagger}, Tetsuya Hosooka{ddagger}, Nakayuki Honma¶, and Masato Kasuga{ddagger}

From the {ddagger}Division of Diabetes, Digestive and Kidney Diseases, Department of Clinical Molecular Medicine, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan and Pharmaceutical Research Laboratories, Kirin Brewery Co. Ltd., Takasaki, Gunma 370-1295, Japan

Received for publication, September 16, 2003 , and in revised form, January 6, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The adhesion receptor SHPS-1 activates the protein-tyrosine-phosphatase SHP-2 and thereby promotes integrin-mediated reorganization of the cytoskeleton. SHPS-1 also contributes to cell-cell communication through association with CD47. Although functional alteration of SHPS-1 is implicated in cellular transformation, the role of the CD47-SHPS-1 interaction in carcinogenesis has been unclear. A soluble SHPS-1 ligand (CD47-Fc) has now been shown to bind to Melan-a non-tumorigenic melanocytes but not to syngeneic B16F10 melanoma cells. Treatment of B16F10 cells with 1-deoxymannojirimycin, which prevents N-glycan processing, restored the ability of SHPS-1 derived from these cells to bind CD47-Fc in vitro, indicating that aberrant N-glycosylation of SHPS-1 impairs CD47 binding in B16F10 cells. CD47-Fc inhibited the migration of Melan-a cells but not that of B16F10 cells. However, a monoclonal antibody that reacts with SHPS-1 on both Melan-a and B16F10 cells inhibited the migration of both cell types similarly. CD47 binding induced proteasome-mediated degradation of SHPS-1 in a tyrosine phosphorylation-independent manner. Furthermore, overexpression of SHPS-1 reduced the level of tyrosine phosphorylation of focal adhesion kinase, and this effect was reversed by CD47 binding. These results suggest that CD47 binds to and thereby down-regulates SHPS-1 on adjacent cells, resulting in inhibition of cell motility. Resistance to this inhibitory mechanism may contribute to the highly metastatic potential of B16 melanoma.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Various adhesion molecules expressed on the surface of mammalian cells, including members of the integrin receptor and cadherin families and of the immunoglobulin superfamily, have been characterized. These proteins are fundamental to cell growth, differentiation, and migration, and their dysregulation has been implicated in the pathogenesis of cancer, inflammation, and neurodegeneration.

SHPS-1, also known as SIRP{alpha}1 (1), BIT (2), MFR (3), and p84 neural adhesion molecule (4), is a transmembrane glycoprotein of the Ig superfamily and is abundant in neural and myeloid tissues (5–7). The cytoplasmic region of SHPS-1 contains two immunoreceptor tyrosine-based inhibitory motifs, which bind and activate the Src homology 2 domain-containing protein-tyrosine-phosphatases SHP-1 and SHP-2 in a phosphorylation-dependent manner (1, 5, 8). The extracellular region of this protein comprises three Ig-like domains, of which the most amino-terminal Ig V-like domain associates with the ligand CD47 (9–11).

CD47, which was originally identified in association with {alpha}v{beta}3 integrin and hence referred to as integrin-associated protein (12), is also a member of the Ig superfamily, possessing an Ig V-like extracellular domain, five putative membrane-spanning segments, and a short cytoplasmic tail (11). CD47 and SHPS-1 appear to constitute a cell-cell communication system (the CD47-SHPS-1 system) that contributes to a variety of physiological processes, including phagocytosis of red blood cells by macrophages (13), macrophage multinucleation (14), T cell activation (15, 16), and neutrophil transmigration (17).

SHPS-1 negatively or positively regulates intracellular signaling initiated either by tyrosine kinase-coupled receptors for growth factors or by cell adhesion to extracellular matrix proteins (18, 19). With the use of mice lacking most of the cytoplasmic region of SHPS-1 (20, 21), we recently showed that this protein plays an important role both in integrin-mediated cytoskeletal reorganization and in cell migration. In addition, studies with dominant negative or loss-of-function mutant proteins have established SHP-2 as a signaling component essential for cell adhesion and migration as well as cytoskeletal organization (22–26). SHPS-1 thus likely promotes cell migration by facilitating regulation of the cytoskeleton by SHP-2 at the cell membrane.

Expression of SHPS-1 has been shown to be down-regulated in subsets of human leukemic myeloid cells (10), human breast cancer tissue (27), and fibroblasts transformed by various oncogene products (28). Furthermore, forced expression of SHPS-1 suppressed the transformed phenotype of glioblastoma cells (29) as well as inhibited both anchorage-independent growth of v-Src-transformed cells (28) and peritoneal dissemination of these cells in nude mice (27). Together, these observations have implicated SHPS-1 in cellular transformation. It has remained unclear, however, whether (and, if so, how) functional alteration of the CD47-SHPS-1 system contributes to carcinogenesis.

To investigate the possible role of CD47-SHPS-1 interaction in malignant cellular transformation, we have now characterized and compared SHPS-1 proteins expressed in the mouse nontumorigenic melanocyte Melan-a (30) and melanoma B16F10 (31) cell lines. Our results show that SHPS-1 expressed on the surface of B16F10 cells is impaired in the ability to bind a soluble ligand (CD47-Fc fusion protein) as a result of aberrant N-glycosylation. Whereas CD47-Fc substantially inhibited the motility of Melan-a cells, it had no such effect on that of B16F10 cells, suggesting that resistance to this inhibitory mechanism might underlie the highly metastatic potential of B16 melanoma.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Expression Vectors, Antibodies, and Reagents—The cDNA encoding a mutant SHPS-1 (SHPS-1-4F), in which all four tyrosine residues in the cytoplasmic region (Tyr408, Tyr432, Tyr449, and Tyr473) are replaced by phenylalanine, was generated by site-directed mutagenesis with the full-length mouse SHPS-1 cDNA (6) as a template and a Transformer Site-directed Mutagenesis kit (Clontech). The wild-type and mutant SHPS-1 cDNAs were inserted separately into the EcoRI and NotI sites of the pTracerCMV vector (Invitrogen). The full-length mouse CD47 cDNA was excised from pIAP328 (32) and subcloned into pTracerCMV. The pRc/CMV vector encoding hemagglutinin (HA)1 epitope-tagged mouse focal adhesion kinase (FAK) was provided by S. Hanks (Vanderbilt University). The pcDNA3 vector encoding Myc-tagged mouse SHP-2 was provided by H. Ohnishi (Gunma University).

Rabbit polyclonal antibodies to SHP-2 (5) and a rat monoclonal antibody (mAb) to mouse SHPS-1 (20) were described previously. A rat mAb to CD47 (MIAP301), normal rat IgG, and horseradish peroxidase-conjugated mAb PY20 to phosphotyrosine were obtained from Santa Cruz Biotechnology; rabbit polyclonal antibodies to SHPS-1 and to FAK were from Upstate Biotechnology Inc.; a mouse mAb to tubulin was from Sigma; goat antibodies to rat IgG, fluorescein isothiocyanate-conjugated goat antibodies to human IgG, and the Fc fragment of human IgG were from Cappel; Cy2-conjugated goat antibodies to rat IgG were from Amersham Biosciences; and goat antibodies to the Fc fragment of human IgG were from Jackson ImmunoResearch. The mAb 9E10 to the Myc tag and mAb 12CA5 to the HA tag were purified from the culture supernatants of mouse hybridoma cells.

Tunicamycin, 1-deoxymannojirimycin, and chloroquine were purchased from Sigma. MG132 was from Calbiochem.

Cells and Transfection—Melan-a and B16F10 cells were maintained in modified Eagle's medium (MEM) (Sigma) supplemented with 10% fetal bovine serum (Invitrogen) and containing or not containing 12-O-tetradecanoylphorbol-13-acetate (200 ng/ml) (Sigma), respectively. Chinese hamster ovary (CHO) cells stably expressing H-Ras (CHO-Ras cells) (33) were provided by S. Shirahata (Kyushu University). CHO cells stably expressing either wild-type mouse SHPS-1 (CHO-SHPS-1-WT cells) or the SHPS-1-4F mutant (CHO-SHPS-1-4F cells) as well as CHO-Ras cells stably expressing wild-type mouse CD47 (CHO-Ras-CD47 cells) were generated as described previously (34) with the use of Zeocin (500 µg/ml) (Invitrogen) as a selection reagent. The resulting CHO-derived lines and parental CHO-K1 cells were maintained in Ham's F-12 medium (Sigma) supplemented with 10% fetal bovine serum. Transient transfection of CHO cells (~4 x 105 cells per 60-mm dish) was performed with the use of a LipofectAMINE Transfection kit (Invitrogen) or a CellPhect Transfection kit (Amersham Biosciences).

Generation of a Soluble SHPS-1 Ligand—To prepare a CD47-Fc fusion protein, we first obtained by PCR a cDNA that encodes the Fc portion of human IgG1, as described previously (35). The PCR product was then digested with SpeI and NotI and ligated into the corresponding sites of the pEFneo vector (Invitrogen), yielding pEFneoFc76. A cDNA encoding the extracellular domain of mouse CD47 (amino acids 1–161) was amplified by PCR with pIAP328 as the template, the sense primer 5'-TAATGAATTCGAGATGTGGCCCTTGGCGGC-3', and the antisense primer 5'-TAGCACTAGTCTTTTCATTTGGAGAAAACCACGAA-3'. The PCR product was digested with EcoRI and SpeI and inserted into the corresponding sites of pEFneoFc76. The resulting construct was digested with EcoRI and NotI, and the released fragment was then subcloned into the corresponding sites of pTracerCMV, yielding pTracerCMV-mCD47-Fc. CHO-Ras cells were transfected with this latter plasmid and subjected to selection with Zeocin as described above. Several cell lines that produced the CD47-Fc fusion protein were identified by immunoblot analysis of culture supernatants with antibodies to human IgG, and were cultured in {alpha}MEM (Sigma) supplemented with 2 mM L-glutamine, 10 mM HEPES-NaOH (pH 7.6), and 10% fetal bovine serum. The CD47-Fc protein was then purified from the culture supernatants by column chromatography on HiTrap Protein G-Sepharose (Amersham Biosciences).

Flow Cytometry—Cell surface expression of SHPS-1 and the binding of CD47-Fc to cells were examined by flow cytometry. In brief, cells were detached from culture dishes by treatment with 0.01% EDTA and then washed in phosphate-buffered saline (PBS). The cells (0.5 x 106 to 1 x 106 in 0.1 ml of PBS) were then incubated for 30 min on ice with either a mAb to SHPS-1 (10 µg/ml), normal rat IgG (10 µg/ml), CD47-Fc (20 µg/ml), or the Fc fragment of human IgG (20 µg/ml), after which they were washed twice with PBS and incubated for 30 min on ice with appropriate secondary antibodies (10 µg/ml). After washing twice with PBS, the stained cells were suspended in 1 ml of PBS and analyzed with a FACSCalibur flow cytometer (BD Biosciences). Data were processed with CellQuest software (BD Biosciences).

Cell Migration Assay—Cell migration was assessed with a Boyden chamber assay. In brief, polyvinylpyrrolidine-free polycarbonate filters (pore size, 8 µm; Neuroprobe) coated with fibronectin (10 µg/ml) (Sigma) were placed over the lower wells of a Boyden multiwell chemotactic chamber that had been filled with serum-free MEM. Cells (1.5 x 105 in 0.2 ml of serum-free MEM) were added to each of the upper wells. The chamber was placed in a humidified incubator containing 5% CO2 and incubated for 2–4 h at 37 °C. Cells that had migrated through the filter were fixed in methanol, washed with PBS, and exposed to Giemsa stain (Nakalai Tesque) for 15 s. The number of migrated cells was counted in at least six fields with a microscope fitted with a grid eyepiece and at a total magnification of x200.

Immunoprecipitation, Lectin Binding, and Immunoblot Analysis— Cells in one 60-mm dish were lysed on ice in 1 ml of ice-cold lysis buffer (20 mM Tris-HCl (pH 7.6), 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40) containing 5 mM NaF, 1 mM phenylmethylsulfonyl fluoride, aprotinin (10 µg/ml), and 1 mM sodium vanadate. The cell lysates were centrifuged at 10,000 x g for 15 min at 4 °C, and the resulting supernatants were incubated for 3 h at 4 °C with antibody-coupled protein G-Sepharose beads (20 µl of beads; Amersham Biosciences). The supernatants were also incubated for 1 h at 4 °C in the presence of 1 mM MnCl2 and 1 mM MgCl2 with agarose beads conjugated to Galanthus nivalis agglutinin (20 µl of beads; EY Laboratories). Both types of beads were then washed three times with lysis buffer and suspended in Laemmli sample buffer, and the associated proteins were resolved by SDS-PAGE. Immunoblot analysis was performed with the ECL detection system (Amersham Biosciences).

Deglycosylation of SHPS-1 in Vitro—N-Linked oligosaccharides were removed from SHPS-1 by N-glycosidase F digestion. Prior to digestion cell lysates were incubated for 5 min at 100 °C in the presence of 0.5% SDS and 1% {beta}-mercaptoethanol. The sample was then incubated with N-glycosidase F (40 units/ml; Roche Applied Science) for 12 h at 37 °C.

Statistical Analysis—Data are presented as means ± S.E. The significance of differences between independent means was assessed by Student's t test. A p value of <0.05 was considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Differential N-Glycosylation of SHPS-1 in Mouse Nontumorigenic Melanocytes and Malignant Melanoma Cells—Immunoblot analysis revealed that, among various cell types examined, Melan-a cells, an immortal line of pigmented melanocytes, and mouse macrophages expressed the largest amounts of SHPS-1 (data not shown). The Melan-a line was derived from normal epidermal melanoblasts and is nontumorigenic (30). Furthermore, Melan-a cells are syngeneic with the B16 melanoma line and its sublines, thus providing a control for studies of the cellular and molecular basis of melanoma malignancy (30). Melan-a cells and highly metastatic B16F10 melanoma cells (31) were found to express similar amounts of SHPS-1. However, the electrophoretic mobility of this protein in the latter cells was reduced compared with that apparent in the former cells (Fig. 1A). This difference in electrophoretic mobility was abolished by prior exposure of the cells to tunicamycin, which prevents the attachment of N-glycans to cellular proteins (Fig. 1A, left panel). Treatment with the mannosidase inhibitor 1-deoxymannojirimycin, which blocks the processing of N-linked high mannose-type oligosaccharides, also abolished this difference (Fig. 1A, right panel). SHPS-1 derived from the two cell lines, when digested with recombinant N-glycosidase F, exhibited similar electrophoretic mobility (Fig. 1B). These results indicate that SHPS-1 is differentially N-glycosylated in Melan-a and B16F10 cell lines.



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  		FIG. 1.
Aberrant N-glycosylation of SHPS-1 in B16F10 melanoma cells. A, Melan-a and B16F10 cells were incubated for 48 h in the absence (-) or presence (+) of tunicamycin at a concentration of 2.5 or 1 µg/ml, respectively (left panel). These cells were also incubated for 24 h in the presence of 1 mM 1-deoxymannojirimycin (DMJ)(right panel). Cell lysates (20 µg of protein) prepared were subjected to immunoblot analysis with polyclonal antibodies to SHPS-1 ({alpha}SHPS-1). B, lysates of Melan-a or B16F10 cells were treated (or not) with N-glycosidase F for 12 h at 37 °C and then subjected to immunoblot analysis with a mAb to SHPS-1. C, lysates of Melan-a or B16F10 cells were incubated with agarose beads conjugated to G. nivalis agglutinin lectin. Bead-bound proteins as well as cell lysates were subjected to immunoblot analysis with polyclonal antibodies to SHPS-1 under nonreducing conditions. The positions of native and modified forms of SHPS-1, as well as of molecular size standards (in kilodaltons), are indicated.

 
Electrophoresis under nonreducing conditions revealed that SHPS-1 expressed in both cell lines migrated as two major bands (Fig. 1C). The faster migrating forms exhibited higher affinity for G. nivalis agglutinin lectin, which specifically recognizes terminal mannose residues (36), than did the slower migrating forms (Fig. 1C). Given that terminal mannose residues are usually exposed by folding intermediates of type I glycoproteins in the early steps of N-glycan processing, these faster migrating forms may correspond to immature SHPS-1. The reduced ability of the slower migrating forms of SHPS-1 to bind to G. nivalis agglutinin lectin indicated that they had undergone further N-glycan processing and represent the mature protein that is transported to the cell surface. A substantial difference in electrophoretic mobility of SHPS-1 between the two cell types was apparent selectively for the slower migrating forms. Furthermore, the lower mobility form of SHPS-1 in B16F10 cells bound to G. nivalis agglutinin lectin with much lower affinity than did that in Melan-a cells. These results suggested that SHPS-1 on the surface of the two cell types possesses distinct N-glycan structures.

Impaired Binding of a Soluble SHPS-1 Ligand to B16F10 Melanoma Cells—Differences in the N-glycan structure of SHPS-1 might be expected to affect binding affinity for the ligand CD47. We therefore next examined whether the binding of a soluble version of this ligand, a recombinant CD47-Fc fusion protein (the extracellular domain of mouse CD47 fused to the Fc portion of human IgG), differed between B16F10 and Melan-a cells. Flow cytometry with a mAb that reacts with the extracellular portion of mouse SHPS-1 revealed no marked difference in the surface expression of SHPS-1 between the two cell types (Fig. 2A). Although CD47-Fc bound significantly to Melan-a cells, however, this SHPS-1 ligand exhibited virtually no binding to the surface of B16F10 cells (Fig. 2B).



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  		FIG. 2.
Impaired binding of a soluble SHPS-1 ligand to the surface of B16F10 cells. A, detached Melan-a and B16F10 cells were incubated with a mAb to SHPS-1 (solid trace) or with control rat IgG (broken trace). Immune complexes were then detected with Cy2-conjugated goat antibodies to rat IgG and flow cytometry. B, detached cells were incubated with the CD47-Fc fusion protein (solid trace) or with the Fc fragment of human IgG (broken trace). Complexes were then detected with fluorescein isothiocyanate-conjugated goat antibodies to human IgG and flow cytometry.

 
SHPS-1 derived from Melan-a cells, but not that from B16F10 cells, bound CD47-Fc in vitro (Fig. 3A). In contrast, SHPS-1 from the two cell lines after treatment with 1-deoxymannojirimycin bound similar amounts of CD47-Fc (Fig. 3B). SHPS-1 from both cell lines exposed to tunicamycin also bound CD47-Fc to similar extents (Fig. 3C). Sequencing of the products generated by reverse transcription and PCR analysis of SHPS-1 mRNA from the two cell lines revealed no differences in the coding sequence for the amino-terminal Ig V-like domain of SHPS-1 (data not shown). Together, these results indicate that the impaired binding of CD47-Fc by B16F10 cells results, at least in part, from aberrant N-glycosylation of SHPS-1.



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  		FIG. 3.
Restoration of CD47 binding to SHPS-1 of B16F10 cells in vitro. Melan-a and B16F10 cells were either left untreated (A) or incubated in the presence of 1-deoxymannojirimycin (DMJ) for 24 h (B) or tunicamycin for 48 h (C). Cell lysates were then prepared and incubated with protein G-Sepharose beads coupled with CD47-Fc or with a control Fc fragment, after which bead-bound proteins were subjected to immunoblot analysis with a mAb to SHPS-1 (A and B, upper panels) or with polyclonal antibodies to SHPS-1 (C, upper panel). Cell lysates were also probed with the same antibodies to verify the presence of equal amounts of SHPS-1 in each assay (lower panels). Incubation without cell lysate (right-most lane) revealed that CD47-Fc was responsible for the nonspecific immunoreactive material recognized by the antibodies to SHPS-1 in C.

 
Ligation of SHPS-1 Inhibits Cell Migration—To investigate the possible relation between the impairment in CD47 binding to SHPS-1 on B16F10 cells and the malignant phenotype of these cells, we examined the biological consequences of CD47-SHPS-1 interaction. Given the role of SHPS-1 in cytoskeletal reorganization (20, 21) and the fact that B16F10 cells are highly motile whereas Melan-a cells are not (30), we focused our attention on the effect of CD47 binding to SHPS-1 on cell migration. Quantitative analysis with a Boyden chamber assay revealed that the number of Melan-a cells that migrated through a membrane coated with fibronectin was reduced by ~20% in the presence of CD47-Fc (Fig. 4A). Cross-linking of CD47-Fc by antibodies to human IgG increased the inhibitory effect of this ligand on the motility of Melan-a cells by an additional 10%. In contrast, CD47-Fc had no marked effect on the migration of B16F10 cells in the absence or presence of cross-linking antibodies. These results suggest that the binding of CD47 to surface SHPS-1 reduces the motility of cells expressing the latter protein. Given that the mAb to SHPS-1 recognized equally well the antigen expressed on the surface of both cell lines (Fig. 2A), it, unlike CD47-Fc, might be expected to act as a functional ligand in B16F10 cells. Indeed, treatment with this mAb reduced the migration of Melan-a cells by ~22% and that of B16F10 cells by ~25% (Fig. 4B). Cross-linking of the mAb increased its inhibitory effect on the motility of both cell types by an additional 5%. CD47-Fc exists as a dimer under nonreducing conditions (data not shown), presumably as a result of the formation of a disulfide bond between the Fc portions of the two molecules and consistent with previous observations (35, 37). Thus, CD47-Fc, like the mAb to SHPS-1, might engage surface SHPS-1, and this engagement appears to be responsible for the inhibition of cell migration.



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  		FIG. 4.
Inhibition of cell migration by ligation of surface SHPS-1. A, detached Melan-a and B16F10 cells were transferred to porous membranes that had been both coated with fibronectin and placed in a Boyden multiwell chamber. Cells were then allowed to migrate for 4 (Melan-a) or for 2 h (B16F10) at 37 °C in the presence of CD47-Fc (20 µg/ml) or control Fc fragment (20 µg/ml), each in the absence (upper panels) or presence (lower panels) of goat antibodies to human IgG (100 µg/ml). Cells that had migrated during the assay were stained with Giemsa solution and counted. B, migration assays were performed in the presence of a mAb to SHPS-1 (10 µg/ml) or control rat IgG (10 µg/ml), each in the absence (upper panels) or presence (lower panels) of goat antibodies to rat IgG (100 µg/ml). Data are means ± S.E. of triplicate determinations from three independent experiments. *, p < 0.05; **, p < 0.01 versus corresponding control value.

 
Binding of CD47 to Cells Down-regulates SHPS-1 Expression—SHPS-1 promotes cell migration by recruiting SHP-2 to the cell membrane and thereby activating its phosphatase activity (20), suggesting that CD47 binding to cells might inhibit cell motility by abrogating this function of SHPS-1. We observed that the expression level of SHPS-1 in Melan-a cells incubated with CD47-Fc for 6 h was substantially lower than that apparent in cells incubated with the control Fc fragment for a similar period (Fig. 5A, upper panel). In contrast, the abundance of SHPS-1 in B16F10 cells incubated with CD47-Fc was comparable with that observed for control cells (Fig. 5A, lower panel). Exposure to CD47-Fc did not substantially affect the expression of tubulin in both cell lines, arguing against a toxic effect of the soluble ligand (Fig. 5A).



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  		FIG. 5.
Down-regulation of SHPS-1 expression as a result of CD47 engagement. A, detached Melan-a (upper panel) or B16F10 (lower panel) cells (4 x 105 in 1 ml of MEM) were transferred to 12-well culture plates and incubated with CD47-Fc (20 µg/ml) or with control Fc fragment (20 µg/ml) for 6 h in the presence of goat antibodies to human IgG (100 µg/ml). Cell lysates were then prepared and subjected to immunoblot analysis with a mAb to SHPS-1 or to tubulin ({alpha}Tubulin). The amount of SHPS-1 was quantified by scanning densitometry with the NIH Image program and is expressed as a percentage of the value for cells incubated with the control Fc fragment. B, detached Melan-a (upper panel) or B16F10 (lower panel) cells (1 x 106 in 1 ml of MEM) were mixed with CHO-Ras (CHO) or CHO-Ras-CD47 (CD47) cells (1 x 106 in 1 ml of MEM) and then cultured in 60-mm dishes for 5 h in the absence or presence of a mAb (MIAP301) to CD47 (5 µg/ml), as indicated. The amount of SHPS-1 was then quantified as in A and expressed as a percentage of the value for cells exposed to CHO-Ras cells. Data in A and B are means ± S.E. of triplicate determinations from three independent experiments. Representative immunoblot data are also shown.

 
Given that Melan-a cells express both SHPS-1 and CD47 (data not shown), it was possible that the inhibitory effect of CD47-Fc on SHPS-1 expression in these cells resulted from disruption of the interaction between CD47 and SHPS-1 present on the same cells or on adjacent cells, rather than directly from the engagement of surface SHPS-1. To exclude this possibility, we generated cells that express wild-type mouse CD47 (CHO-Ras-CD47 cells). Flow cytometry revealed that these cells, but not the parental CHO-Ras cells, expressed substantial amounts of CD47 on the cell surface (data not shown). The abundance of SHPS-1 in Melan-a cells cocultured with CHO-Ras-CD47 cells for 5 h was markedly reduced compared with that apparent in cells cocultured with CHO-Ras cells (Fig. 5B, upper left panel). Again, this effect was not likely due to toxicity given that expression of tyrosinase-related protein-1, a melanocyte-specific protein, was also unaffected (data not shown). A mAb that reacts with the extracellular region of mouse CD47 blocked the down-regulation of SHPS-1 expression in Melan-a cells by CHO-Ras-CD47 cells (Fig. 5B, upper right panel), indicating that the down-regulation was mediated by CD47-SHPS-1 interaction. In contrast to these observations with Melan-a cells, exposure to CHO-Ras-CD47 cells had no substantial effect on the amount of SHPS-1 in B16F10 cells (Fig. 5B, lower panel). These results are thus consistent with the notion that engagement of surface SHPS-1 by CD47-Fc or by CD47 on the surface of adjacent cells, rather than disruption of the CD47-SHPS-1 interaction, results in down-regulation of SHPS-1 expression.

CD47 Binding Induces Proteasome-dependent, Tyrosine Phosphorylation-independent Degradation of SHPS-1—To determine whether tyrosine phosphorylation of SHPS-1 is required for its CD47-induced down-regulation, we generated CHO cell lines that express either wild-type SHPS-1 (CHO-SHPS-1-WT cells) or an SHPS-1 mutant in which all four cytoplasmic tyrosine residues are replaced by phenylalanine (CHO-SHPS-1-4F cells). This mutant protein did not undergo tyrosine phosphorylation in the transfected cells (data not shown). CHO-SHPS-1-WT and CHO-SHPS-1-4F cells expressed similar amounts of SHPS-1 immunoreactivity on the cell surface as revealed by flow cytometry (Fig. 6A). They also bound CD47-Fc to similar extents (Fig. 6A), suggesting that cytoplasmic tyrosine residues of SHPS-1 do not play an important role in the interaction with CD47. Expression of the SHPS-1 mutant, like that of the wild-type protein, was markedly reduced on culture of the corresponding cells with CHO-Ras-CD47 cells (Fig. 6B). Tyrosine phosphorylation thus appeared dispensable for CD47-induced down-regulation of SHPS-1.



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  		FIG. 6.
Tyrosine phosphorylation-independent, proteasome-mediated degradation of SHPS-1 induced by CD47. A, CHO-SHPS-1-WT cells, CHO-SHPS-1-4F cells, or CHO-K1 cells transfected with the corresponding empty vector (CHO-CMV cells) were detached and incubated either with a mAb to SHPS-1 (solid trace) or with control rat IgG (broken trace)(upper panels). Alternatively, the cells were incubated with CD47-Fc (solid trace) or with control Fc fragment (broken trace) (lower panels). All cells were then analyzed by flow cytometry with appropriate secondary antibodies. B, detached CHO-SHPS-1-WT or CHO-SHPS-1-4F cells (1 x 106 in 1 ml of MEM) were mixed with CHO-Ras or CHO-Ras-CD47 cells (1 x 106 in 1 ml of MEM) and then cultured in 60-mm dishes for 3 h, after which cell lysates were prepared and subjected to immunoblot analysis with polyclonal antibodies to SHPS-1. C, CHO-SHPS-1-WT cells were incubated for 2 h at 37 °C in the absence or presence of 100 µM chloroquine, 10 mM NH4Cl, or 40 µM MG132, after which they were washed with PBS and processed for coculture experiments as in B.

 
The reduction in SHPS-1 expression induced by CD47 binding might have been attributable either to reduced biosynthesis or to an increased rate of degradation, although the rapidity of the effect suggested the latter possibility. To distinguish between these possibilities, we examined the effects of inhibitors of protein degradation by lysosomes or the proteasome. Prior treatment of CHO-SHPS-1-WT cells with chloroquine or NH4Cl, both of which inhibit lysosomal function, had no marked effect on the ability of CHO-Ras-CD47 cells to suppress SHPS-1 expression (Fig. 6C). In contrast, MG132, a reversible peptide aldehyde that blocks proteasomal activity, substantially attenuated the effect of CD47 (Fig. 6C). None of these reagents affected the expression of SHPS-1 in CHO-SHPS-1-WT cells cocultured with CHO-Ras cells (Fig. 6C). These results suggest that CD47 binding induces the degradation of SHPS-1 by the proteasome.

CD47-SHPS-1 Interaction Regulates Tyrosine Phosphorylation of FAK—FAK plays an important role in cell migration (38, 39) and is regulated by SHP-2 (22, 23, 40). To investigate further the molecular basis of the regulation of cell migration by CD47-SHPS-1 interaction, we determined whether this interaction affects the tyrosine phosphorylation of FAK. As expected, the amount of SHPS-1 bound to SHP-2 markedly decreased on exposure to CHO-Ras-CD47 cells (Fig. 7A). CHO-SHPS-1-WT, CHO-SHPS-1-4F, and parental CHO-K1 cells were next subjected to transient transfection with an expression vector for HA-tagged FAK and then cocultured with CHO-Ras-CD47 or CHO-Ras cells. The phosphorylation status of the recombinant FAK protein was subsequently quantitated by immunoprecipitation with a mAb to the HA tag and immunoblot analysis of the resulting precipitates with a mAb to phosphotyrosine. CHO-K1 cells cocultured with either CHO-Ras or CHO-Ras-CD47 cells exhibited similar substantial levels of FAK phosphorylation (Fig. 7B). The extent of FAK phosphorylation in CHO-SHPS-1-WT cells cocultured with CHO-Ras cells was less than that apparent in CHO-K1 cells. However, exposure to CHO-Ras-CD47 cells increased FAK phosphorylation in CHO-SHPS-1-WT cells to a level similar to that observed in CHO-K1 cells. In contrast, CHO-SHPS-1-4F cells cocultured with CHO-Ras or CHO-Ras-CD47 cells exhibited a level of FAK phosphorylation even greater than that apparent in CHO-K1 cells (Fig. 7B), consistent with the recent observation that rat SHPS-1 with mutations equivalent to those in SHPS-1-4F acts in a dominant negative manner (41). These results suggest that the amount of intact SHPS-1 is inversely related to the extent of tyrosine phosphorylation of FAK. Furthermore, CD47 binding might increase FAK phosphorylation, at least in part, through the down-regulation of SHPS-1.



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  		FIG. 7.
Effect of CD47-SHPS-1 interaction on tyrosine phosphorylation of FAK. A, CHO-SHPS-1-WT cells were transiently transfected with 5 µg of pcDNA3 encoding Myc-tagged SHP-2. After 24 h, the transfected cells (1 x 106 in 1 ml of MEM) were detached, mixed with CHO-Ras or CHO-Ras-CD47 cells (2 x 106 in 1 ml of MEM), and incubated in 60-mm dishes for 3 h. The cells were then lysed and subjected to immunoprecipitation (IP) with mAb 9E10 to the Myc tag ({alpha}Myc), and the resulting precipitates were subjected to immunoblot analysis with a mAb to SHPS-1. Duplicate immunoprecipitates were probed with polyclonal antibodies to SHP-2 ({alpha}SHP-2) to verify the presence of equal amounts of SHP-2 in each lane. Cell lysates were also probed with the same antibodies. B, CHO-K1, CHO-SHPS-1-WT, or CHO-SHPS-1-4F cells were transiently transfected with 1 µg of pRc/CMV vector encoding HA-tagged FAK. The transfected cells were processed for coculture experiments as in A and subjected to immunoprecipitation with mAb 12CA5 to the HA tag ({alpha}HA). The resulting precipitates were subjected to immunoblot analysis with horseradish peroxidase-conjugated mAb PY20 to phosphotyrosine ({alpha}PY). Duplicate precipitates were probed with polyclonal antibodies to FAK ({alpha}FAK) to verify the presence of equal amounts of FAK in each lane. The amount of tyrosine-phosphorylated FAK (FAK-P) was quantified by scanning densitometry with the NIH Image program, normalized to the amount of total FAK protein, and expressed as a percentage of the value for CHO-K1 cells cocultured with CHO-Ras cells. Data are representative of those obtained in three independent experiments.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
We have shown that the binding of a soluble SHPS-1 ligand (CD47-Fc) to B16F10 mouse melanoma cells was greatly reduced compared with that apparent with Melan-a nontumorigenic melanocytes, even though the two cell lines express similar amounts of SHPS-1 on their surface. SHPS-1 derived from B16F10 cells also bound CD47-Fc in vitro to a much lesser extent than did SHPS-1 from Melan-a cells. Furthermore, CD47-Fc inhibited the migration of Melan-a cells, whereas it had no marked effect on that of B16F10 cells. These results suggest that SHPS-1 expressed on the surface of B16F10 cells is impaired in the ability to bind CD47 on adjacent cells and that this defect renders the melanoma cells resistant to CD47-induced inhibition of cell motility.

We detected marked differences in both electrophoretic mobility and the affinity for G. nivalis agglutinin lectin, indicative of differential glycosylation, between SHPS-1 proteins expressed in Melan-a and B16F10 cells. Nonglycosylated forms of SHPS-1 from the two cell lines exposed to tunicamycin equally bound CD47-Fc in vitro. Furthermore, inhibition of N-glycan processing by 1-deoxymannojirimycin treatment restored the ability of SHPS-1 in B16F10 cells to bind CD47-Fc. These results provide evidence that aberrant N-glycosylation within the extracellular region of SHPS-1 is responsible for the observed reduced level of binding of CD47-Fc to B16F10 cells. Consistent with this notion, differential galactosylation of SHPS-1 has been shown to be a determinant of its cellular binding specificity in neuronal and hematopoietic cells (42). The activity of {beta}1,4-galactosyltransferase I, which catalyzes galactosylation of SHPS-1 (42), is increased at the surface of several invasive murine melanoma cell lines, including B16F10 (43, 44). Excessive galactosylation of SHPS-1 mediated by this enzyme may thus account for the reduced level of CD47-Fc binding to the surface of B16F10 melanoma cells. However, we cannot exclude the possibility that another mechanism also contributes to this reduced binding, given that CD47 potentially interacts with additional surface molecules such as thrombospondin-1 (45).

Monoclonal antibodies to SHPS-1 as well as soluble fusion proteins containing the extracellular domain of CD47 inhibit the migration of neutrophils across an epithelial cell layer, although the mechanism of this effect has been unclear (17). We have now shown that CD47-Fc inhibited the migration of Melan-a cells but not that of B16F10 cells, whereas an antibody to SHPS-1, which recognizes equally well the protein expressed on both Melan-a and B16F10 cells, reduced the motility of both cell types to a similar extent. During the course of this study, Motegi et al. (46) showed that SHPS-1 ligands inhibit migration of human melanoma cells. Our results thus provide further support for the notion that ligation of surface SHPS-1 inhibits cell migration. The impaired ability of SHPS-1 on B16F10 cells to bind CD47-Fc thus likely renders these cells resistant to CD47-induced inhibition of motility. Both flow cytometry and immunohistochemistry have shown that CD47 is abundant in keratinocytes (data not shown). Given the important role of these cells in control of the melanocyte phenotype in the epidermis (47, 48), the motility of SHPS-1-expressing melanocytes is likely limited by contact with adjacent CD47-expressing keratinocytes. Impaired binding of SHPS-1 on melanoma cells to CD47 on keratinocytes might abrogate this keratinocyte-mediated inhibitory mechanism and thereby promote melanoma cell invasion and metastasis.

The migration of immortalized fibroblasts that express an SHPS-1 mutant protein lacking most of the cytoplasmic region was markedly impaired, suggesting that the formation of a complex between SHPS-1 and SHP-2 is required for cell migration (20). Given that Melan-a and B16F10 cells both express SHP-2 but not SHP-1,2 the dissociation of SHP-2 from SHPS-1 (i.e. functional inactivation of SHP-2) might underlie the inhibition of Melan-a cell migration induced by the binding of CD47. Consistent with this idea, we showed that the binding of CD47 to SHPS-1 resulted in the proteasome-mediated degradation of SHPS-1. Tyrosine phosphorylation of SHPS-1 was shown to increase on incubation with CD47-expressing erythrocytes but not by exposure to erythrocytes deficient in CD47 (13). It is therefore possible that CD47 binding affects SHPS-1 differentially in different cell types.

The activity of SHP-2 is required for tyrosine dephosphorylation of FAK either in suspended cells (22) or in cells treated with insulin-like growth factor (23) or ephrins (40). Dephosphorylation of FAK, in turn, is both required and sufficient for the epidermal growth factor-induced increase in tumor cell motility, invasion, and metastasis (49). These observations have suggested that impaired dephosphorylation of FAK contributes to the defect in cell migration associated with loss of SHP-2 function. Consistent with this hypothesis, we have now shown that overexpression of wild-type SHPS-1 (i.e. functional activation of SHP-2), but not that of an SHPS-1 mutant that does not bind SHP-2, resulted in a reduction in the level of tyrosine phosphorylation of FAK; furthermore, this effect was reversed by the inactivation of SHP-2 that presumably accompanied the CD47-induced down-regulation of SHPS-1. Given that CD47 has been proposed to form dimers in cis (12, 50), engagement of SHPS-1 by CD47 dimers on adjacent cells likely results in proteasomal degradation of SHPS-1, inactivation of SHP-2, and inhibition of the tyrosine dephosphorylation of FAK, of the subsequent disassembly of focal adhesions, and eventually, of cell migration. In addition to FAK, the small GTPase Rho has been implicated in SHP-2 regulation of cell migration (51–53). It is therefore also possible that inhibition of cell migration by CD47-SHPS-1 interaction is mediated by regulation of Rho.

In conclusion, we have shown that the ability of SHPS-1 to bind CD47 is impaired as a result of aberrant N-glycosylation in B16F10 melanoma cells. Our results thus reveal a new example of a functional alteration in cell-cell communication that may contribute to the highly motile and invasive phenotype of malignant melanoma.


   FOOTNOTES
 
* This work was supported by a grant-in-aid for scientific research from the Ministry of Education, Science, Sports, and Culture of Japan and by a grant-in-aid from the Research for the Future Program of the Japan Society for the Promotion of Science. 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. Back

§ To whom correspondence should be addressed: Division of Diabetes, Digestive and Kidney Diseases, Dept. of Clinical Molecular Medicine, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan. Tel.: 81-78-382-5861; Fax: 81-78-382-2080; E-mail: noguchi{at}med.kobe-u.ac.jp.

1 The abbreviations used are: HA, hemagglutinin epitope; FAK, focal adhesion kinase; mAb, monoclonal antibody; MEM, modified Eagle's medium; CHO, Chinese hamster ovary; PBS, phosphate-buffered saline; WT, wild type. Back

2 T. Ogura and T. Noguchi, unpublished data. Back


   ACKNOWLEDGMENTS
 
We thank T. Horikawa for providing Melan-a and B16F10 cells, F. Lindberg for pIAP328, and T. Matozaki for helpful discussions.



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