Osteopontin increases CD44 expression and cell adhesion in RAW 264.7 murine leukemia cells

Immunology Letters 95 (2004) 109–112

Short communication

Osteopontin increases CD44 expression and cell adhesion in RAW 264.7 murine leukemia cells Carlos E. Marroquin, Laura Downey, Hongtao Guo, Paul C. Kuo∗ Department of Surgery, Duke University Medical Center, DUMC, 110 Bell Bldg., Box 3522, Durham, NC 27710, USA Received 17 April 2004; received in revised form 1 June 2004; accepted 6 June 2004 Available online 2 July 2004

Abstract Background: Osteopontin (OPN) is an inducible cell attachment protein which binds ␣v ␤3 -integrin and CD44 receptors to promote tumor metastasis. We hypothesized that OPN alters expression of its CD44 receptor to promote neoplastic cell migration. Methods: RAW264.7 cells were stimulated with OPN (0–10 nM) for 0–12 hours to determine the time- and concentration-dependence of CD44 protein and mRNA expression. In selected instances, a competitive ligand for the ␣v ␤3 -integrin, GRGDSP (50 nM), or an inhibitor of protein synthesis, anisomycin (10 ␮g/ml), was added. Cell adhesion to hyaluronan was assayed with the crystal violet assay. Results: OPN upregulates plasma membrane total CD44 protein in a concentration-(ANOVA P = 0.001) and time-dependent (ANOVA P = 0.001) fashion. CD44v6 is not altered. Cell adhesion to hyaluronate increases in parallel with CD44 expression. Steady state mRNA levels for CD44 are not altered by OPN. 5 nM OPN increases CD44 protein half-life from 105 ± 11 minutes to 278 ± 15 minutes. (P < 0.03) Blockade of either ␣v ␤3 -integrin ablates the OPN-dependent increase in CD44. Conclusions: These data indicate that OPN increases plasma membrane CD44 expression and cell adhesion by binding to its ␣v ␤3 -integrin receptor. We conclude that OPN may promote tumor metastatic behavior by CD44 expression. © 2004 Elsevier B.V. All rights reserved. Keywords: Metastasis; Hyaluronate; Extracellular matrix

Osteopontin (OPN) is a secreted glycoprotein that is rich in aspartate and sialic-acid residues and contains functional domains that mediate cell–matrix interactions and cellular signaling through binding with integrin and CD44 receptors [1]. OPN has multiple molecular functions which mediate cell adhesion, chemotaxis, angiogenesis, prevention of apoptosis and anchorage-independent growth of tumor cells [2–4]. A substantial body of data has linked OPN with the regulation of metastatic spread by malignant cells. However, the molecular mechanisms which defines the role of OPN in tumor metastasis are incompletely understood. We and others have previously demonstrated that OPN upregulates CD44v6 isoform expression and in vitro cell adhesion in HepG2 hepatoma cells [5,6]. It is unknown whether this is a generalized response of tumor cells to OPN stimulation. To address the potential generalized nature of this response to OPN, we use murine RAW 264.7 cells to examine the hypothesis that OPN alters the expression of its CD44 cell ∗ Corresponding author. Tel.: +1-919-668-1856; fax: +1-919-684-8716. E-mail address: [email protected] (P.C. Kuo).

0165-2478/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.imlet.2004.06.001

surface receptor to promote a metastatic phenotype, as determined by in vitro adhesion assays. We find that, although OPN upregulates plasma membrane total CD44 protein in a concentration- and time-dependent-fashion. CD44v6 protein is not altered. Cell adhesion to hyaluronate increases in parallel with CD44 expression. Steady-state mRNA levels for CD44 are not altered by OPN. 5 nM OPN increases CD44 protein half-life from 105 ± 11 to 278 ± 15 min (P < 0.03). Blockade of ␣v ␤3 -integrin receptor binding ablates the OPN-dependent increase in CD44 half-life. In conjunction with our findings in HepG2 cells, this suggests that OPN regulation of tumor cell adhesion may act in a generalized fashion through its CD44 receptor. RAW 264.7 and ANA-1 murine leukemia cells were grown as monolayer cultures in modified Eagle’s medium (DMEM) with the addition of 10% fetal bovine serum (FBS). The ANA-1 immortalized murine macrophage cell line are derived from infecting normal bone marrow cells of C57BL/6 mice with the murine recombinant J2 retrovirus containing the v-myc and v-raf oncogenes. In selected instances, the cells were treated for various incubation periods (0, 2, 4, 8 and 12 h) with murine OPN (0, 2, 5 and

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10 nM). Protein half-life determinations were performed by addition of anisomycin (10 ␮g/ml) changed every 3–4 days, and the cells were maintained at 37 ◦ C in a humidified atmosphere containing 5% CO2 (10 ␮g/ml) following 12 h of OPN (5 nM) incubation. For immunoblot 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% Na2 HPO4 and 0.024% KH2 PO4 , pH 7.4) and centrifuged at 12,000 × g for 10 min at 4 ◦ C. Protein concentration was determined by absorbance at 650 nm using protein assay reagent (Bio-Rad, Hercules, CA). Cell lysates (50 ␮g/lane) were separated by 12% SDS-PAGE and the products were electrotransferred to PVDF membrane (Amersham Pharmacia, Piscataway, NJ). Blocked membranes were incubated with antibody directed against CD44 (R&D Systems, Minneapolis, MN) and ␤-actin (goat polyclonal Ab, Santa Cruz Biotechnology) and incubated with horseradish peroxidase-conjugated secondary antibody. For northern blotting, [32 P]dATP-labeled probes for CD44 (exons 1–5, nt 179–849) were constructed based upon GenBank accession no. NM000610. cDNA probes were constructed to bridge two contiguous exons and were prepared by random primer labeling. Hybridization was performed at 42 ◦ C for 18 h in ULTRAhyb hybridization buffer (Ambion, Austin, TX). ␤-Actin was utilized as the housekeeping gene. Quantification was performed using a Phosphorimager (Molecular Dynamics Storm 840). Pulse-chase studies were performed to determine rate of synthesis of CD44 in the setting of OPN (5 nM) stimulation. Cells were subcultured at a density of 1.0 × 105 cells/well. After 24 h, cells were washed with PBS and then were incubated with 1 ml of FBS-, methionineand cysteine-free DMEM supplemented with 100 ␮Ci/ml of l-[35 S]methionine and l-[35 S]cysteine in vitro cell labeling mixture and incubated for 0, 3, 6 and 12 h. A total of 500 ␮l of cell lysate (100 ␮g total protein) was immunoprecipitated with antibody directed against murine CD44 (R&D Systems). The immunoprecipitates were washed, solubilized, subjected to SDS-PAGE and 35 S incorporation determined. Cell adhesion to hyaluronate (HA) was determined in the presence and absence of 5 nM OPN. 2 × 105 cells were added to each well and the 96-well plate incubated at 37 ◦ C for 60 min in serum-free medium. Non-adhered cells were removed by washing. Adhered cells were fixed with 1.0% glutaraldehyde and stained with 0.1% (w/v) crystal violet solution. Absorbance readings were recorded at 570 nm. The results obtained were corrected by the subtraction of background staining of the underlying matrix. RAW 264.7 cells were exposed to OPN (0, 2, 5 and 10 nM) for periods of 0, 2, 4, 8 and 12 h. Previous work has demonstrated elevated OPN serum levels of 8–15 nM in patients with metastatic liver, breast and prostate cancer [5]. Plasma membrane CD44 protein expression was determined using immunoblot analysis, and densitometry was measured relative to a CD44 standard (Fig. 1). CD44 plasma membrane protein expression was upregulated in a concentration- (ANOVA P = 0.001) and time-dependent

Fig. 1. OPN-dependent plasma membrane CD44 protein expression. CD44 protein expression was determined using immunoblot analysis, and densitometry was measured relative to a CD44 standard. CD44 plasma membrane protein expression was upregulated in a concentration- (ANOVA, P = 0.001) and time-dependent (ANOVA, P = 0.001) manner.

(ANOVA P = 0.001) manner. Incubation with OPN (5 nM) was associated with a three-fold increase in CD44 expression at 8 h. Parallel experiments performed to determine plasma membrane CD44v6 protein expression in response to OPN demonstrated no change. In subsequent experiments, an OPN concentration of 5 nM and an incubation time of 8 h were utilized. Northern blot analysis was performed to determine whether OPN increases steady-state CD44 mRNA levels in parallel with plasma membrane CD44 protein expression. A radiolabelled probe for CD44 (nt-179 to nt-849; Genbank NM000610) was constructed to bridge two contiguous exons. Following 8 h of incubation, normalized CD44 mRNA levels did not differ among cells exposed to OPN at concentrations of 0, 2, 5 and 10 nM (data not shown). This data suggests that OPN does not alter CD44 gene transcription. Plasma membrane CD44 protein half-life was then determined in the presence of OPN (5 nM) and the protein synthesis inhibitor, anisomycin (10 ␮g/ml). Pulse chase experiments were performed using l-[35 S]methionine and l-[35 S]cysteine followed by immunoprecipitation of murine CD44 protein (Fig. 2). Semi-log plots determined that protein half-life of CD44 increased from 105 ± 11 to 278 ± 15 min in the presence of OPN (5 nM) (P < 0.03). These experiments were then repeated in the presence of GRGDSP (50 nM; Sigma Corp), a competitive ligand for OPN binding to its ␣v ␤3 integrin receptor. In the presence of GRGDSP, plasma membrane CD44 protein half-life was not altered by incubation with OPN (5 nM). These data suggest that OPN mediated prolongation of CD44 protein half-life is mediated via the ␣v ␤3 integrin receptor.

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Fig. 2. Semi-log plot of pulse-chase assays of CD44 protein half-life. Laser densitometric analysis was performed relative to ␤-actin protein. Data are expressed as means ± S.D. Data represent a total of three experiments.

To determine a potential functional role for OPN-mediated increase in CD44 expression, in vitro assays of RAW 264.7 and ANA-1 cell adhesion to the CD44 ligand, hyaluronate (HA), were performed (Fig. 3). Incubation in the presence of OPN (5 nM) for 8 h resulted in a statistically significant four-fold and six-fold increase in adhesion of both RAW and ANA cells to HA, respectively. To confirm the role of CD44, cells were incubated in the presence of OPN and monoclonal blocking antibody to CD44 (R&D Systems,

Fig. 3. In vitro assay of RAW 264.7 cell adhesion to the CD44 ligand, hyaluronate (HA). GRGDSP (50 nM) is a competitive ligand for OPN binding to its ␣v ␤3 integrin receptor (∗ P < 0.01 vs. unstimulated cells).

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Minneapolis, MN). Blockade of CD44 ablated the OPN mediated increase in HA adhesion. These data suggest that the increased RAW and ANA cell adhesion noted in the presence of OPN is mediated via CD44 function. In addition, incubation of cells with OPN + GRGDSP abolished the increased cell adhesion noted in the presence of OPN alone. OPN is a ligand for the ␣v ␤ integrin and CD44 families of receptors. OPN-integrin binding was shown to mediate migration and invasion of tumor cells [7–9]. The CD44 glycoproteins are ubiquitously expressed, cell-surface adhesion molecules that mediate cell–matrix and cell–cell interactions [10,11]. The principal ligand for CD44 is hyaluronic acid. OPN–CD44 interactions appear to be RGD-independent [10,11]. Available data suggest that OPN can activate CD44-mediated pathways to promote cell survival, chemotaxis, homing and adhesion, each potentially enhancing metastatic behavior. Clinically, a correlation between high levels of OPN protein expression and malignant invasion was established when OPN was demonstrated within tumor cells and in the surrounding stroma of numerous human cancers [12,13]. Supporting this link was the significant increase in plasma concentration of OPN in patients with metastatic disease compared to normal plasma [12,13]. Subsequently, increased OPN expression has been associated with tumor invasion, progression or metastasis in cancers of the breast, stomach, lung, prostate, liver and colon [12,13]. We have previously demonstrated that OPN increases HepG2 cell expression of CD44v6, although in this current instance CD44v6 was unaltered in RAW 264.7 cells [5]. CD44 is a transmembrane cell adhesion molecule that is the major cell surface receptor for HA. Multiple high molecular weight isoforms (CD44v) of the core molecule (CD44s) may be generated through alternative splicing of 10 variable exons which encode part of the extracellular domain. Upregulation of Cd44s and CD44v expression and their impact on metastatic biology characterize multiple human malignancies, including colorectal, breast, pancreatic, gastric and melanoma cancers [5,6]. It is clear that the bulk of experimental data indicates that OPN interacts with CD44s and CD44v isoforms to convey a metastatic phenotype. In our setting, CD44s appears to mediate the in vitro functional correlate of OPN simulation. The molecular mechanisms which define the role of OPN in tumor metastasis are incompletely understood. In this study using murine RAW 264.7 cells, we examine the hypothesis that OPN alters the extent of expression of its CD44 cell surface receptor to promote a metastatic phenotype, as determined by in vitro adhesion assays to HA. As we have examined only HA dependent adhesion, the strong possibility exists that additional mechanisms of action might also exist in parallel to those that are CD44-dependent. Nevertheless, our results suggest that OPN binds to its ␣v ␤3 integrin receptor to enhance plasma membrane CD44 expression and increase cell adhesion. In conjunction with our findings in HepG2 cells, this suggests that OPN regulation of tumor cell

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adhesion may act in a generalized fashion through its CD44 receptor. Acknowledgements

[7]

Supported by NIH AI44629 and GM65113 grants to PCK. [8]

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