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J. Biol. Chem., Vol. 278, Issue 31, 28593-28606, August 1, 2003

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Osteopontin Stimulates Cell Motility and Nuclear Factor B-mediated Secretion of Urokinase Type Plasminogen Activator through Phosphatidylinositol 3-Kinase/Akt Signaling Pathways in Breast Cancer Cells^*

Riku Das, Ganapati H. Mahabeleshwar and Gopal C. Kundu 

From the National Center for Cell Science (NCCS), NCCS Complex, Pune 411 007, India

Received for publication, April 3, 2003 , and in revised form, May 24, 2003.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
We have recently reported that osteopontin (OPN) induces nuclear factor B (NFB)-mediated promatrix metalloproteinase-2 activation through IB/IKK signaling pathways and that curcumin (diferulolylmethane) down-regulates these pathways (Philip, S., and Kundu, G. C. (2003) J. Biol. Chem. 278, 14487–14497). However, the molecular mechanism by which upstream kinases regulate the OPN-induced NFB activation and urokinase type plasminogen activator (uPA) secretion in human breast cancer cells is not well defined. Here we report that OPN induces the phosphatidylinositol 3'-kinase (PI 3'-kinase) activity and phosphorylation of Akt in highly invasive MDA-MB-231 and low invasive MCF-7 cells. The OPN-induced Akt phosphorylation was inhibited when cells were transfected with a dominant negative mutant of the p85 domain of PI 3-kinase (p85) and enhanced when cells were transfected with an activated form of PI 3-kinase (p110CAAX), indicating that PI 3'-kinase is involved in Akt phosphorylation. OPN enhances the interaction between IB kinase (IKK) and phosphorylated Akt. OPN also induces NFB activation through phosphorylation and degradation of IB by inducing the IKK activity. However, both pharmacological (wortmannin and LY294002) and genetic (p85) inhibitors of PI 3'-kinase inhibited OPN-induced Akt phosphorylation, IKK activity, and NFB activation through phosphorylation and degradation of IB. OPN also enhances uPA secretion, cell motility, and extracellular matrix invasion. Furthermore, cells transfected with p85 or the super-repressor form of IB suppressed the OPN-induced uPA secretion and cell motility, whereas cells transfected with p110CAAX enhanced these effects. Pretreatment of cells with PI 3-kinase inhibitors or NFB inhibitory peptide (SN-50) reduced the OPN-induced uPA secretion, cell motility, and invasion. To our knowledge, this is first report that OPN induces NFB activity and uPA secretion by activating PI 3'-kinase/Akt/IKK-mediated signaling pathways and further demonstrates a functional molecular link between OPN-induced PI 3'-kinase-dependent Akt phosphorylation and NFB-mediated uPA secretion, and all of these ultimately control the motility of breast cancer cells.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Cell migration and extracellular matrix invasion are two of the major steps in embryonic development (1, 2) and wound healing and cancer cell metastasis (3, 4). However, the exact molecular mechanisms that regulate these processes are not well defined. In the past several investigators have shown that urokinase type plasminogen activator (uPA)¹ plays a major role in the regulation of cancer cell motility, extracellular matrix (ECM) invasion, and metastasis by degrading the ECM proteins (5–7). Current investigations have focused on the understanding of molecular mechanism(s) by which osteopontin (OPN), an ECM protein, regulates uPA secretion and controls motility in human breast cancer cells.

OPN is a non-collagenous, sialic acid-rich, and glycosylated phosphoprotein (8, 9). This protein has a functional thrombin cleavage site and is a substrate for tissue transglutaminase (9). It has an N-terminal signal sequence, a highly acidic region consisting of nine consecutive aspartic acid residues, and a GRGDS cell adhesion sequence predicted to be flanked by the -sheet structure (10). OPN binds with several integrins and CD44 variants in an RGD sequence-dependent and -independent manner. This protein is involved in normal tissue-remodeling processes such as bone resorption, angiogenesis, wound healing, and tissue injury as well as certain diseases such as restenosis, atherosclerosis, tumorigenesis, and autoimmune diseases (11–13). OPN expression is up-regulated in several cancers and reported to associate with tumor progression and metastasis (14–16). OPN causes cell adhesion and migration, ECM invasion, and cell proliferation by interacting with its receptor _v3 integrin in various cell types (12). Integrins are non-covalently associated, heterodimeric, cell-surface glycoproteins with - and -subunits. Integrins are a superfamily of transmembrane glycoproteins found predominantly on the surface of leukocytes that mediate cell-cell and cell-substratum interactions. Until today, 12 -subunits, 8 -subunits, and 20 -heterodimers were documented in the literature (17).

Cell migration, a major step in cancer metastasis, is often associated with the activation of phosphatidylinositol (PI) 3-kinase (7, 18). PI 3'-kinase is consisting of a catalytic subunit p110 (, , and ) or p110, and a regulatory subunit p85 (, , p55, and p101) (19). PI 3'-kinase is activated by a number of growth factors. One of the downstream target molecule of PI 3'-kinase is Akt. Akt is a serine threonine kinase and also known as protein kinase B (PKB) or RAC-PK (related to A and C protein kinase). Akt is a cellular homolog of v-akt oncogene (20). Akt is activated by factors that stimulate PI 3'-kinase activity in cells such as thrombin, platelet-derived growth factor, and insulin (21). The activation of Akt is also occurred by stress factor, and that is independent of the PI 3'-kinase-mediated pathway (22, 23). In PI 3'-kinase-dependent pathway, phosphatidylinositol 3,4-bisphosphate, a product of PI 3'-kinase directly binds to the pleckstrin homology domain of Akt and leads to its activation (24, 25). However, the complete activation of Akt requires its phosphorylation on serine and threonine residues (26). Akt regulates cell cycle progression, growth factor-mediated cell survival, and cell migration. It promotes cell survival by phosphorylation and inactivation of Bad and Caspase-9 (27, 28). Several metastatic tumors express higher level of Akt (29). Previous data indicated that the PI 3'-kinase/Akt pathway is critically involved in anchorage-independent growth of tumor cells, which is one of the important steps of cancer metastasis (30). The mechanism by which OPN regulates PI 3'-kinase activity and controls cell motility in human breast cancer cells is not well defined.

The NFB family consists of several members, including p65, p50, RelB, and c-Rel molecules (31). The activity of NFB is tightly controlled by its inhibitor, the IB family of proteins (32). These inhibitory proteins bind to NFB dimers, hiding their nuclear localization sequence resulting in cytoplasmic retention of NFB (33). Upon stimulation, IB is phosphorylated and degraded via the ubiquitination and proteasome-mediated pathway, permitting activation and nuclear import of NFB where it binds to cognate sequence in promoter regions of multiple genes. There are other less explored pathways by which NFB translocates into the nucleus through tyrosine phosphorylation of IB (34). IKK (IB kinase) is a multisubunit protein kinase, the activation of which phosphorylated IB. Constitutive activation of NFB has been detected in lymphomas, melanomas, and breast cancers (35–39). The signaling pathways by which OPN regulates Akt phosphorylation followed by activations of IKK and NFB in human breast cancer cells are not clearly understood.

uPA is a member of serine protease that interacts with the uPA receptor (uPAR) and facilitates the conversion of inert zymogen plasminogen into widely acting serine protease plasmin and activation of metalloproteinases (40, 41). These proteases then degrade the surrounding matrix components (collagen, fibronectin, and laminin) and allow cancer cells to migrate to the distant sites. uPA is also involved in cell adhesion and chemotaxis (42, 43). It is well documented that uPA plays a significant role in tumor growth and metastasis (6, 7). NFB-responsive element is present in the promoter region of uPA, which plays a key role in cancer metastasis. However, the molecular mechanism by which OPN induces NFB-mediated uPA secretion and regulates cell migration and ECM invasion in breast cancer cells is not well documented.

In this study, we demonstrate that OPN induced the PI 3'-kinase activity and phosphorylation of Akt in human breast cancer cells. OPN also enhanced nuclear translocation of p65 subunit of NFB, NFB-DNA binding, and NFB transactivation through phosphorylation and degradation of IB by inducing the IKK activity. Moreover, OPN induced uPA secretion, ECM invasion, and cell motility in these cells. The OPN-induced NFB transactivation, uPA secretion, and cell motility were suppressed when both these cells were transfected with super-repressor form of IB or p85 and enhanced when cells were transfected with p110CAAX suggesting that PI 3-kinase is involved in these processes. Pretreatment of cells with PI 3'-kinase inhibitors (wortmannin and LY294002), _v3 integrin antibody, or NFB inhibitory peptide (SN-50) reduced the OPN-induced uPA secretion, cell motility, and invasion. Taken together, these data demonstrate that OPN enhances the cell motility and induces NFB-mediated uPA secretion through PI 3'-kinase/Akt/IKK-mediated signaling pathways.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Materials—The rabbit polyclonal anti-phospho-Akt1 (Ser-437), anti-p65 subunit of NFB (anti-p65), anti-NFB p65 X TransCruz, anti-IB, anti-IKK/, anti-actin, mouse monoclonal anti-phosphotyrosine antibody, anti-phospho IB, goat polyclonal anti-Akt1/2, and IB recombinant protein were purchased from Santa Cruz Biotechnology. Mouse monoclonal anti-uPA antibody and normal rabbit IgG were obtained from Oncogene. Mouse monoclonal anti-human _v3 integrin antibody was from Chemicon International. LY294002, SN-50, and SN-50M were obtained from Calbiochem. Wortmannin was from Sigma. LipofectAMINE Plus, GRGDSP, and GRGESP were obtained from Invitrogen. The dual luciferase reporter assay system and NFB consensus oligonucleotide were from Promega. Boyden-type cell migration chambers were obtained from Corning and BioCoat Matrigel^TM invasion chambers were from Collaborative Biomedical. [-³²P]ATP was purchased from Board of Radiation and Isotope Technology (Hyderabad, India). The human OPN was purified from milk as described previously (44) and used throughout these studies. All other chemicals were analytical grade.

Cell Culture—The MDA-MB-231 and MCF-7 cells were purchased from ATCC (Manassas, VA). Both MDA-MB-231 and MCF-7 cells were cultured in Dulbecco's modified Eagle's medium. The medium was supplemented with 10% fetal calf serum, 100 units/ml penicillin, 100 µg/ml streptomycin, and 2 mM glutamine in a humidified atmosphere of 5% CO₂ and 95% air at 37 °C.

DNA Transfection—HA-Delta.p85 in pCMV (p85) and Myc.p110.CAAX in pSG5 (p110CAAX) cDNA constructs were generous gifts from Dr. Alex Toker (Tufts University, Boston, MA). Both MCF-7 and MDA-MB-231 cells were split 12 h prior to transfection in Dulbecco's modified Eagle's medium containing 10% fetal calf serum. These cells were transiently transfected with HA-Delta.p85 (p85) or Myc.p110.CAAX cDNA using LipofectAMINE Plus according to the manufacturer's instructions. Briefly, p85 or p110CAAX cDNA (8 µg) was mixed with Plus reagent, and then cDNA reagent Plus was incubated with LipofectAMINE. The LipofectAMINE Plus cDNA complex was added to the cells and incubated further at 37 °C for 12 h. The control cells received LipofectAMINE Plus alone. The cell viability was detected by a trypan blue dye exclusion test. After incubation, the medium was removed, and the cells were refed with fresh medium and maintained for an additional 12 h. In another experiments, these cells were individually transfected with super-repressor form of IB cDNA fused downstream to a FLAG epitope in an expression vector (pCMV4) (a kind gift from Dr. Dean Ballard, Vanderbilt University School of Medicine) under the same conditions as described above. These transfected cells were used for the NFB activity by luciferase reporter gene assay, detection of uPA expression by Western blot analysis and cell migration. The p85-transfected cells were also used for Akt phosphorylation, IKK activity, and IB phosphorylation studies.

Western Blot Analysis—To delineate the role of OPN in regulation of Akt phosphorylation, both MCF-7 and MDA-MB-231 cells were treated with 5 µM OPN at 37 °C for 0–90 min. In separate experiments, cells were individually pretreated with PI 3'-kinase inhibitor (0–100 nM wortmannin or 0–10 µM LY294002), anti-_v3 integrin antibody (0–20 µg/ml), or with RGD peptide (0–10 µM GRGDSP or GRGESP) for 1 h and then treated with 5 µM OPN at 37 °C for 30 min in MCF-7 cells and 15 min in MDA-MB-231 cells. Cells were lysed in lysis buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% Nonidet P-40, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 5 mM iodoacetamide, 2 mM phenylmethylsulfonyl fluoride, 20 µg/ml leupeptin, and 2 mM EDTA) containing 25 mM NaF and 2 mM Na₃VO₄. The cleared lysates were collected by centrifugation at 12,000 x g for 15 min at 4 °C. The protein concentration in the lysate was measured by Bio-Rad protein assay. The lysates containing equal amounts of total proteins were resolved by SDS-PAGE. The proteins were electrotransferred from gel to nitrocellulose membrane. The membrane was incubated with rabbit polyclonal anti-phospho Akt antibody and incubated further with anti-rabbit horseradish peroxidase-conjugated IgG. The membrane was washed and detected by the enhanced chemiluminescence (ECL) detection system (Amersham Biosciences) according to the manufacturer's instructions. The membrane was reprobed with goat polyclonal anti-Akt antibody to ensure equal protein loading. In separate experiments, both these cells were individually transfected with a dominant negative mutant of the p85 domain of PI 3-kinase (p85) or the activated form of PI 3-kinase (p110CAAX) in the presence of LipofectAMINE Plus and then stimulated with OPN. Cells were lysed in lysis buffer. The lysates containing equal amounts of total proteins were resolved by SDS-PAGE, and the levels of both phospho- and non-phospho Akt were detected by Western blot analysis as described previously.

To check the effect of OPN in regulation of IB serine/threonine phosphorylation and degradation in breast cancer cells, both these cells were either treated with 5 µM OPN for 0–90 min or pretreated with LY294002 (10 µM) or wortmannin (100 nM) for 1 h and then treated with 5 µM OPN for 30 min in MCF-7 and 15 min in MDA-MB-231 cells at 37 °C. In other experiments, cells were transfected with p85 cDNA and then treated with OPN as described above. Cells were lysed in lysis buffer containing 25 mM NaF and 2 mM Na₃VO₄. The equal amounts of total proteins in the lysates were separated by SDS-PAGE and detected by Western blot using mouse anti-phospho-IB antibody. The same blots were reprobed with rabbit anti-IB antibody. As loading controls, the expression of actin was also analyzed by reprobing the blots with anti-actin antibody and detected by using the ECL detection system.

To investigate the role of OPN on uPA secretion in both MCF-7 and MDA-MB-231 cells, both these cells were individually treated with various concentrations of OPN (0–5 µM) for 24 h. In separate experiments, cells were pretreated with anti-_v3 integrin antibody (0–20 µg/ml), RGD peptide (0–10 µM GRGDSP or GRGESP), PI 3'-kinase inhibitor (0–100 nM wortmannin and 0–10 µM LY294002), NFB inhibitory peptide (0–100 µg/ml SN-50 or SN-50M) for 1 h and then treated with 5 µM OPN for additional 24 h at 37 °C. These cells were lysed, and cell lysates containing equal amounts of total proteins were subjected to Western blot analysis using mouse monoclonal anti-uPA antibody. In separate experiments, both these cells were individually transfected with dominant negative mutant of PI 3-kinase (p85), the activated form of PI 3-kinase (p110CAAX), or the super-repressor form of IB in the presence of LipofectAMINE Plus and then treated with 5 µM OPN for 24 h. Cells were lysed in lysis buffer. The level of uPA in these lysates was detected by Western blot analysis. As loading controls, the expression of actin was also detected by reprobing the blot with rabbit anti-actin antibody.

Immunoprecipitation—To check whether OPN regulates the interaction between IKK and phosphorylated Akt, MCF-7, and MDA-MB-231 cells were treated with 5 µM OPN at 37 °C for 30 and 15 min, respectively. In another experiments, these cells were pretreated with LY294002 (10 µM) or wortmannin (100 nM) for 1 h or transfected with p85 cDNA and then treated with 5 µM OPN as described above. Cells were lysed in lysis buffer (20 mM Tris-HCl (pH 8.0), 137 mM NaCl, 15% v/v glycerol, 1% Nonidet P-40, 2 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 1 µg/ml leupeptin, 1 µg/ml pepstatin, 25 mM -glycerophosphate, 2 mM benzamidine, 25 mM NaF, 10 mM pNPP, 2 mM Na₃VO₄) and clarified by centrifugation at 12,000 x g for 15 min at 4 °C. The cell lysates containing equal amounts of total proteins were immunoprecipitated with goat anti-Akt antibody. The half of the immunoprecipitated samples was resolved by SDS-PAGE, and the level of IKK was detected by Western blot analysis using rabbit anti-IKK antibody. The remaining half of the samples was resolved by SDS-PAGE, and the serine-phosphorylated Akt was detected by Western blot analysis using rabbit anti-phospho-Akt (serine-specific) antibody. The same blots were reprobed with goat anti-Akt antibody. Similarly, in other experiments, the treated cells were lysed in lysis buffer and immunoprecipitated with rabbit anti-IKK antibody. The half of the immunoprecipitated samples were used to detect the levels of phospho and non-phospho Akt by Western blot analysis using anti-phospho-Akt and anti-non-phospho-Akt antibody, respectively. The other half of the samples were resolved by SDS-PAGE and IKK level was detected by Western blot analysis using anti-IKK antibody.

In Vitro Kinase Assay—To check the role of OPN in regulation of PI 3'-kinase activity, both MCF-7 and MDA-MB-231 cells were treated with 5 µM OPN for 0–60 min at 37 °C. In separate experiments, cells were pretreated with anti-_v3 integrin antibody (20 µg/ml) for 1 h and then treated with 5 µM OPN for 30 min in MCF-7 and for 15 min in MDA-MB-231 cells. Cells were lysed in lysis buffer, and the protein concentration in the lysates was measured by Bio-Rad protein assay. The PI 3'-kinase assay was performed as described previously (7). Briefly, cell lysates were immunoprecipitated with mouse monoclonal anti-phosphotyrosine antibody, and the immunoprecipitated samples were incubated in kinase assay buffer (25 mM Hepes (pH 7.4), 10 mM MgCl₂, and 1 mM EDTA) containing phosphatidylinositol (0.25 mg/ml), 100 mM ATP, and 15 µCi of [-³²P]ATP and incubated at 30 °C for 10 min. The reaction was terminated by addition of acidified chloroformmethanol (2:1). Lipids were extracted according to the procedure as described previously (45) and separated on oxalate-treated plastic TLC plates using a solvent system consisting of chloroform, methanol, and 20% methylamine (65:35:10, v/v). The spots corresponding to the position of radioactive phosphatidylinositol phosphate (PIP) were visualized by autoradiography.

To detect the effect of OPN on IKK activity, MCF-7 and MDA-MB-231 cells were treated with 5 µM OPN at 37 °C for 30 and 15 min, respectively. The IKK assay was performed as described previously (46). The cells were lysed in lysis buffer; equal amounts of total proteins in the lysates were immunoprecipitated with rabbit anti-IKK/ antibody as described above. Half of the immunoprecipitated samples were incubated with recombinant IB (4 µg) in kinase assay buffer (20 mM Hepes (pH 7.7), 2 mM MgCl₂, 10 µM ATP, 3 µCi of [-³²p]ATP, 10 mM -glycerophosphate, 10 mM NaF, 10 mM pNPP, 300 µM Na₃VO₄, 1 mM benzamidine, 2 µM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 1 µg/ml leupeptin, 1 µg/ml pepstatin, and 1 mM DTT) at 30 °C for 1 h. The kinase reactions were stopped by addition of SDS-sample buffer. The samples were resolved by SDS-PAGE, dried, and autoradiographed. The remaining half of the immunoprecipitated samples were subjected to SDS-PAGE and analyzed by Western blot using anti-IKK/ antibody. In separate experiments, both these cells were either transfected with p85 cDNA or pretreated with LY294002 (10 µM) or wortmannin (100 nM) then treated with OPN (5 µM). These cells were used for detection of IKK activity under the same conditions as described previously.

Nuclear and Cytoplasmic Extracts and Western Blot—Both MCF-7 and MDA-MB-231 cells were treated with 5 µM OPN for 4 h at 37 °C. The nuclear extracts were prepared as described (46). Briefly, the cells were scraped, washed with phosphate-buffered saline (pH 7.4), resuspended in hypotonic buffer (10 mM Hepes (pH 7.9), 1.5 mM MgCl₂, 10 mM KCl, 0.2 mM phenylmethylsulfonyl fluoride, and 0.5 mM dithiothreitol), and allowed to swell on ice for 10 min. Cells were homogenized in a Dounce homogenizer. The nuclei were separated by spinning at 3300 x g for 5 min at 4 °C. The supernatant was used as cytoplasmic extract. The nuclear pellet was extracted in nuclear extraction buffer (20 mM Hepes (pH 7.9), 0.4 M NaCl, 1.5 mM MgCl₂, 0.2 mM EDTA, 25% glycerol, 0.5 mM phenylmethylsulfonyl fluoride, and 0.5 mM DTT) for 30 min on ice and centrifuged at 12,000 x g for 30 min. The supernatant was used as a nuclear extract. The protein concentrations in the supernatants of both nuclear and cytoplasmic extracts were measured by the Bio-Rad protein assay. The nuclear and cytoplasmic extracts (30 µg) were resolved by SDS-PAGE, and the level of p65 was detected by Western blot analysis using rabbit anti-p65 antibody.

EMSA—EMSA was performed as described previously (46). Both MCF-7 and MDA-MB-231 cells were treated with 5 µM OPN for 0–6 h. The nuclear extracts (10 µg) were incubated with 16 fmol of ³²P-labeled double-stranded NFB oligonucleotide (5'-AGT TGA GGG GAC TTT CCC AGG C-3') in binding buffer (25 mM Hepes (pH 7.9), 0.5 mM EDTA, 0.5 mM DTT, 1% Nonidet P-40, 5% glycerol, and 50 mM NaCl) containing 2 µg of polydeoxyinosinic deoxycytidylic acid (poly(dI-dC)). The DNA-protein complex was resolved on a native polyacrylamide gel and analyzed by autoradiography. For supershift assay, the OPN-treated nuclear extracts were incubated with anti-p65 antibody for 30 min at room temperature and analyzed by EMSA.

NFB Luciferase Reporter Gene Assay—The semiconfluent cells (MCF-7 and MDA-MB-231) grown in 24-well plates were transiently transfected with a luciferase reporter construct (pNFB-Luc) containing five tandem repeats of the NFB binding site (a generous gift from Dr. Rainer de Martin, University of Vienna, Vienna, Austria) using LipofectAMINE Plus reagent (Invitrogen). The transfection efficiency was normalized by cotransfecting the cells with pRL vector (Promega) containing a full-length Renilla luciferase gene under the control of a constitutive promoter. After 24 h of transfection, the cells were treated with varying concentrations of OPN (0–5 µM) for 6 h or pretreated with anti-_v3 integrin antibody (20 µg/ml), wortmannin (100 nM), and LY294002 (10 µM) for 1 h and then treated with OPN (5 µM) for an additional 6 h at 37 °C. In other experiments, both MCF-7 and MDA-MB-231 cells were individually transfected with HA-Delta.p85, Myc.p110.CAAX, super-repressor form of IB, and ASOPN (OPN-specific phosphorothioate-linked antisense oligonucleotide) in the presence of pNFB-Luc and then treated with OPN (5 µM) for 6 h. Cells were harvested in passive lysis buffer (Promega). The luciferase activities were measured by luminometer (Lab Systems) using the dual luciferase assay system according to the manufacturer's instructions (Promega). Changes in luciferase activity with respect to control were calculated.

Cell Migration Assay—The migration assay was conducted using Transwell cell culture chamber according to the standard procedure as described previously (7, 44, 46). Briefly, the confluent monolayers of MCF-7 or MDA-MB-231 cells were harvested with trypsin-EDTA and centrifuged at 800 x g for 10 min. The cell suspension (5 x 10⁵ cells/well) was added to the upper chamber of the prehydrated polycarbonate membrane filter. The lower chamber was filled with fibroblast condition medium, which acted as a chemoattractant. Purified OPN (0–5 µM) was added to the upper chamber. In another experiments, MCF-7 or MDA-MB-231 cells were individually pretreated with anti-_v3 integrin antibody (0–20 µg/ml), GRGDSP or GRGESP (0–10 µM), PI 3'-kinase inhibitors (0–100 nM wortmannin or 0–10 µM LY294002), NFB inhibitory peptides (0–100 µg/ml SN-50 or SN-50M), and monoclonal anti-uPA antibody (0–20 µg/ml) at 37 °C for 6 h. In other experiments, cells were individually transfected with super-repressor form of IB, p85, or p110CAAX and used for migration assay. OPN (5 µM) was used in the upper chamber. After treatment, these cells were incubated in a humidified incubator in 5% CO₂ and 95% air at 37 °C for 16 h. The non-migrated cells on the upper side of the filter were scraped, and the filter was washed. The migrated cells in the reverse side of the filter were fixed with methanol and stained with Giemsa. The migrated cells on the filter were counted under an inverted microscope (Olympus). The experiments were repeated in triplicate. Preimmune IgG served as nonspecific control.

Chemoinvasion Assay—The chemoinvasion assay was performed using Matrigel^TM-coated invasion chamber as described (44, 46). MCF-7 or MDA-MB-231 cell suspension (5 x 10⁵cells/well) was added to the upper portion of the prehydrated Matrigel^TM-coated chamber. The lower chamber was filled with fibroblast condition medium, which acted as chemoattractant. Purified OPN (5 µM) was added to the upper chamber. In separate experiments, MCF-7 or MDA-MB-231 cells were individually pretreated with anti-_v3 integrin antibody (20 µg/ml), PI 3'-kinase inhibitors (100 nM wortmannin or 10 µM LY294002), or NFB inhibitory peptide (100 µg/ml SN-50) at 37 °C for 6 h. OPN (5 µM) was added to the upper chamber. The cells were incubated at 37 °C for 16 h. The non-migrating cells and Matrigel^TM from the upper side of the filter were scraped and removed using a moist cotton swab. The invaded cells in the lower side of the filter were stained with Giemsa and washed with phosphate-buffered saline (pH 7.6). The invaded cells were then counted, and photomicrographs were taken under the inverted microscope (Olympus). The experiments were repeated in triplicate. Preimmune IgG served as nonspecific control.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
OPN Induces PI 3'-Kinase Activity and Akt Phosphorylation—Because we have reported earlier that OPN induces NFB activity through phosphorylation and degradation of IB by activating IKK (46), we sought to determine whether any upstream kinases such as PI 3'-kinase/Akt play any role in OPN-induced NFB activation by inducing IKK activity in breast cancer cells. Accordingly, both MCF-7 and MDA-MB-231 cells were treated with 5 µM OPN in basal medium for 0–60 min at 37 °C. The lysates containing an equal amount of total proteins were immunoprecipitated with anti-phosphotyrosine antibody. The immunoprecipitated samples were used for the PI 3'-kinase assay. The PIP was separated by TLC and visualized by autoradiography. The maximum PI 3'-kinase activity was found at 15–30 min in both MCF-7 and MDA-MB-231 cells (Fig. 1, A and B, lanes 1–5). Pretreatment of cells with anti-_v3 integrin antibody suppressed the OPN-induced PI 3'-kinase activity in both these cells (panels A and B, lane 6) suggesting that OPN induces PI 3'-kinase activity through the integrin-mediated pathway. The bands were analyzed densitometrically (Kodak Digital Science), and the -fold changes were calculated.

View larger version (42K): [in this window] [in a new window]   FIG. 1. OPN induces PI 3'-kinase activity (A and B) and Akt phosphorylation (C–H) in MCF-7 and MDA-MB-231 cells. A and B, PI 3'-kinase activity. Both MCF-7 (A) and MDA-MB-231 (B) cells were treated with 5 µM OPN for 0–60 min. Equal amounts of total proteins from the untreated or treated lysates of MCF-7 or MDA-MB-231 cells were immunoprecipitated with mouse monoclonal anti-phosphotyrosine antibody, and the immunocomplexes were assayed for their ability to phosphorylated PI to PIP using [-32P]ATP at 30 °C for 10 min. The PIP was resolved by TLC and autoradiographed (lanes 1–5). In separate experiments, cells were pretreated with anti-v3 integrin antibody (20 µg/ml) and treated with OPN, and cell lysates were used for PI 3'-kinase assay. Note that maximum OPN-induced PI 3'-kinase activity was observed at 30 min in MCF-7 and at 15 min in MDA-MB-231 cells. v3 integrin antibody suppressed the OPN-induced PI 3'-kinase activity in both these cells. C and D, serum-starved cells were treated with 5 µM OPN for 0–90 min; lysates containing equal amounts of total proteins were resolved by SDS-PAGE and subjected to Western blot analysis using anti-phospho Akt antibody (lanes 1–6, upper panels). The same blots were reprobed with anti-Akt antibody (lanes 1–6, lower panels). Arrows indicate the phospho- and non-phospho-specific Akt bands. E and F, both these cells were individually pretreated with two different concentrations of v3 integrin antibody, GRGDSP, GRGESP, LY294002, and wortmannin and then treated with 5 µM OPN. The cells were lysed, and lysates containing equal amounts of total proteins were analyzed by Western blot using anti-phospho or anti-non-phospho Akt antibodies. Lane 1, control; lane 2, 5 µM OPN; lane 3, 10 µg/ml v3 integrin antibody and 5 µM OPN; lane 4, 20 µg/ml v3 integrin antibody and 5 µM OPN; lane 5, 5 µM GRGDSP and 5 µM OPN; lane 6, 10 µM GRGDSP and 5 µM OPN; lane 7, 5 µM GRGESP and 5 µM OPN; lane 8, 10 µM GRGESP and 5 µM OPN; lane 9, 5 µM LY294002 and 5 µM OPN; lane 10, 10 µM LY294002 and 5 µM OPN; lane 11, 10 nM wortmannin and 5 µM OPN; and lane 12, 100 nM wortmannin and 5 µM OPN. G and H, cells were transiently transfected with a dominant negative p85 domain of PI 3'-kinase (p85) or activated form of p110 domain of PI 3'-kinase (p110CAAX) in the presence of LipofectAMINE Plus and then treated with 5 µM OPN. The levels of phospho Akt and Akt in the cell lysates were detected by Western blot analysis. Lane 1, control; lane 2, 5 µM OPN; lane 3, transfected with p85 and treated with 5 µM OPN; and lane 4, transfected with p110CAAX and treated with 5 µM OPN. Upper panel shows the phospho-specific Akt, and the lower panel indicates non-phospho-specific Akt bands. All of these bands in A–H were quantified by densitometric analysis, and the -fold changes are calculated. The results shown here represent three experiments exhibiting similar effects.

To ascertain the role of OPN on Akt phosphorylation, both these cells were treated with 5 µM OPN for 0–90 min at 37 °C, the cell lysates containing equal amounts of total proteins were resolved by SDS-PAGE, and the level of phosphorylated Akt was detected by Western blot analysis using anti-phospho-Akt antibody (Ser-473). The data revealed that the maximum level of OPN-induced Akt phosphorylation occurred at 30 min in MCF-7 cells and at 15 min in MDA-MB-231 cells (Fig. 1, C and D, upper panels, lanes 1–6) and also suggested that Ser-473 residue is crucial for phosphorylation of Akt. The same blots were reprobed with anti-Akt antibody and the data showed that there was no change in expression of non-phospho Akt in both these cells upon treatment with OPN confirming the equal loading of the samples (lower panels, lanes 1–6). To check whether OPN-induced Akt phosphorylation occurred through the PI 3'-kinase-mediated pathway, both these cells were either transfected with p85 or CAAXp110 of PI 3'-kinase in the presence of LipofectAMINE Plus or pretreated with PI 3'-kinase inhibitor (wortmannin or LY294002) and then treated with OPN. To further delineate whether _v3 integrin or the RGD/RGE peptide is involved in OPN-induced Akt phosphorylation; cells were pretreated with anti-_v3 integrin antibody or with RGD/RGE peptide (GRGDSP or GRGESP) and then treated with OPN. The level of phosphorylated Akt was detected by Western blot analysis. The data demonstrated that PI 3'-kinase inhibitor (wortmannin or LY294002), integrin antibody, and RGD (GRGDSP) but not RGE (GRGESP) peptide suppressed the OPN-induced Akt phosphorylation in these cells (Fig. 1, E and F, upper panels, lanes 1–12). Similarly, p85 inhibited but CAAXp110 enhanced the OPN-induced Akt phosphorylation suggesting that PI 3'-kinase is involved in Akt phosphorylation (Fig. 1, G and H, upper panels, lanes 1–4), and it occurred through the integrin-mediated pathway. The level of non-phospho Akt was unchanged in transfected or treated cells (Fig. 1, E–H, lower panels). Western blot data were quantified by densitometric analysis, and the -fold changes were calculated. These results further suggested that OPN binds with _v3 integrin receptor and regulates Akt phosphorylation through the PI 3'-kinase-dependent pathway.

OPN Stimulates the Interaction between Phosphorylated Akt and IKK—To determine if OPN has any role in regulating the interaction between phosphorylated Akt and IKK, both these cells were treated with 5 µM OPN. The cells were lysed and immunoprecipitated with goat polyclonal anti-Akt antibody. Half of the immunoprecipitated samples were analyzed by Western blot analysis using anti-IKK/ antibody, and the remaining half of the samples were immunoblotted by anti-phospho-Akt antibody. The same blots were reprobed with anti-Akt antibody to ensure that the basic level of Akt was the same. The results indicated that OPN induces the interaction between IKK/ (Fig. 2, A and B, upper panel, lane 2) and phosphorylated Akt (middle panel, lane 2) compared with control (lane 1). LY294002 and wortmannin suppressed the OPN-induced interaction between IKK/ and phosphorylated Akt in both MCF-7 and MDA-MB-231 cells (upper and middle panels, lanes 3 and 4). The cells transfected with p85 followed by treatment with OPN showed significant inhibition of this interaction (lane 5). As expected, the level of non-phospho Akt remained unchanged in both these cells (lower panel).

View larger version (29K): [in this window] [in a new window]   FIG. 2. Effect of OPN on the interaction between IKK and phosphorylated Akt. A–D, both MCF-7 (A and C) and MDA-MB-231 (B and D) cells were either treated with 5 µM OPN or pretreated with PI 3'-kinase inhibitor (10 µM LY294002 or 100 nM wortmannin) and then treated with 5 µM OPN or transfected with p85 and then treated with 5 µM OPN. A and B, cell lysates containing equal amounts of total proteins were immunoprecipitated with anti-Akt antibody. Half of the samples were immunoblotted with anti-IKK/ antibody. The remaining half of the samples were analyzed by Western blot using anti-phospho Akt antibody. The same blots were reprobed with anti-Akt antibody. C and D, cell lysates containing equal amounts of total proteins were immunoprecipitated with anti-IKK/ antibody. Half of the samples were analyzed by Western blot using anti-non-phospho and phospho Akt antibodies. The remaining half of the samples were immunoblotted with anti-IKK/ antibody. Lane 1, control; lane 2, 5 µM OPN; lane 3, 10 µM LY294002 and 5 µM OPN; lane 4, 100 nM wortmannin and 5 µM OPN; lane 5, transfected with p85 and treated with 5 µM OPN; lane 6, treated with OPN and immunoprecipitated with normal goat/rabbit IgG; and lane 7, treated with OPN and then direct Western blot with anti-IKK, anti-phospho Akt or anti-Akt antibody. All of these bands in A–D were quantified densitometrically, and the -fold changes were calculated. The data shown represent three experiments exhibiting similar effects.

To further confirm this interaction, cells were treated with OPN; the lysates were immunoprecipitated with anti-IKK/ antibody, and the levels of Akt, phospho-Akt, or IKK were detected by Western blot analysis using their specific antibodies. The results showed that OPN induces the interaction between IKK and Akt (Fig. 2, C and D, upper and middle panels, lane 2) compared with control (lane 1) in these cells. LY294002, wortmannin, or p85 inhibited this interaction (upper and middle panels, lanes 3–5) in these cells. As expected, the level of IKK/ remained identical (lower panel). As control, both IKK- and Akt-specific bands were detected when cell lysates were used for direct Western blot analysis using their specific antibodies (Fig. 2, A–D, lane 7). As expected, no IKK/- or Akt-specific band was found when cell lysates were immunoprecipitated with normal goat/rabbit IgG (lane 6). All these bands were quantified densitometrically, and the -fold changes were calculated.

OPN Enhances the IB Phosphorylation by Inducing IKK Activity—To delineate whether OPN has any effect on phosphorylation of IB through modulating the activation of IKK in breast cancer cells, both these cells were treated with 5 µM OPN. The cells were lysed and immunoprecipitated with anti-IKK/ antibody. Half of the immunoprecipitated samples were used for kinase assay using recombinant IB as substrate. The radiolabeled, phosphorylated IB-specific band was detected in OPN-treated cells, demonstrating that OPN induces the IKK activity (Fig. 3, A and B, upper panels, lane 2). The IKK activity was dramatically reduced when cells were either transfected with p85 (lane 3) or pretreated with LY294002 (lane 4) or wortmannin (lane 5), suggesting that OPN-induces IKK activity through the PI 3'-kinase-dependent pathway in breast cancer cells. A low level of IKK activity was detected in untreated cells (lane 1). The protein bands were quantified by densitometric analysis, and -fold changes were calculated. The OPN-induced IKK activity was almost 4.4- and 5.2-fold higher compared with control in MCF-7 and MDA-MB-231 cells, respectively. The remaining half of the immunoprecipitated samples were analyzed by Western blot using anti-IKK/ antibody. Fig. 3 (A and B) showed the identical level of expression of IKK, suggesting that equal amount of IKK was used in this assay (lower panels).

View larger version (13K): [in this window] [in a new window]   FIG. 3. OPN stimulates the IKK activity. Both MCF-7 (A) and MDA-MB-231 (B) cells were either treated with 5 µM OPN or pretreated with PI 3'-kinase inhibitor (10 µM LY294002 or 100 nM wortmannin) and then treated with 5 µM OPN or transfected with p85 and then treated with 5 µM OPN. Cell lysates containing equal amounts of total proteins were immunoprecipitated with anti-IKK/ antibody, and half of the immunoprecipitated samples were used for kinase assay using IB as substrate (upper panels of A and B). The remaining half of the immunoprecipitated samples were analyzed by Western blot using anti-IKK/ antibody (lower panels of A and B). Lane 1, control; lane 2, 5 µM OPN; lane 3, transfected with p85 and then treated with 5 µM OPN; lane 4, 10 µM LY294002 and 5 µM OPN; and lane 5, 100 nM wortmannin and 5 µM OPN. All these bands were quantified by densitometric analysis, and the -fold changes were calculated. The results shown here represent three experiments exhibiting similar effects.

To check the effect of OPN on IB phosphorylation and degradation, both MCF-7 and MDA-MB-231 cells were treated with 5 µM OPN for 0–90 min and lysed. The lysates containing equal amounts of total proteins were resolved by SDS-PAGE, and phosphorylated IB was detected by Western blot analysis using anti-phospho-IB antibody. The maximum level of OPN-induced IB phosphorylation was detected in 30 min in MCF-7 cells (Fig. 4A, upper panel) and in 15 min in MDA-MB-231 cells (Fig. 4B, upper panel). The level of phospho-IB was reappeared in 90 min in both MCF-7 and MDA-MB-231 cells (upper panels of A and B). The blots were reprobed with anti-IB antibody, and the data indicated that the maximum OPN-induced degradation was observed in 30 min in both MCF-7 and MDA-MB-231 cells (middle panels of A and B). After that, IB synthesis was reactivated possibly by NFB in 60 min (middle panels). The reduced level of phosphorylated IB at 60 min in MCF-7 and MDA-MB-231 cells indicates that the rate of degradation exceeded the rate of IB phosphorylation at this time point (upper panels of A and B).

View larger version (29K): [in this window] [in a new window]   FIG. 4. OPN induces IB phosphorylation and degradation. A–D, both MCF-7 (A and C) and MDA-MB-231 (B and D) cells were either treated with 5 µM OPN for 0–90 min (A and B) or pretreated with PI 3'-kinase inhibitor (10 µM LY294002 or 100 nM wortmannin) for 1 h and then treated with 5 µM OPN (C and D) or transfected with p85 and then treated with 5 µM OPN (C and D) as described under "Experimental Procedures." Cell lysates containing equal amounts of total proteins were analyzed by Western blot using anti-phospho IB antibody. The same blots were reprobed with anti-IB or anti-actin antibody. Note that maximum IB phosphorylation occurred at 30 min in MCF-7 and at 15 min in MDA-MB-231 cells, whereas maximum degradation was observed at 30 min in both these cells. PI 3'-kinase inhibitor blocks the OPN-induced IB phosphorylation and degradation in MCF-7 and MDA-MB-231 cells. The blots were analyzed densitometrically, and the values were normalized to actin. The relative values of phospho-IB or IB in terms of -fold changes are indicated. The data shown represent three experiments exhibiting similar effects.

In separate experiments, cells were pretreated with LY294002 (10 µM) or wortmannin (100 nM) for 1 h and then treated with OPN (5 µM) for 30 min in MCF-7 and for 15 min in MDA-MB-231 cells. In other experiments, both these cells were transfected with p85 in the presence of LipofectAMINE Plus and then treated with OPN (5 µM). The cells were lysed, and equal amounts of total proteins from the lysates were resolved by SDS-PAGE and analyzed by Western blot using anti-phospho-specific IB or anti-IB antibody as described above. The results indicated that both PI 3'-kinase inhibitor (LY294002 or wortmannin) and p85 inhibited OPN-induced IB phosphorylation and degradation in these cells (upper and middle panels of C and D) suggesting that OPN induces IB phosphorylation and degradation through the PI 3'-kinase-dependent pathways. As loading controls, all these blots were reprobed with anti-actin antibody (lower panels of A–D). The bands were quantified by densitometry, and the values were normalized with respect to actin expression. The -fold changes, as compared with control, were calculated.

OPN Induces Translocation of the p65 Subunit of NFB into the Nucleus—To check whether OPN induces the NFB translocation in low invasive (MCF-7) and highly invasive (MDA-MB-231) breast cancer cells, both these cells were treated with 5 µM OPN for 4 h at 37 °C. The nuclear and cytoplasmic fractions were prepared from the untreated and treated cells. The levels of p65 in these fractions were analyzed by Western blot using rabbit anti-p65 antibody (Fig. 5, A and B). In the OPN-untreated cells, the p65 was localized mostly in the cytoplasm (lane 1) instead of the nucleus (lane 2), whereas in the OPN-treated cells, it was translocated from the cytoplasm (lane 3) to the nucleus (lane 4). The Western blot data were quantified densitometrically, and the -fold changes as compared with control were calculated.

View larger version (35K): [in this window] [in a new window]   FIG. 5. OPN enhances nuclear accumulation of p65 subunit of NFB, NFB-DNA binding, and NFB activation. A and B, nuclear translocation of p65 into nucleus by Western blot analysis. Both MCF-7 (A) and MDA-MB-231 (B) cells were treated with or without OPN (5 µM) for 4 h. The nuclear and cytoplasmic extracts from untreated and treated cells were immunoblotted with rabbit polyclonal anti-p65 antibody. In the untreated cells, p65 was detected in the cytoplasm (lane 1) but not in the nucleus (lane 2). In the OPN-treated cells, p65 translocated from cytoplasm (lane 3) into the nucleus (lane 4). The levels of p65 were quantified by densitometric analysis, and the -fold changes are calculated. C and E, electrophoretic mobility shift assay. MCF-7 (C) and MDA-MB-231 (E) cells were treated with 5 µM OPN for 0–6 h. The nuclear extracts were prepared and analyzed by EMSA as described under "Experimental Procedures." The arrow indicates the NFB-specific bands. Note that maximum NFB binding was observed at 4 h in both these cells (lanes 1–4). D and F, supershift assay. The nuclear extracts from OPN-treated cells were incubated with anti-p65 antibody and analyzed by EMSA. Lane 1, with 5 µM OPN; lane 2, with 5 µM OPN and anti-p65 antibody. The results shown in A–F represent three experiments exhibiting similar effects. G and H, luciferase reporter gene assay. MCF-7 (G) and MDA-MB-231 (H) cells were transiently transfected with luciferase reporter construct (pNFB-Luc) with LipofectAMINE Plus. Transfected cells were either stimulated with various doses of OPN (0–5 µM) for 6 h or pretreated with anti-v3 integrin antibody (20 µg/ml) or PI 3'-kinase inhibitor (10 µM LY294002 and 100 nM wortmannin) and then treated with 5 µM OPN. In other experiments, cells were individually transfected with p85, p110CAAX, the super-repressor form of IB, or ASOPN in the presence of pNFB-Luc and then treated with 5 µM OPN. The cell lysates were used to measure the luciferase activity. The values were normalized to Renilla luciferase activity. The -fold changes were calculated, and mean ± S.E. of triplicate determinations are plotted.

OPN Stimulates NFB-DNA Binding and NFB Transactivation—Cells were treated with 5 µM OPN for 0–6 h, and nuclear extracts were prepared and used for EMSA using ³²P-labeled NFB oligonucleotides. Fig. 5 (C and E) showed the maximum NFB-DNA binding in 4 h in both MCF-7 (panel C, lanes 1–4) and MDA-MB-231 (panel E, lanes 1–4) cells. Whether the band (panels C and E) obtained by EMSA in OPN-treated cells is indeed NFB, the nuclear extracts were incubated with anti-p65 antibody and then analyzed by EMSA. Fig. 5 (D and F) showed the shift of the NFB-specific band to a higher molecular weight when the nuclear extracts were treated with anti-p65 antibody, suggesting that the OPN-activated complex consists of the p65 subunit in MCF-7 (panel D, lanes 1 and 2) and MDA-MB-231 (panel F, lanes 1 and 2) cells.

To detect whether OPN stimulates NFB transcriptional activity and whether _v3 integrin and PI 3'-kinase are involved in this process, a luciferase reporter gene assay was performed. MCF-7 and MDA-MB-231 cells were transfected with a pNFB luciferase reporter construct (pNFB-Luc) in the presence of LipofectAMINE Plus. Transfected cells were either treated with OPN (0–5 µM) or pretreated with anti-_v3 integrin antibody (20 µg/ml), LY294002 (10 µM), or wortmannin (100 nM) and then treated with OPN (5 µM). In separate experiments, cells were individually transfected with p85, p110CAAX, a super-repressor form of IB, or ASOPN in the presence of pNFB-Luc and then treated with OPN (5 µM). The transfection efficiency was normalized by cotransfecting the cells with pRL vector. Changes in luciferase activity with respect to control were calculated. The -fold changes were calculated, and the means of triplicate determinations were plotted. The data demonstrated that OPN stimulated the NFB transcriptional activity in a dose-dependent manner and that _v3 integrin antibody and PI 3'-kinase inhibitor (LY294002 and wortmannin) suppressed the OPN-induced NFB activity (Fig. 5, G and H). Similarly, p85, a super-repressor form of IB, and ASOPN inhibited and CAAXp110 enhanced the OPN-induced NFB activity in these cells (panels G and H). These data further suggested that both catalytic (p110) and regulatory (p85) subunits of PI 3'-kinase are involved in OPN-induced NFB transactivation through the _v3 integrin-mediated pathway.

OPN Induces PI 3'-Kinase and NFB-dependent uPA Secretion—To delineate whether OPN plays any role in uPA secretion, both MCF-7 and MDA-MB-231 cells were treated individually with varying concentrations of OPN (0–5 µM). The cells were lysed, and the lysates containing equal amounts of total proteins were resolved by SDS-PAGE and analyzed by Western blot using mouse monoclonal anti-uPA antibody. The data indicated that OPN enhanced the uPA secretion in a dose-dependent manner in both MCF-7 and MDA-MB-231 cells (Fig. 6, A and B, lanes 1–5).

View larger version (41K): [in this window] [in a new window]   FIG. 6. Effect of OPN on uPA secretion. A–D, MCF-7 (A and C) and MDA-MB-231 (B and D) cells were treated with OPN (0–5 µM) for 24 h (A and B). Cells were also pretreated with anti-v3 integrin antibody (0–20 µg/ml), GRGDSP or GRGESP peptide (0–10 µM), PI 3'-kinase inhibitor (0–10 µM LY294002 or 0–100 nM wortmannin), or NFB inhibitory peptide (SN-50 or SN-50M (0–100 µg/ml)) for 1 h and then treated with 5 µM OPN for additional 24 h (C and D). The level of uPA in the lysates was detected by Western blot analysis using anti-uPA antibody. The arrow indicates the uPA-specific band. The same blots were reprobed with anti-actin antibody. The constitutive expression of uPA was significantly higher in MDA-MB-231 cells compared with MCF-7 cells. Note that OPN induced uPA secretion (A and B, lanes 1–5). v3 integrin antibody, GRGDSP peptide, PI 3'-kinase inhibitors, and NFB inhibitory peptide suppressed OPN-induced uPA secretion (C and D, lanes 1–16). GRGESP and SN-50M had no effect on OPN-induced uPA secretion (C and D, lanes 1–16). The bands were quantified by densitometric analysis, and -fold changes were calculated. The results shown represent three experiments exhibiting similar effects.

To examine whether OPN-induced uPA secretion occurred through _v3 integrin/PI 3'-kinase/NFB-mediated pathways, both these cells were individually treated with anti-_v3 integrin antibody, RGD peptide (GRGDSP or GRGESP), PI 3'-kinase inhibitors (LY294002 or wortmannin), and NFB inhibitory peptide (SN-50 or SN-50M). The treated cell lysates containing equal amounts of total proteins were separated by SDS-PAGE and immunoblotted with mouse monoclonal anti-uPA antibody. The level of OPN-induced uPA expression was reduced significantly when both these cells were individually treated with _v3 integrin antibody (Fig. 6, C and D, lanes 3 and 4), GRGDSP (lanes 5 and 6), LY294002 (lanes 9 and 10), wortmannin (lanes 11 and 12), and SN-50 (lanes 13 and 14) compared with cells treated with OPN alone (lane 2). No changes of uPA secretion were observed in cells treated with GRGESP (lanes 7 and 8) or SN-50M (lanes 15 and 16). As expected, a very low level of uPA was observed in OPN-untreated cells (lane 1). The constitutive expression of uPA was significantly higher in MDA-MB-231 cells compared with MCF-7 cells (Fig. 6, A–D). As loading controls, all these blots were reprobed with anti-actin antibody (Fig. 6, A–D, lower panels).

In separate experiments, cells were also individually transfected with p85, CAAXp110, or a super-repressor form of IB in the presence of LipofectAMINE Plus and then treated with OPN as described above. The results indicated that both p85 (Fig. 7, A and B, lane 3) and that super-repressor form of IB (C and D, lane 3) suppressed and CAAXp110 (A and B, lane 4) enhanced the OPN-induced uPA secretion compared with OPN-treated (A–D, lane 2) cells. The low level of the uPA-specific band was detected in untreated cells (A–D, lane 1). As loading controls, all these blots were reprobed with anti-actin antibody (Fig. 7, A–D, lower panels).

View larger version (18K): [in this window] [in a new window]   FIG. 7. PI 3'-kinase and IB are involved in uPA secretion responding to OPN. A–D, MCF-7 (A and C) and MDA-MB-231 (B and D) cells were individually transfected with p85, p110CAAX, or the super-repressor form of IB and then treated with 5 µM OPN for 24 h. The cell lysates containing equal amounts of total proteins were analyzed by Western blot using anti-uPA antibody. The same blots were reprobed with anti-actin antibody. Note that OPN-induced uPA secretion (A–D, lane 2) was blocked by both p85 domain of PI 3'-kinase (A and B, lane 3) and the super-repressor form of IB (C and D, lane 3) but enhanced by p110CAAX (A and B, lane 4) compared with control (A–D, lane 1). The bands were quantified by densitometric analysis, and -fold changes were calculated. The results shown represent three experiments exhibiting similar effects.

In all of these experiments, the uPA-specific bands were quantified by densitometric analysis, and the values of -fold changes were determined (Figs. 6 and 7). These results suggested that OPN induces uPA secretion via the _v3 integrin-mediated pathway and further demonstrated that PI 3'-kinase and NFB are involved in this process.

PI 3'-Kinase, NFB, and uPA Play Crucial Roles in OPN-induced _v3 Integrin-mediated Cell Migration and Chemoinvasion—Be