Osteopontin: it's role in regulation of cell motility and nuclear factor kappa B-mediated urokinase type plasminogen activator expression

Cancer progression depends on an accumulation of metastasis supporting cell signaling molecules that target signal transduction pathways and ultimately gene expression. Osteopontin (OPN) is one such chemokine like metastasis gene which plays a key signaling event in regulating the oncogenic potential of various cancers by controlling cell motility, invasiveness and tumor growth. We have reported that OPN stimulates tumor growth and nuclear factor kappaB (NFkappaB)-mediated promatrix metalloproteinase-2 (pro-MMP-2) activation through IkappaBalpha/IKK (IkappaBalpha kinase) signaling pathway in melanoma cells. Urokinase type plasminogen activator (uPA), a widely acting serine protease degrades the ECM components and plays a pivotal role in cancer progression. However, the molecular mechanism by which upstream kinases regulate the OPN-induced NFkappaB activation and 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/PKB (protein kinase B) in highly invasive (MDA-MB-231) and low invasive (MCF-7) breast cancer cells. The OPN-induced Akt phosphorylation was inhibited when cells were transfected with dominant negative mutant of p85 domain of PI 3'-kinase (Deltap85) indicating that PI 3'-kinase is involved in Akt phosphorylation. OPN enhances the interaction between IkappaBalpha kinase (IKK) and phosphorylated Akt. OPN also induces NFkappaB activation through phosphorylation and degradation of IkappaBalpha by inducing the IKK activity. OPN also enhances uPA secretion, cell motility and ECM-invasion. Furthermore, cells transfected with Deltap85 or super-repressor form of IkappaBalpha suppressed the OPN-induced uPA secretion and cell motility. Pretreatment of cells with PI 3'-kinase inhibitors or NFkappaB inhibitory peptide (SN50) reduced the OPN-induced uPA secretion, cell motility and ECM-invasion. Taken together, OPN induces NFkappaB 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 NFkappaB-mediated uPA secretion, and all of these ultimately control the motility and invasiveness of breast cancer cells.     

Nuclear factor-inducing kinase plays a crucial role in osteopontin-induced MAPK/IkappaBalpha kinase-dependent nuclear factor kappaB-mediated promatrix metalloproteinase-9 activation

We have recently demonstrated that osteopontin (OPN) induces nuclear factor kappaB (NFkappaB)-mediated promatrix metalloproteinase-2 activation through IkappaBalpha/IkappaBalpha kinase (IKK) signaling pathways. However, the molecular mechanism(s) by which OPN regulates promatrix metalloproteinase-9 (pro-MMP-9) activation, MMP-9-dependent cell motility, and tumor growth and the involvement of upstream kinases in regulation of these processes in murine melanoma cells are not well defined. Here we report that OPN induced alpha(v)beta(3) integrin-mediated phosphorylation and activation of nuclear factor-inducing kinase (NIK) and enhanced the interaction between phosphorylated NIK and IKKalpha/beta in B16F10 cells. Moreover, NIK was involved in OPN-induced phosphorylations of MEK-1 and ERK1/2 in these cells. OPN induced NIK-dependent NFkappaB activation through ERK/IKKalpha/beta-mediated pathways. Furthermore OPN enhanced NIK-regulated urokinase-type plasminogen activator (uPA) secretion, uPA-dependent pro-MMP-9 activation, cell motility, and tumor growth. Wild type NIK, IKKalpha/beta, and ERK1/2 enhanced and kinase-negative NIK (mut NIK), dominant negative IKKalpha/beta (dn IKKalpha/beta), and dn ERK1/2 suppressed the OPN-induced NFkappaB activation, uPA secretion, pro-MMP-9 activation, cell motility, and chemoinvasion. Pretreatment of cells with anti-MMP-2 antibody along with anti-MMP-9 antibody drastically inhibited the OPN-induced cell migration and chemoinvasion, whereas cells pretreated with anti-MMP-2 antibody had no effect on OPN-induced pro-MMP-9 activation suggesting that OPN induces pro-MMP-2 and pro-MMP-9 activations through two distinct pathways. The level of active MMP-9 in the OPN-induced tumor was higher compared with control. To our knowledge, this is the first report that NIK plays a crucial role in OPN-induced NFkappaB activation, uPA secretion, and pro-MMP-9 activation through MAPK/IKKalpha/beta-mediated pathways, and all of these ultimately control the cell motility, invasiveness, and tumor growth.     

Osteopontin induces AP-1-mediated secretion of urokinase-type plasminogen activator through c-Src-dependent epidermal growth factor receptor transactivation in breast cancer cells

We have recently reported that osteopontin (OPN) stimulates cell motility and nuclear factor kappaB-mediated secretion of urokinase-type plasminogen activator (uPA) through phosphatidylinositol 3-kinase/Akt signaling pathways in breast cancer cells (Das, R., Mahabeleshwar, G. H., and Kundu, G. C. (2003) J. Biol. Chem. 278, 28593-28606). However, the role(s) of OPN on AP-1-mediated uPA secretion and cell motility and the involvement of c-Src/epidermal growth factor receptor (EGFR) in these processes in breast cancer cells are not well defined. In this study we report that OPN induces alpha(v)beta(3) integrin-mediated c-Src kinase activity in both highly invasive (MDA-MB-231) and low invasive (MCF-7) breast cancer cells. Ligation of OPN with alpha(v)beta(3) integrin induces kinase activity and tyrosine phosphorylation of EGFR in MDA-MB-231 and wild type EGFR-transfected MCF-7 cells, and this was inhibited by the dominant negative form of c-Src (dn c-Src) indicating that c-Src kinase plays a crucial role in this process. OPN induces association between alpha(v)beta(3) integrin and EGFR on the cell membrane in a macromolecular form with c-Src. Furthermore, OPN induces alpha(v)beta(3) integrin/EGFR-mediated ERK1/2 phosphorylation and AP-1 activation. Moreover, dn c-Src also suppressed the OPN-induced phosphatidylinositol (PI) 3-kinase activity in these cells indicating that c-Src acts as master switch in regulating MEK/ERK1/2 and phosphatidylinositol 3-kinase/Akt signaling pathways. OPN-induced ERK phosphorylation, AP-1 activation, uPA secretion, and cell motility were suppressed when cells were transfected with dn c-Src or pretreated with alpha(v)beta(3) integrin antibody, c-Src kinase inhibitor (pp2), EGFR tyrosine kinase inhibitor (PD153035), and MEK-1 inhibitor (PD98059). To our knowledge, this is the first report that OPN induces alpha(v)beta(3) integrin-mediated AP-1 activity and uPA secretion by activating c-Src/EGFR/ERK signaling pathways and further demonstrates a functional molecular link between OPN-induced integrin/c-Src-dependent EGFR phosphorylation and ERK/AP-1-mediated uPA secretion, and all of these ultimately control the motility of breast cancer cells.     

Nuclear factor inducing kinase: A key regulator in osteopontin- induced MAPK/IκB kinase dependent NF-κB-mediated promatrix metalloproteinase-9 activation

Osteopontin (OPN) is a secreted, non-collagenous, sialic-acid rich, glycosylated adhesive phospho- protein. Several highly metastatic transformed cells synthesized a higher level of OPN compared with non-tumorigenic cells. We have recently reported that OPN induces nuclear factor-κB (NF-κB)-mediated promatrix metalloproteinase-2 activation through IκBα/IKK signaling pathways. However, the molecular mechanism(s) by which OPN regulates pro-matrix metalloproteinase-9 (pro-MMP-9) activation and involvement of upstream kinases in regulation of these processes that ultimately control cell motility and tumor growth in murine melanoma cells are not well defined. Here we report that OPN induces αvβ3 integrin-mediated phosphorylation and activation of nuclear factor inducing kinase (NIK) and enhances the interaction between phosphorylated NIK and IκBα kinase α/β (IKKα/β) in B16F10 cells. Moreover, NIK is involved in OPN-induced phosphorylations of MEK-1 and ERK1/2 in these cells. OPN induces NIK-dependent NF-κB activation through ERK/IKKα/β-mediated pathways. Furthermore, OPN enhances NIK-regulated urokinase-type plasminogen activator (uPA) secretion, uPA-dependent pro-MMP-9 activation, and cell motility. Pretreatment of cells with anti-MMP-2 antibody along with anti-MMP-9 antibody drastically inhibited the OPN-induced cell migration and chemoinvasion, whereas cells pretreated with anti-MMP-2 antibody had no effect on OPN-induced pro-MMP-9 activation suggesting that OPN induces pro-MMP-2 and pro-MMP-9 activations through two distinct pathways. Taken together, NIK acts as crucial regulator in OPN-induced MAPK/IKK-mediated NF-κB-dependent uPA secretion and MMP-9 activation thereby controlling melanoma cell motility and chemoinvasion. An erratum to this article is available at .     

JNK1 differentially regulates osteopontin-induced nuclear factor-inducing kinase/MEKK1-dependent activating protein-1-mediated promatrix metalloproteinase-9 activation

We have recently demonstrated that nuclear factor-inducing kinase (NIK) plays a crucial role in osteopontin (OPN)-induced mitogen-activated protein kinase/I kappa B alpha kinase-dependent nuclear factor kappa B (NF kappa B)-mediated promatrix metalloproteinase-9 activation (Rangaswami, H., Bulbule, A., and Kundu, G. C. (2004) J. Biol. Chem. 279, 38921-38935). However, the molecular mechanism(s) by which OPN regulates NIK/MEKK1-dependent activating protein-1 (AP-1)-mediated promatrix metalloproteinase-9 activation and whether JNK1 plays any role in regulating both these pathways that control the cell motility are not well defined. Here we report that OPN induces alpha v beta3 integrin-mediated MEKK1 phosphorylation and MEKK1-dependent JNK1 phosphorylation and activation. Overexpression of NIK enhances OPN-induced c-Jun expression, whereas overexpressed NIK had no role in OPN-induced JNK1 phosphorylation and activation. Sustained activation of JNK1 by overexpression of wild type but not kinase negative MEKK1 resulted in suppression of ERK1/2 activation. But this did not affect the OPN-induced NIK-dependent ERK1/2 activation. OPN stimulated both NIK and MEKK1-dependent c-Jun expression, leading to AP-1 activation, whereas NIK-dependent AP-1 activation is independent of JNK1. OPN also enhanced JNK1-dependent/independent AP-1-mediated urokinase type plasminogen activator (uPA) secretion, uPA-dependent promatrix metalloproteinase-9 (MMP-9) activation, cell motility, and invasion. OPN stimulates tumor growth, and the levels of c-Jun, AP-1, urokinase type plasminogen activator, and MMP-9 were higher in OPN-induced tumor compared with control. To our knowledge this is first report that OPN induces NIK/MEKK1-mediated JNK1-dependent/independent AP-1-mediated pro-MMP-9 activation and regulates the negative crosstalk between NIK/ERK1/2 and MEKK1/JNK1 pathways that ultimately controls the cell motility, invasiveness, and tumor growth.     

Interferon-dependent activation of the serine kinase PI 3'-kinase requires engagement of the IRS pathway but not the Stat pathway

Several signaling pathways are activated by interferon alpha (IFNalpha) in hematopoietic cells, including the Jak-Stat and the insulin receptor substrate (IRS) pathways. It has been previously shown that IFNalpha activates the phosphatidylinositol (PI) 3'-kinase via an interaction of the p85 subunit of PI 3'-kinase with IRS proteins. Other studies have proposed that Stat-3 also functions as an adapter for p85. We sought to identify the major pathway that regulates IFNalpha activation of the PI3'-kinase in hematopoietic cells. Our data demonstrate that IFNalpha induces the interaction of p85 with IRS-1 or IRS-2, but not Stat-3, in various hematopoietic cell lines in which IRS-1 and/or IRS-2 and Stat-3 are activated by IFNalpha. In addition, inhibition of PI 3'-kinase activity by preincubation of cells with the PI 3'-kinase inhibitor LY294002 does not affect IFN-dependent formation of SIF complexes that contain Stat-3. To determine whether phosphorylation of tyrosine residues in the IFN receptor is required for activation of the PI 3'-kinase, we performed studies using mouse L929 fibroblasts transfected with mutated human IFNAR1 and/or IFNAR2 subunits of the Type I IFN receptor, lacking tyrosine phosphorylation sites. The serine kinase activity of the PI-3K was activated by human IFNalpha in these cells, suggesting that phosphorylation of the Type I IFN receptor is not essential for PI3K activation. We then determined whether IFNalpha activates the Akt kinase, a known downstream target for PI 3'-kinase that mediates anti-apoptotic signals. Akt was activated by insulin or IGF-1, but not IFNalpha, in the IFNalpha-sensitive U-266 myeloma cell line. Altogether, our data establish that the IRS pathway and not the Stat pathway, is the major pathway regulating engagement of PI 3'-kinase in hematopoietic cells. Furthermore, the selective activation of Akt by insulin/IGF-1 suggests the existence of distinct regulatory activities of PI3'-kinase in growth factor versus interferon signaling.     

Dissociation of apoptosis from proliferation, protein kinase B activation, and BAD phosphorylation in interleukin-3-mediated phosphoinositide 3-kinase signaling

Interleukin-3 (IL-3) acts as both a growth and survival factor for many hemopoietic cells. IL-3 treatment of responsive cells leads to the rapid and transient activation of Class IA phosphoinositide-3-kinases (PI3Ks) and the serine/threonine kinase Akt/protein kinase B (PKB) and phosphorylation of BAD. Each of these molecules has been implicated in anti-apoptotic signaling in a wide range of cells. Using regulated expression of dominant-negative p85 (Deltap85) in stably transfected IL-3-dependent BaF/3 cells, we have specifically investigated the role of class IA PI3K in IL-3 signaling. The major functional consequence of Deltap85 expression in these cells is a highly reproducible, dramatic reduction in IL-3-induced proliferation. Expression of Deltap85 reduces IL-3-induced PKB phosphorylation and activation and phosphorylation of BAD dramatically, to levels seen in unstimulated cells. Despite these reductions, the levels of apoptosis observed in the same cells are very low and do not account for the reduction in IL-3-dependent proliferation we observe. These results show that Deltap85 inhibits both PKB activity and BAD phosphorylation without significantly affecting levels of apoptosis, suggesting that there are targets other than PKB and BAD that can transmit survival signals in these cells. Our data indicate that the prime target for PI3K action in IL-3 signaling is at the level of regulation of proliferation.     

1,25-dihydroxyvitamin D3 induces biphasic NF-kappaB responses during HL-60 leukemia cells differentiation through protein induction and PI3K/Akt-dependent phosphorylation/degradation of IkappaB

1,25-dihydroxyvitamin D(3) (VD(3)) induces differentiation in a number of leukemia cell lines and under various conditions is able to either stimulate or inhibit nuclear factor kappa B (NF-kappaB) activity. Here we report a time-dependent biphasic regulation of NF-kappaB in VD(3)-treated HL-60 leukemia cells. After VD(3) treatment there was an early approximately 4 h suppression and a late 8-72 h prolonged reactivation of NF-kappaB. The reactivation of NF-kappaB was concomitant with increased IKK activities, IKK-mediated IkappaBalpha phosphorylation, p65 phosphorylation at residues S276 and S536, p65 nuclear translocation and p65 recruitment to the NF-kappaB/vitamin D responsive element promoters. In parallel with NF-kappaB stimulation, there was an up-regulation of NF-kappaB controlled inflammatory and anti-apoptotic genes such as TNFalpha, IL-1beta and Bcl-xL. VD(3)-triggered reactivation of NF-kappaB was associated with PI3K/Akt phosphorylation. PI3K/Akt antagonists suppressed VD(3)-stimulated IkappaBalpha phosphorylation as well as NF-kappaB-controlled gene expression. The early approximately 4 h VD(3)-mediated NF-kappaB suppression coincided with a prolonged increase of IkappaBalpha protein which require de novo protein synthesis, lasted for as least 72 h and was insensitive to MAPK, IKK or PI3K/Akt inhibitors. Our data suggest a novel biphasic regulation of NF-kappaB in VD(3)-treated leukemia cells and our results may have provided the first molecular explanation for the contradictory observations reported on VD(3)-mediated immune-regulation.     

Transformation of mouse fibroblasts by Jaagsiekte sheep retrovirus envelope does not require phosphatidylinositol 3-kinase

Jaagsiekte sheep retrovirus (JSRV) is the causative agent of ovine pulmonary adenocarcinoma, a transmissible lung cancer of sheep. The envelope of JSRV may have oncogenic properties, since it can morphologically transform mouse NIH 3T3 cells and other fibroblast lines. Recently, we found that the cytoplasmic tail of the envelope transmembrane (TM) protein is necessary for transformation, and in particular a consensus binding motif (YXXM) for phosphatidylinositol 3-kinase (PI3K) is important. Moreover, JSRV-transformed cells show phosphorylation (activation) of Akt/protein kinase B, a downstream target of PI3K. In these studies, we directly tested for the involvement of PI3K in transformation by JSRV. Contrary to expectations, four different experiments indicated that PI3K is not necessary for JSRV-induced transformation: (i) cotransfection with a dominant negative truncated form of the PI3K regulatory subunit (Deltap85) did not affect transformation frequency, (ii) cells stably expressing Deltap85 showed the same frequencies of transformation as parental NIH 3T3 cells, (iii) fibroblasts established from double-knockout mice lacking PI3K p85alpha and p85beta could be transformed with JSRV envelope, and (iv) incubation of cells with the PI3K inhibitor LY294002 did not specifically inhibit transformation, nor did the drug reverse transformation of JSRV-transformed cells. One alternate explanation for the lack of transformation by YXXM mutants could be that they were defective in intracellular trafficking. However, confocal microscopy of epitope-tagged envelope proteins of both wild-type and nontransforming YXXM mutants showed a cell surface or plasma membrane localization. While PI3K is not required for JSRV-induced transformation of NIH 3T3 cells, the downstream target Akt kinase was found to be activated (phosphorylated) in JSRV-transformed PI3K-negative cells. Therefore, JSRV envelope can induce PI3K-independent phosphorylation of Akt.     

Membrane-targeted phosphatidylinositol 3-kinase mimics insulin actions and induces a state of cellular insulin resistance.

Phosphatidylinositol (PI) 3-kinase plays an important role in various insulin-stimulated biological responses including glucose transport, glycogen synthesis, and protein synthesis. However, the molecular link between PI 3-kinase and these biological responses is still unclear. We have investigated whether targeting of the catalytic p110 subunit of PI 3-kinase to cellular membranes is sufficient and necessary to induce PI 3-kinase dependent signaling responses, characteristic of insulin action. We overexpressed Myc-tagged, membrane-targeted p110 (p110(CAAX)), and wild-type p110 (p110(WT)) in 3T3-L1 adipocytes by adenovirus-mediated gene transfer. Overexpressed p110(CAAX) exhibited approximately 2-fold increase in basal kinase activity in p110 immunoprecipitates, that further increased to approximately 4-fold with insulin. Even at this submaximal PI 3-kinase activity, p110(CAAX) fully stimulated p70 S6 kinase, Akt, 2-deoxyglucose uptake, and Ras, whereas, p110(WT) had little or no effect on these downstream effects. Interestingly p110(CAAX) did not activate MAP kinase, despite its stimulation of p21(ras). Surprisingly, p110(CAAX) did not increase basal glycogen synthase activity, and inhibited insulin stimulated activity, indicative of cellular resistance to this action of insulin. p110(CAAX) also inhibited insulin stimulated, but not platelet-derived growth factor-stimulated mitogen-activated protein kinase phosphorylation, demonstrating that the p110(CAAX) induced inhibition of mitogen-activated protein kinase and insulin signaling is specific, and not due to some toxic or nonspecific effect on the cells. Moreover, p110(CAAX) stimulated IRS-1 Ser/Thr phosphorylation, and inhibited IRS-1 associated PI 3-kinase activity, without affecting insulin receptor tyrosine phosphorylation, suggesting that it may play an important role as a negative regulator for insulin signaling. In conclusion, our studies show that membrane-targeted PI 3-kinase can mimic a number of biologic effects normally induced by insulin. In addition, the persistent activation of PI 3-kinase induced by p110(CAAX) expression leads to desensitization of specific signaling pathways. Interestingly, the state of cellular insulin resistance is not global, in that some of insulin's actions are inhibited, whereas others are intact.