Published online before print April 27, 2005
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Department of Biochemistry, Biosciences Institute, University College Cork, Ireland
1 Correspondence: Department of Biochemistry, Biosciences Institute, University College Cork, Cork, Ireland. E-mail: t.cotter{at}ucc.ie
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Key Words: chronic myeloid leukemia • cellular signaling • gene expression
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Bcr-Abl reportedly activates many signaling pathways, including the phosphatidylinositol-3 kinase (PI-3K)/AKT pathway [13 ]. This PI-3K activity is required for the proliferation of CML cells. Other downstream effectors of Bcr-Abl include Ras [14 ] and Myc [15 ]. The Janus kinase/signal transducer and activator of transcription pathway (STAT) has also been shown to play a key role in human CML [16 ], and STATs are the only transcription factors known to be regulated by tyrosine phosphorylation. It is hoped that the identification of genes associated with Bcr-Abl expression will potentially lead to new molecular targets for therapy.
Osteopontin (OPN) is a secreted, acidic phosphoprotein, which binds to various cell-surface receptors including integrins, e.g., 
vß3 [17
] and CD44 [18
]. One well-documented function of OPN is as a cell adhesion molecule [19
, 20
]. Its expression has been shown to be increased by phorbol esters, growth factors, and hormones [21
]. It has also been demonstrated that OPN is involved in many disease states including atherosclerosis [22
], kidney disease [23
], and the growth and survival of metastatic cells [24
]. Other studies indicate increased levels of OPN in the blood of patients with metastatic disease [25
]. However, the function of OPN in tumorigenesis is unclear. In addition, the upstream signaling pathways regulating OPN expression in response to various stimuli are poorly characterized.
Here, we demonstrate that OPN expression is up-regulated transcriptionally in response to high levels of p210 Bcr-Abl. We also describe a systematic evaluation of the major signaling molecules required for this up-regulation of OPN. Analysis of the mitogen-activated protein kinase (MAPK) cascade reveals that this pathway is required for OPN expression in our model of CML. Additionally, we demonstrate involvement of atypical protein kinase C (aPKC) and PI-3K. We determine that activity of PI-3K and aPKC is required for optimal activation of Raf-1, even in the presence of active Ras. The requirement of aPKC activity represents a novel pathway in the up-regulation of OPN. As such, this may represent a potentially globally important pathway in the induction of OPN expression in other disease states. These data not only provide insights into the regulation of OPN but also identify key signaling cascades initiated by the oncogene Bcr-Abl.
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
pseudosubstrate inhibitor, and LY294002, purchased from Calbiochem (Merck Biosciences, Nottingham, UK).
Cell lysis and immunoblotting
Cells were lysed in radio immunoprecipitation assay (RIPA) buffer (50 mM Tris-HCl, pH 7.4, 1% Nonidet P-40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EGTA, 1 mM Na3VO4, 1 mM NaF, 1 µg/ml antipain, 1 µg/ml aprotinin, 1 µg/ml chymostatin, 0.1 µg/ml leupeptin, 1 µg/ml pepstatin, and 100 µM phenylmethylsulfonyl fluoride). The lysates were centrifuged at 20,000 g (4°C) for 15 min to remove insoluble debris. Equivalent amounts of protein, as determined by the Bio-Rad protein assay, were resolved using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membrane (Schleicher and Schuell, Dassel, Germany). All secondary antibodies were peroxidase-conjugated, and proteins were detected using enhanced chemiluminescence (Amersham Biosciences). Antibodies included anti-c-Abl (AB-3; Calbiochem); antiphosphotyrosine (PY20; Transduction Laboratories, San Diego, CA); anti-OPN (R&D Systems, Abingdon, UK); anti-Ras (clone RAS10), anti-MAPK kinase (MEK), and anti-Raf-1 (Upstate Biotechnology Ltd., Milton Keynes, UK); anti-nPKC, also detects PKC
/
(Santa Cruz Biotechnology, Heidelberg, Germany); antiphospho-AKT (ser 473) and anti-AKT (Cell Signaling Technology); and anti ß-actin (Sigma-Aldrich).
Measurement of apoptosis [phosphatidyl serine (PS) exposure]
The exposure of PS on the extracellular surface of the plasma membrane was monitored by the binding of annexin V-fluorescein isothiocyanate (FITC), according to the manufacturer’s instructions (IQ Products, Labron Ltd., Dublin, Ireland). Briefly, 5 x 105/ml cells were resuspended in calcium-binding buffer (10 mM Hepes, 2.5 mM CaCl2, 140 mM NaCl) and incubated with annexin V-FITC for 5 min at room temperature in the dark. Cells were incubated with 50 µg/ml propidium iodide (PI) at room temperature before analysis. Fluorescence resulting from FITC and PI was measured at 530 nm (FL1) and 590 nm (FL2), respectively, and analyzed using CellQuest software on a FACScan flow cytometer (Becton Dickinson, Oxford, UK) using an excitation of 488 nm.
Reverse transcriptase-polymerase chain reaction (RT-PCR)
Total cellular RNA was prepared using Tri Reagent (Molecular Research Centre Inc, Dublin, Ireland). Single-stranded cDNA was synthesized according to the Moloney murine leukemia virus (M-MLV) RT protocol (Promega, Southampton, UK). Oligo dT, MgCl2, and RNAsin were also purchased from Promega. Deoxy-nucleoside 5'-triphosphates (dNTPs) were obtained from Sigma-Aldrich. cDNA was amplified using Taq DNA polymerase (Promega), according to the manufacturer’s instructions. PCR reactions were carried out as follows: 95°C for 5 min, 22 cycles of –95°C for 1 min, Tanneal for 1 min, 72°C for 1 min, followed by 72°C for 5 min. Primers included OPN (forward 5'-TTCACAGCCACAAGGACAAG-3', reverse 5'-TGCAGAAGCTTTTGGTTACAA-3', Tanneal, 55°C) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; forward 5'-ACCACAGTCCATGCCATCAC-3', reverse 5'-TCCACCACCCAGTTGCTGTA-3', Tanneal, 58°C). To control for DNA contamination of RNA samples, PCR was also carried out in the absence of RT (see Fig. 1a
). In this case, M-MLV was omitted from the RT reaction. Control reactions were performed initially to ensure linearity of amplification over the number of cycles used.
 View larger version (35K): [in a new window] |
Figure 1. Expression of p210 Bcr-Abl in 32D cells. (a) Western blot analysis of Bcr-Abl expression in whole cell lysates from 32D parental cells and Bcr-Abl-transfected C2, C4, and C5 cells. Actin antibody-binding demonstrates equal protein loading. (b) Western blot analysis of phosphotyrosine levels in total cell lysates from 32D, untreated C4 cells (Unt), and C4 cells treated with 1 µM STI571 (STI) for 48 h. (c) Untreated 32D and C4 cells and C4 cells treated for 48 h with 1 µM STI571 were assessed for apoptotic cell death by annexin V/PI staining. Percentages in the bottom-right quadrants indicate annexin V-positive, PI-negative cells, and percentages in the top-right quadrants indicate annexin V-positive, PI-positive cells. 32D cells treated for 48 h with 5 µg/ml VP16 are included as a positive control for high levels of apoptotic cell death. Data are representative of three independent experiments.
|
Kinase assays
Ras activity was measured using the Ras activation assay kit from Upstate Biotechnology Ltd. and following the manufacturer’s instructions. Guanosine 5'-triphosphate (GTP)-bound Ras from cell lysates was "pulled down" using the glutathione S-transferase fusion protein corresponding to the Ras-binding domain (RBD) of Raf-1, bound to agarose. The presence of active Ras was detected by Western blotting using anti-Ras antibody. Raf-1 kinase assays were performed using the Raf-1 kinase cascade assay kit from Upstate Biotechnology Ltd., according to the manufacturer’s instructions. Raf-1 was immunoprecipitated from cell lysates, and kinase activity was measured using MEK, extracellular signal-regulated kinase (ERK), and myelin basic protein (MBP) as sequential substrates. MEK kinase assays were performed essentially as Raf-1 kinase assays with some modifications. MEK was immunoprecipitated from cell lysates, and kinase activity was measured using ERK and MBP as sequential substrates. aPKC activity was assessed according to Takeda et al. [26
]. Briefly, aPKC was immunoprecipitated from whole cell lysates, and kinase activity was assessed using MBP as a substrate. Assay products for Raf-1, MEK, and aPKC assays were assessed using P81 phosphocellulose paper (Upstate Biotechnology Ltd.) and counting on a Beckman Coulter LS 6500 scintillation counter.
Measurement of cell death
Cell death was assessed by PI uptake on a FACScan (Becton Dickinson) flow cytometer at 590 nm (FL2). Cells were incubated with 5 µg/ml PI at room temperature prior to analysis. The criteria for cell death, as measured by flow cytometry, were based on changes in light-scattering properties of dead cells as a result of cell shrinkage and increased granularity and also, permeability to PI.
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
 View larger version (9K): [in a new window] |
Figure 2. Bcr-Abl expression leads to increased levels of OPN mRNA and protein. (a) RT-PCR experiments, using cDNA from 32D, untreated C4 cells, and C4 cells treated for 48 h with 1 µM STI571 (STI), show expression levels of OPN in the presence (+RT) and absence (–RT) of RT. Levels of GAPDH-PCR product demonstrate equal quantities of cDNA. (b) Western blot analysis of OPN expression in total cell lysates from 32D, C2, C4, and C5 cells. Actin antibody-binding demonstrates equal protein loading. (c) Western blot analysis of OPN expression in cell lysates from untreated C4 cells (Unt) and C4 cells treated for 48 h with 1 µM STI571. (d) Western blot analysis of OPN expression in barium citrate precipitates from culture media of 32D, untreated C4 cells, and C4 cells treated for 48 h with 1 µM STI571.
|
OPN is reportedly a secreted protein. Barium citrate precipitation was used to precipitate OPN from CM obtained from 32D and C4 cells and C4 cells treated with STI571. High levels of OPN could be precipitated from the CM of untreated C4 cells alone with undetectable levels in the media from 32D- and STI571-treated C4 cells (Fig. 2d) .
The up-regulation of OPN is dependent on Ras activity
The ability of high levels of Bcr-Abl to up-regulate the expression of OPN represents a novel and potentially important downstream affect of this oncogene. As such, we aimed to determine the pathways and signaling molecules required for this induction. A potential role for Ras was first examined, as OPN expression in other models has previously been shown to be dependent on Ras [30
], and also, as Ras is well-documented as a target of Bcr-Abl [14
, 31
, 32
]. Western blot analysis revealed that C4 cells express higher levels of Ras than do 32D cells and that this increase in expression is Bcr-Abl tyrosine kinase-dependent, as it can be reversed by treatment with STI571. Treatment of C4 cells with 10 µM FTS for 48 h does not significantly reduce Ras expression (Fig. 3a
). C4 cells also display a higher level of Ras activity than 32D cells, as determined by a Ras activity assay (Fig. 3b)
. It is shown that this increase in Ras activity seen in C4 cells can be reversed by STI571 treatment and also by treatment with 10 µM FTS (Fig. 3b)
. As shown in Figure 3a
and b, levels of Ras protein and activity are increased in C4 cells in a Bcr-Abl-dependent manner. These results indicate that Bcr-Abl up-regulates this pathway at two levels—first, by increasing expression of Ras and then, by leading to its increased activation. Treatment with FTS blocked the induction of OPN expression in response to Bcr-Abl (Fig. 3c)
, indicating that Ras activity is essential for its up-regulation.
 View larger version (10K): [in a new window] |
Figure 3. The up-regulation of OPN is dependent on Ras activity. (a) Western blot analysis of Ras expression in total cell lysates from 32D, untreated C4 cells (Unt), and C4 cells treated for 48 h with 1 µM STI571 (STI) or 10 µM FTS. Actin antibody-binding demonstrates equal protein loading. (b) Cell lysates from (a) were incubated with Raf-1 RBD agarose-conjugated beads. Bound proteins were denatured by boiling in reducing sample buffer and then resolved by SDS-PAGE, followed by Western blotting to detect Ras-GTP using a monoclonal antibody to Ras. (c) Samples from (a) were harvested and separated by electrophoresis on a 10% polyacrylamide gel. The resulting membrane was immunoblotted using antibodies to OPN.
|
 View larger version (11K): [in a new window] |
Figure 4. The up-regulation of OPN is dependent on MEK activity. (a) Western blot analysis of MEK expression in total cell lysates from 32D, untreated C4 cells (Unt), and C4 cells treated for 48 h with 1 µM STI571 (STI) or for 24 h with 5 µM UO126 (ERK1/2-specific MEK1/2 inhibitor). Actin antibody-binding demonstrates equal protein loading. (b) Cell lysates from (a) were immunoprecipitated with MEK antibodies, and kinase activity was determined using an in vitro kinase assay in which recombinant ERK and MBP were used. MEK activity was determined quantitatively by scintillation counting of the P81 phosphocellulose squares spotted with the assay product. Background counts were subtracted from each sample, and the resultant net counts were expressed as a percentage of the values obtained for C4 cells. Data are presented as the mean and standard deviation of the mean for independent experiments (n=4). Statistical analysis was performed using an unpaired Student’s t-test (***, P<0.005, compared with levels of activity in C4 cells). (c) Western blot analysis of OPN expression in total cell lysates from samples used in (a).
|
 View larger version (11K): [in a new window] |
Figure 5. The up-regulation of OPN is dependent on Raf-1 activity. (a) Western blot analysis of Raf-1 expression in total cell lysates from 32D, untreated C4 cells (Unt), and C4 cells treated for 48 h with 1 µM STI571 (STI) or for 24 h with 400 µM 8-bromo-cAMP (Raf-1 inhibitor). Actin antibody-binding demonstrates equal protein loading. (b) Cell lysates from (a) were immunoprecipitated with Raf-1 antibodies, and kinase activity was determined using an in vitro kinase assay. Raf activity was determined by scintillation counting. Background counts were subtracted from each sample, and the resultant net counts were expressed as a percentage of C4 cells. Data are presented as the mean and standard deviation of the mean for independent experiments (n=4). Statistical analysis was performed using an unpaired Student’s t-test (**, P<0.01; ***, P<0.005, compared with levels of activity in C4 cells). (c) Western blot analysis of OPN expression in total cell lysates from samples used in (a).
|
 View larger version (9K): [in a new window] |
Figure 6. Increasing Raf-1 activity in C4 cells leads to a further increase in OPN expression. (a) Lysates from untreated 32D and C4 cells or cells treated for 24 h with 5-Iodo were subjected to immunoprecipitation with Raf-1 antibodies, and kinase activity was determined using an in vitro kinase assay. Data are presented as the mean and standard deviation of the mean for independent experiments (n=4). Statistical analysis was performed using an unpaired Student’s t-test (***, P<0.005, compared with levels of activity in untreated cells). (b) Lysates from (a) were immunoprecipitated with MEK antibodies, and kinase activity was determined using an in vitro kinase assay. Data are presented as the mean and standard deviation of the mean for independent experiments (n=4). Statistical analysis was performed using an unpaired Student’s t-test (***, P<0.005, compared with levels of activity in untreated cells). (c) Western blot analysis of OPN levels in lysates from untreated C4 cells and C4 cells treated with the indicated concentration of 5-Iodo for 24 h. Actin antibody-binding demonstrates equal protein loading.
|
The up-regulation of OPN is dependent on aPKC, which lies upstream of Raf-1 and MEK
PKC has been shown previously to be involved in the up-regulation of OPN [37
]. PKC represents a family of at least 11 members that can be divided into three distinct classes: classical PKC (cPKC), nPKC, and aPKC. cPKC and nPKC members are activated by diacylglycerol (DAG) and phorbol esters in vitro and in vivo [38
, 39
]. In contrast, aPKC members require neither DAG nor phorbol esters for activation [40
, 41
]. There are currently two members of the aPKC subfamily, and . The murine ortholog of PKC is termed PKC. In particular, the aPKC isoform has been shown to activate Raf-1 directly in the absence of Ras in Rat-1 fibroblasts [42
]. We investigated the possibility that a signaling pathway involving aPKC is activated in C4 cells. Western blot analysis showed that C4 cells express higher levels of aPKC than 32D cells, and STI571 treatment confirmed that this up-regulation was dependent on Bcr-Abl tyrosine kinase activity. Treatment with the myristoylated PKC pseudosubstrate peptide (10 µM for 24 h) did not lead to any significant alteration in the expression levels of aPKC in C4 cells (Fig. 7a
). We also found that the basal level of aPKC activity in C4 cells is approximately fourfold greater than that of 32D cells (Fig. 7b)
. Following treatment with STI571, a large decrease in aPKC activity was seen, indicating that the kinase activity of Bcr-Abl is necessary for its activation. Treatment with the myristoylated PKC pseudosubstrate peptide (10 µM for 24 h) also inhibited aPKC activity in C4 cells (Fig. 7b)
. This peptide has been shown to inhibit PKC activity by interacting with the substrate-binding pocket in the catalytic domain [43
, 44
]. However, as the sequence of this peptide from PKC is identical to the corresponding sequence in PKC/ [45
], it may be assumed that this PKC pseudosubstrate peptide acts as a general aPKC inhibitor. Once again, it is shown that Bcr-Abl up-regulates aPKC at the level of expression and activation, suggesting that this may be a common feature of Bcr-Abl signaling. Western analysis of whole cell lysates revealed that inhibition of aPKC activity in C4 cells led to a down-regulation in OPN expression (Fig. 7c)
, confirming that its activity is required for the induction of OPN in these cells.
 View larger version (17K): [in a new window] |
Figure 7. The up-regulation of OPN is dependent on aPKC, which lies upstream of Raf-1 and MEK. (a) Western blot analysis of aPKC expression in total cell lysates from 32D, untreated C4 cells (Unt), and C4 cells treated for 48 h with 1 µM STI571 (STI) or for 24 h with 10 µM PKC pseudosubstrate inhibitor. Actin antibody-binding demonstrates equal protein loading. (b) Cell lysates from (a) were immunoprecipitated with aPKC antibodies, and kinase activity was determined using an in vitro kinase assay. aPKC activity was determined by scintillation counting. Background counts were subtracted from each sample, and the resultant net counts were expressed as a percentage of the values obtained for C4 cells. Data are presented as the mean and standard deviation of the mean for independent experiments (n=4). Statistical analysis was performed using an unpaired Student’s t-test (**, P<0.01; ***, P<0.005, compared with levels of activity in C4 cells). (c) Western blot analysis of OPN expression in total cell lysates from samples used in (a). (d) Untreated 32D and C4 cells and cells treated with 10 µM PKC pseudosubstrate inhibitor for 24 h were subjected to immunoprecipitation with Raf-1 antibodies, and kinase activity was determined using an in vitro kinase assay. Data are presented as the mean and standard deviation of the mean for independent experiments (n=4). Statistical analysis was performed using an unpaired Student’s t-test (**, P<0.01; ***, P<0.005, compared with levels of activity in untreated cells). (e) Lysates from (d) were immunoprecipitated with MEK antibodies, and kinase activity was determined using an in vitro kinase assay. Data are presented as the mean and standard deviation of the mean for independent experiments (n=4). Statistical analysis was performed using an unpaired Student’s t-test (*, P<0.05; ***, P<0.005, compared with levels of activity in untreated cells).
|
pseudosubstrate inhibitor also led to a reduction in Raf-1 activity to undetectable levels (Fig. 7d)
and to a downstream reduction in MEK activity levels (Fig. 7e)
. This indicates that the pathway from aPKC to Raf-1 and MEK is active in the parental cell line. We have also shown that 32D cells have significantly lower levels of aPKC activity than do C4 cells, and this may explain our inability to activate Raf-1 in 32D cells using a compound 5-Iodo, which led to a strong activation in C4 cells. This potentially highlights the importance of increased expression as well as activation of signaling molecules downstream of Bcr-Abl.
Ras lies upstream of aPKC activation
Our results thus far demonstrate that in response to Bcr-Abl tyrosine kinase activity, expression of the OPN gene is activated. We have shown that the signaling pathways involved require Ras, PKC (specifically aPKC), MEK, and Raf-1 activities and that aPKC lies upstream of Raf-1 activation, which leads to downstream activation of MEK. This led us to question where Ras lies in this signaling pathway. We considered two possibilities: First, Ras and aPKC are required for activation of Raf-1, and second, Ras is not, in fact, required for the activation of Raf directly but lies upstream and is responsible for the activation of aPKC. To investigate the latter possibility, untreated C4 cells and C4 cells treated with 10 µM FTS for 48 h were analyzed for aPKC activity. We found that inhibition of Ras activity led to a significant reduction in aPKC activity (Fig. 8
). This indicates that in response to Bcr-Abl expression, Ras is activated, leading to a downstream activation of the aPKC, Raf-1, and MEK. This does not rule out the possibility that signals from Ras directly and aPKC are required for Raf-1 activation. We also found that treatment of 32D cells with 10 µM FTS led to a significant reduction in aPKC activity (Fig. 8)
. This indicates that the same pathway exists in the parental cell line.
 View larger version (9K): [in a new window] |
Figure 8. Ras lies upstream of aPKC activation. Cell lysates from untreated 32D and C4 cells and cells treated for 48 h with 10 µM FTS were immunoprecipitated with aPKC antibodies, and kinase activity was determined using an in vitro kinase assay. Data are presented as the mean and standard deviation of the mean for independent experiments (n=4). Statistical analysis was performed using an unpaired Student’s t-test (*, P<0.05; ***, P<0.005, compared with levels of activity in untreated cells).
|
depends on phosphatidylinositol phosphate 3 (PIP3), produced mainly by PI-3K [46
]. It has also been extensively reported that PI-3K activity is increased in response to Bcr-Abl tyrosine kinase activity [13
, 31
, 47
]. To confirm that this is the case, in our system, we performed Western blot analysis to study levels of AKT phosphorylation (ser 473) in 32D and C4 cells (Fig. 9a
). We show that this increase in p-Akt is Bcr-Abl-dependent by treatment with STI571 and that it is PI-3K-dependent by treatment for 48 h with the PI-3K inhibitor LY294002 (Fig. 9a) .
 View larger version (19K): [in a new window] |
Figure 9. The up-regulation of OPN is dependent on PI-3K, which lies upstream of aPKC. Western blot analysis of phosphor-AKT (p-Akt; ser 473) levels in total cell lysates from 32D, untreated C4 cells (Unt), and C4 cells treated for 48 h with 1 µM STI571 (STI) or 20 µM LY294002 (LY). AKT antibody-binding demonstrates equal protein loading. (b) Untreated 32D and C4 cells and cells treated with 20 µM LY294002 for 48 h were immunoprecipitated with aPKC antibodies, and kinase activity was determined using an in vitro kinase assay. Data are presented as the mean and standard deviation of the mean for independent experiments (n=4). Statistical analysis was performed using an unpaired Student’s t-test (**, P<0.01; ***, P<0.005, compared with levels of activity in untreated cells). (c) Western blot analysis of OPN levels in whole cell lysates from samples used in (a) above. Actin antibody-binding demonstrates equal protein loading. (d) Cell lysates from (a) were incubated with Raf-1 RBD agarose-conjugated beads. Bound proteins were denatured by boiling in reducing sample buffer and then resolved by SDS-PAGE, followed by Western blotting to detect Ras-GTP using a monoclonal antibody to Ras.
|
These results indicate that the pathway involved in OPN expression response to Bcr-Abl involves activation of Ras by Bcr-Abl and subsequent downstream activation of PI-3K, aPKC, Raf-1, and MEK (Fig. 10 ).
 View larger version (28K): [in a new window] |
Figure 10. Proposed mechanism of up-regulation of OPN by Bcr-Abl. This figure illustrates the pathway by which OPN expression is induced in a model of Bcr-Abl expression. Bcr-Abl activates Ras, which leads to the activation of a signaling cascade involving PI-3K, aPKC, Raf-1, and MEK. This ultimately leads to the transcriptional activation of the OPN gene and subsequent expression and secretion of the OPN protein.
|
pseudosubstrate inhibitor results in increased levels of death in response to VP16 treatment. All of these treatments also lead to the reduction of OPN expression in C4 cells.
 View larger version (8K): [in a new window] |
Figure 11. OPN expression correlates with drug resistance. 32D, C2, C4, and C5 cells and C4 cells pretreated for 48 h with 1 µM STI571 (STI) or 10 µM FTS or for 24 h with 5 µM UO126 or 10 µM PKC pseudosubstrate inhibitor prior to treatment for 24 h with 5 µg/ml VP16. Levels of cell death were assessed by PI staining. Data are presented as the mean and standard deviation of the mean for independent experiments (n=4). Statistical analysis was performed using an unpaired Student’s t-test (*, P<0.05; ***, P<0.005, compared with levels of cell death in C4 cells).
|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
The work reported here used a model system of CML, whereby normal 32D cells were transfected with p210 Bcr-Abl. This model allows us to compare directly the effects of different levels of Bcr-Abl expression between the cell lines. Although this model is subject to the limitations of any cell line model, in all experiments, we have used the Bcr-Abl-specific inhibitor STI571 to confirm that all differences seen are in fact a result of Bcr-Abl tyrosine kinase activity. The data presented here demonstrate for the first time that OPN expression is induced (at the level of mRNA and secreted protein) in a leukemic cell line expressing high levels of p210 Bcr-Abl. Our inability to detect OPN in a cell line expressing lower levels of Bcr-Abl suggests that its expression is correlated with disease progression, as has been shown to be the case for many solid tumors. Results from this lab have previously demonstrated that although low levels of Bcr-Abl are sufficient to confer growth-factor independence on 32D cells, higher levels of Bcr-Abl expression are required for drug resistance [12 ]. As such, the C2 cells have a phenotype intermediate between that of 32D and C4 cells, being growth factor-independent but still susceptible to drug-induced apoptosis. The results presented here suggest that levels of Bcr-Abl, sufficient to induce drug resistance, are required for the induction of OPN expression. The potential importance of OPN up-regulation in CML prompted us to begin to define the signaling pathways by which the oncogene Bcr-Abl regulates OPN expression. Our results establish that high levels of Bcr-Abl, typically seen in CML patients in the blast-crisis stage of disease, result in the up-regulation of signaling pathways already existing in the cell with the ultimate induction of OPN expression. We show that the pathway involved requires the increased activity of Ras, PI-3K, aPKC, Raf-1, and MEK.
It has been shown that increased levels of OPN mRNA can be a result of increased mRNA stability [52 ]. This is unlikely to be the case in C4 cells, as the parental cell line 32D does not express detectable levels of OPN mRNA. It has also been demonstrated that the up-regulation of OPN in some cases is dependent on Ras [53 ]. It has been shown that this induction may be direct, via a Ras-activated enhancer, in the OPN promoter [54 ]. Here, we report that in response to Bcr-Abl expression, Ras activity is required for the induction of OPN expression. It is unlikely that this induction is direct in our model, as we have also shown that multiple signaling molecules are involved downstream of Ras.
The MAPK signaling cascade has also been implicated in OPN regulation in various systems including induction of OPN by inorganic phosphate in osteoblasts [33 ] and induction by 12-O-tetradecanoylphorbol 13-acetate (TPA) in HL-60 cells [55 ] and also in JB6 cells [56 ], in response to injury of rat arterial muscle cells [57 ] and to angiotensin II in cardiac cells [58 ]. The fact that the MAPK cascade has been implicated in the induction of OPN in response to so many varying stimuli may suggest that activation of the MAPK cascade is a crucial component in the regulation of OPN expression, regardless of the stimulus. These data support our findings that the expression of OPN in response to Bcr-Abl is dependent on MEK activity. As we have shown that Ras activity is required for OPN expression, we investigated the possibility that Raf-1 activity is also required, as Raf-1 is widely accepted to lead to MEK phosphorylation and activation in response to its own activation by Ras. The requirement for Raf-1 activity in our system represents a novel finding, as Raf-1 has not been implicated previously in the up-regulation of OPN.
The results presented here also identify the requirement for an atypical member of the PKC family of serine/threonine kinases in the up-regulation of OPN. To date, 11 PKC isozymes have been identified. They are classified into three groups based on their structure and mode of regulation. The conventional cPKCs are activated by calcium and DAG; the nPKCs can be activated by DAG alone (i.e., they do not require calcium); and the activators for the aPKCs remain unknown. There are currently two members of the aPKC subfamily, and . The murine ortholog of PKC is termed PKC. Members of the PKC family have been reported to be involved in OPN regulation, including induction by TPA in HL-60 [55
] and JB6 cells [56
]. These studies suggest that nPKC isoforms 
and 
are required for OPN expression. Beck and Knecht [33
] demonstrated that a PKC isoform other than cPKCs is required for induction of OPN by inorganic phosphate in osteoblasts. We used a PKC pseudosubstrate inhibitor to show that in response to Bcr-Abl, aPKC is required for OPN expression. This represents the first demonstrated link between aPKC and OPN. Further investigation is required to determine which of the aPKC isoforms is specifically required. A study by Murray and Fields [59
] has shown that K562 cells (a cell line derived from a patient in blast-crisis CML) express PKC but undetectable levels of PKC. This group has also shown that PKC activation in K562 cells requires Bcr-Abl activity [60
] and furthermore, that Bcr-Abl regulates PKC transcription [61
]. All of these data support our finding that expression and activity of aPKC are increased in C4 cells in a Bcr-Abl-dependent manner. This may suggest that the aPKC/ isoform is most likely to be involved in Bcr-Abl oncogenesis and indeed, in the up-regulation of OPN, although further investigation will be required to confirm this.
We have shown that Raf-1 activation in 32D and C4 cells occurs via aPKC. We have not ruled out the possibility that other signaling pathways also result in the activation of Raf-1 in these cells; however, we have shown that aPKC activation of Raf-1 is essential for the induction of OPN. Mas et al. [62
] have demonstrated previously the activation of Raf-1 by PKC. They demonstrated that in a human lymphocytic cell line (U937), the anthracycline danorubicin leads to increased Raf-1 and MEK activities via a PKC-mediated pathway. To date, however, there have been no reports of Raf-1 activation by PKC/. Mas et al. [62
] showed that the activation of PKC was sensitive to the PI-3K inhibitor Wortmannin. The activation of PKC/ by PI-3K has also been demonstrated [63
]. We have shown that in downstream signaling of Bcr-Abl, PI-3K is in fact responsible for the activation of aPKC. Our results also place Ras upstream of aPKC activation. Both aPKC isoforms have been reported previously to act as Ras effectors [64
65
66
]. Inhibition of PI-3K had no affect on Ras activation, suggesting that Ras lies upstream of PI-3K in this pathway.
PKC and the MAPK pathway have been implicated in the up-regulation of OPN in a number of different cell lines and in response to various stimuli. This may suggest that these pathways are a common requirement for induction of OPN expression, although the particular PKC isozyme involved appears to be cell type-/stimulus-specific. There is also some discrepancy as to whether these signaling molecules form a single pathway or two individual pathways, and both are required for OPN expression. Our studies suggest that in a Bcr-Abl model, a single pathway is involved, with aPKC lying upstream of Raf-1. Similarly, Chang et al. [56
] demonstrated a single pathway with PKC lying upstream of MEK, although in this case, a nPKC isozyme ( and/or ) was involved. Atkins et al. [55
] demonstrated a similar single pathway. Beck and Knecht [33
], however, have shown that PKC and MAPK form two distinct pathways in their model of OPN expression. This difference may again be dependent on the cell type or the stimulus used by the different groups.
The function of OPN, in tumor and in normal cells, remains poorly understood. The fact that OPN expression could only be detected in response to high levels of Bcr-Abl suggests that its role may be in disease progression or drug resistance. Our results demonstrate that levels of OPN correlate with the drug resistance of cells, suggesting that this may be its function in Bcr-Abl-mediated oncogenesis. We find that treatment of C4 cells with STI571 completely abolishes the drug resistance of these cells, indicating the crucial nature of Bcr-Abl tyrosine kinase activity. Treatment with Ras, MEK, and aPKC inhibitors reduces OPN expression in C4 cells and also sensitizes them to drug-induced apoptosis. However, levels of death seen in response to pretreatment with FTS and UO126 are not as high as those seen in 32D and C2 cells, suggesting that other pathways may also be involved in drug resistance. Our results indicate that these pathways lie downstream of aPKC, as pretreatment with an inhibitor of aPKC blocks all resistance to VP16 treatment in C4 cells.
In summary, the data presented here establish for the first time that OPN expression is induced in a model of leukemia, specifically by high levels of p210 Bcr-Abl. We have used this model to determine the specific signaling pathways involved in this up-regulation. The signaling pathway appears to be initiated by Ras downstream of Bcr-Abl, leading to the subsequent activation of PI-3K, aPKC, Raf-1, and MEK. These findings should prove valuable in the understanding of signaling downstream of Bcr-Abl and also in the up-regulation of OPN. To date, it has been shown that OPN is up-regulated as tumors become more aggressive and metastasize. The finding that OPN is also induced in a model of late-stage, aggressive leukemia may suggest a role for OPN in oncogenesis in general.
Received November 14, 2004; revised February 24, 2005; accepted March 22, 2005.
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
v ß 1 and v ß 5 J. Biol. Chem. 270,26232-26238
-1) Science 271,509-512[Abstract]
and protein kinase C- mediate RAS-independent activation of MAP kinase by a Gi protein-coupled receptor EMBO J. 18,386-395[CrossRef][Medline]
B mediates vß3 integrin-induced endothelial cell survival J. Cell Biol. 141,1083-1093 subspecies from rat brain: its structure, expression, and properties Proc. Natl. Acad. Sci. USA 86,3099-3103, expressed dominantly in an undifferentiated mouse embryonal carcinoma cell line and also in many tissues and cells J. Biol. Chem. 269,12677-12683 and Raf-1 Biochem. J. 325,303-307[Medline] substrate. Connection between cell surface signaling and nucleus in PC12 cells J. Biol. Chem. 272,31130-31137, an atypical isoform of protein kinase C derived from insulin-secreting cells J. Biol. Chem. 268,24296-24302 (PKC): activation mechanisms and cellular functions J. Biochem. (Tokyo) 133,1-7 protects human leukemia cells against drug-induced apoptosis J. Biol. Chem. 272,27521-27524 activity is necessary for Bcr-Abl-mediated resistance to drug-induced apoptosis J. Biol. Chem. 274,3927-3930 (PKC) transcription via an Elk1 site in the PKC promoter J. Biol. Chem. 279,9400-9408 mediated Raf-1/extracellular-regulated kinase activation by daunorubicin Blood 101,1543-1550 through phosphatidylinositol 3-kinase EMBO J. 15,788-798[Medline] is required for Ras transformation and colon carcinogenesis in vivo J. Cell Biol. 164,797-802 in Ras-mediated transcriptional activation of vascular permeability factor/vascular endothelial growth factor expression J. Biol. Chem. 276,2395-2403This article has been cited by other articles:
 |
|
 |
|
  C. H. Jamieson Chronic Myeloid Leukemia Stem Cells Hematology, January 1, 2008; 2008(1): 436 - 442. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Juric, N. J. Lacayo, M. C. Ramsey, J. Racevskis, P. H. Wiernik, J. M. Rowe, A. H. Goldstone, P. J. O'Dwyer, E. Paietta, and B. I. Sikic Differential Gene Expression Patterns and Interaction Networks in BCR-ABL-Positive and -Negative Adult Acute Lymphoblastic Leukemias J. Clin. Oncol., April 10, 2007; 25(11): 1341 - 1349. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 V. Schapira, G. Lazer, and S. Katzav Osteopontin Is an Oncogenic Vav1- but not Wild-type Vav1-Responsive Gene: Implications for Fibroblast Transformation. Cancer Res., June 15, 2006; 66(12): 6183 - 6191. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
