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J. Biol. Chem., Vol. 281, Issue 26, 17670-17680, June 30, 2006
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-Peptide by Metal-dependent Up-regulation of Metalloprotease Activity*
¶1¶||**2||**3¶
From the
Departments of Pathology and ||Biochemistry and the ¶Centre for Neuroscience, University of Melbourne, Victoria 3010, Australia and the Mental Health Research Institute and the **Bio21 Molecular Biology and Biotechnology Institute, Parkville, Victoria 3052, Australia
Received for publication, March 16, 2006 , and in revised form, April 28, 2006.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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-peptide (A) metabolism in cell culture. Treatment of Chinese hamster ovary cells overexpressing amyloid precursor protein with CQ and Cu2+ or Zn2+ resulted in an 
85–90% reduction of secreted A-(1–40) and A-(1–42) compared with untreated controls. Analogous effects were seen in amyloid precursor protein-overexpressing neuroblastoma cells. The secreted A was rapidly degraded through up-regulation of matrix metalloprotease (MMP)-2 and MMP-3 after addition of CQ and Cu2+. MMP activity was increased through activation of phosphoinositol 3-kinase and JNK. CQ and Cu2+ also promoted phosphorylation of glycogen synthase kinase-3, and this potentiated activation of JNK and loss of A-(1–40). Our findings identify an alternative mechanism of action for CQ in the reduction of A deposition in the brains of CQ-treated animals and potentially in Alzheimer disease patients. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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-peptide (A), which is cleaved from the membrane-bound amyloid precursor protein (APP) (1). Aggregated or oligomeric A can induce neurotoxicity through pathways involving free radical production and increased neuronal oxidative stress (2). Among the factors capable of promoting A aggregation in vivo, recent evidence supports a central role for biometals such as Cu2+ and Zn2+ in this process (3).
An important factor in controlling A accumulation in AD patients is the activity of A-degrading enzymes. Recent studies have identified several candidate proteases that may contribute to catabolism of A in the brain. Neprilysin, insulin-degrading enzyme, angiotensin-converting enzyme, and matrix metalloproteases (MMPs) have all demonstrated A-degrading activity in vitro and/or in vivo (4–6). Reduced activity of these or other A-degrading proteases with age may play a role in promoting accumulation and deposition of A in AD patients. Development of strategies to enhance clearance of A may lead to novel therapeutic treatments for AD patients.
Promoting A clearance may be achieved through modulating metal sequestration or metal-protein interactions. 5-Chloro-7-iodo-8-hydroxyquinoline or clioquinol (CQ), a disused antibiotic, has received considerable attention as a potential metal ligand in AD and Parkinson disease patients (7–9). Preliminary studies revealed that CQ rapidly and potently dissolved aggregates of synthetic or AD brain-derived A in vitro (10). In subsequent animal studies, a 9-week oral treatment with CQ resulted in a 49% reduction of A levels and significantly increased Cu2+ and Zn2+ levels in brains of Tg2576 mice (10). Small clinical trials of CQ have demonstrated a significant slowing of cognitive decline together with a lowering of plasma A-(1–42) levels in a subset of AD patients compared with matched placebo controls (8).
The mechanism of action by CQ was suggested to be via metal sequestration, resulting in A dissolution. However, CQ could also act by alternative pathways involving modulation of cellular biometal metabolism, APP expression, or A processing (11). To investigate this, Chinese hamster ovary (CHO) cells overexpressing APP were treated with CQ in the presence or absence of physiological levels of biometals. When CQ was added to cells in the presence of Cu2+ or Zn2+, the secreted levels of A-(1–40) and A-(1–42) were dramatically reduced. Analogous effects were seen in N2a neuroblastoma cells. Subsequent investigation revealed that this effect was associated with uptake of Cu2+ and Zn2+ and loss of A through increased MMP-mediated degradation. These findings identify a novel mechanism for the therapeutic efficacy of CQ in which CQ·Cu2+ or CQ·Zn2+ complexes promote A degradation.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Generation of APP-transfected CHO and N2a Neuroblastoma Cells—APP-CHO and APP-N2a neuroblastoma cells were generated by expressing the 695-amino acid APP cDNA in the pIRESpuro2 expression vector (Clontech). Cells were transfected using Lipofectamine 2000 and cultured in RPMI 1640 medium supplemented with 1 mM glutamine and 10% fetal bovine serum (all from Invitrogen, Mount Waverley, Victoria). Transfected cells were selected and maintained using 7.5 µg/ml puromycin (Sigma).
Exposure of Cells to CQ and Metals—APP-overexpressing cells were passaged at a ratio of 1:6 and grown in 6- or 12-well plates for 2–3 days before experiments. CQ was prepared as a 10 mM stock solution in Me2SO and added to serum-free RPMI 1640 medium supplemented with puromycin as described above. Basal metal levels in the medium were 0.5, 1.3, and 2.1 µM for Cu2+, Zn2+, and Fe2+, respectively, as determined by inductively coupled plasma mass spectrometry (ICP-MS). Additional metals were added (10 µM unless stated otherwise), and the medium was briefly mixed by aspiration prior to addition to cells. Control cultures were treated with vehicle (Me2SO) alone. Inhibitors of phosphoinositol 3-kinase (PI3K) (LY-294,002 and wortmannin), JNK (SP600125), MEK1/2 (PD 98,059), p38 (SB 203580), GSK3 (GSK Inhibitor IX), and metalloproteases (GM 6001, phosphoramidon, thiorphan, bestatin, MMP-2 Inhibitor I, and MMP-9 Inhibitor I) were prepared as 10 mM stock solutions in Me2SO and added at the indicated concentrations. Ascorbate, MnTMPyP, bacitracin, BPS, LiCl, and MMP-3 Inhibitor I were prepared as 10 mM solutions in distilled H2O. Serine/cysteine protease inhibitor mixture (EDTA-free) was prepared as a 10x solution in distilled H2O. Where stated, vector only-transfected or wild-type (non-APP-overexpressing) cells were exposed to synthetic human A-(1–40) with or without CQ, metals, and inhibitors (see below). Cultures were incubated for up to 6 h, and conditioned media were taken for measurement of A levels by enzyme-linked immunosorbent assay (ELISA). Cell viability was determined by lactate dehydrogenase release following kit instructions (Promega Corp., Annandale, New South Wales, Australia). For immunoblotting, cells were harvested into PhosphoSafe extraction buffer (Novagen) containing Protease Inhibitor Cocktail III (Calbiochem) and stored at –80 °C until used. Alternatively, cells were washed three times with phosphate-buffered saline (PBS) and harvested for analysis of metal levels by ICP-MS.
ICP-MS—Cells were treated with CQ and/or metals for 6 h unless stated otherwise and washed three times with Chelex 100-treated PBS (pH 7.4). Cells were scraped into PBS; an aliquot was taken for protein determination (protein microassay, Bio-Rad); and the remaining cells were collected by centrifugation at 14,000 rpm for 2 min in a Hermle microcentrifuge (Labnet International, Inc., Edison, NJ). Metal levels were determined in cell pellets by ICP-MS as described previously (12) and converted to ng of metal/mg of protein.
Degradation of Synthetic A-(1–40)—Human A-(1–40) was purchased from the W. M. Keck Laboratory (Yale University, New Haven, CT) and dissolved in Me2SO at 1 mg/ml. The dissolved peptide was further diluted into Chelex 100-treated distilled H2O at 100 ng/ml before addition to vector only-transfected CHO cell cultures in serum-free medium at 10 ng/ml without aging. In separate experiments, A-(1–40) was also added to N2a mouse neuroblastoma, SH-SY5Y human neuroblastoma, or HeLa human epithelial cells in serum-free Opti-MEM I (Invitrogen). After 6 h (with or without addition of inhibitors and 10 µM each CQ, Cu2+, or CQ and Cu2+), the medium was collected, and the remaining A-(1–40) levels were determined by ELISA.
Double Antibody Capture ELISA for A Detection—A levels were determined in culture medium using the DELFIA® double capture ELISA (PerkinElmer Life Sciences, Melbourne, Australia). 384-Well plates (Greiner Bio-One GmbH, Frickenhausen, Germany) were coated with monoclonal antibody G210 in 15 mM Na2CO3 and 35 mM NaHCO3 (pH 9.6) for A-(1–40) detection. Plates were washed with PBS containing 0.05% Tween and blocked with 0.5% (w/v) casein. Biotinylated monoclonal antibody WO2 (A-(5–8) epitope) and the culture medium or A standards were added (50 µl) to each well and incubated overnight at 4 °C. Plates were washed with PBS containing 0.05% Tween, and streptavidin-labeled europium (PerkinElmer Life Sciences) was added. The plates were washed; enhancement solution (PerkinElmer Life Sciences) was added; and the plates were read in a Wallac VICTOR2 plate reader with excitation at 340 nM and emission at 613 nM. A-(1–40) and A-(1–42) standards and samples were assayed in triplicate. The values obtained from the triplicate wells were used to calculate the A concentration (expressed as ng/ml) based on the standard curve generated on each plate. We observed a good correlation between ELISA results and Western blot analysis of A levels in CQ·Cu2+-treated cultures. As the ELISA offered quantitative data on A levels, we chose this as the preferred method for assessing changes to secreted A levels.
Western Blot Analysis of Protein Expression and Phosphorylation—Cell lysates prepared in PhosphoSafe extraction buffer were mixed with SDS sample buffer (Novex) and separated on 12% Tris/glycine/SDS-polyacrylamide gels (Novex). Western blotting of A in the conditioned medium was performed using 10–20% Tris/Tricine gels. Proteins were transferred to polyvinylidene difluoride membranes and blocked with milk solution in Tris-buffered saline/Tween before immunoblotting for total or phospho-specific proteins. Membranes were probed for 1 h with antiserum against A (antibody WO2), C-terminal APP (antibody 369), or full-length APP (antibody 22C11) at 1:2000 dilution and with horseradish peroxidase-conjugated rabbit anti-mouse or goat anti-rabbit secondary antibody at 1:5000 dilution. For detection of signal transduction molecules, membranes were probed with polyclonal antiserum against actin, JNK, phospho-JNK, p38, phospho-p38, ERK1/2, phospho-ERK1/2, Akt, phospho-Akt, GSK3, phospho-GSK3
/, MMP-2, or MMP-6 at 1:5000 dilution. Horseradish peroxidase-conjugated goat anti-rabbit secondary antiserum was used at 1:10,000 dilution. Blots were developed by chemiluminescence (ECL Advance, Amersham Biosciences) and imaged on a GeneGnome chemiluminescence imager (Syngene, Cambridge, UK). We found that the expression of total levels of kinases (Akt, JNK, ERK, and p38) was unaffected by metal uptake in APP-CHO cells. In contrast, actin, tubulin, and other proteins normally used for equalizing protein loading were found to be altered depending on metal levels.5 Therefore, equal sample loading and protein transfer were assessed by consistency of total kinase protein levels rather than unrelated protein levels on immunoblots.
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MMP Assays—The activity of MMPs in the conditioned medium and cell lysates was determined using an EnzoLyte MMP fluorometric assay kit (AnaSpec, Inc., San Jose, CA). Briefly, the conditioned medium or cell lysates (freshly extracted without protease inhibitors) were incubated with MMP-specific peptide substrates following the kit instructions. The substrates used were QXL520-
Abu-Pro-Cha-Abu-S-methyl-L-cysteine-His-Ala-Dab(5-FAM)-Ala-Lys-HN2 (where Abu is -aminobutyric acid, Cha is D-cyclohexylalanine, Dab is 2,4-diaminobutyric acid, and 5-FAM is 5-carboxyfluorescein; broad-spectrum substrate), QXL520-Pro-Leu-Ala-Leu-Trp-Ala-Arg-Lys(5-FAM)-NH2 (MMP-1), 5-FAM-Pro-LeuAla-Nva-Dap(QXL520)-Ala-Arg-NH2 (where Nva is norvaline and Dap is diaminopropionic acid; MMP-2), QXL520-Pro-Tyr-Ala-Tyr-Trp-Met-Arg-Lys(5-FAM)-NH2 (MMP-3), QXL520-Pro-Leu-Gly-Met-Trp-Ser-Arg-Lys(5-FAM)-NH2 (MMP-2/9), and QXL520-Pro-Leu-Ala-Tyr-Trp-Ala-Arg-Lys(5-FAM)-NH2 (MMP-8). No MMP-9-specific substrate was available. Cleavage of substrates by MMPs removed the quenching effect of QXL520 on 5-carboxyfluorescein, resulting in increased fluorescence with excitation at 490 nM and emission at 535 nm.
Statistical Analysis—All data described in graphical representations are means ± S.E. unless stated from a minimum of three separate experiments. Results were analyzed using Student's two-tailed t test.
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Levels in Vitro—We examined whether CQ affects A generation in APP-CHO cells. Treatment of APP-CHO cultures with 10 µM CQ alone for 6 h induced no significant change to A-(1–40) levels in the culture medium (Fig. 1A). Interestingly, 10 µM Cu2+ alone for 6 h induced a 35% increase in A-(1–40) levels (p < 0.01) (Fig. 1A).
When cultures were exposed to 10 µM CQ and 10 µM Cu2+, we observed a potent reduction (85%) of secreted A-(1–40) levels (p < 0.0001) (Fig. 1A). An analogous effect was observed upon treatment with CQ and 10 µM Zn2+ (Fig. 1A). No significant changes were observed in A-(1–40) levels when cells were treated with CQ plus Fe2+ (Fig. 1A). Potent inhibition of secreted A-(1–42) levels also occurred with CQ·Cu2+-treated cells (Fig. 1B). However, as A-(1–42) levels in APP-CHO cells were near the detection limit of the ELISA, subsequent analysis of A was restricted to A-(1–40). The loss of secreted A upon treatment with CQ·Cu2+ was confirmed by immunoblot analysis of the conditioned medium (Fig. 1C) and surface-enhanced laser desorption ionization mass spectrometry (data not shown).
Inhibition of A Can Be Induced by Low Concentrations of CQ and Cu2+—To examine the potency of CQ in inhibiting secreted A levels, we treated cultures with 0.1–50 µM CQ with or without 10 µM Cu2+ for 6 h. A-(1–40) was significantly decreased at 0.1 and 1.0µM CQ plus Cu2+ (Fig. 2A). We also examined the effects of different concentrations of CQ on Cu2+ uptake in APP-CHO cells. 0.1 µM CQ induced an increase of 25-fold in cellular Cu2+ levels (Fig. 2B). Increasing CQ concentrations resulted in further elevation of cellular Cu2+ levels, reaching 112-fold (at 50 µM) compared with control levels (Fig. 2B). The ability of low concentrations of CQ to increase cellular Cu2+ levels correlated with the potent reduction of secreted A levels by CQ·Cu2+ (Fig. 2A). Although CQ has been reported to optimally bind Cu2+ at a ratio of 2:1 (13), our titration studies showed no significant differences in Cu2+ uptake and inhibition of A levels upon varying the CQ/Cu2+ ratios.
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levels, cultures were exposed to 10 µM CQ with different concentrations of Cu2+. 0.1 µM added Cu2+ significantly inhibited A levels (Fig. 2C). Higher concentrations of added Cu2+ further decreased secreted A levels (Fig. 2C). We also examined the time course of A inhibition by CQ plus Cu2+ (10 µM each). We observed an initial decrease in A levels from 30 to 60 min after addition of CQ·Cu2+. A greater loss of A was observed from 60 to 120 min after treatment (Fig. 2D). Examination of cellular metal levels revealed a 22-fold increase in Cu2+ after a 10-min exposure to CQ·Cu2+ (Fig. 2E). Cu2+ levels increased further at each time point, reaching a maximum level of 103-fold at 360 min (Fig. 2E).
Loss of A by CQ·Cu2+ Does Not Correlate with Cellular APP Levels—To further understand how CQ·Cu2+ mediates A loss, we determined whether there is a corresponding loss in APP expression. Exposure to CQ alone or to CQ·Cu2+ reduced both APP expression and secretion (Fig. 2F). However, as shown in Fig. 1A, only CQ·Cu2+ inhibited secreted A levels. Interestingly, there was a reduction in the -C-terminal 83-amino acid fragment of APP (C83) upon CQ·Cu2+ treatment, although no changes in APP -C-terminal 99-amino acid fragment (C99) expression were found (Fig. 2F). This was consistent with our observation that the activity of BACE1 (beta-site APP-cleaving enzyme 1) in APP-CHO membrane preparations was unchanged after treatment with CQ·Cu2+. Likewise, analysis of COS-7 cells transfected with a C-terminal APP construct (APP C99) (14) revealed no effect on -secretase cleavage of APP C99 by CQ·Cu2+.5 These findings demonstrate that the loss of secreted A upon treatment with CQ·Cu2+ is unlikely to result from altered APP processing.
Loss of Secreted A by CQ·Cu2+ Is Mediated through Activation of JNK and ERK—Metal ligands can stimulate MAPK pathways (15, 16). To examine whether the effects of CQ·Cu2+ on A occur via these pathways, we treated cultures with CQ and Cu2+ or Zn2+ (10 µM each) and measured activation of JNK, p38, and ERK1/2 in cell lysates. CQ with Cu2+ or Zn2+ induced substantial activation of JNK and ERK1/2, with moderate activation of p38 (Fig. 3A).
We then examined whether activation of these MAPK pathways is involved in the inhibitory action of CQ and metals on secreted A levels. The JNK inhibitor SP600125 resulted in significant inhibition of JNK phosphorylation (Fig. 3B) and a significant elevation of A-(1–40) levels compared with CQ·Cu2+ alone (p < 0.001) (Fig. 3B). The ERK1/2 phosphorylation inhibitor PD 98,059 (5 µM) prevented ERK activation after exposure to CQ·Cu2+ (Fig. 3C) and significantly inhibited A loss (p < 0.001) (Fig. 3C). In contrast, the p38 inhibitor SB 203580 or the broad-spectrum protein kinase inhibitor staurosporine had no restorative effect on A levels (Fig. 3, D and E).
JNK can be activated in response to cell stresses such as generation of reactive oxygen species or through growth factor-mediated pathways (17). Therefore, we examined whether JNK phosphorylation is mediated by generation of reactive oxygen species in the CQ·Cu2+-treated cultures. APP-CHO cells were exposed to CQ·Cu2+ together with the reactive oxygen species scavenger MnTMPyP (200 µM) or the antioxidant ascorbate (1 mM). Treatment with these antioxidants did not inhibit JNK phosphorylation or prevent A loss in CQ·Cu2+-treated cultures (Fig. 3F). This is consistent with Zn2+ inducing effects analogous to those of Cu2+, as Zn2+ is a redox-inactive metal and should not directly stimulate reactive oxygen species generation. Therefore, the results strongly suggest that activation of JNK by CQ·Cu2+ is not mediated through metal-induced oxidative stress.
Inhibition of A by CQ·Cu2+ Requires Activation of the PI3K-Akt-GSK3 Pathway—Modulation of GSK3, a downstream target of PI3K and Akt activation, changes A production in APP-CHO cells (18). Therefore, we examined whether the PI3K-Akt-GSK3 pathway is associated with the loss of A production in CQ·Cu2+-treated cells. Treatment of cells with CQ and Cu2+ (10 µM each) for 6 h resulted in significant activation of Akt (Fig. 4A). Co-treatment of cultures with the specific PI3K inhibitor LY-294,002 inhibited Akt phosphorylation induced by CQ·Cu2+ and significantly abrogated the decrease in secreted A levels (p < 0.0001) (Fig. 4A).
Treatment of cultures with 10 µM CQ·Cu2+ for 6 h increased the phosphorylated forms of GSK3/, and this effect was blocked by LY-294,002 (Fig. 4A). There was also a small increase in total GSK3 levels in CQ·Cu2+-treated cultures, which may partially account for the increased levels of phosphorylated GSK3.
We then investigated whether PI3K-Akt-GSK3 activation is upstream of MAPK activation. Treatment of cultures with 25 µM LY-294,002 (or 10 nM wortmannin; data not shown) inhibited phosphorylation of Akt as well as phosphorylation of both JNK and ERK1/2 (Fig. 4B). Conversely, treatment of cultures with inhibitors of JNK and ERK1/2 phosphorylation (SP600125 and PD 98,059 respectively) did not inhibit Akt phosphorylation (data not shown). These data demonstrate that PI3K-Akt activation is upstream of JNK and ERK activation.
Activation of the PI3K-Akt and JNK Pathways Alone Is Not Sufficient for Loss of A—As inhibitors of PI3K and JNK pathways blocked the loss of A by CQ·Cu2+, we examined whether nonspecific up-regulation of these pathways also results in loss of A in APP-CHO cells. Cultures exposed to 25–100 µM Cu2+ (without CQ) for 6 h revealed potent activation of Akt, whereas 50 and 100 µM Cu2+ also induced phosphorylation of GSK3 and JNK (Fig. 4C). Moreover, cultures treated with the apoptotic agent cisplatin (200 µM) for 6 h revealed activation of JNK but not Akt (Fig. 4C). However, neither of these treatments (Cu2+ or cisplatin) reduced secreted A levels, demonstrating that the PI3K-Akt and JNK pathways are necessary, but insufficient alone, for the loss of A in APP-CHO cells.
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loss in the medium (Fig. 5B). To confirm the potentiating effect of GSK3 phosphorylation on JNK activation, we treated cultures with 1 µM CQ and 10 µM Cu2+ in the presence of GSK Inhibitor IX (10 or 25 µM). This increased phosphorylation of GSK3 and JNK compared with CQ·Cu2+ alone (Fig. 5C). The increased GSK3 and JNK phosphorylation correlated with a down-regulation of A levels in the culture medium (Fig. 5C). These results provide strong evidence that increased phosphorylation of GSK3 in CQ·Cu2+-treated cultures promotes activation of JNK and leads to loss of secreted A.
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—Exposure of APP-CHO cells to CQ·Cu2+ for 6 h resulted in morphological changes consistent with altered cell adhesion (detachment of cells), but without an obvious role for cytotoxicity or oxidative stress (Fig. 3F). To examine this, we measured adhesion of cells to a collagen type IV matrix after treatment with 10 µM CQ and Cu2+. As shown in Fig. 6A, CQ·Cu2+ inhibited APP-CHO cell adhesion to collagen type IV by 50%. The loss of adhesion could be prevented by treatment with SP600125 or LY-294,002 (Fig. 6A). As loss of cell adhesion is commonly associated with activation of metalloproteases (19), we treated cells with broad-spectrum metalloprotease inhibitors. GM 6001 (10 µM), BPS (500 µM), and MMP Inhibitor I (20 µM) significantly inhibited CQ·Cu2+-mediated loss of cell adhesion to collagen type IV (Fig. 6A).
To determine whether metalloproteases mediate A loss, APP-CHO cells were treated with a range of metalloprotease inhibitors, and A levels were measured after exposure to CQ·Cu2+. All metalloprotease inhibitors tested except thiorphan (neprilysin inhibitor) significantly inhibited the decrease in secreted A levels induced by CQ·Cu2+ (Fig. 6B). To confirm that the loss of secreted A was mediated through increased metalloprotease-mediated degradation rather than altered APP processing, vector only-transfected CHO cell cultures were exposed to 10 ng/ml synthetic human A-(1–40) for 6 h with or without CQ·Cu2+. Measurement of A-(1–40) levels in the conditioned medium revealed 0.89 ± 0.11 ng/ml remaining in the control medium after 6 h, indicating substantial clearance by cell uptake and/or degradation (Fig. 6C). Exposure of cultures to 10 µM CQ or 10 µM Cu2+ increased the levels of synthetic A-(1–40) remaining in the medium after 6 h (Fig. 6C). However, treatment of cultures with CQ·Cu2+ significantly decreased A-(1–40) levels by 56% compared with controls and by 77% compared with CQ alone (Fig. 6C). Interestingly, this effect was prevented by co-treatment of cultures with LY-294,002 (25 µM) or inhibitors of metalloproteases (Fig. 6C). The results clearly support a role for PI3K-mediated metalloprotease degradation of A as the primary cause of A loss in cultures treated with CQ·Cu2+.
CQ·Cu2+ Induces Up-regulation of MMP-2 and MMP-3 through Activation of the PI3K and JNK Pathways—The efficacy of GM 6001 and MMP Inhibitor I against loss of A and cell adhesion strongly supported a role for up-regulation of MMPs in CQ·Cu2+-treated cultures. Therefore, we measured the activity of MMPs in cells treated with CQ·Cu2+ using MMP-specific fluorescent substrates. MMP assays of cell lysates or the conditioned medium after treatment with CQ·Cu2+ for 6 h revealed a significant elevation of the specific activities of MMP-2 and MMP-3 (Fig. 7A). No significant changes were observed in the activities of MMP-1, MMP-8, and MMP-9. Western blot analysis of cell lysates with antisera to MMP-2 and MMP-9 confirmed the results from the fluorescent substrate assay. Both latent and activated forms of MMP-2 were up-regulated in cultures exposed to CQ·Cu2+, whereas MMP-9 revealed only a minimum change (Fig. 7A, inset).
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Interestingly, the inhibitors of MMP-2 and MMP-3 significantly abrogated the loss of A-(1–40) caused by CQ·Cu2+ (Fig. 7C). These effects were consistent with a previous report that both MMP-2 and MMP-3 can cleave A at several sites (10). In fact, surface-enhanced laser desorption ionization analysis of medium from our control cultures revealed A cleavage products consistent with MMP-2-mediated degradation.5 Unfortunately, few A fragments of any size were observed in CQ·Cu2+-treated cultures. This suggested that further degradation of the A cleavage fragments may be occurring in these cultures, possibly through activation of aminopeptidases. This was supported by inhibition of A loss by co-treatment with bestatin (aminopeptidase inhibitor) (Fig. 6B).
Finally, we examined alternative cell types for their ability to degrade A when exposed to CQ·Cu2+. Treatment of APP-overexpressing N2a murine neuroblastoma cells with 10 µM CQ and 10 µM Cu2+ for 6 h reduced A levels from 1.1 to 0.4 ng/ml (p < 0.001), whereas CQ or Cu2+ alone had no significant effect (Fig. 8A). In addition, non-transfected N2a, SH-SY5Y human neuroblastoma, and HeLa human epithelial cells were treated with 10 µM CQ and Cu2+ together with 10 ng/ml synthetic human A-(1–40). Measurement of A levels in the conditioned medium after 6 h revealed significantly reduced synthetic A-(1–40) levels in all cell types after treatment with CQ·Cu2+, and this could be prevented by co-treatment with GM 6001 (Fig. 8B). These results demonstrate that CQ·Cu2+ can modulate secreted A levels via metalloproteases in different cell types, including neuroblastoma cells.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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levels in vitro. Whereas treatment of APP-expressing cells with CQ alone had little effect on A levels in the culture medium, treatment with CQ complexed to Cu2+ or Zn2+ dramatically decreased extracellular A levels. We have shown that this effect is closely related to the ability of CQ to mediate substantial increases in cellular Cu2+ or Zn2+ levels, resulting in selective up-regulation of MMP activity.
Interestingly, we found that even low concentrations of CQ or Cu2+ (0.1–1 µM each) could induce a significant loss of A after only 6 h. The potency with which CQ·Cu2+ inhibited A underscores the potential physiological relevance of our findings. A recent study reported human plasma levels of CQ at 13–25 µM during small phase clinical trials (8). CQ levels in the brain may reach 20% of serum levels, which equates to 2.6–5 µM (20). These concentrations were well within the range of CQ levels found to inhibit A in our cultures if sufficient Cu2+ or Zn2+ was available. Cu2+ levels can range from 1.7 µM in the extracellular space of the brain to 250 µM in the synaptic cleft, whereas Zn2+ is also highly abundant in the brain, with synaptic levels reaching 300 µM (21). Further investigation is required to determine whether CQ can transport other metals (i.e. nickel or cobalt) into cells and, if so, whether similar effects on APP metabolism are induced.
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The mechanism of action by CQ·Cu2+ is via activation of the PI3K-Akt pathway and subsequent phosphorylation of JNK and ERK1/2. Although it is common to view PI3K-Akt and JNK/p38 as opposing pathways leading to cell survival and apoptosis, respectively (24), JNK activation can also be potentiated through PI3K activation (25, 26), as we have demonstrated here. Activation of both PI3K-Akt and JNK pathways has been reported in AD brain tissue, although the downstream consequences of this activity are not clear (27, 28).
PI3K is normally activated in response to cell stresses or growth factors, and metals can activate PI3K in some cell culture models (29). Particularly intriguing was our finding that activation of Akt and JNK by treatment with high Cu2+ levels alone (without CQ) or cisplatin had no effect on secreted A levels, demonstrating that, although up-regulation of these pathways is required, by themselves, they are not able to decrease secreted A levels. It is possible that, after exposure to CQ and metals, elevation of Cu2+ (or Zn2+) levels in certain subcellular compartments results in specific modulation of multiple metal-dependent pathways, including PI3K activation (Fig. 9). This is consistent with reports that Zn2+ can activate gene expression by a PI3K- and JNK-dependent process (30). Alternatively, elevated metal levels could promote release of growth factors or other ligands that, in turn, activate PI3K, MAPK, and additional pathways. Interestingly, stimulation of MAPK pathways by metal-mediated growth factor release has been reported in lung epithelial cells (16, 31).
A common downstream signaling pathway controlled by PI3K activation involves phosphorylation of Akt and subsequent inhibition of GSK3 through phosphorylation (32). Treatment of cultures with LY-294,002 blocked phosphorylation of both Akt and GSK3/ by CQ·Cu2+. Inhibition (phosphorylation) of GSK3 can result in abrogation of A production in APP-transfected cells (18), consistent with our findings. However, we found that phosphorylation of GSK3/ correlated closely with increased JNK phosphorylation. Using inhibitors of GSK3 (LiCl and GSK Inhibitor IX), we demonstrated that increased phosphorylation of GSK3 potentiated JNK activation and subsequent A loss. The mechanism behind this is not clear. As phosphorylation of GSK3 leads to its inactivation, the data suggest that activated GSK3 may inhibit or reduce JNK activation by certain stimuli. Similar effects have been reported previously, where a loss of GSK3 activity potentiated JNK activation by growth factors but not by cell stress (17, 33). This is consistent with our data suggesting that MAPK pathways are activated by CQ·Cu2+ via non-oxidative mechanisms. Down-regulation of GSK3 activity by CQ·Cu2+ could also affect tau phosphorylation, and this should be investigated in appropriate neuronal cell models.
Activation of cell signaling pathways by CQ·Cu2+ culminated in up-regulation of MMP activity and degradation of extracellular A. Fig. 9 shows that the order of events are activation of PI3K-Akt, followed by phosphorylation of GSK3 as well as JNK and ERK. Inhibition of these kinases (Akt, JNK, and ERK) blocked activation of MMPs, so they are upstream of MMP activation. Moreover, inhibitors of the kinases and MMPs blocked the loss of A, demonstrating that A loss is downstream of these events. MMP activation is often associated with pathological changes to the cellular microenvironment in the brain, including tumor cell tissue invasion and migration and breakdown of blood-brain barrier permeability during cerebral ischemia. However, MMP activation can also have beneficial functions in the brain, including angiogenesis following ischemia and during axon guidance.
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. However, we investigated only CQ·Cu2+ complexes in detail, and it remains to be determined whether CQ·Zn2+ complexes mediate activation of the same MMPs.
Importantly, MMP-2 and MMP-3 levels can be increased through stimulation of the PI3K-Akt and MAPK (JNK and ERK) pathways (36, 37), which is consistent with our findings in CQ·Cu2+-treated cells. Nonspecific Akt and JNK phosphorylation was unable to induce the decrease in secreted A levels, indicating that CQ-delivered Cu2+ has a more complex effect on cell signaling pathways, resulting in up-regulation of MMPs. For example, there are a number of soluble inducers of MMP-2 and MMP-3, including transforming growth factor- and epidermal growth factor, that could be released upon exposure to CQ and metals.
A can be degraded in vitro and in vivo by several proteases, including metalloproteases, neprilysin, insulin-degrading enzyme, and MMPs (4, 6, 38). These metalloproteases may have important roles in clearance of A in the brain, whereas reduced activity in AD patients could promote amyloid deposition. As thiorphan (neprilysin inhibitor) had no effect on A levels in CQ·Cu2+-treated cultures, increased neprilysin activity is unlikely to be involved in A
