Department of 1Biochemistry & Molecular Biology, Keck School of Medicine, University of Southern California, Los Angeles, California; and 2Center for Aging, University of Rochester, Rochester, New York
Submitted 20 September 2004 ; accepted in final form 16 February 2005
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ABSTRACT |
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amyloid peptide; chemokine receptor 5; small inhibitory ribonucleic acid
Immunohistochemical studies (32) show increased presence of chemokine macrophage inflammatory protein (MIP)-1 in a subpopulation of reactive astrocytes and MIP-1
in neurons of AD brain than those of controls. Moreover, their studies (32) showed that chemokine receptor (CCR)3 and -5 are present on microglial cells of both control and AD brain but with an increased expression on reactive microglia in AD. Because CCR5 is expressed on monocytes and certain lymphocytes, and is activated by the
-chemokines [MIP-1
, MIP-1
, and regulated on activation normal T-expressed and presumably secreted (RANTES)] (25), we hypothesized that the increased expression of CCR5 on microglial cells or their precursors (monocytes/macrophages) could occur as a result of activation by A
peptides. Moreover, these activated monocytes may transmigrate across the brain vasculature in response to
-chemokines (MIP-1
, MIP-1
, and RANTES) elaborated by microglia and astrocytes in the brain.
The role of CCR5 and its ligands in human immunodeficiency virus (HIV) infection has been the subject of much scrutiny (1, 23, 24). Studies (1, 28) have shown that CCR5 serves as the main coreceptor for the entry of HIV in monocytes/macrophages and humans having a 32-base pair (bp) mutation in the CCR5 gene (CCR532 mutant allele) are generally resistance to HIV-1 infection. In vitro studies of peripheral blood lymphocytes show decreased cell surface expression of CCR5 in response to TNF- (13) and LPS (7). However, relatively less is known of the mechanism by which amyloid peptides induce the expression of CCR5 in monocytes/macrophages, the progenitor cells of microglia.
In the present study, we show that A peptides (A
140 and A
142) at pathophysiological concentrations (125 nM), as found in the plasma of AD individuals (14), cause cellular signaling in THP-1 monocytic cells and peripheral blood monocytes to increase the gene expression of CCR5. The cellular signaling for increased expression of CCR5 in response to A
involves activation of Raf kinase and MAP kinase (ERK1/ERK2). We (10) recently showed that A
at submicromolar concentration causes activation of transcription factor AP-1 and Egr-1 but not of NF-
B and cAMP response element binding protein (CREB) in THP-1 monocytes and peripheral blood monocytes (PBM). The presence of a putative Egr-I consensus sequence in the CCR5 promoter indicated that this transcription factor could regulate expression of CCR5. In the present study, we show for the first time by EMSA, transfection of THP-1 cells with truncated CCR5 gene promoter construct, and chromatin immunoprecipitation analysis that Egr-1 binds in vitro and in vivo to the newly identified consensus sequence of the CCR5 promoter. Moreover, transfection with small inhibitory RNA (siRNA) for Egr-1 mRNA abrogates A
-induced CCR5 expression. In addition, we show that A
-induced CCR5 expression in THP-1 monocytes plays a role in chemotaxis in response to
-chemokines (MIP-1
and RANTES) as well as to amyloid peptide. We demonstrate that transfection of THP-1 monocytes with Egr-1 siRNA abrogates chemotaxis in response to MIP-1
. For the first time, these studies demonstrate the importance of Egr-1 transcription factor in the regulation of CCR5 expression in monocytes.
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METHODS |
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Reagents.
PD-98059, U-0126, and genistein were purchased from BIOMOL (Plymouth Meeting, PA). GW-5074, SB-203580, and SP-600125 were obtained from Tocris Cookson (Ellisville, MO). Rabbit anti-Egr-1 (SC-110X) and goat anti-SP-1 (SC-59X) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Monoclonal antibody to CCR5, goat anti-MIP-1 and RANTES were obtained courtesy of NIH AIDS Reagent and Reference Program, operated by McKesson Bioservices (Rockville, MD). Recombinant human MIP-1
and goat anti-MIP-1
were obtained from R&D Systems (Minneapolis, MN). All other reagents, unless otherwise specified, were purchased from Sigma.
Cell culture and isolation of peripheral blood monocytes. The THP-1 promonocytic cell line obtained from ATCC (Manassas, VA) was cultured in RPMI 1640 containing 10% heat-inactivated fetal calf serum, as described earlier (10). On the day of the experiment, THP-1 cells (1 x 106 cells/ml) were cultured in serum-free RPMI 1640 for 46 h. PBMs were isolated from blood collected in EDTA as the anticoagulant, as previously described (10).
RNase protection assay. THP-1 monocytes were treated with amyloid peptides for various time periods and total RNA was isolated with TriZOL reagent (Invitrogen, Carlsbad, CA). RNase protection assays (RPA) were performed using custom made multiprobe templates for CCR5, CCR2a, and CCR2b and the housekeeping genes L32 and GAPDH (Pharmingen, San Diego, CA), as previously described (10). The intensity of bands corresponding to CCR5, CCR2a, CCR2b, and GAPDH were analyzed using a gel documentation system (model 2000, Alpha Imager, San Leandro, CA). Values were expressed as relative expression of mRNA normalized to the means of L32 and GAPDH mRNA values.
EMSA for transcription factors Egr-1.
THP-1 cells (5.0 x 106 cells) were treated with A140 (125 nM) for varying time periods (15240 min) and nuclear extracts were prepared as described previously (10). The oligonucleotide probes used for CCR5-Egr-1 (putative Egr-1 binding site in CCR5 promoter) were as follows: 5'-(GTC CCT ATA TGG GGC GGG GGT GGG GGT GTC T)-3' and 3'-(CAG GGA TAT ACC CCG CCC CCA CCC CCA CAG A)-5', which were synthesized at Norris Cancer Center Microchemical core facility of University of Southern California-Keck School of Medicine (Los Angeles, CA). Probes were 5' end labeled with 100 µCi of [
-32P] ATP using T4-polynucleotide kinase (10). The DNA binding reaction mixture contained nuclear proteins (24 µg), [32P]-labeled double-stranded oligonucleotide probe (
50,000 cpm), and 2 µg of poly dI-dC. To demonstrate specificity of DNA-protein interaction, 50-fold excess of unlabeled double-stranded oligonucleotide probe was added to the nuclear extract before the addition of radiolabeled probe. In supershift assays, nuclear extracts were preincubated for 20 min at room temperature with 2 µg of antibody specific to each transcription factor, before the addition of radiolabeled probe. The DNA-protein complex was then size fractionated from the free DNA probe by electrophoresis in a 4% nondenaturing polyacrylamide gel. The gel was dried and exposed to X-ray film.
Transient transfection of THP-1 cells and luciferase activity assay. The firefly luciferase reporter gene plasmids of CCR5 promoter (PA-3; 731 to +33 regions) employed was kindly provided by Dr. Sunil Ahuja (San Antonio, TX). Mummidi and coworkers (23) have previously described their preparation and features. THP-1 cells (23 x 106 cells/well) were cultivated in 6-well chambers. The reporter gene constructs were transiently transfected in THP-1 cells by using Lipofectamine reagent (Invitrogen, Carlsbad, CA). Transfection efficiency was normalized by cotransfecting THP-1 cells with CCR-5 promoter-luciferase constructs (10 µg/well) and 0.5 µg of Renilla luciferase vector (pRL-CMV; obtained from Promega, Madison, WI). Alternatively, THP-1 cells cotransfected with 10 µg of the promoter less vector pGL3-Basic (Promega) and 0.5 µg of pRL-CMV was used as a negative control. After 2 days of transfection, the cells were pelleted, washed in Dulbecco's PBS, and lysed in 1x passive lysis buffer (Promega). The protein concentration in the cell lysates was determined by the Bradford method. The firefly and Renilla luciferase activities in the lysates were determined according to the manufacturer's instructions (Dual-Luciferase Reporter Assay System, Promega) utilizing a luminometer (Berthold Technologies, Oakridge, TN). The relative luciferase activity in each sample was determined as follows: X = firefly luciferase activity of CCR-5 promoter construct ÷ Renilla luciferase activity of pRL-CMV construct; Y = firefly luciferase activity of promoter less vector pGL3-basic ÷ Renilla luciferase activity of pRL-CMV vector; Z = X ÷ Y and relative luciferase activity is expressed as Z ÷ micrograms of protein in the lysate sample.
Chromatin immunoprecipitation assay.
THP-1 cells (5 x 106 cells) were serum starved for 6 h, followed by treatment with A140 for the indicated time period. Chromatin immunoprecipitation assay (ChIP) analysis was performed as described previously (26). Briefly, after stimulation of cells with A
, cells were washed with PBS and then cross-linked with 1% formaldehyde at room temperature for 10 min. Cells were lysed, sonicated, and supernatants were then recovered by centrifugation of lysate at 12,000 rpm for 10 min at 4°C. The supernatant was diluted fourfold in a dilution buffer (1% Triton X-100, 2 mM EDTA, 150 mM NaCl and 20mM Tris·HCl, pH 8.1), followed by the addition of 2 µg of sheared salmon sperm DNA, 2.5 µg of preimmune serum, and 20 µl of protein A-Sepharose (50% slurry). The contents were kept at 4°C for 2 h. The precleared supernatant was immunoprecipitated by adding antibody (2 µg/ml) to either Egr-1 or SP-1, 2 µg of sheared salmon-sperm DNA, and 20 µl of protein A-Sepharose (50% slurry) and incubated at 4°C for 1216 h. After several washings, the protein was digested with proteinase K (10 µg/ml) for 1 h. The cross-linking between DNA and protein was reversed by incubating the immunoprecipitate at 65°C overnight. DNA was phenol-chloroform extracted, ethanol precipitated, air dried, and dissolved in 50 µl of buffer composed of 10 mM Tris·HCl, pH 8.0, and 1 mM EDTA. A DNA sample (5 µl) was subjected to PCR amplification utilizing primers (5'-CCA GCA GCA TGA CTG CAG TT-3', forward primer; 5'-GCT AAT TGC TGG TGC TTG GAG-3' reverse primer) corresponding to the promoter region of CCR-5 (from 847 to 603, respective to the transcription start site).
Flow cytometry analysis.
THP-1 cells (5 x 106 cells) were incubated with A140 for indicated time period (14 h). Cells were collected, washed with ice-cold PBS, and resuspended in PBS at a concentration of 1 x 106 cells/ml. An antibody (5 µg/ml) was added to these cells to either CCR5 or CCR2b, and the contents were incubated at 4°C for 60 min. Cells were washed in ice-cold PBS, followed by incubation with FITC-conjugated goat antimouse IgG. These cells were washed with PBS and fixed at room temperature with 2% paraformaldehyde for 15 min. Fixed cells were washed three times in PBS and analyzed for CCR5 surface expression by flow cytometry. Gated acquisition of monocytes (10,000 events) was performed based on forward and side-scatter parameters.
Synthesis of siRNA duplexes for Egr-1 mRNA. The 22 nucleotide sequence of Egr-1 siRNA was derived from human Egr-1 mRNA sequence (Genebank Accession No. GI: 5420378) and was targeted to the coding region 12371258 relative to the start codon of Egr-1 gene. Egr-1 siRNA and scrambled Egr-1 siRNA (scEgr-1 siRNA) were synthesized as previously described (10).
Transient transfection of THP-1 cells with Egr-1 siRNA duplex. THP-1 cells were transfected with Egr-1 siRNA duplex utilizing lipofectamine (10). Briefly, Egr-1 siRNA or scEgr-1 siRNA (0.25 or 0.5 µg/ml) was incubated in 100 µl of serum-free DMEM containing 10 µl of lipofectamine (Invitrogen) for 15 min, followed by the addition to THP-1 cells. After 4872 h of transfection, cells were harvested and used for further experiments.
Chemotaxis assay.
Chemotaxis was assayed in 96-well plates (Neuro Probe, Gaithersburg, MD) with Transwell inserts of 5-µm pore size. Briefly, THP-1 monocytes were washed and resuspended in serum free RPMI-1640 medium and 1 x 105 cells/50 µl were then loaded into an insert of the Boyden chamber. Chemotaxis medium (30 µl of serum-free RPMI-1640 medium) containing indicated amounts of chemokines or A was placed in the bottom compartment. After 2 h of incubation at 37°C in a 5% CO2 incubator, cells were scraped from the top chamber and washed with PBS (100 µl) to remove nonmigrated cells. This was followed by the addition of PBS containing 2 mM EDTA to the top chamber and incubation at 4°C for 15 min. Cells that had migrated into the lower compartment of the Boyden chamber were counted in five microscopic high-power fields (x40) with the use of an Olympus IMT-2 microscope. Where indicated, THP-1 cells were pretreated with A
140 for 4 h, followed by a wash with serum-free medium, and used directly in the chemotaxis assay. In addition, the effect of neutralizing antibody against chemokines was determined by adding antibody to the chemoattractant protein in the lower compartment of the chemotaxis chamber. Where indicated, A
-treated THP-1 cells were preincubated with antibody to chemokine receptor (CCR5 and CCR2b) for 1 h at room temperature. Each sample was tested in triplicate.
Statistical analysis.
Statistical analysis of the responses obtained from control and A-treated monocytic cells was carried out by one-way ANOVA utilizing Instat 2 (GraphPad, San Diego, CA) software program. The effects of inhibitors on A
-induced responses were analyzed by comparing the response of THP-1 cells in the presence and absence of inhibitor. Dunnett's test was used for multiple comparisons. P values <0.05 were considered as significant.
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RESULTS |
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Chromatin immunoprecipitation assay demonstrates binding of Egr-1 to the CCR5 promoter.
To determine whether Egr-1 binds to the native chromatin in THP-1 monocytes, we performed chromatin immunoprecipitation assay (ChIP) assays on chromatin obtained from THP-1 cells, which were pretreated with A140 for 30 and 60 min. Chromatin samples were immunoprecipitated with antibody to either Egr-1 or SP-1. DNA recovered from the antibody-bound fractions and DNA from input chromatin (before immunoprecipitation) were analyzed by semiquantitative PCR-using primers (as shown in boxed region of Fig. 5A) corresponding to the promoter region of CCR-5 (from 847 to 603 relative to the transcription start site). As shown in Fig. 5B, THP-1 cells treated with A
140 for 30 and 60 min exhibited increased amplification of PCR product, corresponding to the expected length (244 bp), with maximum Egr-1 chromatin binding activity at 30 min (Fig. 5B, lane 2). Both PD-98059 (Fig. 5B, lane 4) and SP-600125 (Fig. 5B, lane 5) reduced the in vivo Egr-1 chromatin binding activity in THP-1 cells treated with A
140 by
75%. As a positive control, LPS increased Egr-1 binding to CCR5 promoter region (Fig. 5B, lane 6). Because the SP-1 binding element (705 to 698) and Egr-1 binding element (702 to 693) in CCR-5 promoter (847 to 603) overlap, as indicated in Fig. 5A, we evaluated the SP-1 binding status to native chromatin derived from untreated and A
-treated THP-1 cells. The data show (Fig. 5B, bottom) a PCR product corresponding to expected length (244 bp) in chromatin derived from untreated THP-1 cells, which were immunoprecipitated with antibody to SP-1. However, treatment with A
modestly reduced amplification of PCR product (244 bp) at 30- and 60-min time periods (Fig. 5B, lanes 2 and 3, respectively). Both, PD-98059 (Fig. 5B, bottom, lane 4) and SP-600125 (Fig. 5B, bottom, lane 5) did not affect SP-1 chromatin binding activity in THP-1 cells treated with A
140. Moreover, LPS also did not affect SP-1 binding to CCR5 promoter region (Fig. 5B, bottom, lane 6). Figure 5B, bottom, shows amplification of input DNA before immunoprecipitation. There is no change in the amplification of the input DNA in all the samples. Taken together, these data show the effect of A
is specific for Egr-1 binding to CCR5 promoter region in vivo.
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DISCUSSION |
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Next, we examined the A-mediated cell signaling mechanism leading to the increased CCR5 mRNA expression. We show that A
140-induced expression of CCR5 mRNA is inhibited more than 90% by genistein (a protein tyrosine kinase inhibitor), PD-98059 and U-0126 (inhibitors of MEK1/2), and GW 5074 (a potent and specific inhibitor of c-Raf kinase). Furthermore, we observed that SP-600125, a specific inhibitor of c-Jun NH2 terminal kinase, reduced CCR5 mRNA expression by
60%. However, SB-203580 (a p38 MAP kinase inhibitor) augmented A
-induced expression of CCR5 mRNA, although in transfection studies with CCR5-luc promoter SB-203580 did not alter CCR5 promoter activity. To address this discrepancy, further studies are required to delineate the role of p38MAP kinase, utilizing transfection with either dominant negative p38 MAP kinase (MEK3/MEK6) constructs or siRNA approach. In our previous study (10), we showed that A
caused phosphorylation of tyrosine residues in a subset of proteins and phosphorylation of ERK-1/ERK-2 but not of p38 MAPK in THP-1. Taken together, these results suggest that A
-induced cellular signaling for the expression of CCR5 involves activation of protein tyrosine kinase, c-Raf kinase, and MAPKK/MEK in THP-1 monocytes, as illustrated in Fig. 12A. However, the nature of putative receptor (e.g., RAGE, SR-A, CD36, CD47, and FRP-2) involved in the nonfibrillar and fibrillar form of A
-mediated signaling in monocytes and microglia remains controversial (6, 18, 34, 35).
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A computer-aided analysis of CCR5 promoter [the promoter sequence analysis reported by Mummidi et al. (23)] resulted in the identification of the cis-element GCGGGGGTG at positions 702 to 694, which closely resembles the bona fide Egr-1 binding sequence (GCGGGGGCG) with a change to T from C at position 8. It is pertinent to note that the macrophage colony-stimulating factor (M-CSF) gene promoter has a GCGGGGGAG sequence at position 273 to 265 that were found to be an Egr-1-binding site (29). Second, we show by EMSA analysis that there was a clear electrophoretic shift due to binding of Egr-1 protein to oligonucleotide probes corresponding to either the putative Egr-1 binding element (GCGGGGGTG) present in CCR5 promoter (present study) or to the bona fide Egr-1 oligonucleotide (GCGGGGGCG) in the previous study (10). A subsequent supershift analysis demonstrated that the A-induced transcription factor interacting with the oligonucleotide was Egr-1 but not SP-1. These results clearly demonstrate that nuclear Egr-1 interacts with the CCR5 promoter region.
A further proof that Egr-1 binds to the promoter region of the CCR5 gene was obtained by studying the effect of A in THP-1 cells transfected with truncated CCR5 promoter (731 to +33) firefly luciferase construct. These studies indicated that the A
responsive region of the CCR5 promoter is localized within the 731 to +33 regions, which contains a putative Egr-1 binding site and a SP-1 cis acting element. Finally, chromatin immunoprecipitation (ChIP) analysis demonstrated that Egr-1 binds in vivo to the CCR5 promoter and that interaction with this transcription factor increases after A
treatment. Moreover, pharmacological inhibitors, which attenuated A
-induced CCR5 expression, also reduced Egr-1 binding to CCR5 promoter in ChIP assay.
Finally, we observed that transfection of Egr-1 siRNA but not scrambled Egr-1 siRNA (scEgr-1 siRNA) in THP-1 cells caused >75% reduction in A140-mediated CCR5 expression. However, the mRNA expression of CCR2a and CCR2b was unaltered in THP-1 cells transfected with either Egr-1 siRNA or scEgr-1 siRNA. These results further corroborate our contention that CCR5 expression but not CCR2a and CCR2b expression is regulated by Egr-1 transcription factor. We previously (10) reported that A
causes activation of ERK-1/2, which in turn results in phosphorylation of Elk-1. A parallel pathway involving activation of c-Jun NH2 terminal kinase also occurs, which causes phosphorylation of Elk-1 and AP-1 complex. Both of these pathways merge at Elk-1 phosphorylation resulting in the activation of Egr-1, as illustrated in Fig. 12A.
Because CCR5 is a cognate receptor for -chemokines (MIP-1
and RANTES), we hypothesized that this receptor could play a role in mediating chemotaxis of THP-1 monocytes. We show that A
-activated monocytes, which exhibit increased surface expression of CCR5 but not of CCR2b, undergo chemotaxis in response to a chemoattractant gradient generated by chemokines (MIP-1
and RANTES) as well as to A
140, although the extent of chemotaxis in response to amyloid (A
140) was relatively lower compared with these chemokines. Here, we show that A
-activated monocyte chemotaxis to MIP-1
is reduced in the presence of antibody to CCR5 but unaffected by antibody to CCR2b. Moreover, THP-1 monocytes transfected with Egr-1 siRNA, followed by treatment with A
, which has been shown to reduce CCR5 expression, resulted in attenuated chemotaxis to MIP-1
. These results indicate that chemotaxis of monocytes to MIP-1
requires cognate receptor CCR5 expressed on monocytes.
In conclusion, this is the first report, to our knowledge, showing that inhibition of Egr-1 transcription factor expression by Egr-1 siRNA can block A-mediated upregulation of CCR5 expression and concomitant chemotaxis of THP-1 monocytes. This is further supported by our finding of an Egr-1 like binding site in the promoter region of CCR5. We speculate that the increased presence of A
peptides in plasma of AD patients (14) upregulates the surface expression of CCR5 on monocytes, which may facilitate their migratory response to the chemokines elaborated from activated microglia in brain parenchyma of AD. Both of these processes might be acting together (Fig. 12B) to promote the transmigration of monocytes across the BBB. Taken together, our studies show that downregulation of CCR5 gene expression by either Egr-1 siRNA or pharmacological agents (e.g., curcumin) (9), which reduce CCR5 expression, may provide a novel therapeutic approach to ameliorate the inflammation-induced progression of AD. It is pertinent to note that a 32-bp deletion in CCR5 gene (CCR532 mutant allele) results in a nonfunctional receptor, which has been shown to confer resistance to HIV infection and displays protective effect toward certain inflammatory diseases (3). However, a recent study by Combarros et al. (3), conducted in a small sample of AD patients in Spain, show that the CCR532 allele neither influences the risk for AD nor modifies the age at which the onset of disease occurs, indicating that the CCR532 allele is not a preventive factor for AD. Thus additional in vivo studies are warranted to determine the role of CCR5 in chemotaxis of monocytes across BBB in AD. Recently, several therapeutic strategies, such as immunization with amyloid peptides (16), which promote efflux of soluble A
from brain to the periphery, have emerged, although the role of CCR5 in reducing amyloid burden in these studies is unknown. Our studies thus provide one avenue, among several approaches, to ameliorate amyloid peptide mediated inflammation and neurodegeneration in AD. This is supported by our finding that curcumin, a safe natural product, which suppresses CCR5 and cytochemokines expression in monocytes (9) in vitro has been shown to reduce levels of cytokines and plaque burden in Alzheimer's transgenic mice (17).
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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