P2Y2 Nucleotide Receptors Enhance α-Secretase-dependent Amyloid Precursor Protein Processing
2005; Elsevier BV; Volume: 280; Issue: 19 Linguagem: Inglês
10.1074/jbc.m500219200
ISSN1083-351X
AutoresJean M. Camden, Ann M. Schrader, Ryan E. Camden, Fernando A. González, Laurie Erb, Cheikh I. Seye, Gary A. Weisman,
Tópico(s)Genetics and Neurodevelopmental Disorders
ResumoThe amyloid precursor protein (APP) is proteolytically processed by β- and γ-secretases to release amyloid β, the main component in senile plaques found in the brains of patients with Alzheimer disease. Alternatively, APP can be cleaved within the amyloid β domain by α-secretase releasing the non-amyloidogenic product sAPPα, which has been shown to have neuroprotective properties. Several G protein-coupled receptors are known to activate α-secretase-dependent processing of APP; however, the role of G protein-coupled nucleotide receptors in APP processing has not been investigated. Here it is demonstrated that activation of the G protein-coupled P2Y2 receptor (P2Y2R) subtype expressed in human 1321N1 astrocytoma cells enhanced the release of sAPPα in a time- and dose-dependent manner. P2Y2 R-mediated sAPPα release was dependent on extracellular calcium but was not affected by 1,2-bis(2-aminophenoxy)ethane-N,N,N,-trimethylammonium salt, an intracellular calcium chelator, indicating that P2Y2 R-stimulated intracellular calcium mobilization was not involved. Inhibition of protein kinase C (PKC) with GF109203 or by PKC down-regulation with phorbol ester pre-treatment had no effect on UTP-stimulated sAPPα release, indicating a PKC-independent mechanism. U0126, an inhibitor of the mitogen-activated protein kinase pathway, partially inhibited sAPPα release by UTP, whereas inhibitors of Src-dependent epidermal growth factor receptor transactivation by P2Y2 Rs had no effect. The metalloprotease inhibitors phenanthroline and TAPI-2 and the furin inhibitor decanoyl-Arg-Val-Lys-Arg-chloromethylketone also diminished UTP-induced sAPPα release. Furthermore, small interfering RNA silencing of an endogenous adamalysin, ADAM10 or ADAM17/TACE, partially suppressed P2Y2R-activated sAPPα release, whereas treatment of cells with both ADAM10 and ADAM17/TACE small interfering RNAs completely abolished UTP-activated sAPPα release. These results may contribute to an understanding of the non-amyloidogenic processing of APP. The amyloid precursor protein (APP) is proteolytically processed by β- and γ-secretases to release amyloid β, the main component in senile plaques found in the brains of patients with Alzheimer disease. Alternatively, APP can be cleaved within the amyloid β domain by α-secretase releasing the non-amyloidogenic product sAPPα, which has been shown to have neuroprotective properties. Several G protein-coupled receptors are known to activate α-secretase-dependent processing of APP; however, the role of G protein-coupled nucleotide receptors in APP processing has not been investigated. Here it is demonstrated that activation of the G protein-coupled P2Y2 receptor (P2Y2R) subtype expressed in human 1321N1 astrocytoma cells enhanced the release of sAPPα in a time- and dose-dependent manner. P2Y2 R-mediated sAPPα release was dependent on extracellular calcium but was not affected by 1,2-bis(2-aminophenoxy)ethane-N,N,N,-trimethylammonium salt, an intracellular calcium chelator, indicating that P2Y2 R-stimulated intracellular calcium mobilization was not involved. Inhibition of protein kinase C (PKC) with GF109203 or by PKC down-regulation with phorbol ester pre-treatment had no effect on UTP-stimulated sAPPα release, indicating a PKC-independent mechanism. U0126, an inhibitor of the mitogen-activated protein kinase pathway, partially inhibited sAPPα release by UTP, whereas inhibitors of Src-dependent epidermal growth factor receptor transactivation by P2Y2 Rs had no effect. The metalloprotease inhibitors phenanthroline and TAPI-2 and the furin inhibitor decanoyl-Arg-Val-Lys-Arg-chloromethylketone also diminished UTP-induced sAPPα release. Furthermore, small interfering RNA silencing of an endogenous adamalysin, ADAM10 or ADAM17/TACE, partially suppressed P2Y2R-activated sAPPα release, whereas treatment of cells with both ADAM10 and ADAM17/TACE small interfering RNAs completely abolished UTP-activated sAPPα release. These results may contribute to an understanding of the non-amyloidogenic processing of APP. P2 nucleotide receptors modulate a wide range of physiological responses following their activation by extracellular nucleotides (1Ralevic V. Burnstock G. Pharmacol. Rev. 1998; 50: 413-492PubMed Google Scholar, 2Schwiebert E.M. Zsembery A. Biochim. Biophys. Acta. 2003; 1615: 7-32Crossref PubMed Scopus (370) Google Scholar). The G protein-coupled P2Y2 receptor (P2Y2R) 1The abbreviations used are: P2Y2R, P2Y2 receptor; Aβ, amyloid-β peptide; ADAM, a disintegrin and metalloprotease; APP, amyloid-β precursor protein; BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N,-trimethylammonium salt; CMK, decanoyl-Arg-Val-Lys-Arg-chloromethylketone; EGF, epidermal growth factor; EGFR, EGF receptor; ERK, extracellular signal-regulated kinase; GPCR, G protein-coupled receptor; MAPK, mitogen-activated protein kinase; PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate; sAPPα, ectodomain of APP generated by α-secretase; siRNA, small interfering RNA; TNF-α, tumor necrosis factor-α; TACE, TNF-α converting enzyme; TAPI-2, TNF-α protease inhibitor.1The abbreviations used are: P2Y2R, P2Y2 receptor; Aβ, amyloid-β peptide; ADAM, a disintegrin and metalloprotease; APP, amyloid-β precursor protein; BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N,-trimethylammonium salt; CMK, decanoyl-Arg-Val-Lys-Arg-chloromethylketone; EGF, epidermal growth factor; EGFR, EGF receptor; ERK, extracellular signal-regulated kinase; GPCR, G protein-coupled receptor; MAPK, mitogen-activated protein kinase; PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate; sAPPα, ectodomain of APP generated by α-secretase; siRNA, small interfering RNA; TNF-α, tumor necrosis factor-α; TACE, TNF-α converting enzyme; TAPI-2, TNF-α protease inhibitor. subtype is fully activated by equivalent concentrations of ATP or UTP (3Lustig K.D. 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In the present study we demonstrate that activation of a P2Y2 nucleotide receptor expressed in the human 1321N1 astrocytoma cells stimulates ADAM10-mediated APP processing and ADAM17/TACE-mediated APP processing, leading to the release of sAPPα. Because nucleotide release from damaged cells in the central nervous system would be expected to activate P2Y2Rs, these findings suggest a novel neuroprotective role for P2Y2R-mediated APP processing in neurodegenerative disorders, including Alzheimer disease. Materials—All chemicals and reagents were purchased from Sigma unless stated otherwise. Cell culture medium, fetal bovine serum, and G418 were obtained from Invitrogen. U0126, AG1478 and the pyrazole pyrimidine-type 2 inhibitor (PP2) were purchased from Calbiochem. TNF-α protease inhibitor (TAPI-2) was purchased from Peptides International (Louisville, KY), and decanoyl-Arg-Val-Lys-Arg-chloromethylketone (CMK) was purchased from Bachem (Torrance, CA). Cell Culture—Human 1321N1 astrocytoma cells stably transfected with human P2Y2R cDNA (5Parr C.E. Sullivan D.M. Paradiso A.M. Lazarowski E.R. Burch L.H. Olsen J.C. Erb L. Weisman G.A. Boucher R.C. Turner J.T. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 3275-3279Crossref PubMed Scopus (357) Google Scholar) were cultured in Dulbecco's modified Eagle's medium supplemented with 5% (v/v) fetal bovine serum, 100 units/ml penicillin, 100 μg/ml streptomycin, and 500 μg/ml G418 and maintained at 37 °C in a humidified atmosphere of 5% CO2 and 95% air. Cells transfected with a pLXSN expression vector served as a negative control. sAPPα Release—Cells were plated on 12-well culture dishes and grown until ∼80–90% confluence. Cells were washed twice with a modified Krebs-HEPES buffer (15 mm HEPES, pH 7.4, 120 mm NaCl, 4 mm KCl, 1.2 mm MgSO4, 1.2 mm KH2PO4, 1 mm CaCl2, and 10 mm d-glucose) and then incubated on a rocking platform in a humidified incubator at 37 °C for specified times with 200 μl of buffer containing compounds as indicated in the Fig. 4 legend. When indicated, cells were pretreated with inhibitors for 30 min prior to the addition of nucleotides. At the end of the incubation period, the medium was collected, centrifuged at 12,000 × g for 1 min to remove cellular debris, and the protein concentration of the supernatant was determined by the Lowry method (49Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar). 80 μg of supernatant protein was diluted 1:4 with 5× Laemmli buffer, boiled for 3 min, subjected to electrophoresis on 7.5% SDS-polyacrylamide mini-gels, and transferred to nitrocellulose membranes. Membranes were blocked for 1 h with 5% (w/v) nonfat dry milk in TBST (Tris-buffered saline containing 0.1% (v/v) Tween 20) and immunoblotted overnight at 4 °C in 3% (w/v) bovine serum albumin with 0.02% (w/v) sodium azide in TBST with a 6E10 monoclonal antibody (1:1000 dilution; Senetek, Maryland Heights, MO) that recognizes residues 1–17 of the Aβ domain of sAPPα (50Kim S.K. Miller D. Sapienza V. Chen C.M. Bai C. Grundke-Iqbal I. Currie J. Wisniewski H. Neurosci. Res. Commun. 1990; 2: 121-130Google Scholar). Membranes were washed three times during a 45-min period with TBST and incubated with peroxidase-linked goat anti-mouse IgG antibody (1:1000 dilution; Santa Cruz Biotechnology, Santa Cruz, CA) at room temperature for 1 h. After three more washes with TBST, the membrane was subjected to chemiluminescence, and the protein bands detected on x-ray film were quantified using a computer-driven scanner and Quantity One software (Bio-Rad). The amount of sAPPα generated was expressed as a percentage of untreated controls. All experiments were performed in duplicate and repeated at least three times. Analysis of variance and unpaired Student's t test was used to determine statistical significance (p < 0.05). Measurement of the Intracellular Calcium Concentration—The intracellular free calcium concentration ([Ca2+]i) was measured by dual excitation spectrofluorometric analysis of cell suspensions loaded with fura-2 (6Turner J.T. Weisman G.A. Camden J.M. Am. J. Physiol. 1997; 273: C1100-C1107Crossref PubMed Google Scholar) and BAPTA as described previously (51Gendron F.-P. Biomed. Res. 2003; 14: 47-61Google Scholar). Cells were assayed in modified Krebs-HEPES buffer containing 0.1% (w/v) bovine serum albumin. The intracellular calcium concentration was calculated by the method of Grynkiewicz et al. (52Grynkiewicz G. Poenie M. Tsien R.Y. J. Biol. Chem. 1985; 260: 3440-3450Abstract Full Text PDF PubMed Scopus (80) Google Scholar). siRNA Targeting of ADAM10 and ADAM17/TACE Genes—Transfection of cells with siRNA duplexes (Integrated DNA Technologies, Coralville, IA) to inhibit endogenous ADAM10 or ADAM17/TACE expression was performed using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. Briefly, 0.7 μg of each of the three ADAM10 or ADAM17/TACE siRNA duplexes was incubated with cells in serum-free medium for 6 h and then replaced with normal growth media. Sequences of the siRNA duplexes were AUUCGUAGGUUGAAAUGUCdTdT, UUCCAUUUCCACAAAUAGGdTdT, and AGCCAUUACAUAUUCCUUCdTdT (ADAM10) and AGUUUGCUUGGCACACCUUdTdT, AGUAAGGCCCAGGAGUGUdTdT, and CAUAGAGCCACUUUGGAGAdTdT (ADAM17/TACE). Cells were assayed for sAPPα release 48 h after siRNA transfection, and silencing of the targeted protein expression was confirmed by Western analysis of cell lysates as described above for the detection of sAPPα by using goat anti-human ADAM17/TACE antibody (1:1000 dilution; Santa Cruz Biotechnology) or goat anti-human ADAM10 antibody (1:1000 dilution; Sigma). Blots were stripped and reprobed with goat anti-human actin antibody (1: 1000 dilution; Cytoskeleton, Denver, CO) to verify the equivalence of protein loading per lane. P2Y2R Activation Stimulates the Release of sAPPα—UTP (100 μm) caused a time-dependent increase in sAPPα (∼120 kDa) release from human 1321N1 astrocytoma cells stably transfected with P2Y2R cDNA (1321N1-P2Y2 cells) that was significantly greater than basal sAPPα release (Fig. 1, A and B). UTP also caused a dose-dependent increase in sAPPα from 1321N1-P2Y2 cells with an EC50 value of 2.5 μm (Fig. 2), which is characteristic of P2Y2R activation (5Parr C.E. Sullivan D.M. Paradiso A.M. Lazarowski E.R. Burch L.H. Olsen J.C. Erb L. Weisman G.A. Boucher R.C. Turner J.T. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 3275-3279Crossref PubMed Scopus (357) Google Scholar, 6Turner J.T. Weisman G.A. Camden J.M. Am. J. Physiol. 1997; 273: C1100-C1107Crossref PubMed Google Scholar, 53Garrad R.C. Otero M.A. Erb L. Theiss P.M. Clarke L.L. González F.A. Turner J.T. Weisman G.A. J. Biol. Chem. 1998; 273: 29437-29444Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 54Turner J.T. Redman R.S. Camden J.M. Landon L.A. Quissell D.O. Am. J. Physiol. 1998; 275: C367-C374Crossref PubMed Google Scholar). In contrast, UTP-induced sAPPα release was undetectable in untransfected cells or in cells transfected with the expression vector only (data not shown).Fig. 2UTP induces a dose-dependent release of sAPPα from 1321N1-P2Y2 cells. Cells were stimulated for 2 h with the indicated concentration of UTP (0.1–100 μm), and sAPPα release into the medium was analyzed as described under "Experimental Procedures." UTP-induced sAPPα release is expressed as a percentage increase over basal levels. Data points represent the means ± S.E. of results from four experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT) UTP-induced sAPPα Release Is Independent of PKC Activation—The PKC activator phorbol myristate acetate (PMA; 1 μm) also caused a time-dependent release of sAPPα, similar to that caused by UTP (Fig. 1A). Because P2Y2R activation causes the phospholipase C-dependent stimulation of PKC (1Ralevic V. Burnstock G. Pharmacol. Rev. 1998; 50: 413-492PubMed Google Scholar), we determined whether P2Y2R-mediated sAPPα release is dependent on activation of PKC. Therefore, 1321N1-P2Y2 cells were pre-treated for 30 min with GF109203 (10 μm), an inhibitor of PKC, followed by incubation with UTP (100 μm) or PMA (1 μm) for an additional 2 h. GF109203 completely inhibited PMA-activated release of sAPPα but had no effect on UTP-induced release (Fig. 3A). Furthermore, PKC down-regulation by overnight treatment with PMA (1 μm) abolished PMA-stimulated sAPPα release but had no effect on the UTP-induced sAPPα release (Fig. 3B). Thus, UTP-induced sAPPα release is independent of P2Y2R-mediated activation of PKC. UTP-induced sAPPα Release Is Dependent on Extracellular Calcium but Not P2Y2R-mediated Intracellular Calcium Mobilization—Activation of G protein-coupled P2Y2Rs with UTP in 1321N1-P2Y2 cells is known to cause increases in [Ca2+]i through the Gαq-dependent stimulation of phospholipase C, the generation of inositol-1,4,5 triphosphate, and the release of calcium from inositol-1,4,5 triphosphate-sensitive calcium stores (12Weisman G.A. Turner J.T. 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Reprod. 2000; 6: 435-442Crossref PubMed Scopus (37) Google Scholar). Therefore, we examined the role of calcium signaling pathways in UTP-induced sAPPα release. In the absence of extracellular calcium, UTP-stimulated sAPPα release was completely inhibited (Fig. 4A). In contrast, PMA-stimulated sAPPα release was unaffected by the absence of extracellular calcium, suggesting the existence of both calcium entry-dependent and -independent pathways for stimulating sAPPα release. Unexpectedly, introduction into 1321N1-P2Y2 cells of the intracellular calcium chelator BAPTA, which suppresses P2Y2R-mediated increases in [Ca2+]i (Fig. 4B), had no effect on sAPPα release induced by UTP (Fig. 4A). These data suggest that sAPPα release due to P2Y2R activation is independent of increases in cytosolic calcium. Consistent with this conclusion, neither ionomycin, a calcium ionophore, nor thapsigargin, an inhibitor of the plasma membrane Ca2+-ATPase, caused an increase in sAPPα release (data not shown). Inhibition of MAPK (ERK1/2) Partially Inhibits UTP-stimulated sAPPα Release—Previous studies in our laboratory have shown that UTP activates ERK1/2 phosphorylation in 1321N1-P2Y2 cells, in part via the Src-dependent transactivation of the EGF and platelet-derived growth factor receptors (14Liu J. Liao Z. Camden J. Griffin K.D. Garrad R.C. Santiago-Perez L.I. Gonzalez F.A. Seye C.I. Weisman G.A. Erb L. J. Biol. Chem. 2004; 279: 8212-8218Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar), consistent with Src-dependence of ERK1/2 phosphorylation by other GPCRs (56Luttrell L.M. Della Rocca G.J. van Biesen T. Luttrell D.K. Lefkowitz R.J. J. Biol. Chem. 1997; 272: 4637-4644Abstract Full Text Full Text PDF PubMed Scopus (428) Google Scholar, 57Cao W. Luttrell L.M. Medvedev A.V. Pierce K.L. Daniel K.W. Dixon T.M. Lefkowitz R.J. Collins S. J. Biol. Chem. 2000; 275: 38131-38134Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar). Therefore, we investigated the role of the MAPK/EGF receptor (EGFR) pathway in UTP-stimulated sAPPα release. U0126, an inhibitor of MAPK/ERK kinase (MEK) whose substrates are ERK1/2, partially inhibited (∼40%) sAPPα release induced by UTP but not by PMA (Fig. 5). Incubation of cells with the Src inhibitor pyrazole pyrimidine-type 2 (10 μm) or the EGFR kinase inhibitor AG1478 (10 μm) had no effect on the UTP-stimulated or PMA-stimulated sAPPα release (data not shown). These results suggest that UTP-stimulated sAPPα release in 1321N1-P2Y2 cells is partially dependent on phosphorylation of ERK1/2 but independent of Src and EGFR activation. Inhibitors of Metalloproteases and Furin, the Proprotein Convertase, Decrease UTP-induced sAPPα Release—Disintegrin metalloproteases, including ADAM10 and ADAM17/TACE, catalyze the shedding of the ectodomain of APP and other transmembrane proteins (58Allinson T.M. Parkin E.T. Turner A.J. Hooper N.M. J. Neurosci. Res. 2003; 74: 342-352Crossref PubMed Scopus (379) Google Scholar). We determined that P2Y2R-mediated sAPPα release was inhibited by phenanthroline, a broad
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