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• W1992463891 abstract "Programmed cell death (pcd) may take the form of apoptosis or of nonapoptotic pcd. Whereas cysteine aspartyl-specific proteases (caspases) mediate apoptosis, the mediators of nonapoptotic cell death programs are much less well characterized. Here we report that alternative, nonapoptotic pcd induced by the neurokinin-1 receptor (NK1R) activated by its ligand Substance P, is mediated by a MAPK phosphorylation cascade recruited by the scaffold protein arrestin 2. The activation of the protein kinases Raf-1, MEK2, and ERK2 is essential for this form of nonapoptotic pcd, leading to the phosphorylation of the orphan nuclear receptor Nur77. NK1R-mediated cell death was inhibited by a dominant negative form of arrestin 2, Raf-1, or Nur77, by MEK1/2-specific inhibitors, and by RNA interference directed against ERK2 or MEK2 but not ERK1 or MEK1 and against Nur77. The MAPK pathway is also activated in neurons in primary culture undergoing NK1R-mediated death, since the MEK inhibitor PD98059 inhibited Substance P-induced death in primary striatal neurons. These results suggest that Nur77, which is regulated by a MAPK pathway activated via arrestin 2, modulates NK1R-mediated nonapoptotic pcd. Programmed cell death (pcd) may take the form of apoptosis or of nonapoptotic pcd. Whereas cysteine aspartyl-specific proteases (caspases) mediate apoptosis, the mediators of nonapoptotic cell death programs are much less well characterized. Here we report that alternative, nonapoptotic pcd induced by the neurokinin-1 receptor (NK1R) activated by its ligand Substance P, is mediated by a MAPK phosphorylation cascade recruited by the scaffold protein arrestin 2. The activation of the protein kinases Raf-1, MEK2, and ERK2 is essential for this form of nonapoptotic pcd, leading to the phosphorylation of the orphan nuclear receptor Nur77. NK1R-mediated cell death was inhibited by a dominant negative form of arrestin 2, Raf-1, or Nur77, by MEK1/2-specific inhibitors, and by RNA interference directed against ERK2 or MEK2 but not ERK1 or MEK1 and against Nur77. The MAPK pathway is also activated in neurons in primary culture undergoing NK1R-mediated death, since the MEK inhibitor PD98059 inhibited Substance P-induced death in primary striatal neurons. These results suggest that Nur77, which is regulated by a MAPK pathway activated via arrestin 2, modulates NK1R-mediated nonapoptotic pcd. Programmed cell death (pcd) 1The abbreviations used are: pcd, programmed cell death; SP, substance P; NK1R, neurokinin 1 receptor; RNAi, RNA interference; MAP, mitogen-activated protein; MAPK, MAP kinase; JNK, c-Jun N-terminal kinase; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; Rp-8-Br-cAMPS, 8-bromoadenosine 3′,5′-cyclic monophosphorothioate, Rp-isomer; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ERK, extracellular signal-regulated kinase; siRNA, small interfering RNA; GFP, green fluorescent protein; TES, 2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic acid; NGF, nerve growth factor. 1The abbreviations used are: pcd, programmed cell death; SP, substance P; NK1R, neurokinin 1 receptor; RNAi, RNA interference; MAP, mitogen-activated protein; MAPK, MAP kinase; JNK, c-Jun N-terminal kinase; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; Rp-8-Br-cAMPS, 8-bromoadenosine 3′,5′-cyclic monophosphorothioate, Rp-isomer; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ERK, extracellular signal-regulated kinase; siRNA, small interfering RNA; GFP, green fluorescent protein; TES, 2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic acid; NGF, nerve growth factor. is a form of cell death in which the cell plays an active role in its own demise. Although pcd has often been equated with apoptosis, it has become increasingly clear that nonapoptotic forms of pcd also exist (1Schweichel J.U. Z. Anat. Entwicklungsgesch. 1972; 136: 192-203Crossref PubMed Scopus (23) Google Scholar, 2Schweichel J.U. Merker H.J. Teratology. 1973; 7: 253-266Crossref PubMed Scopus (558) Google Scholar, 3Schwartz L.M. BioEssays. 1991; 13: 389-395Crossref PubMed Scopus (82) Google Scholar, 4Clarke P.G. Anat. Embryol. 1990; 181: 195-213Crossref PubMed Scopus (1528) Google Scholar, 5Lockshin R.A. Williams C.M. J. Insect Physiol. 1964; 10: 643-649Crossref Scopus (259) Google Scholar, 6Pilar G. Landmesser L. J. Cell Biol. 1976; 68: 339-356Crossref PubMed Scopus (223) Google Scholar, 7Oppenheim R.W. Annu. Rev. Neurosci. 1991; 14: 453-501Crossref PubMed Scopus (2750) Google Scholar, 8Oppenheim R.W. Trends Neurosci. 1985; 17: 487-493Abstract Full Text PDF Scopus (254) Google Scholar, 9Cunningham T.J. Int. Rev. Cytol. 1982; 74: 163-186Crossref PubMed Scopus (272) Google Scholar, 10Turmaine M. Raza A. Mahal A. Mangiarini L. Bates G.P. Davies S.W. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8093-8097Crossref PubMed Scopus (380) Google Scholar, 11Dal Canto M.C. Gurney M.E. Am. J. Pathol. 1994; 145: 1271-1279PubMed Google Scholar, 12Sperandio S. de Belle I. Bredesen D.E. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 14376-14381Crossref PubMed Scopus (777) Google Scholar, 13Majno G. Joris I. Am. J. Pathol. 1995; 146: 3-15PubMed Google Scholar). For example, certain developmental cell deaths, such as “autophagic” cell death (1Schweichel J.U. Z. Anat. Entwicklungsgesch. 1972; 136: 192-203Crossref PubMed Scopus (23) Google Scholar, 2Schweichel J.U. Merker H.J. Teratology. 1973; 7: 253-266Crossref PubMed Scopus (558) Google Scholar, 3Schwartz L.M. BioEssays. 1991; 13: 389-395Crossref PubMed Scopus (82) Google Scholar, 4Clarke P.G. Anat. Embryol. 1990; 181: 195-213Crossref PubMed Scopus (1528) Google Scholar, 5Lockshin R.A. Williams C.M. J. Insect Physiol. 1964; 10: 643-649Crossref Scopus (259) Google Scholar) and “cytoplasmic” cell death (2Schweichel J.U. Merker H.J. Teratology. 1973; 7: 253-266Crossref PubMed Scopus (558) Google Scholar, 4Clarke P.G. Anat. Embryol. 1990; 181: 195-213Crossref PubMed Scopus (1528) Google Scholar, 6Pilar G. Landmesser L. J. Cell Biol. 1976; 68: 339-356Crossref PubMed Scopus (223) Google Scholar, 7Oppenheim R.W. Annu. Rev. Neurosci. 1991; 14: 453-501Crossref PubMed Scopus (2750) Google Scholar, 8Oppenheim R.W. Trends Neurosci. 1985; 17: 487-493Abstract Full Text PDF Scopus (254) Google Scholar, 9Cunningham T.J. Int. Rev. Cytol. 1982; 74: 163-186Crossref PubMed Scopus (272) Google Scholar), do not resemble apoptosis. Furthermore, neurodegenerative diseases such as Huntington's disease and amyotrophic lateral sclerosis demonstrate neuronal cell death that does not fulfill the criteria for apoptosis (10Turmaine M. Raza A. Mahal A. Mangiarini L. Bates G.P. Davies S.W. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8093-8097Crossref PubMed Scopus (380) Google Scholar, 11Dal Canto M.C. Gurney M.E. Am. J. Pathol. 1994; 145: 1271-1279PubMed Google Scholar) but instead resembles a recently described form of pcd dubbed paraptosis (12Sperandio S. de Belle I. Bredesen D.E. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 14376-14381Crossref PubMed Scopus (777) Google Scholar). Ischemia-induced cell death may also display a nonapoptotic morphology, referred to as “oncosis” (13Majno G. Joris I. Am. J. Pathol. 1995; 146: 3-15PubMed Google Scholar).The biochemical basis for these alternative morphological forms of cell death remains largely unknown. Understanding the mechanisms for these forms would have potentially important implications for the understanding of evolutionary aspects of cell death programs, developmental cell death, neurodegeneration, and cancer therapeutics and for the design of novel therapeutic agents for diseases featuring these alternative forms of cell death.Cell death has been divided into two main types: pcd, in which the cell plays an active role, and passive (necrotic) cell death. The pcd observed during development and tissue homeostasis has been classified morphologically into three main types: type 1, also known as nuclear or apoptotic; type 2 or autophagic; and type 3, also referred to as cytoplasmic (4Clarke P.G. Anat. Embryol. 1990; 181: 195-213Crossref PubMed Scopus (1528) Google Scholar).Apoptosis is the best characterized type of pcd, in which the cells display membrane blebbing, flipping of phosphatidylserine in the plasma membrane (14Fadok V.A. Voelker D.R. Campbell P.A. Cohen J.J. Bratton D.L. Henson P.M. J. Immunol. 1992; 148: 2207-2216PubMed Google Scholar), nuclear fragmentation, and activation of a family of cell suicide cysteine proteases referred to as caspases (15Yuan J. Shaham S. Ledoux S. Ellis H.M. Horvitz H.R. Cell. 1993; 75: 641-652Abstract Full Text PDF PubMed Scopus (2234) Google Scholar, 16Thornberry N.A. Lazebnik Y. Science. 1998; 281: 1312-1316Crossref PubMed Scopus (6133) Google Scholar). The biochemical activation of apoptosis occurs through two general pathways: the intrinsic pathway, originating from mitochondrial release of cytochrome c and associated activation of caspase-9, and the extrinsic pathway, originating from the activation of cell surface death receptors such as Fas and resulting in the activation of caspase-8 or -10 (17Salvesen G.S. Dixit V.M. Cell. 1997; 91: 443-446Abstract Full Text Full Text PDF PubMed Scopus (1932) Google Scholar). A third general pathway, originating from the endoplasmic reticulum and resulting in the activation of caspase-12 and -9, has also recently been described (18Yuan J. Yankner B.A. Nat. Cell Biol. 1999; 1: E44-45Crossref PubMed Scopus (40) Google Scholar, 19Rao R.V. Hermel E. Castro-Obregon S. del Rio G. Ellerby L.M. Ellerby H.M. Bredesen D.E. J. Biol. Chem. 2001; 276: 33869-33874Abstract Full Text Full Text PDF PubMed Scopus (544) Google Scholar, 20Rao R.V. Castro-Obregon S. Frankowski H. Schuler M. Stoka V. Del Rio G. Bredesen D.E. Ellerby H.M. J. Biol. Chem. 2002; 277: 21836-21842Abstract Full Text Full Text PDF PubMed Scopus (435) Google Scholar, 21Rao R.V. Peel A. Logvinova A. del Rio G. Hermel E. Yokota T. Goldsmith P.C. Ellerby L.M. Ellerby H.M. Bredesen D.E. FEBS Lett. 2002; 514: 122-128Crossref PubMed Scopus (499) Google Scholar, 22Morishima N. Nakanishi K. Takenouchi H. Shibata T. Yasuhiko Y. J. Biol. Chem. 2002; 277: 34287-34294Abstract Full Text Full Text PDF PubMed Scopus (792) Google Scholar).Much less is known about the biochemical mediators of type 2 and type 3 programmed cell death. Type 2 (autophagic) cell death can be activated in some cases by Ras (23Chi S. Kitanaka C. Noguchi K. Mochizuki T. Nagashima Y. Shirouzu M. Fujita H. Yoshida M. Chen W. Asai A. Himeno M. Yokoyama S. Kuchino Y. Oncogene. 1999; 18: 2281-2290Crossref PubMed Scopus (208) Google Scholar), whereas the molecular activation of type 3 cell death is unknown. Recently, it was noted that the binding of the undecapeptide neurotransmitter Substance P (SP) to its receptor, neurokinin-1 receptor (NK1R), induces a nonapoptotic form of pcd resembling type 2 or 3 morphologically and characterized by cytoplasmic vacuolation, lack of caspase activation, lack of inhibition by caspase inhibitors (benzyloxycarbonyl-VAD-fluoromethyl ketone and Boc-aspartyl fluoromethyl ketone) and by Bcl-xL, lack of nuclear fragmentation or membrane blebbing (Fig. 1), and a requirement for new gene transcription and translation (24Castro-Obregon S. Del Rio G. Chen S.F. Swanson R.A. Frankowski H. Rao R.V. Stoka V. Vesce S. Nicholls D.G. Bredesen D.E. Cell Death Differ. 2002; 9: 807-817Crossref PubMed Scopus (59) Google Scholar).The development of specific agonists and antagonists for NK1R has supported a role for NK1R in numerous biological processes, such as the transmission of pain in the spinal cord. In the central nervous system, it also regulates cardiovascular and respiratory function and is involved in activating the emetic reflex. NK1R also regulates several behavioral responses and has recently been implicated in depression and schizophrenia. It appears to be involved in a wide variety of functions due to its ability to modulate the release of other neurotransmitters, such as excitatory amino acids (25Quartara L. Maggi C.A. Neuropeptides. 1998; 32: 1-49Crossref PubMed Scopus (263) Google Scholar).SP also seems to play an important role in pathological states in which neural cell death occurs, such as status epilepticus and ischemia. For example, SP-null mice demonstrate resistance to excitotoxin-induced seizures, with an associated reduction in neuronal death (26Liu H. Cao Y. Basbaum A.I. Mazarati A.M. Sankar R. Wasterlain C.G. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12096-12101Crossref PubMed Scopus (104) Google Scholar). Similarly, treatment with an antagonist for NK1R inhibits seizures and reduces kainic acidinduced cell death in the CA1 region of the hippocampus (27Zachrisson O. Lindefors N. Brene S. Brain Res. Mol. Brain Res. 1998; 60: 291-295Crossref PubMed Scopus (37) Google Scholar). Furthermore, in a model of focal cerebral ischemia, administration of an NK1R antagonist reduced infarct volume and improved neurological function (28Yu Z. Cheng G. Huang X. Li K. Cao X. Neuroreport. 1997; 8: 2117-2119Crossref PubMed Scopus (49) Google Scholar). Taken together, these observations suggest that NK1R may be a mediator of cell death in vivo.In the current report, we demonstrate that SP/NK1R-induced cell death is mediated by a MAP kinase activation pathway involving Raf-1, MEK2, and extracellular signal-regulated protein kinase 2 (ERK2), upon recruitment by the scaffold protein arrestin 2, in both neuronal and nonneuronal cells. The activation of ERK2 leads to the phosphorylation of Nur77, whose activity is essential for the progression to cell death.MATERIALS AND METHODSNeuronal Primary CulturePrimary striatal cultures were prepared from 17-day-old Sprague-Dawley rat embryos (B&K, Fremont, CA). The tissue was dissected, minced, and trypsinized for 5 min using 0.25% trypsin (Cellgro). After the addition of 10% horse serum to inhibit the trypsin, the cell suspension was triturated 15–20 times with a 10-ml syringe and centrifuged for 5 min at 800 relative centrifugal force. The pellet was resuspended in MEM-PAK (University of California-San Francisco Cell Culture facility), supplemented with 2.02 mg of glucose, 2 mm GlutaMax (Invitrogen), and penicillin/streptomycin (100 units/ml). The suspension was filtered through a 70-μm cell strainer, and the final culture medium contained 5% horse serum (Invitrogen). 3–4 × 105 cells/cm2 were seeded onto either poly-d-lysine-precoated eight-well chamber slides (BD Biosciences) or 96-well plates precoated with 50 μg/ml poly-d-lysine (Sigma) in water. After a 30-min incubation, unattached cells were removed together with the medium and replaced with glucose-enriched MEM-PAK plus 5% horse serum. The cultures were incubated at 37 °C in 95% air, 5% carbon dioxide with 95% humidity. Cultures were used for experiments between day 1 and day 4 when glial contamination was at a minimum. 0.1 nm to 100 μm SP (Sigma) was added 24–48 h after seeding in the presence of 2.5% horse serum. Viability was quantified by trypan blue staining of the total cell population.cAMP QuantificationTotal cellular cAMP was measured using the Biotrak nonacetylation enzyme immunoassay system (Amersham Biosciences), following the manufacturer's instructions. Forskolin (Sigma) was added at 10 μm.Imaging of [Ca2+]c in Single CellsSingle cell imaging was performed in a Merlin imaging facility (Olympus America) using an Olympus IX70 inverted epifluorescence microscope equipped with a × 40 oil immersion objective and a Spectramaster monochromator (Life Science Resources, Cambridge, UK) (excitation, 340 and 380 nm; emission, >505 nm). Cells were loaded with 3 μm Fura-2/AM (from TEF LABS) for 30 min in incubation medium containing 120 mm NaCl, 3.5 mm KCl, 1.3 mm CaCl2, 0.4 mm KH2PO4, 20 mm TES buffer, 5 mm NaHCO3, 1.2 mm Na2SO4, 15 mm glucose, 1.2 mm MgCl2, pH adjusted to 7.4 with NaOH. 30 μg/ml bovine serum albumin was also added. Experiments were performed in a nonperfusing thermostatted chamber (37 °C) with incubation medium without bovine serum albumin (71Castilho R.F. Hansson O. Ward M.W. Budd S.L. Nicholls D.G. J. Neurosci. 1998; 18: 10277-10286Crossref PubMed Google Scholar). 2 μm ionomycin (Sigma) and 10 μm EGTA (Sigma) were added at the times indicated in Fig. 1B.InhibitorsProtein kinase inhibitors were added 30 min before 100 nm SP exposure at the following concentrations: 10 μm adenosine 3′,5′-cyclic monophosphorothioate, 8-bromo-, Rp-isomer, sodium salt (Rp-8-Br-cAMPS), which is a protein kinase A inhibitor; 50 nm calphostin C, which is a protein kinase C inhibitor; 10 μm PD 98059 and 10 μm U0126, which are MEK1/2 inhibitors; 10 μm SB203580, which is a p38 inhibitor; and 10 μm SP600125, which is a JNK inhibitor. These compounds were obtained from Calbiochem. The caspase inhibitor Boc-Asp(Ome)CH2F was obtained from Enzyme Systems Products (Livermore, CA).NK1R Mutants, Plasmids, and TransfectionsNK1R mutants were created using the QuikChange™ strategy (Stratagene, La Jolla, CA), using the following sequences for the oligonucleotides: 5′-GTGGACCTGGCCTTCGCTCAGGCCTGCATGGCTGCATTC-3′ for the NK1RE78Q mutation and 5′-GGAAATGAAATCCACCCGATACCTCTAGGCATATTCAAGGCATGC-3′ for NK1RΔ342 truncation. Human embryonic kidney 293T cells were grown in high glucose Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% fetal bovine serum (Sigma) and penicillin/streptomycin (100 units/ml) (Invitrogen). The cultures were incubated at 37 °C in 95% air and 5% carbon dioxide with 95% humidity. Transient transfection was performed using Superfect (Qiagen) as described by the manufacturer. Briefly, 2 × 105 cells/well were seeded into 35-mm wells 16 h prior to transfection. Superfect-DNA complexes were added at a 5 μl/2 μg ratio for 3 h; then the medium was replaced, and after 24 h, SP was added. Expression of each construct in the transient transfections was determined by Western blot and efficiency of transient transfections by immunocytochemistry (see Fig. 11). Transient transfection efficiencies were in all cases >80%, which was compatible with previously reported use of the same system (see Ref. 72Castro-Obregon S. del Rio G. Chen S. Frankowski H. Bredesen D.E. Cell Death Differ. 2002; 9: 807-817Crossref PubMed Scopus (61) Google Scholar; see Figs. 4 and 6 therein). The dominant negative constructs evaluated were shown not to reduce NK1R expression (see Fig. 11). 24–48 h after the addition of SP, cell death was quantified as follows. The media containing floating cells were collected and centrifuged for 5 min at 2000 relative centrifugal force, the pellet was resuspended in PBS/trypan blue (1:10), and the blue cells were counted using a hematocytometer.Fig. 11The efficiency of the transfection of the NK1R plasmid is not affected by the co-transfection with plasmids encoding proteins that interfere with the death pathway activated by SP/NK1R. HEK293T cells were co-transfected at a ratio of 1:3 with the plasmid encoding NK1R and the following plasmids: GFP (A), dominant negative arrestin 2 (B), Nur77ΔDBD-GFP (C), Nur77ΔN152-GFP (D), or the empty vector (E). The transfected cells were identified by immunocytochemistry using an anti-NK1R antibody (the secondary antibody was Cy3-labeled (red)) or an anti-arrestin 2 antibody (the secondary antibody was coupled to fluorescein isothiocyanate (green)) or by GFP expression. The Nur77 mutants are fused to GFP. The nucleus was stained with 4′,6-diamidino-2-phenylindole (DAPI). The images were magnified × 400.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 4Expression of dominant negative Raf-1 inhibits SP/NK1R induction of cell death. Death induced by SP treatment of 293T cells transfected with NK1R was blocked by co-transfection of the Raf-1 dominant-negative mutant, c-Raf-C4. 24 h following co-transfection of empty vector (Control), NK1R and empty vector (NK1R+Control) or NK1R and c-Raf-C4 (NK1R+DN-Raf), SP was added to 293T cells, and cell death was quantified after 48 h by trypan blue exclusion. To eliminate any potential proapoptotic effects of c-Raf-C4 expression, all 293T cells were treated with the pancaspase inhibitor Boc-Asp(Ome)CH2F (35 μm) prior to transfection, a treatment shown previously to have no effect on nonapoptotic pcd mediated by NK1R. The error bars represent S.D. (number of independent experiments, n = 3). *, p < 0.025 calculated by paired t test analysis.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 6RNAi directed against ERK2 or MEK2, but not ERK1 or MEK1, inhibits SP/NK1R-induced death. A, 293T cells were transfected with NK1R and a siRNA specific for the indicated genes, and the cell death in response to SP was evaluated after 24 h. The error bars represent S.D. (number of duplicated independent experiments, n = 3). ***, p < 0.001, calculated by two-way analysis of variance with Bonferroni post hoc test. B, Western blots show the specific inhibition by the siRNAs used. Left panel, siRNA targeting GAPDH or luciferase (Luc), blot development with anti-GAPDH. Central panel, specific inhibition of ERK1 with siRNA targeting ERK1, or ERK2 with siRNA targeting ERK2, whereas siRNAs targeting other genes had no effect on them; the blot was developed with anti-ERK1/2. Right panel, specific inhibition of MEK1 with siRNA targeting MEK1, but not MEK2, as revealed with an antibody with higher affinity for MEK1 than for MEK2 (MEK1); the siRNA targeting MEK2 reduced the amount of protein detected with an antibody that recognizes both MEK1 and MEK2; therefore, the band shown is likely to correspond to MEK1 that was not silenced by siMEK2 (MEK1 and MEK2 overlap in their gel migration) (MEK1/2).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Nur77 plasmids were described by Li et al. (52Li H. Kolluri S.K. Gu J. Dawson M.I. Cao X. Hobbs P.D. Lin B. Chen G. Lu J. Lin F. Xie Z. Fontana J.A. Reed J.C. Zhang X. Science. 2000; 289: 1159-1164Crossref PubMed Scopus (585) Google Scholar).Western Blot AnalysisFor the transfected HEK293T cells, the cells were washed with cold PBS after incubation with SP for the time indicated and homogenized in lysis buffer (150 μm NaCl, 1% Triton X-100, 50 μm Tris-HCl, pH 8.0, 2 mm Na3VaO3, 20 mm NaF, 20 mm glycero-2-phosphate, proteinase inhibitor mixture (Roche Applied Science)). Cytoplasmic extracts were collected after a 10-min centrifugation at 14,000 relative centrifugal force. Protein was quantified by Bradford assay, and electrophoresis of equal amounts of total protein was performed on SDS-polyacrylamide gels. Separated proteins were transferred to polyvinylidene fluoride membranes at 4 °C for Western blot analysis. Membranes were probed with a 1:1000 dilution of anti-ERK1/2 or anti-phospho-ERK1/2, (BioSource, Camarillo, CA); a 1:8000 dilution of anti-GAPDH (Research Diagnostics, Flanders, NJ); 1:1000 dilution of anti-MEK1 monoclonal antibody (StressGen Biotechnologies Corp., Victoria, Canada); a 1:1000 dilution of anti-MEK1/2, anti-p38, anti-phospho-p38, anti-JNK1/2, anti-phospho-JNK1/2, or anti-phosphothreonine monoclonal antibody (Cell Signaling Technology, Inc., Beverly, MA); or a 1:200 dilution anti-Nur77 (M-210) (Santa Cruz Biotechnology, Inc., Santa Cruz CA). The membranes were incubated in the appropriate horseradish peroxidase-coupled secondary antibody for 1 h followed by enhanced chemiluminescence detection of the proteins with ECL reagent (Amersham Biosciences). The immunoprecipitations were carried out using superparamagnetic Microbeads conjugated to protein A, following the manufacturer's instructions (MACS; Miltenyi Biotec, Auburn, CA).RNA Interference (RNAi)Synthesis—The siRNA was generated by in vitro transcription using the Silencer siRNA Construction Kit (Ambion, Austin, TX), following the manufacturer's instructions. Two regions were targeted for each gene, which resulted in similar levels of silencing with either siRNA, so both were combined. The accession numbers given below are from GenBank™. The regions targeted were ERK1 (accession number X60188) 245–265 and 1018–1038; ERK2 (accession number HUMERK2A) 466–486 and 1225–1245; MEK1 (accession number NM_002755) 241–261 and 1216–1236; MEK2 (accession number HUMMEK2NF) 562–582 and 1165–1185; Nur77 (accession number NM_173157) 943–953 and 1845–1865. The siRNA targeting GAPDH was synthesized using the oligonucleotides provided by the kit. An siRNA control sequence was synthesized targeting the region 153–173 of firefly luciferase gene from the plasmid pGL2-control (accession number X65324).siRNA Transfection—Human embryonic kidney 293T cells (105 cells/well in 12-well plates) were grown in high glucose Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (Sigma) with no antibiotics for 16 h. Then NK1R cDNA was transfected as described above. 24 h later, the siRNA specific for each target gene was transfected with TransIT-TKO reagent (Mirus Corp., Madison, WI) according to the manufacturer's instructions. Briefly, for one well, 2 μl of TransIT-TKO and 20 nm siRNA (two target sequences for each gene were combined) were diluted with OPTI-MEM (Invitrogen) to make a final volume of 100 μl, incubated 15 min at room temperature, added to the cells, and incubated for 4 h at 37°C. Then 1 ml of fresh medium was added to each well and incubated for an additional 20 h. After that, 100 nm SP was administered for 24 h, and the samples were collected for Western and cell death assays. Alternatively, the siRNA was transfected with TransMessenger (Qiagen, Valencia, CA) following the manufacturer's instructions. Briefly, for one well (5 × 104 cells/well, 24-well format), 1.6 μl of Enhancer R and 100 nm siRNA were diluted in buffer EC to obtain a final volume of 100 μl. After 10 min at room temperature, 4 μl of TransMessenger reagent were mixed gently and incubated for another 10 min at room temperature, and then the complex was diluted with 100 μl of OPTI-MEM. The media were removed from the wells, and the complex mixture was added to the cells and incubated for 3 h at 37 °C, after which time the media were replaced with fresh media and incubated for an additional 20 h. To estimate the efficiency of the transfection, the siRNAs for luciferase (as a sequence irrelevant for the mammalian genome) and ERK2 were chemically synthesized and fluorescently labeled with fluorescein isothiocyanate (Xeragon-Qiagen, Valencia, CA).Microarray Generation and ProcessingCustom DNA microarrays were prepared from a Research Genetics human cDNA library (Invitrogen), by amplifying material using PCR from 8432 different cDNAs under standard conditions. This was facilitated through use of a volumetric robot (Genesis, RSP150; Tecan, Durham, NC) and several quality controls to verify the amplification of unique products for each gene, including gel electrophoretic sizing of all amplicons. Amplified products were transferred to 384-well plates and purified using commercially available kits (Millipore Corp., Billerica, MA). The purified PCR products were transferred to 384-well print plates (Genetix, Beaverton, OR), dried, and resuspended in 6 μl of print buffer (50% Me2SO in 0.04× SSC). High density spotted cDNA microarrays were generated on commercially available glass slides (MWG Epoxy; MWG Biotech Inc., High Point, NC) with a commercial microarrayer (Omnigene; GeneMachines, San Carlos, CA) at the Genomics Facility of the Buck Institute. For each printed array, there were a total of 976 buffer controls, which facilitated differentiating signal from noise. The total number of analyzable genes per array, after removing PCR failures and genes with nondetectable expression, was 7483.Total RNA was prepared from four independent experiments of HEK293T cells transfected with NK1R and treated (or not) with SP for 30 min, 3 h, or 6 h. The RNA quality was assessed by measuring the size distribution on an Agilent Bioanalyzer (Agilent Technologies, Palo Alto, CA) and by measuring the spectrophotometric 260/280 ratio (>1.8). RNA was labeled with either Cy3 or Cy5 using a commercially available kit (3DNA array 50, Genisphere Inc., Mahwah, NJ).Microarray Data Acquisition and AnalysisAfter hybridization and washing at high stringency, chips were scanned using a Packard Bioscience scanner (Hewlett Packard, Palo Alto, CA), using ScanArray Express (version 1.1) (PerkinElmer Life Sciences). The scans were carried out using a PMT laser setting of 75 and power of 80. The resulting TIFF files were then analyzed using Quantarray 3 (PerkinElmer Life Sciences) using fixed circle and mean intensity for spot and background quantification. Data were LOWESS-normalized using the GeneTraffic software suite (Iobion Informatics, La Jolla, CA). For identifying statistically significant differential expression in the different treatment groups, eight hybridizations per gene (technical duplicates plus experimental duplicates) were analyzed. A list of the genes whose expression presented a mean log2 ratio (experimental/reference) of ≥0.6, with a coefficient of variance (COV = S.D./mean log2 ratio) of <30% were included, giving a total of 91 differentially regulated genes out of 7483.RESULTSSP/NK1R Death Signaling Is Independent of G-protein Activation—NK1R is a G-protein-coupled receptor that is connected by various second messengers to a wide variety of effector mechanisms to modulate cellular function. Three appare" @default.
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• W1992463891 date "2004-04-01" @default.
• W1992463891 modified "2023-10-01" @default.
• W1992463891 title "Alternative, Nonapoptotic Programmed Cell Death" @default.
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