Retrograde Transport of Transcription Factor NF-κB in Living Neurons
2001; Elsevier BV; Volume: 276; Issue: 15 Linguagem: Inglês
10.1074/jbc.m009253200
ISSN1083-351X
AutoresHenning Wellmann, Barbara Kaltschmidt, Christian Kaltschmidt,
Tópico(s)NF-κB Signaling Pathways
ResumoThe mechanism by which signals such as those produced by glutamate are transferred to the nucleus may involve direct transport of an activated transcription factor to trigger long-term transcriptional changes. Ionotropic glutamate receptor activation or depolarization activates transcription factor NF-κB and leads to translocation of NF-κB from the cytoplasm to the nucleus. We investigated the dynamics of NF-κB translocation in living neurons by tracing the NF-κB subunit RelA (p65) with jellyfish green fluorescent protein. We found that green fluorescent protein-RelA was located in either the nucleus or cytoplasm and neurites, depending on the coexpression of the cognate inhibitor of NF-κB, IκB-α. Stimulation with glutamate, kainate, or potassium chloride resulted in a redistribution of NF-κB from neurites to the nucleus. This transport depended on an intact nuclear localization signal on RelA. Thus, in addition to its role as a transcription factor, NF-κB may be a signal transducer, transmitting transient glutamatergic signals from distant sites to the nucleus. The mechanism by which signals such as those produced by glutamate are transferred to the nucleus may involve direct transport of an activated transcription factor to trigger long-term transcriptional changes. Ionotropic glutamate receptor activation or depolarization activates transcription factor NF-κB and leads to translocation of NF-κB from the cytoplasm to the nucleus. We investigated the dynamics of NF-κB translocation in living neurons by tracing the NF-κB subunit RelA (p65) with jellyfish green fluorescent protein. We found that green fluorescent protein-RelA was located in either the nucleus or cytoplasm and neurites, depending on the coexpression of the cognate inhibitor of NF-κB, IκB-α. Stimulation with glutamate, kainate, or potassium chloride resulted in a redistribution of NF-κB from neurites to the nucleus. This transport depended on an intact nuclear localization signal on RelA. Thus, in addition to its role as a transcription factor, NF-κB may be a signal transducer, transmitting transient glutamatergic signals from distant sites to the nucleus. For short-term signals (e.g. synaptic activity) to trigger long-term changes, differential gene expression is required (1Nguyen P.V. Abel T. Kandel E.R. Science.. 1994; 265: 1104-1107Google Scholar, 2Kang H. Schuman E.M. Science.. 1996; 273: 1402-1406Google Scholar, 3Frey U. Morris R.G. Nature.. 1997; 385: 533-536Google Scholar, 4Martin K.C. Casadio A. Zhu H., E.Y. Rose J.C. Chen M. Bailey C.H. Kandel E.R. Cell.. 1997; 91: 927-938Google Scholar). This raises the issue of determining the signaling systems that translate short-term signals to changes in gene expression. Two possible mechanisms are a signal transducer that is retrograde-transported and that subsequently transmits information to a transcription factor, or a transcription factor that independently can fulfill both functions. There is evidence for each mechanism in different systems. With regard to the former mechanism, the nerve growth factor TrkA receptor functions in complex with nerve growth factor as a retrograde signal transducer, connecting extracellular signals at distant sites with nuclear gene expression via phosphorylation of the transcription factor CREB1 (5Riccio A. Pierchala B.A. Ciarallo C.L. Ginty D.D. Science.. 1997; 277: 1097-1100Google Scholar). NF-κB is a transcription factor that may act via the latter mechanism. Recently, it was reported that potentiated synapses are marked with a molecular tag that may sequester relevant proteins necessary for changes in gene expression (3Frey U. Morris R.G. Nature.. 1997; 385: 533-536Google Scholar). NF-κB is present in synaptic compartments (6Kaltschmidt C. Kaltschmidt B. Baeuerle P.A. Mech. Dev... 1993; 43: 135-147Google Scholar, 7Guerrini L. Blasi F. Denis D.S. Proc. Natl. Acad. Sci. U. S. A... 1995; 92: 9077-9081Google Scholar, 8Meberg P.J. Kinney W.R. Valcourt E.G. Routtenberg A. Mol. Brain Res... 1996; 38: 179-190Google Scholar, 9Suzuki T. Mitake S. Okumura-Noji K. Yang J.P. Fujii T. Okamoto T. Neuroreport.. 1997; 8: 2931-2935Google Scholar, 10Kaltschmidt B. Deller T. Frotscher M. Kaltschmidt C. Neuroreport.. 2000; 11: 839-844Google Scholar) and rapidly activated independent of protein synthesis (11Baeuerle P.A. Baltimore D. Cell.. 1988; 53: 211-217Google Scholar), making this factor a likely candidate as a synaptic tag (3Frey U. Morris R.G. Nature.. 1997; 385: 533-536Google Scholar). NF-κB is present in many neuronal cell types (for review, see Ref. 12O'Neill L.A.J. Kaltschmidt C. Trends Neurosci... 1997; 20: 252-258Google Scholar) and, in neurons, can be constitutively active (13Rattner A. Korner M. Walker M.D. Citri Y. EMBO J... 1993; 12: 4261-4267Google Scholar, 14Kaltschmidt C. Kaltschmidt B. Neumann H. Wekerle H. Baeuerle P.A. Mol. Cell. Biol... 1994; 14: 3981-3992Google Scholar, 15Schmidt-Ullrich R. Memet S. Lilienbaum A. Feuillard J. Raphael M. Israel A. Development.. 1996; 122: 2117-2128Google Scholar) or activated by a variety of stimuli such as glutamate (7Guerrini L. Blasi F. Denis D.S. Proc. Natl. Acad. Sci. U. S. A... 1995; 92: 9077-9081Google Scholar, 16Kaltschmidt C. Kaltschmidt B. Baeuerle P.A. Proc. Natl. Acad. Sci. U. S. A... 1995; 92: 9618-9622Google Scholar, 17Grilli M. Pizzi M. Memo M. Spano P. Science.. 1996; 274: 1383-1385Google Scholar) and amyloid β-peptide (18Kaltschmidt B. Uherek M. Volk B. Baeuerle P.A. Kaltschmidt C. Proc. Natl. Acad. Sci. U. S. A... 1997; 94: 2642-2647Google Scholar, 19Kaltschmidt B. Uherek M. Wellmann H. Volk B. Kaltschmidt C. Proc. Natl. Acad. Sci. U. S. A... 1999; 96: 9409-9414Google Scholar). A physiological role was defined for NF-κB in neuroprotection against amyloid β-peptide (19Kaltschmidt B. Uherek M. Wellmann H. Volk B. Kaltschmidt C. Proc. Natl. Acad. Sci. U. S. A... 1999; 96: 9409-9414Google Scholar) and oxidative stress and glutamate (20Lezoualc'h F. Sagara Y. Holsboer F. Behl C. J. Neurosci... 1998; 18: 3224-3232Google Scholar, 21Mattson M.P. Goodman Y. Luo H. Fu W. Furukawa K. J. Neurosci. Res... 1997; 49: 681-697Google Scholar). Depending on the context, NF-κB might also be involved in neurodegeneration (22Post A. Holsboer F. Behl C. J. Neurosci... 1998; 18: 8236-8246Google Scholar). To date, five mammalian NF-κB DNA-binding subunits are known: p50, p52, RelA (p65), c-Rel, and RelB (23Baeuerle P.A. Baltimore D. Cell.. 1996; 87: 13-20Google Scholar, 24Baldwin A.J. Annu. Rev. Immunol... 1996; 14: 649-683Google Scholar). The important role of the transactivating RelA subunit is apparent in relAknockout mice, for which there is a high rate of embryonic mortality. Inhibitory subunits are IκB-α, IκB-β, IκB-γ (p105), IκB-δ (p100), and IκB-ε (25Whiteside S.T. Israel A. Semin. Cancer Biol... 1997; 8: 75-82Google Scholar). Within the nervous system, heteromeric NF-κB is most frequently composed of two DNA-binding subunits (e.g. p50 or RelA) that either are constitutively active or form a complex with the inhibitory subunit IκB-α (6Kaltschmidt C. Kaltschmidt B. Baeuerle P.A. Mech. Dev... 1993; 43: 135-147Google Scholar, 7Guerrini L. Blasi F. Denis D.S. Proc. Natl. Acad. Sci. U. S. A... 1995; 92: 9077-9081Google Scholar, 13Rattner A. Korner M. Walker M.D. Citri Y. EMBO J... 1993; 12: 4261-4267Google Scholar, 14Kaltschmidt C. Kaltschmidt B. Neumann H. Wekerle H. Baeuerle P.A. Mol. Cell. Biol... 1994; 14: 3981-3992Google Scholar, 16Kaltschmidt C. Kaltschmidt B. Baeuerle P.A. Proc. Natl. Acad. Sci. U. S. A... 1995; 92: 9618-9622Google Scholar, 18Kaltschmidt B. Uherek M. Volk B. Baeuerle P.A. Kaltschmidt C. Proc. Natl. Acad. Sci. U. S. A... 1997; 94: 2642-2647Google Scholar, 26Kaltschmidt C. Kaltschmidt B. Henkel T. Stockinger H. Baeuerle P.A. Biol. Chem. Hoppe-Seyler.. 1995; 376: 9-16Google Scholar). Interactive ankyrin repeats of IκB-α can physically block the nuclear localization signal (NLS) on the RelA subunit (27Malek S. Huxford T. Ghosh G. J. Biol. Chem... 1998; 273: 25427-25435Google Scholar, 28Jacobs M.D. Harrison S.C. Cell.. 1998; 95: 749-758Google Scholar), preventing transport of the complex into the nucleus. Activation of NF-κB results in the degradation of IκB-α, which in turn exposes the NLS, allowing NF-κB to be transported into the nucleus (29Baeuerle P.A. Henkel T. Annu. Rev. Immunol... 1994; 12: 141-179Google Scholar). Thus, the specific post-translational regulation of NF-κB and its synaptic distribution support the idea that NF-κB functions both as a transcription factor in the nucleus, where it can function as a molecular switch for turning on gene expression, and as an immediate retrograde signal transducer, which unifies signal perception at distant sites (dendrites, axons, and synapses) (6Kaltschmidt C. Kaltschmidt B. Baeuerle P.A. Mech. Dev... 1993; 43: 135-147Google Scholar, 12O'Neill L.A.J. Kaltschmidt C. Trends Neurosci... 1997; 20: 252-258Google Scholar,30Kaltschmidt C. Kaltschmidt B. Trends Neurosci... 1998; 21: 106Google Scholar). It is not known, however, if NF-κB undergoes retrograde transport upon activation. In this study, we examined whether activated NF-κB RelA was transported from distant sites (neurites) to the nucleus in living cells using jellyfish green fluorescent protein (GFP) fusion technology to attach a fluorescent tag to the RelA subunit of NF-κB. To analyze the transport of NF-κB, a fusion protein was constructed that consisted of RelA and a GFP mutant optimized for maximal light emission (EGFP). This new technique offers the opportunity to image both the distribution and interactions of the protein in living cells. In contrast to intracellular antibody staining, using this technique, living cells can be observed over time, and small structures can be labeled, e.g. the label in dendritic spines can be greatly enhanced following overexpression of the GFP protein (31Fischer M. Kaech S. Knutti D. Matus A. Neuron.. 1998; 20: 847-854Google Scholar). Hippocampal neurons were cultured from embryonic day 17 or 18 rats as described by Banker and Cowan (32Banker G.A. Cowan W.M. Brain Res... 1977; 126: 397-425Google Scholar) and detailed by de Hoop et al. (33de Hoop M.J. Meyn L. Dotti C.G. Cell Biology: A Laboratory Handbook.Academic Press. 1998; 1: 154-163Google Scholar). One day prior to preparation of the hippocampal neurons, neurobasal medium (Life Technologies, Inc.) supplemented with B27 (1:50; Life Technologies, Inc.) and 0.5 mml-glutamine was conditioned by astrocyte co-culture. Dissected hippocampi were treated with trypsin (2 mg/ml; Sigma) in Hanks' balanced saline solution without calcium or magnesium and then with soybean trypsin inhibitor (1 mg/ml). Neurons were dissociated by strokes with a fire-polished Pasteur pipette and were suspended in minimal essential medium with Earle's salts containing 1 mm pyruvate, 26 mmNa2HCO3, 2 mml-glutamine, 20 mm KCl, and 10% heat-inactivated horse serum. Cells (2 × 104cells/cm2) were plated on glass coverslips coated with polyethyleneimine (1:1000 in borate buffer, pH 7.2; Sigma). After 3 h, the cells adhered and were moved to astrocyte co-cultures with conditioned neurobasal medium. Biolistic experiments were performed after neurons had been maintained in culture for at least 7 days. At this age, the cultures contain mainlyN-methyl-d-aspartic acid type synapses clustered in varicosities (34Liao D. Zhang X. O'Brien R. Ehlers M.D. Huganir R.L. Nat. Neurosci... 1999; 2: 37-43Google Scholar). HEK 293 cells (American Type Culture Collection) were seeded on glass coverslips (104 cells/cm2) in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 100 unit/ml penicillin, and 0.1 mg/ml streptomycin. The following day, calcium phosphate transfection was performed as described previously (26Kaltschmidt C. Kaltschmidt B. Henkel T. Stockinger H. Baeuerle P.A. Biol. Chem. Hoppe-Seyler.. 1995; 376: 9-16Google Scholar); and 24 h later, cells were fixed with 4% paraformaldehyde. Luciferase assays were performed as described previously (14Kaltschmidt C. Kaltschmidt B. Neumann H. Wekerle H. Baeuerle P.A. Mol. Cell. Biol... 1994; 14: 3981-3992Google Scholar), and standardization was performed using Renilla reniformisluciferase together with Photinus pyralis luciferase (Dual-Luciferase, Promega, Mannheim, Germany) as suggested by the manufacturer. On day 8 in culture, hippocampal neurons were treated with either 100 or 500 μm glutamate or 100 μm kainate in glia-conditioned medium for 5 min. After two washing steps with neurobasal medium, cells were incubated at 37 °C for 1, 1.5, or 2 h. The cells were then fixed for 2 min in ethanol and for 5 min in 3.7% formaldehyde. After washing with phosphate-buffered saline, the cells were incubated in 5% goat serum for 30 min at room temperature, followed by two 5-min washes with phosphate-buffered saline. Cells were then incubated with anti-RelA monoclonal antibody (1:50 dilution; Roche Molecular Biochemicals, Mannheim). Anti-RelA antibody was detected by Cy3-conjugated anti-mouse IgG (1:400 dilution; Dianova, Hamburg, Germany); staining was performed subsequently; and detection was finished when incubation with the next primary antibody was started. Cells incubated with secondary antibodies, but without primary antibodies, were used as a control. The state of RelA activation in these cultures was tested using the anti-RelA monoclonal antibody because this antibody is commercially available and is specific for the activated form of the RelA subunit (26Kaltschmidt C. Kaltschmidt B. Henkel T. Stockinger H. Baeuerle P.A. Biol. Chem. Hoppe-Seyler.. 1995; 376: 9-16Google Scholar). Immunoreactivity with this antibody is detectable only after the activation of NF-κB (16Kaltschmidt C. Kaltschmidt B. Baeuerle P.A. Proc. Natl. Acad. Sci. U. S. A... 1995; 92: 9618-9622Google Scholar, 18Kaltschmidt B. Uherek M. Volk B. Baeuerle P.A. Kaltschmidt C. Proc. Natl. Acad. Sci. U. S. A... 1997; 94: 2642-2647Google Scholar, 35Carter B.D. Kaltschmidt C. Kaltschmidt B. Offenhauser N. Bohm-Matthaei R. Baeuerle P.A. Barde Y.A. Science.. 1996; 272: 542-545Google Scholar, 36Bethea J.R. Castro M. Keane R.W. Lee T.T. Dietrich W.D. Yezierski R.P. J. Neurosci... 1998; 18: 3251-3260Google Scholar). A pcDNA3 expression vector (Invitrogen, Leek, Netherlands), a pEGFP-1 vector (CLONTECH, Palo Alto, CA), a cytomegalovirus-driven human RelA expression vector (37Schmitz M.L. Baeuerle P.A. EMBO J... 1991; 10: 3805-3817Google Scholar), and a cytomegalovirus-driven human IκB-α vector (38Zabel U. Henkel T. Silva M.S. Baeuerle P.A. EMBO J... 1993; 12: 201-211Google Scholar) were used. A pcDNA3-EGFP expression plasmid was generated usingBamHI/NotI restriction of pEGFP-1 and sticky-end ligation of the purified fragment into pcDNA3. The pcDNA3-EGFP-RelA expression plasmid was constructed by recombinant polymerase chain reaction using Pwo/Taq DNA polymerases (Expand High Fidelity PCR system, Roche Molecular Biochemicals). pEGFP-1 was used as the template for polymerase chain reaction to amplify an EGFP fragment (769 base pairs) with forward primer A (5′-AAGCTTCGCCACCATGGTGAGCAAGGGCGAGGAGCTGTTC-3′) and reverse primer B (5′-GAAGATGAGGGGGAACAGTTCGTCGGCCCCGGCCCCCTTGTACAGCTCGTCCATGCCGAGAGTGAT-3′). At the 5′-end, the fragment carried a HindIII site and a Kozak consensus sequence (CGCCACC) and, at the 3′-end, a sequence encoding (Gly-Ala)2 and the first 30 base pairs from 5′-RelA. Using forward primer C (5′-GGCCGACGAACTGTTCCCCCTCATCTTCC-3′) and reverse primer D (5′-GGATCCTTAGGAGCTGATCTGAC-3′), a RelA fragment containing the first 361 amino acids including transactivation domain TA3, but without TA1 and TA2 (37Schmitz M.L. Baeuerle P.A. EMBO J... 1991; 10: 3805-3817Google Scholar), was amplified using a 29-base pair overlap with the 3′-end of the EGFP fragment, with the pCMV-RelA expression plasmid used as a template. This strategy was chosen to avoid potential toxicity via the induction of neurotoxic NF-κB target genes. The 3′-end of the RelA fragment carried a stop codon and a BamHI restriction site. The recombinant fragment was ligated into the HindIII/BamHI site of the pcDNA3 vector. The NLS of the pcDNA3-EGFP-RelA expression vector was mutated by site-directed mutagenesis polymerase chain reaction (QuikChange site-directed mutagenesis kit, Stratagene, La Jolla, CA). Lys287, Lys290, and Arg291 were mutated to threonine residues (forward primer, 5′-GTCACCGGATTGAGGAGACACGTACAACGACATATGAGACCTTCAAG-3′; and reverse primer, 5′-CTTGAAGGTCTCATATGTCGTTGTACGTGTCTCC TCAATCCGGTGAC-3′). Mutations were verified by sequencing. HEK 293 cells were grown on 10-cm culture dishes; and 1 day after calcium phosphate precipitation, the expression of EGFP-RelA and EGFP-(NLSmut)RelA was detected. Cells were treated with buffer containing 20 mm HEPES, pH 7.9, 350 mm NaCl, 20% glycerol, 1% Nonidet P-40, 1 mmMgCl2, 0.5 mm EDTA, 0.1 mm EGTA, 0.5 mm dithiothreitol, 2 mmphenylmethylsulfonyl fluoride, and 0.1% aprotinin, and the protein concentration was determined using Bradford dye reagent. Extracts were frozen in liquid nitrogen and stored at −80 °C until used. Extracts containing 25 μg of protein were boiled for 5 min in sample buffer and separated on an SDS-gradient polyacrylamide minigel (4, 8, and 12%; Novel, Heidelberg, Germany). Proteins were transferred at 1.5 mA/cm2 for 1 h onto polyvinylidene difluoride membrane (Roche Molecular Biochemicals). Chromogenic detection of alkaline phosphatase-labeled antibodies was performed with a chromogenic Western Blotting kit (Roche Molecular Biochemicals) as recommended by the manufacturer. Nonspecific binding was blocked using 1% blocking solution for 30 min, and the blot was washed twice with 2× buffer containing 50 mm Tris base, 150 mm NaCl, and 0.1% (v/v) Tween 20, pH 7.5, for 10 min. Primary antibodies to GFP (0.4 μg/ml) and RelA (26Kaltschmidt C. Kaltschmidt B. Henkel T. Stockinger H. Baeuerle P.A. Biol. Chem. Hoppe-Seyler.. 1995; 376: 9-16Google Scholar) were used (both from Roche Molecular Biochemicals). Electrophoretic mobility shift assay was performed as described previously (6Kaltschmidt C. Kaltschmidt B. Baeuerle P.A. Mech. Dev... 1993; 43: 135-147Google Scholar). Oligonucleotides (Promega) encompassing the NF-κB-binding site and the Sp1-binding site were labeled at the 5′-end with γ-32P and T4 polynucleotide kinase. Binding reactions (20 μl) contained 20 μg of protein, 1 μg of poly(dI-dC), 15,000 cpm labeled DNA, and probe buffer (20 mm HEPES, pH 7.9, 100 mm KCl, 20% glycerol, 0.25% Nonidet P-40, 0.5 mm EDTA, 2 mm dithiothreitol, and 0.1 mm phenylmethylsulfonyl fluoride). Binding reactions were initiated by the addition of a DNA-binding mixture to the nuclear extracts at room temperature. For supershift analysis, anti-RelA antibody (Santa Cruz Biotechnology, Heidelberg) was incubated for 20 min at room temperature with the 32P-labeled oligonucleotide probes. Reaction mixtures were resolved by electrophoresis on a 4% polyacrylamide gel in 0.5× Tris borate/EDTA (1× Tris borate/EDTA = 89 mm Tris base, 89 mm boric acid, and 2 mm EDTA, pH 7.2). Gels were dried and exposed to x-ray film. For competition experiments,32P-labeled oligonucleotide probes were mixed with a 10-fold excess of unlabeled competitor oligonucleotides prior to incubation with the nuclear extracts. Gold particles were coated, and cartridges were prepared as described by the manufacturer (Bio-Rad) with slight modifications (39Wellmann H. Kaltschmidt B. Kaltschmidt C. J. Neurosci. Methods. 1999; : 9255-9264Google Scholar). Briefly, 30 μg of gold (1 μm in diameter) was suspended in 60 μl of 0.05 mspermidine (Sigma, Deisenhofen, Germany), and 60 μl of DNA (0.5 μg/μl) was added. DNA was precipitated on the particles using 60 μl of 2 m CaCl2. DNA-coated particles were suspended in 4 ml of a polyvinylpyrrolidone solution (0.05 mg/ml absolute ethanol; Sigma). Cartridges were prepared for use with the Helios Gene Gun (Bio-Rad) with a helium pressure of 100 p.s.i. Hippocampal neurons were imaged 24 h after biolistic transfection at 30 °C in custom-built observation chambers (courtesy of Prof. Dr. Rainer Greger) using an Axiovert 100 microscope (Carl Zeiss, Jena, Germany), high numerical aperture, oil immersion lenses, and a fluorescein isothiocyanate filter set (Carl Zeiss). Images were captured using a 2-s exposure every 20 min on Eastman Kodak 3200 ASA color slide films using light from a 50-watt mercury lamp (HBO50, Carl Zeiss). Care was taken to minimize exposure to the light. Cellular photodamage was prevented by perfusion via gravity feed with α-tocopherol (0.1 mm)-supplemented glia-conditioned medium after each exposure (31Fischer M. Kaech S. Knutti D. Matus A. Neuron.. 1998; 20: 847-854Google Scholar). Glutamate (100 or 500 μm) or kainate (50 or 100 μm) was added to the glia-conditioned medium for 5 min. Cells were washed twice, and movements of EGFP-RelA and EGFP-(NLSmut)RelA were observed for 2 h. Quantification of fluorescence intensities was performed as described previously (40Parent C.A. Blacklock B.J. Froehlich W.M. Murphy D.B. Devreotes P.N. Cell.. 1998; 95: 81-91Google Scholar), but without background subtraction, using IP-Lab-Spectrum software (Scanalytics, Fairfax, VA). In brief, GFP fluorescence over distance was measured for untreated conditions (I 0) and after treatment (I) as values obtained after integrating the area under the plotted fluorescence intensities. For statistical evaluation, relative values (I/I 0 × 100) obtained from different neurites and experiments were pooled and analyzed as the experimental group containing EGFP-RelA versus the control group containing EGFP-RelA with a mutated NLS, using the Wilcoxon rank sum test. Pseudo-color images were created using NIH Image Version 1.61. To analyze the distribution of NF-κB, a GFP fusion protein containing RelA with its nuclear localization signal was constructed (Fig. 1 A). This construct contains only one weak transactivation domain, TA3 (37Schmitz M.L. Baeuerle P.A. EMBO J... 1991; 10: 3805-3817Google Scholar), to avoid potential toxicity via induction of NF-κB target genes or via squelching (41Gill G. Ptashne M. Nature.. 1988; 334: 721-724Google Scholar) of other signal transduction pathways. The feasibility of using this GFP-RelA fusion protein to analyze nuclear transport was tested in HEK 293 cells (Fig. 1 B). The EGFP-RelA fusion protein was found exclusively in the nucleus (Fig.1 B), whereas unfused EGFP was randomly distributed across all cellular compartments, including the nucleus and cytoplasm (Fig.1 B). Overexpression of IκB-α resulted in the exclusion of EGFP-RelA from the nucleus (Fig. 1 B), consistent with the idea that IκB-α regulates the location of EGFP-RelA in the cell. In contrast, distribution of unfused EGFP was not influenced by the coexpression of IκB-α (Fig. 1 B). To biochemically characterize the fusion proteins, transfected HEK 293 cell extracts were separated on SDS gels and analyzed using Western blotting (Fig. 2 A). A band corresponding to the predicted protein molecular mass was detected with antibodies directed against either the RelA portion (Fig.2 A, lane 5) or the EGFP portion (lane 1) of the fusion protein. A second, faster migrating band was present that might be a degradation product. The specificity of the immunolabeling was tested via the expression of EGFP (Fig.2 A, lane 3), which was recognized only by the antibody to GFP and not by the antibody to RelA (lane 7). Similarly, overexpressed RelA was not detected by the antibody to GFP (Fig. 2 A, lane 2), but was detected by the antibody to RelA (lane 6). Mock-transfected cells did not react specifically with either antiserum (Fig. 2 A,lanes 4 and 8). The DNA-binding characteristics of the EGFP-RelA fusion protein were analyzed by electrophoretic mobility shift assay (Fig. 2 B). Using a κ enhancer probe, the binding of EGFP-RelA was examined. The probe bound strongly to EGFP-RelA (Fig. 2 B, lane 1). Supershifting with the antibody to RelA verified the identity of the EGFP-RelA complexes (Fig. 2 B, lane 2). Antibody binding to EGFP-RelA was specifically competitively inhibited by unlabeled NF-κB oligonucleotide (Fig. 2 B, lane 4), but not by the nonspecific Sp1 oligonucleotide (lane 3). The transcriptional capability of the EGFP-RelA fusion protein was analyzed using an NF-κB-dependent luciferase vector (Fig.3, A and B). The fusion protein activated NF-κB-dependent transcription, whereas the control reporter, containing mutated NF-κB sites, was not activated (Fig. 3 A). Transcription mediated by EGFP-RelA was inhibited when IκB-α was coexpressed. In general, however, EGFP-RelA exhibited a lower transactivation capability compared with that of RelA (Fig. 3 B) due to the fact that this fusion protein contains only one weak transactivation domain (TA3) (37Schmitz M.L. Baeuerle P.A. EMBO J... 1991; 10: 3805-3817Google Scholar). Deletion of strong transactivation domains was done to avoid potential toxicity via the induction of NF-κB target genes (42Kaltschmidt B. Sparna T. Kaltschmidt C. Antioxidants Redox Signal... 1999; 1: 129-144Google Scholar) or via squelching (41Gill G. Ptashne M. Nature.. 1988; 334: 721-724Google Scholar) of other signal transduction pathways. Taken together, the biochemical and cell biological data indicate that the behavior of the fusion protein was sufficiently similar to that of endogenous RelA to warrant using this EGFP-RelA construct to examine transport in living neurons. We constructed an EGFP-RelA fusion protein with a mutated NLS containing three point mutations (38Zabel U. Henkel T. Silva M.S. Baeuerle P.A. EMBO J... 1993; 12: 201-211Google Scholar) (Fig.4 A). To study the mechanisms of transport into the nucleus of hippocampal neurons (Fig.4 B, left panels) or HEK 293 cells (right panels), cells were transfected as indicated. EGFP-RelA was localized primarily in the nucleus (Fig. 4 B), whereas EGFP-RelA with a mutated NLS was localized primarily in the cytoplasm, suggesting that an intact NLS is essential for a nuclear distribution of EGFP-RelA. Low levels of EGFP-RelA with a mutated NLS were detected in the nucleus, which might be due to additional auxiliary nuclear localization sequences (28Jacobs M.D. Harrison S.C. Cell.. 1998; 95: 749-758Google Scholar). This effect was observed in both hippocampal neurons and HEK 293 cells, suggesting that the distribution of NF-κB may be regulated via this mechanism in both cell types. To activate the reconstituted EGFP-RelA complex via endogenous signal transduction pathways, it was necessary to avoid overloading the system with an overexpression of IκB-α. Therefore, it was essential to determine the least amount of IκB-α necessary to keep the EGFP-RelA fusion protein in the cytoplasm (Fig.5). The amount of the EGFP-RelA expression plasmid was kept constant, and different amounts of IκB-α expression plasmid were added. The ratios were calculated based on the molecular masses of the expression plasmids. For both hippocampal neurons (Fig. 5, a, c, ande) and HEK 293 cells (b, d, andf), increasing the concentration of IκB-α resulted in changes in the distribution of EGFP-RelA, from primarily nuclear (a and b), to nuclear or cytoplasmic for each specific cell (c and d), and finally to primarily cytoplasmic (e and f). In the cytoplasm, EGFP-RelA was observed within puncta. Based on these data, a ratio of 1:0.2 (EGFP-RelA/IκB-α) was used in the time course experiments described below. We determined whether endogenous NF-κB was activated by glutamate in a hippocampal neuron/glia co-culture (32Banker G.A. Cowan W.M. Brain Res... 1977; 126: 397-425Google Scholar, 33de Hoop M.J. Meyn L. Dotti C.G. Cell Biology: A Laboratory Handbook.Academic Press. 1998; 1: 154-163Google Scholar). Without treatment, cells exhibited no nuclear staining, whereas following treatment with glutamate, nuclear staining was visible (data not shown). To examine whether treatment with the glutamate agonist kainate activated RelA in these cultured cells, as previously observed in cerebellar granule cells (16Kaltschmidt C. Kaltschmidt B. Baeuerle P.A. Proc. Natl. Acad. Sci. U. S. A... 1995; 92: 9618-9622Google Scholar), we also examined the effects of kainate. A 5-min treatment with 100 μm kainate resulted in a robust activation of RelA after a delay of 90 min (Fig. 6). The use of a monoclonal antibody specific for the activated form of NF-κB allowed us to detect RelA in the neuronal nuclei induced by a brief 5-min kainate treatment. In addition, residual amounts of activated NF-κB could still be detected in neurites 90 min after a kainate pulse (Fig. 6, right panels), similar to thein vivo situation of hippocampal granule cells (10Kaltschmidt B. Deller T. Frotscher M. Kaltschmidt C. Neuroreport.. 2000; 11: 839-844Google Scholar). In processes of hippocampal granule cells, activated NF-κB is present, most likely due to permanent neuronal activity in these cells in vivo. Control cultures incubated with secondary (but not primary) antibody did not exhibit detectable staining (data not shown). Thus, the hippocampal cultures used here are very well suited to study transport of RelA. To investigate the stimulus-dependent transport of EGFP-RelA from neurites to the nucleus, cultures were treated with 100 mm KCl for 5 min (Fig. 7). Note that under control conditions, EGFP-RelA was localized in long neurites and in puncta resembling varicosities (Fig. 7, upper panel). Ninety min after treatment with KCl, EGFP-RelA was redistributed from neurites and the soma to the nucleus. Thus, we wished to test other stimuli for the capability to induce NF-κB redistribution from neurites to the nucleus. Therefore, we used time-lapse microscopy. To follow the transport of EGFP-RelA from neurites to the nucleus over time, cultures were treated with 500 μm glutamate after an adaptation time of several minutes following transfer to the imaging chamber (Fig. 8 A). After treatment, a gradual tra
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