Sp1-binding Elements in the Promoter of RAGE Are Essential for Amphoterin-mediated Gene Expression in Cultured Neuroblastoma Cells
1998; Elsevier BV; Volume: 273; Issue: 47 Linguagem: Inglês
10.1074/jbc.273.47.30870
ISSN1083-351X
AutoresJianfeng Li, Xiaoqin Qu, Ann Marie Schmidt,
Tópico(s)Neuroscience of respiration and sleep
ResumoReceptor for AGE (RAGE) and the polypeptide amphoterin are highly expressed and co-localized in neurons of the developing central nervous system of the rat. In vitro, the interaction of amphoterin with neuronal RAGE induces neurite outgrowth. We tested the hypothesis that interaction of amphoterin with neuronal cells enhances RAGE expression, thereby providing a mechanism by which amphoterin-mediated regulation of RAGE might contribute to promotion of neurite growth and spreading. Incubation of cultured neuroblastoma cells with amphoterin resulted in increased transcription and translation of RAGE, a process largely inhibited in the presence of anti-RAGE IgG but not by nonimmune IgG. To begin to delineate molecular mechanisms underlying these findings, we identified multiple putative binding elements within the 5′-flanking region of the RAGE gene for Sp1, a transcription factor that has been critically linked to the process of normal development. DNase I footprinting and electrophoretic mobility shift assays demonstrated multiple functional Sp1-binding sites within the region −245 to −40 of the RAGE promoter. Transient transfection of cultured SK-N-SH neuroblastoma cells with chimeric 5′-deletion constructs linked to luciferase reporter revealed that the region containing Sp1-binding elements did not contribute uniquely to basal expression of the RAGE gene. Simultaneous mutation of the multiple Sp1-binding elements in this region did not affect basal promoter function; however, promoter responsiveness to amphoterin was markedly attenuated. These results point to Sp1-dependent mechanisms underlying amphoterin-mediated increases in RAGE expression in neuroblastoma cells and further link amphoterin-RAGE interaction to development of the nervous system. Receptor for AGE (RAGE) and the polypeptide amphoterin are highly expressed and co-localized in neurons of the developing central nervous system of the rat. In vitro, the interaction of amphoterin with neuronal RAGE induces neurite outgrowth. We tested the hypothesis that interaction of amphoterin with neuronal cells enhances RAGE expression, thereby providing a mechanism by which amphoterin-mediated regulation of RAGE might contribute to promotion of neurite growth and spreading. Incubation of cultured neuroblastoma cells with amphoterin resulted in increased transcription and translation of RAGE, a process largely inhibited in the presence of anti-RAGE IgG but not by nonimmune IgG. To begin to delineate molecular mechanisms underlying these findings, we identified multiple putative binding elements within the 5′-flanking region of the RAGE gene for Sp1, a transcription factor that has been critically linked to the process of normal development. DNase I footprinting and electrophoretic mobility shift assays demonstrated multiple functional Sp1-binding sites within the region −245 to −40 of the RAGE promoter. Transient transfection of cultured SK-N-SH neuroblastoma cells with chimeric 5′-deletion constructs linked to luciferase reporter revealed that the region containing Sp1-binding elements did not contribute uniquely to basal expression of the RAGE gene. Simultaneous mutation of the multiple Sp1-binding elements in this region did not affect basal promoter function; however, promoter responsiveness to amphoterin was markedly attenuated. These results point to Sp1-dependent mechanisms underlying amphoterin-mediated increases in RAGE expression in neuroblastoma cells and further link amphoterin-RAGE interaction to development of the nervous system. receptor for AGE advanced glycation end products analysis of variance bovine serum albumin fetal bovine serum electrophoretic mobility shift assay phosphate-buffered saline polymerase chain reaction enzyme-linked immunosorbent assay 1,4-piperazinediethanesulfonic acid glyceraldehyde-3-phosphate dehydrogenase interleukin 6. The receptor for AGE (RAGE),1 a member of the immunoglobulin superfamily of cell-surface molecules, interacts with a range of ligands (1Schmidt A.M. Vianna M. Gerlach M. Brett J. Ryan J. Kao J. Esposito C. Hegarty H. Hurley W. Clauss M. Wang F. Pan Y.C. Tsang T.C. Stern D. J. Biol. Chem. 1992; 267: 14987-14997Abstract Full Text PDF PubMed Google Scholar, 2Neeper M. Schmidt A.M. Brett J. Yan S.D. Wang F. Pan Y.C. Elliston K. Stern D. Shaw A. J. Biol. Chem. 1992; 267: 14998-15004Abstract Full Text PDF PubMed Google Scholar). In pathophysiologic circumstances, advanced glycation end products (AGEs) result from nonenzymatic glycation and oxidation of proteins and lipids and accumulate in conditions such as diabetes, renal failure, and amyloidoses (3Brownlee M. Cerami A. Vlassara H. N. Engl. J. Med. 1988; 318: 1315-1320Crossref PubMed Scopus (2317) Google Scholar, 4Miyata T. Oda O. Inagi R. Iida Y. Araki N. Yamada Y. Horiuchi S. Taniguchi N. Maeda K. Kinoshita T. J. Clin. Invest. 1992; 92: 1243-1252Crossref Scopus (409) Google Scholar). AGEs interact with RAGE in a manner linked to perturbation of vascular and inflammatory cells (5Schmidt A.M. Yan S.D. Brett J. Mora R. Stern D. J. Clin. Invest. 1993; 92: 2155-2168Crossref Scopus (269) Google Scholar, 6Yan S.D. Schmidt A.M. Anderson G. Zhang J. Brett J. Zou Y.S. Pinsky D. Stern D. J. Biol. Chem. 1994; 269: 9889-9897Abstract Full Text PDF PubMed Google Scholar, 7Wautier J.-L. Wautier M.P. Schmidt A.M. Anderson G.M. Zoukourian C. Capron L. Chappey O. Yan S.D. Brett J. Guillausseau P.J. Stern D. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7742-7746Crossref PubMed Scopus (323) Google Scholar, 8Schmidt A.-M. Hori O. Chen J. Li J.F. Crandall J. Zhang J. Cao R. Yan S.D. Brett J. Stern D. J. Clin. Invest. 1995; 96: 1395-1403Crossref PubMed Scopus (809) Google Scholar, 9Schmidt A.M. Weidman E. Lalla E. Yan S.D. Hori O. Cao R. Brett J. Lamster I. J. Periodontal Res. 1996; 31: 508-515Crossref PubMed Scopus (206) Google Scholar). In addition, RAGE interacts with amyloid β-peptide, consequences of which include enhanced neuronal and microglial oxidant stress and dysfunction (10Yan S.D. Chen X. Fu J. Chen M. Zhu H. Roher A. Slattery T. Nagashima M. Morser J. Migheli A. Nawroth P. Godman G. Stern D. Schmidt A.M. Nature. 1996; 382: 685-691Crossref PubMed Scopus (1783) Google Scholar, 11Yan S.-D. Zhu H. Fu J. Yan S.-F. Roher A. Tourtellotte W. Rajavashisth T. Chen X. Stern D. Schmidt A.-M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5296-5301Crossref PubMed Scopus (400) Google Scholar). These processes may be important in the pathogenesis of neurodegenerative disorders such as Alzheimer's disease. The expression of RAGE is strikingly enhanced in cells affected in settings associated with increased accumulation of AGEs and amyloid β-peptide (12Abel M. Ritthaler U. Zhang Y. Deng Y. Schmidt A.M. Greten J. Sernau T. Wahl P. Andrassy K. Ritz E. Stern D. Nawroth P.P. Nephrol. Dial. Transplant. 1995; 10: 1662-1667PubMed Google Scholar, 13Soulis T. Thallas V. Youssef S. Gilbert R.E. McWilliam B.G. Murray-McIntosh R.P. Cooper M.E. Diabetologia. 1997; 40: 619-628Crossref PubMed Scopus (198) Google Scholar, 14Schmidt A.M. Yan S.D. Stern D. Nat. Med. 1995; 1: 1002-1004Crossref PubMed Scopus (124) Google Scholar). We recently identified that an important means by which the expression of RAGE might be regulated in inflammatory-type milieu such as diabetic vasculature and wounds, and Alzheimer brain, is via two functional NF-kB-binding elements located with the 5′-flanking region of the RAGE gene (15Li J. Schmidt A.M. J. Biol. Chem. 1997; 272: 16498-16506Abstract Full Text Full Text PDF PubMed Scopus (442) Google Scholar). Transfection of 5′-deletion luciferase reporter gene constructs containing two mutated NF-kB-binding sites into either endothelial cells or vascular smooth muscle cells significantly diminished RAGE promoter responsiveness to prototypic inflammatory stimuli such as lipopolysaccharide (15Li J. Schmidt A.M. J. Biol. Chem. 1997; 272: 16498-16506Abstract Full Text Full Text PDF PubMed Scopus (442) Google Scholar). Our studies have also identified that the expression of RAGE is enhanced in the developing central nervous system, a setting in which excessive accumulation of AGEs and amyloid β-peptide is not likely (16Hori O. Brett J. Slattery T. Cao R. Zhang J. Chen J. Nagashima M. Lundh E.R. Vijay S. Nitecki D. Morser J. Stern D. Schmidt A.M. J. Biol. Chem. 1995; 270: 25752-25761Abstract Full Text Full Text PDF PubMed Scopus (1007) Google Scholar). Indeed, the increased expression of RAGE in neurons of the cerebrum, hippocampus, and cerebellum co-localized with that of the polypeptide amphoterin. In in vitro studies, amphoterin bound neuronal RAGE in a dose-dependent manner and mediated neurite outgrowth. In the presence of blockade of neuronal RAGE, employing either anti-RAGE F(ab′)2 or soluble RAGE, the extracellular two-thirds of RAGE (17Schmidt A.-M. Hasu M. Popov D. Zhang J.-H. Yan S.-D. Brett J. Cao R. Kuwabara K. Costache G. Simionescu N. Simionescu M. Stern D. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8807-8811Crossref PubMed Scopus (279) Google Scholar, 18Wautier J.-L. Zoukourian C. Chappey O. Wautier M.P. Guillausseau P.J. Cao R. Hori O. Stern D. Schmidt A.M. J. Clin. Invest. 1996; 97: 238-243Crossref PubMed Scopus (492) Google Scholar), amphoterin-mediated neurite outgrowth was inhibited (16Hori O. Brett J. Slattery T. Cao R. Zhang J. Chen J. Nagashima M. Lundh E.R. Vijay S. Nitecki D. Morser J. Stern D. Schmidt A.M. J. Biol. Chem. 1995; 270: 25752-25761Abstract Full Text Full Text PDF PubMed Scopus (1007) Google Scholar). We hypothesized that ligation of neuronal RAGE by amphoterin in the developing nervous system might be, at least in part, one means by which the expression of RAGE is enhanced in that setting. Such findings would suggest contributory mechanisms by which amphoterin-mediated regulation of RAGE promotes outgrowth of neurites. Since examination of the promoter of RAGE revealed multiple putative binding elements for Sp1, a transcription factor essential for early embryonic development (19Marin M. Karis A. Visser P. Grosveld F. Philipsen S. Cell. 1997; 89: 619-628Abstract Full Text Full Text PDF PubMed Scopus (440) Google Scholar, 20Saffer J.D. Jackson S.P. Annarella M.B. Mol. Cell. Biol. 1991; 11: 2189-2199Crossref PubMed Scopus (483) Google Scholar), we tested the hypothesis that amphoterin-mediated activation of Sp1 might be important in regulation of RAGE expression in developing neurons. In this study, we demonstrate that interaction of amphoterin with RAGE on cultured neuroblastoma cells up-regulates transcription and translation of RAGE. The presence of functional Sp1-binding elements within the promoter of RAGE appears necessary for this outcome. These findings further suggest a role for amphoterin-RAGE interaction in the development of the nervous system and implicate RAGE as a gene whose regulation in the process of neuronal development is, at least in part, mediated by Sp1. SK-N-SH cells were obtained from American Type Tissue Corporation (Rockville, MD). SK-N-SH cells are derived from human neuroblastoma, and their neuronal origin has been extensively characterized (21Biedler J.L. Helson L. Spengler B.A. Cancer Res. 1973; 33: 2643-2652PubMed Google Scholar). Cells were grown in Medium 199 containing fetal bovine serum (FBS) (10%) (Life Technologies, Inc.) in the presence of penicillin (100 units/ml) and streptomycin (100 μg/ml) in humidified incubators containing CO2 (5%). Monospecific, polyclonal anti-human RAGE IgG was used to prepare an ELISA for the detection of human RAGE. SK-N-SH cells were cultured in Medium 199 containing FBS (1%) for 16 h and then completely starved of serum for another 24 h. At the end of that time, cells were exposed to the indicated amount of recombinant rat amphoterin prepared, purified, and characterized as described previously (16Hori O. Brett J. Slattery T. Cao R. Zhang J. Chen J. Nagashima M. Lundh E.R. Vijay S. Nitecki D. Morser J. Stern D. Schmidt A.M. J. Biol. Chem. 1995; 270: 25752-25761Abstract Full Text Full Text PDF PubMed Scopus (1007) Google Scholar) or bovine serum albumin (BSA) (Sigma) for 24 h. Cells (1.5 × 106) were washed with PBS and lysed in buffer containing Tris, 0.02 m, pH 7.4; NaCl, 0.1m; phenylmethylsulfonyl fluoride, 0.002 m; and octyl-β-glucoside, 1% at 4 °C for 4 h and then centrifuged at 11,000 × g for 20 min. The supernatant was collected and protein concentration determined using the Bradford reagent (Bio-Rad). The supernatant was then diluted with appropriate amounts of sodium bicarbonate/carbonate buffer, pH 9.8, in order to coat equal amounts of protein (0.1 ml) onto each well of a plastic dish (Nunc Maxisorp). After 16 h at 4 °C, unbound sites on the plate were blocked in PBS buffer containing 1% BSA. Wells were then incubated with rabbit anti-RAGE IgG (34 μg/ml) (prepared and characterized as described previously; see Ref. 16Hori O. Brett J. Slattery T. Cao R. Zhang J. Chen J. Nagashima M. Lundh E.R. Vijay S. Nitecki D. Morser J. Stern D. Schmidt A.M. J. Biol. Chem. 1995; 270: 25752-25761Abstract Full Text Full Text PDF PubMed Scopus (1007) Google Scholar) and sites of primary antibody binding visualized using goat anti-rabbit IgG with peroxidase conjugate (Sigma). A standard curve was prepared using human RAGE previously prepared and purified from a baculovirus expression system. The limit of detection in the assay is 0.05 in each case. Amphoterin stimulation is as follows: in the setting of stimulation with amphoterin, the presence of single mutations (lines 2, 7 and 11) or double mutations (lines 3, 4 and 8) not involving Sp1 V, no significant effect on relative luciferase activity compared with wild-type (pGL-245) was noted,p > 0.05. When Sp1 V was mutated either singly (line 13) or in double mutations (lines 5, 9, and 12), relative luciferase activity decreased significantly compared with that of pGL-245; p < 0.01 in each case. When triple mutations were generated without affecting Sp1 V (line 6), no significant effect was noted on relative luciferase activity compared with pGL-245, p > 0.05. When triple mutations including Sp1 V were generated (lines 10, 14, and 15), significant reduction in relative luciferase activity was observed compared with that of pGL-245,p < 0.01. When all Sp1-binding sites were simultaneously mutated (Sp1M, line 16), a significant decrease in relative luciferase activity compared with that of pGL-245 was observed, p < 0.01.View Large Image Figure ViewerDownload Hi-res image Download (PPT) SK-N-SH cells were grown and maintained as described above. Transfection of the cells was performed using LipofectAMINE (Life Technologies, Inc.) according to the manufacturer's instructions. Briefly, 2 × 105 cells were plated onto the wells of tissue culture plates (6-wells, Corning, Corning, NY) 1 day prior to transfection. The DNA mixture for transfection was composed of test plasmid (2 μg) and pSV-β-galactosidase (0.5 μg) which served as internal control to normalize activities of luciferase. Cells were exposed to the mixture of LipofectAMINE and plasmid for 5 h. Following removal of the LipofectAMINE/plasmid mixture, fresh medium was added and the incubation continued for 48 h. Prior to studies of basal-stimulated function of the RAGE promoter, cells were washed with PBS and placed in medium as above, except that serum was omitted for 24 h prior to further studies. Where indicated, stimulus (amphoterin or BSA) was added to the medium 24 h prior to harvesting. Luciferase activity was determined using standard reagents (Promega) and measured in a luminometer (Wallac, Gaithersburg, MD) as directed, and results were standardized for β-galactosidase activity. Statistical analysis was performed using ANOVA (analysis of variance). Increased expression of RAGE in the developing neurons of the central nervous system co-localizes with high levels of expression of amphoterin. In order to determine if interaction of amphoterin with RAGE, at least in part, mediated this phenomenon, we tested the ability of amphoterin to regulate the expression of RAGE. Incubation of cultured neuroblastoma cells with amphoterin for 24 h revealed an ≈6.1-fold increase in mRNA for RAGE (Fig. 1 A, lane 4), when compared with incubation with BSA (Fig. 1 A, lane 5). The effects of amphoterin were mediated by interaction with cell-surface RAGE as demonstrated by suppression of amphoterin-stimulated RAGE expression in the presence of anti-RAGE IgG (Fig. 1 A, lane 2). In contrast, preincubation of neuroblastoma cells with nonimmune IgG prior to exposure to amphoterin had no effect (Fig. 1 A, lane 3). Incubation of SK-N-SH cells with anti-RAGE IgG alone had no effect on expression of RAGE (Fig. 1 A, lane 1). The enhanced mRNA for RAGE by amphoterin was dose-dependent. Maximal levels (6.1-fold enhancement) of mRNA for RAGE were noted in the presence of amphoterin, 40 μg/ml (Fig. 1 B). Nuclear run-on transcription assay revealed that amphoterin-mediated increase in mRNA for RAGE was due to enhanced transcription (Fig. 1 C). Consistent with enhanced mRNA for RAGE in amphoterin-treated cultured neuroblastoma cells, increased cellular RAGE antigen was detected by ELISA in a dose-dependent manner (Fig. 2 A). Compared with cells treated with BSA, an ≈5.2-fold increase (p < 0.01) in RAGE protein was noted in the presence of amphoterin. That these effects were mediated by RAGE was demonstrated by inhibition of amphoterin-mediated expression of RAGE in the presence of anti-RAGE IgG, but not nonimmune IgG (Fig. 2 B). Incubation of SK-N-SH cells with anti-RAGE IgG alone was without effect. Increased cell-surface RAGE in the presence of amphoterin was further confirmed by flow cytometry and immunohistochemistry (data not shown). Taken together, these data indicated that amphoterin modulated expression of RAGE in cultured neuroblastoma cells in a RAGE-dependent manner. In our previous studies, we identified two putative binding sites for the transcription factor Sp1 (designated Sp1 II and Sp1 V) (Fig. 3) in the promoter of RAGE using computer analysis (Genetics Computer Group; 23) by matching consensus sequences for Sp1 with those in the RAGE promoter. To determine if the putative Sp1-binding sites within the promoter of RAGE were functional, DNase I footprinting analysis was performed. By using purified human Sp1 and 32P-labeled RAGE promoter fragment from −720 to +11, protection of Sp1 sites (II and V) was demonstrated (Fig. 4 A). Surprisingly, three more protected areas were noted (Sp1 I, Sp1 III, and Sp1 IV (Fig. 3). Comparison of the core nucleotide sequences comprising these footprints with the Sp1 consensus reported by Bucher (25Bucher P.J. Mol. Biol. 1990; 212: 563-578Crossref Scopus (969) Google Scholar) revealed nearly 100% identity, or one nucleotide difference (Fig. 4 B). In order to determine if amphoterin induced activation of Sp1 DNA binding activity in neuroblastoma cells, SK-N-SH cells were treated with BSA, amphoterin, or amphoterin in the presence of anti-RAGE IgG or nonimmune IgG. Nuclear extracts were prepared and EMSA performed employing either Sp1 consensus probe or specific Sp1-binding site probes synthesized
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