Enhanced IP₃R promoter activity with TNF-α addition. Constructs containing the −582 to +190 region of the human type-1 IP₃R promoter driving the expression of firefly luciferase were utilized to determine the ability of TNF-α to enhance IP₃R transcription. A, luciferase activity was tested at varying time points after 100 ng/ml TNF-α addition to obtain a temporal profile of regulation. B, deletion constructs were utilized to determine which region of the promoter was responsive to TNF-α regulation. Consistent luciferase activity observed 6 h after cytokine addition from each of the constructs suggested the −9 to +190 region as the site of regulation. Transfection efficiency was monitored by the co-transfection of a Renilla luciferase construct. n = 6 for each group. Error bars represent 1 S.D. and * indicates a p value < 0.01 obtained by a Bonferroni Multiple Comparison.

Site-specific activation of the IP₃R promoter by ΔMEKK1. The regulation of the human type-1 IP₃R promoter with JNK activation was further analyzed using luciferase constructs containing wild type and mutated versions of the putative AP-1/SP-1 sites identified within the IP₃R promoter. A, 771-bp nucleotide sequence of the full-length human IP₃R promoter is shown, and the sequences corresponding to the location of the putative JNK-regulated sites are underlined. Luciferase reporter constructs that contain wild-type or mutated versions of the three predicted AP-1/SP-1 binding sites centered at −252 (B), +59 (D), and +125 bp (C) within human IP₃R promoter upstream of the luciferase gene were co-transfected with either the ΔMEKK1 expression plasmid or hcRed control plasmid, and luciferase assays were performed 24 h later. Error bars represent 1 S.E. n = 6 for each group, and * indicates a p value < 0.01 by one-way ANOVA.

c-Jun-DN fails to block ΔMEKK1 enhancement of IP₃R promoter activity. A, Neuro2A cells were co-transfected with c-Jun, a dominant-negative form of c-Jun (cJun-DN), and/or ΔMEKK1 expression plasmid along with a plasmid harboring concatenated AP-1 consensus binding sequence upstream of a luciferase reporter gene. Luciferase expression was assayed 24 h after transfection. B, Neuro2A cells were co-transfected with a c-Jun, c-Jun-DN, and/or ΔMEKK1 expression plasmid along with plasmid harboring the human IP₃R promoter sequence upstream of a luciferase reporter gene. An hcRed reporter gene-expressing plasmid was included as an internal control, and firefly luciferase activity was measured 24 h after transfection. C, Neuro2A cells were transfected with a c-Jun or c-Jun-DN along with the human IP₃R promoter reporter gene. Cultures were then incubated in the presence of 100 ng/ml TNF-α. Luciferase expression was assayed 6 h later. Error bars represent 1 S.E. * indicates a p value < 0.01 obtained by a Bonferroni Multiple Comparison test after significance was determined by one-way ANOVA.

Dominant-negative SP-1 eliminates the ability of TNF-α to regulate the IP₃R promoter and enhance IP₃R-mediated Ca²⁺ signals. Neuro2A cells were transiently transfected with pSP1-DN, a plasmid expressing a dominant-negative SP-1 fused to GST, or a control GST-nuclear localization signal construct (pEBGN). A, Neuro2A cells were co-transfected with pSP1-DN or pEBGN and the full-length IP₃R promoter luciferase construct, and subsequently incubated with TNF-α for 6 h. Relative luciferase activity is shown. B and C, Neuro2A cells transfected as described above were subjected to Ca²⁺ imaging with Fura2, 24 h after TNF-α incubation. Sample traces from individual neurons and collective data obtained from coverslip averages is illustrated. p values were obtained by a Bonferroni Multiple Comparison test after significance was determined by one-way ANOVA. Box plot parameters: dots represent coverslip averages, the center of the box is the mean of the data, the edge of the box is 1 S.E., whiskers are 1 S.D.

Enhanced IP₃R promoter activity with JNK activation. To identify which signaling pathway was primarily responsible for enhanced IP₃R promoter activity following TNF-α exposure, ΔMEKK1 and p65 expression plasmids were employed to separately activate JNK and NFκB signaling, respectively. A, plasmid harboring a concatenated AP-1 consensus binding sequence upstream of a luciferase reporter gene, pGL3-AP1, was utilized to confirm JNK-related activation by the ΔMEKK1 expression plasmid. B, second plasmid that contained a concatenated NFκB consensus binding sequence upstream of a luciferase reporter gene, pGL3-NFκB, was utilized to confirm NFκB-related activation by the p65 expression plasmid. C, luciferase reporter constructs that contained progressively shorter segments of the human IP₃R promoter upstream of the luciferase gene were co-transfected with either the ΔMEKK1 or NFκB expression plasmids and luciferase assays were performed 24-h later. Transient co-transfection of a control plasmid, hcRed, or the NFκB p65 expression construct with any of these IP₃R promoter deletion plasmids into Neuro2A cells did not significantly affect luciferase activity output, whereas co-transfection of the ΔMEKK1 construct resulted in significant enhancement of luciferase reporter gene expression from each of the three reporter plasmids. Error bars represent 1 S.E., and * indicates a p value < 0.01 by one-way ANOVA.

EMSA analyses indicate SP-1 binds to the +59-bp site within the human IP₃R promoter. An EMSA was performed to ascertain transcription factor binding to a radioactively labeled 42-bp probe containing the putative +59-bp AP-1/SP-1 site identified within the human IP₃R promoter. A, increasing concentrations of untreated Neuro2A nuclear extracts (lanes 2–4), obtained by a salt gradient method, were utilized to examine transcription factor binding to 0.5 ng of ³²P-labeled probe. Protein binding is indicated by retardation in DNA probe migration and is designated in the figure by the bound complex arrow. Molar excess of wild-type control probe (lanes 5–7) was added to compete away the probe/protein interaction to illustrate the specificity of the shifted band. B, binding to a mutated probe was also assayed and the absence of a bound complex (lanes 2–4) indicated an elimination of transcription factor binding. To further test the binding affinity of the mutant probe, we assayed the ability of a molar excess of mutant cold probe to compete away the bound complex formed with the wild-type probe (lanes 5–7). C, during the binding reaction, 1 μg of a nonspecific IgG (lanes 1 and 2), c-Fos (lanes 3 and 4), or SP-1 antibody (lanes 5 and 6) was used to evaluate the respective antibodies ability to bind the transcription factor and compete with probe binding.

TNF-α enhances Ca²⁺ mobilization in Neuro2A cells. Neuro2A cells were treated with TNF-α for 24 h in the presence and absence of the JNK inhibitor (SP600125), and Ca²⁺ imaging was performed. A, traces of the emitted fluorescence after 340/380 nm excitation with bradykinin stimulation, an IP₃-generating agonist, were formed by averaging the responses from each Neuro2A cell in the field of view. SP600125 (10 μm) was added to the culture medium 1 h before TNF-α addition. B, coverslip averages for each treatment group (n = 8) are presented in bar graph form. C, traces are shown depicting enhanced responses in the presence of the JNK activator, ΔMEKK. D, overall averages are presented in bar graph form. E and F, Neuro2A cells were loaded with ryanodine (100 μm) to block the RyR and subjected to Ca²⁺ imaging. E, trace is shown illustrating the absence of a caffeine-induced Ca²⁺ signal, indicating successful block of the RyR. Box plot parameters: dots represent coverslip averages, the center of the box is the mean of the data, the edge of the box is one S.E., whiskers are one S.D. F, similar cytokine-induced enhancement in bradykinin signals suggested RyR coupling is not responsible for the effect. G and H, Ca²⁺ stores were measured using the cyclopiazonic acid leak method (in 300 μm La³⁺-reduced Ca²⁺ external solution), and total Ca²⁺ released by the area under the curve method is shown. I, total RNA from TNF-α-treated and untreated cultures was isolated at 0, 4, 8, and 12 h after cytokine addition. cDNA was subsequently prepared and quantitative real-time RT-PCR performed to determine the steady-state levels of the type-1 IP₃R mRNA with TNF-α treatment. Transcript expression changes are depicted as fold-change as compared with values obtained from the 0-h control time point. J, total RNA from TNF-α-treated and control cultures was isolated at 8 h after cytokine addition, but in the absence or in the presence of SP600125. Transcript expression changes are depicted as fold-change as compared with values obtained from the untreated control condition. K and L, type-1 IP₃R protein levels were analyzed via Western blotting and OD measurements are shown. Error bars represent 1 S.E., and p values were obtained using two-tailed Student's t-test.