Unknown,Transcriptomics,Genomics,Proteomics

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PACAP and TNF regulate discrete population of genes in bovine chromaffin cells

ABSTRACT: The bovine chromaffin cell (BCC) is a unique modelM-bM-^@M-^Ta highly homogeneous and accessible neuroendocrine cellM-bM-^@M-^Tin which to study gene regulation through first messenger-initiated signaling pathways that are specific to post-mitotic cells. BCCs were treated with tumor necrosis factor (TNF) or pituitary adenylate cyclase activating polypeptide (PACAP), two critical regulators of neural cell transcriptional programming during inflammation that act on TNFR2 and PAC1 receptors, respectively, in post-mitotic neuroendocrine cells. Transcripts which were significantly up regulated by either or both first messenger were identified from microarray analysis using two bovine oligonucleotide arrays (Affymetrix and Agilent) followed by statistical analysis with Partek Genomic suite. Microarray data were combined from the two arrays using qRT-PCR sampling validation, and the first-messenger transcriptome derived from TNF and PACAP signaling were compared. More than 90 percent of the genes up regulated either by TNF or PACAP were specific to a single first messenger. BioBase suite, DIRE and Opossum were used to identify common promoter/enhancer response elements that control the expression of TNF- or PACAP-stimulated genes. Bioinformatic analysis revealed that distinct groups of transcription factors control the expression of genes up regulated by either TNF or PACAP . Most of the genes up regulated by TNF contained response elements for members of the Rel transcription factor family, suggesting TNF-TNFR2 signaling mainly through the NF-kB signaling pathway. On the other hand, the PACAP regulated genes showed no enrichment for any single response element, containing instead response elements for combinations of transcription factors allowing activation through multiple signaling pathways, including cAMP, calcium and ERK, in neuroendocrine cells. Pharmacological strategies for mimicking neuroprotection by either PACAP or TNF in the context of CNS injury or degeneration in disease might focus on individual downstream gene activation pathways to achieve greater specificity in vivo. For our analysis we have used both Affymetrix and Agilent arrays to identify the genes that are regulated up or down by neuropeptide PACAP and TNF-alpha. On Affyemetrix platform we performed technical repeats with 3 arrays with untreated samples, 3 arrays with samples from PACAP treatment, and 3 array with samples from TNF-alpha treatment. On the Agilent platform we performed technical repeats with 3 arrays for each treatment hybridizing Cy3-labelled RNA from untreated samples and Cy5-labelled RNA from either TNF-alpha or PACAP treated samples.

ORGANISM(S): Bos taurus

SUBMITTER: Babru Samal

PROVIDER: E-GEOD-24070 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:The bovine chromaffin cell (BCC) is a unique model—a highly homogeneous and accessible neuroendocrine cell—in which to study gene regulation through first messenger-initiated signaling pathways that are specific to post-mitotic cells. BCCs were treated with tumor necrosis factor (TNF) or pituitary adenylate cyclase activating polypeptide (PACAP), two critical regulators of neural cell transcriptional programming during inflammation that act on TNFR2 and PAC1 receptors, respectively, in post-mitotic neuroendocrine cells. Transcripts which were significantly up regulated by either or both first messenger were identified from microarray analysis using two bovine oligonucleotide arrays (Affymetrix and Agilent) followed by statistical analysis with Partek Genomic suite. Microarray data were combined from the two arrays using qRT-PCR sampling validation, and the first-messenger transcriptome derived from TNF and PACAP signaling were compared. More than 90 percent of the genes up regulated either by TNF or PACAP were specific to a single first messenger. BioBase suite, DIRE and Opossum were used to identify common promoter/enhancer response elements that control the expression of TNF- or PACAP-stimulated genes. Bioinformatic analysis revealed that distinct groups of transcription factors control the expression of genes up regulated by either TNF or PACAP . Most of the genes up regulated by TNF contained response elements for members of the Rel transcription factor family, suggesting TNF-TNFR2 signaling mainly through the NF-kB signaling pathway. On the other hand, the PACAP regulated genes showed no enrichment for any single response element, containing instead response elements for combinations of transcription factors allowing activation through multiple signaling pathways, including cAMP, calcium and ERK, in neuroendocrine cells. Pharmacological strategies for mimicking neuroprotection by either PACAP or TNF in the context of CNS injury or degeneration in disease might focus on individual downstream gene activation pathways to achieve greater specificity in vivo.

Project description:The neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) is considered to be a potential therapeutic agent for prevention of cerebral ischemia. Ischemia is a most common cause of death after heart attack and cancer causing major negative social and economic consequences. - This study was designed to investigate the effect of PACAP38 injection intracerebroventrically in a mouse model of permanent middle cerebral artery occlusion (PMCAO) along with corresponding sham control that used 0.9% saline injection. Ischemic and non-ischemic brain tissues were sampled at 6 and 24 hours post-treatment. - Following behavioral analyses to confirm whether the ischemia has occurred, we investigated the genome-wide changes in gene and protein expresison using DNA microarray chip (4x44K, Agilent) and one-/two-dimensional gel electrophoresis (1-/2-DGE) coupled with matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS), respectively. - Our results revealed numerous changes in the transcriptome of ischemic (ipsilateral) hemisphere treated with PACAP38 over the saline-injected SHAM control hemisphere. In parallel, 2-DGE analysis revealed a highly expressed protein spot in the ischemic region that was identified as dihydropyrimidinase-related protein 2 (DPYL2). The DPYL2, also known as Crmp2, is a marker for the activated neuronal defense mechanisms, and interestingly, PACAP strongly suppresed this induction. - This study provides the first inventory of PACAP influenced gene and protein targets in ischemic hemisphere, and provides new targets for thereaupetic interventions. The PMCAO model mice were generated as described previously. Briefly, the mice were anesthetized with 4% sevoflurane (induction) and 2% sevoflurane (maintenance) in a 30% O2 and 70% N2O gas mixture via a face mask. An incision was then made in the cervical skin followed by opening of the salivary gland, and the right common carotid artery was visualized. A midline cervical incision was made to expose the external carotid artery. The intraluminal filament technique was used, to generate the PMCAO model. - The PACAP38 (1 M-BM-5l containing 1 pmol) or 1 M-BM-5l of saline (0.9% NaCl) was injected intracerebroventrically, immediately after PMCAO. PACAP38 (Peptide Institute Inc., Osaka, Japan; supplier temperature was -20M-BM-:C and was stored at -80M-BM-:C) was dissolved in, and diluted M-CM-^W10 times with 0.9% NaCl just before use. - In the sham control animals, following exposure of the external carotid artery, the wound was sutured. The PACAP38 or saline was injected intracerebroventrically in the same concentrations as above. After injections, the animals were returned to their cages. - A total of eight groups were prepared: four groups of three, seven, four and five mice in the PMCAO plus PACAP38 and PMCAO plus saline cohorts at 6 and 24 h after operation, respectively, and five mice each in the control (sham) plus PACAP and saline groups at 6 and 24 h after operation, respectively. - We used three mice each in PMCAO groups that exhibited neurological grades G1 and G2 and three mice each at random in sham groups for the subsequent downstream analysis. Six or 24 hours post-injection of PACAP38 or saline, the mice were removed from their cages, decapitated, and their brains carefully removed on ice. The left (contralateral) and right (ipsilateral; ischemic) hemispheres were dissected and placed in 2 mL Eppendorf tubes, which were then quickly immersed in liquid nitrogen before being stored in -80M-BM-:C prior to further analysis. - For analysis of gene components, the tissues were ground to a very fine powder with liquid nitrogen, used for total RNA extraction, quantity and quality check, followed by cDNA synthesis, and RT-PCR. A mouse 4 x 44K whole genome oligo DNA microarray chip (G4122F, Agilent Technologies, Palo Alto, CA, USA) was used for global gene expression analysis using the ipsilateral (ischemic) hemisphere. Briefly, total RNA (900 ng; 300 ng each replicate pooled together) was labeled with either Cy3 or Cy5 dye using an Agilent Low RNA Input Fluorescent Linear Amplification Kit (Agilent). Fluorescently labeled targets of control (sham) as well as treated (PMCAO) samples with PACAP38 or without PACAP38 (saline) were hybridized to the same microarray slide with 60-mer probes. - Here, we compared the PMCAO plus PACAP38 injected mice to PMCAO plus saline, i.e. the ipsilateral brain region of the PMCAO mice was compared with the same right hemisphere of the control mice. Similarly, Sham control plus PACAP38 injected mice to Sham control plus saline were analyzed to identify only the PACAP38 influenced genes. A flip labeling (dye-swap or reverse labeling with Cy3 and Cy5 dyes) procedure was followed to nullify the dye bias associated with unequal incorporation of the two Cy dyes into cDNA.

Project description:PURPOSE: The goal of this study was to determine the gene expression networks regulated by tumor necrosis factor receptor 2 (TNFR2, or Tnfrsf1b) and to evaluate their potential bearing on immune cell subsets and inflammatory bowel disease (IBD). METHODS: mRNA-seq was performed on isolated distal colons from TNFR2-knockout and wildtype mice. Differentially expressed transcripts were compared to human ulcerative colitis microarray datasets on Gene Expression Omnibus and to mouse immunological expression datasets at the Immunological Genome Project. RESULTS: We identified 252 mouse transcripts whose expressions were significantly altered by the loss of TNFR2. The majority of these transcripts (228 of 252, ~90%) were downregulated in TNFR2-/- colons. TNFR2-regulated genes were able to positively discriminate between ulcerative colitis patients and healthy individuals with ~80% accuracy. Many TNFR2-regulated genes were also highly expressed in CD8+ T cells. CONCLUSIONS: Downregulation of TNFR2 is associated with a gene expression profile that is prominent in IBD and supportive of the role of CD8+ T cells in IBD pathogenesis. MANUSCRIPT ABSTRACT: Increased tumor necrosis factor (TNF) production has been associated with inflammatory bowel disease (IBD), and anti-TNF therapy is a common therapeutic for this patient population. However, the role of TNF or its receptors (TNFR1 and TNFR2) in the immunopathogenesis of inflammatory bowel disease (IBD) remains unclear. Here we report that TNFR2 is protective in spontaneous (IL-10 knockout) and chemically (azoxymethane/dextran sodium sulfate)-induced mouse models of colitis and colitis-associated cancer. Mechanistically, TNFR2-deficiency in hematopoietic cells significantly increased incidence and severity of colitis and colitis-associated cancer characterized by a selective expansion of CD8+ T cells. We identified TNFR2-regulated genes in the colon that were specific for CD8+ T cells, interacted with multiple IBD risk genes, and are important regulators of CD8+ T cell biology. TNFR2 regulated CD8+ T-cell-specific genes that act as genetic susceptibility modifiers for IBD to mitigate the development of a pro-colitogenic milieu. Antibody-mediated depletion of CD8+ T cells prevented colonic inflammation and significantly reduced pathology in IL10-/-/TNFR2-/- deficient mice. Furthermore, adoptive transfer of TNFR2-/- naïve CD8+ T cells resulted in more severe disease than with wildtype naïve CD8+ T cells. Our findings provide insight into the disease modifier role of TNFR2 in the immunopathogenesis of IBD through the modulation of CD8+ T cell responses and support future investigation of this therapeutic target, especially in the subset of IBD patients with CD8+ T-cell dysfunction. Total RNA from distal colons of 8 week-old male wildtype C57Bl/6 and TNFR2-/- mice (n=3 each) was isolated using the PureLink RNA kit (Ambion, Life Technologies). RNA samples were submitted to the Genomic Services Lab at the HudsonAlpha Institute for Biotechnology (Huntsville, AL) for multiplex library preparation, mRNA enrichment, and sequencing. Sequencing was performed to an average depth of 50M paired-end 50bp reads per sample (HiSeq, Illumina, San Diego, CA). Data files containing raw reads were aligned to the mouse genome using Tophat2/Bowtie2. Alignments were assembled into transcript representations with Cufflinks, and statistical tests for differential expression were performed with Cuffdiff 2. An adjusted P value < 0.05 (q<0.05) from the Cuffdiff 2 output was used as the cutoff for statistical significance.

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PACAP and TNF regulate discrete population of genes in bovine chromaffin cells

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