Project description: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.

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:Pro-inflammatory cytokines, such as tumor necrosis factor-alpha (TNFalpha) and interleukin-1beta/alpha (IL1beta/alpha) modulate catecholamine secretion, and long-term gene regulation, in chromaffin cells of the adrenal medulla. Interleukin-6 (IL6), also released during inflammation, affects transcriptional responses in primary chromaffin cells, and may coordinate immune and autonomic adrenomedullary responses via an autocrine mechanism, as TNFalpha itself strongly induces IL6 expression in chromaffin cells, which in turn express receptors responsive to IL6. We have examined the signaling mechanisms employed by IL6 to affect tyrosine hydroxylase (TH) enzymatic activation, and adrenomedullary gene transcription, in cultured bovine chromaffin cells. IL6 caused acute tyrosine/threonine phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), and serine phosphorylation of signal transducer and activator of transcription 3 (STAT3), as do several other first messengers acting on the chromaffin cell, including histamine, nicotine and angiotensin II. IL6 uniquely activated tyrosine phosphorylation of STAT3. Consistent with a short-term ERK1/2 activation, IL6 treatment caused prompt regulation of TH phosphorylation, and up-regulation of genes encoding secreted proteins of the adrenal medulla including galanin, vasoactive intestinal peptide (VIP), gastrin releasing peptide (GRP) and parathyroid hormone-like hormone (PTHLH). We further examined the effects of IL6 treatment on the entire bovine chromaffin cell transcriptome. Of 90 genes up-regulated by IL6, only 16 of which are known targets of IL6 in the immune system. The remaining genes likely represent a combination of novel IL6/STAT3 targets, targets of ERK1/2 shared by other first messengers, and, potentially, IL6-dependent genes activated in a secondary cascade via transcription mediated by IL6-induced transcription factors, such as HIF-1alpha. Notably, genes induced by IL6 represent a cohort with a profile that includes both neuroendocrine-specific genes, including several that are activated by G-protein couple receptor (GPCR) signaling pathways initiated by histamine and pituitary adenylate cyclase-activating polypeptide (PACAP), and some transcripts also activated by cytokines including interferon-alpha (INFalpha and TNFalpha. These results suggest an integrative role for IL6 in overall fine-tuning of the chromaffin cell response to a wide range of physiological and paraphysiological stressors, particularly when immune and endocrine stimuli converge in the adrenal medulla.

Project description:We investigated the effects of PACAP treatment, and endogenous PACAP deficiency, on infarct volume, neurological function, and the cerebrocortical transcriptional response in a mouse model of stroke, middle cerebral artery occlusion (MCAO). PACAP-38 administered i.v. or i.c.v. one hour after MCAO significantly reduced infarct volume, and ameliorated functional motor deficits measured 24 hours later in wild-type mice. Infarct volumes and neurological deficits (walking faults) were both greater in PACAP-deficient than in wild-type mice, but treatment with PACAP reduced lesion volume and neurological deficits in PACAP-deficient mice to the same level of improvement as in wild-type mice. A 35,546-clone mouse cDNA microarray was used to investigate cortical transcriptional changes associated with cerebral ischemia in wild-type and PACAP-deficient mice, and with PACAP treatment after MCAO in wild-type mice. 229 known (named) transcripts were increased (228) or decreased (1) in abundance at least 50% following cerebral ischemia in wild-type mice. 49 transcripts were significantly up-regulated only at one hour post-MCAO (acute response transcripts), 142 were up-regulated only at 24 hours post-MCAO (delayed response transcripts) and 37 transcripts were up-regulated at both times (sustained response transcripts). More than 50% of these are transcripts not previously reported to be associated with brain ischemia responses. Many of the acutely regulated neurotrauma-associated transcripts, but a larger percentage of the delayed ones, require endogenous PACAP for up-regulation by ischemia, suggesting a more prominent role for PACAP in later response to injury than in the initial response, and consistent with a neuroprotective role for PACAP in late response to injury and neuroprotective efficacy when given post-MCAO. Putative injury effector transcripts regulated by PACAP include ãÑ-actin, midline 2, and metallothionein 1. Potential neuroprotective transcripts include several demonstrated to be PACAP-regulated in other contexts. Prominent among these were transcripts encoding the PACAP-regulated gene Ier3, and the neuropeptides enkephalin, substance P (tachykinin 1), and neurotensin. Keywords: transcript identification design

Project description:We investigated the effects of PACAP treatment, and endogenous PACAP deficiency, on infarct volume, neurological function, and the cerebrocortical transcriptional response in a mouse model of stroke, middle cerebral artery occlusion (MCAO). PACAP-38 administered i.v. or i.c.v. one hour after MCAO significantly reduced infarct volume, and ameliorated functional motor deficits measured 24 hours later in wild-type mice. Infarct volumes and neurological deficits (walking faults) were both greater in PACAP-deficient than in wild-type mice, but treatment with PACAP reduced lesion volume and neurological deficits in PACAP-deficient mice to the same level of improvement as in wild-type mice. A 35,546-clone mouse cDNA microarray was used to investigate cortical transcriptional changes associated with cerebral ischemia in wild-type and PACAP-deficient mice, and with PACAP treatment after MCAO in wild-type mice. 229 known (named) transcripts were increased (228) or decreased (1) in abundance at least 50% following cerebral ischemia in wild-type mice. 49 transcripts were significantly up-regulated only at one hour post-MCAO (acute response transcripts), 142 were up-regulated only at 24 hours post-MCAO (delayed response transcripts) and 37 transcripts were up-regulated at both times (sustained response transcripts). More than 50% of these are transcripts not previously reported to be associated with brain ischemia responses. Many of the acutely regulated neurotrauma-associated transcripts, but a larger percentage of the delayed ones, require endogenous PACAP for up-regulation by ischemia, suggesting a more prominent role for PACAP in later response to injury than in the initial response, and consistent with a neuroprotective role for PACAP in late response to injury and neuroprotective efficacy when given post-MCAO. Putative injury effector transcripts regulated by PACAP include Ã£Ã-actin, midline 2, and metallothionein 1. Potential neuroprotective transcripts include several demonstrated to be PACAP-regulated in other contexts. Prominent among these were transcripts encoding the PACAP-regulated gene Ier3, and the neuropeptides enkephalin, substance P (tachykinin 1), and neurotensin. In the hybridization method employed, equal amounts (2-5Ã£Ãg/15.5 Ã£Ãl) of total RNA obtained from brain tissue of different experimental groups were separately labeled with two different fluorescent dyes (Cy3 or Cy5) and applied on the same NIMH Mouse 36K cDNA microarray chip containing 13217 distinct Entrez gene IDs (formerly known as LocusLink) and 15888 distinct unigene IDs, the remainders are incompletely annotated IMAGE clones. Probe preparation and hybridization were performed as follows. The total RNA (15-50 Ã£Ãg in 15.5 Ã£Ãl) was incubated with amine-modified random primer (2 Ã£Ãg/Ã£Ãl, 2 Ã£Ãl) and RNase inhibitor (5 units/Ã£Ãl, 1 Ã£Ãl) at 70 Ã¤aC for 10 min. Primer-RNA solution was then incubated at 42 Ã¤aC for 2 hr with the reverse transcriptase mix containing 5X first-strand buffer, 50X aa-dUTP/dNTPs (25 mM dATP, 25 mM dGTP, 25 mM dCTP, 15 mM dTTP and 10 mM aminoallyl dUTP), 0.1 M DDT, and Superscript II reverse transcriptase. The cDNA were labeled with NHS-ester Cy3 or Cy5 dye in the presence of 1 M sodium bicarbonate. Array slides placed in a hybridization chamber (Corning, Corning, NY) were incubated at 42 Ã¤aC for 16-24 hr, and successively washed with 0.5X SSC, 0.01% SDS, and 0.06X SSC at room temperature for 10 min each. Arrays were scanned with a GenePix 4000A scanner (Axon, Foster City, CA), and the resulting images were analyzed using IPlab (Fairfax, VA) and a FileMaker Pro 5 (Santa Clara, CA) relational database designed by Zedong Chen, NHGRI. Following background subtraction and normalization, a calculated ratio of Cy3 to Cy5 signal intensities was used to define the relative increase or decrease of a particular transcript. Ratios were calculated only from those spots with a combined ratio quality above 0.3.

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Effect of PACAP treatment on Bovine chromaffin cells

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