Discovery of Pituitary Adenylate Cyclase‐Activating Polypeptide‐Regulated Genes through Microarray Analyses in Cell Culture and In Vivo

Abstract

Pituitary adenylate cyclase-activating polypeptide (PACAP) is an evolutionarily well conserved neuropeptide with multiple functions in the nervous, endocrine, and immune systems. PACAP provides neuroprotection from ischemia and toxin exposure, is anti-inflammatory in gastric inflammatory disease and sepsis, controls proliferative signaling pathways involved in neural cell transformation, and modulates glucohomeostasis. PACAP-based, disease-targeted therapeutics might thus be both effective and benign, enhancing homeostatic responses to behavioral, metabolic, oncogenic, and inflammatory stressors. PACAP signal transduction employs synergistic regulation of calcium and cyclic adenosine monophosphate (cAMP), and noncanonical activation of both calcium- and cAMP-dependent processes. Pharmacological activation of PACAP signaling should consequently have highly specific effects even in vivo. Here, a combined cellular biochemical, pharmacologic, transcriptomic, and bioinformatic approach to understanding PACAP signal transduction by identifying PACAP target genes with oligonucleotide- and cDNA-based microarray is described. Calcium- and cAMP-dependent PACAP signaling pathways for regulation of genes encoding proteins required for neuritogenesis, changes in cell morphology, and cell survival have been traced in PC12 cells. Pharmacological experiments have linked gene expression to cell physiological responses in this system, in which gene silencing can also be employed to confirm the functional significance of induction of specific transcripts. Differential transcriptional responses to metabolic, ischemic, and other stressors in wild type compared to PACAP-deficient mice establish in principle which PACAP-responsive transcripts in culture are PACAP-dependent in vivo. Bioinformatic approaches aid in creating a pipeline for identifying neuropeptide-regulated genes, validating their cellular functions, and defining their expression in the context of neuropeptide signaling physiology, required for discovery of new targets for drug action.

Acute Response of the Hippocampal Transcriptome Following Mild Traumatic Brain Injury After Controlled Cortical Impact in the Rat

“…A first-messenger network analysis was conducted using IPA to identify candidate upstream regulators of the CCI-induced transcripts altered in expression following mCCI, using internal quality and post hoc bioinformatic controls established for transcriptome analysis in our earlier studies, and adapted for the Affymetrix Rat 2.0 ST Array (Ait-Ali et al 2010;Chen et al 2006;Eiden et al 2008;Samal et al 2007;Samal and Eiden 2008). Of the dataset of 193 upregulated 1 transcripts uploaded into the IPA tool, 190 were recognized by IPA and used as gene target molecules in the IPA first-messenger network analysis.…”

Pituitary Adenylate Cyclase-Activating Polypeptide and Its Receptors: 20 Years after the Discovery

“…PACAP also enhances chromogranin A gene expression (Taupenot et al, 1998); activates the transcription of transfected neuropeptide Y, NPY-Y1 receptor gene, and proenkephalin A (Colbert et al, 1994;Monnier and Loeffler, 1998); and regulates genes bearing a CRE or 12-O-tetradecanoylphorbol 13-acetate response element motif via the cAMP/PKA and PLC/inositol 1,4,5-trisphosphate pathways (Schadlow et al, 1992;Monnier and Loeffler, 1998;Bournat and Allen, 2001). Microarray studies have provided a comprehensive view of the genes activated by PACAP in PC12 cells (Vaudry et al, 2002b;Grumolato et al, 2003b;Ishido and Masuo, 2004;Eiden et al, 2008;Ravni et al, 2008). Many of the known genes and proteins regulated by PACAP are associated with neuritogenesis [i.e., DISC1-binding zinc-finger protein or early growth response 1 Ravni et al, 2008)], hormone secretion [i.e., selenoprotein T (Grumolato et al, 2003a(Grumolato et al, , 2008], cell growth [i.e., growth arrest specific 1 or cyclin B2 (Vaudry et al, 2002b)], and cell survival [i.e., caspase3 or serum/glucocorticoid regulated kinase (Lebon et al, 2006;Ravni et al, 2006aRavni et al, , 2008Samal et al, 2007)].…”

Neuropeptides, Growth Factors, and Cytokines: A Cohort of Informational Molecules Whose Expression Is Up-Regulated by the Stress-Associated Slow Transmitter PACAP in Chromaffin Cells

“…The PC12 cell response to PACAP is a compound one—the initial phase of growth arrest and neurite extension takes the cells to a neuronal phenotype, followed by terminal differentiated into a final neurochemical phenotype (Rausch et al 1988). We noted in our 2008 study that secretory proteins were generally not induced by PACAP in PC12 cells: only when transfecting PC12 cells with a ‘physiological’ level of PAC1 (PC12_bPAC1hop cells) was one of these more ‘fully differentiated’ marker, tachykinin precursor 1 (Tac1), expressed (Eiden et al 2008). We examined the entire cohort of secretory protein-encoding transcripts in the PC12_bPAC1hop cell transcriptome induced by 6 h of treatment with PACAP-38 (100 nM).…”

“…We noticed in our previous studies that secretory proteins known to be present, and in some cases induced by PACAP, in primary chromaffin cells were neither expressed, nor induced by PACAP in the PC12 cell line (Vaudry et al 2002; Samal et al 2007; Eiden et al 2008; Ghzili et al 2008). The PC12 cell response to PACAP is a compound one—the initial phase of growth arrest and neurite extension takes the cells to a neuronal phenotype, followed by terminal differentiated into a final neurochemical phenotype (Rausch et al 1988).…”