Research Report
Pituitary adenylate cyclase-activating polypeptide (PACAP) mimics neuroendocrine and behavioral manifestations of stress: Evidence for PKA-mediated expression of the corticotropin-releasing hormone (CRH) gene

https://doi.org/10.1016/j.molbrainres.2005.03.016Get rights and content

Abstract

The physiologic response to stress is highly dependent on the activation of corticotropin-releasing hormone (CRH) neurons by various neurotransmitters. A particularly rich innervation of hypophysiotropic CRH neurons has been detected by nerve fibers containing the neuropeptide PACAP, a potent activator of the cAMP-protein kinase A (PKA) system. Intracerebroventricular (icv) injections of PACAP also elevate steady-state CRH mRNA levels in the paraventricular nucleus (PVN), but it is not known whether PACAP effects can be associated with acute stress responses. Likewise, in cell culture studies, pharmacologic activation of the PKA system has stimulated CRH gene promoter activity through an identified cAMP response element (CRE); however, a direct link between PACAP and CRH promoter activity has not been established. In our present study, icv injection of 150 or 300 pmol PACAP resulted in robust phosphorylation of the transcription factor CREB in the majority of PVN CRH neurons at 15 to 30 min post-injection and induced nuclear Fos labeling at 90 min. Simultaneously, plasma corticosterone concentrations were elevated in PACAP-injected animals, and significant increases were observed in face washing, body grooming, rearing and wet-dog shakes behaviors. We investigated the effect of PACAP on human CRH promoter activity in αT3-1 cells, a PACAP-receptor expressing cell line. Cells were transiently transfected with a chloramphenicol acetyltransferase (CAT) reporter vector containing region − 663/+124 of the human CRH gene promoter then treated for with PACAP (100 nM) or with the adenylate cyclase activating agent, forskolin (2.5 μM). Both PACAP and forskolin significantly increased wild-type hCRH promoter activity relative to vehicle controls. The PACAP response was abolished in the CRE-mutant construct. Pretreatment of transfected cells with the PKA blocker, H-89, completely prevented both PACAP- and forskolin-induced increases in CRH promoter activity. Furthermore, CREB overexpression strongly enhanced PACAP-mediated stimulation of hCRH promoter activity, an effect which was also lost with mutation of the CRE. Thus, we demonstrate that icv PACAP administration to rats under non-stressed handling conditions leads to cellular, hormonal and behavioral responses recapitulating manifestations of the acute stress response. Both in vivo and in vitro data point to the importance of PACAP-mediated activation of the cAMP/PKA signaling pathway for stimulation of CRH gene transcription, likely via the CRE.

Introduction

Parvocellular corticotropin-releasing hormone (CRH) neurons in the hypothalamic paraventricular nucleus (PVN) provide a major common pathway for endocrine, autonomic and behavioral responses to stress [25]. In response to stressful stimuli, these neurons secrete CRH (and arginine vasopressin, a supplementary secretagogue) into the hypophysial portal system to stimulate pituitary adrenocorticotropic hormone (ACTH) secretion, which in turn increases the biosynthesis and release of glucocorticoids from the adrenal cortex.

The characterization of neural inputs to parvocellular CRH neurons has suggested roles for catecholamines, glutamate, GABA and neuropeptides in the integration of signals influencing CRH neuronal function in the PVN [26]. A particularly robust innervation to CRH neurons is provided by nerve fibers containing the neuropeptide, pituitary adenylate cyclase-activating polypeptide (PACAP) [38]. Extrahypothalamic brain regions, such as the extended amygdala and lower brainstem that may contribute to the augmentation of the stress response, have also been identified as sites of PACAPergic innervation [34], [35], [36], [55]. The physiologic relevance of the hypothalamic PACAP–CRH interaction has been supported by the finding that intracerebroventricular (icv) injection of PACAP increases steady-state CRH mRNA levels in the PVN, as evaluated at 4 h post-injection. This effect is blocked by co-administration of a selective PACAP receptor antagonist [20]. Conversely, administration of the PACAP antagonist alone downregulates steady state CRH mRNA levels, suggesting a tonic regulatory role for PACAP on hypophysiotropic CRH neurons [20]. Nevertheless, it has not been determined whether PACAP effects can be linked to acute stress responses in the rat brain.

PACAP is a member of the secretin/glucagon/vasoactive intestinal peptide (VIP) family and exists as either a 38-amino acid peptide (PACAP38) or N-terminally truncated form, PACAP27, of which PACAP38 is the predominant form in the brain [4], [42], [43]. PACAP acts via binding to G-protein-coupled receptors termed PAC1, VPAC1 and VPAC2 according to their relative affinity for PACAP and VIP [24]. Although originally described as an activator of the cAMP-dependent protein kinase A (PKA) system, PACAP also may stimulate phospholipase C and calcium signaling pathways [50], [51], [64]. Within the brain, the highest concentration of PACAP and the PACAP selective PAC1 receptors are present in the hypothalamus, including the parvocellular and magnocellular subdivisions of the PVN, which are critical for mediating neuroendocrine responses [29], [47], [63].

Both in vitro and in vivo experiments have defined the PKA system as a critical modulator of CRH biosynthesis and secretion. Pharmacologic activation of the cAMP/PKA system stimulates CRH gene expression in cultured primary hypothalamic cells as well as a wide variety of heterologous cell lines [1], [66], [78]. Similarly, local microinjection of the cAMP analogue, 8-Br-cAMP, into the PVN increases endogenous CRH mRNA levels [28]. This cAMP response is mediated, at least in part, via binding of the transcription factor CREB (cyclic AMP response element binding protein) to a classical cyclic AMP response element (CRE) located at position −221 position in the human gene [16], [61]. Mutation of the hCRH-CRE prevents CREB binding and substantially blunts PKA responsiveness of the CRH gene promoter. Of note, an increase in the activated form of CREB, phosphorylated CREB (PCREB), coincides with an increase in de novo synthesized CRH transcripts in the PVN following acute stress [32]. Thus, the summary of these findings strongly suggests a link between the PKA system, CREB phosphorylation, and the activation of CRH neurons.

The aim of the present study was to advance the hypothesis that PACAP is a physiologically important regulator of the central limb of the HPA axis using both in vivo and in vitro approaches. We first investigated whether icv administration of PACAP under stress-free handling conditions would result in activation of the CRH neurons of the PVN and whether this treatment would mimic stress-like behavioral and corticosterone responses. Second, we used an immortalized cell culture system to characterize the molecular mechanisms by which PACAP regulates CRH gene transcription with a focus on the PKA system.

Section snippets

Animals

These experiments were performed on adult male Sprague–Dawley rats (n = 52, Taconic Farms, Germantown, New York) weighing 210–230 g at the beginning of the study. The animals were housed individually in cages under standard environmental conditions (light between 06:00 and 18:00 h; temperature, 22 ± 1 °C; rat chow and water available ad libitum). All experimental protocols were reviewed and approved by the Institutional Animal Care and Use Committees at the Tufts-New England Medical Center

Dose–response and time-course characteristics of icv PACAP-induced PCREB immunoreactivity in the PVN

The phosphorylation of CREB, as detected by immunolabeling for PCREB, was first surveyed at the 30-min post-injection time point. In control (aCSF vehicle-treated) animals, PCREB labeling was restricted to neuronal cell nuclei primarily residing in the magnocellular regions of the PVN with only a few nuclei exhibiting PCREB immunoreactivity in the parvocellular regions of the PVN (Fig. 1A). This pattern corresponds to the distribution of PCREB observed in earlier studies of non-stressed animals

Discussion

PACAP was originally isolated from ovine hypothalamic tissue based on its ability to stimulate adenylate cyclase activity and intracellular Ca2+ in anterior pituitary, glial and neuronal cells [3], [42], [43]. Converging lines of evidence have suggested that PACAP may serve as a neurotransmitter or neuromodulator as well as a protective substance for the survival of injured or developing neurons [2], [65], [73], [74].

Central injections of PACAP in rodents elicit a number of responses such as

Acknowledgments

This work was supported by NIH Grant R01 HD38089 (to L.M.H.), NIH Grant R01 MH63320 (to G.L.) and National Science Foundation Grant IBN 0080793 (to G.L.). We thank Dr. Qinheng Huang for his help with the corticosterone radioimmunoassay.

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