Greenwood, M. P., Greenwood, M., Mecawi, A. D. S., Antunes- Rodrigues, J., Paton, J. F. R., & Murphy, D. (2016). Rasd1, a small G protein with a big role in the hypothalamic response to neuronal activation. Molecular Brain, 9(1), [1]. https://doi.org/10.1186/s13041- 015-0182-2 Publisher's PDF, also known as Version of record License (if available): CC BY Link to published version (if available): 10.1186/s13041-015-0182-2 Link to publication record in Explore Bristol Research PDF-document © 2016 Greenwood et al. Open Access: This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. University of Bristol - Explore Bristol Research General rights This document is made available in accordance with publisher policies. Please cite only the published version using the reference above. Full terms of use are available: http://www.bristol.ac.uk/red/research-policy/pure/user-guides/ebr-terms/ Greenwood et al. Molecular Brain (2016) 9:1 DOI 10.1186/s13041-015-0182-2 RESEARCH Open Access Rasd1, a small G protein with a big role in the hypothalamic response to neuronal activation Michael P. Greenwood1*, Mingkwan Greenwood1, Andre S. Mecawi2,3,4, José Antunes-Rodrigues2, Julian F. R. Paton5 and David Murphy1,3 Abstract Background: Rasd1 is a member of the Ras family of monomeric G proteins that was first identified as a dexamethasone inducible gene in the pituitary corticotroph cell line AtT20. Using microarrays we previously identified increased Rasd1 mRNA expression in the rat supraoptic nucleus (SON) and paraventricular nucleus (PVN) of the hypothalamus in response to increased plasma osmolality provoked by fluid deprivation and salt loading. RASD1 has been shown to inhibit adenylyl cyclase activity in vitro resulting in the inhibition of the cAMP-PKA-CREB signaling pathway. Therefore, we tested the hypothesis that RASD1 may inhibit cAMP stimulated gene expression in the brain. Results: We show that Rasd1 is expressed in vasopressin neurons of the PVN and SON, within which mRNA levels are induced by hyperosmotic cues. Dexamethasone treatment of AtT20 cells decreased forskolin stimulation of c-Fos, Nr4a1 and phosphorylated CREB expression, effects that were mimicked by overexpression of Rasd1, and inhibited by knockdown of Rasd1. These effects were dependent upon isoprenylation, as both farnesyltransferase inhibitor FTI-277 and CAAX box deletion prevented Rasd1 inhibition of cAMP-induced gene expression. Injection of lentiviral vector into rat SON expressing Rasd1 diminished, whereas CAAX mutant increased, cAMP inducible genes in response to osmotic stress. Conclusions: We have identified two mechanisms of Rasd1 induction in the hypothalamus, one by elevated glucocorticoids in response to stress, and one in response to increased plasma osmolality resulting from osmotic stress. We propose that the abundance of RASD1 in vasopressin expressing neurons, based on its inhibitory actions on CREB phosphorylation, is an important mechanism for controlling the transcriptional responses to stressors in both the PVN and SON. These effects likely occur through modulation of cAMP-PKA-CREB signaling pathway in the brain. Background While the SON contains a homogenous population of The hypothalamo-neurohypophyseal system (HNS) is MCN, the PVN is divided into MCN and parvocelluar the source of the neuropeptide hormone arginine vaso- neurons (PCNs). The PCNs form part of the pressin (AVP). AVP is synthesised in magnocellular neu- hypothalamo-pituitary-adrenal (HPA) axis that mediates rons (MCN) of the supraoptic nucleus (SON) and the stress response. In response to restraint stress, AVP paraventricular nucleus (PVN) and is transported ante- and corticotropin releasing hormone (CRH) are released rogradely to terminals in the posterior pituitary gland. A from the PCN axon terminals in the median eminence rise in plasma osmolality increases secretion of AVP into into the portal vasculature [2–4] that supplies the anterior the blood stream where it promotes water reabsorption pituitary to stimulate the release of adrenocorticotropin at the kidney [1]. hormone [5, 6], and, subsequently, glucocorticoids from the adrenal cortex. These secretory responses are accom- panied by transcriptional increases in Avp and Crh in * Correspondence: [email protected] PCN by stress [7–9] and Avp in MCN of the hypothal- 1School of Clinical Sciences, University of Bristol, Bristol BS1 3NY, UK Full list of author information is available at the end of the article amus by osmotic stress [10]. © 2016 Greenwood et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Greenwood et al. Molecular Brain (2016) 9:1 Page 2 of 17 The signaling mechanisms governing transcriptional transcription [26, 27]. Thus, Rasd1 has the capacity to increases in Crh and Avp are believed to involve cAMP influence transcriptional events either indirectly, by inhi- activation of the protein kinase A (PKA) pathway and biting AC activity at the cell membrane, or directly, the subsequent phosphorylation of cAMP response through modulation of activity at the level of the element binding protein (CREB) [11]. It is known promoter. that both hyperosmotic and restraint stress increase Using microarrays we identified increased Rasd1 ex- the abundance of phosphorylated CREB, a process pression in the rat and mouse SON in response to that occurs within minutes of stimulation in MCN hyperosmotic stress (dehydration) [28, 29]. Rasd1 and PCNs [8, 9, 12, 13]. Stress induced transcrip- mRNA expression has previously been described in tional increases can be short-lived, particularly for the mouse PVN and SON but its function in these Crh and Avp in PCNs, as the subsequent increase in brain nuclei is not known [30, 31]. Only in the supra- circulating levels of glucocorticoid following stress, chiasmatic nucleus (SCN) of the brain has Rasd1 ex- through its interactions with glucocorticoid receptors pression been interrogated thoroughly [32]. Rasd1 (GR) within these neurons [14], rapidly dampens this cycles with a circadian rhythm that oscillates in anti- transcriptional response. This feedback by glucocorti- phase with many of the cAMP inducible mRNAs in coids has been reported to inhibit CREB phosphoryl- the SCN [31] including Avp. Interestingly, in the ation in PCNs [12], through a proposed unknown SCNs of Rasd1 knockout mice, Cheng et al. [26] re- intermediate intracellular signaling molecule regulat- ported higher cAMP levels alongside increased CREB ing cAMP [7]. Less is known about inhibitory inputs phosphorylation compared to wild-type mice. We thus controlling the transcriptional response to osmotic hypothesised that Rasd1 may control the transcription stressinMCNofthePVNandSONwherephos- of cAMP inducible genes, and ultimately their down- phorylated CREB levels also increase [13]. MCN of stream targets, Avp and Crh, in the hypothalamus. the SON express GRs [15], and expression has been Using in vitro and in vivo approaches we demonstrate shown to increase during hypoosmotic stress [16], in- that Rasd1 negatively regulates the hypothalamic tran- dicating that glucocorticoid negative feedback is a scriptional response to stimulation by hyperosmotic possible route for regulation, though studies suggest stress in the rat. considerably lower levels of this receptor compared to PCNs of the PVN [14, 17]. Nonetheless, we rea- soned that a glucocorticoid inducible gene might be Results important for regulating transcriptional feedback in- Rasd1 mRNA expression in the rat hypothalamus in hibition in both MCN and PCNs. response to different stressors Our candidate was Rasd1 (dexamethasone inducible We first investigated the time course of Rasd1 mRNA Ras protein 1, Dexras1), a member of the Ras family of expression in the PVN and SON of control, dehydrated monomeric G proteins that was first identified as a (DH) and salt loaded (SL) rats using qPCR (Fig. 1a). dexamethasone (DEX) inducible gene in the pituitary Rasd1 mRNA expression was significantly higher in the corticotroph cell line AtT20 [18]. A putative glucocortic- PVN and SON of 1 day (day) DH and 1 day SL rats oid response element identified by Kemppainen and col- compared with control rats. Increasing the duration of leagues [19] in the 3’ flanking region of the human hypertonic stress to 3 days DH and 7 days SL further in- Rasd1 gene was shown to confer rapid responsiveness to creased the magnitude of this response in both PVN and glucocorticoids by reporter assay. Indeed, the peripheral the SON. When drinking water was