Cell Mol Neurobiol (2010) 30:14511457 DOI 10.

1007/s10571-010-9567-z

REVIEW PAPER

Stress and Adrenergic Function: HIF1a, a Potential Regulatory Switch

Dona Lee Wong T. C. Tai David C. Wong-Faull Robert Claycomb Brenda J. Siddall Rose Ann Bell Richard Kvetnansky

Received: 19 May 2010 / Accepted: 2 September 2010 / Published online: 3 November 2010 Springer Science+Business Media, LLC 2010

Abstract Stress elicits adrenal epinephrine and cortisol release into the bloodstream to initiate physiological and behavioral responses to counter and overcome stress, the classic ght or ight response (Cannon and De La Paz, Am J Physiol 28:6470, 1911). Stress and the stress hormone epinephrine also contribute to the pathophysiology of illness, e.g., behavioral disorders, cardiovascular disease, and immune dysfunction. Epinephrine itself is regulated by stress through its biosynthesis by phenylethanolamine N-methyltransferase (PNMT, EC 2.1.1.28). Single and repeated immobilization (IMMO) stress in rats stimulates adrenal PNMT mRNA and protein expression via the transcription factors, Egr-1 and Sp1. Moderate hypoxic stress increases PNMT promoter-driven gene expression and endogenous PNMT mRNA and protein in PC12 cells.
A commentary to this article can be found at doi: 10.1007/s10571-010-9607-8. D. L. Wong (&) T. C. Tai D. C. Wong-Faull R. Claycomb Department of Psychiatry, Harvard Medical School, Laboratory of Molecular and Developmental Neurobiology, McLean Hospital, 115 Mill Street, MRC Rm 116, Mail Stop 144, Belmont, MA 02478, USA e-mail: dona_wong@hms.harvard.edu D. L. Wong T. C. Tai B. J. Siddall R. A. Bell Department of Psychiatry and Behavioral Sciences, The Nancy Pritzker Laboratory of Developmental and Molecular Neurobiology, Stanford University School of Medicine, Stanford, CA 94306, USA T. C. Tai Division of Medicine, Northern Ontario School of Medicine, Laurentian University, Sudbury, ON P3E 2C6, Canada R. Kvetnansky Institute of Experimental Endocrinology, Slovak Academy of Sciences, Bratislava, Slovakia

Induction is initiated through cAMP and PLC signaling, with PKA, PKC, PI3K, ERK1/2 MAPK, and p38 MAPK continuing downstream signal transduction, followed by activation of HIF1a, Egr-1, and Sp1. While functional Egr-1 and Sp1 binding sites exist within the proximal PNMT promoter, a putative hypoxia response element is a weak HIF binding site. Yet, HIF1a overexpression increases PNMT promoter-driven luciferase activity and endogenous PNMT. When the Egr-1 or Sp1 sites are mutated, HIF1a does not stimulate the PNMT promoter. siRNA knock down of Egr-1 or Sp1 prevents promoter activation while siRNA knock down of HIF1a inhibits Egr-1 and Sp1 induction. Findings suggest that hypoxia activates the PNMT gene indirectly via HIF1a stimulation of Egr-1 and Sp1. Thus, for stress-induced illnesses where adrenergic dysfunction is implicated, HIF1a may be an onoff switch regulating adrenergic responses to stress and a potential target for therapeutic intervention. Keywords Stress Phenylethanolamine N-methyltransferase Transcriptional control Egr-1 Sp1 HIF1a

Introduction Stress, whether physiological, psychological, social or due to disruptors of cardiovascular/metabolic function, causes activation of the hypothalamicpituitaryadrenal (HPA) axis and release of the stress hormones, epinephrine, and cortisol into the circulation. In the short-term response to stress, behavioral and physiological responses are initiated to cope with the stress, posturing the organism to confront the stress or ee. These actions represent the classic ght or ight response described by Walter Cannon, with

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homeostasis being the resting norm (Cannon and De La Paz 1911). Hans Selye introduced the concept of stress, responses of the body to perturbations from the norm, and the notion of adaptation, which must occur to overcome the stress and ensure the organisms survival (Selye 1975). Current stress theory extends well beyond the concepts put forth by Cannon, who focused on the adrenal medulla, and Selye, who focused on the pituitaryadrenocortical axis. The reader is referred to the recent comprehensive review provided by Kvetnansky et al. (2009). In brief, irrespective of origin, all types of stress interfere with homeostasis. It can be of short or long duration, single, repeated, or chronic. Two main components comprise the stress axis: a hormonal component constituted by the HPA axis and orchestrated through the actions of corticotropin releasing hormone (CRH), adrenocorticotropic hormone (ACTH), and the adrenocortical hormones, cortisol or corticosterone; and a neural component constituted by the hypothalamus, intermediolateral column in the spinal cord, peripheral autonomic nerves, the sympathetic neurotransmitter norepinephrine, and the adrenomedullary neurohormone/ neurotransmitter epinephrine (see Kvetnansky et al. 2009, Fig. 8). Rapid inhibition of stressful events is necessary to sustain stress responsiveness and minimize the adverse effects of stress. Delayed inhibition and prolonged stress can increase susceptibility to illness, including physical and behavioral disorders, e.g., cardiovascular disease, diabetes, allergy, and immune dysfunction, cancer, schizophrenia, Alzheimers disease, and depression (Frieri 2003; Guglielmotto et al. 2009; Halliwill 2003; Hop et al. 2004; Lorita et al. 2002; Lundberg 2005; Mikhailenko et al. 2009; Rybnikova et al. 2008; Schmidt-Kastner et al. 2006; Semenza 2000b; Shuin et al. 2006). Furthermore, early exposure to stress may have long-term consequences for growth and development, with the central nervous system being a particularly vulnerable target (Ducsay et al. 2007; Mamet et al. 2002; Marco et al. 2010). Epinephrine, which primes acute stress responsiveness, may also contribute adversely to well-being with prolonged elevation. Thus, understanding how stress affects epinephrine expression and the consequences of elevated epinephrine under conditions of extended and recurring stress is important. As described above, stress can lead to dysfunction of the cardiovascular, immune, and nervous systems, all of which are affected by alterations in epinephrine. Circulating epinephrine is primarily derived from the adrenal medulla, wherein catecholamine biosynthesis occurs. The major catecholamine released from the chromafn cells of the adrenal medulla is epinephrine. Neurons in the C1, C2, and C3 regions of the brainstem also produce epinephrine, which can contribute to modulation of stress responses in both the central and peripheral nervous systems through afferent and efferent neural circuitry (Kvetnansky et al. 2009).

In this article, the authors describe the current knowledge about the mechanisms by which stress may regulate adrenal medullary epinephrine via its biosynthesis by phenylethanolamine N-methyltransferase (PNMT, EC 2.1.1.28) by extending our earlier ndings on IMMO stress in rats through investigation of hypoxic stress effects in PC12 cells.

Epinephrine Biosynthesis Catecholamine biosynthesis is initiated from L-phenylalanine by conversion to L-tyrosine or L-tyrosine ingested through the diet. Tyrosine hydroxylase (TH) adds a hydroxyl group onto the 6-carbon aromatic ring ortho to the existing hydroxyl group to produce L-dihydroxylphenylalanine (L-DOPA), which is then converted to L-dopamine through decarboxylation of the 2-chain aliphatic carbon moiety by DOPA decarboxylase (DDC). The a-carbon adjacent to the aromatic ring is then hydroxylated by dopamine b-hydroxylase (DBH) to generate L-norepinephrine. In turn, L-norepinephrine is converted to L-epinephrine by methylation of the amino group substituted on the bcarbon of the aliphatic side chain, a process catalyzed by phenylethanolamine N-methyltransferase (PNMT), using as co-substrate and methyl donor, S-adenosylmethionine (AdoMET).

Regulation of the PNMT Gene PNMT, the nal enzyme in epinephrine production, was originally identied by Axelrod in 1962 (Axelrod 1962). With the continued advancement in molecular biologic technology since the 1970s, much information has been acquired about the structure, function, and regulation of PNMT. In the rat, transcriptional control of the PNMT gene is predominantly orchestrated through the proximal 1 kb of the PNMT promoter (Ross et al. 1990), which includes binding sites for the transcription factors such as early growth response protein 1 (Egr-1) (Ebert et al. 1994), stimulatory protein 1 (Sp1) (Ebert and Wong 1995), activator protein 2 (AP-2) (Ebert et al. 1998; Wong et al. 1998b), the glucocorticoid receptor (GR) (Ross et al. 1990; Tai et al. 2002), cMyc-associated zinc nger protein (MAZ) (Her et al. 1999; Her et al. 2003), and a glial cell missinglike factor (GCMl) (Tai and Wong 2003). These transcriptional activators of PNMT play important roles in hormonal and neural regulation of PNMT, leading to changes not only in mRNA expression but in protein expression as well (Morita et al. 1996; Morita and Wong 1996; Tai et al. 2002; Wong et al. 1993, 1996, 1998a, 2002; Wong and Tai 2002). A schematic of the rat PNMT promoter is shown in Fig. 1.

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Cell Mol Neurobiol (2010) 30:14511457 Fig. 1 Schematic of the rat PNMT promoter. The binding site for the various transcription factors, GR, AP-2, GCMl, Egr-1, Sp1, and MAZ (for abbreviations, see text), identied for PNMT are depicted in the proximal -893 bp of PNMT promoter

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Stress Activation of PNMT In Vivo The two components of the stress axis, which contribute to stress regulation of adrenal PNMT, include adrenocortical outow from the HPA axis, and preganglionic sympathetic splanchnic innervation to the adrenal medulla (Kvetnansky et al. 2009). The HPA axis is regulated via a negative feedback loop (Herman and Cullinan 1997; Ziegler and Herman 2002). Corticosteroids are auto-regulated to limit steroid production and terminate glucocorticoid-initiated responses, thereby restricting the adverse effects of their overexpression. Synthesis is initiated in the brain through activation of CRH and arginine vasopressin (AVP) production and release from the paraventricular nucleus (PVN) of the hypothalamus. ACTH synthesis and release, in turn, is activated in the anterior lobe of the pituitary gland. Wending its way through the portal system to the adrenal cortex, ACTH initiates corticosteroid production in the adrenal cortex. As glucocorticoids accumulate, further production is limited directly via negative feedback to the anterior pituitary and PVN and indirectly via transynaptic activation of the hippocampus (see Herman and Cullinan 1997 and Ziegler and Herman 2002 for details of the HPA axis circuitry). The adrenal medulla is a modied component of the sympathetic nervous system. Chromafn cells receive preganglionic innervation via the splanchnic nerve, which stimulates the release of epinephrine. Two major transmitters released from the splanchnic nerve are acetylcholine and pituitary adenylate cyclase-activating polypeptide (PACAP). Epinephrine has been shown to be controlled through its biosynthesis by glucocorticoid, acetylcholine, and PACAP regulation of PNMT via transcriptional, translational, post-transcriptional, and/or post-translational events (Evinger et al. 1992, 1994; Morita et al. 1996; Morita and Wong 1996; Stachowiak et al. 1988, 1990; Tonshoff et al. 1997; Wong et al. 1992a, b, 1993, 1995, 1996, 1998a; 2002; Wong and Tai 2002). Corticosteroids activate PNMT production through activation of glucocorticoid response elements in the PNMT promoter to initiate PNMT gene transcription and via controlling the production of the co-substrate and methyl donor, S-adenosylmethionine, to regulate PNMT degradation. Acetylcholine and PACAP induce PNMT synthesis through activation of muscarinic

and nicotinic receptors and PACAP type I receptors on the chromafn cell membranes. It is well established that stress regulates the expression of the catecholamine biosynthetic enzymes (Kvetnansky et al. 2009). However, variability in their responses is apparent and highly associates with the nature and intensity of the specic stressful stimulus. For example, TH and DBH are very responsive to cold stress, whereas PNMT shows less susceptibility to the same. In contrast, IMMO stress markedly activates TH, DBH, and PNMT expression. As described above, the major peripheral source of stress-evoked epinephrine circulating in the bloodstream is the adrenal medulla. Viskupic et al. (1994) demonstrated in rats that single and repeated IMMO rapidly and markedly elevated PNMT mRNA in the medulla. These changes depend on an intact pituitaryadrenocortical axis as hypophysectomy abrogates induction. We have further shown that changes in mRNA, at least in part, represent activation of PNMT gene expression (Tai et al. 2007). IMMO elevates nuclear levels of Egr-1 and Sp1 mRNA and protein and the generation of Egr-1 and Sp1 protein/ DNA-binding complex in adrenal chromafn cells of rats subjected to single or multiple IMMO (1, 6, and 7 IMMOs) for 30 or 120 min. These changes lead to induction of PNMT mRNA and cytosolic protein in the chromafn cells. However, with delayed euthanasia after 7 days of repeated IMMO, the magnitudes of change in PNMT protein and PNMT mRNA do not match, suggesting that desensitization to the stress may have initiated and/or that dissociation of transcriptional or post-transcriptional regulatory mechanisms may be occurring.

Regulatory Mechanisms Underlying Stress Activation of PNMT Studying stress axis regulatory responses in the intact organism is difcult at best, given the complicated physiology and compensatory mechanisms associated with such responses. As our ultimate goal was to reveal mechanisms by which stress regulates epinephrine via PNMT and thereby can result in adrenergic dysfunction and lead to illness, a cell model system was clearly required for

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investigations to proceed. Evinger et al. (2002) had shown that hypoxic stress-induced expression of a rat PNMT promoter-driven reporter gene in immortalized mouse pheochromocytoma cells, which required corticosteroids for survival. We had preliminary ndings demonstrating that hypoxia-induced PNMT expression in PC12 cells (established from rat pheocromocytoma) (Greene and Tischler 1976) as well and via PNMT transcriptional activators that we had previously associated with PNMT gene induction. While little is known about the effects of hypoxia on adrenal medullary function, hypoxia is a cellular stressor in vivo, and one which does target the adrenal and other components of the stress axis. Developmentally, fetal exposure to hypoxia in sheep markedly reduces TH, DBH, and PNMT in the adrenal medulla and results in lower circulating epinephrine and epinephrine/PNMTexpressing cells (Coulter et al. 1998; Ducsay et al. 2007). Hypoxia alters hypothalamic gene expression, mRNA expression of genes in the anterior pituitary, and adrenal cortex, and reduces arterial O2 saturation and body temperature (Bruder et al. 2008). Thus, ndings derived from examining the effects of hypoxic stress in PC12 cells may indeed be relevant to hypoxic stress effects on PNMT in the chromafn cells in vivo. Our aim was to work with cells closely associated with the adrenal medulla and a homologous system (single species) with the intent that the cells would express a similar complement of factors as rat chromafn cells and minimize the possibility of species-dependent PNMT responses to hormonal and neural stimuli evidenced in earlier studies (Tai et al. 2001). Furthermore, we wanted a cell culture system, which did not depend on corticosteroids for cell maintenance, since PNMT is glucocorticoid-regulated and mechanistically understanding its glucocorticoid dependency is critical to the full scope of our investigations (Ross et al. 1990; Tai et al. 2002). Thus, we chose to examine the effects of moderate hypoxia (5% O2) in PC12 cells. PC12 cells transfected with a PNMT promoter luciferase reporter gene construct, pGL3RP893, harboring the proximal -893 bp of rat PNMT promoter, were exposed to hypoxia for durations up to 24 h. Hypoxia induced a 4.5-fold rise in luciferase reporter gene expression, which peaked at 6 h and was sustained at this elevated maximum for up to 24 h. In contrast to the ndings of Evinger et al., promoter activity did not increase with decreasing oxygen. Rather, it markedly declined, consistent with previous reports that oxygen reduction commits cells to apoptotic death (Hop et al. 2004; Santra et al. 2008). It may be that in the mouse pheochromocytoma cells, glucocorticoid dependency for propagation and survival may serve in a neuroprotective capacity in addition to stimulating PNMT promoter-driven gene expression via GR activation of the promoter.

Hypoxia can activate a variety of signaling cascades beginning with cAMP-protein kinase A (PKA) and phospholipase C (PLC). When PC12 cells were pre-treated with selective signaling pathway inhibitors, followed by exposure to 5% O2 for 24 h, not only did cAMP, PKA, and PLC appear to be associated with the hypoxic responsiveness of the PNMT promoter but also protein kinase C (PKC), phosphoinositide 3-kinase, ERK1/2 mitogen-activated protein kinase (ERK1/2 MAPK), and p38 mitogen-activated protein kinase (p38 MAPK) as demonstrated by MDL12,330A, H89, U73122, GF109203X, LY294002, U0126, and SB203580, respectively. Activation of these signal transducers resulted in stimulation of two PNMT transcriptional activators, Egr-1 and Sp1, as well as a transcription factor common to other hypoxia-sensitive genes, HIF1a (Tai et al. 2009, 2010). Elevation of mRNA (1 h), protein and proteinDNA-binding complex was observed for all the three transcription factors. Transcriptional activation of HIFa is considered an unusual mode for its regulation since HIFa protein is constitutively expressed, and its intracellular concentrations usually regulated via prolyl hydroxylation, ubiquitinylation, and proteosome shunting for degradation (Hop et al. 2004; Semenza 2000a). Downstream of transcription factor stimulation, induction of PNMT occurred with increased PNMT mRNA and cytosolic protein apparent. Maximum stimulation of mRNA and protein occurred at 6 h. The latter was a bit surprising, for PNMT protein, which given its slow turnover, usually reaches peak responses to a stimulus at 24 h. Consistent with the role of Egr-1 and Sp1 in hypoxiainduced activation of PNMT, mutation of their binding sites in the PNMT promoter (-165 and -45 bp and -168 and -48 bp), either independently or in tandem, or inclusion of Egr-1 or Sp1 siRNA (100 ng/ml) completely abrogated PNMT promoter responsiveness. Furthermore, while hypoxia activated all the signaling enzymes targeted by the signaling inhibitors described earlier, the same inhibitors prevented Egr-1, Sp1, and HIF1a mRNA and nuclear protein induction and consequently, PNMT mRNA, and cytosolic protein induction.

HIF1a, a Switch in Adrenergic Responses to Stress HIF1a forms a heterodimer with another member of the HIF family of proteins, HIF1b, which upon binding to a HIF consensus site, stimulates transcription (Hop et al. 2004; Semenza 2000a). Overexpression of HIF1a, a truncated HIF1a containing only the O2-dependent activation domain, or HIF1b also induced the PNMT promoter and the endogenous PNMT gene. However, overexpression of HIF1a in PC12 cells transfected with PNMT promoter luciferase reporter gene constructs harboring either mutated

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Egr-1- or Sp1-binding elements (21 or 5% O2) did not increase luciferase reporter gene activity above basal levels (Tai et al. 2009). Furthermore, gel mobility shift assays demonstrated that a HIF consensus element within the proximal -893 bp of PNMT promoter was a weak binding site at best (-282 bp). Yet, overexpression of HIF1a increased PNMT mRNA and protein. These ndings indicated that HIF1a might be functioning indirectly as an onoff switch activating adrenergic responses to stress. When stress stimulates HIF1a, it, in turn, induces Egr-1 and Sp1 expression, and thereby PNMT under conditions of hypoxia. If HIF1a does act indirectly to stimulate PNMT, then its overexpression should induce Egr-1 and Sp1, while inclusion of HIF1a siRNA under conditions of hypoxia should have the opposite effect. Indeed, elevating HIF1a by transfection of its expression construct into PC12 cells markedly increased Egr-1 and Sp1 mRNA, and nuclear protein while transfection of HIF1a siRNA in PC12 cells completely inhibited PNMT promoter activation and stimulation of Egr-1 and Sp1 protein (Tai et al. 2009, 2010). Thus, not only does HIF1a act as a regulatory onoff switch to stimulate Egr-1 and Sp1, but these two transcription factors, once activated, also work cooperatively to stimulate PNMT. We now have preliminary evidence that HIF1a protein expression is elevated in adrenal chromafn cells of rats subjected to IMMO stress or treated with PACAP. These ndings are consistent with hypoxia acting as a stressor and PACAP as a neurotransmitter released from the splanchnic nerve to induce PNMT expression via induction of HIF1a
Fig. 2 Schematic of potential role of hypoxic stress and HIF1a in stress-regulated PNMT expression. This schematic is based on earlier and current knowledge of hypoxia effects on the HPA axis and preganglionic sympathetic innervation to the adrenal medulla, as well as our previous ndings and those that we have presented here

We have previously shown that cholinergic activation induces PNMT via Egr-1 activation (Morita et al. 1996) although we do not know at present if HIF1a plays a role in the latter. Hypoxia elevates circulating corticosteroids (Krugers et al. 2000) and may thereby lead to GR activation and PNMT gene induction directly through upstream GREs. We propose that hypoxic induction of PNMT may arise via HPA axis regulation of glucocorticoids and via pre-ganglionic sympathetic innervation through PACAP and acetylcholine. Figure 2 provides a schematic of the proposed role of hypoxia and HIF1a in stress induction of PNMT and epinephrine biosynthesis in the chromafn cell.

Conclusions The ndings of this study demonstrate that hypoxic stress in PC12 cells may provide a model for revealing molecular mechanisms associated with stress-induced activation of PNMT and epinephrine in adrenal chromafn cells. Consistent with earlier results from IMMO stress studies in rats, both Egr-1 and Sp1 were induced and they, in turn, through cooperative interaction, stimulate PNMT gene and protein expression. In addition, however, hypoxic stress also elevated HIF1a. This transcription factor does not appear to directly stimulate the PNMT gene promoter in the case of the proximal -893 bp of promoter sequence. Rather, it seems to work indirectly via activation of Egr-1 and Sp1. Preliminary studies show that IMMO stress and PACAP treatment also activates HIF1a in the adrenal

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Cell Mol Neurobiol (2010) 30:14511457 Guglielmotto M, Aragno M, Autelli R, Giliberto L, Novo E, Colombatto S, Danni O, Parola M, Smith MA, Perry G, Tamagno E, Tabaton M (2009) The up-regulation of BACE1 mediated by hypoxia and ischemic injury: role of oxidative stress and HIF1a. J Neurochem 108:10451056 Halliwill JR (2003) Hypoxic regulation of blood ow in humans. Skeletal muscle circulation and the role of epinephrine. Adv Exp Med Biol 543:223236 Her S, Bell RA, Bloom AK, Siddall BJ, Wong DL (1999) Phenylethanolamine N-methyltransferase gene expression: Sp1 and MAZ potential for tissue specic expression. J Biol Chem 274:86988707 Her S, Claycomb R, Tai TC, Wong DL (2003) Regulation of the rat phenylethanolamine N-methyltransferase gene by transcription factors Sp1 and MAZ. Mol Pharmacol 64:11801188 Herman JP, Cullinan WE (1997) Neurocircuitry of stress: central control of the hypothalamo-pituitary-adrenocortical axis. TINS 20:7884 Hop G, Ogunshola O, Gassmann M (2004) HIFs and tumors causes and consequences. Am J Physiol Regul Integr Comp Physiol 286:R608R623 Krugers HJ, Maslam S, Korf J, Joels M (2000) The corticosterone synthesis inhibitor metyrapone prevents hypoxia/ischemiainduced loss of synaptic function in the rat hippocampus. Stroke 21:11621172 Kvetnansky R, Sabban EL, Palkovits M (2009) Catecholaminergic systems in stress: structural and molecular genetic approaches. Physiol Rev 89:535606 Lorita J, Escalona N, Faraudo S, Soley M, Ramirez I (2002) Effects of epidermal growth factor on epinephrine-stimulated heart function in rodents. Am J Physiol Heart Circ Physiol 283:18871895 Lundberg U (2005) Stress hormones in health and illness: the roles of work and gender. Psychoneuroendocrinology 30:10171021 Mamet J, Peyronnet J, Roux J-C, Perrin D, Cottet-Emard J-M, Pequignot J-M, Lagercrantz H, Dalmaz Y (2002) Long-term prenatal hypoxia alters maturation of adrenal medulla in rat. Pediatr Res 51:207214 Marco EM, Macri S, Laviola G (2010) Critical age windows for neurodevelopmental psychiatric disorders: evidence from animal models. Neurotox Res. doi:10.1007/s12640-010-9205-z Mikhailenko VA, Butkevish IP, Bagaeva TR, Makukhina GV, Otellin VA (2009) Short- and long-term inuences of hypoxia during early postnatal period of development on behavioral and hormonal responses in rats. Neurosci Lett 464:214217 Morita K, Wong DL (1996) Role of Egr-1 in cholinergic stimulation of phenylethanolamine N-methyltransferase promoter. J Neurochem 67:13441351 Morita K, Bell RA, Siddall BJ, Wong DL (1996) Neural stimulation of Egr-1 messenger RNA expression in rat adrenal gland: Possible relation to phenylethanolamine N-methyltransferase gene regulation. J Pharmacol Exp Ther 279:379385 Ross ME, Evinger MJ, Hyman SE, Carroll JM, Mucke L, Comb M, Reis DJ, Joh TH, Goodman HM (1990) Identication of a functional glucocorticoid response element in the phenylethanolamine N-methyltransferase promoter using fusion genes introduced into chromafn cells in primary culture. J Neurosci 10:520530 Rybnikova EA, Samoilov MO, Mironova VI, Tyulkova EI, Pivina SG, Vataeva LA, Ordyan NE, Abritalin EY, Kolchev AI (2008) The possible use of hypoxic preconditioning for the prophylaxis of post-stress depressive episodes. Neurosci Behav Physiol 38:721726 Santra M, Santara S, Zhang J, Chopp M (2008) Ectopic decorin expression up-regulates VEGF expression in mouse cerebral endothelial cells via activation of the transcription factors Sp1, HIF1a, and Stat3. J Neurochem 105:324337

medulla of rats. In vivo siRNA strategies are now targeted at demonstrating that knock down of Egr-1, Sp1, or HIFa protein in the adrenal medulla of rats subjected to IMMO will similarly prevent PNMT induction in response to the stress. If so, then the role of HIF1a may be to serve as an onoff switch for the stress responsiveness of PNMT and epinephrine, providing a target for therapeutic intervention in stress-elicited illness.
Acknowledgments This study was supported by The Spunk Fund, Inc., the Sobel-Keller Research Fund, McLean Hospital, and the Emerald Foundation, Inc. (DLW), and Slovak Grants, APVV-0148-06 and VEGA 2/0133/08 (RK).

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