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Nongenomic cortisol signaling in fish
Accepted Manuscript Nongenomic cortisol signaling in fish Chinmayee Das, Marwa Thraya, Mathilakath M. Vijayan PII: DOI: Reference:
S0016-6480(18)30049-2 https://doi.org/10.1016/j.ygcen.2018.04.019 YGCEN 12918
To appear in:
General and Comparative Endocrinology
Received Date: Revised Date: Accepted Date:
16 January 2018 12 April 2018 14 April 2018
Please cite this article as: Das, C., Thraya, M., Vijayan, M.M., Nongenomic cortisol signaling in fish, General and Comparative Endocrinology (2018), doi: https://doi.org/10.1016/j.ygcen.2018.04.019
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Mini-review
Nongenomic cortisol signaling in fish1
Chinmayee Das*, Marwa Thraya*, and Mathilakath M. Vijayan# Department of Biological Sciences, University of Calgary, 2500 University Drive NW, Calgary, Alberta T2N 1N4, Canada
1
Presented at the ICCE18 meeting in Lake Louise, Alberta, Canada
#
Corresponding author. E-mail address: [email protected] (M.M Vijayan)
*Contributed equally
Das et al Abstract Glucocorticoids are critical regulators of the cellular processes that allow animals to cope with stressors. In teleosts, cortisol is the primary circulating glucocorticoid and this hormone mediates a suite of physiological responses, most importantly energy substrate mobilization that is essential for acute stress adaptation. Cortisol signaling has been extensively studied and the majority of work has been on the activation of the glucocorticoid receptor (GR), a ligand-bound transcription factor, and the associated downstream transcriptional and protein responses. Despite the role of this hormone in acute stress adaptation, very few studies have examined the rapid effects of this hormone on cellular function. The nongenomic corticosteroid effects, which are rapid (seconds to minutes) and independent of transcription and translation, involve changes to second-messenger pathways and effector proteins, but the primary receptors involved in this pathway activation remain elusive. In teleosts, a few studies suggested the possibility that GR located on the membrane may be initiating a rapid response based on the abrogation of this effect with RU486, a GR antagonist. However, studies have also proposed other signaling mechanisms, including a putative novel membrane receptor and changes to membrane biophysical properties as initiators of rapid signaling in response to cortisol stimulation. Emerging evidence suggests that cortisol activates multiple signaling pathways in cells to bring about rapid effects, but the underlying physiological implications on acute stress adaptation are far from clear.
Keywords Glucocorticoid receptor, teleosts, metabolism, stress response
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Das et al 1. Introduction Glucocorticoids are steroid hormones involved in the regulation of an extensive range of physiological processes, including intermediary metabolism, immunity, growth, osmoregulation, neuronal function, and behaviour (Aluru and Vijayan, 2009; Borski, 2000; Faught and Vijayan, 2016; Mommsen et al., 1999; Wendelaar Bonga, 1997). In teleosts, cortisol is the primary glucocorticoid in circulation and is produced by the interrenal cells (analogous to the adrenal cortex in mammals) localized primarily in the head kidney region in response to a stressor (Mommsen et al., 1999; Wendelaar Bonga, 1997). The release of this steroid is under the control of the hypothalamus-pituitary-interrenal (HPI) axis (akin to the hypothalamus-pituitary-adrenal (HPA) axis in mammals) to mediate cellular functions essential for stress adaptation (Mommsen et al., 1999; Wendelaar Bonga, 1997). For example, excess cortisol enhances liver metabolic capacity, including activation of hepatic gluconeogenesis to fuel the increased energy demand associated with stressor exposure and re-establish homeostasis (Aluru and Vijayan, 2009, 2007; Mommsen et al., 1999). Corticosteroids exert wide-spread cellular effects on target tissues through two distinct mechanisms known as the genomic and nongenomic steroid signaling pathways. The genomic pathway, which has been extensively characterized, involves the transfer of cortisol across the cell membrane and binding to intracellular receptors of the nuclear steroid receptor superfamily, to either positively or negatively regulate target gene transcription and protein synthesis (Bury et al., 2003; Faught and Vijayan, 2016; Mommsen et al., 1999; Prunet et al., 2006; Stolte et al., 2006). As this process leads to de novo protein synthesis in altering cellular function, genomic signaling is slower to respond, but longer lasting (Borski, 2000; Mommsen et al., 1999; Wendelaar Bonga, 1997). Nongenomic signaling, on the other hand, is rapid (seconds to
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Das et al minutes) as effector molecules are modulated by activation of membrane-mediated secondary signaling cascades (Borski, 2000; Borski et al., 2002), including cAMP, Ca2+, and protein kinases. The major difference between the two pathways is that while the genomic pathway involves steroid-mediated activation of gene transcription and translation, the nongenomic signaling-mediated effects are independent of gene regulation (Lösel and Wehling, 2003). Therefore, time of onset and activation of the rapid signaling pathway in the presence of transcriptional and translational inhibitors are frequently used to confirm nongenomic steroid signaling (Borski, 2000; Groeneweg et al., 2011; Losel et al., 2003). While nongenomic steroid signaling and its physiological implications have garnered a lot of attention for reproductive steroids in teleost models (Thomas, 2012, 2003), the rapid effects of corticosteroids and its role in stress adaptations are far from clear (Borski, 2000; Faught and Vijayan, 2016). Although nongenomic corticosteroid actions have been reported in non-teleost models involved in rapid hormone regulation and stress adjustments, including the nervous, cardiovascular, and immune systems (select studies summarized in Table 1), such studies in teleosts are few and far between (Table 2). Here, we synthesize the studies that have been carried out in fishes pertaining to rapid effects of cortisol to develop hypotheses about the mode of action of this stress steroid and its role in acute stress adaptation.
2. Mechanisms of rapid cortisol action In mammals, rapid glucocorticoid effects have been implicated in various tissues and cellular system functions, including cardiovascular, immune, central nervous system, skeletal muscle, and liver (Table 1). However, the mechanisms that mediate these actions remain elusive. The teleost GR and mineralocorticoid (MR) receptors have been cloned, sequenced, and
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Das et al extensively reviewed (Prunet et al., 2006; Stolte et al., 2006; Sturm et al., 2011). Briefly in teleosts, there are two isoforms of GR (GR1 and GR2) (Bury et al., 2003), apart from zebrafish, which has only one GR (Alsop and Vijayan, 2009), as well as a splice variant GRß (Schaaf et al., 2008). Fish also express a single MR receptor, but the role of MR in mediating cortisol actions is not well understood in teleosts (Colombe et al., 2000; Faught and Vijayan, 2016; Prunet et al., 2006; Sturm et al., 2011). Although studies have proposed a putative membrane-bound receptor mediating rapid activation of signaling pathways in response to glucocorticoid stimulation, to date, a membranebound receptor for corticosteroid action has not been cloned and sequenced in any animal model (Faught and Vijayan, 2016; Tasker et al., 2006). There has only been a single report of a partial purification of a glucocorticoid receptor from the brain of the rough-skinned newt, Taricha granulosa (Evans et al., 2000). Using cortisol-sepharose affinity resin, this study provided novel insight that a membrane-GR isolated from neuronal membranes is a glycoprotein characterized as having a mass of 63 kDa, making this distinct from the intracellular GR, and binds cortisol with high affinity (Evans et al., 2000). Membrane binding sites for glucocorticoids have been reported in different tissues, including mouse lymphocytes (Gametchu, 1987), chicken liver (Trueba et al., 1987), rodent pituitary (Koch et al., 1977), and amphibian brain (Orchinik et al., 1991). However, less is known in fish. Only a single study has reported cortisol binding to membranes in the tissues of the Mozambique tilapia, Oreochromis mossambicus (Johnstone et al., 2013). It was found that the glucocorticoid membrane binding activity was present in liver (high affinity; Kd = 9.5 nM) and kidney (low affinity; Kd = 30.08 nM) (Johnstone et al., 2013). While the specific binding of cortisol on the membrane has not been looked at, preliminary results from our laboratory by
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Das et al immunodetection using western blotting with antibodies to trout GR and zebrafish MR suggest the presence of these receptors on plasma membranes of rainbow trout liver. However, the mechanism of action of these receptors in bringing about rapid nongenomic signaling is currently unknown. Based on the literature available, we are proposing four working hypotheses on the mode of action of cortisol in mediating rapid effects in teleosts (Fig. 1). One is that corticosteroids mediate changes in the biophysical property of the plasma membrane leading to the activation of intracellular signaling pathways (Dindia et al., 2012; Whiting et al., 2000). For instance, cortisol increased membrane fluidity in DPPC (dipalmitoylphosphatidylcholine) liposomes (Ghosh et al., 1996), and the production of eicosanoids in rabbit cardiac muscle endothelial tissues was inhibited due to membrane ordering effects (Gerritsen et al., 1991). A rapid effect of cortisol on membrane fluidity was revealed in rainbow trout (Oncorhynchus mykiss) plasma membranes, including altered anisotropy within 30 min of cortisol treatment in vitro (Dindia et al., 2012). While the mechanism bringing about biophysical changes in unclear, atomic force microscopy revealed membrane domain shifts in response to cortisol in trout plasma membrane monolayers (Dindia et al., 2012). This rapid change in membrane biophysical properties, including alterations to lipid rafts, can lead to mechanotransduction-related activation of downstream signaling pathways (Vigh et al., 2007). However, the mechanisms by which changes in membrane biophysical properties by cortisol are transduced into rapid cellular effects remains to be elucidated. The second hypothesis involves the activation of membrane (novel) receptors on the cell surface, independent of GR, initiating rapid downstream signaling cascades as shown for the mineralocorticoid aldosterone in mammalian and non-mammalian models (Lösel et al., 2002). It
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Das et al is hypothesized that these novel membrane proteins may belong to the GPCR family as was seen for the nongenomic signaling of sex steroid in fishes (reviewed in Thomas et al., 2006). This is supported by the rapid activation of second messengers, including cAMP and Ca2+ (Fig. 2) (Borski et al., 1991; Espinoza et al., 2017; Hyde et al., 2004; Vijayan et al., 2017), as well as rapid phosphorylation of substrate proteins of the protein kinase A (PKA), B (PKB), and C (PKC) pathways with membrane impermeable cortisol in trout hepatocytes (Dindia et al., 2012). The third working hypothesis revolves around the possibility that modulation of the intracellular receptors or translocation of these receptors to the plasma membrane may lead to the activation of rapid signaling (Groeneweg et al., 2011; Lösel and Wehling, 2003). Support for cortisol signaling by GR was seen from studies that showed abolishment of rapid cortisolmediated responses by RU486 (Espinoza et al., 2017; Roy and Rai, 2009). Further support for this observation comes from preliminary studies showing accumulation of GR close to the plasma membrane within 5 min of cortisol exposure to trout hepatocytes (Fig. 3). This leads us to propose that the rapid tissue responses to GR activation (Gross and Cidlowski, 2008) may involve rapid translocation of intracellular GR to the cell periphery, but the mechanisms involved remains to be determined. Overall, it appears that cortisol rapidly alters intracellular GR dynamics, and the GR translocation to the plasma membrane may be involved in the activation of rapid cell signaling by cortisol. The fourth working hypothesis is that rapid cortisol mode of action involves altering intracellular Ca2+ levels in fish cells by modulating plasma membrane-bound Ca2+ channels, independently of membrane receptor(s). An increase in intracellular Ca2+ levels appears to be a key second messenger for the rapid glucocorticoid response in vertebrate models (Beato and Klug, 2000; Karst et al., 2002), but this is far from clear in fish (Hyde et al., 2004). Nongenomic
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Das et al glucocorticoid actions have been previously shown to be responsible for intracellular Ca2+ spikes in different cell lines (Qiu et al., 1998; Sze and Iqbal, 1994; Wagner et al., 1999). Studies have reported that glucocorticoid addition in cells causes an intracellular Ca2+ wave with varying order of magnitude, peak, and amplitude (Berridge et al., 2000). In addition, glucocorticoids have shown an increase in selectivity for L-type Ca2+ channels over N-type channels in the mouse hippocampus (Chameau et al., 2007). The only study that has examined this in fish showed that cortisol rapidly reduced intracellular Ca2+ and this involved suppression of voltagegated Ca2+ channel activity in the prolactin cells of the Mozambique tilapia (Hyde et al., 2004). Preliminary results from our laboratory showed that cortisol stimulates a rapid (within one min) rise in intracellular Ca2+ in rainbow trout hepatocytes, and this response was also mimicked by the membrane impermeable cortisol-BSA (Fig. 2), suggesting the activation of membrane receptor(s). Also, emerging evidence seems to suggest that cortisol may be favoring rapid influx of extracellular Ca2+, supporting the activation of plasma membrane Ca2+ channels (Vijayan et al., 2017). Taken together, these results suggest that rapid action of cortisol in fish cells involves modulation of intracellular Ca2+ levels, but the response may be cell-specific. This rapid influx of Ca2+ in response to cortisol stimulation may also be involved in the GR translocation seen in trout hepatocytes (Fig. 3), but this needs to be tested. Overall, there appears to be multiple mechanisms by which cortisol may rapidly activate and/or inhibit downstream signaling pathways to mediate an integrated response for acute stress adaptation.
3. Nongenomic physiological actions of cortisol 3.1 Salinity acclimation
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Das et al One of the first studies that explored rapid cortisol action in teleosts was in the pituitary of the Mozambique tilapia where cortisol suppressed prolactin release within 20 min by inhibiting the cAMP and Ca2+ signaling pathways (Borski et al., 2002, 1991; Hyde et al., 2004). As cyclohexamide did not abolish this inhibiting effect, the results suggested an effect independent of protein synthesis and, therefore, not involving the classical GR (Borski et al., 2002). This led to the proposal that a membrane-associated receptor may be involved, as the effect was also mimicked by the membrane impermeable form of cortisol-21 hemisuccinate coupled to bovine serum albumin (BSA) (Borski et al., 2001). Subsequently, in the same species, rapid activation of Na+/K+ and Ca2+ ATPase activities was reported in gill tissue in response to cortisol and corticosterone treatment both in vivo and in vitro, and these effects were insensitive to actinomycin-D, a transcriptional inhibitor, further supporting nongenomic activation (Sunny and Oommen, 2001). Also, cortisol rapidly induced MAPK activity through modulation of extracellular signal-regulated kinase (ERK1/2) in the gills of this species providing a mechanistic underpinning for the rapid modulation of ion transporters by cortisol (Kiilerich et al., 2011a). This study along with the observation that cortisol rapidly inhibits prolactin secretion from the pituitary suggests a key nongenomic role for cortisol in salinity acclimation in euryhaline species. This would be particularly telling to species inhabiting estuaries and would entail daily and rapid switching of ion transporters when moving from hyperosmotic to hypoosmotic environments and vice versa. However, the precise mechanisms of action remain to be elucidated in these species. 3.2 Metabolic stress response Although considered a key target in cortisol action (Mommsen et al., 1999), little has been reported with regards to rapid actions of cortisol in teleost liver (Table 2). Studies that examined
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Das et al the effect on liver showed rapid changes in second messengers, and changes in the activities of enzymes involved in intermediary metabolism (Dindia et al., 2012; Faught and Vijayan, 2016; Sunny et al., 2002; Vijayan et al., 2017). For instance, it has been shown that cortisol decreases the activities of liver malic enzyme, glucose-6-phosphatase, and isocitrate dehydrogenase within 5-l0 min, with effects persistent in the presence of actinomycin-D in vitro (Sunny et al., 2002). Mozambique tilapia injected with cortisol and a transcriptional inhibitor in a short-term in vivo study also showed similar results within 30 min of hormone addition, further suggesting a role in regulating lipid metabolism and possibly promoting gluconeogenesis (Sunny et al., 2002). More recent studies examined cortisol action on membrane fluidity in rainbow trout hepatocytes (Dindia et al., 2013, 2012). The results suggest that cortisol increased membrane fluidity and changed membrane topography within 10 min of cortisol treatment (Dindia et al., 2012). This effect corresponded with rapid phosphorylation of substrate proteins by PKA, PKB, and PKC (Dindia et al., 2012). The changes observed with cortisol were similar to that seen with benzyl alcohol, a known membrane fluidizer (Dindia et al., 2012), leading to the proposal that changes in membrane biophysical properties may be an initiator of downstream stress signaling pathways in trout. This was followed up with an in vivo experiment that showed acute stress elevated plasma cortisol levels altered hepatic membrane fluidity within 30 min, a response that was blocked by metyrapone, an inhibitor of cortisol synthesis (Dindia et al., 2013). Furthermore, the stressorinduced cortisol elevation rapidly activated ERK1/2 MAPK and increased phosphorylation of PKC and PKA substrate proteins (Dindia et al., 2013), similar to that observed in cortisol treated hepatocytes in vitro (Dindia et al., 2012). These results suggest that cortisol action may also occur independent of membrane receptor activation by rapidly altering the membrane fluidity,
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Das et al and stimulating mechanotransduction pathways, but the mechanisms are far from clear. These studies are the only one of its kind to investigate the physiological effects of rapid cortisol signaling and its implications on peripheral liver function and cellular signaling pathways in fish. The emerging results seem to suggest that the rapid effect of cortisol may facilitate utilization of glucose for endogenous liver use to cope with the increased energy demand associated with stress (Dindia, 2013). Also, the results tend to suggest that the rapid effect of cortisol during stress may also involve reducing the glucose output seen with epinephrine activation (Fabbri and Moon, 2016; Reid et al., 1992), thereby facilitating partitioning of energy substrates for endogenous use by the liver (Dindia, 2013). These results led to the hypothesis that the rapid action of cortisol may involve liver energy substrate repartitioning to cope with the enhanced energy demand associated with acute stressor exposure. 3.3 Muscle, cardiovascular, and innate immune systems Recently, myotubules from rainbow trout showed that cortisol and cortisol-BSA rapidly induce reactive oxygen species (ROS) mediated by the activities of NADPH oxidase and phospholipase A2 (Espinoza et al., 2017). ROS production was blocked by the GR antagonist, RU486, but not by the MR antagonist, RU28318, suggesting a membrane-GR may be participating in this rapid glucocorticoid signaling (Espinoza et al., 2017). In addition, cortisolBSA showed a significant increase in the phosphorylation of ERK1/2 and cAMP response element binding protein (CREB) within 15 min of treatment, suggesting rapid activation of stress-signaling pathways in the muscle in response to stressor-mediated cortisol stimulation (Espinoza et al., 2017). However, the physiological implication of this rapid activation of cellular stress-signaling pathways in the muscle remains to be determined. While studies have shown that muscle metabolism is a key target for cortisol action during stress (Faught et al., 2016;
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Das et al Mommsen et al., 1999), the mechanisms of rapid action and the pathways involved in acute stress adaptation needs to be investigated. Information pertaining to nongenomic cortisol signaling is still in its infancy in teleost cardiovascular and innate immune systems (Table 2). A report on nongenomic signaling in the cardiovascular system of rainbow trout showed that cortisol rapidly increased coronary vasoconstriction in less than 15 min (Agnisola et al., 2004). As a result, cortisol-induced vasoconstriction may negatively affect blood circulation under stressed conditions (Agnisola et al., 2004). Rapid cortisol actions were also observed in the spotted murrel (Channa punctatus) within 15 min, including suppression of phagocytosis in the spleen, and a more profound inhibition at 1 h post-hormone treatment (Roy and Rai, 2009). Data from this study points to the activation of a membrane-bound GR, as well as the cAMP-PKA pathway in mediating the rapid responses to cortisol, as inhibitors of GR, adenylate cyclase, and PKA completely abolished those responses (Roy and Rai, 2009). Overall, these results highlight the possibility that the nongenomic response may also involve signaling by GR translocated to the membrane and/or other proteins that share a common epitope with GR.
4. Conclusions and future directions This review was intended to highlight the significance of rapid nongenomic signaling in allowing fishes to cope with stress. Taken together, the nongenomic mode of action of cortisol is diverse, and while the mechanisms are not fully understood, we propose four modes of nongenomic actions of cortisol in fish (Fig. 1). Based on our current understanding of rapid cortisol actions in fish, future studies should be targeted at investigating the mechanisms by which these rapid responses are taking place, and their effects in relation to the delayed actions
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Das et al of glucocorticoids (Groeneweg et al., 2011). Recent developments in gene knockout technology, including CRISPR/Cas9 will allow us to clearly differentiate the effect of nongenomic signaling mediated by novel membrane receptors from those mediated by GR activation. Development of a GR knockout model will also clearly highlight the role of this receptor in mediating rapid cortisol signaling during acute stress adaptation. Another major development in this research area would be the discovery of a novel membrane receptor for cortisol, as this will have huge implications in our understanding of stress coping mechanisms. Furthermore, since glucocorticoids are widely used as clinical immunosuppressive and anti-inflammatory agents, an insight into nongenomic corticosteroid actions and their underlying interaction with different downstream effectors would help to elucidate the cross-talk between the stress and immune response pathways, and will have applications in aquaculture for reducing stress-mediated inflammatory responses. The role of caveolin-1 as a mechanosensor and membrane scaffolding protein is well established in mammalian systems (Okamoto et al., 1998; Van Deurs et al., 2003; Vernocchi et al., 2013), but its role in teleost stress signaling is far from clear. Studies on caveolin-1 involvement in GR mediated rapid signaling has been reported (Matthews et al., 2008), along with observations on GR association with membrane lipid rafts (Jain et al., 2005), but the mechanisms leading to signal transduction is still unclear. We hypothesize that the interaction of GR with caveolin-1 on the plasma membrane may be critical for the transduction of rapid nongenomic signaling in fish cells. From a physiological stand-point, it is important to have a working hypothesis of the significance of the rapid response due to cortisol on stress adaptation. As the fight-or flight response is usually associated with the sympathetic nervous system activation, the role for
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Das et al cortisol was thought to do more with metabolic recovery, including sustaining the glucose release that was activated by epinephrine (Faught et al., 2016; Mommsen et al., 1999; Vijayan et al., 2010). While it seems reasonable to propose that the rapid cortisol-mediated response during stress may play a key role in the fight-or flight response, including the modulation of catecholamine signaling (Mommsen et al., 1999), direct evidence to this is still lacking in animal models. However, the rapid changes in intracellular stress-related signaling pathways seen with corticosteroid stimulation (Dindia et al., 2012, 2013; Espinoza et al., 2017; Vijayan et al., 2017) suggests a key role for this stress steroid in acute stress adaptation, but the target tissue responses and their implications in restoring homeostasis are far from clear.
Acknowledgements Funding for the studies in this mini-review was supported by the Natural Sciences and Engineering Research Council of Canada Discovery Grant to MMV.
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Jain, S., Li, Y., Kumar, A., Sehgal, P.B., 2005. Transcriptional signaling from membrane raftassociated glucocorticoid receptor. Biochem. Biophys. Res. Commun. 336, 3–8. Johnstone, W.M., Mills, K.A., Alyea, R.A., Thomas, P., Borski, R.J., 2013. Characterization of membrane receptor binding activity for cortisol in the liver and kidney of the euryhaline teleost, Mozambique tilapia (Oreochromis mossambicus). Gen. Comp. Endocrinol. 192, 107–114. Karst, H., Nair, S., Velzing, E., Rumpff‐van Essen, L., Slagter, E., Shinnick‐Gallagher, P., Joëls, M., 2002. Glucocorticoids alter calcium conductances and calcium channel subunit expression in basolateral amygdala neurons. Eur. J. Neurosci. 16, 1083–1089. Kiilerich, P., Milla, S., Sturm, A., Valotaire, C., Chevolleau, S., Giton, F., Terrien, X., Fiet, J., Fostier, A., Debrauwer, L., Prunet, P., 2011a. Implication of the mineralocorticoid axis in rainbow trout osmoregulation during salinity acclimation. J. Endocrinol. 209, 221–235. Kiilerich, P., Tipsmark, C.K., Borski, R.J., Madsen, S.S., 2011b. Differential effects of cortisol and 11-deoxycorticosterone on ion transport protein mRNA levels in gills of two euryhaline teleosts, Mozambique tilapia (Oreochromis mossambicus) and striped bass (Morone saxatilis). J. Endocrinol. 209, 115–126. Koch, B., Lutz Bucher, B., Briaud, B., Mialhe, C., 1977. Glucocorticoid binding to plasma membranes of the adenohypophysis. J. Endocrinol. 73, 399–400. Lowenberg, M., Tuynman, J., Bilderbeek, J., Gaber, T., Buttgereit, F., Van Deventer, S., Peppelenbosch, M., Hommes, D., 2005. Rapid immunosuppressive effects of glucocorticoids mediated through Lck and Fyn. Blood 106, 1703–1710. Long, F., Wang, Y.-X., Liu, L., Zhou, J., Cui, R.-Y., Jiang, C.-L., 2005. Rapid nongenomic inhibitory effects of glucocorticoids on phagocytosis and superoxide anion production by macrophages. Steroids 70, 55–61. Lösel, R., Feuring, M., Wehling, M., 2002. Non-genomic aldosterone action: From the cell membrane to human physiology, in: Journal of Steroid Biochemistry and Molecular Biology. pp. 167–171. Lösel, R., Wehling, M., 2003. Nongenomic actions of steroid hormones. Nat. Rev. Mol. Cell Biol. 4, 46-56. Losel, R.M., Falkenstein, E., Feuring, M., Schultz, A., Tillmann, H.-C., Rossol-Haseroth, K., Wehling, M., 2003. Nongenomic steroid action: controversies, questions, and answers. Physiol. Rev. 83, 965–1016. Matthews, L., Berry, A., Ohanian, V., Ohanian, J., Garside, H., Ray, D., 2008. Caveolin mediates rapid glucocorticoid effects and couples glucocorticoid action to the
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Das et al antiproliferative program. Mol. Endocrinol. 22, 1320–1330. Mommsen, T.P., Vijayan, M.M., Moon, T.W., 1999. Cortisol in teleosts:dynamics, mechanisms of action, and metabolic regulation. Rev. Fish Biol. Fish. 9, 211–268. Okamoto, T., Schlegel, A., Scherer, P.E., Lisanti, M.P., 1998. Caveolins, a family of scaffolding proteins for organizing “preassembled signaling complexes” at the plasma membrane. J. Biol. Chem. 273, 5419-5422. Orchinik, M., Murray, T.F., Moore, F.L., 1991. A corticosteroid receptor in neuronal membranes. Science 252, 1848–1851. Prunet, P., Sturm, A., Milla, S., 2006. Multiple corticosteroid receptors in fish: From old ideas to new concepts. Gen. Comp. Endocrinol. 147, 17-23. Qiu, J., Lou, L.G., Huang, X.Y., Lou, S.J., Pei, G., Chen, Y.Z., 1998. Nongenomic mechanisms of glucocorticoid inhibition of nicotine-induced calcium influx in PC12 cells: Involvement of protein kinase C. Endocrinology 139, 5103–5108. Reid, S.D., Moon, T.W., Perry, S.F., 1992. Rainbow trout hepatocyte beta-adrenoceptors, catecholamine responsiveness, and effects of cortisol. Am. J. Physiol. 262, R794-9. Roy, B., Rai, U., 2009. Genomic and non-genomic effect of cortisol on phagocytosis in freshwater teleost Channa punctatus: An in vitro study. Steroids 74, 449–455. Samarasinghe, R.A., Di Maio, R., Volonte, D., Galbiati, F., Lewis, M., Romero, G., DeFranco, D.B., 2011. Nongenomic glucocorticoid receptor action regulates gap junction intercellular communication and neural progenitor cell proliferation. Proc. Natl. Acad. Sci. U. S. A. 108, 16657–16662. Schaaf, M.J.M., Champagne, D., van Laanen, I.H.C., van Wijk, D.C.W. a, Meijer, A.H., Meijer, O.C., Spaink, H.P., Richardson, M.K., 2008. Discovery of a functional glucocorticoid receptor beta-isoform in zebrafish. Endocrinology 149, 1591–9. Stolte, E.H., Verburg van Kemenade, B.M.L., Savelkoul, H.F.J., Flik, G., 2006. Evolution of glucocorticoid receptors with different glucocorticoid sensitivity. J. Endocrinol. 190, 17-28. Sturm, A., Colliar, L., Leaver, M.J., Bury, N.R., 2011. Molecular determinants of hormone sensitivity in rainbow trout glucocorticoid receptors 1 and 2. Mol. Cell. Endocrinol. 333, 181–189. Sunny, F., Lakshmy, P.S., Oommen, O. V., 2002. Rapid action of cortisol and testosterone on lipogenic enzymes in a fresh water fish Oreochromis mossambicus: Short-term in vivo and in vitro study. Comp. Biochem. Physiol. Biochem. Mol. Biol. 131, 297-304. Sunny, F., Oommen, O. V., 2001. Rapid action of glucocorticoids on branchial ATPase activity
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Das et al in Oreochromis mossambicus: An in vivo and in vitro study. Comp. Biochem. Physiol. - B Biochem. Mol. Biol. 130, 323–330. Sze, P.Y., Iqbal, Z., 1994. Regulation of calmodulin content in synaptic plasma membranes by glucocorticoids. Neurochem. Res. 19, 1455–1461. Tasker, J.G., Di, S., Malcher-Lopes, R., 2006. Minireview: Rapid glucocorticoid signaling via membrane-associated receptors. Endocrinology. 147, 5549-5556. Thomas, P., 2012. Rapid steroid hormone actions initiated at the cell surface and the receptors that mediate them with an emphasis on recent progress in fish models. Gen. Comp. Endocrinol. 175, 367-383. Thomas, P., 2003. Rapid, nongenomic steroid actions initiated at the cell surface: Lessons from studies with fish. Fish Physiol. Biochem. 28, 3–12. Thomas, P., Dressing, G., Pang, Y., Berg, H., Tubbs, C., Benninghoff, A., Doughty, K., 2006. Progestin, estrogen and androgen G-protein coupled receptors in fish gonads. Steroids. 71, 310-316. Trueba, M., Guantes, J.M., Vallejo, a I., Sancho, M.J., Marino, a, Macarulla, J.M., 1987. Characterization of cortisol binding sites in chicken liver plasma membrane. Int. J. Biochem. 19, 957–62. Van Deurs, B., Roepstorff, K., Hommelgaard, A.M., Sandvig, K., 2003. Caveolae: Anchored, multifunctional platforms in the lipid ocean. Trends Cell Biol. 13, 92-100. Vernocchi, S., Battello, N., Schmitz, S., Revets, D., Billing, A.M., Turner, J.D., Muller, C.P., 2013. Membrane Glucocorticoid Receptor Activation Induces Proteomic Changes Aligning with Classical Glucocorticoid Effects. Mol. Cell. Proteomics 12, 1764–1779. Vigh, L., Nakamoto, H., Landry, J., Gomez-Munoz, A., Harwood, J.L., Horvath, I., 2007. Membrane regulation of the stress response from prokaryotic models to mammalian cells, in: Annals of the New York Academy of Sciences. pp. 40–51. Vijayan, M., Das, C., Wildering, W.C., 2017. Nongenomic action of cortisol in rainbow trout hepatocytes. FASEB J. 31, 1005–1009. Vijayan, M.M., Aluru, N., Leatherland, J.., 2010. Stress response and the role of cortisol, in: Fish Diseases and Disorders. pp. 182–201. Wagner, P.G., Jorgensen, M.S., Arden, W.A., Jackson, B.A., 1999. Stimulus-secretion coupling in porcine adrenal chromaffin cells: acute effects of glucocorticoids. J. Neurosci. Res. 57, 643–50. Wendelaar Bonga, S.E., 1997. The stress response in fish. Physiol. Rev. 77, 591–625.
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Whiting, K.P., Restall, C.J., Brain, P.F., 2000. Steroid hormone-induced effects on membrane fluidity and their potential roles in non-genomic mechanisms. Life Sci. 67, 743–757.
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Das et al Figure Legends Fig. 1: Proposed model for nongenomic cortisol action. We are proposing four modes of action by which cortisol elicits rapid stress signaling. 1. Biophysical changes to lipid bilayer as a result of cortisol intercalation; 2. Activation of a novel membrane receptor; 3. Activation of the GR that is on the plasma membrane; 4. Opening of Ca2+ channels leading to the rise in intracellular calcium. We are hypothesizing that GR interaction with caveolin-1 is essential for membrane-mediated GR signaling, while the mechanism(s) leading to the opening of Ca2+ channel by cortisol is unknown. We hypothesize that the rise in intracellular Ca2+ with cortisol mediates the translocation of GR to the membrane. Abbreviations: GR - glucocorticoid receptor, PKA - protein kinase A, PKB - protein kinase B, PKC - protein kinase C, MAPK - mitogenactivated protein kinase, iCa2+ - intracellular calcium, Cav-1 - caveolin-1.
Fig. 2: Intracellular Ca2+ waves in trout hepatocytes. A representative image showing that cortisol rapidly elevates intracellular Ca2+ levels in trout hepatocytes. Hepatocytes were treated with cortisol (100 ng/mL) or cortisol-BSA (100 ng/mL) and preloaded with ratiometric dye, FURA-2AM. Rise in Ca2+ is shown using ImageJ-16 colors LUT. Cells with a red core indicate Ca2+ rich areas while cells in blue indicate low Ca2+ at the intracellular level. Bar indicate 12.5 μm.
Fig. 3: Glucocorticoid receptor dynamics in trout hepatocytes. A representative image of rapid localization of GR around the plasma membrane in rainbow trout hepatocytes within 5 min of cortisol treatment. Trout hepatocytes were exposed to stress levels of cortisol (100 ng/mL) and the GR localized by immunofluorescence. Cells were fixed and immunostained with rabbit
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Das et al polyclonal antibody raised against trout GR (green; in-house antibody), and the nuclei visualized by DAPI staining (blue). White arrowheads indicate the intracellular presence of GR, while the yellow arrowheads indicate the localization of GR predominantly at the plasma membrane. Bar indicates 100 μm.
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Das et al Table 1: Nongenomic cortisol signaling in select mammalian models and tissues. Abbreviations: cAMP - cyclic adenosine monophosphate, ACTH - adrenocorticotropic hormone, GR - glucocorticoid receptor, MAPK - mitogen activated protein kinase, GJIC - gap junction intracellular communication, PI3K - phosphatidyl inositol-3 kinase, PKB - protein kinase B, ERK1/2 - extracellular signal regulated kinase 1/2, PKC - protein kinase C, JNK - c-Jun N-terminal.
Treatment
Species
Tissue
Key Findings
Reference
Dexamethasone
Mouse
Pituitary
Inhibition of cAMP and ACTH by G-protein
(Iwasaki et al., 1997)
Brain
Reduced GJIC via MAPK dependent phosphorylation of connexin-43 protein
(Samarasinghe et al., 2011)
Heart
Reduced inflammation by endothelial nitric oxide synthase via PI3K-PKB pathway
(Hafezi-Moghadam et al., 2002)
Corticosterone
Cortisol
Macrophages Reduced phagocytosis
(Long et al., 2005)
Amphibian
Brain
Suppression of sexual behaviours; membrane GR
(Coddington et al., 2007; Evans et al., 2000)
Rat
Liver
Increase glycogenolysis via glycogen phosphorylase through Ca2+ signaling
(Gomez-Muñoz et al., 1989)
Human
Lymphocytes Inhibition of Lck and Fyn kinase; decrease PKB, PKC, ERK, p38, and JNK activities
23
(Lowenberg et al., 2005)
Das et al Table 2: Nongenomic cortisol signaling in fish. Abbreviations: cAMP - cyclic adenosine monophosphate, PKA - protein kinase A, PKB - protein kinase B, PKC - protein kinase C, ERK1/2 - extracellular signal-regulated protein kinase, CREB - cAMP response element binding protein, ROS - reactive oxygen species.
Treatment
Species
Cortisol
Oreochromis mossambicus
Oncorhynchus mykiss
Channa punctatus
Tissue
Key Findings
Reference
Pituitary
Suppression of prolactin release by Ca2+ and cAMP inhibition
(Borski et al., 2002, 1991; Hyde et al., 2004)
Gill
Increase Na+/K+ ATPase and Ca2+ ATPase activity; increase ERK1/2 phosphorylation
(Kiilerich et al., 2011b; Sunny and Oommen, 2001)
Liver
Decrease malic enzyme, glucose-6-phosphate, isocitrate dehydrogenase activities;
(Sunny et al., 2002)
Liver & kidney
Putative glucocorticoid membrane site
(Johnstone et al., 2013)
Heart
Increase coronary vasoconstriction
(Agnisola et al., 2004)
Liver
Increase plasma membrane fluidity; increase phosphorylation of PKA, PKB, PKC substrates
(Dindia et al., 2013, 2012)
Skeletal myotubules
Increase ROS; increase ERK1/2 and CREB phosphorylation; increase pgc1a expression
(Espinoza et al., 2017)
Macrophages
Reduced phagocytosis by adenylate cyclase-cAMP (Roy and Rai, 2009) pathway
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Das et al
Extracellular Ca
2+
CORTISOL
Extracellular
1.
2.
4.
3. Cav-1
Novel receptor?
GR
GR
Cytoplasm Translocation
GR iCa
PKA, PKB, PKC, MAPK
Cellular responses
Fig. 1
25
2+
2+
Ca channel
Das et al
Control
Cortisol
Cortisol-BSA
High Ca
2+
0 min
1 min 2+
Low Ca
Fig. 2
26
Das et al GR
DAPI
Control
Cortisol
Fig. 3
27
Merged
Das et al Highlights
Cortisol stimulates rapid nongenomic effects in fishes
The rapid effects may be mediated by membrane GR or novel membrane proteins
The rapid effects may involve alterations in membrane biophysical properties
The rapid effect may involve cortisol-mediated elevation in intracellular calcium levels.
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S0016-6480(18)30049-2 https://doi.org/10.1016/j.ygcen.2018.04.019 YGCEN 12918
To appear in:
General and Comparative Endocrinology
Received Date: Revised Date: Accepted Date:
16 January 2018 12 April 2018 14 April 2018
Please cite this article as: Das, C., Thraya, M., Vijayan, M.M., Nongenomic cortisol signaling in fish, General and Comparative Endocrinology (2018), doi: https://doi.org/10.1016/j.ygcen.2018.04.019
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Mini-review
Nongenomic cortisol signaling in fish1
Chinmayee Das*, Marwa Thraya*, and Mathilakath M. Vijayan# Department of Biological Sciences, University of Calgary, 2500 University Drive NW, Calgary, Alberta T2N 1N4, Canada
1
Presented at the ICCE18 meeting in Lake Louise, Alberta, Canada
#
Corresponding author. E-mail address: [email protected] (M.M Vijayan)
*Contributed equally
Das et al Abstract Glucocorticoids are critical regulators of the cellular processes that allow animals to cope with stressors. In teleosts, cortisol is the primary circulating glucocorticoid and this hormone mediates a suite of physiological responses, most importantly energy substrate mobilization that is essential for acute stress adaptation. Cortisol signaling has been extensively studied and the majority of work has been on the activation of the glucocorticoid receptor (GR), a ligand-bound transcription factor, and the associated downstream transcriptional and protein responses. Despite the role of this hormone in acute stress adaptation, very few studies have examined the rapid effects of this hormone on cellular function. The nongenomic corticosteroid effects, which are rapid (seconds to minutes) and independent of transcription and translation, involve changes to second-messenger pathways and effector proteins, but the primary receptors involved in this pathway activation remain elusive. In teleosts, a few studies suggested the possibility that GR located on the membrane may be initiating a rapid response based on the abrogation of this effect with RU486, a GR antagonist. However, studies have also proposed other signaling mechanisms, including a putative novel membrane receptor and changes to membrane biophysical properties as initiators of rapid signaling in response to cortisol stimulation. Emerging evidence suggests that cortisol activates multiple signaling pathways in cells to bring about rapid effects, but the underlying physiological implications on acute stress adaptation are far from clear.
Keywords Glucocorticoid receptor, teleosts, metabolism, stress response
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Das et al 1. Introduction Glucocorticoids are steroid hormones involved in the regulation of an extensive range of physiological processes, including intermediary metabolism, immunity, growth, osmoregulation, neuronal function, and behaviour (Aluru and Vijayan, 2009; Borski, 2000; Faught and Vijayan, 2016; Mommsen et al., 1999; Wendelaar Bonga, 1997). In teleosts, cortisol is the primary glucocorticoid in circulation and is produced by the interrenal cells (analogous to the adrenal cortex in mammals) localized primarily in the head kidney region in response to a stressor (Mommsen et al., 1999; Wendelaar Bonga, 1997). The release of this steroid is under the control of the hypothalamus-pituitary-interrenal (HPI) axis (akin to the hypothalamus-pituitary-adrenal (HPA) axis in mammals) to mediate cellular functions essential for stress adaptation (Mommsen et al., 1999; Wendelaar Bonga, 1997). For example, excess cortisol enhances liver metabolic capacity, including activation of hepatic gluconeogenesis to fuel the increased energy demand associated with stressor exposure and re-establish homeostasis (Aluru and Vijayan, 2009, 2007; Mommsen et al., 1999). Corticosteroids exert wide-spread cellular effects on target tissues through two distinct mechanisms known as the genomic and nongenomic steroid signaling pathways. The genomic pathway, which has been extensively characterized, involves the transfer of cortisol across the cell membrane and binding to intracellular receptors of the nuclear steroid receptor superfamily, to either positively or negatively regulate target gene transcription and protein synthesis (Bury et al., 2003; Faught and Vijayan, 2016; Mommsen et al., 1999; Prunet et al., 2006; Stolte et al., 2006). As this process leads to de novo protein synthesis in altering cellular function, genomic signaling is slower to respond, but longer lasting (Borski, 2000; Mommsen et al., 1999; Wendelaar Bonga, 1997). Nongenomic signaling, on the other hand, is rapid (seconds to
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Das et al minutes) as effector molecules are modulated by activation of membrane-mediated secondary signaling cascades (Borski, 2000; Borski et al., 2002), including cAMP, Ca2+, and protein kinases. The major difference between the two pathways is that while the genomic pathway involves steroid-mediated activation of gene transcription and translation, the nongenomic signaling-mediated effects are independent of gene regulation (Lösel and Wehling, 2003). Therefore, time of onset and activation of the rapid signaling pathway in the presence of transcriptional and translational inhibitors are frequently used to confirm nongenomic steroid signaling (Borski, 2000; Groeneweg et al., 2011; Losel et al., 2003). While nongenomic steroid signaling and its physiological implications have garnered a lot of attention for reproductive steroids in teleost models (Thomas, 2012, 2003), the rapid effects of corticosteroids and its role in stress adaptations are far from clear (Borski, 2000; Faught and Vijayan, 2016). Although nongenomic corticosteroid actions have been reported in non-teleost models involved in rapid hormone regulation and stress adjustments, including the nervous, cardiovascular, and immune systems (select studies summarized in Table 1), such studies in teleosts are few and far between (Table 2). Here, we synthesize the studies that have been carried out in fishes pertaining to rapid effects of cortisol to develop hypotheses about the mode of action of this stress steroid and its role in acute stress adaptation.
2. Mechanisms of rapid cortisol action In mammals, rapid glucocorticoid effects have been implicated in various tissues and cellular system functions, including cardiovascular, immune, central nervous system, skeletal muscle, and liver (Table 1). However, the mechanisms that mediate these actions remain elusive. The teleost GR and mineralocorticoid (MR) receptors have been cloned, sequenced, and
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Das et al extensively reviewed (Prunet et al., 2006; Stolte et al., 2006; Sturm et al., 2011). Briefly in teleosts, there are two isoforms of GR (GR1 and GR2) (Bury et al., 2003), apart from zebrafish, which has only one GR (Alsop and Vijayan, 2009), as well as a splice variant GRß (Schaaf et al., 2008). Fish also express a single MR receptor, but the role of MR in mediating cortisol actions is not well understood in teleosts (Colombe et al., 2000; Faught and Vijayan, 2016; Prunet et al., 2006; Sturm et al., 2011). Although studies have proposed a putative membrane-bound receptor mediating rapid activation of signaling pathways in response to glucocorticoid stimulation, to date, a membranebound receptor for corticosteroid action has not been cloned and sequenced in any animal model (Faught and Vijayan, 2016; Tasker et al., 2006). There has only been a single report of a partial purification of a glucocorticoid receptor from the brain of the rough-skinned newt, Taricha granulosa (Evans et al., 2000). Using cortisol-sepharose affinity resin, this study provided novel insight that a membrane-GR isolated from neuronal membranes is a glycoprotein characterized as having a mass of 63 kDa, making this distinct from the intracellular GR, and binds cortisol with high affinity (Evans et al., 2000). Membrane binding sites for glucocorticoids have been reported in different tissues, including mouse lymphocytes (Gametchu, 1987), chicken liver (Trueba et al., 1987), rodent pituitary (Koch et al., 1977), and amphibian brain (Orchinik et al., 1991). However, less is known in fish. Only a single study has reported cortisol binding to membranes in the tissues of the Mozambique tilapia, Oreochromis mossambicus (Johnstone et al., 2013). It was found that the glucocorticoid membrane binding activity was present in liver (high affinity; Kd = 9.5 nM) and kidney (low affinity; Kd = 30.08 nM) (Johnstone et al., 2013). While the specific binding of cortisol on the membrane has not been looked at, preliminary results from our laboratory by
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Das et al immunodetection using western blotting with antibodies to trout GR and zebrafish MR suggest the presence of these receptors on plasma membranes of rainbow trout liver. However, the mechanism of action of these receptors in bringing about rapid nongenomic signaling is currently unknown. Based on the literature available, we are proposing four working hypotheses on the mode of action of cortisol in mediating rapid effects in teleosts (Fig. 1). One is that corticosteroids mediate changes in the biophysical property of the plasma membrane leading to the activation of intracellular signaling pathways (Dindia et al., 2012; Whiting et al., 2000). For instance, cortisol increased membrane fluidity in DPPC (dipalmitoylphosphatidylcholine) liposomes (Ghosh et al., 1996), and the production of eicosanoids in rabbit cardiac muscle endothelial tissues was inhibited due to membrane ordering effects (Gerritsen et al., 1991). A rapid effect of cortisol on membrane fluidity was revealed in rainbow trout (Oncorhynchus mykiss) plasma membranes, including altered anisotropy within 30 min of cortisol treatment in vitro (Dindia et al., 2012). While the mechanism bringing about biophysical changes in unclear, atomic force microscopy revealed membrane domain shifts in response to cortisol in trout plasma membrane monolayers (Dindia et al., 2012). This rapid change in membrane biophysical properties, including alterations to lipid rafts, can lead to mechanotransduction-related activation of downstream signaling pathways (Vigh et al., 2007). However, the mechanisms by which changes in membrane biophysical properties by cortisol are transduced into rapid cellular effects remains to be elucidated. The second hypothesis involves the activation of membrane (novel) receptors on the cell surface, independent of GR, initiating rapid downstream signaling cascades as shown for the mineralocorticoid aldosterone in mammalian and non-mammalian models (Lösel et al., 2002). It
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Das et al is hypothesized that these novel membrane proteins may belong to the GPCR family as was seen for the nongenomic signaling of sex steroid in fishes (reviewed in Thomas et al., 2006). This is supported by the rapid activation of second messengers, including cAMP and Ca2+ (Fig. 2) (Borski et al., 1991; Espinoza et al., 2017; Hyde et al., 2004; Vijayan et al., 2017), as well as rapid phosphorylation of substrate proteins of the protein kinase A (PKA), B (PKB), and C (PKC) pathways with membrane impermeable cortisol in trout hepatocytes (Dindia et al., 2012). The third working hypothesis revolves around the possibility that modulation of the intracellular receptors or translocation of these receptors to the plasma membrane may lead to the activation of rapid signaling (Groeneweg et al., 2011; Lösel and Wehling, 2003). Support for cortisol signaling by GR was seen from studies that showed abolishment of rapid cortisolmediated responses by RU486 (Espinoza et al., 2017; Roy and Rai, 2009). Further support for this observation comes from preliminary studies showing accumulation of GR close to the plasma membrane within 5 min of cortisol exposure to trout hepatocytes (Fig. 3). This leads us to propose that the rapid tissue responses to GR activation (Gross and Cidlowski, 2008) may involve rapid translocation of intracellular GR to the cell periphery, but the mechanisms involved remains to be determined. Overall, it appears that cortisol rapidly alters intracellular GR dynamics, and the GR translocation to the plasma membrane may be involved in the activation of rapid cell signaling by cortisol. The fourth working hypothesis is that rapid cortisol mode of action involves altering intracellular Ca2+ levels in fish cells by modulating plasma membrane-bound Ca2+ channels, independently of membrane receptor(s). An increase in intracellular Ca2+ levels appears to be a key second messenger for the rapid glucocorticoid response in vertebrate models (Beato and Klug, 2000; Karst et al., 2002), but this is far from clear in fish (Hyde et al., 2004). Nongenomic
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Das et al glucocorticoid actions have been previously shown to be responsible for intracellular Ca2+ spikes in different cell lines (Qiu et al., 1998; Sze and Iqbal, 1994; Wagner et al., 1999). Studies have reported that glucocorticoid addition in cells causes an intracellular Ca2+ wave with varying order of magnitude, peak, and amplitude (Berridge et al., 2000). In addition, glucocorticoids have shown an increase in selectivity for L-type Ca2+ channels over N-type channels in the mouse hippocampus (Chameau et al., 2007). The only study that has examined this in fish showed that cortisol rapidly reduced intracellular Ca2+ and this involved suppression of voltagegated Ca2+ channel activity in the prolactin cells of the Mozambique tilapia (Hyde et al., 2004). Preliminary results from our laboratory showed that cortisol stimulates a rapid (within one min) rise in intracellular Ca2+ in rainbow trout hepatocytes, and this response was also mimicked by the membrane impermeable cortisol-BSA (Fig. 2), suggesting the activation of membrane receptor(s). Also, emerging evidence seems to suggest that cortisol may be favoring rapid influx of extracellular Ca2+, supporting the activation of plasma membrane Ca2+ channels (Vijayan et al., 2017). Taken together, these results suggest that rapid action of cortisol in fish cells involves modulation of intracellular Ca2+ levels, but the response may be cell-specific. This rapid influx of Ca2+ in response to cortisol stimulation may also be involved in the GR translocation seen in trout hepatocytes (Fig. 3), but this needs to be tested. Overall, there appears to be multiple mechanisms by which cortisol may rapidly activate and/or inhibit downstream signaling pathways to mediate an integrated response for acute stress adaptation.
3. Nongenomic physiological actions of cortisol 3.1 Salinity acclimation
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Das et al One of the first studies that explored rapid cortisol action in teleosts was in the pituitary of the Mozambique tilapia where cortisol suppressed prolactin release within 20 min by inhibiting the cAMP and Ca2+ signaling pathways (Borski et al., 2002, 1991; Hyde et al., 2004). As cyclohexamide did not abolish this inhibiting effect, the results suggested an effect independent of protein synthesis and, therefore, not involving the classical GR (Borski et al., 2002). This led to the proposal that a membrane-associated receptor may be involved, as the effect was also mimicked by the membrane impermeable form of cortisol-21 hemisuccinate coupled to bovine serum albumin (BSA) (Borski et al., 2001). Subsequently, in the same species, rapid activation of Na+/K+ and Ca2+ ATPase activities was reported in gill tissue in response to cortisol and corticosterone treatment both in vivo and in vitro, and these effects were insensitive to actinomycin-D, a transcriptional inhibitor, further supporting nongenomic activation (Sunny and Oommen, 2001). Also, cortisol rapidly induced MAPK activity through modulation of extracellular signal-regulated kinase (ERK1/2) in the gills of this species providing a mechanistic underpinning for the rapid modulation of ion transporters by cortisol (Kiilerich et al., 2011a). This study along with the observation that cortisol rapidly inhibits prolactin secretion from the pituitary suggests a key nongenomic role for cortisol in salinity acclimation in euryhaline species. This would be particularly telling to species inhabiting estuaries and would entail daily and rapid switching of ion transporters when moving from hyperosmotic to hypoosmotic environments and vice versa. However, the precise mechanisms of action remain to be elucidated in these species. 3.2 Metabolic stress response Although considered a key target in cortisol action (Mommsen et al., 1999), little has been reported with regards to rapid actions of cortisol in teleost liver (Table 2). Studies that examined
9
Das et al the effect on liver showed rapid changes in second messengers, and changes in the activities of enzymes involved in intermediary metabolism (Dindia et al., 2012; Faught and Vijayan, 2016; Sunny et al., 2002; Vijayan et al., 2017). For instance, it has been shown that cortisol decreases the activities of liver malic enzyme, glucose-6-phosphatase, and isocitrate dehydrogenase within 5-l0 min, with effects persistent in the presence of actinomycin-D in vitro (Sunny et al., 2002). Mozambique tilapia injected with cortisol and a transcriptional inhibitor in a short-term in vivo study also showed similar results within 30 min of hormone addition, further suggesting a role in regulating lipid metabolism and possibly promoting gluconeogenesis (Sunny et al., 2002). More recent studies examined cortisol action on membrane fluidity in rainbow trout hepatocytes (Dindia et al., 2013, 2012). The results suggest that cortisol increased membrane fluidity and changed membrane topography within 10 min of cortisol treatment (Dindia et al., 2012). This effect corresponded with rapid phosphorylation of substrate proteins by PKA, PKB, and PKC (Dindia et al., 2012). The changes observed with cortisol were similar to that seen with benzyl alcohol, a known membrane fluidizer (Dindia et al., 2012), leading to the proposal that changes in membrane biophysical properties may be an initiator of downstream stress signaling pathways in trout. This was followed up with an in vivo experiment that showed acute stress elevated plasma cortisol levels altered hepatic membrane fluidity within 30 min, a response that was blocked by metyrapone, an inhibitor of cortisol synthesis (Dindia et al., 2013). Furthermore, the stressorinduced cortisol elevation rapidly activated ERK1/2 MAPK and increased phosphorylation of PKC and PKA substrate proteins (Dindia et al., 2013), similar to that observed in cortisol treated hepatocytes in vitro (Dindia et al., 2012). These results suggest that cortisol action may also occur independent of membrane receptor activation by rapidly altering the membrane fluidity,
10
Das et al and stimulating mechanotransduction pathways, but the mechanisms are far from clear. These studies are the only one of its kind to investigate the physiological effects of rapid cortisol signaling and its implications on peripheral liver function and cellular signaling pathways in fish. The emerging results seem to suggest that the rapid effect of cortisol may facilitate utilization of glucose for endogenous liver use to cope with the increased energy demand associated with stress (Dindia, 2013). Also, the results tend to suggest that the rapid effect of cortisol during stress may also involve reducing the glucose output seen with epinephrine activation (Fabbri and Moon, 2016; Reid et al., 1992), thereby facilitating partitioning of energy substrates for endogenous use by the liver (Dindia, 2013). These results led to the hypothesis that the rapid action of cortisol may involve liver energy substrate repartitioning to cope with the enhanced energy demand associated with acute stressor exposure. 3.3 Muscle, cardiovascular, and innate immune systems Recently, myotubules from rainbow trout showed that cortisol and cortisol-BSA rapidly induce reactive oxygen species (ROS) mediated by the activities of NADPH oxidase and phospholipase A2 (Espinoza et al., 2017). ROS production was blocked by the GR antagonist, RU486, but not by the MR antagonist, RU28318, suggesting a membrane-GR may be participating in this rapid glucocorticoid signaling (Espinoza et al., 2017). In addition, cortisolBSA showed a significant increase in the phosphorylation of ERK1/2 and cAMP response element binding protein (CREB) within 15 min of treatment, suggesting rapid activation of stress-signaling pathways in the muscle in response to stressor-mediated cortisol stimulation (Espinoza et al., 2017). However, the physiological implication of this rapid activation of cellular stress-signaling pathways in the muscle remains to be determined. While studies have shown that muscle metabolism is a key target for cortisol action during stress (Faught et al., 2016;
11
Das et al Mommsen et al., 1999), the mechanisms of rapid action and the pathways involved in acute stress adaptation needs to be investigated. Information pertaining to nongenomic cortisol signaling is still in its infancy in teleost cardiovascular and innate immune systems (Table 2). A report on nongenomic signaling in the cardiovascular system of rainbow trout showed that cortisol rapidly increased coronary vasoconstriction in less than 15 min (Agnisola et al., 2004). As a result, cortisol-induced vasoconstriction may negatively affect blood circulation under stressed conditions (Agnisola et al., 2004). Rapid cortisol actions were also observed in the spotted murrel (Channa punctatus) within 15 min, including suppression of phagocytosis in the spleen, and a more profound inhibition at 1 h post-hormone treatment (Roy and Rai, 2009). Data from this study points to the activation of a membrane-bound GR, as well as the cAMP-PKA pathway in mediating the rapid responses to cortisol, as inhibitors of GR, adenylate cyclase, and PKA completely abolished those responses (Roy and Rai, 2009). Overall, these results highlight the possibility that the nongenomic response may also involve signaling by GR translocated to the membrane and/or other proteins that share a common epitope with GR.
4. Conclusions and future directions This review was intended to highlight the significance of rapid nongenomic signaling in allowing fishes to cope with stress. Taken together, the nongenomic mode of action of cortisol is diverse, and while the mechanisms are not fully understood, we propose four modes of nongenomic actions of cortisol in fish (Fig. 1). Based on our current understanding of rapid cortisol actions in fish, future studies should be targeted at investigating the mechanisms by which these rapid responses are taking place, and their effects in relation to the delayed actions
12
Das et al of glucocorticoids (Groeneweg et al., 2011). Recent developments in gene knockout technology, including CRISPR/Cas9 will allow us to clearly differentiate the effect of nongenomic signaling mediated by novel membrane receptors from those mediated by GR activation. Development of a GR knockout model will also clearly highlight the role of this receptor in mediating rapid cortisol signaling during acute stress adaptation. Another major development in this research area would be the discovery of a novel membrane receptor for cortisol, as this will have huge implications in our understanding of stress coping mechanisms. Furthermore, since glucocorticoids are widely used as clinical immunosuppressive and anti-inflammatory agents, an insight into nongenomic corticosteroid actions and their underlying interaction with different downstream effectors would help to elucidate the cross-talk between the stress and immune response pathways, and will have applications in aquaculture for reducing stress-mediated inflammatory responses. The role of caveolin-1 as a mechanosensor and membrane scaffolding protein is well established in mammalian systems (Okamoto et al., 1998; Van Deurs et al., 2003; Vernocchi et al., 2013), but its role in teleost stress signaling is far from clear. Studies on caveolin-1 involvement in GR mediated rapid signaling has been reported (Matthews et al., 2008), along with observations on GR association with membrane lipid rafts (Jain et al., 2005), but the mechanisms leading to signal transduction is still unclear. We hypothesize that the interaction of GR with caveolin-1 on the plasma membrane may be critical for the transduction of rapid nongenomic signaling in fish cells. From a physiological stand-point, it is important to have a working hypothesis of the significance of the rapid response due to cortisol on stress adaptation. As the fight-or flight response is usually associated with the sympathetic nervous system activation, the role for
13
Das et al cortisol was thought to do more with metabolic recovery, including sustaining the glucose release that was activated by epinephrine (Faught et al., 2016; Mommsen et al., 1999; Vijayan et al., 2010). While it seems reasonable to propose that the rapid cortisol-mediated response during stress may play a key role in the fight-or flight response, including the modulation of catecholamine signaling (Mommsen et al., 1999), direct evidence to this is still lacking in animal models. However, the rapid changes in intracellular stress-related signaling pathways seen with corticosteroid stimulation (Dindia et al., 2012, 2013; Espinoza et al., 2017; Vijayan et al., 2017) suggests a key role for this stress steroid in acute stress adaptation, but the target tissue responses and their implications in restoring homeostasis are far from clear.
Acknowledgements Funding for the studies in this mini-review was supported by the Natural Sciences and Engineering Research Council of Canada Discovery Grant to MMV.
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Whiting, K.P., Restall, C.J., Brain, P.F., 2000. Steroid hormone-induced effects on membrane fluidity and their potential roles in non-genomic mechanisms. Life Sci. 67, 743–757.
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Das et al Figure Legends Fig. 1: Proposed model for nongenomic cortisol action. We are proposing four modes of action by which cortisol elicits rapid stress signaling. 1. Biophysical changes to lipid bilayer as a result of cortisol intercalation; 2. Activation of a novel membrane receptor; 3. Activation of the GR that is on the plasma membrane; 4. Opening of Ca2+ channels leading to the rise in intracellular calcium. We are hypothesizing that GR interaction with caveolin-1 is essential for membrane-mediated GR signaling, while the mechanism(s) leading to the opening of Ca2+ channel by cortisol is unknown. We hypothesize that the rise in intracellular Ca2+ with cortisol mediates the translocation of GR to the membrane. Abbreviations: GR - glucocorticoid receptor, PKA - protein kinase A, PKB - protein kinase B, PKC - protein kinase C, MAPK - mitogenactivated protein kinase, iCa2+ - intracellular calcium, Cav-1 - caveolin-1.
Fig. 2: Intracellular Ca2+ waves in trout hepatocytes. A representative image showing that cortisol rapidly elevates intracellular Ca2+ levels in trout hepatocytes. Hepatocytes were treated with cortisol (100 ng/mL) or cortisol-BSA (100 ng/mL) and preloaded with ratiometric dye, FURA-2AM. Rise in Ca2+ is shown using ImageJ-16 colors LUT. Cells with a red core indicate Ca2+ rich areas while cells in blue indicate low Ca2+ at the intracellular level. Bar indicate 12.5 μm.
Fig. 3: Glucocorticoid receptor dynamics in trout hepatocytes. A representative image of rapid localization of GR around the plasma membrane in rainbow trout hepatocytes within 5 min of cortisol treatment. Trout hepatocytes were exposed to stress levels of cortisol (100 ng/mL) and the GR localized by immunofluorescence. Cells were fixed and immunostained with rabbit
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Das et al polyclonal antibody raised against trout GR (green; in-house antibody), and the nuclei visualized by DAPI staining (blue). White arrowheads indicate the intracellular presence of GR, while the yellow arrowheads indicate the localization of GR predominantly at the plasma membrane. Bar indicates 100 μm.
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Das et al Table 1: Nongenomic cortisol signaling in select mammalian models and tissues. Abbreviations: cAMP - cyclic adenosine monophosphate, ACTH - adrenocorticotropic hormone, GR - glucocorticoid receptor, MAPK - mitogen activated protein kinase, GJIC - gap junction intracellular communication, PI3K - phosphatidyl inositol-3 kinase, PKB - protein kinase B, ERK1/2 - extracellular signal regulated kinase 1/2, PKC - protein kinase C, JNK - c-Jun N-terminal.
Treatment
Species
Tissue
Key Findings
Reference
Dexamethasone
Mouse
Pituitary
Inhibition of cAMP and ACTH by G-protein
(Iwasaki et al., 1997)
Brain
Reduced GJIC via MAPK dependent phosphorylation of connexin-43 protein
(Samarasinghe et al., 2011)
Heart
Reduced inflammation by endothelial nitric oxide synthase via PI3K-PKB pathway
(Hafezi-Moghadam et al., 2002)
Corticosterone
Cortisol
Macrophages Reduced phagocytosis
(Long et al., 2005)
Amphibian
Brain
Suppression of sexual behaviours; membrane GR
(Coddington et al., 2007; Evans et al., 2000)
Rat
Liver
Increase glycogenolysis via glycogen phosphorylase through Ca2+ signaling
(Gomez-Muñoz et al., 1989)
Human
Lymphocytes Inhibition of Lck and Fyn kinase; decrease PKB, PKC, ERK, p38, and JNK activities
23
(Lowenberg et al., 2005)
Das et al Table 2: Nongenomic cortisol signaling in fish. Abbreviations: cAMP - cyclic adenosine monophosphate, PKA - protein kinase A, PKB - protein kinase B, PKC - protein kinase C, ERK1/2 - extracellular signal-regulated protein kinase, CREB - cAMP response element binding protein, ROS - reactive oxygen species.
Treatment
Species
Cortisol
Oreochromis mossambicus
Oncorhynchus mykiss
Channa punctatus
Tissue
Key Findings
Reference
Pituitary
Suppression of prolactin release by Ca2+ and cAMP inhibition
(Borski et al., 2002, 1991; Hyde et al., 2004)
Gill
Increase Na+/K+ ATPase and Ca2+ ATPase activity; increase ERK1/2 phosphorylation
(Kiilerich et al., 2011b; Sunny and Oommen, 2001)
Liver
Decrease malic enzyme, glucose-6-phosphate, isocitrate dehydrogenase activities;
(Sunny et al., 2002)
Liver & kidney
Putative glucocorticoid membrane site
(Johnstone et al., 2013)
Heart
Increase coronary vasoconstriction
(Agnisola et al., 2004)
Liver
Increase plasma membrane fluidity; increase phosphorylation of PKA, PKB, PKC substrates
(Dindia et al., 2013, 2012)
Skeletal myotubules
Increase ROS; increase ERK1/2 and CREB phosphorylation; increase pgc1a expression
(Espinoza et al., 2017)
Macrophages
Reduced phagocytosis by adenylate cyclase-cAMP (Roy and Rai, 2009) pathway
24
Das et al
Extracellular Ca
2+
CORTISOL
Extracellular
1.
2.
4.
3. Cav-1
Novel receptor?
GR
GR
Cytoplasm Translocation
GR iCa
PKA, PKB, PKC, MAPK
Cellular responses
Fig. 1
25
2+
2+
Ca channel
Das et al
Control
Cortisol
Cortisol-BSA
High Ca
2+
0 min
1 min 2+
Low Ca
Fig. 2
26
Das et al GR
DAPI
Control
Cortisol
Fig. 3
27
Merged
Das et al Highlights
Cortisol stimulates rapid nongenomic effects in fishes
The rapid effects may be mediated by membrane GR or novel membrane proteins
The rapid effects may involve alterations in membrane biophysical properties
The rapid effect may involve cortisol-mediated elevation in intracellular calcium levels.
28