Membrane-initiated steroid signaling (MISS): genomic steroid action starts at the plasma membrane.Plasma membrane PM steroid recognition sites are thought to be responsible only for rapid, low t medication side effects responses without any link to the nuclear receptor-mediated genomic effects of steroids. This site interacts also with particular gestagens progesterone, P; medroxyprogesterone, MP; ethynodiol, Ethy and estrogens ethinylestradiol, EE 2 ; mestranol membrane initiated steroid signaling, which do not bind to the nuclear GC receptor GR. Selected steroids were tested for their ability to induce or inhibit luc expression. MH cells respond stronger than CC-1 to luc inducing steroids. The thiol reactive mesylate-derivatives of B, Dex membrane initiated steroid signaling Cortex induced to a considerably lesser extent than the free or BSA-steroids.
Chemical control over membrane-initiated steroid signaling with a DNA nanocapsule | PNAS
The estrogen receptor and glucocorticoid receptor are members of the nuclear receptor superfamily that can signal using both non-genomic and genomic transcriptional modes. Though genomic modes of signaling have been well characterized and several behaviors attributed to this signaling mechanism, the physiological significance of non-genomic modes of signaling has not been well understood. This has partly been due to the controversy regarding the identity of the membrane ER mER or membrane GR mGR that may mediate rapid, non-genomic signaling and the downstream signaling cascades that may result as a consequence of steroid ligands binding the mER or the mGR.
Both estrogens and glucocorticoids exert a number of actions on the hypothalamus, including feedback. This review focuses on the various candidates for the mER or mGR in the hypothalamus and the contribution of non-genomic signaling to classical hypothalamically driven behaviors and changes in neuronal morphology.
It also attempts to categorize some of the possible functions of non-genomic signaling at both the cellular level and at the organismal level that are relevant for behavior, including some behaviors that are regulated by both estrogens and glucocorticoids in a potentially synergistic manner. Lastly, it attempts to show that steroid signaling via non-genomic modes may provide the organism with rapid behavioral responses to stimuli.
Nuclear receptor ligands such as estrogen and glucocorticoids signal via both non-genomic and genomic pathways within cells. Once bound to their cognate ligands, these receptors act as ligand-activated transcription factors in the nucleus by binding to specific enhancer elements such as the estrogen response element ERE 1 and glucocorticoid response element GRE 2 in the promoters of genes.
Both receptors have a modular structure, with a conserved DNA-binding domain, multiple transactivation domains, and a C-terminal ligand-binding domain 3 , 4. In the case of corticosterone-mediated rapid actions, treatment of neurons with dexamethasone, a synthetic glucocorticoid, rapidly induced the nuclear localization of the GR 9 , 10 , an effect potentiated by the inhibition of p38MAPK Extracts from rat hippocampal synaptoneurosomes showed a reduction in Akt and ERK phosphorylation within 30 min in response to pharmacological inhibition of the GR by RU 12 , suggesting that the classical nuclear receptor was required for non-genomic signaling in the hippocampus.
Apart from kinase activation, dexamethasone-mediated negative feedback at the corticotropin releasing hormone CRH neuron was also rapid, consisting of suppression of the excitatory drive to the CRH neuron, mediated by endocannabinoids acting as a retrograde messenger to the presynaptic glutamatergic neuron 13 , an effect mimicked with a membrane-limited dexamethasone conjugated to bovine serum albumin Dex-BSA Hence, non-genomic signaling by steroid hormones is extra-nuclear signaling that is initiated by the endogenous ligand within minutes, in contrast to the hours required to detect transcriptionally regulated proteins.
Central to this concept of non-genomic signaling that is typically demonstrated by the use of membrane-limited conjugates 14 , is the idea of a receptor that initiates such signaling from the plasma membrane.
This review aims to describe the current candidates for the mER and the mGR that mediate rapid non-genomic signaling from the plasma cell membrane as well as focus on rapid actions that are relevant for hypothalamically driven behaviors that are dependent on estrogens but that have a glucocorticoid-regulated component.
We concentrate on the hypothalamus since this is a classically steroid-responsive area of the brain and is critical for several estrogen-dependent behaviors For more general reviews on rapid actions of estrogens and glucocorticoids in the CNS, including tools that are typically used to elucidate membrane-initiated non-genomic effects, the reader is referred to 7 , 14 , 17 — What is a good definition for a mER or mGR?
Previously, Micevych et al. Most studies described below show evidence of the candidate mER at the plasma membrane and its ability to rapidly within minutes modulate rapid signaling pathways such as kinase regulation or calcium flux.
A few studies also demonstrate the regulation of the candidate ER at the membrane. Caveolin proteins are highly conserved structural proteins that are necessary and sufficient for the existence of caveolae, a subset of lipid rafts 40 that comprise a restricted compartment for signaling molecules associated with the plasma membrane. Apart from serving as adaptors, the binding of caveolins to the ER also increases palmitoylation of the ER, a process by which palmitate, a C16 fatty acid is added to an internal cysteine via a thioester bond.
The NLS refers to the nuclear localization signal since nuclear localization of this receptor can occur rapidly and is a non-genomic effect. Hence, the continued presence of biotin in this preparation would argue that the receptor was internalized and protected against glutathione action.
The regulation in male mice is unknown and is under investigation in our laboratory. In astrocytes, only the 66 kDa form is thought to be interact with mGluR1 43 and the interaction, signaling properties, and function, if unique, of the 52 kDa variant remain unknown. A 36 kDa variant lacking the AF-1 and AF-2 domains with a unique C-terminus was detected in human breast cancer cells, coded for by a unique promoter located in intron 1 of the Esr1 gene 59 and can be detected by a specific antibody.
The function and expression pattern of the ER-X is unknown though it was upregulated after ischemic injury 63 in the adult. If it is not present in the adult hypothalamus, it is unlikely to be a receptor that mediates normal behavior that is dependent on the hypothalamus Several reviews 7 , 68 , 69 , 71 — 74 detail the studies on this STX-activated receptor.
Though GPR30 was not detected at the plasma membrane using surface biotinylation in hypothalamic astrocytes 38 , GPR30 was localized to the post-synaptic density in the hippocampus and associates with PSD through its C-terminal tail Immuno-electron microscopy analysis of rat hippocampi also revealed membrane localization with no intracellular staining However, it can be detected intracellularly in normal mammary gland epithelial cells 80 and colocalized with a marker of the Golgi apparatus in primary cultures of the hippocampus Also, overexpressed GPR30 in COS7 cells exhibits localization at the endoplasmic reticulum and the Golgi 82 , suggesting that cell type can determine localization.
In addition, HeLa cells transfected with FLAG-GPR30 show mostly staining at the membrane while cells transfected with GFP-GPR30 show staining mainly in the endoplasmic reticulum [ 79 ], suggesting that inclusion of molecular tags may interfere with proper intracellular trafficking, possibly confounding the interpretation of experiments relying on ectopic expression of GPR Studies describing the regulation of internalization of GPR30 have also yielded conflicting results.
When FLAG-GPR30 was ectopically expressed in HeLa cells, receptor endocytosis was ligand dependent and internalized GPR30 occupied a diffuse cytoplasmic localization consistent with classical receptor recycling or proteasomal degradation pathways In human embryonic kidney HEK cells, GPR30 internalization was not only ligand-dependent but also occurs constitutively While most GPCRs are either recycled to the plasma membrane via endosomes or degraded in lysosomes to limit excessive signaling, GPR30 rapidly accumulates in the perinuclear compartment via clathrin-coated vesicles It is thought that this Rab dependent accumulation is in the trans-Golgi network and degradation occurs via an ubiquitin-proteasome-mediated pathway, unlike the endosomal degradation or recycling to the surface that is typical for other GPCRs Since this pathway for internalization and degradation is rapid and constitutive, Cheng et al.
Furthermore, although GPR30 protein was readily detectable in both the microsomal and plasma membrane subcellular fractions of breast cancer cell lines, only GPR30 in the plasma membrane fraction bound ligand and activated G-protein signaling, suggesting that only membrane-associated GPR30 protein is functional However, GPR30 activation in COS7 cells initiated intracellular calcium mobilization and nuclear accumulation of PIP3 via EGFR transactivation 82 via a non-membrane-initiated signaling mechanism since calcium flux was not replicated by membrane restricted estradiol derivatives These discrepant results have yet to be reconciled.
However, while plasma membrane association of other steroid hormone receptors, including ER, is dependent on palmitoylation, mutation of the homologous sequence in GR did not affect membrane localization 93 , suggesting that other mechanisms must tether mGR to the plasma membrane. In the brain, most of the rapid effects of glucocorticoids have been confined to the hippocampus and to the hypothalamus.
Membrane glucocorticoid receptors were first observed in the synaptic plasma membrane fractions SPM of rat brain via [3H]-corticosterone binding assays Hypothalamic SPM had a higher binding capacity than hippocampal or cortical SPM, which is in contrast to corticosterone binding of the iGR, which is lower in the hypothalamus and much higher in cerebral cortex and hippocampus Synaptosomal fractions from rat hippocampus contained plasma membrane-associated GR 95 and GR immunoreactivity was observed at the plasma membranes and vesicle membranes of the hypothalamus 96 while in the CA1, GR was shown in the spine The hypothalamo—pituitary—adrenal HPA axis is also subject to rapid negative feedback at the level of the hypothalamus Bath application of glucocorticoids to rat hypothalamic slices caused a rapid suppression of glutamate-mediated excitatory synaptic currents onto CRH neurons that was decreased by a CB1 receptor antagonist Hence, in the PVN, rapid negative feedback by glucocorticoids on post-synaptic CRH neurons is exerted by a combination of suppression of presynaptic glutamatergic neurons and excitation of presynaptic GABAergic neurons.
As is evident in the preceding sections, several questions about the identity of the mER and mGR remain. Different ER proteins might be mERs in different tissues 14 or different proteins might be mERs in the same tissue at different times to generate divergent rapid signaling outcomes within the same cell or different tissues that is congruent with incoming stimuli.
Again, this would change the response of the cell to stimuli, depending on the mER present. The significance of these spatially separated and functionally opposing ER populations may be to modulate the hypothalamic and hippocampal neuron response to estrogen and prevent runaway signaling. In the ventral hippocampus in male mice, Hart et al. Whether signaling from both G-protein subunits is concomitant or if one signaling pathway predominates, under certain physiological conditions, is not known.
Lastly, the model generated by the existing literature is that the mER or mGR is at the inner leaflet of the membrane, associated with intrinsic membrane proteins or scaffolds such as caveolin. However, surface biotinylation experiments imply that some part of the mER or mGR is exposed to the extracellular mileu Though an abundance of hydrophobic amino acids in the ligand-binding domain of the mER predicts that this domain might insert into the plasma membrane , no study has demonstrated this in the CNS.
Though kinase regulation and calcium flux has been shown to occur rapidly when cells are exposed to the hormone in a number of tissues and the idea of non-genomic signaling more accepted than ever before, the relevance of non-genomic signaling for behavior has remained murky. Estrogen is critical in the display of female sex behavior in rodents by acting on the hypothalamus; retrograde tract tracing using the pseudorabies virus PRV injected into the lumbar muscles labeled the lordosis circuit, in particular, the plexus of oxytocin fibers in the vl-VMH A candidate could be the GPR30 receptor, which is expressed in the vl-VMH; the role of this receptor in spinogenesis in the hypothalamus is not known.
Rapid effects on spinogenesis and spine morphology following GR activation have been observed in the hippocampus, but not in the hypothalamus. CA1 neurons from male rat hippocampal slices treated with dexamethasone for 1 h demonstrated a translation-independent increase in spine density, which was lost with co-application of dexamethasone with either the GR antagonist RU or the NMDA receptor blocker MK The proportion of mushroom-shaped and thin-type spines was also increased following GR activation Does non-genomic signaling play a role in the increase in spine density by estrogens or by glucorticoids?
Though EB can increase filopodial spines in the ARH within 4 h of treatment, mushroom shaped, more stable spines require time frames in excess of 20 h and parallels the time frames required for the full display of lordosis behavior, suggesting that rapid non-genomic signaling is insufficient for formation of stable spines Both ERK and PI3K signaling are implicated in the increase in spine density, though the formation of mature mushroom spines also appears to require transcription Though there are no reports of glucocorticoid-mediated regulation of spine density in the hypothalamus, suppression of PKA, PKC, MAPK, or PI3K signaling completely blocked GR-mediated spinogenesis in CA1 neurons, suggesting that GR signals through convergent kinase pathways to increase actin polymerization, which would allow for spine changes In CA1 neurons, treatment with the synthetic glucocorticoid dexamethasone increased p-cofilin levels within 30 min, similar to the rapid induction seen in the ARH by EB The consequences of spine disruption for other behaviors such as aggressive behavior or male sex behavior see Male Sex Behavior are also unknown.
Though this suggests that non-genomic signaling may be sufficient for an increase in spine density at least in the hippocampus and cortex, this has to be confirmed with experiments that utilize a transcription inhibitor.
A similar rapid effect of estrogen on spine density in the hypothalamus has not been demonstrated. A series of elegant experiments from the laboratory of Micevych delineated a microcircuit consisting of neuronal afferents from the ARH to the mPOA This transient inhibition of lordosis initiated by MOR internalization is removed 30 h later by progesterone Apart from these receptors, the localization of the GPR30 in the hypothalamus and the ability of G-1, an agonist at the GPR30 to facilitate lordosis in female ovariectoimized mice argues that GPR30 signaling is also important, at least in mice.
Since Kow et al. This appetitive sexual behavior was also blocked by vorozole, administered icv 15 min before behavioral testing This implies that neuroestrogens that are rapidly generated in response to stimuli such as the sight of a female signal rapidly to initiate appetitive sexual behavior.
Stimuli-dependent rapid regulation of aromatase could represent a mechanism to locally elevate neuroestrogens, which then increase sex behavior in male quail or in the male rodent. However, contrary to this expectation, aromatase activity decreased rapidly when male quail were exposed to female stimuli but neuroestrogen concentrations itself increased in the mPOA , a nuclei important for male sex behavior.
Similar to female sex behavior, the non-genomic component in male sex behavior is important for the initial approach and mounting aspects of male sex behavior. Castration in male rodents removes both testosterone and estrogens and results in loss of aggression in response to territorial intrusion , In the castrated male rat, EB synergized with dihydrotestosterone DHT to increase aromatase activity The areas of the brain wherein this may occur in the rodent are not clear and are being investigated in our laboratory.
What is the link between glucocorticoids and aggression? Though aggression itself is stressful and leads to secretion of glucocorticoids, dominance in an aggressive encounter typically leads to lowering of the corticosterone level However, pre-fight corticosterone levels have been shown to be associated with aggressiveness in fish and acute treatment with adrenocorticotrophic hormone ACTH increased fighting behavior in male mice while acute treatment with corticosterone 2 min before an encounter increased aggressiveness in male rats and risk assessment in the open field and elevated plus maze tests Supporting the idea that corticosterone is important in regulation the onset of aggressive behavior, acute treatment with GR antagonists , or prevention of glucocorticoid synthesis prevented aggression when presented with social challenge.
Since acute central application of corticosterone increased aggression within 7 min and protein synthesis inhibitors did not decrease it, non-genomic signaling by glucocorticoids in the CNS is most likely required to increase aggression Consistent with this idea, acute corticosterone injection also decreased the magnitude of electrical stimulation to the hypothalamus required to elicit attack In addition, corticosterone administration to rats in established colonies did not change aggression, demonstrating that glucocorticoids promote aggression only in ethologically relevant situations such as territorial intrusion However, chronic elevated levels of glucocorticoids, such as seen in stressed or socially defeated animals decrease aggression and increased submissiveness in hamsters , , mice , and rats , This is thought to be due to genomic signaling by glucocorticoids though the molecular mechanisms by which glucocorticoids facilitate or inhibit aggression are unclear Gonadal status can modify the actions of glucocorticoids on the hypothalamus or can regulate corticosterone levels itself.