Estradiol

Estradiol’s interesting life at the cell’s plasma membrane
J.D. Caldwell, V.M. Gebhart, G.F. Jirikowski PII: S0039-128X(16)00079-9
DOI: http://dx.doi.org/10.1016/j.steroids.2016.03.012
Reference: STE 7949

To appear in: Steroids

Received Date: 17 December 2015
Accepted Date: 14 March 2016

Please cite this article as: Caldwell, J.D., Gebhart, V.M., Jirikowski, G.F., Estradiol’s interesting life at the cell’s plasma membrane, Steroids (2016), doi: http://dx.doi.org/10.1016/j.steroids.2016.03.012

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Estradiol’s interesting life at the cell’s plasma membrane.

Tel.: 540-231-4000

Highlights: Rapid effects of estrogens cannot exclusively be explained be nuclear receptor actions.
Membrane receptors may be distinct from classical steroid receptors Steroid binding globulins are expressed in the central nervous system Expression of steroids binding globulins is in part steroid dependent Cellular accumulation of steroid hormones depends on binding globulins

Keywords: Gonadal steroids, Adrenal hormones, non genomic effects, membrane receptor, steroid binding globulins

Abbreviations:

CBG- Corticosteroid binding globulin, CHO cell- Chinese hamster ovary cell, CNS- central nervous system,
E2- Estradiol,

ER – Estrogen receptor alpha , ER estrogen receptor- beta,
Erk-1 / 2 Extracellular-signal Regulated Kinase, GC- glucocorticoid,
GPR 30 G-protein coupled receptor 30, RBP- Retinol binding protein,
SHBG- Sex hormone binding globulin,

Stra6- Stimulated by retinoic acid 6 transmembrane protein,

The key ovarian steroid in humans, estradiol, is perhaps the most potent single molecule in the human body. It demonstrates a plethora of physiological effects on almost every organ in the body and it seems to have more pathways of physiological action than any other molecule. We are still discovering whole avenues of its effects. Many physicians and endocrinologists devote any attention that they pay to estradiol entirely to its manifold effects in the cell’s nucleus. There is a pharmacology review for preparing students for their medical exams that has this line, “Mechanism of action utilizes intracellular receptors” as the only thing medical students need to know about steroids. In contrast, this review will

focus on some little noted, but still extremely fascinating, effects of estradiol on the cell’s plasma membrane envelope and will suggest there are effects of the membrane back on estradiol. In short, it will review the very interesting life of estradiol at the cell’s plasma membrane.

Well, it’s in all of the textbooks

As indicated above, the currently taught version of how all steroids work is dictated by what endocrinologists call the “Free Steroid Hypothesis”. Briefly, this hypothesis states that steroids are carried in the blood by proteins known as steroid-binding globulins. Around their target organs, these steroids exist in equilibrium with their binding proteins and as in any system in equilibrium, if the free moiety is being drawn down, by for example diffusion into another compartment, more of the bound element is released into the free reservoir. This free moiety of steroids, since everyone knows that steroids are lipophilic, passes freely through the plasma lipid layer of the cell membrane and into the cytosol where it is grabbed by important cytosolic receptors. A number of events may occur at this point, but basically the steroid with its receptor passes into the cell nucleus where it has many, many effects on transcription of genes. In other reviews [1-3] we have argued against the viability of the Free Steroid Hypothesis, and we will not reiterate in this paper either the cogent arguments of numerous researchers or the chronology of our own path to believing that there are fallacies in the Free Steroid Hypothesis.

There may be many places where this model does not work very well, but the most jarringly illogical point is on the inside of the plasma membrane. What could possibly induce a steroid, sitting snuggly in its lipid bilayer to come out of that layer and jump into the aqueous cytosolic environment? The answer to that question is—nothing. No self-respecting steroid would leave the lipid bilayer, without some help, to go into the water inside the cell. Allera and Wildt [4] clearly demonstrated that without some protein element, steroids did not even pass through the lipid bilayer. So, if steroids do not just pass through the cell membrane, what happens to them there? This question is the basis of this entire paper. What happens to the steroid at the level of the cell’s plasma membrane envelope?
Estradiol is being grabbed

One possibility as to what is happening to the ovarian steroid estradiol (E2) at the membrane is that, unlike what is suggested in the Free Steroid Hypothesis, it is grabbed by a membrane-associated receptor. That is the reason that there need to be proteins in the plasma membrane for E2 to move through the membrane. This could be a membrane-associated receptor for E2. Many excellent laboratories have been engaged in trying to determine what this receptor or these receptors are.

One candidate for a grabber of E2 in the cell’s membrane is GPR30. In the 1990s it was found that E2 stimulated cellular functions via second messengers Erk-1 and Erk-2 [5]. This study demonstrated that this effect could be seen in a cell line that did not express either estradiol receptor alpha (ERα) or ERβ suggesting that the membrane-associated GPR30 receptor not only mediates rapid actions of E2, but that it does so without the assistance of either of the intracellular ERs α or β. The possibility that many of the proteins involved in binding and carrying steroids such as E2 interact to mediate even a single action of a steroid is a relevant consideration and some models of such combined

and synergistic action will be presented later in this review. Actions of E2 via GPR30 have been demonstrated in cell lines linked to breast cancer [6] and thyroid cancer [7]. Oddly, however, even though there are antibodies that apparently have specificity for GPR30 at least in blot material [8], there has been no attempt to map the localization of GPR30 in a range of organs. Interestingly, with regard to the issue of interactions among various grabbers of steroids is evidence that GPR30 and ERβ may be found in the same cells [9] where they may interact to mediate E2’s effects.

Martin Kelly has proposed a separate membrane-associated estradiol receptor [10-17]. Their ER has rapid effects that are mediated both by interaction with GABA receptors [13, 18] and associated with opiate neurons [18]. They have found a synthetic agonist that is specific to their ER called ST-X [15, 19]. Unlike GPR30, Kelly has not yet presented evidence that their ER is associated with other known response factors for E2.

What about the inside of the plasma membrane?

It should be mentioned that it is controversial whether GPR30 even has a role in E2 actions. Kang et al. [20] claim that many of the actions attributed to GPR30 are actually mediated by a splice variant of ERα called ERalpha-36. Ellis Levin is a prominent researcher in the field and he has claimed that ERα is selectively moved to the inside of the plasma membrane [21-23]. They have even defined the elements that are required to direct ERα to the inner side of the plasma membrane. In spite of this, Levin does not claim that ERα binds extracellular E2 from this position.

This raises the obvious question, if cells go through all of the energy expenditure to shunt ERs to the membrane, what are they doing there? Although Levin’s laboratory does not claim that ERs bind extracellular E2, other laboratories do. Mermelstein et al. [24, 25] have demonstrated that ERα is shunted to the cell’s membrane where it interacts with glutamate receptors to influence cell excitability. A process known as palmitoylation is essential for this to occur [26]. The Micevych laboratory has also studied the role of ERα at the membrane as well, which they suggest is sometimes mediated by a splice variant of ERα [27]. Their laboratory has done extensive work to examine the role of membrane ERs interacting with the brain’s opiate system [28-30], which they link to central control of female sexual receptivity [30, 31].

Another possibility suggested by the presence of ERs on the inside of the plasma membrane, but not having a function to bind extracellular E2, is that they are there as part of a larger complex of binding proteins and response proteins that either help to internalize E2 or help to mediate some function of E2 at the plasma membrane. This idea will be further explored below.

A collection of proteins in caveolae

Several of the studies cited above suggest that ERs are shunted to the level of the cell membrane, from which site they may function as part of a complex involved in either E2 uptake or intracellular responses to E2. Could the cell membrane really have so many proteins just involved in

uptake of E2? The answer is “yes and it is more crowded in specific areas than even that suggests”. Caveolae are structurally-defined, protein-rich areas of the plasma membrane [32]. There are proteins that are associated with the caveolae and these are called caveolins [32, 33]. Caveolin-1 seems to be most closely associated with E2 receptors ERα and β [33, 34]. There are several tissues in which caveolin-1 and ERβ are particularly closely associated [33]. Levin has described the presence of ERα associated with caveolae [21]. There has been considerable research on the importance of the non- genomic interaction of ERs in the caveolae in the control of nitric oxide production in vascular endothelial cells [35, 36]. Chambliss et al. [37] have made the point that ERβ is very important in caveolae in controlling this response. Therefore, it is clear that caveolae contain a concentration and perhaps complexes of proteins that seem to interact in important ways to control cellular function; for example, maintenance of vascular patency. It also seems clear that something or some things in the caveolae is/are binding E2.

What is estradiol doing in the plasma membrane?

Therefore, it is clear that there are elements associated with the cell’s plasma membrane that bind E2 and it seems likely that these binding elements are concentrated, perhaps with other important proteins, in specific areas of the plasma membrane, such as the caveolae. But, does E2 have any action at the membrane level or are these complexes only involved in delivering E2 to the nucleus? Most early studies of E2 examined its long-term developmental effects. However, Clara Szego had conducted many experiments in the 1970s showing that E2 had receptors in [38] and had actions on the growth of uterine endometrium [39, 40]. Then in the early 1980s her laboratory conducted a series of experiments that turned ideas about the actions of E2 upside down. They found that E2 treatment of uterus in vitro increased the presence of microvilli in endometrial cells within seconds, not hours [41]. They clearly showed that E2 treatment resulted in a very rapid appearance of microvilli that stuck out of the endometrial cells into the uterine lumen. This finding sat fallow for more than a decade, perhaps because there was no model at the time for such a rapid action of E2 and partly because no one could imagine a mechanism by which such a rapid, non-genomic action of steroids could occur.

Kipp and Ramirez [42] suggest a possible mechanism of the very rapid effect of E2 to produce evaginations from the cell’s surface. They found that E2 was bound to one end of the cellular structural elements tubulin (whereas the testicular steroid testosterone was bound at the other end). They demonstrated that from this position, E2 regulated microtubule extension which drives distortion of the cell surface to produce microvilli or any similar cellular evagination. It seems that one non-genomic action of E2 that has tremendous potential to affect basic cellular functions: It is to alter the cell’s shape by making such evaginations. Below we see that such evaginations from neurons have dramatic significance for memory and other brain functions.

Things that stick out of neurons are called processes and can be either dendrites or axons. Perhaps more equivalent to Szego’s endometrial microvilli are tiny projections particularly from dendrites called spines. Woolley’s laboratory has studied E2-induced production of dendritic spines and their relationship to neuronal function [43-45]. However, they have never examined these effects of E2

in the timeframe of seconds. It would be fascinating to examine whether, in fact, E2 induces a very rapid proliferation of numerous spines and that what is most commonly examined after 24 hours are those few spines that remain after many have long since receded. If this is correct, is E2 a trophic factor that encourages outgrowth and exploration out from the neuron? The implications of such neuronal outgrowth are critical for the laying down of new memories [46, 47].

Our laboratory has used the recently available E2 pre-linked to a fluorophor called E2Glow™ [104]. E2Glow™ is designed to maintain the basic chemical characteristics of E2 and thus to be available for binding by SHBG and cytosolic ERs. We have examined the uptake of E2Glow™ into the classically estradiol-sensitive cell line from Chinese hamster ovaries- CHO [48-50]. Figure 1 shows time-lapse confocal micrographs of binding and uptake of E2Glow™ over a period of 30 seconds. Already, after only 10 seconds, in Figure 1A, the CHO cell is almost completely outlined by fluorescence. This probably indicates the initial accumulation of E2 on the plasma membrane of the CHO cell. Such accumulation could be due to binding of E2 to membrane-associated receptors such as GPR30, or membrane- associated ERα or β, or perhaps this represents the binding of E2 associated with SHBG to a receptor for SHBG. Because the CHO cells were raised and kept in serum, which contains steroid-binding globulins [48, 49], it is likely that SHBG in the medium quickly binds up the added E2Glow™ and thus the SHBG may help it bind the exogenous E2Glow™ to the membrane of the cell. Even at the earliest latency shown (see arrow, Figure 1A) there is an indication of the presence of membrane-associated outgrowths that are fluorescing, suggesting that these outgrowths very quickly accumulate and/or bind E2. This outgrowth, which we will call a sustentacle for reasons that will be made clear below, is even brighter and clearer in Figure 1B taken 20 seconds after addition of E2Glow™, perhaps indicating that by 20 seconds, the sustentacle has accumulated even more E2. It should be noted that even at this point, there is very little indication of fluorescence in the middle of the cell perhaps indicating that E2 is just starting to be taken up into the cytoplasm. At this point there is still a large dark area in the middle of the cell, which presumably represents the nucleus. Even in Figure 1C, where the entire cytoplasm seems to be filled with fluor, there is no fluorescence in this area, suggesting that there still has been no translocation of E2 into the nucleus. Therefore, it appears that E2 first accumulates at the cell membrane and most particularly in outgrowths that we are calling sustentacles. Only after considerable accumulation has occurred at the plasma membrane level does the cytoplasm seem to fill with E2 and, even then, no E2 is detectable in the cell nucleus.

SHBG is involved in uptake of E2

Figure 2A gives some idea of the process by which E2Glow™ is taken up into the cell. First, it appears that the addition of 10-5 M unlabeled E2 competes with E2Glow™ for whatever mechanism is involved in internalization of E2 into the cell. This alone suggests that the internalization of E2 involves a saturable receptor-type mechanism rather than passive diffusion across the plasma membrane. In figure 2B it is clear that the addition of antibody to SHBG to the medium blocks or at least delays the uptake of E2Glow™, clearly indicating that SHBG is part of the uptake process for E2 into CHO cells. As indicated above, many laboratories have suggested SHBG is important for E2 uptake, but this is the first

demonstration that blocking SHBG activity in CHO cells affects E2 uptake. Since CHO cells are an important model for cytoplasmic estradiol receptor action, our findings suggest that SHBG is part of a multi-protein system that is responsible for E2 uptake; a system that likely includes membrane- associated ERs. Such a multi-protein system is delineated for another steroid below.

A further indication that SHBG is important for the internalization of E2 is shown in figure 3: Co- localization of SHBG immunostaining and E2Glow™ accumulation occurs within cytoplasmic granules in CHO cells. Combined with the indication that SHBG is necessary for E2 uptake shown in figure 2, this may suggest that SHBG is internalized with E2 bound to it and then the combination can be found in some secretory vesicles [105]. Since we have already demonstrated the presence of SHBG in neuronal axons, it is possible that these organelles are responsible for shunting E2 throughout the cell.

Role of Sustentacles

It might be suggested that the sustentacles that are seen to accumulate E2 in figure 1, may actually have been created by E2. That is, Clara Szego and coworkers suggested that very soon after exposure to E2, endothelial cells of the uterus showed an increased level of microvilli [38, 39, 51]. We suggested above that the Kipp and Ramirez data [42] showing E2 binding to tubulin could be a rapid, non-genomic mechanism whereby microtubules are activated, exactly the sort of structural microtubules that would effect the evagination of cell membranes to make microvilli or sustentacles. Is it possible that the first effect of exposure to E2 is that outgrowths are produced on the surface of the estradiol-sensitive cell that aid in the internalization of E2?

But, how does E2 get into the nucleus after it is accumulated at the outside of the cell? A clue to the answer for this question is in Figure2. Figure 2B was taken 30 sec. after another CHO cell was exposed to E2Glow™, so at a time when an untreated cell would have been filled with cytoplasmic E2. However, these cells had been pretreated with a 1:200 concentration of SHBG antibody. Clearly there is almost no accumulation of E2 in this cell suggesting that blocking of SHBG with antibody dramatically inhibited the entrance of E2Glow™ into the cell. This would indicate that SHBG is a critical factor in the internalization of all E2 into the cell. We have previously demonstrated that fluor-labeled SHBG was internalized into specific cells in the brain [52] within 10 minutes, suggesting that SHBG itself is internalized rather rapidly in some brain cells. So, it is very possible that without SHBG, either in the media surrounding the cell or SHBG on the cell membrane extracellular surface is critical for internalization of E2 into the cell. Further, it is possible that there are specialized outgrowths that we are calling sustentacles, from cells that may concentrate these internalization mechanisms.

What are SHBG and CBG doing in the nose?

We have evidence that steroid-binding globulins such as SHBG and corticosteroid binding globulin (CBG) are found in the brain [53-60]. We have also found evidence that both CBG and SHBG are

found in both the main olfactory epithelium (look this up) and in the epithelium of the vomeronasal organ of rats [59, 61]. We have found these steroid-binding globulins in the nasal mucus of rats [62]) suggesting that they are excreted into the mucus by epithelial olfactory cells in order to bind aerosolized steroids [63, 64]. Ploss et al. [65] showed that this SHBG is made in the olfactory epithelium as shown by RT-PCR and found the presence of SHBG in the axons of sensory olfactory cells suggesting that SHBG, and possibly bound steroids, are moved out of the epithelial cells toward the olfactory bulb. Figure 2 shows a model that we are proposing for the action of CBG in the main olfactory area. In this model CBG is found in the nasal mucus of rats, where it could bind aerosolized steroids such as a fear pheromone [64, 66-68] or a glucocorticoid (GC) or even possibly progestins. CBG with a bound steroid then binds to a putative CBG receptor on the surface of the nasal sensory cell. CBG with its bound steroid is then internalized into the olfactory cell (it is also possible that these cells produce CBG). Our evidence that CBG is found in the axons efferent from these olfactory epithelial cells suggests that CBG (and probably olfactory SHBG) is important in moving the steroid within the cell. It likely moves CBG along the axon and into the olfactory glomerulum where the CBG and its associated steroid may be released to either affect mitral cells in that area or perhaps to enter blood vessels at this point to affect the CNS via the blood system. In this way, the nose can inform the brain, via nervous conduction or via the blood, of the presence of aerosolized steroids in the environment. Although, we are not sure what these steroids are, there is evidence that rodents can detect ovarian-steroid-like pheromones [69-73], which might be bound by SHBG in the mucus. There is also evidence for “fear pheromones” in rodents and humans [63, 64, 66, 74] that are perhaps bound by CBG and passed along to the CNS. Berglund et al. [75] have suggested that lesbian women detect a progesterone-like steroid, which might be bound by CBG.

Above we used the newly created term sustentacles to describe outgrowths from CHO cells that concentrated fluorescence very quickly when E2Glow™ was added to the cell medium. This term is derived from the term sustentacular cells, which are olfactory epithelial cells that are not part of the neurogenic progression [76-78]. Sustentacular cells are characterized by being in the olfactory epithelium, not being of neuronal developmental origin, and in that they have considerable tiny, narrow outgrowths of uncertain purpose. These outgrowths roughly resemble the outgrowths seen in figure 1 that concentrate E2. Therefore, we are naming these outgrowths sustentacles. It is perhaps interesting that we have also identified similar outgrowths, or sustentacles, in neurons near the third ventricle that have SHBG in them but do not produce SHBG [52, 54]. We found that these periventricular cells internalized SHBG, perhaps via their sustentacles [52]. It is perhaps a function of sustentacles that they have SHBG receptors on them and that they, therefore, aid in the internalization of steroids.

Is there another player?

For the uninitiated, all of these various proteins that seem to live at the plasma membrane and interact to affect various steroid-related physiological functions may seem rather dizzying. Well, hold on to your hats. There is at least one more (and perhaps many more) player on this stage. In the Free Steroid Model of steroid action, the steroid binding globulins such as SHBG and CBG carry steroids in the blood and mostly just release them in the vicinity of target cells (purists would say that there is an equilibrium at all times near a target cell wherein removal of steroids from the milieu by diffusion across

the membrane results in continued release from stores [79], they diffuse passively across the plasma membrane and into the cytosol where they are bound by cytoplasmic receptors. One glaringly obvious flaw in this model has always been, once lipophilic steroids are cozily ensconced in the lipid bilayer of the plasma membrane what would possibly coax them to jump out of that layer and into the aqueous environment of the cytosol? As indicated above, many researchers, such as Allera and Wildt [4] claim that this does not happen without the presence of specific proteins for steroids. Above, this review has already delineated many potential proteins that might live at the inner edge of the plasma membrane. Razandi et al. [23] found that ERα was specifically targeted to go there. Mermelstein and Micevych [25, 25, 80, 80, 81] have evidence that ERβ is there. Recent work in this laboratory has suggested that the kind of increase in spines induced by E2 and discussed above is mediated via glutamate receptors [82]. Several researchers have identified either ERα or β in caveolae in plasma membrane [21, 36, 37, 83].

However, we have claimed for some time [2] that steroid-binding globulins are involved in active responses to steroids. We have evidence that they are internalized into neurons and other brain cells in vivo [52] and that they are internalized by hippocampal and fibroblast cells in vitro [52, 84]. Others have evidence of SHBG internalization in other tissues [85-87]. Rosner’s laboratory has presented extensive evidence that SHBG is bound by a putative receptor at the plasma membrane level in the prostate [88- 93]. Above we presented evidence (Figure 2) that E2 is not internalized without functional SHBG present suggesting that not only is SHBG and likely an SHBG receptor found in the membrane, but that these are critical elements in internalization of E2 and perhaps of other steroids. Below is a model for another steroid-binding globulin that may serve, at least for now, as a model for how many steroids are internalized.

Do steroids live in the membrane for a while?

We [2] and others [90, 94-99] have searched for the putative SHBG receptor for some time. However, undoubtedly the best characterized system involving internalization of a steroid by a steroid- binding globulin is that of the Sun laboratory [100-102]. This laboratory first discovered that a membrane-associated protein originally associated with control of cancer [103], called Stra6 was involved in binding of retinol binding protein (RBP) [100-102]. In their model [102] RBP is bound by the Stra6 in the membrane. Then the steroid (retinol) is released into the lipid bilayer, which should be quite commodious for a steroid. Retinol then can remain in the plasma membrane until it is picked up by a protein. One such protein is enumerated by them as “cytosolic RBP”. Therefore, in their model, the steroid-binding globulin is essential to deliver the steroid to the lipid bilayer where the binding globulin itself is bound by a receptor; and a steroid-binding globulin is also essential in getting the steroid actually into the cytoplasm of the cell. This agrees with our evidence that CBG can be found in the axons of olfactory neurons [62] and figure 2. We suggested that steroid-binding globulins are a part of the system that internalizes steroids as well as responsible for moving them around within the cell. Therefore, the Sun model may serve as a model for steroids other than retinol in that it shows the critical nature of steroid-binding globulins in steroid internalization into cells.
Summary

Clearly, we have presented here evidence of a very complex set of mechanisms and proteins involved with various and intricate actions of steroids at the plasma membrane. Steroids do MUCH more at the plasma membrane than simply passing passively through it. They may sit in the membrane; they are bound by numerous proteins in the membrane, including ERs, SHBG, steroid-binding globulin receptors, and perhaps elements of cellular architecture such as tubulin. It also seems likely that the membrane itself responds graphically to the presence of steroids by actually changing its shape as well, perhaps, as accumulating steroids. Clara Szego suggested in the 1980s that actions of E2 at one level would act synergistically with its actions at another level (e.g. membrane actions would complement nuclear actions). Given the sheer number of proteins involved in steroid actions, just at the membrane level, it seems unlikely that every action of a steroid on every potential protein effector will act to the same end. It seems more likely that these multiple effects and sites of effect of steroids contribute to the confusion that exists as to what actions steroids always have. For example, there is confusion with regard to synthetic agents (SERMs etc.) that have different and often opposite actions depending on which organ they act upon. A better understanding of the basic actions of steroids should aid in understanding the variability of their actions.

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Figure Captions

Figure 1. Sequential confocal laser micrographs of a live CHO cell over time after exposure to 10-9 M E2Glow™ demonstrating E2 entry into the cell. At 10 seconds (A) E2 seems to be concentrated around the edges of the cell, possibly indicating early concentration at the plasma membrane. Long moving membrane protrusions, “sustentacles” (white arrows), particularly have concentrated fluorescence and thus E2. At 20 seconds (B) more E2 is concentrated in the plasma membrane with very little in the cytoplasm yet. At this point even more sustentacles are apparent. Finally, after 30 seconds (C) much E2 is found in the cytoplasm as well as around the cell. As yet, however, almost no E2 is found in the nucleus. Scalebar = 5µm

Figure 2. Pretreatment of CHO cells with 10-5 M E2 prevents accumulation and uptake of E2Glow™ (Fig. 2A). Preincubation of CHO cells with an antibody to SHBG (B) prevents movement and extension of sustentacles suggesting that the presence of SHBG is essential for uptake of E2. In both cases, fluorescent E2 is accumulated on the cell membrane, but entry into the cytoplasm is delayed.
Nuclear counterstain with Hoechst nuclear dye. Scalebars= 10 µm

Figure 3. After 10 min of treatment with E2Glow™, fluorescent steroid is accumulated in the nucleus and to a lesser extent in cytoplasm (A). Membrane protrusions are no longer visible. Fluorescence appears diminished due to histological fixation and subsequent immunostaining (B). Immunostaining for SHBG reveals that E2Glow™ is concentrated in SHBG-positive granules as visualized by immunoperoxidase staining (Arrows) indicating a close association of internalized E2 with SHBG. Scalebar = 20µm

Figure 4: CBG in the olfactory system: CBG is expressed in goblet cells of the respiratory mucosa and in the Bowman Glands to enter the nasal mucus and to trap airborne glucocorticoids and possibly other aerosolic steroids. In this model sensory cells express CBG receptors (CBG-R). CBG expressed in mitral cells and in periglomerular cells, where it may aid comparison of systemic (endogenous) GC levels with exogenous GC concentrations. The interaction of olfactory and limbic circuits may be important for controlling social behaviors including dominance and stress response.

Figure

Fig. 1: Sequential confocal laser micrographs of life CHO cell over time after exposure to 10- 9M E2Glow™ demonstrating E2 entry into the cell. At 10 seconds (A) E2 seems to be concentrated around the edges of the cell, possibly indicating early concentration at the plasma membrane. Long moving membrane protrusions, “sustentacles” (white arrows) particularly have concentrated fluorescence and thus E2. At 20 seconds (B) more E2 is concentrated in the plasma membrane with very little in the cytoplasm yet. At this point even more sustentacles are apparent. Finally, after 30 seconds much E2 is found in the cytoplasm as well as around the cell. As yet, however, almost no E2 is found in the nucleus (N). Scale bar = 5µm

A B

Fig. 2: Pretreatment of CHO cells with 10-5M Estradiol prevents accumulation and uptake of Estradiol Glow (Fig. 2A). Preincubarion of CHO cells with an antibody to SHBG prevents movement and extension of sustentacles. The fluorescent steroid is accumulated on the cell membrane, entry into the cytoplasm is delayed. Nuclear counterstain with Hoechst nuclear dye. Scalebars= 5µm

Fig. 3: After 10 min of treatment with E2Glow, the fluorescent steroid is accumulated in the nucleus and to a lesser extent in cytoplasm. Membrane protrusions are not visible anymore. Fluorescence appears dimished due to histological fixation and subsequent immunostaining (Fig.5). Imunostaining for SHBG reveals that E2Glow is concentrated in SHBG positive granules as visualized by immunoperoxidase staining (Arrows) indicating a close association of internalized E2 with SHBG.

Fig. 4: CBG in the olfactory system: CBG is expressed in goblet cells of the respiratory mucosa and in Bowman glands to enter the nasal mucus and to trap air borne GCs. Sensory cells express CBG receptor (CBG-R). CBG expressed in Mitral cells and in periglomerular cells may aid comparison of systemic (endogenous) GC levels with exogenous GC concentrations. The interaction of olfactory and limbic circuits may be important for controlling social behaviors including dominance and stress response.

Abstract: Clearly, we have presented here evidence of a very complex set of mechanisms and proteins involved with various and intricate actions of steroids at the plasma membrane. Steroids do MUCH more at the plasma membrane than simply passing passively through it. They may sit in the membrane; they are bound by numerous proteins in the membrane, including ERs, SHBG, steroid-binding globulin receptors, and perhaps elements of cellular architecture such as tubulin. It also seems likely that the membrane itself responds graphically to the presence of steroids by actually changing its shape as well, perhaps, as accumulating steroids.
Clara Szego suggested in the 1980s that actions of E2 at one level would act synergistically with its actions at another level (e.g. membrane actions would complement nuclear actions). Given the sheer number of proteins involved in steroid actions, just at the membrane level, it seems unlikely that every action of a steroid on every potential protein effector will act to the same end. It seems more likely that these multiple effects and sites of effect of steroids contribute to the confusion that exists as to what actions steroids always have. For example, there is confusion with regard to synthetic agents (SERMs etc.) that have different and often opposite actions depending on which organ they act upon. A better understanding of the basic actions of steroids should aid in understanding the variability of their actions.