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Discuss the evidence for non-genomic actions of steroid hormones.
All natural steroid hormones are made from cholesterol and are modifications of the basic C19 structure. There are 5 distinct classes of steroid hormones: progestogens, glucocorticoids, mineralocorticoids, androgens, oestrogens and the steroid derivative D3. They are all structurally and functionally distinct, and therefore act on their target cells in various ways, with genomic and/or nongenomic actions, as recently discovered. Through these effects, steroids control many physiological processes.
How do steroid hormones act?
Traditionally, it has been thought that steroid hormones enter the cell and bind to classical steroid receptors, located mainly in the cytosol. Ligand binding induces a conformational change in the receptor protein and causes dimerisation, as well as disassociation of heat-shock proteins. These receptors act as transcription factors. In the nucleus, the DNA binding domain (DBD) on the protein binds the hormone response element (HRE), modulating gene transcription. The protein made from the newly synthesised mRNA elicits the genomic response. This process occurs with a time lag of many hours, and the pathway is sensitive to inhibitors such as actinomycin D or cyclohexmide.
However, it has been noted in the past (Hans Selye, 1942) and only recently verified that steroids also have rapid actions through binding steroid receptors located on the plasma membrane (nongenomic actions), much quicker than the above pathway would allow. Association with DNA in the nucleus and the subsequent gene expression is avoided. The receptor types initiate signalling pathways, similar to the actions of peptide hormones. There are 2 main types of peptide hormone receptors: 7TMD receptors linked to the production of a second messenger, or receptors activating a kinase directly. With steroids, the exact structure of these atypical receptors is not yet defined but possibilities of receptor types include GCPRs, ion channels or enzyme-linked receptors. Many of these nongenomic effects appear to involve second messengers such as cAMP and DAG, kinases including PKC and MAPK, and ion fluxes (mainly calcium). This is clearly incompatible with the genomic model described in the past.
Evidence for nongenomic actions
Evidence for their rapid effects is available for steroids of all clones and for a multitude of species and tissues. Nongenomic actions seem to depend on the type of steroid, cells, tissues or species used. Therefore, different pathways and receptors are involved in this speedy process. Tissues traditionally considered as 'non-targets' for genomic actions are vividly found to be regulated by nongenomic mechanisms. Examples of supposed nongenomic actions include rapid oestrogen and aldosterone effects in vascular smooth muscle, and progesterone in the acrosome reaction of sperm.
Hans Seyle provided criteria for defining nongenomic action. These include: the absence of a nucleus, effects that are not obstructed by transcriptional inhibitors, a short time frame, and actions elicited by steroid analogues that do not enter the cell. Seyle's paper was the first to actually detail the nongenomic phenomenon by observing rapid steroid induced effects, such as anaesthesia. This discovery led to development of steroidal anaesthetics used in human medicine and on animals.
Zhu et al were the first to show that their receptor (progestogens) meets all of the criteria for a steroid receptor, including structural plausibility, tissue specificity, membrane localisation, characteristic steroid binding, signalling cascade activation, hormonal regulation and biological relevance.
The pharmacological agonist and antagonist profiles for genomic and nongenomic actions often differ markedly. Antagonists inhibiting genomic effects are inactive towards the nongenomic effects. However, there are only a few selective inhibitors for nongenomic steroid action known at present.
Antibodies have been used to prove their presence, but this approach is sometimes misleading due to crossreactivity and low abundance of antibodies. Various steroids have been shown to bind membrane proteins, but their characterisation has been limited to determination of their molecular weight or antibody identification.
Three types of receptors have been recorded.
- Classical nuclear receptors - directly affect gene transcription
- Classical membrane receptors - nongenomic actions through signalling
- Nonclassical membrane receptors - nongenomic actions though signalling
Classical receptors can either lead to changes in gene expression or have nongenomic actions. Nonclassical receptors merely lead to a direct and rapid physiological response. There is a controversy with regards to the existence and identity of nonclassical receptors, and the question being asked is do they exist, and if so, how do they work?
There is evidence that kinases affect gene transcription through binding to CREB, and that transcription factors are linked to kinase cascades. This phenomenon is called cross-talk. (This is true of both steroid and peptide hormones binding to their respective membrane receptors). In thyroid hormones, following initiation of a signal cascade, other events such as phosphorylation of signal transducers, STATs and p53 take place These events lead to a modulation of gene expression, exerted by a nonclassical pathway. Another example of this is the admission of dexamethasone (a synthetic glucocorticoid), which rapidly stabilises lysosomal membranes within 10 minutes, yet a similar effect is detected 24 hours later, indicating a dual action through classical and nonclassical receptors. A multi-step model for steroid action is an extension of the cross-talk model. It explains and inter-relates these 2 mechanisms.
As mentioned above, in some cases, classical nuclear receptors have been shown to drive nongenomic actions. For example, the classical PR has been shown recently to interact with signalling components. The interaction of PR with Src (a SH3-containing protein) activates Ras and/or Raf1 and ERK/MAPK cascades, which then influence the activity of TFs in the nucleus.
Furthermore, there is evidence of receptor-free direct nongenomic action as can be seen with enzymes, for example in the rapid effect of thyroid hormones on mitochondrial respiration.
Steroid groups and evidence for receptor initiated signalling cascades
- Gonadal steroids
- Vitamin D3
- Thyroid hormones
Progestogens are C21 steroids and traditionally bind to the Progesterone receptor. They are secreted by the developing follicle and corpus luteum (ovary), facilitating the menstrual cycle and maintenance of pregnancy. Nongenomic effects of progesterone have been described in germ cells such as oocytes or sperm.
In March 2003, Zhu et al studied the progestin receptor isolated from Cynoscion nebulosus (spotted seatrout) ovaries, which functions at the cell membrane and sets forth a signal cascade. The rapid response produced is atypical of classical receptors.
Xenopus laevis oocytes arrested in the G2 phase undergo maturation upon progesterone administration. This process can occur in enucleated cells and is insensitive to actinomycin D, indicative of nongenomic action. Progesterone results in inhibition of adenylate cyclase and a decrease in CAMP levels. It is more effective when applied outside the cell rather than the cytoplasm.
XPR (Xenopus isoform of the classical PR) is present in small amounts in the membrane, and seems to activate PIK3 and ERK/MAPK administration of progesterone. The classical PR antagonist RU486 and other steroids do not trigger this mechanism, which indicates atypical receptor forms.
The acrosome reaction is a result of sperm cells responding to progesterone. It has been proven that this response is nongenomic, and therefore involves a nonclassical receptor. However, the involvement of the classical PR in spermatozoa is still unexplained.
Fluorescent progesterone-albumin conjugates have been used to localise membrane receptors. Progesterone-binding proteins are found to be located on the outer surface of sperm. Progesterone effect is reduced by using antibodies against the mPR isolated from liver. There is also evidence that progesterone may have a dual role, through genomic and nongenomic pathways.
Oestrogens are C18 steroids and are made in the ovary and adipose tissue. They are the principal female sex hormones, and have been thought to solely act via the oestrogen receptor. Similar to progesterone, oestrogen has been shown to have nongenomic effects in reproductive cells.
In the late 1970s, Pietras & Szego described the presence of cytoplasmic membrane binding sites for oestrodiol in endometrial and granulosa cells. Rapid increases in calcium concentration have been observed as a result of this receptor-ligand interaction. In granulosa cells, the classical ER inhibitor tamoxifen or typical inhibitors of classical receptors did not block the calcium response.
In addition, oestrodiol has a direct action on the vascular system, inducing relaxation. Oestrodiol activates eNOS, which is then phosphorylated via the P13K-Akt pathway, resulting in increased NO, a vasodilator. Pertussis toxin inhibits this response, which indicates that a GPCR may be involved. There is uncertainty as to whether this receptor is a classical ERa. However the rapid activation of ERK/MAPKs by oestrodiol also occurs in ERa-knockout mice, which points to the involvement of ERß shown to initiate nongenomic steroid effects, or even a nonclassical receptor. Both ER forms have been shown to localise in caveolae (invaginations of the membrane). Double knockout mice are now being studied to rule out possibilities of ERa and ERß involvement.
Androgens (e.g. testosterone), however, are C19 steroids secreted by the Leydig cells of testis and by the adrenal cortex. These are the male sex hormones and act by binding to the nuclear androgen receptor. Binding sites for testosterone have been found on cell membranes of rat osteoblasts, macrophages, T lymphocytes, prostate cells, as well as vascular cells.
Glucocorticoids (cortisol) are secreted by the adrenal cortex, and traditionally bind to the Glucocorticoid receptor. They have a role in the adaptation to stress. The nongenomic effects of glucocorticoids have not been studied thoroughly, but rapid responses are presumably nongenomic.
The actions of corticosterone in roughskin newt Taricha granulosa mimic the rapid stress reaction of courtship behaviour. A membrane glucocorticoid receptor has been assigned, similar to 7 transmembrane opiod receptors. High levels of dexamethasone (a synthetic glucorticoid) rapidly stabilise lysosomal membranes, insensitive to the GR antagonist RU486.
As with oestrogens, glucocorticoids are also shown to activate eNOS nongenomically, mediated by P13K-Akt phosphorylation, and resulting in vasorelaxation.
Mineralocorticoids (aldosterone being the main one) are also C21 steroids and are secreted by the zona glomerulosa of the adrenal cortex, acting on the kidney to promote sodium reabsoption. Genomically, they act by binding to the mineralocorticoid receptor. In the 1960s, it was noted that acute cardiovascular effects of aldosterone occurred almost immediately after its administration in humans. The short time frame suggested nongenomic action.
In vitro studies on control of sodium exchange by aldosterone were carried out on dog erythrocytes (enucleated). This is also indicative of nongenomic action.
Aldosterone induced various responses in mononuclear leukocytes, including activation of the Na+/H+ antiporter, leading to subsequent changes in cellular volume. Inhibitors of classical MRs cannot inhibit this response. Nonetheless, the time scale of 15 minutes does not completely rule out a genomic mechanism.
There is evidence for aldosterone involvement in the regulation of vascular function via ion fluxes. In mice lacking classical MRs, there is evidence for the persistence of Ca2+ and CAMP responses to aldosterone, suggesting a nongenomic mode of action.
In human arteries, aldosterone-induced alkalisation was studied and lead to the notion that a nonclassical receptor with different ligand specificity is involved.
In skeletal muscle, aldosterone produces a rapid and transient Ca2+ increase, maximal after 2 minutes, followed by a slower and more sustained rise. PKC often seems to be involved, since its inhibition seems to cease the response.
In the isolated rat heart, aldosterone has a rapid and positive ionotropic effect (within minutes). Spironlactone (a classical MR inhibitor) does not antagonise this effect.
Vitamin D3 is formed by the action of sunlight on the skin, and acts on the Vitamin D receptor. It has an important role in calcium metabolism.
The active form of Vitamin D (1a25(OH)2D3) has a flexible conformation, unlike other steroids, suggesting the involvement of nonclassical receptors. 1a25(OH)2D3 initiates membrane-lipid hydrolysis, causing the production of 2nd messengers such as InsP3, that in turn release Ca2+ from intestinal intracellular stores (transcaltachia). DAG is also formed, probably activating PKC and followed by rapid activation of Raf and ERK/MAPK. The nongenomic pathway also involves the CAMP-PKA pathway. Inhibitors of PKA block the rapid Ca2+ response. 1a25(OH)2D3 elicits both nongenomic and/or genomic responses. The diastereomer inhibits this effect, but cannot block the genomic action. Cis-locked analogues activate rapid pathways and hardly bind classical receptors.
Many steroids on the nervous system and influence sleep, reaction to stress, mood and memory. Dementia has been correlated with decreased amounts of neurosteroids. Steroids binding the GABA receptor are used in clinical studies. The GABA receptor is the site of action of some barbiturate-like ligands, with a potent depressor effect in the CNS. Clinically used anaesthetics alphaxolone and alphadolone also act on this receptor.
In the nervous system of rodents, progesterone acts on the GABA receptor in the ventral tegmental area (nongenomic action). However, in the ventromedial hypothalamus, progesterone action seems to depend on an intracellular PR (suggesting genomic action).
Thyroid hormones are chemically different from steroids yet their classical receptors belong to the same steroid-receptor superfamily. T3 and T4 stimulate plasma-membrane and sarco/endoplasmic-reticulum Ca2+ ATPase activity, resulting in Ca2+ efflux from the cytosol. This is probably mediate by PKC. The ERK/MAPK signalling cascade is driven by T4.
The importance of nongenomic steroid actions has been underestimated, regardless of early reports. Rapid nongenomic responses are essential when presented with similarly rapid stimuli. These effects have been observed in humans and many other vertebrate and are therapeutically relevant.
However, many studies suffer from technical limitations and quite simply prove the existence of membrane receptors. Further experimentation, such as classical-receptor knockout animals, is required to rule out the possibility of classical receptors being involved and identify nonclassical receptor proteins, if such a thing exists. Clinical trials and animal models are needed. However, with human trials, it is not possible to induce genomic inhibitors. Incorporation of specific 'non-classical' receptors will help identify targets for therapeutic intervention.