Steroid Hormones

Stress

In most situations, stress is harmful. Although there is so-called healthy stress, the emphasis here is on stress events that can lead to damage. There are acute and chronic stresses. Acute stress is short-lived and usually invokes a “flight-or-fight” response. For example, walk through the woods and suddenly see a rattlesnake in your path. Catecholamines would pour out and you would turn and run as fast as possible. After the stressor has disappeared, the internal systems would normalize again; epinephrine, norepinephrine , and cortisol would have entered the bloodstream during the stress and, ultimately, these secretions would normalize again after the stress event is over.

Chronic stress , on the other hand, lasts over a long period with suppression of the “flight-or-fight” response. Long recoveries from surgery, long-term relationship problems, financial worries, and pressured work environment are examples. Long-term stresses can lead to elevated blood pressure, heart disease, suppression of the immune system, and even to diabetes (stress-induced diabetes) as well as other pathologies. Over long periods, stress causes an increase in the level of blood cortisol (also seen during aging ; so, aging itself may constitute a type of stress). Cortisol feeds back negatively on the hippocampus and amygdala of the limbic system that can lead to depression. As this chronic stress continues, the increase in circulating cortisol can cause the hippocampus, via the glucocorticoid receptor (GR), to shrink (cortisol induction of programmed cell death). Since the hippocampus is the center of memory , this also can account for memory losses. Even in acute stress, there can be short-term memory losses that are reversed during recovery. Another factor in the development of depression relates to the fact that part of serotonin’s action is to create a feeling of well-being and cortisol acts to suppress serotonin release . This form of depression is typical in aging.

Nociceptin

Nociceptin (also orphanin FQ ) is a 17-amino acid peptide (FGGFTGARKSARKLAMQ) anxiolytic that experimentally modulates anxiety produced by acute stress. While it is classified as an opioid , it binds to its receptor that is separate from the classical plasma membrane opioid receptors (kappa, mu, and delta); it binds to the nociceptin receptor called NOP1 . The kappa receptor (κ), KOR (another name for the kappa receptor), binds dynorphins (there is “big dynorphin,” dynorphin A and dynorphin B; big dynorphin and dynorphin A are more potent and selective than dynorphin B); the mu receptor (μ), MOR (another name for the mu receptor), binds β-endorphin and endomorphins (endomorphin 1 is YPYF); and the delta receptor (δ), DOR, binds enkephalins (Met-enkephalin is YGGFM and Leu-enkephalin is YGGFL). Remember that in the anterior pituitary, preproopiomelanocortin is the precursor of β- endorphin and Met-enkephalin ( Fig. 15.6 ). The precursor of nociceptin in nociceptinergic neurons is prepronociceptin . It is a polypeptide of 176 amino acids that is degraded by enzymes that cleave at basic amino acid sites into a signal peptide and active peptides: amino acids 1–19 constitute a signal peptide, amino acids 20–95 constitute a spacer peptide, amino acids 98–127 constitute neuropeptide 1 ( nocistatin ), amino acids 130–146 constitute nociceptin, amino acids 149–165 constitute neuropeptide 2 ( Nocil ), and amino acids 169–176 constitute the C-terminal peptide fragment. The NOP is distributed throughout the brain, including the limbic system (e.g., hippocampus and amygdala), and spinal cord and the nociceptin-NOP system are present both in the central nervous system and in the peripheral nervous system where it modulates nociception , the transmission of pain . Nociceptin binds to NOP in neurons where it reduces the activation of adenylate cyclase , reduces the activity of calcium channels , and also opens potassium channels (similarly to the action of opioids).

Responses to Stress

The release of catecholamines in short-term stress and the release of glucocorticoid in long-term stress are shown in Fig. 16.1 .

A stress event , evoked from the external or internal environment, is filtered in the brain by some mechanism, probably sensory gating (involving the thalamus) so that certain stimuli are rejected from further reaction. However, many forms of stress, especially psychological stressors (job insecurity, abuse, marital problems, etc.) or acute stressors (loud noises, sudden fright, etc.), set into motion the short- and long-term effects of stress shown in Fig. 16.1 . Some sort of signal (possibly an electrochemical signal), following a stress event, impacts the hypothalamus and it sends impulses through the spinal cord to preganglionic fibers that innervate the adrenal medulla resulting in the secretion of epinephrine and norepinephrine into the bloodstream. The elevation of these catecholamines in the circulation causes rapid increases in heart rate, blood pressure, and metabolic rate and stimulates the dilation of the bronchioles as well as causing the conversion of glycogen to free glucose for energy use.

The hypothalamus releases corticotropin-releasing hormone ( CRH ) into the portal blood system, enclosed by the stalk, and generates the release of adrenocorticotropin hormone ( ACTH ) from the corticotropic cells of the anterior pituitary. ACTH circulates to the adrenal cortex where it binds and activates its receptor on the plasma membranes of adrenal cortex cells resulting in the synthesis and release of the glucocorticoid cortisol and the mineralocorticoid aldosterone . Aldosterone is also released by acetylcholine during stress and by angiotensin II (Ang II) .

Cortisol circulates in the bloodstream and crosses the plasma membranes (presumably by free diffusion owing to its lipid solubility) and freely passes back out to the extracellular space if there is little receptor available in the cytoplasm of the cell. Target cells, like the hepatocyte, have large amounts of the cytoplasmic receptor and cortisol is sequestered thereby binding to the receptor molecules. Kidney cells also have large amounts of the receptor. Virtually, all the cells in the body have some receptor molecules, except hepatobiliary cells and cells in the space between the anterior and posterior pituitary, so it can be said that some cortisol is taken up by all cells except the two forms indicated. As a result, there is protein and lipid breakdown, the products of which are converted to blood glucose. Also glucose uptake by peripheral tissues is inhibited resulting, in the short term, of about a 10% increase in the blood level of glucose, enough to cause a release of insulin from the pancreatic β-cell. In itself, it is not a harmful response but if continued over a long period, the pressure to release insulin from unrelenting stress events could lead to stress-induced diabetes through exhaustion of the β-cells’ ability to produce and secrete insulin. Also, in prolonged stress, suppression of the immune system and breakdown of muscle protein together with decreased protein synthesis can occur.

Aldosterone , a true stress hormone, causes the retention of sodium by its actions on the kidney and increases blood volume (by increasing water reabsorption) and blood pressure. Whereas cortisol secretion (primarily from zona fasciculata cells of the adrenal cortex) occurs endogenously under the control of serotonin , in stress, its secretion is added to the endogenous level. This accounts for the nearly 1000 times higher circulating level of cortisol (up to 25 μg/dL) compared to aldosterone (up to about 30 ng/dL). Aldosterone is secreted primarily as the result of stress. Abnormally, high-salt ingestion will increase the blood level of aldosterone. In addition to the action of ACTH on the zona glomerulosa cell (outer cells of the adrenal cortex), aldosterone is also released by the action of Ang II and hypokalemia as shown in Fig. 16.2 .

Besides causing the release of aldosterone from the zona glomerulosa , Ang II has many other activities. It constricts blood vessels, especially the efferent arterioles of the kidney causing increased sodium ion and water retention. Sodium retention also is promoted by the direct action of Ang II on proximal tubules. It increases thirst by acting on the hypothalamus and stimulates the secretion of antidiuretic hormone , ADH (AVP) from the posterior pituitary, thus increasing water reabsorption , among other actions. Ang II stimulates tyrosine kinases (pp60 c-src ) focal adhesion kinases and JAK2 , TYK2 kinases that are involved in vasoconstriction of vascular smooth muscle, protooncogene expression, and protein synthesis. The angiotensin precursor is formed in the liver and converted to Ang II in the lungs.

Aldosterone is secreted from the zona glomerulosa cell into the bloodstream and circulates to the tissues. The cellular targets, especially the distal kidney tubular cell , contain cytoplasmic mineralocorticoid receptors (Mrs) to which aldosterone binds (there is a very small percentage of mineralocorticoid-like receptors in the plasma membrane that, when activated, probably play a small role in ion transport; plasma membrane “Mrs” may be proteins different from the classical receptor). The binding of the steroid hormone activates the receptor complex, causing the release of bound heat shock proteins and the dimerization of the monomeric receptor protein. The dimer translocates to the cellular nucleus and binds to the appropriate steroid response element in various promoter elements to activate genes encoding the serum- and glucocorticoid-induced kinase ( Sgk ), the Kirsten-ras A oncogene protein , and CHIF , the corticosteroid hormone–induced factor that stimulates the formation of potassium uptake channels. The actions of aldosterone on the principal cells of the kidney are shown in Fig. 16.3 .

The activation of the ACTH receptor by binding ACTH occurring on the zona fasciculata cell (the intermediate layer of cells in the adrenal cortex) membrane of the adrenal cortex to generate the synthesis of cortisol and its secretion is shown in Fig. 16.4 .

Cortisol is synthesized and released from the fasciculata cell, enters the bloodstream, and is carried through the adrenal medulla to the general circulation. In the medulla the hormone induces the formation of catecholamines by inducing a key enzyme, phenylethanolamine N -methyltransferase . Cortisol circulates to the tissues entering most of the cells; in the target cells, there are many molecules of the cytoplasmic GR that bind cortisol. The retention of cortisol within a cell depends on the concentration of its receptor, the liver and kidney cells having the largest amounts. The receptor complex becomes activated, enters the nucleus as a dimer (some models show the receptor as forming a dimer in the nucleoplasm), and binds to the glucocorticoid response elements (GREs) of various genes to produce many different mRNAs and subsequently their protein counterparts. These events are pictured in Figs. 16.5 and 16.6 .

It is uncertain as to in what compartment, the receptor complex dimerizes since some mechanistic schemes show the receptor entering the nucleus as a dimer, whereas in the illustrations here, the GR enters the nucleus as a monomer. In Fig. 16.6 , examples are shown of the response elements in gene promoters in which the GR dimer is involved: the GRE where the receptor dimer positively transcribes the message for genes like tyrosine aminotransferase ; the STAT (signal transducer and activator of transcription) response element positively regulating the protein, casein and the negative regulator, nGRE , that downregulates the osteocalcin gene; the AP-1 response element where Fos/Jun binds and then the GR binds to c-Jun to downregulate collagenase and the nuclear factor kappaB ( NF- κB) response element where the receptor binds to the subunits of NF - κB to downregulate interleukin (IL)-1β.

The endogenous secretion of cortisol under the control of a serotonergic neuron is highest at about 8:00 a.m. and falls to lower levels during the day; it will rise again to its peak at 8:00 a.m. on the next day. During individual stress events the released cortisol in the blood is added to the circulating cortisol derived from the serotonin-induced endogenous release. The incidence of heart attacks may be associated with the highest endogenous level of cortisol in the blood and many occur in the early morning.

Although these responses to stressors reoccur after each stress event, eventually, an adaptive process must take place allowing the survival of the individual. Such adaptation may occur in the brain in the form of epigenetic modifications in the brain cells’ DNA to allow the expression of genes that are required for the stressed individual to adapt.

Production of High Levels of Cortisol (Cushing’s Disease) and Subnormal Levels of Cortisol (Addison’s Disease)

Normally, in response to serotonin for the endogenous production of cortisol and in response to a stress event, CRH is released from the hypothalamus causing the output of ACTH from the corticotrope of the anterior pituitary. ACTH enters the bloodstream and binds to ACTH receptors on the cells of the zona fasciculata of the adrenal cortex resulting in the synthesis and release of cortisol into the bloodstream. In Cushing’s disease , there is an overproduction of cortisol. The cause is often a tumor in the anterior pituitary that is producing abnormally high amounts of ACTH. This results in overstimulation of the adrenal zona fasciculata cells that synthesize and secrete abnormally high levels of cortisol. The usual negative feedback occurs by cortisol on the CRH neurons and the corticotrope but the tumor itself may not respond to this negative feedback (it may express little or no GR ). A high level of cortisol in the blood is the result. Hypercortisolism , typical of Cushing’s disease, also can be caused by ectopic tumors that produce ACTH and, occasionally, a tumor can produce CRH resulting in the abnormally elevated production of ACTH. Overproduction of ACTH can lead to hyperpigmentation because α-melanocyte-stimulating hormone (α-MSH) is produced from ACTH by cells in the space between the anterior and posterior pituitary. Hypercortisolism generates symptoms of rapid weight gain, especially of the trunk and face and growth of fat pads along the collar bone, excessive sweating, dilation of capillaries often generating purple or red striae (stripes or lines) due to hemorrhage, elevation of blood glucose level that can lead to diabetes , hypertension , and others.

Underproduction of cortisol ( Addison’s disease ) is due to chronic adrenal insufficiency in which there is abnormally low production of adrenal steroids (cortisol and aldosterone ). In this somewhat rare disease, cells of the adrenal cortex may have been destroyed by autoimmunity or by infectious agents or the adrenal cortex can be poorly formed from underdevelopment. There are many symptoms: weight loss, anxiety, nausea, headache, diarrhea, mood swings, sweating, low blood pressure, and hyperpigmentation of the skin (this time caused by insufficient negative feedback by cortisol on the corticotrope so that ACTH is overproduced and its breakdown by cells in the pars intermedia area generates α-MSH). There are symptoms of muscle weakness, lightheadedness, fatigue, fever, muscle and joint pains, vermillion border of the lips, and a low concentration of blood sodium with elevated calcium ions and potassium ions. Addison’s disease is treated by oral administration of adrenal steroids so that a near-normal existence can be obtained.

Adrenal Cortex

The adrenal gland is located within masses of fat above the kidneys. The adrenal has two structures, the outer adrenal cortex and the inner adrenal medulla . The adrenal cortex consists of three layers of cells internal to the outer capsule. The layer directly beneath the capsule is the zona glomerulosa and this layer of cells secretes mainly aldosterone in response to stress signals. The thick middle layer of cells is the zona fasciculata and its main secretion product is cortisol in response to ACTH and indirectly to serotonin (through CRH and to ACTH) forming the endogenous production of cortisol. The innermost layer of the cortex is the zona reticularis and its main secretion products are weak androgens, in particular, dehydroepiandrosterone ( DHEA ) plus some androstenedione . Reactions leading to all of the individual steroid hormones are shown in Fig. 16.7 .

The fetal adrenal gland consists mainly of zona reticularis type cells. The other layers of cells develop near birth.

Aldosterone levels, similar to cortisol, fluctuate during the day because ACTH also releases some aldosterone in addition to cortisol. The stimulation of aldosterone by ACTH is short term, whereas the stimulation of cortisol by ACTH is longer term. Consequently, the blood concentrations of aldosterone are very much smaller than those of cortisol. Also, cortisol levels respond both to the endogenous pathway elicited by serotonin and to stress events, whereas the secretion of aldosterone is mainly the result of stress events. In the absence of ACTH , sodium depletion activates the renin–angiotensin system that elevates the synthesis of aldosterone as shown in Fig. 16.8 .

Details of the action of the hormones Ang II and ACTH and acetylcholine in response to stress are shown in Fig. 16.2 . The hormone angiotensin is derived from the precursor, angiotensinogen (480 amino acid polypeptide), synthesized in the liver. Angiotensinogen is cleaved by the kidney proteolytic enzyme, renin , to produce angiotensin I (10 amino acids). Angiotensin I is further cleaved by the lung enzyme, angiotensin-converting enzyme ( ACE ) to yield an octapeptide-containing amino acid 1–8. This ( right side ) form can be further converted to angiotensin IV (amino acids 3–8) and angiotensin III (amino acids 2–8). Angiotensin I is converted to Ang II , the major form of the hormone , by ACE and Ang II binds to its receptor (AT1R) to produce hypertension , vasoconstriction , and cardiovascular disorders . Alternatively, angiotensin I can be cleaved by angiotensin-converting enzyme 2 (ACE2) to produce angiotensin 1–9 or it can be cleaved by neprilysin to produce angiotensin 1–7. If angiotensin 1–7 binds to the Mas receptor or if Ang II binds to the renal AT2 receptor (AT2R), opposite reactions are obtained (antihypertension, vasodilation, and cardiovascular protection). These reactions are depicted in Fig. 16.9 .

There are abundant physiological effects of Ang II directly and through its stimulation of the secretion of aldosterone . Ang II causes increased blood pressure through its constriction of blood vessels. Ang II has important effects on the kidney microcirculation and acts directly on the proximal tubules to cause sodium retention . Through the release of aldosterone and its effects on the collecting tubules, there are increases in plasma sodium ion concentration and reductions in potassium ions and hydrogen ions. Aldosterone acts on the hypothalamus to cause thirst and the secretion of ADH (AVP). Ang II potently constricts both afferent and efferent arterioles in the kidney microvasculature [its responses can be regulated by both paracrine (from neighboring cells) and autocrine (from its own cell) factors derived from the endothelium and from the macula densa ]. The concentration of Ang II determines the extent of tubuloglomerular feedback activity, especially when the concentration of the protease renin (from the macula densa ) is elevated. The vascular effects of Ang II are primarily mediated by the AT1R. In the fetus, AT2R is expressed in the kidney and is critical for kidney development but seems to be expressed less strongly in the adult. As shown in Fig. 16.9 , the AT2R–Ang II complex can mediate vasodilation.

Regarding the effects of Ang II on the afferent arteriole (blood vessel carrying blood to the glomerulus), it constricts this vessel by stimulating the entry of calcium ions through voltage-sensitive L-type channels , while the constriction of efferent arterioles (blood vessel carrying blood away from the glomerulus) results from the release of calcium ions from intracellular stores and the uptake of calcium ions through voltage-independent channels to cause back pressure at the glomerular capillaries.

Significantly, angiotensin receptors are expressed in the brain, kidney, adrenal, vascular wall, and in the heart. ACE2, related to ACE, is expressed in the endothelium of the coronary, intrarenal vessels, and in the epithelium of the renal tubules. Ang II can stimulate tyrosine kinases [pp60 c-src , focal adhesion, and Janus kinases (JAK2, TYK2)]. These tyrosine kinases may be involved in the vasoconstriction of vascular smooth muscle, protein synthesis, and protooncogene expression.

Aldosterone , released from the zona glomerulosa cell, circulates in the bloodstream and binds to its receptors in the cytoplasm of its cellular targets, principally the distal kidney tubular cell , and exerts the effects shown in Fig. 16.3 . The action of aldosterone causes sodium ion reabsorption from the tubular urine while potassium ion is secreted. Aldosterone signal transduction increases epithelial sodium ion channels (ENaC) in the apical membrane while increasing Na ⁺ /K ⁺ -ATPase in the basolateral membrane and these represent slow responses (taking place about 6–24 hours after aldosterone). The rapid and genomic responses to aldosterone ( Fig. 16.3 ) are the elevation of Sgk , the CHIF , and Kirsten ras and these actions occur in the first 6 hours after aldosterone. Very rapid ion transport responses to aldosterone that occur before gene regulation are possible and could be due to a direct effect on a small fraction of “aldosterone receptors” located in the cell membrane. Fig. 16.10 explains recent information on the genomic and nongenomic effects of aldosterone indicating that the membrane aldosterone receptor may be due to the action of a protein (possibly G protein–coupled receptor 30) that is different from the classical aldosterone Mr that operates genomically.

Rapid responses after 6 hours elevate the number of transport proteins allowing the reabsorption of sodium ion from the tubular urine. The uptake of sodium ions is followed by the passive reabsorption of water to maintain a constant concentration of sodium causing the extracellular volume to expand and increasing blood pressure.

If the plasma volume is decreased ( hypovolemia ), the protease, renin , is secreted from the granular cells of the juxtaglomerular apparatus . Renin degrades liver-expressed angiotensinogen to yield angiotensin I and pulmonary ACE cleaves a dipeptide from the C-terminus of decameric angiotensin I to produce octomeric Ang II ( Fig. 16.9 ). The action of Ang II on the zona glomerulosa cell of the adrenal cortex generates the synthesis and release of aldosterone , the effects of which also increase blood volume and pressure. One of the effects of aldosterone is to cause K ⁺ to move into the extracellular space ( Fig. 16.3 ) and ultimately into the urine. Potassium ion depolarizes the zona glomerulosa cell and because its entry into the cell stimulates the synthesis of aldosterone, the removal of K ⁺ into the urine acts as a negative feedback [there is also a negative effect of the atrionatriuretic hormone (ANH or ANF) on the synthesis of aldosterone].

Structures of Steroid Hormone Receptors

The structures of the glucocorticoid (cortisol) receptor and the mineralocorticoid (aldosterone) receptor are similar; their N-terminal domains, however, are quite different. Fig. 16.11 shows a cartoon depicting the domains of several of the hormone receptors in the steroid hormone receptor gene family .

There are many other receptors assigned to the steroid hormone receptor gene superfamily . In addition to those listed in Fig. 16.11 , the following are also members: thyroid hormone receptors (in addition to TRβ): TRα1 and TRα2; c-erbA1 and Rev-Erb; RARα and RARγ in addition to RARβ listed in the figure; RXRα, RXRβ, and RXRγ (retinoic acid receptors activated by 9- cis -retinoic acid), whereas RARs are activated by all trans-retinoic acid and 9-cis-retinoic acid ; and PPARα, PPARβ, and PPARγ (peroxisome proliferator-activated receptor) that bind fatty acids , including ω6 atherogenic lipids, and fatty acid derivatives; there are numerous synthetic compounds (e.g., thiazolidinediones) that bind with high affinity but all of the physiological ligands are not known.

There are a whole series of receptors that fall into this class that are labeled as orphan receptors ; some of these now have ligands that have been identified (the benzoate X receptor; the pregnane receptor; the liver X receptor; CARβ, a constitutive androstane receptor; FXR, a farnesoid receptor and hSXR, a steroid and xenobiotic receptor). Additionally, there are a number of orphan receptors, the human physiological ligands of which are unknown (original definition of an “orphan” receptor). Among these are the chicken ovalbumin upstream promoter (COUP) receptors (COUP-TFOI/EAR and COUP-TFII/ARP-1), DAX-1, SHP (Src homology 2 domain), nur77/NGF1-B, ERR1 and ERR2, HNF-3 and HNF-4, and SF-1.

The ligand-binding domain ( LBD ) of the GR is shown in Fig. 16.12 . This receptor will be used as a model, although the LBDs of other receptors in this family also have been reported.

The binding of the ligand induces conformational changes in the receptor causing the associated nonreceptor proteins to dissociate ( Fig. 16.5 ). The relative affinities of the receptor for various steroid ligands are presented in Table 16.1 . Remembering that the LBDs of some of the receptors in this class are homologous to a certain extent, some of the ligands listed are bound, not only to the GR, but also to the Mr and to other receptors.

Table 16.1

Compounds Related to Cortisol Which Have Glucocorticoid Activity (Antiinflammatory) and/or Mineralocorticoid Activity (Salt-Retaining).

	Mineralocorticoid	Antiinflammatory
	1	1
	800	1
	800	5–40
	40	0
	0.6	4
	0	5
	0	5–100
	0	10–35
	0	>100

Structures of the ligands for many of the receptors in this class are shown in Fig. 16.13 .

As Table 16.1 shows, there are crossovers in ligand binding between the GR (antiinflammatory) and the Mr (salt-retaining). Both receptors bind cortisol equally well . They both bind prednisolone to differing degrees. While there is crossover in certain binding of ligands, the overall physiological actions of these receptors appear distinct, although, in some cases, the activated receptors may bind to the same gene promoters. Presumably, in addition to common promoters, there exist specific promoters for Mr and for GR. When there is a gene knockout of either of these receptors, different physiological consequences are encountered but death occurred in both cases. When the GR is knocked out in mice, most of the experimental animals die shortly after birth, primarily due to pathologies in the lungs but also from pathologies in the liver, adrenal glands, brain, thymus, and bone marrow (all of these tissues normally express the GR), and there is interference in the feedback regulation of the hypothalamic–pituitary axis because the negative feedback of cortisol requires the activation of GRs in the responding tissues. In the case where a similar knockout of the Mr , by targeted gene disruption, was performed, the newborns die of dehydration as a major effect of renal sodium and water loss with increased potassium ion in plasma, decreased sodium ion concentration, and elevated levels of components of the angiotensin system, namely, renin , Ang II and aldosterone , and elevated expression of renin, angiotensinogen , and Ang II receptor (ACE, angiotensin I-converting enzyme, in the kidney was not affected). It should be remembered that although there exist similarities in the LBDs and in the DNA-binding domains (DBDs), the N-termini of these receptors are widely different and must contribute to the differing actions of the two receptors, perhaps by attracting different activation factors to the Tau1 domain in the N-terminus. The hyperphosphorylation s of GR occur in the N-terminus and regulate the negative charge in this region.

The DBDs of receptors in the steroid receptor gene family have some homology as shown in Fig. 16.10 . In the case of the GR [and the Mr and androgen receptor (AR)], the DBD consists of two zinc fingers. The left zinc finger is closer to the N-terminus and the right zinc finger is downstream from the left zinc finger ( Fig. 16.14 ).

The zinc fingers of the homodimer of GR and other receptors associate with DNA in the major groove as shown in Fig. 16.15 .

Steroid receptors bind to response elements in the promoters of the genes they affect. In the case of the GR, the response element is a sequence of two half-sites: 5′ AGAACAnnnTGTTCT 3′ (or on the complementary strand: 3′ TCTTGTnnnACAAGA 5′) where n is any nucleotide. This is the general response element for Mr, AR, and progesterone receptor (PR) as well as for GR. There are differences in the DNA base contact points when the receptors other than the GR bind to this palindromic sequence (same sequence whether read 5′–3′ on one strand or 5′–3′ on the complementary strand). The GRE is located on a large number of different gene promoters. In many cases the response element is positive and promotes the synthesis of an mRNA that leads to the synthesis of a new protein or, conversely, the response element could be negative and generate repression of specific gene activation. Examples of positive GREs are those located in genes for tyrosine aminotransferase and enzymes of gluconeogenesis. Examples of negative GREs are those that reside in genes for gonadotropin-releasing hormone (GnRH), prolactin, or proopiomelanocortin.

The major receptors of the steroid hormone receptor gene family and their hormone response elements are shown in Fig. 16.16 .

There are four classes of nuclear receptors . Class I (also referred to as type 1) receptors are those located in the cytosol and when the ligand binds to the unliganded receptor complex, ancillary proteins (e.g., heat shock proteins, HSP90) dissociate, homodimerization takes place followed by nuclear translocation and binding to hormone-responsive elements (see Fig. 16.16 ). GR, AR, estrogen receptor (ER), and PR are representatives of class I. Class II receptors are retained in the nucleus with or without ligand and bind to DNA as heterodimers, often with RXR, and are in complex with corepressor proteins in the absence of the ligand. When the ligand binds, the corepressor proteins dissociate and coactivator proteins are recruited. Then, an array of other proteins forms a complex along with RNA polymerase and transcription of DNA follows to form an mRNA. Class III receptors resemble class I because they bind DNA as homodimers that bind to direct repeats instead of inverted repeats. Class IV receptors bind to DNA in the form of dimers or monomers; however, there is only one DBD that binds to a single half-site hormone response element. These distinctions are further clarified in Fig. 16.17 .

The sequences flanking the palindromic (a palindrome is a nucleic acid sequence that is the same whether read 5′–3′ on one strand or 5′–3′ on the complementary strand of a double helix) responsive element (e.g., for the GR or ER) can play a role in the tightness of binding between the responsive element and the receptor.

Coactivators and Corepressors

Coactivator proteins interact directly with certain steroid hormone receptors and stimulate transcription. There are at least 15 coactivators, so far. A summary of several of the coactivator proteins and the receptors to which they bind is shown in Table 16.2 .

Coactivators bind to nuclear steroid receptors after the ligand binds to the receptor and a corepressor protein is dissociated from the receptor complex as shown in Fig. 16.18 .

The major corepressors are NCoR and SMRT as shown in Table 16.3 where it describes with which specific receptors each corepressor interacts as well as additional information.

These corepressors have a histone deacetylase (HDAC)-binding site and function by recruiting HDACs with other proteins to the receptor complexes at the promoter of gene target sequences. Unlike coactivators, corepressors themselves do not appear to have enzymatic activity but they are essential for the full activity of HDAC. When the nuclear receptor, like TR (thyroid receptor) or RAR (retinoic acid receptor) complexed with DNA, binds its ligand, the attached corepressor, NCoR, is dissociated allowing the attachment of coactivator complexes with histone acetyltransferase (HAT) activity to open DNA and enhance transcription.

Physiological Functions of Steroid Hormones From Specific Receptor Knockouts

Members of the steroid and nuclear receptor family are present in various concentrations in many or most tissues of the body. In the case of the GR , it has been shown to reside in the cytosols of all tissues, in various concentrations, except that it is absent from hepatobiliary cells and cells located in the region of the pars intermedia (in the human, there is no specific organ as in lower forms, only scattered cells with the described activities) of the anterior pituitary. Experimentally, it has been possible to “knock out” the genes for certain steroid receptors to create a homozygous knockout mouse . Following the knockout, the physiological effects of the loss in specific hormonal functions on the experimental mouse and its tissues are indicative of the actual roles of the steroids in the body. Knockout of the ERα did not result in death of the newborn but did result in infertility in both male and female animals. The uteri, ovaries, and mammaries of F-1 females were abnormal. Plasma levels of estradiol and LH (luteinizing hormone) were substantially higher than in wild-type animals although FSH (follicle-stimulating hormone) was relatively unaffected. There appeared to be heightened aggression toward other females. In males, lacking the ER, there was abnormal reproductive tract histology, infertile sperm, and smaller testicles than in the wild types. These males have reduced aggressive behavior over the normal wild types. Loss of the ER (and estrogen action), although not lethal, affects both female and male offspring in sexually related physiology and behavior.

There are several reproductive abnormalities in mice lacking the PR . In females the mammary gland does not develop normally and there is hyperplasia of the uterus . This knockout also was not lethal and sexual differentiation was normal, although females were infertile, whereas male fertility was unaffected. Circulating levels of LH were elevated about twofold (lack of negative feedback) but FSH levels were unchanged compared to normals. Thus the lack of progesterone function underscores the role of PRs in the regulation of hypothalamic and pituitary functions in the secretion of gonadotropins.

When the GR is knocked out, the offspring die shortly after birth by damage to the lung where the GR is essential for the development of surfactant . If neonatal animals were to survive in early development (which is not the case), the lack of ability to adapt to stress and environmental change would be lethal. In addition, there are effects on many other tissues, the functions of which rely on glucocorticoids, such as liver, brain, adrenals, thymus, bone marrow, and especially in the negative feedback regulation (via the GR) of the hypothalamic–pituitary axis. Thus cortisol and its receptor are essential for survival.

When the Mr is knocked out, offspring (mice) die within 2 weeks after birth. The animals die from loss of kidney reabsorption of water and sodium ions and they exhibit dehydration, hyperkalemia, loss of circulating sodium, and elevated levels of Ang II, aldosterone, and renin. There were elevations in expression of renin, angiotensinogen, and Ang II receptor (ST1) but the kidney level of angiotensin I-converting enzyme (ACE) was unchanged over normal controls.

AR deficiency is characteristic of the Tfm mouse (X-linked inherited disease) and these mice display the androgen insensitivity syndrome causing male mice to be infertile.