GR and MR are intracellular receptors responsible for binding and mediating
the “ classic ” effects of cortisol and aldosterone, respectively. They belong to
subfamily 3C of a large and diverse family of transcription factors known as
the nuclear receptor family. Other members of subfamily 3C include the pro-
gesterone receptor ( PR ) and the androgen receptor ( AR ). GR and MR share
a high degree of structural homology, refl ecting the structural similarities
between their corticosteroid ligands. The structural homology is highest at the
DNA - binding domain s ( DBD s) (94%) and 56% between the ligand - binding
domain s ( LBD s) [38] . This high degree of homology suggests that the two receptors
are closely associated in evolutionary terms and are most likely descended from a
common ancestral receptor. A polar surface within the ligand - binding pocket of
MR, lacking in GR and other receptors of the family, permits preferential binding
of aldosterone. Nevertheless, the cloning and expression of MR [39] revealed
considerable ligand promiscuity with receptor specifi city being governed by ligand
access.
Unactivated, GR and MR are sequestered in the cytoplasm by complexing with
heat - shock protein ( HSP ). The HSP acts to stop the receptors entering the nucleus
in the absence of an appropriate activation signal. Cortisol and aldosterone circu-
late in plasma bound to plasma proteins, and can easily diffuse through the cell
membrane into the cytoplasm. Here they will act to displace the HSP from their
receptor to allow the formation of a hormone – receptor complex. This changes the
conformation of the receptor, allowing it to form a homodimer, which can now
readily enter the nucleus where it will recognize specifi c hormone response ele-
ment s ( HRE s) associated with target genes, acting as a ligand - dependent transcrip-
tion factor (Figure 1.4 ). Interestingly, a recent study has shown that GR and MR
can form heterodimers [40] , which can translocate to the nucleus: the downstream
effects on DNA transcription of this complex are unknown. HREs are typically
located within a gene enhancer, which can be several kilobases away from the gene
promoter. GR and MR, along with PR and AR, recognize response elements whose
HRE sequence consists of two hexameric half - sites (TGTTCT). Glucocorticoids
can also affect transcription independent of direct DNA binding by interacting
with protein transcription factors [41] . This allows the transcription of genes that
mediate GR - and MR - induced responses to be tightly regulated by appropriate
ligand binding and hormone – receptor complex conformation.
Control of Ligand Access
The two 11 β - hydroxysteroid dehydrogenase ( 11 β HSD ) enzymes, types 1 and 2, are
key determinants of ligand access to GR and MR, respectively. The enzymes
interconvert cortisol (active) and cortisone (inactive), thereby controlling the local
concentrations of glucocorticoids (Figure 1.5 ).
11 β HSD1 is the product of HSD11B1 found on chromosome 1 in both mice
and humans. The enzyme has a wide distribution but its major areas of action, in
terms of both transcript expression and activity, are the liver, adipose and brain
[42] . HSD11B2, found on chromosome 16 in humans and on chromosome 8 in
mice, encodes the second isozyme. 11 β HSD2 has a more limited distribution than
11 β HSD1, being expressed predominantly in aldosterone target tissues such as
the distal nephron and colon [43] . It is also found in the placenta [44, 45] and in
the vascular endothelium [46] . 11 β HSD1 was cloned from the liver [47] and
11BHSD2 from the kidney [48] . Although both enzymes belong to the same super-
family of short - chain alcohol dehydrogenase reductases [49] , sequence comparison
reveals little identity with the exception of the regions encompassing cofactor
binding (NAD or NADP[H]) and the active site [50] . In cell culture systems, both
enzymes are single - chain polypeptides localized to the membrane of the endoplas-
mic reticulum, with opposing orientations of their catalytic sites [51, 52] . In vivo ,
however, homodimerization may provide additional regulation of enzyme activity.
Dimerization supports full activity of 11 β HSD1 [53] , but inactivates 11 β HSD2
[54] .
Although there appears to be no physical association of 11 β HSD1 with GR, the
enzyme governs in vivo the extent to which the receptor is activated by glucocorti-
coids. This occurs by converting inactive cortisone to active cortisol (see Figure
1.5 ) thereby maintaining glucocorticoid signaling at a local level [50] . Due to its
widespread distribution and the lack of specifi c inhibitors, functional dissection
of the role of the enzyme in specifi c tissues is diffi cult. However, signifi cant
advances in our understanding have come from the generation of genetically
modifi ed mice. For example, the 11 β HSD1 null mouse has elucidated major func-
tions for the enzyme in the response to stress and in regulation of the HPA [50] .
For the latter, regeneration of glucocorticoids by the liver appears to be particularly
important [55] . In addition, 11 β HSD1 null mice are resistant to age - related
cognitive impairment [56] , indicating roles in the brain. This literature has recently
been reviewed [50] and the role of 11 β HSD1 in the regulation of metabolism
and cardiovascular function is discussed later in the chapter. These studies have
important implications for human disease, suggesting that 11 β HSD1 is an excit-
ing therapeutic target. This area will no doubt be advanced by the recent crystalli-
zation of the enzyme [57] .
11 β HSD1 null mice have improved glucose tolerance, a favorable lipoprotein
profi le, and increased sensitivity of the liver and fat to insulin [175] . Moreover, on
the obese - prone C57BL/6J background, mice carrying the null mutation were
resistant to the weight gain induced by high - fat feeding [176] . That loss of 11 β HSD1
confers a cardioprotective metabolic profi le is intriguing and most probably results
from a lack of glucocorticoid regeneration in adipocytes. However, these mice also
had an increase calorifi c intake, suggesting that energy expenditure was also
stimulated (Morton et al. , 2004 ).
The generation of a mouse that overexpresses 11 β HSD1 under the control of
an adipocyte - specifi c promotor [58] further highlights the role of the enzyme in
metabolic function. The amplifi cation of glucocorticoids was relatively modest and
confi ned to the adipocyte, circulating corticosterone being normal. Nevertheless,
these transgenic mice developed central obesity, insulin resistance and glucose
intolerance. In addition, these animals were hypertensive due to a chronic activa-
tion of the RAS [59] . This clustering of metabolic and cardiovascular phenotypes
is characteristic of the metabolic syndrome, which is discussed later in this chapter.
The level of enzyme function in the adipocyte appears therefore, to play a critical
role in setting of metabolic profi le.
In vitro , MR can be activated with equal potency both by aldosterone and cortisol
[39] . In vivo , ligand access to MR is determined by colocalization with 11 β HSD2
(Figure 1.6 ). By catalyzing the rapid conversion of cortisol into cortisone (Figure
1.5 ), which does not activate MR, 11 β HSD2 confers upon MR the specifi city to
aldosterone that it inherently lacks [60, 61] . MR and 11 β HSD2 have overlapping
distributions in those tissues classically held to be aldosterone selective [43] . In
addition to protecting MR from activation by glucocorticoids, there is evidence of
a direct association of the proteins [62] and 11 β HSD2 may directly regulate MR
activation by aldosterone.
Inactivating mutations in the gene HSD11B2 cause the Syndrome of Apparent
Mineralocorticoid Excess ( SAME ; OMIM #218030). In this setting, cortisol acti-
vates MR [63 – 67] , resulting in severe hypertension thought to arise from volume
expansion secondary to renal sodium retention [63, 64, 66] . Dexamethasone can
be therapeutically effective [67] as it will suppress endogenous glucocorticoids, but
does not activate MR. In addition, dexamethasone may act as a chaperone and
stabilize mutant enzyme [68] . Nevertheless, neither synthetic glucocorticoid nor
MR blockade has a consistent antihypertensive effect [69] .
SAME has been modeled by targeted disruption of the 11 β HSD2 locus, produc-
ing a mouse in which the cardinal features of the disorder were preserved [70] .
Although animals were born in normal Mendelian ratios, there was high neonatal
mortality in the homozygote null animals, and the remainder were hypertensive
and severely hypokalemic. The RAS was suppressed and plasma aldosterone
was also low [71, 70] . In one patient with SAME, the disorder was fully corrected
by kidney transplant [72] , indicating the disease is of renal origin. In support of
this, 11 β HSD2 null mice have excess renal sodium reabsorption due to activation
of the epithelial sodium channel [71] . Nevertheless, sodium retention was found
to be transient, and the hypertension moves from a renal to a central and ulti-
mately vascular disorder through activation of the sympathetic nervous system
[71] . It is possible that the sympathetic nervous system is activated by the hyper-
natremia that is sustained beyond the period of sodium retention. However,
11 β HSD2 is also expressed in the nucleus of the solitary tract and amygdala of
the mouse brain [73, 74] , regions important to the central of blood pressure. Thus,
SAME may refl ect overactivation of MR in regions other than the kidney. This is
supported by experiments showing that central administration of either aldoste-
rone [75] or 11 β HSD2 inhibitors [76] have a sustained hypertensive effect. In
addition, inhibition (pharmacological or antisense) of 11 β HSD2 sensitizes the
vasculature to both Ang II [77] and catecholamines [78] . Vascular reactivity to
noradrenalin is enhanced in a patient with SAME [79] . The 11 β HSD2 null mice
have endothelial dysfunction, with enhanced sensitivity to vasoactive agents being
underpinned by a reduction in nitric oxide ( NO ) production [46, 80] . However, the
extent of the endothelial dysfunction following targeted disruption of 11 β HSD2 is
dependent on the underlying background strain of the mouse [71] and may not,
therefore, contribute in a major way to altered blood pressure homeostasis.
the “ classic ” effects of cortisol and aldosterone, respectively. They belong to
subfamily 3C of a large and diverse family of transcription factors known as
the nuclear receptor family. Other members of subfamily 3C include the pro-
gesterone receptor ( PR ) and the androgen receptor ( AR ). GR and MR share
a high degree of structural homology, refl ecting the structural similarities
between their corticosteroid ligands. The structural homology is highest at the
DNA - binding domain s ( DBD s) (94%) and 56% between the ligand - binding
domain s ( LBD s) [38] . This high degree of homology suggests that the two receptors
are closely associated in evolutionary terms and are most likely descended from a
common ancestral receptor. A polar surface within the ligand - binding pocket of
MR, lacking in GR and other receptors of the family, permits preferential binding
of aldosterone. Nevertheless, the cloning and expression of MR [39] revealed
considerable ligand promiscuity with receptor specifi city being governed by ligand
access.
Unactivated, GR and MR are sequestered in the cytoplasm by complexing with
heat - shock protein ( HSP ). The HSP acts to stop the receptors entering the nucleus
in the absence of an appropriate activation signal. Cortisol and aldosterone circu-
late in plasma bound to plasma proteins, and can easily diffuse through the cell
membrane into the cytoplasm. Here they will act to displace the HSP from their
receptor to allow the formation of a hormone – receptor complex. This changes the
conformation of the receptor, allowing it to form a homodimer, which can now
readily enter the nucleus where it will recognize specifi c hormone response ele-
ment s ( HRE s) associated with target genes, acting as a ligand - dependent transcrip-
tion factor (Figure 1.4 ). Interestingly, a recent study has shown that GR and MR
can form heterodimers [40] , which can translocate to the nucleus: the downstream
effects on DNA transcription of this complex are unknown. HREs are typically
located within a gene enhancer, which can be several kilobases away from the gene
promoter. GR and MR, along with PR and AR, recognize response elements whose
HRE sequence consists of two hexameric half - sites (TGTTCT). Glucocorticoids can also affect transcription independent of direct DNA binding by interacting
with protein transcription factors [41] . This allows the transcription of genes that
mediate GR - and MR - induced responses to be tightly regulated by appropriate
ligand binding and hormone – receptor complex conformation.
Control of Ligand Access
The two 11 β - hydroxysteroid dehydrogenase ( 11 β HSD ) enzymes, types 1 and 2, are
key determinants of ligand access to GR and MR, respectively. The enzymes
interconvert cortisol (active) and cortisone (inactive), thereby controlling the local
concentrations of glucocorticoids (Figure 1.5 ).
11 β HSD1 is the product of HSD11B1 found on chromosome 1 in both mice
and humans. The enzyme has a wide distribution but its major areas of action, in
terms of both transcript expression and activity, are the liver, adipose and brain
[42] . HSD11B2, found on chromosome 16 in humans and on chromosome 8 in
mice, encodes the second isozyme. 11 β HSD2 has a more limited distribution than
11 β HSD1, being expressed predominantly in aldosterone target tissues such as
the distal nephron and colon [43] . It is also found in the placenta [44, 45] and in
the vascular endothelium [46] . 11 β HSD1 was cloned from the liver [47] and
11BHSD2 from the kidney [48] . Although both enzymes belong to the same super-
family of short - chain alcohol dehydrogenase reductases [49] , sequence comparison
reveals little identity with the exception of the regions encompassing cofactor
binding (NAD or NADP[H]) and the active site [50] . In cell culture systems, both
enzymes are single - chain polypeptides localized to the membrane of the endoplas-
mic reticulum, with opposing orientations of their catalytic sites [51, 52] . In vivo ,
however, homodimerization may provide additional regulation of enzyme activity.
Dimerization supports full activity of 11 β HSD1 [53] , but inactivates 11 β HSD2
[54] .
Although there appears to be no physical association of 11 β HSD1 with GR, the
enzyme governs in vivo the extent to which the receptor is activated by glucocorti-
coids. This occurs by converting inactive cortisone to active cortisol (see Figure
1.5 ) thereby maintaining glucocorticoid signaling at a local level [50] . Due to its
widespread distribution and the lack of specifi c inhibitors, functional dissection
of the role of the enzyme in specifi c tissues is diffi cult. However, signifi cant
advances in our understanding have come from the generation of genetically
modifi ed mice. For example, the 11 β HSD1 null mouse has elucidated major func-
tions for the enzyme in the response to stress and in regulation of the HPA [50] .
For the latter, regeneration of glucocorticoids by the liver appears to be particularly
important [55] . In addition, 11 β HSD1 null mice are resistant to age - related
cognitive impairment [56] , indicating roles in the brain. This literature has recently
been reviewed [50] and the role of 11 β HSD1 in the regulation of metabolism
and cardiovascular function is discussed later in the chapter. These studies have
important implications for human disease, suggesting that 11 β HSD1 is an excit-
ing therapeutic target. This area will no doubt be advanced by the recent crystalli-
zation of the enzyme [57] .
11 β HSD1 null mice have improved glucose tolerance, a favorable lipoprotein
profi le, and increased sensitivity of the liver and fat to insulin [175] . Moreover, on
the obese - prone C57BL/6J background, mice carrying the null mutation were
resistant to the weight gain induced by high - fat feeding [176] . That loss of 11 β HSD1
confers a cardioprotective metabolic profi le is intriguing and most probably results
from a lack of glucocorticoid regeneration in adipocytes. However, these mice also
had an increase calorifi c intake, suggesting that energy expenditure was also
stimulated (Morton et al. , 2004 ).
The generation of a mouse that overexpresses 11 β HSD1 under the control of
an adipocyte - specifi c promotor [58] further highlights the role of the enzyme in
metabolic function. The amplifi cation of glucocorticoids was relatively modest and
confi ned to the adipocyte, circulating corticosterone being normal. Nevertheless,
these transgenic mice developed central obesity, insulin resistance and glucose
intolerance. In addition, these animals were hypertensive due to a chronic activa-
tion of the RAS [59] . This clustering of metabolic and cardiovascular phenotypes
is characteristic of the metabolic syndrome, which is discussed later in this chapter.
The level of enzyme function in the adipocyte appears therefore, to play a critical
role in setting of metabolic profi le.
In vitro , MR can be activated with equal potency both by aldosterone and cortisol
[39] . In vivo , ligand access to MR is determined by colocalization with 11 β HSD2
(Figure 1.6 ). By catalyzing the rapid conversion of cortisol into cortisone (Figure
1.5 ), which does not activate MR, 11 β HSD2 confers upon MR the specifi city to
aldosterone that it inherently lacks [60, 61] . MR and 11 β HSD2 have overlapping
distributions in those tissues classically held to be aldosterone selective [43] . In
addition to protecting MR from activation by glucocorticoids, there is evidence of
a direct association of the proteins [62] and 11 β HSD2 may directly regulate MR
activation by aldosterone.
Inactivating mutations in the gene HSD11B2 cause the Syndrome of Apparent
Mineralocorticoid Excess ( SAME ; OMIM #218030). In this setting, cortisol acti-
vates MR [63 – 67] , resulting in severe hypertension thought to arise from volume
expansion secondary to renal sodium retention [63, 64, 66] . Dexamethasone can
be therapeutically effective [67] as it will suppress endogenous glucocorticoids, but
does not activate MR. In addition, dexamethasone may act as a chaperone and
stabilize mutant enzyme [68] . Nevertheless, neither synthetic glucocorticoid nor
MR blockade has a consistent antihypertensive effect [69] .
SAME has been modeled by targeted disruption of the 11 β HSD2 locus, produc-
ing a mouse in which the cardinal features of the disorder were preserved [70] .
Although animals were born in normal Mendelian ratios, there was high neonatal mortality in the homozygote null animals, and the remainder were hypertensive
and severely hypokalemic. The RAS was suppressed and plasma aldosterone
was also low [71, 70] . In one patient with SAME, the disorder was fully corrected
by kidney transplant [72] , indicating the disease is of renal origin. In support of
this, 11 β HSD2 null mice have excess renal sodium reabsorption due to activation
of the epithelial sodium channel [71] . Nevertheless, sodium retention was found
to be transient, and the hypertension moves from a renal to a central and ulti-
mately vascular disorder through activation of the sympathetic nervous system
[71] . It is possible that the sympathetic nervous system is activated by the hyper-
natremia that is sustained beyond the period of sodium retention. However,
11 β HSD2 is also expressed in the nucleus of the solitary tract and amygdala of
the mouse brain [73, 74] , regions important to the central of blood pressure. Thus,
SAME may refl ect overactivation of MR in regions other than the kidney. This is
supported by experiments showing that central administration of either aldoste-
rone [75] or 11 β HSD2 inhibitors [76] have a sustained hypertensive effect. In
addition, inhibition (pharmacological or antisense) of 11 β HSD2 sensitizes the
vasculature to both Ang II [77] and catecholamines [78] . Vascular reactivity to
noradrenalin is enhanced in a patient with SAME [79] . The 11 β HSD2 null mice
have endothelial dysfunction, with enhanced sensitivity to vasoactive agents being
underpinned by a reduction in nitric oxide ( NO ) production [46, 80] . However, the
extent of the endothelial dysfunction following targeted disruption of 11 β HSD2 is
dependent on the underlying background strain of the mouse [71] and may not,
therefore, contribute in a major way to altered blood pressure homeostasis.
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