*3.5.1.1 ACTH*

Adrenocorticotrophin (ACTH) is a 39 amino acid peptide released from the anterior pituitary in pulsatile and diurnal rhythm with the highest levels in the morning and the lowest at night [23]*.* ACTH exerts its effects by binding to its receptor (ACTH-R), a G-protein-coupled receptor. ACTH acutely stimulates aldosterone secretion but in the long term has an inhibitory effect on *CYP11B2* gene expression and aldosterone levels [24].

Acute stimulation of aldosterone production has suggested to be mediated via activation of StAR protein production. Also, ACTH after binding to its receptor can activate adenylate cyclase, resulting in an increased intracellular cAMP concentration, activation of protein kinase A (PKA), and calcium influx via calcium channels (**Figure 5**) [25]*.*

On the other hand, chronic ACTH stimulation may depress serum aldosterone level as cyclic AMP, the second messenger for ACTH, desensitizes adrenocortical cells to angiotensin II by causing a reduction in the expression of angiotensin II receptors. ACTH may also decrease aldosterone production by stimulating the expression of *CYP11B1* and *CYP17*, thereby resulting in a removal of precursors from the aldosterone pathway and using them to

#### **Figure 5.**

*Intracellular mechanisms of angiotensin II, K+ and ACTH influencing gene expression. Abbreviations: ER, endoplasmic reticulum; AT1-R, angiotensin 1 receptor; PLC, phospholipase C; PKA, protein kinase A; IP3R, inositol triphosphate receptor; IP3, inositol triphosphate; CaMK, calmodulin kinase; SF-1, steroidogenic factor 1; CREB, cAMP regulatory element response element; ACTH, adrenocorticotrophic hormone.*

**23**

*Aldosterone Synthase Gene (*CYP11B2*) Polymorphisms and Enhanced Cardiovascular Risk*

synthesize cortisol. ACTH appears to specifically induce the proliferation of zona fasciculata cells while recruiting and transforming glomerulosa cells into

Under normal circulating ACTH levels, the glomerulosa maintains *CYP11B2* expression by at least two mechanisms. First, angiotensin II inhibits ACTHstimulated cAMP production in glomerulosa but not fasciculata cells. Second, the glomerulosa expresses a type of adenyl cyclase that is inhibited by increasing

Angiotensin II is thought to stimulate aldosterone synthesis as result of sodium depletion and extracellular fluid volume reduction through various intracellular signaling pathways. However, the best characterized pathway is the activation of phospholipase C (**Figure 5**). It is mediated by acting on angiotensin 1 (AT1) receptor, a specific G-protein-coupled receptor that activates phospholipase C. Once activated, phospholipase C hydrolyses phosphatidyl inositol 4,5-biphosphate (PIP2) to 1,4,5 inositol triphosphate (IP3) and 1,2-diacylglycerol (DAG) resulting in release of Ca2+ from intracellular stores and activation of protein kinase C (PKC), respectively. The increased intracellular Ca2+ concentration activates calmodulin and Ca2+/ calmodulin-dependent protein kinases (CaM kinases) [28, 29] to phosphorylate and activate transcription factors as activating transcription factor 1 (ATF-1), cAMPresponse-element binding protein (CREB), nerve growth factor IB (NGFIB), and nuclear receptor related 1 protein (*NURR1*) that combined to specific cis-acting series in the 5′ region of *CYP11B2* [30]. The acute stimulation mediated by angiotensin II can stimulate rapid aldosterone synthesis either de novo or from intermediate compounds in the steroidogenic pathway. However, chronic stimulation may lead to ZG hypertrophy and hyperplasia with increased *CYP11B2* expression and

The level of potassium affects renin secretion as well as having a direct effect on the adrenal cortex to increase aldosterone secretion. Increased

an increase in intracellular Ca2+ and activation of calmodulin kinases with consequent phosphorylation of transcription factors leading to stimulation of *CYP11B2* gene transcription. Potassium signaling is mediated through membrane depolarization, leading to an influx of calcium through T and L-type

As mentioned previously, chronic regulation of steroidogenesis involves transcription of the genes encoding the necessary steroidogenic enzymes. This is mediated by alteration of trans-acting factors that bind to the cis-regulatory elements within the 5′ regulatory regions of the target genes. Investigation of the 5′ regulatory regions of the *CYP11B* gene revealed six cis-acting elements (Ad1–6) (**Figure 6**). The most important cis-elements in the *hCYP11B2* promoter are CRE at −71/−64 (Ad1), Ad5 at −129/−114, Ad4 at −344/−336, and a cis-element termed

NGFIB response element 1 (NBRE-1) −766/−759 (**Figure 7**) [33].

(like angiotensin II) stimulates aldosterone secretion through

[27]*.*

intracellular Ca2+, the second messenger for both angiotensin II and K+

*DOI: http://dx.doi.org/10.5772/intechopen.89133*

subsequent aldosterone secretion [31].

*3.5.1.3 Potassium*

extracellular K<sup>+</sup>

channels (**Figure 5**) [32].

*3.5.2 Transcriptional regulation of CYP11B2 gene*

fasciculata-like cells [26].

*3.5.1.2 Angiotensin II*

*Aldosterone Synthase Gene (*CYP11B2*) Polymorphisms and Enhanced Cardiovascular Risk DOI: http://dx.doi.org/10.5772/intechopen.89133*

synthesize cortisol. ACTH appears to specifically induce the proliferation of zona fasciculata cells while recruiting and transforming glomerulosa cells into fasciculata-like cells [26].

Under normal circulating ACTH levels, the glomerulosa maintains *CYP11B2* expression by at least two mechanisms. First, angiotensin II inhibits ACTHstimulated cAMP production in glomerulosa but not fasciculata cells. Second, the glomerulosa expresses a type of adenyl cyclase that is inhibited by increasing intracellular Ca2+, the second messenger for both angiotensin II and K+ [27]*.*

## *3.5.1.2 Angiotensin II*

*The Recent Topics in Genetic Polymorphisms*

*3.5.1.1 ACTH*

(**Figure 5**) [25]*.*

encoding the steroidogenic enzymes [22].

expression and aldosterone levels [24].

*3.5.1 Signaling pathways that regulate aldosterone production*

The chronic response takes several hours and involves the transcription of the genes

Adrenocorticotrophin (ACTH) is a 39 amino acid peptide released from the anterior pituitary in pulsatile and diurnal rhythm with the highest levels in the morning and the lowest at night [23]*.* ACTH exerts its effects by binding to its receptor (ACTH-R), a G-protein-coupled receptor. ACTH acutely stimulates aldosterone secretion but in the long term has an inhibitory effect on *CYP11B2* gene

Acute stimulation of aldosterone production has suggested to be mediated via activation of StAR protein production. Also, ACTH after binding to its receptor can activate adenylate cyclase, resulting in an increased intracellular cAMP concentration, activation of protein kinase A (PKA), and calcium influx via calcium channels

On the other hand, chronic ACTH stimulation may depress serum aldosterone level as cyclic AMP, the second messenger for ACTH, desensitizes adrenocortical cells to angiotensin II by causing a reduction in the expression of angiotensin II receptors. ACTH may also decrease aldosterone production by stimulating the expression of *CYP11B1* and *CYP17*, thereby resulting in a removal of precursors from the aldosterone pathway and using them to

 *and ACTH influencing gene expression. Abbreviations: ER,* 

*endoplasmic reticulum; AT1-R, angiotensin 1 receptor; PLC, phospholipase C; PKA, protein kinase A; IP3R, inositol triphosphate receptor; IP3, inositol triphosphate; CaMK, calmodulin kinase; SF-1, steroidogenic factor* 

*1; CREB, cAMP regulatory element response element; ACTH, adrenocorticotrophic hormone.*

**22**

**Figure 5.**

*Intracellular mechanisms of angiotensin II, K+*

Angiotensin II is thought to stimulate aldosterone synthesis as result of sodium depletion and extracellular fluid volume reduction through various intracellular signaling pathways. However, the best characterized pathway is the activation of phospholipase C (**Figure 5**). It is mediated by acting on angiotensin 1 (AT1) receptor, a specific G-protein-coupled receptor that activates phospholipase C. Once activated, phospholipase C hydrolyses phosphatidyl inositol 4,5-biphosphate (PIP2) to 1,4,5 inositol triphosphate (IP3) and 1,2-diacylglycerol (DAG) resulting in release of Ca2+ from intracellular stores and activation of protein kinase C (PKC), respectively. The increased intracellular Ca2+ concentration activates calmodulin and Ca2+/ calmodulin-dependent protein kinases (CaM kinases) [28, 29] to phosphorylate and activate transcription factors as activating transcription factor 1 (ATF-1), cAMPresponse-element binding protein (CREB), nerve growth factor IB (NGFIB), and nuclear receptor related 1 protein (*NURR1*) that combined to specific cis-acting series in the 5′ region of *CYP11B2* [30]. The acute stimulation mediated by angiotensin II can stimulate rapid aldosterone synthesis either de novo or from intermediate compounds in the steroidogenic pathway. However, chronic stimulation may lead to ZG hypertrophy and hyperplasia with increased *CYP11B2* expression and subsequent aldosterone secretion [31].

### *3.5.1.3 Potassium*

The level of potassium affects renin secretion as well as having a direct effect on the adrenal cortex to increase aldosterone secretion. Increased extracellular K<sup>+</sup> (like angiotensin II) stimulates aldosterone secretion through an increase in intracellular Ca2+ and activation of calmodulin kinases with consequent phosphorylation of transcription factors leading to stimulation of *CYP11B2* gene transcription. Potassium signaling is mediated through membrane depolarization, leading to an influx of calcium through T and L-type channels (**Figure 5**) [32].

### *3.5.2 Transcriptional regulation of CYP11B2 gene*

As mentioned previously, chronic regulation of steroidogenesis involves transcription of the genes encoding the necessary steroidogenic enzymes. This is mediated by alteration of trans-acting factors that bind to the cis-regulatory elements within the 5′ regulatory regions of the target genes. Investigation of the 5′ regulatory regions of the *CYP11B* gene revealed six cis-acting elements (Ad1–6) (**Figure 6**). The most important cis-elements in the *hCYP11B2* promoter are CRE at −71/−64 (Ad1), Ad5 at −129/−114, Ad4 at −344/−336, and a cis-element termed NGFIB response element 1 (NBRE-1) −766/−759 (**Figure 7**) [33].

#### **Figure 6.**

*Schematic diagram of the CYP11B2 promoter with the cis-elements (Ad1–Ad6).*

#### **Figure 7.**

*Schematic diagram of the most important transcription factor binding sites in the hCYP11B2 5'UTR. Abbreviations: CREB, cAMP regulatory element response element; SF-1, steroidogenic factor 1; COUP-TF, chicken ovalbumin upstream promoter transcription factor; NGFIB, nerve growth factor IB; NURRI, nuclear receptor-related 1 protein; NBRE-1, NGFI-B response element.*

#### *3.5.2.1 Ad1 (CRE)*

The Ad1 element closely resembles a consensus cAMP regulatory element (CRE) site. CREs plays an essential role in cAMP-dependent gene expression of a wide variety of genes. Proteins, such as the CRE-binding protein (CREB) and the highly related activating transcription factors (ATF), bind to CRE sites to initiate transcription. CREB binds to DNA as a dimer and has a conserved region of leucine residues (leucine zipper) at its C terminus that enables dimerization and sequence specific DNA binding [34].

CREB functions not only as a component of a variety of signaling pathways, particularly PKA, but also mitogen-activated protein kinases (MAPKs) and CaMKs. All these pathways mediate CREB-induced transcription by phosphorylating CREB at residue serine 133 [35]. The phosphorylated serine 133 binds another protein referred to as the CREB-binding protein (CBP). CBP is a 265-kDa nuclear protein, which binds to phosphorylated CREB and allows recruitment and stabilization of the RNA polymerase II transcription complex on the promoter of CREB target genes [36].

Using electrophoretic mobility shift assay, the *CYP11B2* CRE has been shown to bind members of the activating transcription factor (ATF-1 and ATF-2) and CREbinding protein (CREB) families. The ability of these transcription factors, particularly ATF-1 and CREB, to enhance transcription is partially regulated by their state of phosphorylation. Thus, one possibility is that activated CaMK I or CaMK IV phosphorylates CREB or ATF-1 leading to increased *CYP11B2* transcription [28].

#### *3.5.2.2 AD4 (SF-1)*

The Ad4 site (CCAAGGTC) is also found to be important in the regulation of the *CYP11B* gene. This Ad4 site or homologous sequences have been identified in the regulatory regions of all other steroid P450 genes (*CYP11A1*, *CYP21*, *CYPI7*, *CYP11B1*,

**25**

*3.5.2.3 AD5*

*3.5.2.4 NBRE*

*Aldosterone Synthase Gene (*CYP11B2*) Polymorphisms and Enhanced Cardiovascular Risk*

*CYP11B2*, and *CYPI9*), suggesting an important functional role in steroidogenesis. An Ad4-binding protein (Ad4BP) has been identified and cloned from bovine adrenal cortex nuclear extract. Ad4BP is a homolog of the steroidogenic factor 1 (SF-1) identi-

SF-1 is an orphan member of the nuclear hormone receptor superfamily, with potential phosphorylation sites for cAMP-dependent kinases, CaMK or PKC, suggesting a role for SF-1 in cAMP-dependent transcription. SF-1 is a 53 kDa protein consisting of a zinc finger domain and ligand binding/dimerization domain. The hydroxyl cholesterols enhance SF-1-dependent transcriptional activity in vitro, suggesting that SF-1 is a ligand-activated receptor. However, this is a controversial

SF-1 is expressed exclusively in steroidogenic tissues and plays an essential role in the development and function of the primary steroidogenic tissues [39]. Within the adrenal, SF-1 has been found to play a key role in the transcriptional regulation of most of the steroid hydroxylase genes (*CYP11A1*, *CYP21*, *CYP11B1*, *CYP17*, and *CYP 19*) as well as three *β HSD* and steroidogenic *acute regulatory protein* (StAR). SF-1 regulation of transcription is mediated by interaction with various co-activator proteins, including steroid receptor coactivator 1 (SRC1), glucocorticoid receptor interacting protein (GRIP), and also through repressors such as dosage-sensitive adrenal hypoplasia congenita of the X chromosome 1 (DAX1) that inhibits SF-1-

The Ad4 or SF-1 site has been identified in all steroid P450s based on sequence alignments, including at position −351/−343 (AGGTCC) of *CYP11B2.* Although this site binds strongly to SF-1, deletion studies suggest that it is not essential for basal or stimulus-induced expression of *CYP11B2*. In fact, co-expression of *CYP11B2* reporter gene constructs with SF-1 has a negative effect on gene transcription, making the regulation of *CYP11B2* different to the other steroidogenic genes, *StAR, CYP11A*, *CYP11B1*, and *CYP 17* that are all induced by SF-1. The effects of both NGFIB and NURRl on *CYP11B2* expression can be inhibited by SF-1 supporting a negative effect of SF-1 [33]*.* Thus, *hCYP11B2* expression is regulated quite differently from genes for other proteins involved in steroid biosynthesis including *StAR*, *CYP11A*, *CYP11B1*, and *CYP17*, which are all positively regulated by SF-1 [41].

Electrophoretic mobility shift assay (EMSA) analysis of the −129/−114 (Ad5) element (CTCCAGCCTTGACCTT) has shown that it binds several nuclear proteins, including SF-1 and another orphan nuclear receptor, chicken ovalbumin upstream promoter transcription factor (COUP-TF). On the bovine *CYP 17* and the mouse *CYP21* gene, COUP-TF and SF-1 bind competitively to a common site. Deletion of this site decreases basal activity by approximately 80% and also reduces

The transcription factors nerve growth factor IB (NGFIB) and nuclear receptor-related 1 protein (NURRl) are members of the NGFIB family of orphan nuclear receptors that bind to a consensus sequence NGFI-B response element (NBRE) (AAAGGTCA). These factors can also bind to the Ad5 element as well as a novel NBRE-1 site at −766/−759. Both these transcription factors increase

site decreases basal expression as well as the response to angiotensin II and K<sup>+</sup>

stimulation. Mutation of the NBRE-1

.

the maximal response to Ca2+ and cAMP stimulation [42].

*CYP11B2* expression by angiotensin II or K<sup>+</sup>

*DOI: http://dx.doi.org/10.5772/intechopen.89133*

finding and needs further clarification [38].

mediated steroidogenesis [40].

fied in the human [37].

*Aldosterone Synthase Gene (*CYP11B2*) Polymorphisms and Enhanced Cardiovascular Risk DOI: http://dx.doi.org/10.5772/intechopen.89133*

*CYP11B2*, and *CYPI9*), suggesting an important functional role in steroidogenesis. An Ad4-binding protein (Ad4BP) has been identified and cloned from bovine adrenal cortex nuclear extract. Ad4BP is a homolog of the steroidogenic factor 1 (SF-1) identified in the human [37].

SF-1 is an orphan member of the nuclear hormone receptor superfamily, with potential phosphorylation sites for cAMP-dependent kinases, CaMK or PKC, suggesting a role for SF-1 in cAMP-dependent transcription. SF-1 is a 53 kDa protein consisting of a zinc finger domain and ligand binding/dimerization domain. The hydroxyl cholesterols enhance SF-1-dependent transcriptional activity in vitro, suggesting that SF-1 is a ligand-activated receptor. However, this is a controversial finding and needs further clarification [38].

SF-1 is expressed exclusively in steroidogenic tissues and plays an essential role in the development and function of the primary steroidogenic tissues [39]. Within the adrenal, SF-1 has been found to play a key role in the transcriptional regulation of most of the steroid hydroxylase genes (*CYP11A1*, *CYP21*, *CYP11B1*, *CYP17*, and *CYP 19*) as well as three *β HSD* and steroidogenic *acute regulatory protein* (StAR). SF-1 regulation of transcription is mediated by interaction with various co-activator proteins, including steroid receptor coactivator 1 (SRC1), glucocorticoid receptor interacting protein (GRIP), and also through repressors such as dosage-sensitive adrenal hypoplasia congenita of the X chromosome 1 (DAX1) that inhibits SF-1 mediated steroidogenesis [40].

The Ad4 or SF-1 site has been identified in all steroid P450s based on sequence alignments, including at position −351/−343 (AGGTCC) of *CYP11B2.* Although this site binds strongly to SF-1, deletion studies suggest that it is not essential for basal or stimulus-induced expression of *CYP11B2*. In fact, co-expression of *CYP11B2* reporter gene constructs with SF-1 has a negative effect on gene transcription, making the regulation of *CYP11B2* different to the other steroidogenic genes, *StAR, CYP11A*, *CYP11B1*, and *CYP 17* that are all induced by SF-1. The effects of both NGFIB and NURRl on *CYP11B2* expression can be inhibited by SF-1 supporting a negative effect of SF-1 [33]*.* Thus, *hCYP11B2* expression is regulated quite differently from genes for other proteins involved in steroid biosynthesis including *StAR*, *CYP11A*, *CYP11B1*, and *CYP17*, which are all positively regulated by SF-1 [41].

#### *3.5.2.3 AD5*

*The Recent Topics in Genetic Polymorphisms*

*Schematic diagram of the CYP11B2 promoter with the cis-elements (Ad1–Ad6).*

**24**

*3.5.2.2 AD4 (SF-1)*

*3.5.2.1 Ad1 (CRE)*

**Figure 6.**

**Figure 7.**

specific DNA binding [34].

The Ad1 element closely resembles a consensus cAMP regulatory element (CRE) site. CREs plays an essential role in cAMP-dependent gene expression of a wide variety of genes. Proteins, such as the CRE-binding protein (CREB) and the highly related activating transcription factors (ATF), bind to CRE sites to initiate transcription. CREB binds to DNA as a dimer and has a conserved region of leucine residues (leucine zipper) at its C terminus that enables dimerization and sequence

*Schematic diagram of the most important transcription factor binding sites in the hCYP11B2 5'UTR. Abbreviations: CREB, cAMP regulatory element response element; SF-1, steroidogenic factor 1; COUP-TF, chicken ovalbumin upstream promoter transcription factor; NGFIB, nerve growth factor IB;* 

*NURRI, nuclear receptor-related 1 protein; NBRE-1, NGFI-B response element.*

CREB functions not only as a component of a variety of signaling pathways, particularly PKA, but also mitogen-activated protein kinases (MAPKs) and CaMKs. All these pathways mediate CREB-induced transcription by phosphorylating CREB at residue serine 133 [35]. The phosphorylated serine 133 binds another protein referred to as the CREB-binding protein (CBP). CBP is a 265-kDa nuclear protein, which binds to phosphorylated CREB and allows recruitment and stabilization of the RNA polymerase II transcription complex on the promoter of CREB target genes [36].

Using electrophoretic mobility shift assay, the *CYP11B2* CRE has been shown to bind members of the activating transcription factor (ATF-1 and ATF-2) and CREbinding protein (CREB) families. The ability of these transcription factors, particularly ATF-1 and CREB, to enhance transcription is partially regulated by their state of phosphorylation. Thus, one possibility is that activated CaMK I or CaMK IV phosphorylates CREB or ATF-1 leading to increased *CYP11B2* transcription [28].

The Ad4 site (CCAAGGTC) is also found to be important in the regulation of the *CYP11B* gene. This Ad4 site or homologous sequences have been identified in the regulatory regions of all other steroid P450 genes (*CYP11A1*, *CYP21*, *CYPI7*, *CYP11B1*,

Electrophoretic mobility shift assay (EMSA) analysis of the −129/−114 (Ad5) element (CTCCAGCCTTGACCTT) has shown that it binds several nuclear proteins, including SF-1 and another orphan nuclear receptor, chicken ovalbumin upstream promoter transcription factor (COUP-TF). On the bovine *CYP 17* and the mouse *CYP21* gene, COUP-TF and SF-1 bind competitively to a common site. Deletion of this site decreases basal activity by approximately 80% and also reduces the maximal response to Ca2+ and cAMP stimulation [42].

#### *3.5.2.4 NBRE*

The transcription factors nerve growth factor IB (NGFIB) and nuclear receptor-related 1 protein (NURRl) are members of the NGFIB family of orphan nuclear receptors that bind to a consensus sequence NGFI-B response element (NBRE) (AAAGGTCA). These factors can also bind to the Ad5 element as well as a novel NBRE-1 site at −766/−759. Both these transcription factors increase *CYP11B2* expression by angiotensin II or K<sup>+</sup> stimulation. Mutation of the NBRE-1 site decreases basal expression as well as the response to angiotensin II and K<sup>+</sup> .

Therefore, the CRE, Ad5, and NBRE-1 sites interact to regulate basal transcription as well as the response to each signaling pathway (cAMP or Ca2+) [33].
