POMC Transcripts

The POMC gene has three RNA transcripts of 1200 (T1), 800 (T2), and 1380 (T3) nucleotides, respectively (Fig. 3-3).

Regulatory Sites for Transcription

The POMC promoter has a number of common elements found in other genes that may contribute to regulation of POMC gene transcription (Fig. 3-4). Owing to the lack of availability of a human corticotrope cell line, the mouse-equivalent cell line (AtT20) has been used to assess POMC transcriptional regulation along with transgenic mouse models,²⁷ although some studies have utilized primary human pituitary cells from surgical procedures.²⁸

The T1 and T3 mRNA transcripts of POMC are initiated from a TATA box that is located close to the start of exon 1.

The correct spatial, temporal, and hormonal regulation of POMC transcription in the pituitary is conferred by two promoter regions immediately 5′ of exon 1. These regions are between −314 and −276 bp and between −67 and −27 bp in the human POMC gene.27,29,30

Hypothalamic and CNS-specific expression of POMC has been shown to require DNA control elements distal to those required for POMC expression in the pituitary. A 13 kb region immediately 5′ of the POMC gene has been demonstrated to control CNS and hypothalamic expression in transgenic mice.³¹ A 4-kb distal region of the POMC gene situated −13 to −9 kb in mouse and −11 to −7 kb in human from POMC exon 1 has been shown to contain two neuronal-specific enhancer regions capable of directing POMC expression in the arcuate nucleus of the hypothalamus.¹² These two neuronal POMC enhancer regions (nPE1 and nPE2) have been shown by deletion studies to be individually capable of driving POMC transcription in the arcuate nucleus and have also been demonstrated to be inactive in the pituitary, implying that there is a modular independence between the promoter regions used to control pituitary and hypothalamic POMC transcription.¹²

The expression of the short POMC mRNA transcript (T2) seems to be regulated by “GC box” promoter sequences located in the 3′ end of intron 2.¹⁹

Expression in the Pituitary

In humans, expression of POMC is most abundant in the corticotrope cells of the anterior pituitary, and in healthy subjects, these cells are the only ones that express the gene at high levels.32,33 POMC is one of the top 10 most abundant transcripts in the pituitary gland.³⁴ The main POMC mRNA expressed in the pituitary is the T1 transcript with a size of 1200 nt. POMC mRNA is also detected in the intermediate lobe of the pituitary, which is present during fetal life in humans and is found in other species such as the mouse and rat.^35–37

Pituitary expression is conferred by the 5′ flanking region of the gene25,27,38–40 (see Fig. 3-4), but there does not appear to be a specific element sufficient to direct high-level transcription, as for example, in the prolactin gene, where the pituitary-specific transcription factor Pit-1 binds to multiple sites to direct transcription. Rather, in the case of the POMC gene, there appears to be a requirement for integrity of the promoter.

The region just 5′ to the start site close to the TATA box confers basal expression and contains the binding sites for PO-B (at −27 nucleotides) and NUR 77 (−67 nucleotides). Further upstream, in the central region of the pituitary promoter, there is a response element which binds the homeobox protein Pitx1⁴¹ and the related Pitx2.⁴² During development, Pitx1/2 play an important role in corticotrope development and in the development of the anterior pituitary in general.^42–45 Close to the response element which binds Pitx1, there is a binding site for the T box factor, Tpit, which acts in synergy with Pitx1 and is required for expression of the POMC gene and for terminal differentiation of the pituitary corticotrope lineage.⁴⁶ Tpit functions as an activator of transcription by recruiting SRC/p160 co-activators to its cognate DNA target in the POMC promoter.⁴⁷

Evidence for the importance of the role of Tpit comes from Tpit-deficient mice, which represent a model of isolated ACTH deficiency, and from humans with Tpit gene mutations which are associated at high frequency with early-onset isolated ACTH deficiency (IAD).48,49 One cause of neonatal death has been identified as congenital IAD associated with mutations of the Tpit gene.⁵⁰ Different Tpit mutations have been associated with IAD, but all these mutations are likely to manifest functionally through disruption of DNA-protein and protein-protein interactions. In one case, a mutation of the Tpit gene (M86R) has been shown to inhibit the binding of other DNA-bound proteins, ultimately leading to loss of recruitment of the p160 coactivator SRC-2.⁵¹

Other evidence for the importance of Tpit relates to bone morphogenic proteins (BMP) 4 and 2, which are signaling molecules associated with early organogenesis and cell differentiation of the pituitary. BMP4 stimulation leads to the recruitment of activated phospho-Smad1 by the POMC promoter through “tethered” interactions with Pitx1 and Tpit, thus reducing their transcriptional activity and resulting in repression of POMC transcription.⁵²

The distal region of the pituitary promoter cannot confer activity independently of the central region but does contain a binding site (Ebox_neuro) for NeuroD/1A acting as a heterodimer with other basic helix-loop-helix (bHLH) factors and synergizing with Pitx.40,53 The Ebox_neuro element of the distal pituitary promoter, and a nearby Nur response element (NurRE), are required to modulate expression of POMC throughout pituitary development.⁵⁴ A member of the T-box gene family, Tbx19, a pituitary development–specific transcription factor, has been shown to have a putative binding site at −310 nt, close to the Ebox_neuro site. However, this protein may also exert POMC transcriptional effects synergistically with other pituitary-specific transcription factors.⁵⁵

Expression in Other Tissues

POMC is also expressed, but at a much lower level, in other tissues such as the arcuate nucleus of the hypothalamus, skin, testis, ovary, placenta, duodenum, liver, kidney, adrenal medulla, lung, thymus, heart, and lymphocytes.14,19,22,25,56–59 Extrapituitary POMC mRNA is frequently expressed as the 1200 nt T1 transcript (similar to that in the pituitary), for example in the hypothalamus, skin, and placenta. However, POMC mRNA from extracranial tissues can also have a preponderance of the shorter 800 nt T3 transcript.^58,60,61 As indicated earlier, this shorter T2 transcript could not give rise to mature POMC and would lack a signal sequence, so its physiologic role is unclear. It may be that low-level expression of longer POMC transcripts (T1 and T3) account for POMC transcription and expression even when there is high expression of the short transcript, for example as demonstrated in the testes.^20,60,62

POMC expression and regulation in the skin is now well established.14,63,64 Current evidence would suggest that POMC transcription in the skin is regulated by the region immediately 5′ of exon 1 in keratinocytes⁶⁴ and melanocytes.⁶⁵

Expression in Pituitary Tumors

In corticotrope adenomas that give rise to pituitary-dependent Cushing’s disease, POMC is the most highly expressed gene,⁶⁶ similar to that in the normal pituitary.^34,67 The loss of retinoblastoma tumor-suppressor protein (Rb) expression has been linked to pituitary corticotrope tumor progression.⁶⁸ It has been shown that Rb is a transcriptional activator of POMC by bridging between NeuroD and Nurr77 and potentiating interactions between Nurr77 and the p160 co-activator SRC-2.^69,70

Expression in Nonpituitary Tumors

Tumors giving rise to the ectopic ACTH syndrome produce an mRNA transcript of 1200 bp, similar to that found in the pituitary, and approximately 20% of tumors express a larger transcript of 1400 to 1500 bp.⁶⁷ This larger transcript seems to be under the regulation of a promoter region located at −392 and −432 bp relative to the conventional start site.^21,71–73 Analysis of this domain in the human small cell lung carcinoma cell line DMS-79 showed that it binds the E2F family of trans-acting factors.⁷⁴

The expression of this promoter in the ectopic ACTH syndrome suggests loss of the tight tissue-specific expression. This promoter is embedded within a CpG island which has been shown to be unmethylated in a number of tumors giving rise to the ectopic ACTH syndrome and in the POMC-expressing small cell lung carcinoma cell line, DMS-79.⁷⁵ In contrast, the CpG island was methylated in normal nonexpressing tissues.²⁷

Recently it has been shown that hypomethylation of the POMC promoter in thymic carcinoid tumors correlates with POMC overexpression and the ectopic ACTH syndrome. The region of the POMC promoter that underwent change in methylation status was shown to correspond to the E2F binding region of the POMC promoter.⁷⁶

Regulation of the POMC Gene in the Pituitary

Numerous factors are known to regulate POMC gene expression in the pituitary, but perhaps the most important are CRH and glucocorticoids (Fig. 3-5). Expression of the POMC gene appears to be predominantly controlled at the level of gene transcription.⁷⁷

Regulation of the POMC Gene in Other Tissues

POMC expression can occur in a wide range of nonpituitary tissues, as described earlier, although in many of these extrapituitary tissues, the shorter POMC transcript predominates, leading to extremely low levels of protein.¹⁹ However, there are several tissues where there is significant POMC expression, and subtle differences occur in regulation compared to the pituitary.

Regulation of the POMC Gene in Tumors

POMC

In 1973, high-molecular-weight forms of ACTH were identified in human plasma,¹⁴¹ mouse pituitary cells,^142,143 and human tumors,¹⁴⁴ which led to predictions of the presence of a precursors of ACTH.

Expression of the POMC gene leads to synthesis of the preprohormone POMC. This protein undergoes proteolytic cleavage at dibasic amino acid residues, which generates a series of small molecules, including ACTH¹⁴⁰ (Fig. 3-8). Processing of POMC to its constituent peptides varies in a tissue-specific fashion, in that both the nature of the processing and the degree of processing varies in different tissues. This results in different groups of peptides being secreted from different tissues, although the exact ratios of the constituent peptides and precursors are still not fully understood.

ACTH

The ACTH peptide consists of 39 amino acids, is a single polypeptide chain, and has a molecular weight of 4.5 kD¹⁴⁵ (see Fig. 3-8). The N-terminal 12 amino acids are highly conserved between species, thus reflecting the importance of this region for biological activity. In comparison with the human sequence, ACTH in other mammals has only one or two substitutions, which are in the region of amino acids 24 to 39. In birds, amphibians, and fish, although the N-terminal sequence is conserved, the ACTH sequence is more variable, particularly between amino acids 24 and 39. The melanocyte-stimulating hormone (MSH) sequence His-Phe-Arg-Trp is found at ACTH 6–9 and is termed α-MSH. Identical sequences are present in β-lipotropin (as β-MSH) and N-pro-opiomelanocortin (N-POC) (as γ-MSH). Given that these three forms of MSH bind different melanocortin receptors, it is thought that the surrounding amino acids influence their specific activity.

α-Melanocyte-Stimulating Hormone

α-MSH consists of ACTH 1–13 and is derived from ACTH 1–39 by proteolysis at the C-terminal, which is followed by C-terminal amidation and N-terminal acetylation. α-MSH is produced predominantly by melanotrope cells in the intermediate lobe of the pituitary, particularly in species such as the rat and mouse. The adult human pituitary does not have a distinct intermediate lobe, and therefore this is not a source of α-MSH in humans. In addition, α-MSH is not thought to be produced in the anterior lobe. Therefore, it is not clear whether α-MSH circulates in humans under normal circumstances.146,147

CLIP

Corticotropin-like intermediate lobe peptide (CLIP) consists of ACTH 18–39 and is produced during the cleavage that generates α-MSH. Because this process occurs primarily in the intermediate lobe of the pituitary, which is not present in humans, CLIP is not thought to circulate in humans under normal circumstances.

N-Pro-opiomelanocortin

Also called N-pro-opiomelanocortin, N-POC (see Fig. 3-8) comes from the N-terminal sequence of POMC, and in humans, it is a 76–amino acid peptide with an MSH sequence in the mid-region.¹⁴⁸ The peptide has a tryptophan residue at the N terminus and two disulfide bridges linking cysteines 2 to 24 and 8 to 20, which are thought to be important for the sorting signal that directs POMC to the regulated pathway.¹⁴⁹ N-POC can also undergo N-glycosylation at Asn65 and O-glycosylation at Thr45.

γ-Melanocyte-Stimulating Hormone

γ₁-MSH is found at position 51 to 62 of human N-POC and has sequence homology with α-MSH. There are C-terminally extended forms of γ₁-MSH, which are called γ₂-MSH (51–63) and γ₃-MSH (51–76).

Joining Peptide

Joining peptide is found between N-POC and ACTH and is a 30–amino acid peptide, amidated at the C terminus. It was isolated from human pituitaries in 1981¹⁴⁸ and has been shown to circulate in humans in the form of homodimers.¹⁵⁰

β-Lipotropin

β-Lipotropin lies at the C terminus of POMC and can be cleaved to γ-lipotropin (which contains the β-MSH sequence at its C terminus) and β-endorphin (see Fig. 3-8). In the human anterior pituitary, cleavage appears to be limited, inasmuch as the main form of this peptide in the human circulation is β-lipotropin, with very little β-endorphin.^151,152

β-Endorphin

This 31–amino acid peptide contains the sequence for met-enkephalin as the first five amino acids at its N terminus. β-Endorphin can undergo N-acetylation, which is thought to be a tissue-specific effect, and C-terminally truncated peptides have been found, such as α-endorphin (β-endorphin 1–16), γ-endorphin (β-endorphin 1–17), and δ-endorphin (β-endorphin 1–27).

Processing

After translation of POMC mRNA into peptide, a series of processing stages are needed for release of the constituent peptides.112,114,130,153 The N-terminal signal sequence involved in movement of the peptide into the endoplasmic reticulum is no longer required and is removed at an early phase of posttranslational modification. Subsequently, POMC undergoes glycosylation and phosphorylation in the Golgi apparatus before transport to secretory vesicles, where it undergoes cleavage into its constituent peptides. The ACTH-related peptides are stored in dense core secretory granules and released from the cell on stimulation (e.g., by CRH), as in the stress response (Fig. 3-9). The prohormone, POMC, is found in the human circulation¹⁵⁴ and could be released from the constitutive pathway perhaps as an “overflow” mechanism.

N-Glycosylation and Phosphorylation

These events occur in the Golgi apparatus before cleavage of the peptides. γ-MSH has the sequence Asn-X-Ser, which can be glycosylated on the Asn residue, and in mouse POMC, N-glycosylation of the CLIP sequence can occur. Some evidence indicates phosphorylation of serine 31 in ACTH, although the significance of this finding is unclear.¹⁵⁵

Anterior Pituitary

In the human anterior pituitary, POMC is cleaved to give pro-ACTH, which is then cleaved to ACTH, N-POC, and joining peptide (see Fig. 3-8). Interestingly, the ACTH precursors, POMC and pro-ACTH, are found in the human circulation with ACTH, N-POC, joining peptide, and β-lipotropin.¹⁵⁴ That very little β-endorphin appears to be present indicates that processing of β-lipotropin is minimal (Fig. 3-10). However, reports can be confounded by the fact that in some β-endorphin assays, the antibodies also detect β-lipotropin. In the rat and sheep anterior pituitary, some ACTH is also processed to des-acetyl-α-MSH and α-MSH.¹¹²

Studies in the mouse pituitary tumor cell line, AtT20, suggest that cleavage is sequential, starting with the C terminus of ACTH.¹⁴⁰ However, the same pair of basic amino acids is found between ACTH and β-lipotropin, joining peptide and ACTH, ACTH 1–16 and ACTH 17–39, and γ-lipotropin and β-endorphin. Therefore, the adjacent amino acids and peptide folding must influence the sequential processing.

Intermediate Lobe

In the rodent intermediate lobe, POMC is found in melanotrope cells and undergoes more comprehensive processing to give the smaller fragments α-, β-, and γ-MSH, CLIP, and β-endorphin. The protease responsible for this cleavage is PC2. After endopeptidase cleavage, ACTH 1–17 is further modified by an exopeptidase that removes amino acids from the C terminus to give ACTH 1–13. Subsequently, ACTH 1–13 undergoes N-acetylation and C-terminal amidation.

Hypothalamus

POMC is produced primarily in the neurons of the hypothalamic arcuate nucleus where the peptides are central to regulation of food intake and energy balance¹¹⁴ (see Fig. 3-6). POMC is also expressed in the median eminence and ventromedial border of the third ventricle and much smaller amounts in the tractus solitarius. Processing is different from the anterior pituitary, in that smaller peptides characteristic of the neurointermediate lobe are produced.¹⁶¹ However, most of the studies are limited to the rat hypothalamus. In these extracts, high-performance liquid chromatographic separation of peptides suggests that ACTH is processed to CLIP and that des-acetyl-α-MSH is detected rather than α-MSH, thus indicating that N-terminal acetylation is limited. β-Endorphin 1–31 is found in the rat hypothalamus, again suggesting more extensive processing.^114,112,162 PC1 and PC2 expression in the hypothalamus are increased by leptin and further regulated by the transcription factor Nescient Helix-Loop-Helix (Nhlh)-2, suggesting possible physiologic control of POMC peptide function at the level of peptide processing.¹⁶³

POMC has also been detected in cerebrospinal fluid (CSF), although whether it originates from the pituitary or hypothalamus is uncertain. Evidence from changes in precursors and ACTH in rat CSF in relation to food intake and obesity suggest that they originate from the hypothalamus.¹¹⁸ In human CSF, the POMC precursor peptide has been shown to occur at high concentrations and predominates over ACTH when molar ratios are compared.¹⁶⁴ However, several of the POMC peptides can be detected in CSF.^165,166

Other Tissues

POMC peptides have been detected in the thyroid, pancreas, gastrointestinal tract, placenta, testis, ovary, adrenal gland, and immune system.¹¹² However, in comparison to the pituitary, other tissues produce only low levels of POMC peptides.

POMC peptides are also produced in the skin (see Fig. 3-7). The first peptide to be detected, α-MSH, was found, by immunostaining, to predominate in human melanocytes, but ACTH has also been detected in human keratinocytes.¹⁶⁷ A role for POMC peptides in hair pigmentation is also suggested by two patients with inherited mutations in POMC that prevented synthesis of the ACTH/α-MSH region; both patients had red hair pigmentation.¹¹ The presence of PC1 and PC2 has been demonstrated in melanocytes and keratinocytes, along with functional POMC processing.^63,168 The possibility of extracellular peptide processing of POMC by other peptidases has been demonstrated in dermal cells.¹⁶⁰

Processing of POMC in the placenta results in significant amounts of POMC secretion along with ACTH, β-LPH, α-MSH, and β-endorphin.22,129

Pituitary Tumors

In patients with pituitary-dependent Cushing’s syndrome, the processing of precursors to ACTH appears to be relatively normal as judged by the molar ratios of these peptides in plasma.¹⁵² However, the molar ratio of precursors to ACTH is much higher for corticotrope macroadenomas, thus suggesting that processing is impaired.¹⁶⁹ In pituitary corticotrope adenomas not associated with Cushing’s syndrome, defective PC1 expression may result in an increase of secreted unprocessed POMC.¹⁷⁰

Extrapituitary Tumors

Data on tumor extracts suggest that most extrapituitary tumors causing the ectopic ACTH syndrome do not process the prohormone efficiently. In an early study, analysis of tumor tissue from patients without clinical features of hormone excess identified a high-molecular-weight form of ACTH, and this purified material could be cleaved to mature ACTH (4.5 kD) by the action of trypsin. The ACTH immunoreactivity was found to have no biological activity and was assumed to be due to ACTH precursors.¹⁷¹

Evidence that processing is impaired in tumors from patients with the ectopic ACTH syndrome also comes from the elevated levels of ACTH precursors in plasma and the high ratio of precursors to ACTH.152,172 Identification of ACTH precursors predominating in the circulation of patients with clinically apparent Cushing’s syndrome suggests that these precursors may have some activity at the ACTH receptor or are processed at the adrenal. Most of these patients had clinically obvious small cell carcinoma of the lung. However, patients with highly differentiated, slowly growing tumors, typically bronchial carcinoids, have lower but nevertheless elevated levels of ACTH precursors.¹⁷³ CLIP has also been detected in four tumor extracts from patients with carcinoid tumors,¹⁷⁴ thus suggesting that some tumors may process POMC in the manner of the neurointermediate lobe. It is not yet clear whether the same tumors give rise to increased precursors and smaller fragments or whether processing varies in different tumors.

ACTH and Its Receptor

The major role of ACTH is to stimulate steroidogenesis in the adrenal cortex, which results in the synthesis and release of cortisol in humans and corticosterone in rodents. In pathologic conditions, it is evident that ACTH can increase the production of adrenal androgens and aldosterone; however, under physiologic situations these pathways are regulated by other factors. Long-term overexpression of ACTH can cause adrenal cell proliferation,175,176 although peptides from the N-terminal of POMC have also been implicated in this process. ACTH is thought to have a role in adrenal cortical development,^177,178 particularly since ACTH replacement in POMC knockout mice causes normal adrenal development.¹⁷⁹

In situations with prolonged ACTH excess such as Nelson’s syndrome, Addison’s disease, and ectopic ACTH syndrome, skin pigmentation can occur and is thought to be due to ACTH binding through its MSH sequence to melanocortin receptors in the skin, although whether the skin pigmentation results from cleavage of ACTH to MSH peptides is unclear. ACTH receptors are also present on human mononuclear leukocytes and have been identified on other rat and mouse immune cells, which suggests that ACTH may have a role in immune function.¹³⁰

ACTH Receptors and Signaling

The ACTH 1–39 sequence is most potent in stimulating steroidogenesis, but ACTH 1–24 is also known to have full agonist activity in certain systems. It is clear that the ACTH 1–13 sequence is involved in binding and activation, but ACTH 6–24 has been shown to have some steroidogenic activity.

ACTH binds to the melanocortin-2 receptor (MC2R),¹⁸⁰ which has been identified in human adrenal glands,^181,182 although in rat and ovine adrenocortical cells, there is a suggestion that low-affinity ACTH binding sites are also present. Binding of ACTH to human receptors requires calcium¹⁸¹ and occurs with a K_d of approximately 2.0 nmol/L. However, ACTH at 0.01 nmol/L causes maximal steroidogenesis, and therefore only a small number of the predicted 3500 sites per cell need to be occupied to achieve this activity. ACTH 1–16 is the minimal peptide required for binding to the human MC2R and signaling; the presence of a broad binding pocket in human MC2R, utilizing both conserved and unique amino acid residues, may be the reason why α-MSH was not able to bind hMC2R.¹⁸³

The ACTH receptor is a member of the melanocortin receptor family, which all have similar seven-membrane-spanning domains and are G-protein coupled.¹⁸² On binding to its receptor, ACTH stimulates cAMP production,¹⁸¹ and cAMP in turn stimulates a cAMP-dependent protein kinase that activates the steroidogenic pathway. Calcium is also involved in ACTH stimulation of cAMP in human adrenal cells.

Corticostatins, low-molecular-weight inhibitors of ACTH-induced steroidogenesis, are thought to act by preventing ACTH binding to its receptor, although their physiologic role is unclear.

ACTH Effects on the Adrenal

ACTH acts at a number of levels to increase cortisol production. On binding to its receptor, it stimulates lipoprotein uptake, activates hydrolysis of cholesterol, and increases transport of cholesterol to mitochondria. Importantly, ACTH also regulates cholesterol side-chain cleavage, which is the rate-limiting step in steroidogenesis and results in the production of pregnenolone. This activity takes place in the inner membrane of the mitochondria and is catalyzed by cytochrome P450 side-chain cleavage enzyme.¹⁸⁴

Longer stimulation by ACTH will eventually result in down-regulation of the ACTH receptor, but it is known to cause increased transcription of enzymes in the steroidogenic pathway and can result in adrenal cell proliferation.

α-Melanocyte-Stimulating Peptide

In most mammals, α-MSH is produced in the melanotrope cells of the neurointermediate lobe, but because these cells are absent from the human pituitary, it is unlikely that this peptide has a role as a secreted peptide in humans. In mice, α-MSH acting at the MC-1 receptor causes changes in coat color, and in frogs, it affects skin pigmentation. It is also thought that locally produced α-MSH peptides stimulate melanogenesis in human skin.¹⁶⁷ Recently α-MSH has been shown to have immunoregulatory functions in the human hair follicle¹⁸⁵ as well as cytoprotective activity against UVB-induced apoptosis and DNA damage in the skin.^65,186

α-MSH-related peptides produced in the arcuate nucleus of the hypothalamus and acting at the MC-4 receptor in the paraventricular nucleus of the hypothalamus are important in the regulation of food intake and energy balance and are the principal mediators of the effects of leptin.¹⁶¹ The role of POMC peptides is evidenced by a number of inherited deletions in the POMC gene which are associated with obesity.^11,115,187 Recently, antagonists of α-MSH function have been shown to increase food intake in mice when injected peripherally, thus demonstrating the importance of α-MSH in the regulation of appetite and energy balance.¹⁸⁸

The antiinflammatory and immunomodulatory properties of α-MSH have resulted in the hypothesis that α-MSH and its cognate receptors might present potential antiinflammatory treatment options.189,190

N-Pro-opiomelanocortin and Joining Peptide

N-POC has been reported to potentiate ACTH-induced steroidogenesis in human and rat adrenocortical cells, and it is thought that the γ₃-MSH region, from mid- to C-terminal of N-POC, is responsible for this activity. It has also been shown that N-POC 1–48 and not the γ₃-MSH region stimulates adrenal growth after unilateral adrenalectomy in the rat.¹⁹¹ N-POC 1–28 has also been shown to decrease adrenal steroidogenesis in opposition to ACTH.¹⁹² Since N-POC circulates intact, it has been proposed that cleavage of N-POC occurs at the adrenal gland, and a serine protease capable of cleaving N-POC has been identified in the outer adrenal cortex.¹⁹³ In addition, N-POC stimulates the release of aldosterone from human adrenal tumor cells.^194,195

The role of joining peptide is unclear. It has been suggested that it is the adrenal androgen-stimulating hormone, but subsequently, several reports have shown that joining peptide lacks the ability to increase adrenal androgens.¹⁹⁶

β-Lipotropin and β-Endorphin

β-Lipotropin was named because of its lipolytic activity, and it was suggested that the β-MSH sequence in the mid-region was responsible for this activity. Subsequently, most studies have concentrated on this peptide as a precursor of β-endorphin.

Data are conflicting regarding whether β-endorphin circulates in human plasma.151,154 It may have a more important role when released locally in the brain because, when administered, it has opiate-like analgesic activity associated with the met-enkephalin sequence at its N terminus, and mice lacking β-endorphin exhibit absence of stress-induced analgesia.¹⁹⁷ β-Endorphin has also been shown to affect sexual behavior and learning. In the skin, β-endorphin modifies human hair follicle physiology via its ability to up-regulate melanogenesis, dendricity, and proliferation in melanocytes.¹⁹⁸

ACTH Precursors

It has proved difficult to get a clear indication of ACTH precursor bioactivity because of problems in obtaining pure preparations of the peptides and the limitations of available bioassays. POMC itself is thought to have relatively little biological activity,¹⁷¹ whereas pro-ACTH was shown to be equipotent with ACTH in a rat adrenal cell bioassay or 8% to 33% as potent in a cytochemical ACTH bioassay.¹⁹⁹ Nothing is currently known about the binding of POMC and pro-ACTH to the ACTH receptor (MC2-R) and the other MSH receptors (MC3-R, MC5-R).²⁰⁰ However, at the MC4-R, the receptor for α-MSH in the brain, β-MSH and ACTH bind MC4-R with similar affinity to α-MSH, and POMC itself functions with low potency.²⁰¹ Also, at the MC1-R which is considered to be the receptor for α-MSH in the skin, again ACTH and α-MSH have similar affinity, and POMC can function at low potency.⁶³ The possible biological effect of POMC was examined in human pigment cells, where functional activity was shown but only at concentrations in excess of 10⁻⁷ M, considerably higher than the concentrations of POMC released from the cells (∼1 × 10⁻¹⁰ M).⁶³ However, it is possible that POMC is degraded extracellularly to ACTH-like peptides.

Because ACTH precursors are present in the circulation at concentrations greater than those of ACTH,¹⁵⁴ it would be valuable to examine the agonist/antagonist activity of the precursors at the other human melanocortin receptors. In patients with hyperpigmentation related to post-adrenalectomy Cushing’s disease, concentrations of both ACTH and ACTH precursors correlated with pigmentation scores.²⁰² The interaction of ACTH precursor peptides with the recently cloned receptor MC4-R found exclusively in the brain may also be of interest, inasmuch as concentrations of ACTH precursors in CSF are 100-fold those of ACTH (414 versus 3.2 pmol/L).¹⁶⁴

Some information regarding in vivo POMC bioactivity can be gained from clinical studies. If patients with the ectopic ACTH syndrome produce ACTH precursors in preference to ACTH,¹⁵² it must either have biological activity when present at very high levels or be cleaved to ACTH at the level of the adrenal as previously suggested.¹⁹¹

Glucocorticoids

Glucocorticoids exert a classic feedback inhibitory effect on the production of CRH and ACTH (Fig. 3-11). Therefore, ACTH stimulation of cortisol release from the adrenal directly determines the concentration of cortisol feeding back to inhibit the ACTH release from the pituitary. However, there is also regeneration of cortisol from cortisone by the enzyme 11β-hydroxysteroid dehydrogenase (11β-HSD1) in tissues such as the liver and adipose,^203–205 and this will influence circulating cortisol concentrations, particularly in conditions such as obesity.

There are multiple ways in which glucocorticoids negatively regulate the activity of the HPA axis, and their interactions are not fully understood. Much is known about the molecular mechanisms whereby the glucocorticoid receptor acts as a transcription factor to activate gene transcription, but less is known about specific mechanisms of glucocorticoid inhibition of the human POMC gene (see section on regulation of POMC gene expression) or about the early effects which must involve nongenomic mechanisms. These interactions have been grouped into fast, intermediate, and slow feedback, based on the timing of the phenomena.

Corticotropin-Releasing Hormone

CRH is an important physiologic activator of ACTH. This is evidenced by mice with inactivating mutations in the CRH gene which die at birth with dysplastic lungs, preventable by prenatal maternal glucocorticoids.²¹² Thus CRH activation of ACTH is necessary to provide sufficient glucocorticoids for lung development. Both CRH and arginine vasopressin (AVP) are considered important for activation of pituitary ACTH, but CRH is a more potent secretagogue in rat and horse, whereas AVP is thought to be more potent in sheep. In all cases, there is a marked synergism, with CRH functioning permissively, while AVP is the main dynamic signal.

CRH stimulates ACTH secretion from dispersed pituitary cells in a sustained manner, initially causing the release of preformed peptide but simultaneously stimulating peptide synthesis (see Fig. 3-9). In humans, a biphasic response to exogenous CRH reflects these two mechanisms of action.²¹³

The effects of CRH on the levels of ACTH precursors in the human circulation have been examined only during petrosal sinus sampling, which is used as a diagnostic test in patients with suspected Cushing’s syndrome. In this test, CRH is given intravenously, and ACTH peptides are measured in the petrosal sinuses draining the pituitary. In this situation, the increase in ACTH is much greater than the increase in precursors, which suggests that CRH is stimulating release of processed ACTH from secretory granules.¹⁵⁴

Hypothalamic CRH is subject to regulation by multiple afferent signals and will in turn influence ACTH release. These signals include upregulation by catecholamines via β- and α₁-adrenoceptors, serotonin (5-HT) acting via the 5-HT1A, 5-HT2A, and 5-HT2C receptors,²¹⁴ acetylcholine acting through both muscarinic and nicotinic receptors, and the cytokines interleukin 1 (IL-1) and IL-6 possibly acting by generation of prostaglandins. In addition, CRH expression may be inhibited by glucocorticoids, catecholamines via α₂-receptors, and γ-aminobutyric acid (GABA) released by neuronal input from the hippocampus and amygdala.

Vasopressin

Vasopressin (AVP) is synthesized in the same cells of the paraventricular nucleus of the hypothalamus as CRH (see Fig. 3-11). The two peptides are released concurrently from the median eminence into the hypophysial portal system. In addition, AVP reaches portal blood from the supraoptic nucleus. AVP exerts weak, direct stimulation on ACTH release but powerfully synergizes with CRH. In vivo evidence indicates a role for AVP in stress-induced ACTH secretion.^215,216 In contrast to CRH, which acts via protein kinase A, AVP acts by stimulation of protein kinase C. AVP also increases the cAMP response to CRH in isolated pituicytes, which suggests multiple sites of interaction between the two signaling cascades.

Other Regulatory Factors

L-Dopa and serotonin both increase ACTH secretion by means of neuronal release into the paraventricular nucleus of the hypothalamus.217–219 Pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal polypeptide (VIP) both enhance ACTH secretion, but while PACAP directly stimulates pituitary ACTH, VIP promotes release of CRH. The role of these two peptides is probably most relevant in regulating HPA responses to inflammatory and cold stressors.²²⁰

In contrast, GABA inhibits ACTH when released from hippocampal afferents to the hypothalamus by inhibiting release of CRH and AVP.²²¹ Atrial natriuretic peptide (ANP) has been shown to decrease ACTH secretion and inhibit CRH gene expression. In comparison, opiate receptor agonists inhibit ACTH release, probably by effects at the hypothalamic or hippocampal level, although it has been reported that met-enkephalin can directly inhibit ACTH release at the level of the corticotrope. Oxytocin inhibits CRH stimulated ACTH secretion in humans, but in rats, oxytocin stimulates ACTH, probably by binding to AVP receptors.

Cytokines and Growth Factors

Cytokines are pleiotropic polypeptides released from immune cells in response to inflammation, infection, and tissue injury.222,223 Proinflammatory cytokines stimulate the HPA axis in vivo,²²⁴ and while some act via CRH, several cytokines, including interleukin 2 (IL-2), interferons, and the gp130 cytokine family (IL-6, leukemia inhibitory factor [LIF], oncostatin M) act at the pituitary.

Integrated Control of ACTH Secretion

Three tiers of control subserve the regulation of ACTH secretion (see Fig. 3-11).

Differential Regulation of POMC and ACTH

The relative concentrations of POMC and ACTH in the circulation will depend not only on regulatory mechanisms influencing expression of the POMC gene but also on precursor processing and mechanisms of secretion from the corticotrope cells. Evidence from studies with the mouse corticotrope adenoma cell line AtT20 suggests that in the absence of stimulation, corticotrope cells release newly synthesized POMC.²⁴⁶ Therefore, the levels of POMC and ACTH in the circulation at any given time could well vary because of the differing regulatory mechanisms.

Circadian Rhythmicity

The circadian rhythm for ACTH originates from a primary “clock” which is located in the suprachiasmatic nucleus. Neuronal afferents from this nucleus feed into the paraventricular nucleus of the hypothalamus and regulate CRH expression. ACTH secretion is pulsatile, and the circadian rhythm is generated by variation in the amplitude of the pulses rather than variation in pulse frequency. Therefore, the amplitude of ACTH pulses during peak secretion is fourfold higher than during the ACTH nadir. Peak levels of ACTH, and concordantly cortisol, are reached at 6 am, decline during the day to 4 pm, and then further decline to a nadir between 11 pm and 3 am (Fig. 3-12). The 6 am peak is reached after an abrupt increase in ACTH secretion. Although all the circulating POMC peptides show a diurnal variation and peak at the same time, their decline occurs at different rates, probably conferred by different circulatory half-lives and/or variation in extrapituitary processing.

Pulsatility

ACTH is secreted in a pulsatile manner which is reflected in the pulsatile release of cortisol. However, the analysis of this is very much dependent on the sampling techniques. In humans, studies have shown 12 or 40 pulses per 24 hours, depending on the sampling frequency.247,248 There are fewer pulses during the nadir of ACTH secretion, and there are more pulses, with greater peak amplitude and higher mean level, in males.²⁴⁹ The pulsatility may be a mechanism for overcoming desensitization of the ACTH receptor and may reflect pulsatile release of CRH.

The Stress Response

The HPA axis is stimulated by a number of different types of stress. These include exercise, acute illness, surgical stress, hemorrhage, and hypoglycemia. Infection which activates the immune system causes release of cytokines which in turn stimulate the HPA axis. This network of interactions between the immune system and the HPA axis provides a mechanism whereby activation of the immune system is inhibited by glucocorticoid feedback on the immune cells, leading to the well-recognized immune suppression, which limits the overall response. Chronic stress, as for example in depression, also activates the HPA axis, but this persistent activation can be attributed to failures in the normal feedback regulatory loops.

In response to stress, peripheral and central signals are integrated by the pituitary to modulate adrenal glucocorticoid production. Several lines of evidence suggest a unifying hypothesis linking hypothalamic releasing factors, activation of peripheral cytokine cascades, and intrapituitary cytokine expression with pituitary-mediated modulation of the systemic inflammatory response.247,250

An acute septic insult provokes a local inflammatory response, with coordinated and sequential activation of a series of proinflammatory cytokines238,251,252 and neural and bacterial toxin signals that activate the HPA axis.²⁵³ Initially, peripheral activation of local and distal tumor necrosis factor (TNF) expression is followed by IL-1, IL-6, and LIF.²³⁸ A number of the proinflammatory cytokines exert most if not all their activities at the hypothalamus, notably IL-6 acting on hypothalamic AVP²³⁷ and IL-1 and TNF acting on CRH.²²⁶ Hypothalamic LIF mRNA is also up-regulated by lipopolysaccharide (LPS) treatment, a well-recognized model of gram-negative septic shock.²⁴¹

Other cytokines clearly act at the pituitary, notably LIF.242,254 The pituitary is also a site of de novo cytokine synthesis, and thus in addition to the circulating, peripherally derived cytokines, an intrapituitary network of cytokines is established in the acute phase of septic shock. IL-1β and LIF are up-regulated by LPS,^241,255 and macrophage migration inhibitory factor is acutely released from pituicytes in vitro and in vivo in response to LPS.²⁵⁶ In addition, intrapituitary IL-6 is up-regulated by IL-1.^238,257

The pituitary response to septic shock involves cytokines such as IL-6 and LIF, which limit the inflammatory response. These cytokines cause activation of the HPA axis and increased glucocorticoid production, thus limiting the extent of the inflammatory response and protecting against lethality. The increase in intrapituitary LIF stimulates POMC expression and strongly potentiates CRH action on the corticotrope.242,244 The key role of HPA activation in limiting the lethal effects of unrestrained activation of proinflammatory cytokine cascades is underscored by the poor performance of the CRH knockout mouse exposed to endotoxin.²⁵⁸ IL-1, TNF, and IL-6 have also been shown by some but not all studies to exert direct effects on pituitary ACTH secretion.^227,259,260

A further level of action of the cytokines is to antagonize the negative feedback loop of adrenal glucocorticoids on hypothalamic CRH expression and pituitary POMC secretion.²⁵³ The pattern of acute proinflammatory cytokines induced by septic shock opposes effective glucocorticoid signaling,^261,262 in part by activation of the NF-κB nuclear transcription factor, which inhibits glucocorticoid receptor action.^263–265

Stress-associated disorders, such as melancholic depression, are characterized by persistent activation of the HPA axis. In this situation, there are multiple feedback loops which activate central CRH pathways, including down-regulation of the glucocorticoid receptor which prevents the normal negative feedback on the HPA axis, resulting in the vicious circle of continued activation of the HPA.²⁶⁶

ACTH and ACTH Precursors

ACTH was one of the first peptides to be measured by radioimmunoassay and presented a significant challenge because of the difficulty in generating high-affinity antisera and in labeling ACTH. The development of sensitive immunoradiometric assays for ACTH has improved the reliability of ACTH measurement.173,267 The assays are based on a labeled monoclonal antibody that usually binds the N-terminal region of ACTH and a solid-phase antibody that recognizes a different sequence in ACTH. Because binding of both antibodies is required to generate a signal, the assay does not recognize α-MSH or CLIP. However, it is not always clear whether current ACTH assays recognize the ACTH precursors.

Recent years have seen a shift toward the measurement of ACTH by two-site immunometric assays in preference to radioimmunoassay. This change provides many benefits, including improved sensitivity, speed, reproducibility, and parallel results. The high sample throughput and wide working range of these assays make them ideal for measuring samples taken during inferior petrosal sinus sampling, a test that is fast becoming an important component in the diagnosis of pituitary tumors secreting ACTH.

However, we must recognize that in some clinical situations, the use of an assay that is highly specific for ACTH 1–39 may be insufficient or misleading. In one patient with the ectopic ACTH syndrome, shown by chromatography to be producing high-molecular-weight ACTH precursors, the ACTH concentration was very low when measured by immunoradiometric assay.²⁶⁷ To ensure that patients with the ectopic ACTH syndrome are flagged by an ACTH assay, it is important that the ACTH precursors have a high degree of cross-reactivity in the ACTH assay or that a separate specific assay for ACTH precursors be available.

Detection of ACTH precursors in plasma was first demonstrated in normal subjects after stimulation with metyrapone,²⁶⁸ and it was later observed after insulin-induced hypoglycemia.²⁶⁹ However, complex chromatographic techniques were required to separate ACTH precursors from ACTH. Clearly, this approach cannot be used for large numbers of patient samples and would not provide a quantitative assessment of the concentrations of ACTH precursors in plasma.

Direct measurement of ACTH precursors was made possible by the development of a two-site immunoradiometric assay for the ACTH precursors POMC and pro-ACTH.²⁷⁰ The assay is based on a labeled monoclonal antibody that binds within the ACTH region of POMC and a solid-phase antibody that recognizes N-POC (see Fig. 3-8). Because binding of both antibodies is required to generate a signal, the assay does not detect ACTH. With this assay, the concentrations of ACTH precursors in normal subjects were found to be 5 to 40 pmol/L, which is equivalent to or greater than the concentrations of ACTH, N-POC, β-lipotropin, and β-endorphin.¹⁵⁴ Measurement of ACTH precursors in patients with ectopic ACTH syndrome has indicated that the precursors are present at much higher concentrations than is ACTH.^172,173 A similar approach has been used to measure POMC in aggressive ACTH-secreting tumors.²⁷¹

Other POMC-Derived Peptides

The development of radioimmunoassays and/or immunoradiometric assays for N-POC, γ-MSH, α-MSH, β-lipotropin, and β-endorphin has proved extremely valuable in understanding the production and action of POMC peptides. The development of specific immunometric assays that distinguish circulating levels of β-lipotropin and β-endorphin has shown that β-lipotropin is the main form in human plasma and that relatively little β-endorphin is secreted.¹⁵¹ Nevertheless, questions relating to the relative molar ratios of the family of POMC peptides are still unanswered.