Regulation of Prohormone Convertases in Hypothalamic Neurons

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Endocrinology 148(9):4191– 4200 Copyright © 2007 by The Endocrine Society doi: 10.1210/en.2007-0173

Minireview: Regulation of Prohormone Convertases in Hypothalamic Neurons: Implications for ProThyrotropin-Releasing Hormone and Proopiomelanocortin Eduardo A. Nillni Division of Endocrinology, Department of Medicine, Brown Medical School/Rhode Island Hospital, and Department of Molecular Biology, Cell Biology, and Biochemistry, Brown University, Providence, Rhode Island 02903 Recent evidence demonstrated that posttranslational processing of neuropeptides is critical in the pathogenesis of obesity. Leptin or other physiological changes affects the biosynthesis and processing of many peptides hormones as well as the regulation of the family of prohormone convertases responsible for the maturation of these hormones. Regulation of energy balance by leptin involves regulation of several proneuropeptides such as proTRH and proopiomelanocortin. These proneuropeptide precursors require for their maturation proteolytic cleavage by the prohormone convertases 1 and 2 (PC1/3 and PC2). Because biosynthesis of mature peptides in response to leptin requires prohormone processing, it is hypothesized that leptin might regulate hypothalamic PC1/3 and PC2 expression, ultimately leading to coordinated processing of prohormones into mature peptides. Leptin has been shown to increase PC1/3 and PC2 promoter activities,

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HE BIOSYNTHESIS OF mammalian neuropeptide hormones follows the principles of the prohormone theory, which begins with an mRNA translation process into a large, inactive precursor polypeptide, followed by a limited posttranslational proteolysis to release different products of processing (1). Posttranslational modifications of a given prohormone are a requirement for conversion of the polypeptide from the proform state to its active final peptide. This is achieved through differential processing by the action of different members of the family of prohormone convertases (PCs), which results in biological and functional diversity within the central nervous system as well as in endocrine cells outside the central nervous system. The PCs are a family of seven subtilisin/kexin-like endoproteases including furin, PC1 (also known as PC3), PC2, PC4, PACE4, PC5-A (also First Published Online June 21, 2007 Abbreviations: ARC, Arcuate nucleus; CLIP, corticotrophin-like intermediate lobe peptide; CP, carboxypeptidase; DVC, dorsal vagal complex; HPT, hypothalamic-pituitary-thyroid; LPH, lactase-phlorizin hydrolase; MC-R, melanocortin receptor; Nhlh, nescient helix-loop-helix; N2KO, Nhlh2 knockout mouse; NPY, proneuropeptide Y; NTS, nucleus of the solitary tract; PAM, peptidylglycine ␣-amidating monooxygenase enzyme; PC, prohormone convertase; POMC, proopiomelanocortin; PSTAT3, phosphorylated STAT3; PVN, paraventricular nucleus; STAT, signal transducer and activator of transcription. Endocrinology is published monthly by The Endocrine Society (http:// www.endo-society.org), the foremost professional society serving the endocrine community.

and starvation of rats, leading to low serum leptin levels, resulted in a decrease in PC1/3 and PC2 gene and protein expression in the paraventricular and arcuate nucleus of the hypothalamus. Changes in nutritional status also changes proopiomelanocortin processing in the nucleus of the solitary tract, but this is not reversed by leptin. The PCs are also physiologically regulated by states of hyperthyroidism, hyperglycemia, inflammation, and suckling, and a recently discovered nescient helix-loop-helix-2 transcription factor is the first one to show an ability to regulate the transcription of PC1/3 and PC2. Therefore, the coupled regulation of proneuropeptide/processing enzymes may be a common process, by which cells generate more effective processing of prohormones into mature peptides. (Endocrinology 148: 4191– 4200, 2007)

known as PC6-A), its isoform PC5-B (also known as PC6-B), PC7 (also known as LPC), and PC8 (also known as SPC7) (2– 8). The structures of these serine proteinases resemble those of both the bacterial subtilisins and yeast kexin (9 –11). These enzymes cleave at the C-terminal side of single, paired or tetra basic amino acid residue motifs (12), followed by removal of remaining basic residue(s) by a carboxypeptidase (CP) E and CPD (13–15). The selective expression of PC1/3 and PC2 in endocrine and neuroendocrine cells suggests that they are important in prohormone processing (2, 5, 10, 16). PC1/3 and PC2 have been shown to process proTRH (17–20), proinsulin (12, 21, 22), proenkephalin (23), prosomatostatin (24, 25), proGHRH (26, 27), proopiomelanocortin (POMC) (28, 29), proproneuropeptide Y (NPY) (30), prococaine- and amphetamine-induced transcript (31) and proneurotensin (32) to various intermediates and end products of processing. Among the seven members of the PC family thus far cloned, PC1/3 and PC2 are specifically found in neuronal and endocrine cells containing secretory granules (2, 5, 10). Their involvement in the processing of neuropeptide precursors has been suggested by the finding that PC1/3 and PC2 transcripts and protein products are widely distributed in different areas of the brain, including cerebral cortex, hippocampus, and hypothalamus (16, 33). Within the hypothalamus, these enzymes display an extensive overlapping pattern of expression (16, 33). The critical role of PC1/3 and PC2 in prohormone processing is underscored by studies

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Endocrinology, September 2007, 148(9):4191– 4200

from animals lacking the genes encoding PC1/3 (34) and PC2 (35), as well as 7B2 (36, 37), a neuropeptide essential for the maturation of PC2. Disruption of the gene-encoding mouse PC1/3 results in a syndrome of severe postnatal growth impairment and multiple defects in processing many hormone precursors, including hypothalamic proGHRH to mature GHRH, pituitary POMC to ACTH, islet proinsulin to insulin, and intestinal proglucagon to glucagon-like peptide-1 and -2. PC1/3⫺/⫺ mice are normal at birth but display impaired postnatal growth and are about 60% of normal size at 10 wk. They lack mature GHRH, have low pituitary GH and hepatic IGF-I mRNA levels, and phenotypically are smaller than a normal mouse (34). However, a second mouse model of PC1/3 deficiency generated by random mutagenesis was recently reported (38). This mouse has a missense mutation in the PC1/3 catalytic domain (N222D) that leads to obesity with abnormal proinsulin processing and multiple endocrine deficiencies. Although there was defective proinsulin processing leading to glucose intolerance, neither insulin resistance, nor diabetes developed despite obesity. The apparent key factor in the induction of obesity was impaired autocatalytic activation of mature PC1/3, causing reduced production of hypothalamic ␣-MSH. This is the first published characterization of a Pc1 mutation in a model organism that mimics human PC1 deficiency (38). For example, a patient with a compound heterozygous mutation in the PC1/3 gene resulting in production of nonfunctional

Nillni • Minireview

PC1/3 had severe childhood obesity (39). An analogous obese condition was found in a patient with a defect in POMC processing (40). Because PC1/3 and PC2 are essential for the processing of a variety of proneuropeptides, alterations in the expression and protein biosynthesis of PC1/3 and PC2 are likely to have profound effects on neuropeptide homeostasis. Because the PCs process many proneuropeptide hormones with opposite effects on energy balance such as proTRH and POMC on one side, and proNPY and proagouti-related protein on the other, it is difficult to precisely determine in mouse genetic models which phenotypes are altered due to defects or changes in PC activity. However, one could speculate that with high levels of leptin in the fed state, active processing of POMC and proTRH occur by the up-regulated PCs, whereas simultaneously proNPY and proagouti-related protein are inhibited by leptin. This article will review the regulation of proTRH (Fig. 1) and POMC (Fig. 2) posttranslational processing by the PCs in the context of energy balance in which the hormone leptin plays a key role in their regulation. Leptin produced in adipose tissue provides information on energy stores and energy balance to brain centers involved in regulating appetite, energy expenditure, and neuroendocrine function (41– 47). Leptin through its receptor (ObRb) up-regulates the protrh and pomc genes in the paraventricular nucleus (PVN) and arcuate nucleus (ARC), respectively, and induces the release of TRH in vitro and in vivo (48, 49) and the release of the

FIG. 1. This figure depicts a schematic representation of the proTRH polypeptide and its posttranslational processing. The TRH progenitor sequence is indicated by black rectangles (five identical copies). Arrows indicate where PC1/3, PC2, CPE/D, and PAM produce their enzymatic cleavages. The numbers situated below the intact proTRH represent the location of pair of basic residues at each side of the TRH progenitor sequence. SS, Signal sequence.



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PC1/3, Initial cleavage PRO-OPIOMELANOCORTIN (POMC, ~31kDa)

β-LPH

PRO-ACTH PC1/3

FIG. 2. This figure depicts a schematic representation of the POMC polypeptide and its posttranslational processing. Arrows indicate where PC1/3 and PC2 produce their enzymatic cleavages. N-POC, Amino terminal peptide; JP, joining peptide.

ACTH PC N-POC

JP

PC2 ACTH1-17

CLIP

PC γ-LPH

β-END

CPE γ−MSH

˜ MSH DA -α

β−MSH

N-acetyltransferase

α˜ MSH

␣-MSH peptide from many neuronal projections located in different regions of the brain, inside and outside the hypothalamus (50 –53). In a series of studies investigating the possible role of leptin in the regulation of neuropeptide processing, it was reported for the first time the regulatory role of leptin on PC1/3 and PC2 in the PVN (54), ARC of the hypothalamus, and the nucleus of the solitary tract (NTS) (55), strongly suggesting the existence of a coordinated regulation between peptide expression and processing by the PCs (54, 55). ProTRH

The hypothalamic-pituitary-thyroid (HPT) axis plays an important role in the maintenance of metabolic homeostasis in response to alterations in the external environment, and TRH, produced in the hypothalamus, is recognized as a key hormone responsible for HPT regulation. TRH, synthesized from a larger inactive precursor, proTRH (26 kDa), is posttranslationally modified in the PVN (primarily in the parvocellular division) of the hypothalamus primarily by the action of PC1/3 and secondarily by PC2 (18, 20, 56). The intermediate products of proTRH generated from these enzymatic cleavages are further subjected to additional modifications by exopeptidases, such as CPE or CPD, to remove the C-terminal basic amino acids (15). TRH-gly, the immediate precursor to TRH (pGlu-His-Pro-NH2; thyroliberin), is then amidated at its carboxyl terminus by the action of peptidylglycine ␣-amidating monooxygenase enzyme (PAM) (57) (see Fig. 1). TRH neurons in the PVN project to the median eminence, in which they are in close proximity to the capillaries of the hypophysial-portal system. TRH released in these capillaries stimulates the biosynthesis and secretion of TSH from the pituitary (58, 59), which in turn stimulates biosynthesis of the thyroid hormones, T4 and T3 and their release from the thyroid gland. The maintenance of euthyroidism is dependent on a highly regulated balance of positive and negative feedback, in which TRH positively regulates TSH (58, 59), and thyroid hormones suppress preproTRH expression and TSH secretion (60, 61). An original work published in 1997 (20) and additional

supporting data provided unequivocal evidence for the role of PC1/3 and PC2 in the processing of proTRH (17, 18, 20, 56). Rat preproTRH (29 kDa) composed of 255 amino acids contains an N-terminal 25-amino acid leader sequence, five copies of the TRH progenitor sequence Gln-His-Pro-Gly flanked by paired basic amino acids (Lys-Arg or Arg-Arg), four non-TRH peptides lying between the TRH progenitor sequences, an N-terminal flanking peptide, and a C-terminal flanking peptide (62, 63). The N-terminal flanking peptide (preproTRH25–50-R-R-preproTRH53–74) is further cleaved at the C-terminal side of the arginine pair site to render preproTRH25–50 and preproTRH53–74, thus yielding a total of seven non-TRH peptides. Initial processing of proTRH occurs in the TGN at the prepro152–153-TRH-158 –159 site to generate the 15- and 10-kDa peptides (Fig. 1). In subsequent steps, the 15-kDa N-terminal intermediate moiety of proTRH is processed to a 9.5-kDa peptide followed a continuous processing until the end products are generated in secretory granules. It is proposed that the processing of the remaining 10-kDa C-terminal fragment produces the 5.4-kDa C-terminal peptide preproTRH208 –255 early in the secretory pathway (64), and the remaining intermediate form is further processed to end products in secretory granules (65– 68). Only two sites, thus far, have been identified to be processed by PC2. The pFE22 peptide is further cleaved to two smaller forms of about 1.6 (preproTRH178 –184, pFQ7 ) and 0.84 (preproTRH186 –199) kDa (68). The extended form of pEH24, TRHpEH24 is processed to generate TRH and pEH24 (20) (Fig. 1). Recent studies using the PC1/3 and PC2 null mice developed in Donald Steiner’s laboratory provided novel insights regarding the processing of several peptides hormones (34, 35, 69) including proTRH. The results on proTRH confirmed the initial in vitro finding that attributes a primary role for PC1/3 in the processing of proTRH (20), whereas PC2 has a specific role in cleaving TRH from its extended forms (68). PC1/3 null mice show a dramatic decrease in the biosynthesis of all proTRH-derived peptides analyzed, including TRH and its proform, TRHGly. However, proTRH is still processed to its end products suggesting, as demonstrated in our laboratory and others (20) in earlier studies, that PC2 and

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potentially furin can compensate for the lack of PC1/3 in the processing of proTRH (Nillni et al., unpublished results). In the PC2 null mice, although TRH and TRHGly showed some decrease as compared with the wild-type animals, the concentrations of N-terminal peptides preproTRH25–50 and preproTRH83–106 did not change. This indicates that PC1/3 is the primary enzyme involved in the processing of proTRH at the pairs of basic residues flanking the TRH sequence including the sequence Arg51-Arg52, which does not contain TRH (see Fig. 1). Preliminary evidence seems to indicate that the specific biological end points, such as thyroid axis activity through TSH and T3/4, are unchanged in the PC1/3 and PC2 null mice. This is probably because of compensatory responses by either enzyme to ensure enough production of proTRH-derived peptides to satisfy physiological needs in a steady-state condition. It remains to be studied what effect the mouse Pc1 (N222D) with an obese phenotype will have in the processing of proTRH. However, the real challenge for the thyroid axis occurs when animals are physiologically perturbed such as during cold stress or starvation, situations in which more TRH peptide is necessary to increase the output of the thyroid axis. For example, the role of CPE in processing has been elucidated in Cpefat/Cpefat mice, which lack functional CPE resulting from a naturally occurring mutation (70). Using this mouse, it was found that hypothalamic TRH was depressed by at least 75%, compared with wild-type controls (15), and that CPD was probably responsible for the generation of 21% of the TRH produced in the hypothalamus of these animals (15). These animals cannot maintain their body temperatures when exposed to cold, resulting from hypothalamic TRH depletion and a reduction in thyroid hormone levels (15). Mice with disruption of the gene-encoding PC2 appear to be normal at birth. However, they exhibit a small decrease in rate of growth. They also have chronic fasting hypoglycemia and a reduced blood glucose level during an ip glucose tolerance test, both of which are consistent with a deficiency of circulating glucagons (69). The processing of proglucagon, prosomatostatin, and proinsulin in the ␣-, ␦-, and ␤-cells of the pancreatic islets is severely impaired in PC2 null mice (71). The mere presence of the PCs in secretory granules is not the only factor ensuring the regulation of prohormone processing. The PCs are also physiologically regulated. ProPC1/3 cleavage occurs rapidly in the secretory pathway, yet its major site of action for most prohormones occurs in secretory granules. PC1/3 undergoes an interesting carboxyl-terminal processing event, which appears to activate the enzyme (72). PC1/3 is endogenously inhibited by the neuroendocrine peptide proSAAS (73, 74). ProSAAS (the endogenous granin-like binding protein for PC1/3) itself is processed as it was shown in studies done using two neuroendocrine cell lines expressing this protein: the AtT-20 mouse pituitary corticotrophic line and the PC12 rat adrenal phaeochromocytoma line (75). Various smaller forms of proSAAS were detected, including peptides designated as little SAAS, PEN, and big LEN (75). Two of the peptides identified represent novel C-terminally truncated forms of PEN. ProPC2, however, exhibits comparatively long initial folding times and exits the endoplasmic reticulum without propeptide cleavage and in association with the neuroendocrine-


specific protein 7B2 (76, 77). Once the proPC2/7B2 complex arrives at the trans-Golgi network, 7B2 is internally cleaved into two domains, the 21-kDa peptide and a carboxyl-terminal 31-residue peptide. PC2 propeptide removal occurs in the maturing secretory granules, most likely through autocatalysis, and 7B2 association does not appear to be directly required for this cleavage event (76, 77). However, if proPC2 has not encountered at intracellular level with 7B2, it cannot generate a catalytically active mature species. The molecular mechanism behind the intriguing intracellular association of 7B2 and proPC2 is still unknown but may involve conformational rearrangement or stabilization of a proPC2 conformer mediated by a 36-residue internal segment of 21-kDa 7B2. In fasted animals (low leptin levels) proSAAS protein increases only in the median eminence. This suggests that the SAAS peptide could play a role in the decrease of PC1/3 levels during fasting (54). At the physiological level, the PCs are regulated by states of hyperglycemia (78), inflammation (79), suckling (68), starvation (54), norepinephrine (Perello, M., and E. A. Nillni, unpublished results), and exposure to opioids (Nie, Y., and T. C. Friedman, unpublished results). For example, norepinephrine, one of the main mediators of the cold-induced activators of the thyroid axis, stimulates proTRH processing by increasing the expression of PC1/3 and PC2. Since the discovery of a direct role of leptin on the regulation of proTRH (48, 80), we questioned whether this regulation also involved an active regulation of the PCs. In those previous studies, it was shown that leptin dose-dependently stimulated the increase in proTRH biosynthesis and TRH release in primary cultures of hypothalamic neurons (48). In the same primary cultures, ObRb and proTRH showed to colocalize, which was consistent with the finding of the leptin receptor ObRb in the PVN (81). In addition, leptin produced a 5-fold induction of luciferase activity in CV-1 cells transfected with a TRH promoter-luciferase reporter and the long form of the leptin receptor cDNA (80). [Although the above data are consistent with a direct ability of leptin to promote TRH biosynthesis through actions on TRH neurons, ␣-MSH through the melanocortin system also regulates TRH biosynthesis and release (48, 49, 82), and it is unknown whether ␣-MSH also regulates the PCs.] Further supporting the role of leptin in TRH neurons, fasted rat PVN expresses suppressor of cytokine signaling-3 (which is a sensitive marker of direct leptin action), and a signal transducer and activator of transcription (STAT) response element in the TRH promoter is necessary for leptin’s effect (80). In addition, leptin activates the STAT3 signaling pathway in proTRH neurons (83). The above data are consistent with a direct ability of leptin to promote TRH biosynthesis through actions on TRH neurons. The major breakthrough in those studies was the discovery that leptin regulates prohormone processing by regulating the PCs (54). Because leptin regulation of energy balance works through the activity of several neuropeptides, we hypothesized that leptin might regulate processing enzymes in addition to regulating peptide production. Early studies clearly supported the hypothesis that the regulation of hypophysiotropic TRH biosynthesis by leptin occurs at not only the transcriptional level but also the posttranslational level


through changes in proTRH processing by the action of PC1/3 and PC2 (54). Through the use of in vitro and in vivo approaches, strong evidence implicated leptin in the regulation of PC1 and PC2 expression/protein biosynthesis, and as a consequence of those regulatory changes, the posttranslational processing of proTRH is affected. For example, analysis in 293 T cells and in rats showed that leptin increased PC1/3 and PC2 promoter activity through a STAT3-dependent mechanism (54). Thus, the results from this study demonstrate that leptin couples the up-regulation of preproTRH expression and its protein biosynthesis with the up-regulation of the processing enzymes in a coordinated fashion. We hypothesize that such regulation ultimately leads to more effective processing of leptin-regulated proneuropeptides into mature peptides, such as TRH and ␣-MSH, which are believed to be critical for leptin action. Therefore, it is concluded that transcriptional control of PC1/3 and PC2 gene expression by leptin is another level at which this hormone regulates energy homeostasis, adding a novel key checkpoint that is tightly regulated in the control of energy consumption. Other examples of the regulation of PCs include rats exposed to streptozotocin, whereby it was demonstrated that the diabetic state altered ␣-cell processing of proglucagon to give increased levels of glucagon-like peptide 1 (78). Li and colleagues (84 – 87) also identified regions in PC1/3 and PC2 human promoters that contain putative-negative thyroid hormone response elements and has shown that T3 negatively regulates PC1/3 and/or PC2 expression in rat GH3 cells, rat anterior pituitary, hypothalamus, and cerebral cortex. Changes in the thyroid status also have an effect on the processing of proTRH by altering the PCs (88, 89). Interestingly, these changes in proTRH processing were observed only in hypophysiotropic proTRH neurons. During 6-n-propyl-2-thiouracil-induced hypothyroidism, an increase in PC1/3, PC2, and proTRH was detected in the PVN, whereas induced hyperthyroidism by T3 did not affect PC1/3, PC2, and proTRH expression (88). Thus, regulation of prohormone and enzyme by changes in thyroid status may lead to altered hormonal biosynthesis. The selective coregulation of proTRH processing by thyroid status in the PVN is a novel aspect of the regulation of the HPT axis. Further support for these findings includes the regions in the PC1/3 and PC2 human promoters containing putative-negative thyroid response elements and the fact that T3 negatively regulates PC1/3 and/or PC2 expression in rat GH3 cells, rat anterior pituitary, hypothalamus, and cerebral cortex (84 – 87). These findings suggested the possibility that thyroid hormones could regulate PC1/3 and PC2 expression in specific hypothalamic nuclei by directly regulating proTRH processing. To test this, experiments altering the thyroid hormone status in rats and examining the effects on proTRH processing in the PVN and other extrahypophysiotropic areas were examined. The results clearly showed that a high level of thyroid hormone down-regulates TRH biosynthesis and that this was coupled with the down-regulation of PC1/3 and PC2 in the PVN. Conversely, low levels of thyroid hormone upregulate TRH biosynthesis and PC1/3 levels (88, 89). Such regulation may have important implications in the pathophysiology of hypo- and hyperthyroidism.


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POMC

The central melanocortin system is one of the best-characterized neuronal pathways involved in the regulation of energy balance. The POMC-derived peptide ␣-MSH is well known for inhibiting food intake and increasing energy expenditure (51, 90) by up-regulating, for example, hypophysiotropic proTRH neurons in the PVN (48, 49, 82). Because antagonists of melanocortin 3/4 receptors (MC-Rs) completely block the autonomic, satiety, and metabolic effects of leptin, it is believed that the melanocortin system mediates several central actions of leptin (51, 91). This system is particularly important because mutations in MC-Rs, POMC, or PCs have been associated with obesity in humans and rodents (92). In the ARC, POMC neurons express the leptin receptor ObRb (93), and it is well known that low leptin signaling (e.g. fasting) directly inhibits pomc gene expression, resulting in a decrease in all POMC-derived peptides, including des acetyl ␣-MSH, and administration of leptin can attenuate this response (55, 94 –96). POMC (97, 98) is also generated in the commissural part of the NTS. The NTS contains mainly POMC products generated within the brain stem because it excludes the para brachial nucleus, locus coreuleus, and most parts of the paragigantocellular reticular nucleus, which are recipients of fibers from both ARC and NTS (99, 100). The lateral reticular nucleus and the rostral NTS itself also receive dual innervations; however, the contribution from the ARC seems to be minimal, as established by neuronal fiber-tracing studies (99, 101). Similar to proTRH, POMC follows the intracellular trafficking of a secreted protein through the Golgi complex and ultimately the secretory granules in which the end products of processing are stored before being secreted by exocytosis. During trafficking POMC undergoes a series of posttranslational modifications, resulting in the processing of the precursor to yield the various biologically active POMC-derived peptides. The POMC polypeptide precursor contains eight pairs, and one quadruplet, of basic amino acids, which are the cleavage sites for PC1/3 and PC2 (28). Tissue-specific processing of POMC is one of the best-known examples emphasizing the importance that the expression and activity of PC1/3 and PC2 have on the outcome of POMC products. For example, in the corticotroph cells of the anterior pituitary, only four pairs of basic residues are cleaved, which are all of the Lys-Arg type. These cleavages generate the following six peptides: amino terminal (NT), joining peptide, ACTH, ␤-lactase-phlorizin hydrolase (LPH), a small amount of ␥-LPH, and small amounts of ␤-end (102, 103). On the other hand, in the melanotroph cells of the intermediate lobe of the pituitary in rodents and the hypothalamus, nucleus of the solitary tract, and placenta in man, a different POMC processing occurs. In these tissues most of the pair of basic residues including the tetra basic are cleaved by the PCs. For example, the NT gives rise to the ␥-MSHs, ACTH to ␣-MSH and corticotrophin-like intermediate lobe peptide (CLIP) [or ACTH (18 –39)], and ␤-LPH to ␤-MSH, ␤-end (1–31), and ␤-end (1–27) (103, 104). The key factor involved in the processing of ACTH to ␣-MSH and CLIP in melanotrophs, the ARC, and the NTS is the presence of PC2 (Fig. 2). Further chemical modifications including glycosylation, amidation,

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phosphorylation, acetylation, and sulfation also occur in a cell-specific manner, resulting in an alteration of the biological activity of the peptides (such as acetylation of ␤-end and ␣-MSH). In the ARC, similar to the pars intermedia of the pituitary (105–108), POMC is initially cleaved by PC1/3 to generate proACTH and ␤-lipotrophin. ProACTH is further cleaved by PC1/3 to generate a 16-kDa N-terminal peptide and ACTH. ACTH is further cleaved to generate ACTH (1–17) and CLIP. Then CPE enzyme removes C-terminal basic amino acids from ACTH (1–17), and the PAM enzyme amidates the peptide to generate desacetyl ␣-MSH. Acetylation of ACTH or desacetyl ␣-MSH to acetyl ␣-MSH (109 –111) by the N-acetyltransferase enzyme could occur immediately after the amino terminal side of these peptides becomes available subsequent to the PCs cleavage. Fasting-induced changes in the ARC caused a significant decrease in ACTH and des acetyl ␣-MSH consistent with a decrease in POMC biosynthesis during fasting (55). A similar conclusion can be drawn from the fact that POMC and ACTH levels are reduced in cerebrospinal fluid during fasting (110). This decrease in POMC was associated with a decrease in PC1/3 (55). Leptin administration in fasted rats prevented the fasting-induced decrease in the content of all POMC-related peptides, demonstrating that leptin potently regulates the biosynthesis of POMC in the ARC. Whereas a previous report suggested an increase in acetyl ␣-MSH (the most active form of ␣-MSH) due to leptin action on the yet-undefined N-acetyl-transferase activity (112), studies from different laboratories concluded that leptin is not involved in the N-acetylation of hypothalamic ␣-MSH (55, 109, 113). POMC neurons in the NTS innervate different areas of the brain but not the hypothalamus (51, 99, 100). They send projections primarily within the dorsal vagal complex (DVC), which includes the NTS, and to other structures in the brain stem and medulla (97, 114). The NTS mediates some actions of leptin on energy balance. For example, administration of leptin in the fourth ventricle or directly in the DVC, area postrema, and dorsal motor nucleus inhibits food intake (115). Leptin treatment also increases c-Fos expression in some POMC neurons in the NTS (51). The administration of melanocortin agonists or antagonists directly in the DVC reduces or increases food intake, respectively (116); melanocortin agonists injected in the fourth ventricle increased uncoupling protein-1 expression in the brown fat of rats (117); and MC4-R is highly expressed in brain stem regions, such as the DVC (118). However, it is unclear whether the POMC neurons in the NTS are regulated by changes in energy balance. The biosynthesis of POMC in the NTS is unique and different from the ARC. Similar to the ARC, it produces ␣-MSH through the action of PC2, but the response to fasting and leptin is quite different (55). In the NTS, a prominent peptide of about 28.1 kDa molecular mass, similar in size to POMC has been identified, together with other POMC-derived peptides, including ␣-MSH. Differing from the ARC, during fasting, ACTH and desacetyl ␣-MSH were found to accumulate, whereas at the same time, pomc mRNA decreased. Again, differing from the ARC, these changes were not reversed by leptin (55, 119). However, it is important to point


out that ObRb is present in the NTS (120, 121) and the marker of leptin action Phosphorylated STAT3 (P-STAT3) has been found in the neurons of the NTS of leptin-treated rats (122, 123), and delivery of leptin into the DVC decreases food intake and body weight (121). Other studies did show that peripheral administration of leptin induced P-STAT3 activation in about 30% of the POMC-enhanced green fluorescent protein (EGFP) neurons in the NTS (124). Whereas the phosphorylation of STAT3 is valid and widely used to indicate leptin signaling, P-STAT3 could participate in other signaling pathways independent of the biosynthesis of POMC. Even though all these components of leptin signaling are present in the NTS, leptin does not regulate the pomc gene and POMC-related peptides (55, 119). Similar to previous studies done in the PVN (54), low leptin levels resulted in a decrease in PC1/3 in the ARC and a decrease in PC2 in the NTS. At present, it is unknown whether this selective effect of fasting on the PCs seen in the ARC and NTS may contribute to changes in POMC processing. Still, a proper PC activity on POMC is important in maintaining a proper enzyme-substrate homeostasis. Supporting this view, a recent study showed that PC1/3 and PC2 gene expression and processing of the prohormone was affected in a coordinate fashion during photoperiod changes in seasonal Siberian hamsters to control body weight (125). In that study they compared mRNA levels and protein distribution of PC1/3, PC2, POMC, ACTH, ␣-MSH, ␤-endorphin, and Orexin-A in selected hypothalamic areas of long, short, and natural day. The key finding of that study was that a major part of neuroendocrine body weight control in seasonal adaptation may be effected by posttranslational processing mediated by PC1/3 and PC2, in addition to regulation of gene expression of neuropeptide precursors. Therefore, the coupled regulation of POMC/processing enzymes may be a common process, by which cells generate more effective processing of the prohormone into mature peptides. Table 1 summarizes the effect of fasting and leptin on the PCs in brain regions in which proTRH and POMC are generated. The Basic Helix-Loop-Helix Transcription Factor, Nescient Helix-Loop-Helix (Nhlh)-2, Regulates PC1/3 and PC2

Several transcription factors have been implicated in the regulation of body weight by hypothalamic neurons including steroidogenic factor-1 (126), STAT3 (127), and activator protein-1 (128). However, Nhlh2 was the first one to show an ability to regulate the transcription of PC1/3 and PC2, possibly in conjunction with the leptin-stimulated transcription factor, STAT3. Nhlh2 is a member of the basic helix-loophelix transcription factor family. Similar to other basic helixloop-helix transcription factors, the Nhlh2 protein most likely requires a dimerization partner to modulate the transcription of target genes. The implications of Nhlh2’s role in energy balance became clear after the knockout mouse (N2KO) was developed (129). These animals displayed adult-onset obesity, including fatty deposits in their abdomen, around their gonads, and within their liver (129). The obese phenotype of the N2KO mice was associated with a



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TABLE 1. PC1/3 and PC2 levels in different brain regions producing proTRH and POMC PC1/3

Fed Fasted F⫹L

PC2

POMC mRNA

␣-MSH peptide

TRH mRNA

TRH peptide

PVN

ARC

NTS

PVN

ARC

NTS

ARC

NTS

ARC

NTS

PVN

PVN

ME

⫽ ⫺ ⫹

⫽ ⫺ ?

⫽ NC ?

⫽ ⫺ ⫹

⫽ NC ?

⫽ ⫺ ?

⫽ ⫺ ⫹

⫽ ⫺ -

⫽ ⫺ ⫹

⫽ ⫹ ⫹

⫽ ⫺ ⫹

⫽ ⫺ ⫹

⫽ ⫺ ⫹

ME, Median eminence; ⫽, normal values in fed animals; ⫺, decrease; ⫹, increase; NC, no change; F, testing; L, leptin.

reduced level of POMC-derived peptides, but this reduction in peptide production was not from a reduction in POMC mRNA levels but from reduced peptide processing of POMC. This finding was consistent with 40 – 60% reduction in both PC1/3 and PC2 mRNA in several hypothalamic nuclei of the N2KO mice (130). It was found that preproTRH mRNA and proTRH-derived peptide levels were reduced in N2KO mice and because PC1/3 levels were reduced in the PVN in which proTRH is synthesized, it was concluded that the decrease in TRH peptide synthesis was related to a reduced processing of proTRH caused by less PC1/3 available (130). Altogether, the finding that there is a coordinated regulation between the transcription factor Nhlh2 and PC1/3 and PC2 with a downstream effect on TRH and ␣-MSH could suggest a new level for a potential drug target. A more detailed review on Nhlh2 is described elsewhere [Fox et al. (131)]. Summary

Since the discovery of the PCs in the late 1980s, a new frontier on the propeptide hormone biosynthesis and processing research had come to surface. It is particularly relevant to emphasize the concept that secretion of any particular peptide or neuropeptide hormone from a given cell involves two specific steps. The fist one is the action of a specific secretagogue, inducing the fusion of secretory granules to the plasma membrane of the cell followed by the release of the peptide. The second one, most likely triggered by the first action, is a complex set of enzymatic processes by the PCs on prohormones to generate mature bioactive peptide ready for release from the proper secretory granules. One of the examples described in this review is the action of the hormone leptin on the biosynthesis, processing and secretion of proTRH and POMC prohormones, which also involves the regulation of the PCs responsible for the maturation of these peptide hormones. These sequences of events further support the existence of a coupled regulation of proneuropeptide/processing enzymes as a common process by which cells generate more effective processing of prohormones into mature peptides ready for release. Acknowledgments Received February 6, 2007. Accepted March 26, 2007. Address all correspondence and requests for reprints to: Dr. Eduardo A. Nillni., Division of Endocrinology, Brown Medical School/Rhode Island Hospital, 55 Claverick Street, Third floor, Room 320, Providence, Rhode Island 02903. E-mail: [email protected]. This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases/National Institutes of Health Grant R01

DK58148 and National Institute of Neurological Disorders and Stroke/ National Institutes of Health Grant R01 NS045231. Disclosure Summary: The author has nothing to disclose.

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