integration of neuroendocrine 1 signals that regulate the...

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Molecular Endocrinology, 2006: 1-24 ISBN: 81-308-0100-0 Editor: Patricia Joseph-Bravo

1 Integration of neuroendocrine signals that regulate the activity of hypophysiotropic peptides

Patricia Joseph-Bravo, Antonieta Cote-Vélez and Leonor Pérez-Martínez Departamento de Genética del Desarrollo y Fisiología Molecular, Instituto de Biotecnología, Universidad Nacional Autónoma de México (UNAM), Mexico

Abstract Hypophysiotropic peptides are those, that released fromhypothalamus, control the secretion of pituitary hormones.This review will discuss the different steps involved in regulating the amount of active peptide that reaches its target. Regulatory mechanisms participating in gene expression and post-transcriptional events, release, inactivation, and receptor binding will be analyzed for CRH and TRH. Emphasis will be on the current evidence of the mode of glucocorticoid interaction withtranscription factors modulating gene expression and mRNA stability, possible cross-talk between signalingpathways and, on homologous and heterologous regulationof receptor activity and inactivating processes.

Correspondence/Reprint request: Dr. Patricia Joseph-Bravo, Inst. Biotecnología, UNAM. A.P. 510, Cuernavaca MOR 62271, México. E-mail: [email protected]

Patricia Joseph-Bravo et al. 2

Hypothalamic neurons expressing hypophysiotropic peptides respond to changes of the hormonal milieu and decodify neuronal signals that convey information from several brain regions. These neurons release their products to the portal system that communicates the median eminence with the adenohypophysis, or anterior pituitary, where they control the synthesis and release of several protein hormones; they are thus envisioned as neuroendocrine transducers [1]. The name of these hypophysiotropic or releasing factors arose from the pituitary hormone they control, such as thyrotropin-releasing hormone (TRH) that stimulates the synthesis and release of thyrotropin; corticotropin-releasing hormone (CRH), that of adrenocorticotropin (ACTH); gonadotropin-releasing hormone (GnRH, or LHRH) responsible of luteinizing and folicule stimulating hormones release; growth hormone releasing hormone (GHRH), etc. [2]. All these peptides are synthesized, from larger protein precursors, in the rough endoplasmic reticulum (RER) and are compartamentalized, together with processing enzymes, in the trans-Golgi in vesicles of the regulated secretory pathway [3]. Although hypophysiotropic peptides were isolated from hypothalamus [1] they are also synthesized in other brain regions and some of them, in other organs. In particular, CRH and TRH are present in several hypothalamic nuclei and many regions of the limbic system [4-6]. The hypophysiotropic role of CRH and TRH is however confined only to neurons whose cell bodies are in the paraventricular nucleus of the hypothalamus (PVN) that send projections to the median eminence [7,8]. In response to stress, CRH is released and, through the control of ACTH release, provokes glucorticoids secretion from the adrenal gland [7, 9]; activation of CRH neurons constitutes the initial step in the cascade of hypothalamus-pituitary-adrenal axis (HPA) function. Similarly, TRH by releasing TSH that causes thyroid hormone release, is the main factor responsible for the hypothalamuspituitary-thyroid axis (HPT) function [10,11]. TRH controls also the synthesis and release of prolactin [11]. The release of hypophysial hormones is also modulated by inhibitory factors such as somatostatin (SRIF) and dopamine; another important secretagogue of ACTH is the vasopressin synthesized in the parvocellular neurons of PVN [2,12]. Adequate functioning of these two axes is essential for the organism survival due to their control of glucocorticoids and thyroid hormones, hormones involved in metabolism and energy homeostasis (see chapter 4), development, vascular and immune responses [13,14]. How are these axes regulated? Feeback effects of circulating hormonal levels of their target organ have long been recognized; adrenalectomy or thyroidectomy stimulates the synthesis and release of the hypothalamic and hypophysial hormones while administration of glucocorticoids or thyroid hormones, inhibits. Feedback effects are well observed in long-term paradigms. However, the effect for varying hormonal levels, after acute variations resulting from various stimulations, in tuning HPA or HPT axis, is not completely understood.

Regulation of CRH and TRH activity 3

Depending on the type of stress, whether physical or psychogenic, neurons from brain stem or limbic areas (respectively), impinge on CRH-ergic neurons of the PVN stimulating the synthesis and release of CRH [15,16]. The afferent inputs acting on the PVN will depend on the brain network activated by a particular stimulus. Several receptors for classical neurotransmitter and for neuropeptides have been detected in CRHergic neurons of the PVN: noradrenaline from brain stem nuclei, NPY/AgRP and GABA from the arcuate, local GABA and glutamatergic interneurons, CRH from amygdaloid nuclei and others yet to be characterized [17]. TRHergic neurons are activated in circumstances such as cold exposure, metabolic challenges, suckling, and as for CRH, receive inputs from several brain areas (NA, NPY, somatostatin, GABA) [1, chapter 4]. Peptides are released, by stimulus-secretion coupling, in response to action potentials increasing calcium influx [18].

Figure 1. Diagramatic representation of the pathways involved in modulating activity of a hypophysiotropic peptide, TRH. In the neuronal cell body, circle depicts nucleus, transcription factors GR and CREB that regulate gene expression; the precursor protein is synthesized in rough endoplasmic reticulum. Granules form in the trans-Golgi and travel through the axon, processed TRH is released from nerve terminals into portal vessels to reach the hypophysis, where TRH can bind to TRH receptor R-1 or, be degraded by peptidases present in blood or, PPII in membranes of pituitary cells.


Once released, they act on specific membrane receptors in the pituitary (CRH-R1 or TRH-R1) that belong to the class of G-protein coupled receptors (GPCRs), of seven transmembrane domains, and activate cascades of intracellular pathways modifying the synthesis and release of ACTH, TSH or prolactin [9,19,20]. At the pituitary level, the receptors themselves are regulated by their own ligand and other effectors. The amount of active ligand reaching the receptor can be limited due to inactivation by specific proteins present in blood, or in pituitary cells. In the case of CRH this is achieved by a secreted binding protein (CRH-BP) that competes with the receptor for binding the ligand, inhibiting its action [9,21]; TRH is in contrast inactivated by specific enzymes that cleave the pyroglutamyl residue (one, a membrane ectoenzyme pyrogultamyl aminopeptidase II [PPII]; the other, a soluble serum thyroliberinase [22 and chapter 3]). These inactivating proteins are also subject of regulation by their own ligands and other hormones. The amount of active peptide signaling their target cells will thus be the resultant of biosynthesis (gene expression, precursor processing), release and inactivation, as well as the amount of membrane receptor able to transduce the signal. (Fig. 1). Regulation of biosynthesis A. Transcriptional level Corticotropin releasing hormone CRH is a 41 aminoacid peptide [23] derived from a 196-aminoacid precursor, prepro-CRH, codified from a highly conserved gene that comprises two exons and one intron; pro-CRH sequence is all in exon 2 [24]. The promoter region in the 5’-flanking DNA sequence contains putative regulatory elements (CRE, AP-1, Brn-2, NGF1-B, GRE and ERE) where transcription factors bind (Fig. 2). The cAMP response element (CRE, 5’-TGACGTCA-3’), centered at –224 nucleotide (nt), affects basal promoter activity; cAMP-dependent PKA activation plays a mayor role regulating CRH expression, through phosphorylation of CREB (CREB-P) [25-27]. Several effectors increase the levels of CREB-P in CRH neurons; within minutes of stress stimulation (5-30 min), an increase in the primary transcript of CRH (heterologous nuclear RNA, hnRNA) is observed [9,28]. hnRNA is processed to mRNA that is translocated to the cytoplasm to be translocated into prepro-CRH in RER (Fig. 2). Glucocorticoids inhibit CRH gene expression specifically in the PVN; in other areas as amygdala or supraoptic nucleus, expression is upregulated or unaffected [25]. Considerable research has been performed trying to elucidate the mechanisms of negative glucocorticoid regulation [29]. Glucocorticoids bind to the mineralocorticoid receptor (MR) and to the glucocorticoid receptor (GR) [13]. Given the lower affinity of GR, this receptor is activated only with


Figure 2. Biosynthesis of CRH. Pre-pro CRH gene is illustrated in the diagram showing the position of some elements in the promoter that recognize transcription factors (ERE = estrogen receptor response elemen (re)t; CRE= CREB:cAMP binding protein re; GRE = glucocorticoid receptor GR re; AP-1 = activator protein). Sequences of both DNA strands for site AP1-GRE are depicted. Primary transcript (hnRNA) contains 2 exons; all the sequence of pre-pro-CRH is decodified by exon 2. pro-CRH is processed by the prohormone-convertases PC1 and/or PC2, followed by PCE and finally by PAM, leading to the active peptide of 41 aminoacids with carboxyterminal: ile-ile-NH2. the higher glucocorticoid levels induced by stress; in basal conditions, MR is normally saturated. GR is proposed to be responsible for the negative feedback effects; its mechanisms of activation are dependent on binding to corticosterone, similar to that described for progesterone receptors [13,29, and chapter 2]. The consensus sequence of GRE is AGAACAnnnTGTTC. In the CRH- promoter, sequences resembling half sites are found at –278 to –249 nt that when deleted, result in complete loss of glucocorticoid-dependent repression [30]. In this DNA fragment, the complementary strand presents two AP-1 like sequences (Fig. 2), [31]; the localization of GR and AP-1 binding within a single regulatory element is known as “composite GRE”, where a monomer of GR can heterodimerize with proteins of the AP-1 family as Jun [32]. The


negative effect of glucocorticoids is proposed to depend on direct binding to this site and, on the factors composing the heterodimer AP-1 (cFos, cJun family) that influence differential recruitment of coadaptors, which facilitate or inhibit histone acetylation, and interact with other transcription factors [31,32]. Other group proposes that the effect of GR does not involve direct binding to DNA, but through interactions with CREB bound protein (CRE site located at -228 to -221) [33,34]; transfected cells with CRH-promoter containing deleted and/or mutated potential GR-binding sites increases several fold pro-CRH mRNA levels in response to PKA stimulation but, co-incubation with dexamethasone (a glucocorticoid analog) represses cAMP induced stimulation [34]. A second CRE site is recognized between -213 and –99 bp containing an homeobox response element (CDXRE) at –125 to -118 bp that acts synergistically with factors binding to CRE in response to increased cAMP levels ([35]; this region can be stimulated by glucocorticoids (potential binding site of GR: -202 to -175). The effect of glucocorticoids on CRH gene expression can therefore be inhibitory, stimulatory or with no effect, depending on the particular interactions between transcription factors (some, tissue specific) and the different elements of the CRH promoter. Estrogen stimulation of pro-CRH transcription has been suggested to occur also through protein-protein interactions between estrogen receptors and other transcription factors or their associated complexes [36]; this estrogen effect could explain gender differences found in the stress response. How does this relate to in vivo situations? Conditions that activate HPA as ether exposure or, immobilization, show a transient increase in pro-CRH hnRNA. Using probes containing part of the intron sequence, the levels of recently synthesized RNA are reliably detected; they reflect the immediate changes in transcription, more accurately than measurements of pro-CRH mRNA levels due to the large existing pool of processed mRNA [28,37,38]. Five min of ether exposure increases ACTH, with a peak at 5 min, that is paralleled by increased levels of CRH hnRNA and CREB-P immunoreactivity; mRNA levels return to normal values by 2h. Only after 30 min, increased levels of c-fos mRNA were observed with increased Fos protein at 90 min [28,37]. The transient kinetics of increased expression is also observed after restrain, pro-CRH hnRNA peaks at 30 min and returns to basal levels by 90 min, despite 3h of restrain and sustained high corticosterone levels; this pattern is observed also in adrenalectomized animals replaced with low levels of corticosterone, suggesting that the transient effect in transcription is not due to increased corticosterone levels [39]. Some candidates of this transient regulation are nuclear proteins as ICER, that competes with CREB for transcriptional activators and its synthesis was increased in this paradigm [39]; however, it remains to be demonstrated that an increase of sufficient ICER protein occurs at 60 min, when pro-CRH hnRNA has almost returned to basal levels.


Thyrotropin releasing hormone TRH is a tripeptide derived from a 26 KDa protein precursor [40]. Pro-TRH is codified by one gene whose sequence has been characterized in human (h) [41], rat (r) [42] and mouse (m) [43]. From the three exons present, the first codify for the untranslated 5’ end of mRNA, the second, the sequences codifying for signal peptide and the third, to most of the precursor protein containing 5 repeated sequences of gln-his-pro-gly. In the rat promoter region, the sequence comprised between –547 to +84 confers almost fulltranscriptional activity in transfected cells and transgenic mice have revealed region +6/+84 of rTRH gene to be required for tissue-specific gene expression in vivo [44]. (Fig. 3) All species present response elements for the transcription factors AP-1, CREB, and glucocorticoid receptors (GR). Expression of pro-TRH mRNA is down-regulated by T3 directly in TRHergic cells, but only in PVN and not other hypothalamic nuclei [45-48]. The sequence –59/–52 [TGACCTCA] overlaps a thyroid hormone response element (THRE [AGGTCA]) and is recognized as an important site for thyroid hormone negative feedback (Fig. 3).

Figure 3. Biosynthesis of TRH. Some response elements of TRH gene promoter are depicted (abbreviations as in Fig. 2). Nucleotide sequence of both DNA strands in the region corresponding a composite GRE are shown. Insert corresponds to the final structure of TRH with the amidated carboxyl-end (after PAM) and the pyroglutamil moiety after cyclization of the glutamine by glutamyl cyclase (G.C.).


Transfection of CREB in HTB-11, NIH 3T3 or primary cultures of hypothalamic cells expressing luciferase under the control of hTRH promoter region -242/+58 evokes a 100-800% increase in promoter activity [43,49,50] and blocks negative inhibition by TRβ1-T3 complexes [51,52]. This site has been proposed to be also responsible for CREB stimulation [43,49]; however, we have shown that the site located at -101/-94, containing the conserved critical internal CpG dinucleotide (TGCCGTCA), has higher P-CREB binding from nuclear extracts of hypothalamic cultured cells [50]. CREB involvement on TRH gene transcription has been evidenced in transiently transfected cells expressing luciferase under the control of human or murine hTRH promoters; 8 h incubation with αMSH [49] (that increases phosphorylated CREB [P-CREB];[53]) enhances transcription. The stimulatory effect of αMSH is reduced in presence of triiodothyronine (T3) and transfected thyroid hormone receptor TRβ2 [49]. Glucocorticoids affect also TRH gene expression depending on the dose and length of treatment [54]; stimulated expression is observed after days of incubation with dexamethasone (dex) in CA77 cells [55], in rat anterior pituitary or in primary cultures of diencehalic [56,57], or hypothalamic cells [58] that is time and dose dependent (rapid induction at 1-3 h, 10-6 - 10-8 M; decrement at 10-10 M or, after 24h incubation at 10-8 M [54,58]). An inhibitory effect of glucocorticoids is observed in vivo [59]. Since upregulation of P-CREB follows glucocorticoid depletion [60] the inhibition of TRH transcription is suggested to be due to their inhibitory role on CREB phosphorylation in the PVN [61]. As for the CRH promoter, the one for TRH lacks a canonical GRE consensus; instead, several copies of the strongest half-site [TGTTCT] are located at: -735, -560, or - 210, an inverted half site at –284 [GGTCCAcacTCTTGT], and two other inverted half sites at -275 and –310 [42]. Hela cells transfected with -242/+84 sequence of rTRH promoter, or with deletions between -242/-200 revealed the importance or this region for basal transcription and glucocorticoid response; the site located at -210/-205 binds GR and is responsible for the stimulatory effect of dexamethasone (dex) [55]. This site has the characteristics of a composite GRE since the complementary strand has two sequences similar to AP-1 consensus element (Fig. 3). We have shown that the stimulatory effect of dex or 8Br-cAMP on primary hypothalamic cells is repressed when these drugs are coincubated [50,58], effect observed at the transcriptional level since this occurs also in transfected cells [50]. The stimulatory effect of 8Br-cAMP is mimicked by forskolin treatment or noradrenaline (NA). Furthermore, the binding to oligonucleotides containing the CRE-2 or cGRE, of proteins from nuclear extracts of hypothalamic cells incubated in presence of dex and NA, was diminished compared to that of extracts form stimulated cells with only NA or dex; interference was higher for cGRE at short incubation times (1h) [50].


Other sequences in TRH gene are important regulatory regions. Increased transcription of mouse TRH gene in GH4C1, due to epidermal growth factor, involves sequences -254/-218 and the SP1 site at -130/-83 [62]. A Stat responsive region (-150/-125 in hTRH promoter) that interacts cooperatively with SP-1 mediates leptin receptor signaling [49,63]. As can be seen, TRH share several common features with CRH. Both peptides are synthesized in the parvocellular cells of the PVN and receive noradrenergic afferents that decodify several stressful situations that cause their release. Concomitant to the increased release, increased transcription occurs dependent on increased PKA activity. In vitro, CREB-dependent stimulation can be interfered by glucocorticoids. In the case of pro-TRH, we have also observed a rapid and transient increase of mRNA levels after animals are exposed to cold temperature that releases TSH and corticosterone, mRNA levels increase 2 fold at 60 min, returning to basal levels after 2h, even though the animals remained for 6h at 4°C [64,65]. This contrasts the in vitro situation since pro-TRH mRNA levels remained high in hypothalamic cell cultures stimulated with 8Br-cAMP for several hours [66]. The dynamics of interaction within different intracellular pathways, being activated by different effectors, whether neuronal or hormonal, might be crucial to explain the transient nature of mRNA changes after in vivo stimulation. Hypothalamic primary cultures from rat fetal brain [50,58,67] incubated for 10 min with 10-9M dex, followed by 50 min of NA 10-8M (leaving dex in the incubation medium) produced the same increase as 60 min incubation with NA alone; however, if the order was inverted (10 min of incubation with NA preceded addition of dex), the stimulatory effect was significantly diminished (Fig.4). This points for a fine tuning in the kinetics of the response, where activated transcription factors should probably meet in a precise space and time. Several caveats most be considered in all in vitro studies using transfected cells. An important one is that in vivo, the first event in transcription requires chromatin remodeling, promoted by histone acetylation that releases the DNA from nucleosomes [68]. The compact structure of DNA cannot guarantee that all transcription factors bind to equal efficacy to the gene. This can explain some discrepancies found, between transfected cells vs. primary cultures, in studying the effects of drugs and interaction of transcription factors [50]. Although hypothalamic cultures resemble more this in vivo situation, they also present the inconvenience of having fetal cells as starting material, so the presence of the adequate proportions of intracellular messengers might differ. Additionally, since it is extremely difficult to dissect specific nuclei, as the PVN, from fetal fresh rat brain, the population of TRHergic neurons in culture is mixed with other nuclei that also express TRH (dorsomedial, preoptic area, suprachiastmatic, lateral and anterior hypothalamus) [69]. These paradigms, however, have the advantage of controlling the effectors under study and


provide useful information since in vivo situations have the overall effects of the animal response. Studies with knock out animals also provide important information regarding the relevance of certain molecules but have also shown that pathways and signaling molecules have redundant functions and the effect imposed during development can alter the overall outcome. Conclusions regarding regulation of transcription must thus consider each strategy with caution but, despite their reductionism approach, the ensemble of various experimental designs, can eventually help to elucidate the mechanisms involved.

Figure 4. Effect of noradrenaline (NA) and dexamethasone (Dex, D) on the relative levels of pro-TRH mRNA. Hypothalamic primary cultures of 17 day old rat embryos were incubated for 14 days in Dulbecco modified Eagle’s medium containing 10% fetal bovine serum and supplements described in [67]. On day 14th, half the medium was replaced with fresh medium containing NA (10-8 M) or Dex (D; 10-9M) and incubated for 1h; plates corresponding to the fourth column (D-NA) were incubated for 10 min with dex followed by addition of an aliquot containing NA; the order was inversed in those plates used for column 5. After 1h incubation, plates were rinsed and RNA was extracted from cells. 1ug of RNA was used for RT-PCR semi-quantification of pro-TRH mRNA levels (expressed as the ration of pro-TRH/G3PDH cDNA signal) [50]. n = 6; * p<0.05. Top left: gel of RT-PCR products; upper band =G3PDH, lower band= TRH cDNA. Lower left: Photograph of neuronal culture at 12 DIV (Nomarsky contrast, 20X). Post-transcriptional regulation Increased transcription does not necessarily reflect increased protein or, increased active peptide. After hnRNA is processed in the nucleus and transported to the cytoplasm, the levels of mRNA can be modulated by the rate of its degradation. mRNA stability is influenced by the length of poly(A) tail, the activity of RNA-degrading enzymes (RNAses) and the levels of proteins interacting with mRNAs or with RNAses themselves [70]. Measurement of


these processes is very difficult in vivo but using hypothalamic incubates of previously adrenalectomized rats it has been shown that glucocorticoids transiently increase CRH transcription while decrease the rate of mRNA degradation [71]. Glucocorticoids and other hormones can also decrease mRNA stability of other proteins and tissues although the particular mechanisms might differ. B. Processing of the precursor protein Once translated, the precursor protein is processed. Within the precursor, the sequence that leads to the active peptide is flanked by a pair of basic aminoacids (Lys or Arg); the initial cleavage occurs by the action of prohormone convertases PC1 and/or PC2 at the amino-side of the basic pair followed by their cleavage by carboxypeptidase E. In the case of pro-CRH, whose sequence is present at the last 41 aminoacids of the carboxy terminal of the precursor, only one cleavage is required (Fig.2). For pro-TRH whose first aminoacid is a pyroglutamyl, this corresponds to a glutamine in the precursor that is converted to pyroglutamyl by a glutamyl cyclase. CRH and TRH are both amidated at the carboxyl end; the amino group of a glycine in the precursor is escinded by the peptidylglycine alpha-amidating monooxygenase (PAM) [72]. TRH precursor contains 5 times the gln-his-pro-gly sequence (Fig. 3); in vitro experiments have demonstrated an order sequential cleavage that in some tissues is not complete [73,74]; and this could be due to a differential distribution [75] and/or concentration of PC2 and PC1. A total of seven different peptides of the sequences comprised between the TRH ones arise from this processing and some, like the one present in pro-TRH-(178-199) has physiological actions such as inhibition of ACTH release [76]. The posttranslational processing of hormone precursor proteins is a required step of biological function but can also provide diversity (as for example, POMC processing that yields ACTH and other peptides of different functions: β-lipotropin and β-endorphins [77]). Processing can initiate with the convertases present in RER and golgi [74]. Aggregation of several molecules: the precursor (pro-peptide),convertases, carboxypeptidase, PAM and, in the case of pro-TRH precursor, also pyroglutamyl cyclase, takes place in the trans-Golgi. One of the most accepted theories is that this sorting occurs by signal receptors, that direct trafficking towards the secretory granules of the regulated secretory pathway, being carboxypeptidase E an important one [78,79]. The appropriate amount and activity of these enzymes is thus required to yield adequate processing inside granules. Evidence for coordinated regulation of mRNAs of the precursor and these enzymes is still scarce; selective regulation of PC1 is mediated by glucocorticoids in CRH producing cells [80]; glucocorticoids also induce differential processing of pro-TRH in primary cultures of pituitary cells [74]. Once the enzymes and precursor are in the secretory granule it is


unknown if enzyme activity can be modulated by regulating co-factor concentration inside the granules (Ca++, Cu++ or ascorbic acid for PAM activity [72], or changes in pH, for example). Granules travel from the neuronal soma to the nerve terminal in the median eminence (for hypophysiotropic neurons) at an estimated rate of 5-7 mm/h [81]; in rat, this distance is approximately 3-5 mm [82] so this process would take at least 30 min. Although particular measurements have not been performed it is estimated, by analogy with other systems, that it would take at least 90 min, for a peptide generated by a stimulus-activated gene transcription, to reach the nerve terminal in the median eminence. The kinetics of biosynthesis compared to that of release, in response to a neural stimulus, suggests that stimulus-induced secretagogue gene expression is essentially a recuperative or adaptive action that cannot affect HPA secretory events of short duration [83]. The ideal situation would be to measure the released peptide and increased expression at short times after stimulation; unfortunately this is extremely difficult in vivo (long times of perfusion are sometimes required due to limits of detection sensitivity and, the danger of tissue damage, or the use of anesthetics). Furthermore, not all effectors activating the release of CRH or TRH affect their biosynthesis. Hypothalamic cultures incubated with NA show increased TRH release and mRNA levels while with dexamethasone, only biosynthesis is affected; effects on intracellular peptide levels are detected only after 2h of incubation [50]. The rate of mRNA increase will also depend on the particular intracellular pathways that are activated; for example, phorbol esters increase pro-TRH mRNA levels only after 2h probably depending on c-fos translation in contrast to increased cAMP levels where effects are seen since 30 min [66], coincident with the fast phosphorylation of existing CREB. Neurotransmitters affecting the release at the level of the median eminence, impinging at the nerve terminal, will unlikely affect CREB phosphorylation or other transcription factors directly at the soma and if so, would require slow retrograde communication. In conclusion, although increased levels of mRNA not necessarily lead to increased peptide concentration, when it does, influences posterior events. However, since several of the effectors that cause release, also induce a fast and transient increase of mRNA levels their measurement can reflect activation the neuropeptidergic neuron. Regulation of biological effects A. State of the receptor The biological activities of effector molecules depend on their binding to specific receptors and the intracellular signaling processes. Regulation of receptor number and sensitivity are determinant for cell responsiveness and this is regulated by receptor synthesis, post-translational processing and, targeting to the membrane, as well as the rate of receptor desensitization and


internalization following interaction with the ligand. Desensitization and internalization provides a fast mechanism (seconds) for GPCR regulation of responsiveness; this involves the uncoupling of receptors from their heterotrimeric G proteins, desensitization by phosphorylation and, the internalization (sequestration) of receptors to endosomes that can be reincorporated to the membrane or, targeted to lysosomes for degradation [84]. Two receptors have been characterized for CRH: CRH-R1 and CRH-R2 being the former the main subtype in pituitary corticotrops [19]. Upon CRH binding on CRH-R1, cAMP levels increase, ACTH is released within minutes, and POMC mRNA increases. These processes are both inhibited by glucocorticoids and the velocity of this effect on release (30 min) cannot account for effects on biosynthesis of intracellular proteins but more likely, on pituitary membrane proteins [85]. CRH-R1 binds to CRH with high affinity; the receptor is linked to the cAMP-dependent PKA signaling, and is modulated by divalent cations and guanydil nucleotides; down regulation occurs, only in pituitary, in response to adrenalectomy, corticosterone treatment, and chronic stress [85-87]. CRH-R1 also recognizes sauvagine and urotensin I that stimulate ACTH release in amphibian and fish respectively [19]. Homologous regulation of CRH-R1 occurs at several steps: it is desensitized by phosphorylation which leads to down-regulation; and at the transcriptional and posttranscriptional level. CRH decreases CRH-R1 mRNA in a sustained way in in vitro conditions but only transiently in vivo, where CRH injections or acute stress decrease CRHR1 mRNA levels after 2h, that recover to normal or even increase, by 4h. Translational efficacy of CRH-R1 mRNA is modulated, in pituitary, by regulation of specific binding proteins to complexes of the 5’-untranslated region (UTR). Heterologous regulation is also observed by other effectors regulating PKC-induced phosphorylation of the receptor or, by glucocorticoids [19,86,87]. Adrenalectomy induces a marked down-regulation and desensitization of pituitary CRH receptors, decreasing receptor number and CRH-stimulated adenylate cyclase [87]. Several stress paradigms have shown certain discrepancy between receptor number and desensitization of ACTH responses suggesting that, although required, CRH-R1 receptor number is not a major determinant of corticotroph responsiveness since a small number of receptors is sufficient for full ACTH responses. However, the different modes of regulation permit the rapid changes in CRH receptor content that are necessary to adapt to physiological demands during stress or other conditions [19,86-87]. Two specific receptors with similar binding and affinity characteristics for TRH are expressed in brain, being TRH-R1 the one preferentially found in pituitary, localized in lactotropes and thyrotropes [88]. TRH binds to its receptor in these cells and stimulates PKC and phosphoinositidase activity, leading to increased intracellular calcium, allowing prolactin or TSH release.


Upon ligand binding, receptors are internalized through clathrincoated pits; as for other GPCRs, the C terminal domain interacts with β-arrestin that in turn interacts with clathrin, receptors cluster and undergo endocytosis [88,89]. Pituitary cells exposed for 5 min to TRH show up to 80 % sequestration of the complex of TRH and its receptor [90]; internalization of TRH receptor occurs with a half-time of about 2-3 min; the receptor must be resensitized to respond to a second challenge and this occurs after the ligand dissociates (a slower process due to their high nanomolar affinities). Recycling of the receptor to the membrane occurs, although a small fraction may be sorted to the lysosomal pathway and degraded with each round of endocytosis, lowering the concentration of receptors in the membrane (receptor down regulation) [91]. The pulsatile nature of hypothalamic release avoids the strong down regulation observed when cells are incubated for long time with the agonist or, high concentrations are injected to the animal that can lead to 50-75% receptor loss. Receptor concentration is also defined by its rate of synthesis; transcription is regulated, as seen for the neuropeptides, by activated transcription factors on multiple consensus sequences of each receptor’s promoter, and by regulating mRNA stability. TRH incubation on GH3 cells (a pituitary derived cell line that expresses TRH-R1 receptor and releases prolactin and growth hormone) causes degradation of TRH-R1 mRNA by direct activation of RNAse activity; diminished mRNA levels are observed since 10 min (peak at 30 min). The proposed mechanism involves PKC activation probably phosphorylating a regulatory protein of the RNAse system [92]. The effect of TRH on mRNA degradation requires the 3’ of the TRH-R mRNA that has a AU-rich domain involved in stabilization-destabilization of mRNAs, as well a specific region in the 5’ untranslated region (between nucleotides 3363-3453), that forms a stem like structure that could be recognized by specific proteins, leading to the rapid specific degradation observed after TRH stimulation [92]. Unexpected findings were obtained in transfected HEK-293 cells with full length or C-terminal truncated TRH-R1 cDNA, tagged with green fluorescent protein, that were stimulated with TRH. TRH-R1 fusion protein was upregulated (3-7 fold) after 8 h of incubation with TRH or with PKC activators but the effect was not observed in presence of a proteasome inhibitor suggesting that the receptor protein was prevented from degradation via proteasome [93]. A small (2 fold) increase in mRNA receptor levels was detected after 18h incubation [93]; shorter times are required to distinguish whether cell specificity accounts for the apparent contradiction between increase and decreased mRNA levels or, parallel effects on transcription and mRNA stability occur depending on the time and length of stimulation. Heterologous regulation is observed with hormonal treatments. Thyroid hormones, either in vitro or in vivo, decrease TRH-binding to pituitary membranes [94-95]. TRH-R mRNA levels decrease in pituitary of animals


treated with 0.5 µg T3/100 g BW with a dose-response maximal at 6 µg after 6h; PTU-induced hypothyroidism increased levels only after 4 days of treatment [96]. Consistent with TRH role in prolactin regulation, steroid hormones are also capable of regulating TRH receptor binding and mRNA levels [97]. B. Ligand inactivation Removal of the ligand is a generalized process that contributes to the cell recovery from stimulation and can also influence the amount of active ligand. Dilution in the extracellular medium reduces the concentration of most agonists but specific mechanisms have evolved for different ligands [98]. In the case of neuropeptides, the most common mechanism involves degradation by ectopeptidases of wide specificity. CRH and TRH are particular examples since the mechanism for reduction of CRH biological activity involves a specific binding protein, distinct from the receptors; for TRH, an ectoenzyme with very narrow specificity is involved in its inactivation. CRH binding protein (CRH-BP) is a 37 kDA secreted glycoprotein that binds CRH with equal or greater affinity than CRH-R1 receptor (also binds urocortin). It is synthesized in several brain areas colocalized with CRH or release sites and, in pituitary corticotropes. PKA or PKC activators regulate CRH-BP synthesis and release in cell cultures. Restrain stress or adrenalectomy increase or decrease (respectively) CRH-BP mRNA levels in pituitary. Transgenic mice with increased CRH-BP expression in pituitary or knock out constructs that avoid its expression, showed altered HPA activity and behaviors related with modified CRH levels in the limbic system; the ensemble of these results support the role of this binding protein as a negative regulator of CRH activity [9,21,99]. What remains unknown is the fate of this secreted protein and bound CRH in the extracellular fluid. Although several enzymes capable of degrading TRH in homogenates were described, it is now recognized that the soluble intracellular enzymes do not regulate TRH content in the tissue, or released by in vitro cell fragments [22]. In contrast, a specific ectoenzyme has been characterized that degrades released TRH [22]. An enzyme present in blood called tyroliberinase, has the same biochemical characteristics as PPII and is proposed to arise from the same gene [100]. Both enzymes are subject to stringent regulation. TRH down regulates thyroliberinase and PPII activities, present in blood and pituitary membrane respectively, as well as PPII expression, while thyroid hormones have a stimulatory effect [22,101-103]. In the pituitary, hormonal regulation of PPII seams to mirror that of the receptor since estrogens also present the opposite mode, having a negative influence on PPII while positive on TRH-receptor [104]. (A detailed review of PPII physiology is described in chapter 3).


Conclusions and perspectives Most of the steps involved in defining the amount of ligand able to exert a physiological response appear to be susceptible to regulation. As for most proteins, CRH and TRH biosynthesis is regulated at multiple steps: 1) at the transcriptional level, by interacting nuclear protein factors modulating gene expression; 2) at the level of translation affecting hnRNA processing, exit to cytoplasm, and mRNA stability; and, 3) processing of the precursor. Although there are not precise examples in regard to alternate mechanisms of biosynthesis regulation of hypophysiotropic peptides, recent findings open new possibilities. One of them is the discovery of non-coding RNAs, microRNAs of ~22nt double stranded RNA molecules that can repress translation, or target mRNA degradation; they seem essential regulators of CNS development but might regulate differential transcription in adult brain, in response to neuronal or hormonal effectors (such as increased CREB-P) [105,106]. The other, is the recognition of epigenetic factors; the influence of the perinatal environment in determining maternal behaviour and stress response at adult stages which are then inherited to their offspring for generations. Epigenetic mechanisms involve DNA silencing through the attachment of methyl groups that impede access to transcriptional factors; these patterns of methylation are inherited to the offspring. One important gene, involved in regulation of many others, is the one codifying for GR; some of its regulatory regions show differential degree of methylation [107]. Recognition of the several targets that can be modified, will open new avenues in understanding transcriptional regulation. In summary, the ability of CRH or TRH to act on pituitary target cells depends on the state of their receptor, the amount of protein present in the membrane that can present homologous regulation by previously released ligand and, by the hormonal milieu the animal is exposed to. The concentration of ligand able to bind to the receptor at thepituitary level is regulated in turn by the amount released from the hypothalamus minus the amount inactivated although, lowering ligand concentration could have a dual role, by allowing receptor recovery by inactivating the dissociated ligand from the receptor. The particular effectors involved in regulating each step are just beginning to be recognized but it is evident that more than one signaling molecule can participate and, the same signaling molecule can regulate multiple steps. An example of the later case is the effect of glucocorticoids that up or down regulate TRH and CRH mRNA levels, depending on the dose, time, and tissue; at transcriptional level and regulating mRNA stability, as well as the expression of processing enzymes. Still unknown is if increased biosynthesis leads to an increased release for the following stimulus or it is a process


oriented towards replenishment of lost pools. Furthermore, at a given moment, the peptidergic neuron most decodify a multiplicity of neuronal signals, activated by the changing environment (temperature, stress, feeding [chapter 4]) which are in turn modulated by developmental influences, immediate previous activity, the existing hormonal status, and be susceptible to the pituitary response, also determined by previous influences regulating their receptor status. With all these considerations it is fascinating to understand what the terms homeostasis and allostasis [108] stand for; but also, to consider how many things can go wrong that lead to disease, and envisage new therapies. Acknowledgements The authors want to acknowledge the enthusiastic participation of students and staff that conform the group of Molecular Neuroendocrinology: R.M. Uribe, M.Y. Díaz, A., E. Sánchez, Aguilar-Valles, M.Gutiérrez-Mariscal, A.I.García, V. Trujillo, D. Rebolledo, A. Tusié, C.E. Ramírez, F. Romero, M. Villa, E. Martel. We thank the constant support of the personnel of the administration, library, animal house, and cybernetic, services. Projects have been financed by CONACYT and DGAPA. References 1. Joseph-Bravo, P. 2004, Hypophysiotropic TRH neurons as transducers of energy homeostasis. Endocrinology 145, 4813-4815. 2. Guillemin, R. 2005 Hypothalamic hormones a.k.a. hypothalamic releasing factors.

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