The Thyrotropin-Releasing Hormone-Degrading Ectoenzyme, a Therapeutic Target?

Thyrotropin releasing hormone (TRH: Glp-His-Pro-NH2) is a peptide mainly produced by brain neurons. In mammals, hypophysiotropic TRH neurons of the paraventricular nucleus of the hypothalamus integrate metabolic information and drive the secretion of thyrotropin from the anterior pituitary, and thus the activity of the thyroid axis. Other hypothalamic or extrahypothalamic TRH neurons have less understood functions although pharmacological studies have shown that TRH has multiple central effects, such as promoting arousal, anorexia and anxiolysis, as well as controlling gastric, cardiac and respiratory autonomic functions. Two G-protein-coupled TRH receptors (TRH-R1 and TRH-R2) transduce TRH effects in some mammals although humans lack TRH-R2. TRH effects are of short duration, in part because the peptide is hydrolyzed in blood and extracellular space by a M1 family metallopeptidase, the TRH-degrading ectoenzyme (TRH-DE), also called pyroglutamyl peptidase II. TRH-DE is enriched in various brain regions but is also expressed in peripheral tissues including the anterior pituitary and the liver, which secretes a soluble form into blood. Among the M1 metallopeptidases, TRH-DE is the only member with a very narrow specificity; its best characterized biological substrate is TRH, making it a target for the specific manipulation of TRH activity. Two other substrates of TRH-DE, Glp-Phe-Pro-NH2 and Glp-Tyr-Pro-NH2, are also present in many tissues. Analogs of TRH resistant to hydrolysis by TRH-DE have prolonged central efficiency. Structure-activity studies allowed the identification of residues critical for activity and specificity. Research with specific inhibitors has confirmed that TRH-DE controls TRH actions. TRH-DE expression by ß2-tanycytes of the median eminence of the hypothalamus allows the control of TRH flux into the hypothalamus-pituitary portal vessels and may regulate serum thyrotropin secretion. In this review we describe the critical evidences that suggest that modification of TRH-DE activity in tanycytes, and/or in other brain regions, may generate beneficial consequences in some central and metabolic disorders and identify potential drawbacks and missing information needed to test these hypotheses.

A Glial-Neuronal Circuit in the Median Eminence Regulates Thyrotropin-Releasing Hormone-Release via the Endocannabinoid System.

Based on the type-I cannabinoid receptor (CB1) content of hypophysiotropic axons and the involvement of tanycytes in the regulation of the hypothalamic-pituitary-thyroid (HPT) axis, we hypothesized that endocannabinoids are involved in the tanycyte-induced regulation of TRH release in the median eminence (ME). We demonstrated that CB1-immunoreactive TRH axons were associated to DAGLα-immunoreactive tanycyte processes in the external zone of ME and showed that endocannabinoids tonically inhibit the TRH release in this tissue. We showed that glutamate depolarizes the tanycytes, increases their intracellular Ca2+ level and the 2-AG level of the ME via AMPA and kainite receptors and glutamate transport. Using optogenetics, we demonstrated that glutamate released from TRH neurons influences the tanycytes in the ME. In summary, tanycytes regulate TRH secretion in the ME via endocannabinoid release, whereas TRH axons regulate tanycytes by glutamate, suggesting the existence of a reciprocal microcircuit between tanycytes and TRH terminals that controls TRH release.

A screen for modulators reveals that orexin-A rapidly stimulates thyrotropin releasing hormone expression and release in hypothalamic cell culture.

In the paraventricular nucleus of the mammalian hypothalamus, hypophysiotropic thyrotropin releasing hormone (TRH) neurons integrate metabolic information and control the activity of the thyroid axis. Additional populations of TRH neurons reside in various hypothalamic areas, with poorly defined connections and functions, albeit there is evidence that some may be related to energy balance. To establish extracellular modulators of TRH hypothalamic neurons activity, we performed a screen of neurotransmitters effects in hypothalamic cultures. Cell culture conditions were chosen to facilitate the full differentiation of the TRH neurons; these conditions had permitted the characterization of the effects of known modulators of hypophysiotropic TRH neurons. The major end-point of the screen was Trh mRNA levels, since they are generally rapidly (0.5-3h) modified by synaptic inputs onto TRH neurons; in some experiments, TRH cell content or release was also analyzed. Various modulators, including histamine, serotonin, ß-endorphin, met-enkephalin, and melanin concentrating hormone, had no effect. Glutamate, as well as ionotropic agonists (kainate and N-Methyl-d-aspartic acid), increased Trh mRNA levels. Baclofen, a GABAB receptor agonist, and dopamine enhanced Trh mRNA levels. An endocannabinoid receptor 1 inverse agonist promoted TRH release. Somatostatin increased Trh mRNA levels and TRH cell content. Orexin-A rapidly increased Trh mRNA levels, TRH cell content and release, while orexin-B decreased Trh mRNA levels. These data reveal unaccounted regulators, which exert potent effects on hypothalamic TRH neurons in vitro.

Glucocorticoids curtail stimuli-induced CREB phosphorylation in TRH neurons through interaction of the glucocorticoid receptor with the catalytic subunit of protein kinase A.

PURPOSE: Corticosterone prevents cold-induced stimulation of thyrotropin-releasing hormone (Trh) expression in rats, and the stimulatory effect of dibutyryl cyclic-adenosine monophosphate (dB-cAMP) on Trh transcription in hypothalamic cultures. We searched for the mechanism of this interference. METHODS: Immunohistochemical analyses of phosphorylated cAMP-response element binding protein (pCREB) were performed in the paraventricular nucleus (PVN) of Wistar rats, and in cell cultures of 17-day old rat hypothalami, or neuroblastoma SH-SY5Y cells. Cultures were incubated 1h with dB-cAMP, dexamethasone and both drugs combined; their nuclear extracts were used for chromatin immunoprecipitation; cytosolic or nuclear extracts for coimmunoprecipitation analyses of catalytic subunit of protein kinase A (PKAc) and of glucocorticoid receptor (GR); their subcellular distribution was analyzed by immunocytochemistry. RESULTS: Cold exposure increased pCREB in TRH neurons of rats PVN, effect blunted by corticosterone previous injection. Dexamethasone interfered with forskolin increase in nuclear pCREB and its binding to Trh promoter; antibodies against histone deacetylase-3 precipitated chromatin from nuclear extracts of hypothalamic cells treated with tri-iodothyronine but not with dB-cAMP + dexamethasone, discarding chromatin compaction as responsible mechanism. Co-immunoprecipitation analyses of cytosolic or nuclear extracts showed protein:protein interactions between activated GR and PKAc. Immunocytochemical analyses of hypothalamic or SH-SY5Y cells revealed diminished nuclear translocation of PKAc and GR in cells incubated with forskolin + dexamethasone, compared to either forskolin or dexamethasone alone. CONCLUSIONS: Glucocorticoids and cAMP exert mutual inhibition of Trh transcription through interaction of activated glucocorticoid receptor with protein kinase A catalytic subunit, reducing their nuclear translocation, limiting cAMP-response element binding protein phosphorylation and its binding to Trh promoter.

Focal adhesion kinase is required for actin polymerization and remodeling of the cytoskeleton during sperm capacitation.

Several focal adhesion proteins are known to cooperate with integrins to link the extracellular matrix to the actin cytoskeleton; as a result, many intracellular signaling pathways are activated and several focal adhesion complexes are formed. However, how these proteins function in mammalian spermatozoa remains unknown. We confirm the presence of focal adhesion proteins in guinea pig spermatozoa, and we explore their role during capacitation and the acrosome reaction, and their relationship with the actin cytoskeleton. Our results suggest the presence of a focal adhesion complex formed by ß1-integrin, focal adhesion kinase (FAK), paxillin, vinculin, talin, and α-actinin in the acrosomal region. Inhibition of FAK during capacitation affected the protein tyrosine phosphorylation associated with capacitation that occurs within the first few minutes of capacitation, which caused the acrosome reaction to become increasingly Ca(2+) dependent and inhibited the polymerization of actin. The integration of vinculin and talin into the complex, and the activation of FAK and paxillin during capacitation, suggests that the complex assembles at this time. We identify that vinculin and α-actinin increase their interaction with F-actin while it remodels during capacitation, and that during capacitation focal adhesion complexes are structured. FAK contributes to acrosome integrity, likely by regulating the polymerization and the remodeling of the actin cytoskeleton.

Voluntary exercise adapts the hypothalamus-pituitary-thyroid axis in male rats.

The hypothalamic-pituitary thyroid (HPT) axis modulates energy homeostasis. Its activity decreases in conditions of negative energy balance but the effects of chronic exercise on the axis are controversial and unknown at hypothalamic level. Wistar male rats were exposed for up to 14 days to voluntary wheel running (WR), or pair-feeding (PF; 18% food restriction), or to repeated restraint (RR), a mild stressor. WR and RR diminished food intake; body weight gain decreased in the 3 experimental groups, but WAT mass and serum leptin more intensely in the WR group. WR, but not RR, produced a delayed inhibition of central markers of HPT axis activity. At day 14, in WR rats paraventricular nucleus-pro-TRH mRNA and serum TSH levels decreased, anterior pituitary TRH-receptor 1 mRNA levels increased, but serum thyroid hormone levels were unaltered, which is consistent with decreased secretion of TRH and clearance of thyroid hormones. A similar pattern was observed if WR animals were euthanized during their activity phase. In contrast, in PF animals the profound drop of HPT axis activity included decreased serum T3 levels and hepatic deiodinase 1 activity; these changes were correlated with an intense increase in serum corticosterone levels. WR effects on HPT axis were not associated with changes in the activity of the hypothalamic-pituitary adrenal axis, but correlated positively with serum leptin levels. These data demonstrate that voluntary WR adapts the status of the HPT axis, through pathways that are distinct from those observed during food restriction or repeated stress.

The acute response of the amygdalar TRH system to psychogenic stressors varies dependent on the paradigm and circadian condition.

Central administration of thyrotropin releasing hormone (TRH) reduces anxiety; amygdalar TRH expression is inversely proportional to the anxious behavior displayed in the elevated plus maze performed during the dark phase (EPM-D). To better understand the role of TRH in amygdala function, we evaluated the expression of TRH and the elements involved in its transmission in various stressful paradigms and how they associated with behavior. Wistar male rats were exposed to restraint (RES), EPM, or the open field test (OFT) and sacrificed 0-60 min afterwards; OFT, RES and EPM were performed during the light (L), and OFT during the dark phase. Restraint increased amygdalar levels of proCRH mRNA, without change in proTRH. All paradigms augmented corticosterone release, highest after OFT-L that also enhanced proCRH mRNA levels and decreased those of proTRH. OFT-D activated the TRH system. Levels of anxiety or locomotion were similar in animals tested in light or dark phases but their association with biochemical parameters differed. ProTRH expression and TRH release correlated positively with decreased anxiety in EPM-L and in OFT-D. No association with anxiety was detected in OFT-L where proCRH and proTRH expression correlated with locomotion supporting their involvement in arousal. The responses of TRH amygdalar systems appeared modulated by the extent of the stress response and by the circadian conditions. Increased proTRH expression of animals exposed to OFT-D was specifically observed in the cortical nucleus of the amygdala, area involved in processing fear stimuli; these TRH neurons may thus be part of a circuit with anxiolytic properties.

Creb and Sp/Krüppel response elements cooperate to control rat TRH gene transcription in response to cAMP.

Expression of hypophysiotropic TRH, that controls thyroid axis activity, is increased by cold exposure; this effect is mimicked in rat hypothalamic cells incubated with norepinephrine or cAMP analogs. TRH proximal promoter contains three putative CRE: Site-4 or CRE-1 that overlaps an element recognized by thyroid hormone receptors, CRE-2 with adjacent sequences GC box or CACCC recognized by Sp/Krüppel factors (extended CRE-2), and AP-1 sites flanking a GRE(1/2). To evaluate the role of each element in the cAMP response, these sites were mutated or deleted in rat TRH promoter linked to luciferase gene (TRH-luc) and co-transfected with ß-gal expression vector in various cell lines; C6 cells gave the highest response to forskolin. Basal activity was most affected by mutations or deletion of CRE-2 site, or CACCC (50-75% of wild type-WT). Forskolin-induced 3× stimulation in WT which decreased 25% with CRE-1 or AP-1 deletions, but 50% when CRE-2 or its 5' adjacent GC box was altered. SH-SY5Y cells co-transfected with CREB-expression vector increased dB-cAMP response in the wild type but not in the CRE-2 mutated plasmid; cotransfecting CREB-A (a dominant negative expression vector) strongly diminished basal or cAMP response. Primary cultures of hypothalamic cells transfected with plasmids containing deletions of CRE-1, CRE-2, or extended CRE-2 failed to respond to forskolin when CRE-2 was modified. These results corroborate the CRE-2 site as the main cAMP-response element of rat TRH promoter, not exclusive of transcription factors of hypothalamic cells, and stress the relevance of adjacent Sp-1 sites, important mediators of some metabolic hormones.

The systemic inhibition of nitric oxide production rapidly regulates TRH mRNA concentration in the paraventricular nucleus of the hypothalamus and serum TSH concentration. Studies in control and cold-stressed rats.

Neurons of the paraventricular nuclei of the hypothalamus (PVN) that synthesize the peptide thyrotropin releasing hormone (TRH) control energy homeostasis. Identifying the circuits which regulate these neurons is critical to fully understand integration of metabolic information and the mechanisms that set thyroid hormone levels. We tested the hypothesis that nitric oxide (NO) acutely controls PVN TRH expression and thyrotropin (TSH) secretion by the anterior pituitary. The subcutaneous treatment of rats with N(G)-nitro-L-arginine methyl ester (L-NAME), an inhibitor of NO synthases, enhanced PVN TRH mRNA and medio-basal hypothalamic TRH levels, and reduced serum TSH concentration. Analysis of the effect of a NO donor in primary cultures of hypothalamic or anterior pituitary cells suggested that the effect of NO includes a direct action on hypothalamic neurons. The cold stress-induced increase in TSH release was inhibited by sc L-NAME. Therefore, production of NO may control the activity of the hypothalamus-pituitary-thyroid axis.

Involvement of CRH-R2 receptor in eating behavior and in the response of the HPT axis in rats subjected to dehydration-induced anorexia.

Wistar rats subjected to dehydration-induced anorexia (DIA), with 2.5% NaCl solution as drinking water for 7 days, decrease by 80% their food intake and present some changes common to pair-fed food restricted rats (FFR) such as: weight loss, decreased serum leptin and expression of orexigenic arcuate peptides, increasing the anorexigenic ones and serum corticosterone levels. In contrast, the response of the HPT axis differs: DIA animals have increased TRH expression in PVN and present primary as opposed to the tertiary hypothyroidism of the FFR. Exclusive to DIA is the activation of CRHergic neurons in the lateral hypothalamus (LH) that project to PVN. Since TRH neurons of the PVN contain CRH receptors, we hypothesized that the differences in the response of the HPT axis to DIA could be due to CRH regulating TRHergic neurons. CRH effect was first evaluated on TRH expression of cultured hypothalamic cells where TRH mRNA levels increased after 1h with 0.1nM of CRH. We then measured the mRNA levels of CRH receptors in the PVN of male and female rats subjected to DIA; only those of CRH-R2 were modulated (down-regulated). The CRH-R2 antagonist antisauvagine-30 was therefore injected into the PVN of male rats, during the 7 days of DIA. Antisauvagine-30 induced a higher food intake than controls, and impeded the changes produced by DIA on the HPT axis: PVN TRH mRNA, and serum TH and TSH levels were decreased to similar values of FFR animals. Results corroborate the anorexigenic effect of CRH and show its role, acting through CRH-R2 receptors, in the activation of TRHergic PVN neurons caused by DIA. These new data further supports clinical trials with CRH-R2 antagonists in anorexia nervosa patients.

The PKC and ERK/MAPK pathways regulate glucocorticoid action on TRH transcription.

Biosynthesis of TRH, a neuropeptide involved in energy homeostasis, is modulated by glucocorticoids. TRH mRNA and peptide levels are increased upon incubation of hypothalamic cells with dexamethasone or with cAMP analogs but when combined, a mutual antagonism is observed. These effects are observed at the transcriptional level and on binding of glucocorticoid receptor (GR) or pCREB to the composite GRE (cGRE) and CRE-2 sites of TRH promoter. The present work studied the involvement of PKC and MAPK pathways on the effect of dexamethasone and on its interaction with cAMP signaling in hypothalamic cell cultures. PKC or MEK inhibition abolished dexamethasone-stimulatory effect on TRH mRNA levels, as well as its interference with the stimulatory effect of 8Br-cAMP. Binding of nuclear extracts from hypothalamic or neuroblastoma cells stimulated with dexamethasone or 8Br-cAMP to oligonucleotides containing the CRE or cGRE sites of TRH gene promoter was decreased if cells were preincubated with PKC or MEK inhibitors. Mutations on the AP-1 or the GRE half sites of cGRE showed that GR binds as an heterodimer on cGRE, and PKC or MEK inhibitors diminish binding at the AP-1 site. PKC and ERK signaling thus modulate GR activity and its interaction with CREB or AP-1 at the TRH gene promoter.

Presence of pro-opiomelanocortin mRNA in the rat medial prefrontal cortex, nucleus accumbens and ventral tegmental area: studies by RT-PCR and in situ hybridization techniques.

Pro-opiomelanocortin (POMC) is a large proteic precursor which originates several biologically actives neuropeptides, such as beta-lipotropin (beta-LPH), beta-endorphin (beta-END), adenocorticotropic hormone (ACTH) and alpha-melanocyte-stimulating hormone (alpha-MSH). The arcuate nucleus of the hypothalamus is the main POMC producing cell group in brain and innervates several areas of the limbic system and brainstem. POMC-derived neuropeptides have been related to several motivated and rewarding behaviours, including sexual facilitation, feeding, and drug addiction. However, POMC mRNA has not been detected in regions of the dopaminergic mesocorticolimbic system, which represents the most important reward pathway. The aim of this work was to investigate if POMC mRNA is expressed in the medial prefrontal cortex (mPFC), the nucleus accumbens (NAcc) and the ventral tegmental area (VTA) of the rat. We used the reverse transcriptase reaction coupled to the polymerase chain reaction (RT-PCR). We also used the in situ hybridization technique to study the regional distribution of POMC mRNA in the same regions. We report that RT-PCR amplification of extracted RNA with two different pairs of primers generates the predicted 94bp and 678bp POMC-PCR products. Both the amplification of RNA obtained from the rat glial C-6 cell line (which does not express POMC mRNA) and the omission of reverse transcriptase from the RT reaction of rat brain samples showed no amplification products. We have shown for the first time that the rat medial prefrontal cortex, the nucleus accumbens and the ventral tegmental area contain POMC mRNA. This mRNA is in low concentration, ranging from 21% to 31% with respect to the hypothalamus. In situ hybridization experiments showed that POMC mRNA is homogeneously distributed in these areas. The presence of POMC mRNA in regions of the mesocorticolimbic system could have functional implications in motivated behaviours.