Antonieta Cote-Vélez

A Rapid Interference between Glucocorticoids and cAMP‐Activated Signalling in Hypothalamic Neurones Prevents Binding of Phosphorylated cAMP Response Element Binding Protein and Glucocorticoid Receptor at the CRE‐Like and Composite GRE Sites of Thyrotrophin‐Releasing Hormone Gene Promoter

Glucocorticoids or cAMP increase, within minutes, thyrotrophin‐releasing hormone (TRH) transcription in hypothalamic primary cultures, although this effect is prevented if cells are simultaneously incubated with both drugs. Rat TRH promoter contains a CRE site at −101/−94 bp and a composite GRE element (cGRE) at −218/−197 bp. Nuclear extracts of hypothalamic cells incubated with 8Br‐cAMP or dexamethasone, and not their combination, bind to oligonucleotides containing the CRE or cGRE sequences. Adjacent to CRE are Sp/Krüppel response elements, and flanking the GRE half site, two AP1 binding sites. The present study aimed to identify the hypothalamic transcription factors that bind to these sites. We verified that the effects of glucocorticoid were not mimicked by corticosterone‐bovine serum albumin. Footprinting and chromatin immunoprecipitation (ChIP) assays were used to examine the interaction of cAMP‐ and glucocorticoid‐mediated regulation of TRH transcription at the CRE and cGRE regions of the TRH promoter. Nuclear extracts from hypothalamic cells incubated for 1 h with cAMP or glucocorticoids protected CRE. The GRE half site was recognised by nuclear proteins from cells stimulated with glucocorticoids and, for the adjacent AP‐1 sites, by nuclear proteins from cells stimulated with cAMP or phorbol esters. Protection of CRE or cGRE was lost if cells were coincubated with dexamethasone and 8Br‐cAMP. ChIP assays revealed phospho‐CREB, c‐Jun, Sp1, c‐Fos and GR antibodies bound the TRH promoter of cells treated with cAMP or glucocorticoids; anti:RNA‐polymerase II immunoprecipitated TRH promoter in a similar proportion as anti:pCREB or anti:GR. Recruitment of pCREB, SP1 or GR was lost when cells were exposed simultaneously to 8Br‐cAMP and glucocorticoids. The data show that while pCREB and Sp1 bind to CRE‐2, or GR to cGRE of the TRH promoter, the mutual antagonism between cAMP and glucocorticoid signalling, which prevent their binding to TRH promoter, could serve as a mechanism by which glucocorticoids rapidly suppress cAMP and noradrenaline‐stimulated TRH transcription.

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.

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.

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.

Phosphorylated Cyclic-AMP-Response Element-Binding Protein and Thyroid Hormone Receptor Have Independent Response Elements in the Rat Thyrotropin-Releasing Hormone Promoter: An Analysis in Hypothalamic Cells

Background: Thyrotropin-releasing hormone (TRH) from the hypothalamic paraventricular nucleus (PVN) controls the activity of the hypothalamus-pituitary-thyroid axis. TRH is expressed in other hypothalamic nuclei but is downregulated by 3,3′,5-L-triiodothyronine (T3) exclusively in the PVN. Thyroid hormone receptors (TRs) bind TRH promoter at Site-4 (–59/–52), also proposed to bind phosphorylated cAMP response element-binding protein (pCREB). However, nuclear extracts from 8Br-cAMP-stimulated hypothalamic cells showed no binding to Site-4 and instead to cAMP response element (CRE)-2 (–101/–94). Methods: We characterized, by DNA footprinting and chromatin immunoprecipitation, the sites in the rat (–242/+34) TRH promoter that bind to nuclear factors of hypothalamic primary cultures incubated with 8Br-cAMP and/or T3. Results: In primary cultures of fetal hypothalamic cells, TRH mRNA levels rapidly diminished with 10 nM T3 while they increased by 1 mM 8Br-cAMP (± T3). Site-4 was protected from DNase I digestion with nuclear extracts from T3-incubated cells but not from controls or from those incubated with 8Br-cAMP, which protected CRE-2; T3 + 8Br-cAMP coincubation caused no interference. The region protected by nuclear extracts from cAMP-stimulated cells included sequences adjacent to CRE-2-containing response elements of the SP/Krüppel family. A TRβ2 antibody immunoprecipitated chromatin containing Site-4 but not CRE-2, from cells incubated with T3. A pCREB antibody immunoprecipitated CRE-2 containing chromatin in controls and more in 8Br-cAMP-stimulated cells but none when cells were incubated only with T3. Recruitment of the 2 transcription factors was preserved in cells simultaneously exposed to 8Br-cAMP and T3. Discussion: These results show that pCREB binds to a response element in the TRH promoter (CRE-2) that is independent of Site-4 where TRβ2 is bound; pCREB and TR do not present mutual interference on their binding sites.

An Acute Injection of Corticosterone Increases Thyrotrophin-Releasing Hormone Expression in the Paraventricular Nucleus of the Hypothalamus but Interferes with the Rapid Hypothalamus Pituitary Thyroid Axis Response to Cold in Male Rats

The activity of the hypothalamic‐pituitary‐thyroid (HPT) axis is rapidly adjusted by energy balance alterations. Glucocorticoids can interfere with this activity, although the timing of this interaction is unknown. In vitro studies indicate that, albeit incubation with either glucocorticoid receptor (GR) agonists or protein kinase A (PKA) activators enhances pro‐thyrotrophin‐releasing hormone (pro‐TRH) transcription, co‐incubation with both stimuli reduces this enhancement. In the present study, we used primary cultures of hypothalamic cells to test whether the order of these stimuli alters the cross‐talk. We observed that a simultaneous or 1‐h prior (but not later) activation of GR is necessary to inhibit the stimulatory effect of PKA activation on pro‐TRH expression. We tested these in vitro results in the context of a physiological stimulus on the HPT axis in adult male rats. Cold exposure for 1 h enhanced pro‐TRH mRNA expression in neurones of the hypophysiotrophic and rostral subdivisions of the paraventricular nucleus (PVN) of the hypothalamus, thyrotrophin (TSH) serum levels and deiodinase 2 (D2) activity in brown adipose tissue (BAT). An i.p. injection of corticosterone stimulated pro‐TRH expression in the PVN of rats kept at ambient temperature, more pronouncedly in hypophysiotrophic neurones that no longer responded to cold exposure. In corticosterone‐pretreated rats, the cold‐induced increase in pro‐TRH expression was detected only in the rostral PVN. Corticosterone blunted the increase in serum TSH levels and D2 activity in BAT produced by cold in vehicle‐injected animals. Thus, increased serum corticosterone levels rapidly restrain cold stress‐induced activation of TRH hypophysiotrophic neurones, which may contribute to changing energy expenditure. Interestingly, TRH neurones of the rostral PVN responded to both corticosterone and cold exposure with an amplified expression of pro‐TRH mRNA, suggesting that these neurones integrate stress and temperature distinctly from the hypophysiotrophic neurones.

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.

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

ABSTRACT 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 β1-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 Ca2+ 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. Summary: We describe the role of FAK and focal adhesion proteins in capacitation, acrosome reaction, polymerization and remodeling of actin cytoskeleton, and how inhibition of FAK affects sperm physiology.

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.