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.

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 (T(3)) 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 T(3). RESULTS: In primary cultures of fetal hypothalamic cells, TRH mRNA levels rapidly diminished with 10 nM T(3) while they increased by 1 mM 8Br-cAMP (+/- T(3)). Site-4 was protected from DNase I digestion with nuclear extracts from T(3)-incubated cells but not from controls or from those incubated with 8Br-cAMP, which protected CRE-2; T(3) + 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 TRbeta2 antibody immunoprecipitated chromatin containing Site-4 but not CRE-2, from cells incubated with T(3). 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 T(3). Recruitment of the 2 transcription factors was preserved in cells simultaneously exposed to 8Br-cAMP and T(3). DISCUSSION: These results show that pCREB binds to a response element in the TRH promoter (CRE-2) that is independent of Site-4 where TRbeta2 is bound; pCREB and TR do not present mutual interference on their binding sites.

The role of the secretory immune response in the infection by Entamoeba histolytica.

Intestinal infection with the protozoan parasite Entamoeba histolytica elicits a local immune response with rising of specific secretory IgA (sIgA) antibodies detectable in several compartments associated to mucosa. Anti-amoebic sIgA antibodies have been reported in faeces, saliva, bile and breast milk from dysenteric patients and research trying to elucidate their role in protection has recently intensified. IgA antibodies inhibit the in vitro adherence of E. histolytica trophozoites to epithelial cell monolayers by recognizing several membrane antigens, including the galactose-binding lectin (Gal-lectin), main surface molecule involved in adherence, and the serine and cystein-rich proteins, all of them potential vaccine candidates. In fact, the presence of sIgA anti-Gal lectin in faeces of patients recovered from amoebic liver abscess (ALA) was associated with immunity to E. dispar. Moreover, the combined nasal and intraperitoneal vaccination of C3H/HeJ mice with native and recombinant Gal-lectin protected mice against an intracecal challenge with virulent E. histolytica trophozoites, protection that seemed to be associated with the induction of specific intestinal sIgA antibodies. Therefore, the stimulation of intestinal secretory response by mucosal delivery of amoebic antigens has been positioned as a promising strategy for inducing protection against human amoebiasis.

Dexamethasone represses cAMP rapid upregulation of TRH gene transcription: identification of a composite glucocorticoid response element and a cAMP response element in TRH promoter.

Hypothalamic proTRH mRNA levels are rapidly increased (at 1 h) in vivo by cold exposure or suckling, and in vitro by 8Br-cAMP or glucocorticoids. The aim of this work was to study whether these effects occurred at the transcriptional level. Hypothalamic cells transfected with rat TRH promoter (-776/+85) linked to the luciferase reporter showed increased transcription by protein kinase (PK) A and PKC activators, or by dexamethasone (dex), but co-incubation with dex and 8Br-cAMP decreased their stimulatory effect (as observed for proTRH mRNA levels). These effects were also observed in NIH-3T3-transfected cells supporting a characteristic of TRH promoter and not of hypothalamic cells. Transcriptional regulation by 8Br-cAMP was mimicked by noradrenaline which increased proTRH mRNA levels, but not in the presence of dex. PKA inhibition by H89 avoided 8Br-cAMP or noradrenaline stimulation. TRH promoter sequences, cAMP response element (CRE)-like (-101/-94 and -59/-52) and glucocorticoid response element (GRE) half-site (-210/-205), were analyzed by electrophoretic mobility shift assays with nuclear extracts from hypothalamic or neuroblastoma cultures. PKA stimulation increased binding to CRE (-101/-94) but not to CRE (-59/-52); dex or 12-O-tetradecanoylphorbol-13-acetate (TPA) increased binding to GRE, a composite site flanked by a perfect and an imperfect activator protein (AP-1) site in the complementary strand. Interference was observed in the binding of CRE or GRE with nuclear extracts from cells co-incubated for 3 h with 8Br-cAMP and dex; from cells incubated for 1 h, only the binding to GRE showed interference. Rapid cross-talk of glucocorticoids with PKA signaling pathways regulating TRH transcription constitutes another example of neuroendocrine integration.

A familial syndrome with hypotonia, mental retardation and dysmorphic features resembling Cohen syndrome.

Since its original description (2), many new cases of Cohen syndrome have been reported, most of them showing a quite variable expressivity. Autosomal recessive inheritance is widely accepted (MIM : 216550) (11), however, multiple instances of sporadic cases are observed. From a literature review (52 cases), we could determinate, in order of frequency, the most important clinical traits of the Cohen syndrome. We report here a father and two sons with dysmorphic features resembling Cohen syndrome and transmitting by an autosomal dominant mode.

Molecular characterization of the fragile-X syndrome in the Mexican population.

The fragile X (fra-X) syndrome is the most frequent form of inherited mental retardation. Facial dysmorphism, macroorchidism and a folate-sensitive fragile site on Xq27.3 are commonly associated features. The gene causing this disorder, designated as FMR1, is X-linked and shows an unusual inheritance mode. A multistep amplification of the CGG repeats at the 5' end of the FMR1 gene has been recently identified as the cause of the fra-X syndrome. Different numbers of repeats define three gene forms (normal, premutated and mutated), whose ranges show little variation in the populations studied so far. We analyzed 18 Mexican individuals with the fra-X syndrome, 40 of their relatives (first and second degree), and 76 healthy individuals without antecedents of mental retardation. Southern blot and PCR permitted the assessment of the number of CGG repeats and the methylation state of the FMR1 gene for the normal, premutated, and mutated alleles. The results showed no statistical differences when compared with those from other populations. No cytogenetic expression of the Xq27.3 fragile site in 50% of the affected males and in all the affected and carrier females was observed. This finding emphasizes the necessity of a molecular analysis in fra-X cases and their relatives in order to provide a more adequate genetic counseling.