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.

The biosynthesis of thyrotropin-releasing hormone (TRH) in the hypothalamic paraventricular nucleus (PVN) is subject to neural and hormonal regulations. To identify some of the potential effectors of this modulation, we incubated hypothalamic dispersed cells with dexamethasone for short periods of time (1–3 h) and studied the interaction of this hormone with protein kinase C (PKC) and PKA signaling pathways. TRH mRNA relative changes were determined by the RT-PCR technique. One hour incubation with 10^–10–10^–4 M dexamethasone produced a concentration-dependent biphasic effect: an inhibition was observed on TRH mRNA levels at 10^–10M, an increase above control at 10^–8–10^–6M and a reduction at higher concentrations (10^–5– 10^–4M). The stimulatory effect of 10^–8M dexamethasone on TRH mRNA was essentially independent of new protein synthesis, as evidenced by cycloheximide pretreatment. Changes in TRH mRNA levels were reflected by enhanced TRH cell content. Incubation with a cAMP analogue (8-bromo-cAMP, 8Br-cAMP) or with a PKC activator (12-O-tetradecanoylphorbol-13-acetate, TPA) increased TRH mRNA levels after 1 and 2 h, respectively. An increase in TRH mRNA expression was observed by in situ hybridization of dexamethasone or 8Br-cAMP-treated cells. The interaction of dexamethasone, PKA and PKC signaling pathways was studied by combined treatment. The stimulatory effect of 10^–7M TPA on TRH mRNA levels was additive to that of dexamethasone; in contrast, coincubation with 10^–3M 8-Br-cAMP and dexamethasone diminished the stimulatory effect of both drugs. An inhibition was observed when the cAMP analogue was coincubated with TPA or TPA and dexamethasone. These results demonstrate that dexamethasone can rapidly regulate TRH biosynthesis and suggest a cross talk between cAMP, glucocorticoid receptors and PKC transducing pathways.

Background Neuronal stem cells (NSCs) are promising for neurointestinal disease therapy. While NSCs have been isolated from intestinal musclularis, their presence in mucosa has not been well described. Mucosa-derived NSCs are accessible endoscopically and could be used autologously. Brain-derived Nestin-positive NSCs are important in endogenous repair and plasticity. The aim was to isolate and characterize mucosa-derived NSCs, determine their relationship to Nestin-expressing cells and demonstrate capacity to produce neuroglial networks in vitro and in vivo. Methods Neurospheres were generated from periventricular brain, colonic muscularis (Musc), and mucosa-submucosa (MSM) of mice expressing green fluorescent protein (GFP) controlled by the Nestin promoter (Nestin-GFP). NSCs were also grown as adherent colonies from intestinal mucosal organoids. Their differentiation potential was assessed by immunohistochemistry using glial and neuronal markers. Brain and gut derived neurospheres were transplanted into explants of chick embryonic aneural hindgut to determine their fate. Results Musc- and MSM-derived neurospheres expressed Nestin and gave rise to cells of neuronal, glial and mesenchymal lineage. While Nestin expression in tissue was mostly limited to glia colabelled with glial fibrillary acid protein (GFAP), neurosphere-derived neurons and glia both expressed Nestin in vitro, suggesting Nestin+/GFAP+ glial cells may give rise to new neurons. Moreover, following transplantation into aneural colon, brain- and gut-derived NSCs were able to differentiate into neurons. Conclusions Nestin-expressing intestinal NSCs cells give rise to neurospheres, differentiate into neuronal, glial and mesenchymal lineages in vitro, generate neurons in vivo and can be isolated from mucosa. Further studies are needed exploring their potential for treating neuropathies.