The PKC and ERK/MAPK Pathways Regulate Glucocorticoid Action on TRH Transcription

Abstract

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.

Regulation of TRH neurons and energy homeostasis-related signals under stress

“…Similar cross talk between the PKA and GR intracellular pathways is detected also in cell lines, supporting the hypothesis that the effect is an intracellular event, and not due to the average of differential responses of TRHergic cells from different hypothalamic nuclei present in primary cultures (Cote-Vélez et al 2005). The interference of GCs with the cAMP-stimulatory effect on Trh expression is avoided with inhibitors of the ERK/MAPK pathways (Cote-Vélez et al 2008). These results, although obtained in vitro, support the possibility of fast cross talk between signaling pathways that may alter an immediate response (Fig.…”

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

“…Briefly, 17th-day-gestation dams were anesthetized and their hypothalami dissected taking as limits optic chiasm and lateral sulcus, 2–3 mm depth, as described [29]. Dispersed cells (2 × 10 6 /hypothalamus) were plated on 1.5-µg/ml poly- D -lysine (Sigma) pre-coated dishes (Corning); 6 × 10 5 cells in 15-mm-diameter dishes for RNA purification and 5 × 10 6 cells in 60-mm-diameter dishes for footprinting and ChIP assays.…”

“…Cells were cultured during 18 days in vitro (DIV) when the expression of TRH mRNA [31] and the response to 8Br-cAMP stimulation was maximal [unpubl.]. We have carefully characterized this culture system showing that the presence of a carpet of glia whose reproduction is arrested by cytosine arabinoside, together with the conditioned medium, allows an adequate growth of neurons expressing TRH (4–5% of the total cell population) [7,10,28,29,30,31]. TRH, cyclophilin and glyceraldehyde-3-phosphate dehydrogenase (G3PDH) mRNA levels were first evaluated in cultures treated for 1, 6 or 24 h with 10 –8 or 10 –7 M T 3 (Sigma).…”

Prothyrotropin-releasing Hormone Targets Its Processing Products to Different Vesicles of the Secretory Pathway

“…increase cytosolic Ca 2ϩ (28). NE is a physiological secretagogue that acts via specific receptors using protein kinase A and Ca 2ϩ -dependent intracellular pathways in both neurons and AtT20 cells (35,43). The secretion of N-terminal peptides was only slightly increased by NE and KCl, suggesting that this peptide is not efficiently stored in secretory vesicles (Figs.…”