Molecular effectors and mechanisms involved in the sustained cAMP response mediated by CRHR1.

DOS SANTOS CLARO, P. A.; ARMANDO, N.G.; PIAZZA, V. G.; SILBERSTEIN, S.

Mar del Plata

Congreso; LXIII Reunión Anual de la Sociedad Argentina de Investigacion Clínica; 2018

Sociedad Argentina de Investigacion Clínica.

Corticotropin-releasing hormone (CRH) plays a central role in the stress response and dysregulation of its action is related to the onset of anxiety and depression states. CRH is a high affinity ligand for corticotropin-releasing hormone receptor 1 (CRHR1) whose activation is associated to a sustained increase in cAMP levels and ERK1/2 activation in cellular models that recapitulate contexts of CRHR1 action. In a hippocampal neuronal model, CRHR1-mediated ERK1/2 activation triggered by CRH is biphasic, with an early phase B-Raf and PKA dependent, and a late phase of sustained ERK1/2 phosphorylation dependent on β-Arrestin2 and CRHR1 internalization. The aim of this work is to characterize the mechanisms implicated in the sustained cAMP response to CRH mediated by CRHR1. As a model, mouse hippocampal neuronal cell line HT22 stably expressing CRHR1 (HT22-CRHR1) was used together with the FRET-based biosensor Epac-SH187 to monitor cAMP levels in real time in living cells. By means of pharmacological and genetic tools, we revisited the role of PKA and β-Arrestin and explored PI3K/Akt signaling pathway in our system. In HT22-CRHR1 cells, expression of a β-arrestin dominant negative mutant altered Akt and CREB responses to CRH. Furthermore, Akt activation proved to be dependent on cAMP in HT22-CRHR1 cells as forskolin or 8-CPT-cAMP treatment promoted its phosphorylation. Pharmacological inhibition of PI3K, Akt and PKA triggered a further increase in cAMP levels in CRH-stimulated cells. Besides, PI3K/Akt inhibition led to a decrease in CRH-induced ERK1/2 and CREB activation, similar to what had already been reported when PKA activity was repressed. Moreover, our results showed that PKA inhibition is associated to increased Akt activation in response to CRH. These results suggest that PKA and PI3K/Akt may play a role in the mechanisms involved in CRH-elicited/CRHR1-mediated sustained cAMP response in a hippocampal context. Supported by ANPCyT, CONICET, UBA and FOCEM (COF 03/11).