IFIBYNE   05513
INSTITUTO DE FISIOLOGIA, BIOLOGIA MOLECULAR Y NEUROCIENCIAS
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
Stress and molecular mechanisms of CRH signaling trough CRHR1 in specific areas of the central nervous system.
Autor/es:
BONFIGLIO, J. J.; SILBERSTEIN, S.,; GIACOMINI, D.; REFOJO, D.; DEUSSING, J.,; HOLSBOER, F.; ARZT, E.
Lugar:
Buenos Aires
Reunión:
Simposio; XXXIX Reunión Anual de la Sociedad Argentina de Farmacología Experimental; 2007
Institución organizadora:
Sociedad Argentina de Farmacología Experimental
Resumen:
CRH is the key mediator of the neuroendocrine, autonomic and behavioral responses to stress. Dysregulation of CRH/CRHR1 function in limbic structures and corticotrophs has been shown to be involved in the initiation of pathological stages, anxiety/depression and Cushing disease. We demonstrated that ERK1/2 activation mediates CRH effects in corticotrophs and in the central nervous system. In mice, icv CRH administration in vivo, acting through CRHR1, activates ERK1/2 in specific limbic areas (hippocampus and basolateral amygdala) related to external environment information processing and behavioral aspects to stress. Other regions related to central CRH system but involved in the processing of the ascending visceral information and neuroendocrine-autonomic response to stress, did not show CRH-mediated ERK1/2 activation. We have extended this analysis to a conditional mouse mutant (CRHOE) in which CRH overexpression is restricted to limbic brain.  pERK1/2 levels were assessed in wild type (WT) and CRHOE in basal and after restriction stress.  In absence of stress, pERK1/2 levels in the hippocampus and basolateral amygdala, as well as corticosterone values, were similar in WT and CRHOE, indicating that CRH overexpression in limbic structures does not alter the HPA axis. pERK1/2 signal in the amygdala was decreased in CRHOE under stress. There was no correlation between elevated corticosterone levels caused by stress in both groups and ERK1/2 activation, indicating that glucocorticoids are not responsible for the MAPK regulation in these areas. Binding analysis indicated that decreased levels of pERK1/2 cannot be explained by down-regulation of CRHR1, and suggest that conditions of chronic exposure to CRH would activate inhibitory mechanisms of ERK1/2. We are exploring the signaling network that mediates MAPK activation by CRH in hippocampal cell lines using molecular and pharmacological tools combined with proteomics. We generated stable cell lines expressing CRHR1 that respond to CRH as evaluated with appropriate reporters. Having determined ERK1/2 activation by CRH in this cell line, we started the search for proteins that interact with the intermediate kinase B-Raf that belongs to the canonical MAPK signaling cascade involving MEK1/2, and ERK1/2 in neuronal cells. We performed non-denaturing immunoprecipitations (IPs) in cell lysates from non-stimulated for CRH-stimulated cells. In immunoprecipitates with anti-B-Raf antibodies resolved by SDS/PAGE, a protein band was differentially detected, subjected to mass spec, and identified as vimentin. We investigated if the 14-3-3 proteins, functional signaling modulators expressed in brain, were associated to B-Raf by IPs with anti 14-3-3 antibodies in cell lysates. Our results indicate that 14-3-3 proteins, B-Raf and vimentin associate in a protein complex in CRH-stimulated cell lines of hippocampal origin. Determining the specificity of CRH signaling is crucial to identify new therapeutic targets.