Dissecting CRHR1-mediated pathways via microarray technology

The corticotropin releasing hormone (CRH) system is involved in endocrine, autonomic and behavioural responses to stress. The biological actions of CRH-like neuropeptides are mediated by G-protein-coupled receptors, CRH receptor 1 (CRHR1) and CRHR2. CRHR1 is widely expressed in the mammalian brain and in the pituitary gland. Mice deficient for CRHR1 display decreased anxiety-like behaviour and dysregulation of the hypothalamic-pituitary-adrenal axis. Ligand binding increases the affinity of the CRHR to G-proteins. Binding of a Gαs-protein will activate adenylate cyclase and protein kinase A as well as other cAMP dependent pathways. The reported coupling of additional G-proteins to CRHR1 suggests that also other second messengers are involved in CRHR1-signaling. In order to identify specific target genes of CRHR1-mediated signaling pathways we applied cDNA and oligonucleotide microarray technology using a mouse corticotroph cell line (AtT20) and pituitaries of CRHR1-deficient mice. 102 genes in vitro and more than 400 genes in vivo were found stress- and/or CRH- and CRHR1-dependently regulated. A subset of candidate genes was validated in independent material by quantitative real time PCR. These candidates are involved e.g. in cAMP, mitogen activated protein kinase or epidermal growth factor receptor signaling. In order to examine their functional role the effect of these genes on different known target genes downstream of CRHR1 signaling is analyzed using reporter assays.