Panoply™ Human CRHR1 Over-expressing Stable Cell Line

Corticotropin-releasing hormone receptor 1 (CRHR1) activates atypical soluble adenylate cyclase (sAC) in addition to transmembrane adenylate cyclase (tmAC). Studies have shown that both cAMP sources are required for ERK1/2 phosphorylation triggered by activated G protein-coupled receptor (GPCR) CRHR1 in both neuronal and neuroendocrine settings. Here, researchers reveal that activated CRHR1 promotes growth arrest and neurite elongation in neuronal hippocampal cells (HT22-CRHR1 cells). By characterizing the CRHR1 signaling mechanisms involved in neurite formation, they demonstrate that neurite outgrowth in HT22-CRHR1 cells occurs through an sAC-dependent, ERK1/2-independent signaling cascade. Both tmAC and sAC participate in corticotropin-releasing hormone (CRH)-mediated CREB phosphorylation and c-fos induction, but only the cAMP pool generated by sAC is critical for the neurogenic effects of CRH, further emphasizing the involvement of both cAMP sources downstream of GPCR activation and strengthening the idea that restricted cAMP microdomains may regulate independent cellular processes.

To test whether CRH plays a role in CRHR1 overexpressing HT22 cell (HT22-CRHR1) proliferation, the researchers employed two experimental approaches. First, an area-based growth assay was performed using a "scratch" assay. In this assay, the gap area is likely filled by a combination of cell migration and proliferation. The effect on cell proliferation is considered to be greatest when wound invasiveness is observed after prolonged incubation in the presence of serum, as demonstrated in the experiments (Figures 1a, b). HT22-CRHR1 cell monolayers were scratched to create a cell-free wound area, and the cultures were imaged over time using bright-field microscopy at the indicated times. In the presence of CRH, cell morphology at the scratch edge changed significantly (Figure 1a). Notably, the total cell area covered by cells in the wound was significantly reduced compared to control conditions (Figure 1b). The effect of CRH on growth arrest was measured at various concentrations, and this effect was mimicked using the cell-permeable cAMP analog 8-CPT-cAMP (Figure 1c). CRH and UCN1 stimulation induced growth arrest in HT22-CRHR1 cells at comparable rates (Figure 1d). Researchers then compared the effects of CRH on cells cultured in serum-free medium and in medium with 5% serum at different time points after the addition of CRH (Figure 1e). Although serum clearly stimulated cell proliferation, CRH promoted growth arrest compared to the unstimulated control, regardless of the presence or absence of serum.

Figure 1. CRHR1 activation has a negative effect on cell proliferation. (Inda C, et al., 2017)