BES2005 Poster Presentations Steroids (17 abstracts)
The human adrenal H295R cell line expresses ACTH receptors (MC2R). However, it is generally accepted that ACTH treatment of these cells has little effect on steroid secretion and so forskolin is routinely used to mimic the actions of ACTH. It is unclear whether the ACTH receptors on these cells are functional. The present study was designed to investigate signalling pathways in H295R cells and their activation by ACTH.
Adrenal H295R cells were cultured in 75cm2 flasks in DMEM/F12 medium supplemented with 2% Ultroser SF, 1% ITS+ and 1% penicillin/streptomycin. For experiments cells were seeded into 6-well plates and serum-starved for 24 hours prior to treatments. Cells were treated with stimulants for 2 to 120 minutes for protein analysis and for 3 days for cell growth experiments. Cell growth was estimated using a 3H thymidine incorporation assay and an MTT assay. Cortisol was measured by radioimmunoassay.
ACTH (10pmol/l to 100nmol/l) had no effect on cell number as measured by both 3H thymidine incorporation and MTT assay. Angiotensin II, in contrast caused a significant (p<0.01) increase in cell numbers over the 3-day incubation period. Over 120 minutes ACTH had no effect on cortisol release, but after 2 days caused a significant increase (P<0.001) over basal.
ACTH caused a significant increase in the phosphorylation of both ERK1/2 (P<0.01) and stress-activated protein kinase (SAPK: P<0.05) as measured by Western blotting followed by scanning densitometry. The effects of ACTH on ERK1/2 were mimicked by forskolin, suggesting involvement of cAMP in this response.
In conclusion, the ACTH receptor in H295R cells appears to be functional, and coupled to activation of both MAPK and SAPK pathways, although this activation does not result in increased rates of cell division. It is possible that ACTH has a role in the differentiation of H295R cells, favouring cortisol production.