H295R expression of melanocortin 2 receptor accessory protein results in ACTH responsiveness

Abstract

The H295R adrenocortical cell line is widely used for molecular analysis of adrenal functions but is known to have only modest ACTH responsiveness. The lack of ACTH response was linked to a low expression of its receptor, melanocortin 2 receptor (MC2R). We hypothesized that increasing the MC2R accessory protein (MRAP), which is required to traffic MC2R from the endoplasmic reticulum to the cell surface, would increase ACTH responsiveness. Lentiviral particles containing human MRAP-open reading frame were generated and transduced in H295R cells. Using antibiotic resistance, 18 clones were isolated for characterization. The most ACTH-responsive steroidogenic clone, H295RA, was used for further experiments. Successful induction of MRAP and increased expression of MC2R in H295RA cells was confirmed by quantitative real-time RT-PCR and protein analysis. Treatment with ACTH significantly increased aldosterone, cortisol, and dehydroepiandrosterone production in H295RA cells. ACTH also significantly increased transcript levels for all of the steroidogenic enzymes required to produce aldosterone, cortisol, and dehydroepiandrosterone, as well as MC2R mRNA. Using liquid chromatography/tandem mass spectrometry, we further revealed that the main unconjugated steroids produced in H295RA cells were 11-deoxycortisol, cortisol, and androstenedione. Treatment of H295RA cells with ACTH also acutely increased cAMP production and cellular protein levels for total and phosphorylated steroidogenic acute regulatory protein. In summary, through genetic manipulation, we have developed an ACTH-responsive human adrenocortical cell line. The cell line will provide a powerful in vitro tool for molecular analysis of physiologic and pathologic conditions involving the hypothalamic-pituitary-adrenal axis.

Development of new preclinical models to advance adrenocortical carcinoma research

“…To evaluate the secretome of our newly created cell lines, we performed LS/MS-MS analysis to profile adrenal hormones, and compared to these profiles to that of H295R cells. Whereas H295R cells secreted moderate levels of 11-deoxycortisol in basal conditions, as previously reported (Nanba, et al 2016), CU-ACC1 cells secreted cortisol which is in line with higher expression of CYP11B1 mRNA in CU-ACC1 compared to H295R. No aldosterone secretion was detected in CU-ACC1.…”

GATA transcription factors in testicular adrenal rest tumours

“…Human cell lines, in general, tend to have low (functional) ACTH receptor expression, although several adrenocortical cell lines, including H295R cells, are known to have at least moderate expression of MC2R ( 40 , 41 ). ACTH responsiveness in H295R cells was increased by over-expression of MRAP, resulting in the H295RA cell line, the only human ACTH-sensitive cell line available ( 28 ). We are aware of the limitation of using a differentiated cell line as a model for the involvement of ACTH in foetal steroidogenic cells.…”

“…The Hs181.tes cell line was obtained from American Type Culture Collection (ATCC CRL-7131), while the H295RA cell line was obtained from the University of Michigan ( 28 ). Cells were grown as a monolayer culture, although the H295RA cells tend to grow in clumps.…”

Interaction between Angiotensin II and Insulin/IGF-1 Exerted a Synergistic Stimulatory Effect on ERK1/2 Activation in Adrenocortical Carcinoma H295R Cells

“…Ang II, insulin, and insulin-like growth factor 1 (IGF-1) were shown to play important roles in adrenocortical cells [ 11 – 13 ], and overexpression of IGF-1 receptor was found to be associated with the development of adrenocortical carcinoma [ 14 – 17 ]. In this study, by employing adrenocortical carcinoma H295R cells [ 18 – 22 ], we examined the interaction between Ang II and insulin/IGF-1 in ERK and AKT signaling pathways and expression of steroidogenic enzymes in the cells.…”