title: 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.
authors.affiliation: Departments of Molecular & Integrative Physiology and Internal MedicineUniversity of Michigan, 1150 West Medical Center Drive, Ann Arbor, Michigan 48109, USADivision of MetabolismEndocrinology, and Diabetes, University of Michigan, 1150 West Medical Center Drive, Ann Arbor, Michigan 48109, USA.
authors.forename: Kazutaka
authors.initials: K
authors.lastname: Nanba
authors.affiliation: Departments of Molecular & Integrative Physiology and Internal MedicineUniversity of Michigan, 1150 West Medical Center Drive, Ann Arbor, Michigan 48109, USADivision of MetabolismEndocrinology, and Diabetes, University of Michigan, 1150 West Medical Center Drive, Ann Arbor, Michigan 48109, USA.
authors.forename: Andrew X
authors.initials: AX
authors.lastname: Chen
authors.affiliation: Departments of Molecular & Integrative Physiology and Internal MedicineUniversity of Michigan, 1150 West Medical Center Drive, Ann Arbor, Michigan 48109, USADivision of MetabolismEndocrinology, and Diabetes, University of Michigan, 1150 West Medical Center Drive, Ann Arbor, Michigan 48109, USA.
authors.forename: Adina F
authors.initials: AF
authors.lastname: Turcu
authors.affiliation: Departments of Molecular & Integrative Physiology and Internal MedicineUniversity of Michigan, 1150 West Medical Center Drive, Ann Arbor, Michigan 48109, USADivision of MetabolismEndocrinology, and Diabetes, University of Michigan, 1150 West Medical Center Drive, Ann Arbor, Michigan 48109, USA Departments of Molecular & Integrative Physiology and Internal MedicineUniversity of Michigan, 1150 West Medical Center Drive, Ann Arbor, Michigan 48109, USADivision of MetabolismEndocrinology, and Diabetes, University of Michigan, 1150 West Medical Center Drive, Ann Arbor, Michigan 48109, USA wer@med.umich.edu.
authors.forename: William E
authors.initials: WE
authors.lastname: Rainey
mesh_terms: D000324:Adrenocorticotropic Hormone / Q000502:physiology*; D000450:Aldosterone / Q000096:biosynthesis; D053898:Biosynthetic Pathways; D002460:Cell Line; D015870:Gene Expression; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction
chemical_list: C458099:MRAP protein, human; D008565:Membrane Proteins; D012333:RNA, Messenger; D000450:Aldosterone; D000324:Adrenocorticotropic Hormone
keywords: ACTH; H295R; MC2R; MRAP; cell line