Incorporation of steroidogenic pathways which produce cortisol and aldosterone from cholesterol into nonsteroidogenic cells

Cortisol production from cholesterol requires the activity of four steroid hydroxylases: cholesterol side chain cleavage cytochrome P-450 (P-450_scc), 17α-hydroxylase cytochrome P-450 (P-450_17α), 21-hydroxylase cytochrome P-450 (P-450_c21) and 11β-hydroxylase cytochrome P-450 (P-450₁₁β). We have previously shown that transformed, nonsteroidogenic COS 1 cells derived from monkey kidney are a useful system for expression of various forms of cytochrome P-450. The present study shows that COS 1 cell cultures multiply transfected with six plasmids containing all four steroid hydroxylases, 3β-hydroxysteroid dehydrogenase /Δ^5→4-isomerase (3βHSD) and adrenodoxin produce cortisol and aldosterone when 22(R)-hydroxycholesterol is supplied to the system. When pregnenolone is used as substrate, various intermediate metabolites are detected at different time points further establishing the incorporation of complete functional steroidogenic pathways into the nonsteroidogenic cell cultures. Since the first and the last reactions in these pathways take place in the mitochondrion, the movement of various intermediate metabolites from mitochondrion to endoplasmic reticulum and back to mitchondrion occurs in and between COS 1 cells.