The Search for the Endogenous RORg Ligand and Investigating the Role of Cytochrome B5 in Steroidogenesis and Regulation of the Cyp17a1 Lyase Reaction

RORg is an orphan nuclear receptor important in the regulation of immune development and function. RORg regulated TH17 cells have been implicated in the pathology of various autoimmune diseases including multiple sclerosis and rheumatoid arthritis. Targeting RORg through antagonists has emerged as a novel therapeutic tool in the treatment of autoimmune diseases. Identification of the RORg endogenous ligand would offer insight into RORg regulation. It is currently believed that sterols are the endogenous RORg ligands. In these studies I will show that while sterols can bind RORg, they fail to modulate its activity in-vivo or in-vitro. Endogenously extracted lysophospholipids on the other hand, such as 22:4 LPE, can activate RORg in co-transfection assays and may be RORg ligands. Additionally, 22:4 LPE can increase IL17 production in TH17 cells. My second project involved investigating the role of cytochrome B5 in steroidogenesis. Cyb5 is involved in regulating electron transfer to numerous P450-mono-oxygenases Cyb5's role in human physiology has been confirmed by the discovery of patients with mutations in Cyb5 that present with isolated 17, 20-lyase deficiency, characterized by low plasma androgens and ambiguous external genitalia. To study the consequences of Cyb5 deficiency in an intact animal and in steroidogenic tissue, I generated mice lacking Cyb5 in the Leydig cell by crossing Cyb5fl/fl and CreSF1 animals. I show that the Cyb5-/- animals were born in a normal Mendelian ratio and had normal fertility with no overt phenotype. Testicular histology revealed no differences between Cyb5-/- and WT animals. Homogenates from Cyb5-/- testes had normal progesterone (P)-to-17α-hydroxyprogesterone (17-OHP) conversion but low 17-OHP-to-androstenedione (A) and testosterone (T) metabolism. The ratio of the hydroxylase to lyase activity was observed to be 1.7 in the WT and 4.5 (3-fold higher) in the Cyb5-/- testes due to deficient lyase activity in the knockout animals. However, steroid production was found to be normal in these animals. Exogenous hCG administration gave a large increase in serum steroids for both the Cyb5-/- and WT animals. In the Cyb5-/- animals, this rise was accompanied by the accumulation of 17-OHP in serum, which led to a 17-OHP/(A+T) ratio that was 44-fold in the KOs. Thus these data demonstrate the physiological significance of Cyb5 in the Cyp17a1 lyase reaction.