Studies on 7a-hydroxycholesterol in extrahepatic tissues and cells

Sammanfattning: In human liver, primary bile acids are synthesized from cholesterol via the well-established neutral and also via the recently described acidic pathways. The latter includes27-hydroxycholesterol 7alpha-hydroxylase. Extrahepatic presence and function of this enzymeas well as the possible occurrence of other hydroxylases were investigated in the presentstudy. For this purpose, radio-labeled 27-hydroxycholesterol (27-OHC) and 25-hydroxy-cholesterol (25-OHC) were prepared. Both hydroxysterols were 7a-hydroxylated, andsubsequently oxidized to 7a-hydroxy-3-oxo-A4 steroids, in cultures of human diploidfibroblasts (HDF), rat astrocytes, Schwann cells and neurons, mouse thymic epithelial cells,and the human melanoma cell line SK-MEL-2 but not in virus-transformed fibroblasts(9OVA-VI) and the malignant cell lines WiDr and MDA-231. Some 27-OHC was alsooxidized to 3beta-hydroxy-5-cholestenoic, 3beta,7alpha-dihydroxy-5-cholestenoic and 7alpha-hydroxy-3-oxo-4-cholestenoic acids. In case of 25-hydroxycholesterol, regulation of 7alpha-hydroxylationwas studied and the reaction was found to be stimulated by interleukin-1beta, dexamethasoneand cortisol, and inhibited by metyrapone and RU486. In healthy volunteers, the concentration of 7alpha-hydroxy-3-oxo-4-cholestenoic acidwas found to be significantly higher in peripheral arterial than in hepatic venous blood,indicating an extrahepatic formation of 7alpha-hydroxylated metabolites of cholesterol in vivo. 27-Hydroxycholesterol 7alpha-hydroxylase was present in microsomal preparations fromrat brain. In addition to 25-OHC and 27-OHC, 3beta-hydroxy-5-cholestenoic and 3beta-hydroxy-5-cholenoic acids, dehydroepiandrosterone (DHEA) and pregnenolone were 7alpha-hydroxylated. For 27-OHC, the apparent Km was about 2 myM and Vmax about 15 pmol/min xmg protein. Competition experiments indicated that the same enzyme 7alpha-hydroxylated 27-OHC and 25-OHC which, however, was different from cholesterol 7alpha-hydroxylase. Otherextrahepatic 7alpha-hydroxylase(s) may exist. 25-Hydroxylation was found to be an important reaction in cultures of rat astrocytesand Schwann cells leading to formation of significant amoumts of 7alpha,25-dihydroxy-4-cholesten-3-one. Several 7alpha-hydroxylated metabolites of 27-OHC and 25-OHC, e.g. 7alpha,25-diOHC,27-OHC, 7alpha,27-diOHC and 7alpha,27-dihydroxy-4-cholesten-3-one, were found to be aseffective suppressors of HMG-CoA reductase activity in fibroblasts as 25-OHC. Thesuppression of this activity by 25-OHC and 27-OHC was reduced or abolished by DHEAand pregnenolone. In 90VA-VI cells, 7alpha,27-diOHC but not 7alpha,25-diOHC had suppressoractivity. The results suggest that 7alpha-hydroxylation is not directly involved in the potentialfunction of 27-OHC and 25-OHC as regulators of HMG-CoA reductase activity. Both 25-OHC and 27-OHC were inducers of apoptosis in mouse thymocytes. 7alpha-Hydroxylation of both compounds as well as formation of acidic metabolites of 27-OHCdecreased or abolished the apoptotic effects, suggesting a protective role of these reactionsin hydroxycholesterol-induced apoptosis.Doctoral Thesis, Jie Zhang 1996ISBN 91-628-2138-5