-3 - Sulfuric Acid -25 - Hydroxylation Of Cholesterol Biosynthesis Pathway, Regulation And Function Of Liver Cells, A New Nuclear Oxidation Of Steroids

Cellular cholesterol homeostasis is maintained through the coordinated regulation of cholesterol synthesis, degradation, and secretion. Nuclear receptors for oxygenated cholesterol derivatives(oxysterols) are known to play key roles in the regulation of cholesterol homeostasis. We recently identified a novel sulfatedoxysterol, 5-cholesten-3β, 25-diol 3-sulphate(25HC3S), whose concentration increased dramatically in the mitochondria and the nuclei of primary rat hepatocytes in response to over-expression of cholesterol mitochondria delivery protein, StarD1. This oxysterol was also found in human liver nuclei. Then we synthesized the 25HC3S chemically. In the present study, the metabolic pathway, regulation and functions of the nuclear oxysterol were investigated.In the first part, we studied the biosynthesis pathway of 25HC3S. According to the molecular structure of 25HC3S, we proposed a novel metabolic pathway of nuclear sulfate oxysterol in hepatocytes. The first step is that 25-OH Cholesterol is synthesized by CYP27A1 in mitochondria of hepatocytes. The second step is that 25-OH cholesterol is sulfonated to 25-OH cholesterol sulfate by hydroxysteroid sulfotransferase SULT2B1b in hepatocytes.Mitochondrial cholesterol 27-hydroxylase(CYP27A1) plays an important role in the maintenance of intracellular cholesterol homeostasis. Cholesterol delivery to the mitochondrial inner membrane is believed to be a rate-limiting step for the "acidic" pathway of bile acid synthesis. The present work reports that proteinase K treatment of mitochondria markedly increases CYP27A1 specific activity. With endogenous mitochondrial cholesterol, treatment with proteinase K increased CYP27A1 specific activity by 5-fold. Moreover, the addition of the exogenous cholesterol inβ-cyclodextrin plus proteinase K treatment increased the specific activity by 7-fold. Kinetic studies showed that the increased activity was time, proteinase K and substrate concentration dependent. Proteinase K treatment decreased the apparent Km of CYP27A1 for cholesterol from 400μM to 150μM. Using this new assay, we found that during rat hepatocyte preparation and cell culture, mitochondria gradually lose CYP27A1 activity as compared with mitochondria fleshly isolated from rat liver tissue.We already observed proteinase k stimulated CYP27A1 specific activity dramatically. Proteianses are located in the cells comprehensively, which is around mitochondria too. Incubation of isolated mitochondria with the cytosolic fraction stimulated CYP27A1 specific activity 2-fold. Heat-inactivation of the cytosolic fraction prevented this increase. Isolated crude lysosomal fraction stimulated the production of 27-hydroxycholesterol. However, EDTA and preteinase inhibitor AEBSF suppressed CYP27A1 activity, while CaCl₂ perform the opposite effect. These findings suggest that protelysis is involved in the control of transmembrane mitochndrial cholesterol movement and, therefore, in the "acidic" pathway of bile acid biosynthesis.The present study demonstrates a pathway for synthesis of 25HC3S in hepatocytes. Assays using mitochondria isolated from rats and Cyp27Al^- knockout mice indicated that 25-hydroxycholesterol(25HC) is synthesized by CYP27A1 in mitochondria. Incubation of mitochondrial and cytosol fractions resulted in synthesis of 25HC3S. RT-PCR analysis showed the presence of the hydroxysteroid sulfotransferase 2B1b(SULT2B1b) in the hepatocytes. Its expression was down regulated by 25HC3S but not by 25HC. The current findings suggest that the mitochondria synthesize 25HC, which is subsequently 3β-sulfated to form 25HC3S. A novel biosynthetic pathway of the nuclear oxysterol is proposed in this manuscript.In the second part, we focused on the regulation of the hydroxysteroid sulfotransferases in hepatocytes. Recently, a novel hydroxysteroid sulfotransferase SULT2B1b was cloned and studied. In contrast to the limited tissue distribution of SULT2A1, SULT2B1b was detected in a variety of hormone-responsive tissues including placenta, breast, skin and prostate. During the culture of primary rat hepatocytes, SULT2B1b mRNA and protein levels increased dramatically without any hormone added, meanwile, SULT2A2 and ST-40 mRNA expressions were suppressed significantly. Dexamethasone is important for SULT2A expression, but no effect on SULT2B1b expression. Insulin upregulated SULT2B1b mRNA and protein levels dose-dependently, but did not effect on SULT2A. T4 stimulated SULT2B1b mRNA expression, but not as strong as Insulin. In conclusion, SULT2B1b is a highly regulated hydroxysteroid sulfotransferase, which is different form the well-known SULT2A1.In the third part, we studied the effect of 25HC3S on intracellular lipids metabolism in primary rat hepatocytes. 25HC was studied parallelly as comparison. Addition of varying concentrations of 25HC3S to primary rat hepatocytes markedly inhibited CYP7A1 mRNA expression, which is much stronger than 25HC. 25HC3S shows more potent on inhibiting SREBP-1, and SREBP-2 mRNA expression, subsequently HMG CoA reductase than 25HC. 25HC3S decreases both protein levels of SREBP-1 precursor and mature forms. However, 25-HC increases SREBP-1 precursor and does not affect mature form. These results indicate that 25HC3S, a hydrophilic oxysterol, plays an important role, but in different manner from 25HC in intracellular lipids metabolism in primary rat hepatocytes.In summary, 25HC3S is a functional nuclear oxysterol in hepatocytes. 25-OH Cholesterol is synthesized by CYP27A1 in mitochondria of hepatocytes, and then sulfonated to 25-OH cholesterol sulfate by hydroxysteroid sulfotransferase SULT2B1b in hepatocytes. And the whole pathway is highly regulated.