Studies on human sterol 27-hydroxylase with emphasis on its mechanism of regulation and metabolic consequences of a deficient enzyme

Abstract

Sterol 27-hydroxylase (CYP27A1) is a mitochondrial cytochrome P-450 enzyme present in most animal cells. In hepatocytes, CYP27A1 has an important role in connection with biosynthesis of bile acids from cholesterol. In other cells the enzyme is responsible for a mechanism by which cholesterol can be eliminated by conversion into more polar products that can easily escape from the cells. The above properties are consistent with an antiatherogenic effect of CYP27A1, which is also illustrated by the fact that patients lacking the enzyme develop premature atherosclerosis and xanthomas in tendons and in the brain (Cerebrotendinous Xanthomatosis). Upregulation of CYP27A1 would represent a new strategy to treat atherosclerosis, and thus it was regarded to be important to expand the knowledge about the mechanism of regulation of the enzyme in man. In the first part of this thesis the following findings were made, all of which are consistent with an antiatherogenic effect of CYP27A1:

There was a marked induction of CYP27A1 during differentiation of human monocytes into macrophages, with increased levels of mRNA and production of 27-oxygenated products of cholesterol. This induction has the potential to counteract the effects of induction of scavenger receptors.

The cytokine TGFâ1 was found to have a stimulatory effect on transcription and enzymatic activity of CYP27A1 in monocyte-derived macrophages. This effect was dependent upon responsive elements in the proximal region of the CYP27A1 gene. The possibility is discussed that part of the antiatherogenic effect of TGFâ1 may be mediated by its effects on CYP27A1.

Patients with Pulmonary Alveolar Proteinosis have a local accumulation of cholesterol in the lungs, known to contain particularly high levels of CYP27A1. This accumulation was associated with increased levels of 27- oxygenated steroids in the circulation. This increase may reflect a CYP27A1-mediated defence towards a local accumulation of cholesterol.

In the second part of the thesis a novel unique case of CTX is described, with bilateral Achilles tendon xanthomas as the only visible CTX manifestation. The patient was found to be a heterozygote with one CYP27A1 allele containing a previously not described mutation in exon 8: a two-base substitution (C478A and C479A) possibly disrupting the heme-binding domain of the enzyme. Expression of this mutant in HEK293 cells led to a defective enzyme. Despite extensive sequencing efforts, no other mutation was found in the CYP27A1 gene. Reduced levels of 27-hydroxycholesterol in the plasma of other family members suggest that another genetic defect is present, and that this defect together with the mutation in exon 8 is causing CTX in this patient. The formation of cholesterol and cholestanol containing xanthomas in the brain of CTX patients is difficult to explain in view of the fact that neither cholesterol nor its 5á-saturated analogue cholestanol are likely to pass the blood-brain barrier. Using a model system of the blood-brain barrier, it was shown that bile acid precursor 7á-hydroxy-4-cholesten-3-one, which is accumulated in the circulation of CTX patients to levels more than 100-fold of normal, is passing cultured brain endothelial cells at a rate about 100-fold higher than that of cholesterol and cholestanol. Cultured human astrocytes, microglial cells and neurogenic cells were able to convert 7á-hydroxy-4-cholesten-3-one into cholestanol. This reaction did also occur in human moncyte-derived macrophages. It is suggested that most of the cholestanol present in brain xanthomas of CTX patients accumulate as a consequence of a flux of 7á-hydroxy-4-cholesten-3-one over the blood-brain barrier. The possibility is discussed that the accumulation of cholesterol is secondary to the accumulation of cholestanol.