Expression and localization of sterol 27-hydroxylase (CYP27A1) in monkey retina

Abstract

Sterol 27-hydroxylase (CYP27A1) is a mitochondrial P-450 enzyme with broad substrate specificity for C27 sterols including 7-ketocholesterol (7kCh). CYP27A1 is widely expressed in human tissues but has not been previously demonstrated in the retina. In this study, we examined the expression and localization of CYP27A1 in the monkey retina where it localized mainly to the photoreceptor inner segments. CYP27A1 was also observed in Müller cells with faint immuno staining detected in the RPE and choriocapillaris. We also determined that the 27-hydroxylation of 7-ketocholesterol (27OH7kCh) rendered it non-toxic to cultured RPE cells. Moreover, the 27OH7kCh when mixed with 7-ketocholesterol significantly reduced the toxicity of 7-ketocholesterol. These data, when taken in context of the known functions of CYP27A1 imply that expression in the retina serves to modify the biological activity of oxidized sterols that are either transported or generated locally by photooxidation. Published by Elsevier Ltd. Keywords cholesterol efflux; macula; 7-ketocholesterol; ARPE19; photoreceptors The sterol 27-hydroxylase (CYP27A1:P450c27, Andersson et al., 1989) is a mitochondrial enzyme that performs multiple functions (Javitt, 2002, review): it is known to catalyze bile acid hydroxylations (Russell, 2003, review) and to activate vitamin D (Usui et al., 1990); furthermore, the formation of 27-hydroxycholesterol (27OHCh) by CYP27A1 has been demonstrated to stimulate cholesterol efflux in cultured cells (Escher et al., 2003). CYP27A1 is deficient in individual with the rare disease cerebrotendinous xanthomatosis (CTX) . CTX patients suffer from progressive neurological impairment as well as accelerated atherosclerosis and cataracts (Moghadasian, 2004, review).CYP27A1 readily hydroxylates the oxysterol 7-ketocholesterol (7kCh) to 27-hydroxy-7-ketocholesterol (27OH7kCh) (Brown et al., 2000), which is highly toxic to a variety of cells (Lyons and Brown, 1999) including RPE cells and is the main cholesterol oxide in oxidized low density lipoprotein (Brown et al., 1996) (Brown and Jessup, 1999).The rationale for this study is threefold: (1) CYP27A1 is known to be involved in the cholesterol efflux pathway (Escher et al., 2003), (2) CYP27A1 is known to be involved in the metabolism of 7kCh (Brown et al., 2000;Lyons and Brown, 2001;Jessup and Brown, 2005) and (3) despite the extensive knowledge of cholesterol hydroxylation in the brain (Björkem and Meaney, 2004; Diestschy and Turley, 2004, reviews), there are no published reports concerning CYP27A1 (or other sterol hydroxylases) in the retina. The retina is known to perform de novo synthesis (Fliesler et al., 1993) as well as cholesterol uptake from circulating blood lipoproteins (Elner, 2002;Gordiyenko et al., 2004). However, little is known about the cholesterol efflux or how the retina maintains cholesterol homeostasis. CYP27A1 may be involved in cholesterol efflux by forming 27OHCh which can change the lipid composition of the plasma membrane and/or can alter gene expression by ...

Conversion of 7-ketocholesterol to oxysterol metabolites by recombinant CYP27A1 and retinal pigment epithelial cells

“…The former would be in agreement with the results of our in vitro studies evaluating cholesterol and 7KCh as the substrates for CYP27A1, as well with prior investigations by others showing cytotoxic, pro-infl ammatory, and apoptotic effects of 7KCh on the RPE-derived cells ( 4,15,44 ). 27-Hydroxylation seems to reduce toxicity of 7KCh as demonstrated by survival of the RPE cells treated with 7KCh-27OH at concentrations and time in which 7KCh essentially killed the entire culture ( 15 ). Thus, high 7KCh-27OH/7KCh ratio in a cell would be consistent with attenuated toxicity of 7KCh-27OH relative to 7KCh.…”

“…By using in vitro assays, we demonstrated for the fi rst time that purifi ed recombinant CYP27A1 binds and metabo- compared with the 27COOH/cholesterol ratio may refl ect either more effi cient metabolism of 7KCh relative to cholesterol or less effi cient elimination of 7KCh-27OH relative to 27COOH. The former would be in agreement with the results of our in vitro studies evaluating cholesterol and 7KCh as the substrates for CYP27A1, as well with prior investigations by others showing cytotoxic, pro-infl ammatory, and apoptotic effects of 7KCh on the RPE-derived cells ( 4,15,44 ). 27-Hydroxylation seems to reduce toxicity of 7KCh as demonstrated by survival of the RPE cells treated with 7KCh-27OH at concentrations and time in which 7KCh essentially killed the entire culture ( 15 ).…”

“…Indeed, CYP27A1 is involved in cholesterol elimination from extrahepatic tissues, metabolizes bile acid intermediates in the liver, and activates vitamin D 3 in the kidney (12)(13)(14). CYP27A1 is also a mitochondrial protein whose immunolocalization in the retina overlaps with that of 7KCh: photoreceptor inner segments, ganglion cell layer, and RPE ( 15 ). Furthermore, 7KCh was found to be 27-hydroxylated in human macrophages and HepG2 cells ( 16,17 ); it was suggested to be the substrate for CYP27A1 based on the site of hydroxylation, reduction of its metabolism by the cells treated with several CYP27A1 inhibitors, and absence of 27-hydroxylated 7KCh metabolite in the medium when CYP27A1-defi cient human macrophages were incubated with exogenous radioactive 7KCh ( 16 ).…”

“…Note that normalizations based on total protein do not refl ect local concentrations of CYP27A1 in the neural retina and RPE. The neural retina is a multilayered and multicellular organ with uneven expression of CYP27A1 as assessed by immunohistochemistry ( 15 ), whereas the RPE represents one layer of the same cell type. Therefore, concentrations of CYP27A1 in some retinal cells could be comparable to those in the RPE.…”

Cholesterol oxidation in the retina: implications of 7KCh formation in chronic inflammation and age-related macular degeneration

“…To our knowledge, the only cytochrome P450s reported in the retina that are capable of hydroxylating 7KCh are CYP27A1 ( 39,65 ) and CYP46A1 ( 45 ). CYP27A1 is a mitochondrial protein that can be readily detected in the retina by immunoblot and has been localized by immunofl uorescence ( 65 ). Defi ciency of CYP27A1 is known to cause a rare autosomal recessive disease called Cerebrotendinous Xanthomatosis ( 66 ).…”