7α-Hydroxylation of dehydroepiandrosterone does not interfere with the activation of glucocorticoids by 11β-hydroxysteroid dehydrogenase in E^tC cerebellar neurons

Highlights

• We report that E^tC neuronal cerebellar cells possess 11β-HSD1 and H6PDH activity.

• We examine the interplay of glucocorticoid reduction and DHEA hydroxylation.

• Glucocorticoid activation by 11β-HSD1 is not affected by 7α-hydroxylation of DHEA.

• 7α-OH-DHEA is the sole product of DHEA metabolism by E^tC neuronal cells.

Abstract

The neuroprotective action of dehydroepiandrosterone (DHEA) in the absence of a known specific receptor has been attributed to its metabolism by different cell types in the brain to various steroids, with a preference to its 7-hydroxylated products. The E^tC cerebellar granule cell line converts DHEA almost exclusively to 7α-hydroxy-DHEA (7α-OH-DHEA). It has been postulated that DHEA's 7-OH and 7-oxo metabolites can decrease glucocorticoid levels by an interactive mechanism involving 11β-hydroxysteroid dehydrogenase (11β-HSD). In order to study the relationship of 7-hydroxylation of DHEA and glucocorticoid metabolism in intact brain cells, we examined whether E^tC cerebellar neurons, which are avid producers of 7α-OH-DHEA, could also metabolize glucocorticoids. We report that E^tC neuronal cells exhibit 11β-HSD1 reductase activity, and are able to convert 11-dehydrocorticosterone into corticosterone, whereas they do not demonstrate 11β-HSD2 dehydrogenase activity. Consequently, E^tC cells incubated with DHEA did not yield 7-oxo- or 7β-OH-DHEA. Our findings are supported by the reductive environment of E^tC cells through expression of hexose-6-phosphate dehydrogenase (H6PDH), which fosters 11β-HSD1 reductase activity. To further explore the role of 7α-OH-DHEA in E^tC neuronal cells, we examined the effect of preventing its formation using the CYP450 inhibitor ketoconazole. Treatment of the cells with this drug decreased the yield of 7α-OH-DHEA by about 75% without the formation of alternate DHEA metabolites, and had minimal effects on glucocorticoid conversion. Likewise, elevated levels of corticosterone, the product of 11β-HSD1, had no effect on the metabolic profile of DHEA. This study shows that in a single population of whole-cells, with a highly reductive environment, 7α-OH-DHEA is unable to block the reducing activity of 11β-HSD1, and that 7-hydroxylation of DHEA does not interfere with the activation of glucocorticoids. Our investigation on the metabolism of DHEA in E^tC neuronal cells suggest that other alternate mechanisms must be at play to explain the in vivo anti-glucocorticoid properties of DHEA and its 7-OH-metabolites.

Introduction

It is now well established that dehydroepiandrosterone (DHEA) is produced in the central nervous system independently from its adrenal synthesis [1] and that it has various neuroprotective properties. The many diverse effects of DHEA in the brain, in the absence of a known specific receptor, have been attributed to its metabolic products that are formed by different cell types [2]. For example, in microglia, DHEA is reduced by 17-β-hydroxysteroid dehydrogenase to 5-androstenediol [3], whereas in hippocampal granule cells it is hydroxylated to its 7α form [4]. On the other hand, in astrocytes it is converted to androstenedione (AD) [5], which serves as a precursor to the synthesis of androgens and estrogens; estrogens have been shown to be neuroprotective in hippocampal cells [6], [7] and to enhance learning and memory [rev. by 8]. Collectively, these data suggest the neuroprotection attributed to DHEA is a function of its metabolites, which are intrinsic to distinct cell types.

In hippocampal and cerebellar mouse neuronal cell lines, as well as in rat and mouse brain microsomes, the primary product of DHEA metabolism is 7α-hydroxy-DHEA (7α-OH-DHEA) [4], [9], [10], [11], and to a lesser extent, 7β-OH-DHEA has been reported in rat brain microsomes [12]. In the brain, the conversion of DHEA to 7α-OH-DHEA is primarily attributed to cytochrome P450 CYP7B [13], [14]. The 7-hydroxylated metabolites of DHEA can be neuroprotective [9], [15] and possibly improve memory [16], [17]. Furthermore, in dementia and Alzheimer's disease, levels of CYP7B are lower, which suggests a role for the loss of 7-hydroxylation in these disease states [18].

Although it is important to note that other 7α-hydroxylases are expressed in the brain, i.e. CYP39A1 [19], and liver, i.e. CYP7A [20], thus far, no known P450 enzyme has been identified to be responsible for the conversion of 7α-OH-DHEA to 7β-OH-DHEA [21]. However, it has been shown that 7α-OH-DHEA can be converted to 7β-OH-DHEA through a 7-oxo-DHEA intermediate via 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) [22], [23], the principal activating enzyme of glucocorticoids. This mechanism has yet to be shown to occur in vivo in the brain; additionally, there is no evidence of 7-oxo-DHEA formation in the brain.

The 7α and 7β-OH metabolites of DHEA are known to be more efficacious in their biological actions in various tissues than their parent steroid, DHEA. This is particularly evident in their immunomodulatory activity [10], [24], [25]. Likewise, DHEA and its metabolites have marked anti-stress [26] and anti-glucocorticoid properties [27], suggested to occur via various mechanisms. The inhibition of nuclear translocation of the glucocorticoid receptor has been reported in neurons [28], but not in transfected COS-7 cells [29]. Another proposed mechanism for DHEA's immunomodulatory activity is its involvement in the metabolism of glucocorticoids [rev. by 30]. 11β-HSD1 can convert the inactive glucocorticoid 11-dehydrocorticosterone (11-dehydro-CORT) (cortisone in humans) into corticosterone (CORT) (cortisol in humans) using NADPH as a cofactor; the 11β-HSD2 isoform can perform the reverse reaction using NAD⁺ as a cofactor [rev. by 31]. In vitro, it has been demonstrated that 7α-OH-DHEA can convert to 7β-OH-DHEA through a 7-oxo-DHEA intermediate via the NADP⁺ dependent 11β-HSD and this process is reversed by the addition of NADPH [22], [24]. Furthermore, using microsomal fractions, 7OH-DHEA was shown to inhibit the activity of 11β-HSD, therefore blocking formation of CORT. These data are highly suggestive that DHEA's anti-glucocorticoid activity may occur through a putative competitive inhibitory mechanism of 11β-HSD [23], [30]. In the current study, we aimed to determine if the mouse cerebellar neuronal cell line E^tC, which is an avid producer of 7OH-DHEA, possesses 11β-HSD activity, and use it as a whole-cell system to investigate the DHEA anti-glucocorticoid paradigm.