A steroidogenic pathway for sulfonated steroids: The metabolism of pregnenolone sulfate

https://doi.org/10.1016/j.jsbmb.2014.07.005Get rights and content

Highlights

Abstract

In many tissues sulfonated steroids exceed the concentration of free steroids and recently they were also shown to fulfill important physiological functions. While it was previously demonstrated that cholesterol sulfate (CS) is converted by CYP11A1 to pregnenolone sulfate (PregS), further conversion of PregS has not been studied in detail. To investigate whether a steroidogenic pathway for sulfonated steroids exists similar to the one described for free steroids, we examined the interaction of PregS with CYP17A1 in a reconstituted in-vitro system. Difference spectroscopy revealed a Kd-value of 74.8 ± 4.2 μM for the CYP17A1–PregS complex, which is 2.5-fold higher compared to the CYP17A1–pregnenolone (Preg) complex. Mass spectrometry experiments proved for the first time that PregS is hydroxylated by CYP17A1 at position C17, identically to pregnenolone. A higher Km- and a lower kcat-value for CYP17A1 using PregS compared with Preg were observed, indicating a 40% reduced catalytic efficiency when using the sulfonated steroid. Furthermore, we analyzed whether the presence of cytochrome b5 (b5) has an influence on the CYP17A1 dependent conversion of PregS, as was demonstrated for Preg. Interestingly, with 17OH-PregS no scission of the 17,20-carbon–carbon bond occurs, when b5 is added to the reconstituted in-vitro system, while b5 promotes the formation of DHEA from 17OH-Preg. When using human SOAT-HEK293 cells expressing CYP17A1 and CPR, we could confirm that PregS is metabolized to 17OH-PregS, strengthening the potential physiological meaning of a pathway for sulfonated steroids.

Introduction

CYP11A1 initiates steroid hormone biosynthesis (Fig. 1) through a side-chain cleavage reaction on cholesterol yielding pregnenolone [1], [2] which represents the precursor of all steroid hormones. In a series of reactions where six different cytochromes P450 (CYPs) and three hydroxysteroid dehydrogenases (HSD) participate, mineralocorticoids, glucocorticoids and sex hormones are formed.

In order to induce a biological response, steroid hormones interact with their corresponding receptor and thus e.g., regulation of blood pressure, provision of carbon hydrates or development of secondary sexual characteristics take place in mammalian organisms. Interestingly, sulfonated steroids or sulfated steroids [3] often circulate in mammals in considerably higher concentrations than unconjugated steroids [4], [5], [6]. Dehydroepiandrosterone (DHEA) for example, one of the most abundant steroid in humans, occurs to 99% in its sulfonated form [7], reaching concentrations of up to 10 μM in young adults [8]. Steroid sulfates are generated by sulfonation of free steroids by three different sulfotransferases (SULT1E1, SULT2B1, SULT2A1). These enzymes are widely distributed in mammalian organism, as NCBI EST profiles indicate. Sulfonation of unconjugated steroids seems to contribute to the modulation of the genomic action of steroids. For instance, SULT1E1 is highly active in cultured normal breast epithelial cells compared to tumor cell lines [9]. Since it is known that increased exposure of estrogens increases breast cancer development, sulfonation of estrogens might be a crucial mechanism to impede the danger of excessive estrogenic exposure [9]. In addition, alterations of steroid sulfonation have a severe impact on the development in mammals. DHEA is mainly produced in the adrenals and the gonads. In the gonads DHEA is further metabolized to sex hormones, but in the adrenals, due to the presence of SULT2A1, most of the DHEA is sulfonated. In case of altered DHEA sulfonation, unconjugated DHEA accumulates, yielding to an excess of androgens and thus, virilization occurs in women [10].

On the other hand, excess of sulfonated steroids can lead to diseases, like the X-linked ichtiosis, which is caused by the accumulation of cholesterol sulfate in the epidermis [11].

Cell uptake of steroid sulfates depends on transporters of the SLC or SLCO family [12], [13], [14] as their hydrophilicity caused by the sulfate moiety hinders a passive passage. These transporters are expressed in many tissues, like liver, ovary, adrenal gland and hippocampus [15].

Sulfonated steroids have been regarded for a long time either as an end-product of xenobiotic metabolism designated for renal clearance [16] or as an inactive reservoir for unconjugated, active steroids [17]. Although the knowledge about the physiological role of steroid sulfates is still scarce, investigations about their biological function increased in the last decade. Recent studies showed that sulfonated steroid metabolites are involved in many different processes in mammalian organisms. Cholesterol sulfate (CS) possesses a stabilizing function in cell membranes; it is involved in the regulation of serine proteases and, moreover, in the differentiation of keratinocytes [18]. Recently, DHEAS was shown to induce a non-classical signaling pathway in spermatogenic cells [19]. Pregnenolone sulfate (PregS), on the other hand, represents a reservoir for pregnenolone (Preg), the precursor of mineralo-, and glucocorticoids, as well as sex hormones and it is described to act as neurosteroid modulating a big variety of ion channels, transporters, and enzymes [15]. For example, PregS was demonstrated to inhibit GABA-receptors [20], which play a crucial role in the neuronal network and to modulate N-methyl-d-aspartate (NMDA)-receptors. These receptors, which are essential for neuronal development and synapse formation, are heterodimers formed by several subunits (NR1, NR2A–D, NR3A–B). PregS modulates these receptors by enhancing the generation of NR1/NR2A and NR1/NR2B receptors and inhibiting the formation of NR1/NR2C and NR1/NR2D receptors [21]. Further studies revealed that PregS promotes NMDA-receptor insertion in the cell surface [22] and thus, enhances the function of NMDA-receptors.

Moreover, PregS seems to play an essential role in reproduction as extremely increased concentrations during birth, pregnancy and parturition indicate [15].

It was demonstrated that PregS can be formed from CS through a side-chain cleavage reaction catalyzed by CYP11A1 [23] and seems to be metabolized to further sulfonated steroids as Korte et al. showed using tissue from human adrenals [24]. These findings inevitably lead to the question, whether a steroidogenic pathway for sulfonated steroids exists similar to the one described for free steroids. Trying to elucidate this question, we decided to investigate whether PregS serves as substrate for CYP17A1, a microsomal cytochrome P450 involved in the steroid hormone biosynthesis. CYP17A1 catalyzes the hydroxylation at position C17 of Preg or progesterone (Prog) yielding 17OH-Preg and 17OH-Prog, respectively, and subsequently catalyzes a 17,20-lyase reaction yielding dehydroepiandrosterone (DHEA) and androstenedione (andro) [25] (Fig. 2). In human and bovidae families (sheep, goat, bovine, bison), CYP17A1 exhibits lyase activity only toward delta5-steroids, like Preg and 17OH-Preg [26]. In these mammalian species CYP17A1 hydroxylates Prog at position C17 but 17OH-Prog is not further converted to androstenedione. Compared to the 17-hydroxylase activity, the 17,20-lyase reaction using 17OH-Preg as substrate is weak, but it is strongly enhanced in the presence of cytochrome b5 (b5) [27] or through phosphorylation of CYP17A1 at position 258 of its amino acid chain [28].

Here, we investigated the reaction of CYP17A1 with PregS as substrate in a reconstituted in-vitro system, consisting of recombinantly expressed and purified CYP17A1 and its electron transfer partner CPR, and were able to demonstrate for the first time its conversion to 17OH-PregS, but not to DHEAS.

Section snippets

Materials

Steroids were obtained from Sigma–Aldrich (Taufkirchen, Germany) or from C/D/N Isotopes Inc. (Quebec, Canada). 1,2-Dilauroyl-sn-glycero-3-phosphocholine (DLPC), kanamycin sulfate, arabinose, magnesium chloride, sodium hydroxide, sulfur trioxide triethylamine, sulfatase from Helix pomatia Type H-1 and HPLC-grade acetonitrile were from Sigma–Aldrich (Taufkirchen, Germany). Yeast extract, technical was from Becton, Dickinson and Company (Heidelberg, Germany). Pepton, pancreatically digested and

Construction of recombinant expression plasmids in E. coli

The plasmid pET-17b was utilized to express bovine CYP17A1, b5 and CPR in E. coli. Each cDNA was cloned via the restriction sites NdeI and BamHI into the vector. The cDNAs of b5 and CPR were obtained through amplification from a cDNA library of bovine liver (Zyagen, California, USA). CYP17A1 cDNA was kindly provided by Prof. M. Waterman (Vanderbilt University, Nashville, USA). The amino acid sequence of CYP17A1 is lacking its N-terminal hydrophobic anchor [29] and is extended at the C-terminus

Results and discussion

The levels of sulfonated steroids exceed in many species and tissues considerably the levels of the corresponding unconjugated steroids. In human, the plasma level of PregS varies significantly during life. The highest level is reached after birth with concentrations between 2–3 μM, depending on the gender, and afterwards rapidly decreases in the first year up to concentrations of about 30–50 nM [43]. During and after adrenarche, the PregS level in plasma again increases, achieving concentrations

Summary

In this work we could clearly demonstrate that PregS serves as a substrate for CYP17A1, being converted to 17OH-PregS. Summarizing, we demonstrated for the first time that CYP17A1, which is involved in the steroid hormone biosynthesis, can convert sulfonated steroids in a similar manner as free steroids indicating a potential alternative steroidogenic pathway for sulfonated steroids. As already described, this pathway is initiated by the side-chain cleavage of CS by CYP11A1, yielding PregS [23]

Acknowledgement

The financial support of the Deutsche Forschungsgemeinschaft DFGFOR1369 is gratefully acknowledged.

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