16α-Hydroxyprogesterone: Origin, biosynthesis and receptor interaction

Molecular and Cellular Endocrinology 336 (2011) 92–101 Contents lists available at ScienceDirect Molecular and Cellular Endocrinology journal homepage: www.elsevier.com/locate/mce Review 16␣-Hydroxyprogesterone: Origin, biosynthesis and receptor interaction Karl-Heinz Storbeck, Pieter Swart, Donita Africander, Riaan Conradie, Renate Louw, Amanda C. Swart ∗ Department of Biochemistry, University of Stellenbosch, Stellenbosch 7602, South Africa a r t i c l e i n f o Article history: Received 31 August 2010 Received in revised form 11 November 2010 Accepted 11 November 2010 Keywords: 16␣-Hydroxyprogesterone 16-OHPROG Cytochrome P450 17␣-hydroxylase/17,20-lyase CYP17A1 Progesterone receptor PR a b s t r a c t The metabolism of progesterone (PROG) by cytochrome P450 17␣-hydroxylase/17,20-lyase (CYP17A1) results in the formation of both 17␣-hydroxyprogesterone (17-OHPROG) and 16␣-hydroxyprogesterone (16-OHPROG) in humans. Unlike 17-OHPROG, 16-OHPROG is not metabolised further in steroidogenic tissue. While this metabolite can be readily detected in serum and urine, its physiological role remains unclear. This paper reviews the production of 16-OHPROG by human CYP17A1 by providing insight into the catalysis of PROG by CYP17A1 and highlights the role of Ala105 in the 16␣-hydroxylation reaction. As 16-OHPROG has been putatively linked to reproductive function, we investigated the interaction of this steroid metabolite with both isoforms of the human progesterone receptor (hPR). We show for the first time that 16-OHPROG can bind to both hPR-A and hPR-B and act as an agonist for both receptors. © 2010 Elsevier Ireland Ltd. All rights reserved. Contents 1. 2. 3. 4. 5. 6. 7. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92 Early investigations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93 Catalysis by CYP17A1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93 3.1. Expression of CYP17A1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94 3.2. Linking the 16␣-hydroxylation of PROG to CYP17A1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94 3.3. The role of Ala105 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94 3.4. Kinetic analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95 3.5. The role of cytochrome b5 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95 3.6. Additional sources of 16-OHPROG . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95 Metabolism of 16-OHPROG . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96 Putative physiological functions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97 5.1. Reproductive function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97 5.2. Natriuretic effect . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97 Interaction with the steroid receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99 Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100 1. Introduction Abbreviations: 3␤HSD, 3␤-hydroxysteroid dehydrogenase; 16-OHPROG, 16␣hydroxyprogesterone; 17-OHPREG, 17␣-hydroxypregnenolone; 17-OHPROG, 17␣hydroxyprogesterone; A4, androstenedione; AIS, androgen insensitivity syndrome; AR, androgen receptor; CAH, congenital adrenal hyperplasia; CYP17A1, cytochrome P450 17␣-hydroxylase/17,20-lyase; CYP21A2, cytochrome P450 21-hydroxylase; DHEA, dehydroepiandrosterone; ER, estrogen receptor; GR, glucocorticoid receptor; hPR, human progesterone receptor; hMR, human mineralocorticoid receptor; MR, mineralocorticoid receptor; PRE, progesterone response element; PREG, pregnenolone; PROG, progesterone; wt, wild type. ∗ Corresponding author. Tel.: +27 21 8085861; fax: +27 21 8085863. E-mail address: acwart@sun.ac.za (A.C. Swart). 0303-7207/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.mce.2010.11.016 The production of 16-OHPROG was first documented in human steroidogenic tissue in the 1960s. There is ample evidence that this steroid metabolite is produced in vivo by humans and released into circulation. The potential physiological role of this metabolite has, however, received scant attention up to now and remains unclear. Although the physiological function of this steroid metabolite has to date, not been unraveled, it is likely that 16-OHPROG is a role player in the steroidogenic milieu. Early studies associated 16- K.-H. Storbeck et al. / Molecular and Cellular Endocrinology 336 (2011) 92–101 OHPROG with normal reproductive physiology as well as a number of clinical conditions. The metabolite was shown to be secreted in a cyclic manner during the menstrual cycle and in increasing concentrations during pregnancy, declining rapidly postpartum (Stiefel and Ruse, 1969). In addition, plasma concentrations are reported to be significantly higher in the umbilical cord than in maternal circulation (Den et al., 1979). Elevated 16-OHPROG levels have also been reported in patients diagnosed with Cushing’s disease (Villee et al., 1962; Ward and Grant, 1963; Villee, 1964) and the salt-losing form of congenital adrenal hyperplasia (CAH) (Janoski et al., 1969). While the latter finding hinted at the metabolite interacting with the mineralocorticoid receptor (MR), higher levels of 16-OHPROG may result from cytochrome P450 21-hydroxylase (CYP21A2) deficiency with concomitant decreased aldosterone biosynthesis being the primary cause of the salt loss. Natriuretic effects have, however, been reported in healthy test subjects (Jacobs, 1969). Nonetheless, the interaction of 16-OHPROG with the MR, or other steroid receptors cannot be discounted. In this review we document the detection of 16-OHPROG production in steroidogenic tissue, and in non-traditional steroidogenic tissue. We present studies regarding the metabolism of 16-OHPROG, as well as the physiological role of the metabolite established to date. The 16␣-hydroxylation of PROG is catalysed primarily by CYP17A1, and the catalytic importance of the residue Ala105 in the human enzyme is demonstrated. Since there is increasing evidence that metabolites of PROG may have significant biological effects, we investigated the ability of 16-OHPROG to bind to, and activate both the A and B isoforms of the hPR, which play critical roles in reproductive function. 2. Early investigations The production of 16-OHPROG was reported in 1961 when the metabolite was identified in homogenates as well as in slices of human fetal adrenals upon incubation with PROG (Villee et al., 1961; Lanman, 1961). Subsequent studies showed 16-OHPROG to be one of the major metabolites produced when adult human adrenals from patients diagnosed with Cushing’s disease were incubated with PROG (Villee et al., 1962; Ward and Grant, 1963; Villee, 1964). Villee et al. (1962) showed that 16-OHPROG was the major metabolite formed from the metabolism of 14 C-PROG, together with other metabolites which included 17-OHPROG, 11␤-hydroxyandrostenedione, corticosterone and cortisol — no radioactivity was associated with androstenedione or deoxycorticosterone. The 16␣-hydroxylation of PROG was subsequently confirmed in the fetal adrenal (Villee and Driscoll, 1965). Consistent with this, Ramseyer et al. (1974), using samples of adrenal vein blood from three healthy adults before and after the administration of corticotrophin, showed that the normal adult adrenal is a source of 16-OHPROG and that its secretion could be stimulated by ACTH. A number of studies have also reported on 16-OHPROG production in normal as well as in cryptorchid testes. Griffiths et al. (1963) showed that cryptorchid testes exhibited 16␣-hydroxylase activity towards PROG, while Bloch (1964) identified testosterone, 17-OHPROG and 16-OHPROG as PROG metabolites in fetal testes homogenates. Sharma et al. (1965) detected significant levels of 16-OHPROG when testicular tissue from patients diagnosed with androgen insensitivity syndrome (AIS) was incubated with PROG. The production of this metabolite by normal human testes was later confirmed by Viscelli et al. (1965), although the levels of 16OHPROG were substantially lower than what had been found in testicular tissue obtained from patients diagnosed with cryptorchid testes (Griffiths et al., 1963) or AIS (Sharma et al., 1965). In 1966, the 16-hydroxylase activity was seemingly linked to the enzyme catalysing the 17␣-hydroxylation of PROG when Colla et al. (1966) carried out inhibitory studies using testes obtained from patients 93 diagnosed with AIS. The group showed that the compound, SU9055, was able to inhibit the formation of 16-OHPROG from PROG and that the degree of inhibition was comparable to that of the inhibition of 17␣-hydroxylation of PROG as well as the formation of testosterone. Oshima et al. (1967) subsequently showed that the 16␣-hydroxylase activity was associated with the microsomal fraction in homogenates and subcellular fractions of testes resected from patients with prostate cancer when incubated with PROG. In females, the production of 16-OHPROG was shown to be closely associated with pregnancy and with specific phases of the menstrual cycle. Warren and Salhanick (1961) found that minced ovary tissue from both normal and Stein-Levental ovaries, which contained no corpora lutea, produced 16-OHPROG when incubated with PROG. Zander et al. (1962a) were, however, able to detect endogenous 16-OHPROG from pooled extracts of thirty-eight corpora lutea obtained during the menstrual cycle and pregnancy. Similarly, Huang (1967) confirmed that 16-OHPROG was formed when PROG was metabolised by a corpus luteum which had been removed on day 26 of the menstrual cycle. Measuring 16-OHPROG in urine, Stiefel and Ruse (1969) demonstrated that the secretion of the metabolite increased progressively throughout pregnancy, declining markedly following delivery. They also found that there was a cyclic change in the secretion of the steroid metabolite during the menstrual cycle, with a maximum concentration being reached in the luteal phase, during which PROG levels are also at their maximum. In agreement with this, Sano et al. (1981) later observed similar changes in 16-OHPROG and 17-OHPROG concentrations when follicles and corpora lutea were isolated from the ovaries of twenty-five patients in different stages of the normal menstrual cycle. Earlier studies had shown 16-OHPROG to be present in the umbilical cord (Zander et al., 1962a,b), with Little et al. (1963) reporting that a soluble supernatant fraction of a normal human placental homogenate could catalyse the 16␣-hydroxylation of PROG. It was also demonstrated by Zander (1963) that 3 H-PROG was metabolised when administered in situ, to two midpregnancy fetuses via the umbilical cord vein. The majority of the radioactivity, which included 16-OHPROG, was found in the fetal liver. Subsequently investigators were able to isolate 16-OHPROG after incubations of fetal liver homogenate (Jungmann and Schweppe, 1967) and microsomal preparations (Lisboa and Gustafsson, 1968) with PROG. In contrast, other studies were unable to detect 16OHPROG in the liver after perfusion of previable human fetuses with PROG (Bird et al., 1966; Wilson et al., 1966) — 16-OHPROG was only detected in the adrenals, kidneys, “residual tissues” and perfusate (Bird et al., 1966). Wilson et al. (1966) were unable to detect 16-OHPROG in similar experiments with previable adrenalectomized human fetuses. It is therefore evident from the early studies presented above, that the production of 16-OHPROG is localized primarily in the steroidogenic tissue which includes the adrenal, testes and ovaries. Interestingly, Ruse and Solomon (1966) had found that when 14 C-PROG was administered to a pregnant subject, the urinary 16-OHPROG contained no 14 C-label which clearly indicated that peripheral 16␣-hydroxylation of PROG did not occur, suggesting that the 16-OHPROG produced by the steroidogenic tissue was the direct result of the 16␣-hydroxylation of PROG in this tissue. It was therefore to be expected that the production 16-OHPROG from PROG would be attributed to one of the steroidogenic cytochromes P450 as is demonstrated in Section 3. 3. Catalysis by CYP17A1 CYP17A1 catalyses two distinct mixed-function oxidase reactions. The first is the 17␣-hydroxylation of the C21 steroids, 94 K.-H. Storbeck et al. / Molecular and Cellular Endocrinology 336 (2011) 92–101 pregnenolone (PREG) and PROG. The resulting 17␣-hydroxy derivatives can form the substrates for the 17,20-lyase activity of CYP17A1, which results in the cleavage of the C17–20 bond to produce the respective C19 steroids, dehydroepiandrosterone (DHEA) and androstenedione (A4) (Nakajin and Hall, 1981; Nakajin et al., 1981). There are, however, marked species-dependent differences in the utilization of either 17␣-hydroxypregnenolone (17-OHPREG) or 17-OHPROG as substrates for the 17,20-lyase activity, with the human enzyme favouring 17-OHPREG (Lorence et al., 1990; Swart et al., 1993). A 3.1. Expression of CYP17A1 In humans, CYP17A1 expression has been reported in all the traditional steroidogenic tissues, with the exception of the placenta (Miller, 1998). These include the zona reticularis and the zona fasciculata of the adrenal (Hyatt et al., 1983), the Leydig cells of the testes (Saez, 1994) and the thecal cells of the ovary (Sasano et al., 1989). More recently CYP17A1 expression has been detected in luteinised granulosa cells (Moran et al., 2003). In the human fetus, CYP17A1 expression was detected in the transitional zone and fetal zone of the fetal adrenal throughout gestation (Mesiano et al., 1993; Narasaka et al., 2001). In addition to the classical steroidogenic tissue, CYP17A1 activity or expression has been reported in the human fetal kidney, thymus and spleen (Casey and MacDonald, 1982) as well as the human heart (Kayes-Wandover and White, 2000), kidney (Quinkler et al., 2003) and adipose tissue (Puche et al., 2002). B 3.2. Linking the 16˛-hydroxylation of PROG to CYP17A1 Investigating the catalytic activity of human/bovine CYP17A1 chimeras expressed in nonsteroidogenic COS-1 cells, Lorence et al. (1990) noted that, in addition to its 17␣-hydroxylase activity, human CYP17A1 was able to convert significant levels of PROG to 16-OHPROG, while the bovine enzyme produced negligible levels of 16-OHPROG. Studying the kinetics of PROG metabolism by human CYP17A1 expressed in COS-1 cells, Swart et al. (1993) subsequently showed that both 17␣- and 16␣-hydroxylated products were likely produced from a common active site and that the ratio of 17-OHPROG to 16-OHPROG was approximately 4:1 over a wide range of substrate concentrations. In the same year, Lin et al. (1993) reported the same ratio of 17-OHPROG to 16-OHPROG for human CYP17A1 expressed in COS-1 cells. In addition, Swart et al. (1993) also showed that, in microsomes prepared from fetal adrenal and adult testis, PROG was converted to 17-OHPROG and 16-OHPROG at a ratio of 3.5:1. The addition of antibodies raised against porcine CYP17A1 inhibited both the 17␣and 16␣-hydroxylation of PROG in fetal adrenal microsomes to the same degree, while no inhibition of the 21-hydroxylation of PROG was observed. Similar results were obtained with the inhibitor of cytochrome CYP17A1, ketoconazole. This study therefore provided the first direct evidence that the production of 16-OHPROG in steroidogenic tissue is catalysed by CYP17A1. Using a yeast expression system, Arlt et al. (2002) demonstrated that chimpanzee CYP17A1 had the ability to catalyse the 16␣-hydroxylation of PROG at similar levels as the human enzyme, while baboon and rhesus monkey CYP17A1 exhibited considerably less 16␣-hydroxylase activity. The reported ratios of 17␣-hydroxylase:16␣-hydroxylase activity in human (2.9:1) and chimpanzee (3.3:1) CYP17A1 were eight times higher than in baboon (25:1) and twice as high as in rhesus (5.5:1). Swart et al. (2002) were unable to detect 16-OHPROG when baboon CYP17A1 was expressed in HEK293 cells. In a more recent study, using UPLC-MS to assess steroid metabolites, Storbeck et al. (2008) found that human CYP17A1 metabolised PROG to 17-OHPROG and 16-OHPROG at a ratio of 2:1, while the Fig. 1. Ratio of 17-hydroxylated products to 16-OHPROG produced in the metabolism of 1 ␮M PROG after 8 h by wild types (A) human and (B) baboon CYP17A1 enzymes and their respective mutant constructs expressed in COS-1 cells in the absence and presence of human cytochrome b5 . The Graph Pad Prism® software was used for data manipulations, graphical representations, and statistical analysis. Columns were compared to the respective wild types by a one-way ANOVA, followed by Bonferroni’s multiple comparison test (*P < 0.05; **P < 0.005; ***P < 0.0001). Results are representative of three independent experiments. Reproduced from Swart et al. (2010). 17-OHPROG:16-OHPROG ratios were 7:1 and 22:1 for the baboon and goat enzymes, respectively. 3.3. The role of Ala105 We have recently demonstrated that the ability of the human enzyme to hydroxylate PROG at the C16 position is due to a single residue, Ala105. The chimpanzee enzyme, which also demonstrates significant 16␣-hydroxylase activity towards PROG (Arlt et al., 2002), has an alanine at position 105, while all other mammalian species have a leucine residue at this alignment position. The introduction of the Ala105Leu substitution into human CYP17A1, significantly diminished 16␣-hydroxylase activity, while the converse substitution in the baboon, goat and pig enzymes significantly enhanced 16-OHPROG production by these species as shown in Fig. 1 (Swart et al., 2010). The Ala residue at position 105 lies in the predicted B′ -C domain which meanders in and out of the heme pocket (Graham-Lorence and Peterson, 1996). This domain forms the first of six substrate recognition sites identified for the cytochromes P450 (Gotoh, 1992; Graham-Lorence and Peterson, 1996). We proposed, using homology modelling, that the small Ala K.-H. Storbeck et al. / Molecular and Cellular Endocrinology 336 (2011) 92–101 Table 1 Summary of the kinetics of PROG metabolism by human CYP17A1 constructs expressed in COS-1 cells. Parameter values for the kinetic model constructed in Mathematica (Wolfram, S. Mathematica 7, Wolfram Research, Inc. (http://www.wolfram.com/)) were based on the metabolism of 1.5 ␮M PROG and validated using 2.5 ␮M PROG. PROG to 16-OHPROG Human wt Human A105L a PROG to 17-OHPROG a Km (␮M) Vmax 0.77 0.23 0.281 0.042 Km (␮M) Vmax a 0.77 0.23 0.603 0.339 nmol/h/mg protein. residue in the active site permits the repositioning of the PROG substrate required for the 16␣-hydroxylase reaction, while the larger Leu residue at this position in other species creates a steric hinderance, thus preventing the repositioning of PROG (Swart et al., 2010). Interestingly, although the Leu105Ala substitution increased the 16␣-hydroxylase activity relative to that of the 17␣-hydroxylase and 17,20-lyase activities, the overall metabolism of PROG by the baboon and pig wild type (wt) enzymes was significantly reduced. This is likely a result of the Leu105Ala substitution affecting the position of the adjacent Ser106, which is critical for catalysis. Lin et al. (1993) have previously shown that both Ser106Ala and Ser106Thr substitutions reduced the 17␣-hydroxylation of PROG to less than 30% of the wt, while reducing the 16␣-hydroxylation of PROG to ∼60% of the wt. The authors concluded that the hydroxyl group of the Ser residue may play an important role in positioning the substrate for the 17␣-hydroxylation and 17,20-lyase reactions, but not for the 16␣-hydroxylation reaction. It should however, be noted that other residues and domains may be important for maximal 16␣-hydroxylase activity. Lorence et al. (1990) demonstrated significant increases in the activity of the chimeric human/bovine enzyme containing the N-terminal (including Leu105) of the bovine enzyme, though the 16␣-hydroxylase was still two fold lower than the wt human enzyme. Similarly, the rhesus monkey produces substantially more 16-OHPROG than other species with a Leu residue at position 105 (Arlt et al., 2002). 3.4. Kinetic analysis From the above discussion it is clear that CYP17A1, like a number of cytochromes P450, is able to catalyse multiple hydroxylations of the same substrate in a regio-selective manner. Expressing human CYP17A1 in COS-1 cells, Swart et al. (1993) showed that the Km values for the 16␣- and 17␣-hydroxylation of PROG were not significantly different, but that the Vmax of the enzyme for 17-OHPROG production was 3.8-fold higher than that for 16-OHPROG production. In contrast, while expressing the human enzyme in yeast, Arlt et al. (2002) obtained significantly different Km and Vmax values for the enzyme in the production of 16-OHPROG and 17-OHPROG from PROG. The respective Km and Vmax values were 3.7- and 7.6-fold higher for the production of 17-OHPROG than for 16-OHPROG. The data presented above prompted further investigations into the kinetic constants and the ratios of 16␣- and 17␣-hydroxylated products. We employed kinetic modelling and a random search parameter estimation method (Moles et al., 2003) to investigate the metabolism of 1.5 ␮M PROG (Fig. 2) and determined the kinetic constants shown in Table 1. Validation was performed by analysing the model’s ability to predict the metabolism of 2.5 ␮M PROG in an independent experiment, in which the Vmax values were adjusted with the same scaling factor (data not shown). The ratio of 17␣-hydroxylated products to 16-OHPROG for wt human CYP17A1 and the Ala105Leu mutant construct expressed in COS-1 cells was ∼2 and ∼8, respectively, and remained constant over time 95 and substrate concentrations (Fig. 2). It was therefore assumed that CYP17A1 would have the same Km value (Table 1) for PROG in the 16␣- and 17␣-hydroxylation reactions, as was previously reported by Swart et al. (1993), and confirmed by the constructed kinetic model’s ability to describe the experimental data. The Vmax for 17-OHPROG production, catalysed by human CYP17A1, was approximately two fold greater than that for 16-OHPROG, accounting for the constant ratio of 17␣-hydroxylated products to 16-OHPROG irrespective of substrate concentrations. Similar results were obtained for the Ala105Leu mutant. The Ala105Leu substitution resulted in a 3.3 fold decrease in the Km for PROG and a 1.8 fold decrease in the Vmax for the 17␣-hydroxylation reaction. The rate of the 16␣-hydroxylation, however, decreased 6.7 fold, highlighting the importance of Ala105 for the 16␣-hydroxylation of PROG. The change in catalytic activity observed in the Ala105Leu mutant was not unexpected, as the substitution may influence the position of other amino acid residues important for catalysis, which include the previously mentioned Ser106 (Lin et al., 1993; Swart et al., 2010). In summary, the data presented here indicates that PROG binds at a single site in the active pocket of CYP17A1. The Ala105 residue permits the bound substrate the rotational freedom to pivot slightly, moving either the C17 or C16 perpendicular to the heme. In contrast, the larger Leu residue creates a steric hindrance preventing this rotation and resulting primarily in 17-OHPROG production. 3.5. The role of cytochrome b5 Cytochrome b5 , a small ubiquitous electron transfer protein, augments the 17,20-lyase activity of CYP17A1, while having no influence on the 17␣-hydroxylase activity (Katagiri et al., 1995). Augmentation has been shown to be via an allosteric mechanism, facilitating association between CYP17A1 and P450 oxidoreductase and not through the direct transfer of electrons to the hydroxylated intermediates (Auchus et al., 1998; Geller et al., 1999). Swart et al. (2010) did however, show that cytochrome b5 promoted the 16␣-hydroxylase activity of CYP17A1 constructs containing a Leu residue at position 105. We had previously proposed that the interaction of cytochrome b5 with CYP17A1 alters the three-dimensional structure of CYP17A1 in such a way as to allow the repositioning of the substrate in an orientation that is more favourable for the 16␣-hydroxylase reaction as additional electrons from P450 oxidoreductase are not required for the 16␣-hydroxylase reaction (Storbeck et al., 2007). Interestingly, cytochrome b5 had no significant effect on the catalytic activity of the constructs with an Ala residue at position 105, suggesting that space provided by the Ala residue for the repositioning of the substrate cannot be further enhanced by the allosteric interaction with cytochrome b5 . 3.6. Additional sources of 16-OHPROG The 16␣-hydroxylation of PROG has been reported in the human placenta (Little et al., 1963) and fetal liver (Zander, 1963; Jungmann and Schweppe, 1967; Lisboa and Gustafsson, 1968), however, neither of these tissues express CYP17A1. The metabolism of PROG by human liver microsomes results primarily in 6␤-hydroxylation, though low levels of 16-OHPROG have been detected (Waxman et al., 1988; Yamazaki and Shimada, 1997). Studying individual hepatic cytochromes P450, Waxman et al. (1991) showed that CYP3A3 and CYP3A4 both demonstrate 16␣-hydroxylation activity towards PROG, though this activity is significantly lower than their 6␤-hydroxylation activity, while Yamazaki and Shimada (1997) found that 6␤-, 16␣-, and 2␤-hydroxyprogesterones were major metabolites of PROG metabolism by CYP3A4. While studying the hydroxylation activities of human hepatic cytochromes 96 K.-H. Storbeck et al. / Molecular and Cellular Endocrinology 336 (2011) 92–101 A B C D Fig. 2. Metabolism of 1.5 ␮M PROG by human CYP17A1 and the A105L mutant construct expressed in COS-1 cells. (A) PROG metabolism by human wt CYP17A1, (B) ratio of 17␣-hydroxylated products to 16-OHPROG by human wt CYP17A1, (C) PROG metabolism by mutant construct, and (D) ratio of 17␣-hydroxylated products to 16-OHPROG by mutant construct. Results are representative of three independent experiments. P450 expressed in yeast, Niwa et al. (1998) found that the 6␤hydroxylase activity towards PROG by CYP3A4 was 4.4 times higher than the 16␣-hydroxylase activity. In addition, the authors demonstrated that CYP3A4 had the highest 16␣-hydroxylation activity towards PROG, followed by CYP1A1 and CYP2D6, while the other enzymes used in their study did not demonstrate such activity. A subsequent study by Schwarz et al. (2000) confirmed that CYP1A1 catalysed the 16␣-hydroxylation of PROG at approximately half of the rate of the 6␤-hydroxylation reaction. It is therefore clear that a number of hepatic cytochromes P450 are able to catalyse the 16␣-hydroxylation of PROG, accounting for the detection of 16OHPROG in the fetal liver (Zander, 1963; Jungmann and Schweppe, 1967; Lisboa and Gustafsson, 1968). The expression of a number of hepatic cytochromes P450, including CYP3A4 and CYP1A1, has been demonstrated in the full-term human placenta (Hakkola et al., 1996a) as well as in human placenta in the first trimester of pregnancy (Hakkola et al., 1996b), thereby accounting for the 16␣hydroxylation of PROG which Little et al. (1963) observed in the human placenta. While CYP17A1 is unable to catalyse the 16␣-hydroxylation of PREG (Swart et al., 1993), the hepatic enzymes CYP1A1 and CYP3A4 catalyse this reaction efficiently, with CYP2C19 also demonstrating low 16␣-hydroxylase activity towards PREG (Niwa et al., 1998). 16-OHPREG has been detected in peripheral circulation (Den et al., 1979; Ogawa et al., 1984), umbilical cord plasma (Shibusawa et al., 1978; Den et al., 1979; Kojima et al., 1981) and neonatal circulation (Den et al., 1979; Kojima et al., 1981). After injection of 14 C-labelled 16-OHPREG into the intact feto-placental circulation, Reynolds et al. (1969) isolated labelled 16-OHPROG from the fetal adrenals, liver and residual fetal tissue as well as the placenta suggesting that 16-OHPREG is a precursor to 16-OHPROG. However, measuring urinary steroids after the administration of 3 H-16-OHPREG to healthy subjects, Janoski and Kelly (1969) showed that the contribution of 16-OHPREG to the amount of 16-OHPROG secreted was negligible. The metabolism of 16-OHPREG to 16-OHPROG would likely be catalysed by 3␤-hydroxysteroid dehydrogenase (3␤HSD), though its expression has to date, only been demonstrated in the placenta, skin, and breast tissue (type I) as well as in the adrenal, ovary and testis (type II) (Rheaume et al., 1991; Simard et al., 1996). 4. Metabolism of 16-OHPROG To date, there have been no reports of 16-OHPROG being metabolised in steroidogenic tissue and it would seem that 16OHPROG is an end metabolite of the steroidogenic pathway. Indeed, Oshima et al. (1967) showed that the 16-OHPROG which formed in the homogenates and subcellular fractions of testes resected from patients with prostate cancer accumulated, and was not metabolised further. In the adrenal zona fasciculata 17-OHPROG is metabolised by CYP21A2 to deoxycortisol. We have incubated baboon CYP21A2, which shares 96% amino acid identity with the human enzyme, with both 16- and 17-OHPROG and found that although the enzyme was unable to metabolise 16-OHPROG, 17-OHPROG was readily converted to deoxycortisol (data not K.-H. Storbeck et al. / Molecular and Cellular Endocrinology 336 (2011) 92–101 shown). Although human CYP21A2 may metabolise 16-OHPROG, no metabolites resulting from the 21-hydroxylation of 16-OHPROG have been detected to date. Furthermore, the release of 16-OHPROG into the blood stream has clearly been demonstrated — the metabolite has been detected in the maternal peripheral circulation (Den et al., 1979; Ogawa et al., 1984), in umbilical cord plasma (Zander et al., 1962a,b; Den et al., 1979), in samples of human adrenal vein blood (Ramseyer et al., 1974), in the urine of normal males (Calvin and Lieberman, 1962; Ruse and Solomon, 1966), as well as that of females (Ruse and Solomon, 1966; Stiefel and Ruse, 1969). The production of 16-OHPROG by the steroidogenic tissue and its detection in plasma and urine raises questions on the physiological relevance of this steroid. Putative physiological roles of this metabolite are addressed in Section 5. 5. Putative physiological functions 5.1. Reproductive function The physiological importance of 16-OHPROG remains largely unknown at present. Studies discussed previously, which demonstrated a cyclic change in 16-OHPROG levels during the menstrual cycle (Stiefel and Ruse, 1969; Sano et al., 1981) as well as an increased 16-OHPROG concentration during pregnancy (Stiefel and Ruse, 1969), indicate that the metabolite may play an important role in the reproductive function of humans. To date only a limited number of studies have, however, been performed on this topic. Chatterton and Forbes (1973) used the Hooker-Forbes bioassay system to investigate the fate of intrauterine injection of 3 H-PROG. Substantial metabolism of PROG was shown, while PROG injected in one uterine horn was not detected in the contralateral horn indicating a lack of circulation. The addition of 16-OHPROG (1% by weight to PROG) to the injection mixture antagonized the stromal nuclear response, the end-point of the bioassay. In addition, the injection of 3 H-PROG and 16-OHPROG, in a 1:1 ratio, resulted in a significant increase in the concentration of PROG metabolites. It was concluded that minor increases in 16-OHPROG had a profound effect on the accumulation of the PROG metabolites in the uterus, which were likely responsible for the failure of PROG to elicit the stromal cell nuclear response. Forbes and Taku (1975) implanted pellets of PROG, testosterone or 17␤-estradiol, each with and without the addition of 1% 16-OHPROG, in the right uterine fat mass in ovariectomized and ovariectomized-hysterectomized mice. The study revealed that PROG implants led to a decrease in the diameter of the ovarian and uterine veins, while testosterone and 17␤-estradiol implants led to the veins increasing in diameter. Both the presence of the uterus and the administration of 1% 16-OHPROG were able to partially inhibit the increases in vein size induced by testosterone and 17␤-estradiol, indicating that 16-OHPROG may antagonize the effect of these steroids. However, the administration of 16-OHPROG could not prevent the uterine hypertrophy and urinary bladder distension caused by testosterone and 17␤-estradiol, suggesting that 16-OHPROG may exert tissue-specific effects. Den et al. (1979) found that the concentration of 16-OHPROG, as well as 16-OHPREG and 16-OHDHEA, were substantially higher in the human umbilical cord plasma than in the maternal peripheral circulation. In the umbilical cord plasma 16-OHPROG increased from 15.5 ± 3.2 ng/ml at 24 weeks to 34.3 ± 11.0 ng/ml at term. In addition, 16-OHPROG levels in the umbilical arterial plasma of full term neonates show a rapid decline within the first 24 h, while 16OHPREG levels remain relatively constant for the first five days post partum. More recently, in 66 pregnant women near term, Ogawa et al. (1984) demonstrated that the plasma levels of 16-OHPROG and other steroid metabolites increased as the uterine sensitiv- 97 ity to oxytocin increased, while the levels of PROG decreased significantly. A significant correlation between serum and myometrial concentrations of PROG, 16-OHPROG and the other steroid metabolites including 16-OHPREG, was identified. It was concluded that plasma levels of steroid hormones may be closely related to myometrial sensitivity to oxytocin and the onset of labour. Interestingly, in pregnancies associated with intrauterine growth retardation, the levels of PROG and 16-OHPROG have been reported to be significantly lower. Increased PREG and 16-OHPREG levels in these cases would, however, suggest impaired 3␤HSD activity (Maruyama et al., 1986). It would appear that higher levels of 16-OHPROG are closely associated with abnormal sexual development in males. Inano and Tamaoki (1978) have noted that the accumulation of 16-OHPROG in testicular microsomal fractions competitively (Ki 72 ␮M) inhibited the 17,20-lyase activity of CYP17A1, while the metabolite inhibited 20␣-hydroxysteroid dehydrogenase non-competitively (Ki 52.9 ␮M). The authors concluded that 16-OHPROG specifically inhibited the 17,20-lyase reaction of 17-OHPREG in the course of androgen biosynthesis in vitro, while the CYP17A1 hydroxylase, 3␤-hydroxysteroid dehydrogenase or 17␤-hydroxysteroid dehydrogenase activities were not influenced. The physiological implication of this finding is, however, questionable due to the high Ki values. Although 16-OHPROG is not metabolised further in the testes (Oshima et al., 1967) the metabolite may accumulate in the tissue and thus exert an effect. We have found that 16-OHPROG concentrations of up to 10 ␮M do not inhibit the conversion of 1 ␮M 17-OHPREG to DHEA by human CYP17A1 expressed in COS-1 cells (data not shown). Although the physiological actions of 16-OHPROG have not been fully elucidated, the results presented above demonstrate 16OHPROG to be involved in reproductive function. 5.2. Natriuretic effect Earlier studies undertaken in the 1960s indicated that 16OHPROG may have a natriuretic effect implicating possible interaction with the MR as is discussed in Section 6. Uete and Venning (1963) investigated the effect of 16␣-hydroxylated 4 and 5 -pregnene compounds on the electrolyte secretion in adrenalectomized rats. PROG diminished the sodium-retaining effect of aldosterone without significantly affecting the potassium secretion. 16-OHPROG and 17-OHPROG were, however, less effective than PROG in inhibiting sodium retention. George et al. (1965) showed that ACTH did not significantly affect serum sodium concentrations in patients diagnosed with the sodium-losing form of CAH and produced negligible increases in urinary sodium. Both PROG and 16-OHPROG demonstrated only weak renal sodiumlosing potency. Jacobs (1969) subsequently studied the effect of 16-OHPROG on the sodium, chloride and potassium balance in healthy test subjects. The administration of 16-OHPROG induced a saluresis, unaccompanied by a significant change in the potassium balance or endogenous creatinine clearance, indicating that 16-OHPROG may well be capable of exerting a natriuretic and chloruretic effect. Janoski et al. (1969) subsequently measured the production of 16-OHPROG and 16-OHPREG in two patients with salt-losing CAH. The production rate of 16-OHPROG was 24 and 28 mg/day, whereas only 310 and 315 ␮g of 16-OHPREG was produced during the same period. The rate of 16-OHPROG production decreased to 0.6 and 7.9 mg/day by the administration of suppressive glucocorticoid therapy. More recently, Quinkler et al. (2003) demonstrated that CYP17A1 is expressed in the human kidney and that the incubation of microsomal preparations of this tissue with PROG resulted in 16-OHPROG production. It is therefore possible that 16-OHPROG could accumulate in the kidney and play a role in sodium balance. 98 K.-H. Storbeck et al. / Molecular and Cellular Endocrinology 336 (2011) 92–101 These results indicate that 16-OHPROG has a weak natriuretic effect and that this effect is particularly pronounced in the saltlosing form of CAH, which is characterised by a reduced or absent aldosterone secretion and increased 16-OHPROG concentrations. 6. Interaction with the steroid receptors There is increasing evidence that PROG metabolites may have significant biological effects. To date, the relative actions of 16OHPROG at the cellular level remain unclear. It is known, however, that putative physiological effects of 16-OHPROG, like other steroid hormones, would be mediated through binding to intracellular steroid receptors, thereby regulating an array of genes. The steroid receptor family comprises the PR, glucocorticoid receptor (GR), MR, androgen receptor (AR), and estrogen receptor (ER), which are hormone-activated transcription factors that share a high level of similarity with regards to their structure, as well as their mechanism of action. However, the prediction of the physiological effects of 16-OHPROG in target tissues is not straightforward. These effects may be influenced by the fact that different cells have different levels, as well as different isoforms, of steroid receptors. Thus, a cell’s response to 16-OHPROG may differ depending on the receptor or receptor isoform the steroid binds to. In addition, receptor density in a specific cell has also been reported to determine the biological A B C - - - - - - - - Fig. 3. Interaction of 16-OHPROG with hPR isoforms expressed in COS-1 cells. (A) Competitive whole cell binding of 16-OHPROG to hPR-A and hPR-B: vectors pSG5hPR-A or pSG5hPR-B were expressed and incubated with 10 nM 3 H-PROG in the absence (total binding) and presence of either 10 ␮M unlabelled (non-specific binding) PROG, R5020 (a synthetic progestin), 16-OHPROG and RU468. Specific binding (total binding minus non-specific binding) is plotted. (B) Agonist activity of 16-OHPROG for transactivation via hPR-A and hPR-B: vectors, pSG5hPR-A or pSG5hPR-B, pTAT-PRE-E1b-luc reporter plasmid and the pCMV-␤-galactosidase were expressed and incubated with vehicle (ethanol, EtOH), 10 ␮M PROG, R5020, 16-OHPROG and RU468 for 24 h. Luciferase (luc) activity is shown as fold induction relative to EtOH set as 1. (C) Antagonist activity of 16-OHPROG for transactivation via hPR-A and hPR-B: vectors as in (B) were expressed and incubated with 1 nM PROG alone or in the presence of 10 ␮M R5020, 16-OHPROG or RU468 for 24 h. Relative light units (RLU) are shown with all test compounds relative to 1 nM PROG set as 100%. Results shown are the averages (±SEM) of at least three independent experiments performed in triplicate. Graph Pad Prism® software was used for data manipulations, graphical representations, and statistical analysis. One-way ANOVA, followed by Bonferroni’s multiple comparison test was used for statistical analysis. Statistical significance of differences is indicated by the letters a, b and c, where values which differ significantly from others are assigned a different letter. K.-H. Storbeck et al. / Molecular and Cellular Endocrinology 336 (2011) 92–101 character (agonist or antagonist) and the transcriptional activity (transactivation or transrepression) of steroids (Zhao et al., 2003). Another level of complexity is the presence of plasma membrane steroid receptors that signal by rapid non-genomic mechanisms and crosstalk between various signalling pathways (Turgeon et al., 2004). Considering that 16-OHPROG is a PROG metabolite, and that the primary physiological response of a cell to PROG is mediated by the PR, it is likely that 16-OHPROG mediates some physiological effects via the PR. The PR is expressed in the female reproductive tract, mammary gland, brain, as well as the pituitary gland (Mangal et al., 1997; Soyal et al., 2005). PR-mediated responses of PROG differ depending on the target tissue — in the uterus its actions are anti-proliferative, while in the breast it can both proliferate and differentiate (Richer et al., 2002). The PR-mediated biological responses of PROG are complex since PROG can activate two functional isoforms of the PR, PR-A and PR-B, which are transcribed from two promoters of a single gene (Kastner et al., 1990). Not only do the ratios of the individual isoforms vary in reproductive tissues (Shyamala et al., 1990), but they also have different physiological functions in various target tissues including the ovary, breast and uterus (Hung et al., 1994). Furthermore, PR-A and PR-B can respond to PROG to regulate overlapping and distinct transcriptional activity that are promoter- and cell-specific (Richer et al., 2002; Conneely et al., 2002). Considering these differences in transcriptional activities between PR-A and PR-B, it may be possible that they mediate different physiological responses to PROG, and also to other PR ligands. Indeed, a study in the Ishikawa endometrial cancer cell line showed that the synthetic progestin medroxyprogesterone acetate, like PROG, effectively inhibited growth of this cell line via stably transfected PR-B, but not PR-A (Smid-Koopman et al., 2003). For 16-OHPROG to mediate biological activity via the PR isoforms, whether agonistic or antagonistic activity, binding of 16-OHPROG to the respective isoforms is a prerequisite. We thus assessed whether 16-OHPROG could compete with 3 H-PROG for binding to overexpressed hPR-A and hPR-B. Competitive whole cell binding assays were performed in COS-1 cells transiently transfected with pSG5hPR-A or pSG5hPR-B expression vectors as described by Bamberger et al. (1995). 16-OHPROG binds to and mediates transcriptional activity, as results indicated that, at 10 ␮M, binding of the metabolite was ∼67% relative to 100% PROG for hPR-A, while for hPR-B it was ∼43% (Fig. 3A). The finding that 16-OHPROG binds to both PR isoforms, raised the question whether once bound, 16-OHPROG could activate PR-A and/or PR-B in a manner similar to PROG. We investigated the relative agonist as well as antagonist activity of 16-OHPROG for transcriptional regulation via a progesterone response element (PRE). The COS1 cell line was transiently transfected with a PRE-driven reporter construct containing two copies of the rat tyrosine amino transferase (TAT)-PRE-luc, a promoter luciferase reporter construct, and a copy of the respective PR isoform expression vector. At 10 ␮M 16-OHPROG, like PROG, is an agonist for transactivation via both hPR-A and hPR-B (Fig. 3B). A similar profile was observed at 1 ␮M 16-OHPROG (data not shown). Of note, 16-OHPROG, unlike RU486, a well known PR antagonist, did not display any antagonist properties via the PR isoforms (Fig. 3C). Although the physiological relevance of 16-OHPROG activity at concentrations in the micromolar range is questionable, the results cannot be excluded as it is possible for the metabolite to accumulate in target tissues leading to high concentrations in vivo. Our results suggest that by binding to PR-A or PR-B, 16-OHPROG may exert physiological functions similar to that of PROG. Thus, 16-OHPROG may play a role in various reproductive events, (as discussed in Section 5.1) normal mammary gland development, as well as in the cardiovascular and central nervous systems (Clarke and Sutherland, 99 1990; Mangal et al., 1997; Conneely et al., 2002; Soyal et al., 2005). To the best of our knowledge, only one other study investigated the effects of 16-OHPROG via a steroid receptor (Quinkler et al., 2002). In that study, Quinkler et al. (2002) observed a Ki for 16-OHPROG binding to the human MR (hMR) greater than 1 ␮M, while the Ki for PROG was 1 nM, indicating that PROG has a very high binding affinity for the hMR, while the binding affinity of 16-OHPROG is much lower. Furthermore, no MR antagonist activity was observed for 16-OHPROG up to 1 ␮M, while PROG displayed potent anti-mineralocorticoid activity. The lack of anti-mineralocorticoid activity for 16-OHPROG is surprising, considering that a much earlier study in humans showed that 16OHPROG administered to healthy individuals caused a natriuretic effect similar to that of the MR antagonist, spironolactone (Jacobs, 1969). This study by Jacobs (1969) suggested that 16-OHPROG antagonizes aldosterone-induced effects via the MR. As it is known that receptor density in a specific cell may determine the biological character in terms of agonist or antagonist properties of steroids (Zhao et al., 2003) it may be possible that, in the context of cellular milieu, 16-OHPROG may indeed have anti-mineralocorticoid properties. Although the above studies shed some light on the mechanism of action of 16-OHPROG via the PR and MR, the complexity of downstream effects of PROG metabolites binding to the MR, PR-A and/or PR-B in different progesterone and mineralocorticoid target tissues remains unclear. In addition, it is probable that 16-OHPROG, like PROG, may also mediate effects via receptors located on the membranes of cells, as well as intracellular GR, AR or ER. Indeed, the much earlier study by Forbes and Taku (1975), presented in Section 5.1, eluded to tissue-specific effects of 16-OHPROG, possibly via the AR and ER, as 16-OHPROG inhibited the testosterone- and 17␤estradiol-induced increase in the diameter of ovarian and uterine veins, but could not prevent the uterine hypertrophy and urinary bladder distension caused by testosterone- and 17␤-estradiol. 7. Conclusion PROG plays a central role in steroidogenesis and as such the metabolism of this steroid hormone would impact on downstream physiological events. The hydroxylation of PROG by human CYP17A1 results in a constant ratio of 16-OHPROG and 17OHPROG being synthesized. In adrenal steroidogenesis, PROG and 17-OHPROG are channeled into the mineralocorticoid and glucocorticoid pathways, impacting on the steroid hormone output of the adrenal. 16-OHPROG, on the other hand, is not further metabolised in steroidogenic tissue and upon its release into circulation may bind either intracellular steroid receptors of target tissues or to plasma membrane steroid receptors that signal by rapid non-genomic mechanisms and crosstalk between various signalling pathways. The metabolite may also accumulate in the tissue where it is produced, binding to steroid receptors and eliciting physiological responses. The evidence presented in this review clearly shows the antagonizing action of 16-OHPROG with PROG, testosterone and 17␤-estradiol, in some tissue, but not in others. Indeed, while investigating the interaction of 16-OHPROG with the hPR, we showed that the steroid metabolite binds to and acts as an agonist for both hPR-A and hPR-B. This provides further evidence that 16-OHPROG plays an important physiological role as both hPR isoforms are integrally linked to reproductive function and physiological responses which are tissue specific. In addition, it can also be concluded that, with the conversion of PROG to 16OHPROG in the human kidney, the anti-mineralocorticoid effect of PROG is attenuated. The hydroxylation of PROG at the C16 position in the kidney may therefore indicate a protective function for this 100 K.-H. Storbeck et al. / Molecular and Cellular Endocrinology 336 (2011) 92–101 metabolite since Quinkler et al. (2002) showed PROG to be a hMR antagonist. Although the physiological role of 16-OHPROG has yet to be explored fully, the evidence suggests that this metabolite impacts on the regulation of the electrolyte balance, on both the male and female reproductive systems and on clinical conditions linked to abnormal adrenal steroidogenesis. 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