The present results provide support for the hypothesis that OSTα–OSTβ contributes the transport of conjugated steroids in steroidogenic cells by demonstrating that OSTα and OSTβ are expressed in the specific brain and adrenal gland cells that are involved in steroidogenesis, that PREGS and DHEAS, two important steroid hormone precursors in the periphery and potent neurosteroids in the central nervous system, are relatively high-affinity substrates for OSTα–OSTβ, and that Ostα-deficient mice exhibit altered serum DHEA and DHEAS levels, and [3H]DHEAS distribution.
OSTα-OSTβ is a recently identified organic solute and steroid carrier that plays a central role in the transport of bile acids in the intestine, liver, and kidney, and in regulating the enterohepatic circulation (Ballatori et al. 2009
). Mice that are deficient in Ost
α exhibit a defect in intestinal bile acid absorption, a markedly diminished bile acid pool size, intestinal hypertrophy, growth retardation, a decrease in serum cholesterol and triglyceride levels, and an increase in fecal excretion of neutral sterols (Ballatori et al. 2008
; Rao et al. 2008
). Although these previous studies indicate that Ostα-Ostβ is critical for bile acid homeostasis, they also demonstrate that alternate or compensatory mechanisms are present in the Ostα−/−
mice that allow bile acids to still be absorbed, albeit less efficiently than in wild type animals (Ballatori et al. 2008
; Rao et al. 2008
Because of the major role of OSTα-OSTβ in bile acid homeostasis, most studies to date have focused on its functions in the gastrointestinal tract (Ballatori et al. 2005
; Rao et al. 2008
). However, OSTα and OSTβ are also expressed in many tissues that do not normally handle bile acids (Seward et al. 2003
), indicating that these proteins are likely serving some other roles in these tissues. In particular, OST
α and OST
β mRNA is also expressed in the brain, the adrenal gland, and all major steroidogenic tissues (Seward et al. 2003
; and ), suggesting a potential role in steroid transport in these tissues. The present findings provide support for this hypothesis.
The present results also provide additional insight into the substrate specificity of OSTα-OSTβ. Previous studies demonstrated that a number of bile acids are substrates, and that sulfated bile salts are among the powerful inhibitors of transport (Ballatori et al. 2005
; Seward et al. 2003
; Wang et al. 2001
). The present results demonstrate that sulfated steroids exhibit the strongest inhibitory effect on both mouse Ostα-Ostβ and human OSTα-OSTβ mediated estrone 3-sulfate transport (). Glucuronidated steroids also inhibited transport, although they appeared to be weaker inhibitors. In contrast, the parent steroids and exogenous steroid conjugates with no net charge showed no significant inhibition of OSTα-OSTβ mediated transport, indicating that charge distribution and molecular structure of the molecule may determine its affinity for OSTα-OSTβ.
By direct measurement of transport, the present results also demonstrate that PREGS and DHEAS are substrates for OSTα-OSTβ, but that the parent compounds (PREG and DHEA) do not appear to be substrates. The kinetics of OSTα-OSTβ mediated DHEAS and PREGS transport indicate both high-affinity and low-affinity components for these compounds. The apparent Km value for the high affinity component of DHEAS transport was 1.5±0.4 µM, which is within the range of human blood plasma DHEAS levels (1–5 µM). Plasma levels of PREGS are generally in the nM range, but nanomolar to micromolar concentrations of PREGS have been shown to alter presynaptic or postsynaptic actions in the brain (Gibbs et al. 2006
; Meyer et al. 2002
; Monnet et al. 1995
), and thus the high-affinity component of PREGS transport (apparent Km of 6.9±2.1 µM) may also be physiologically relevant. However, as noted earlier, because OSTα–OSTβ is expected to mediate primarily sterol export from cells, the intracellular concentration is the more meaningful parameter in defining the kinetics, but unfortunately intracellular concentrations of sterols are difficult to assess.
Our observation that OSTα-OSTβ exhibits both low- and high-affinity transport components also provides insight into the molecular mechanisms of transport. Multiple transport components transport may occur when a transporter has more than one substrate binding sites, or when the transporter has a substrate binding site that assumes different conformations depending, for example, on the type and extent of post-translational modifications or on the presence of accessory proteins or other factors (Eisenhaber and Eisenhaber 2007
; Malo and Fliegel 2006
; Putman et al. 2000
; Sauna et al. 2001
; van den Berghe and Klomp 2010
; van der Heide and Poolman 2002
). Additional studies are needed to define whether OSTα-OSTβ contains more than one substrate binding site or whether it exists in different conformations, and to define the physiological relevance of the low affinity transport component.
In addition to OSTα-OSTβ substrate specificity and transport kinetics, the selective localization of these proteins to steroidogenic cells in the brain and adrenal gland provides additional evidence for the hypothesis that OSTα-OSTβ contributes to the disposition of sulfated steroids in these tissues. Previous studies have demonstrated that both the mouse and human brain contain the enzymes needed for synthesizing DHEAS and PREGS, including the steroid sulfotransferase SULT2A1/Sult2a1 (Kimoto et al. 2001
; Maninger et al. 2009
; Mellon and Deschepper 1993
; Ukena et al. 1998
; Zwain and Yen 1999
). In particular, Purkinje cells and hippocampal neurons have been shown to possess steroidogenic enzymes and to produce DHEA, PREG and their sulfated esters. Both of these brain regions are well known for their function in the process of learning and memory, and several studies have suggested that the hippocampus is a site for PREGS action (Akwa et al. 2001
; Flood et al. 1995
). The present results demonstrate these same brain regions are recognized by the OSTα/Ostα and OSTβ/Ostβ antibodies in both human and mouse brain, indicating co-localization of the transporter with the steroid biosynthetic enzymes.
Interestingly, in a recent study designed to gain insight into the function of proteins involved in Purkinje cells degeneration, Lim and coworkers (2006)
used a yeast two-hybrid screen to identify OSTα as one of the proteins that can interact with ataxin-1, a polyglutamine protein of unknown function, whose mutant form causes type 1 spinocerebellar ataxia (SCA1) in humans. Cerebellar Purkinje cells appear to be the major cell type affected in this inherited neurodegenerative disease, and targeted expression of mutant ataxin-1 in Purkinje cells of transgenic mice produces an ataxic phenotype with pathological similarities to the human disease (Clark and Orr 2000
). The significance of the ataxin-1-OSTα protein-protein interaction is presently unknown, but it does provide clues into both the function of ataxin-1 and into the pathogenesis of SCA1.
Although mouse and human brain are both capable of synthesizing DHEA and DHEAS, mice and humans differ markedly in the major site of DHEA and DHEAS synthesis in the periphery and in concentrations of these steroids in blood. In humans, the adrenal gland cells within the zona reticularis
of the cortex serve as the major site of synthesis of circulating DHEA and DHEAS, and serum concentrations of these steroids are quite high, 1–5 µM (Rainey et al. 2002
; Rainey and Nakamura 2008
). In contrast, in the mouse, DHEA and DHEAS are synthesized mainly in the gonads and liver, and mouse blood concentrations are much lower than in humans, 0.01–0.05 µM (Brock and Waterman 1999
; Katagiri et al. 1998
; Kobayashi et al. 2003
). The present results demonstrate that the zona reticularis
of the human adrenal gland cortex is relatively selectively stained by the OSTα and OSTβ antibodies, whereas the expression of Ost
α and Ost
β mRNA in mouse adrenal gland was quite low and Ostα and Ostβ proteins could not be detected in this tissue. Thus, both the strong expression of OSTα-OSTβ in human adrenal gland cells that synthesize DHEA and DHEAS, and conversely, the low expression of these synthetic and putative transport genes in the mouse adrenal gland, are consistent with the hypothesis that this transporter is involved in DHEAS disposition.
It is now well established that Ost
α and Ost
β mRNA expression is regulated by bile acids via the farnesoid X receptor (FXR; Boyer et al. 2006
; Frankenberg et al. 2006
; Landrier et al. 2006
; Lee et al. 2006
). Interestingly, CYP17
and dehydroepiandrosterone sulfotransferase Sult2a1
, which encode for two key enzymes for DHEAS production, are also positively regulated by FXR (Song et al. 2001
). These similar regulatory mechanisms further support the hypothesis that Ostα-Ostβ is responsible for transporting DHEAS. Given that this transporter is also expressed in other steroidogenic tissues, it will be interesting to examine the localization and function of Ostα-Ostβ in these tissues. In particular, previous studies have demonstrated that estrone 3-sulfate and DHEAS, known substrates for Ostα-Ostβ, can be taken up by the testis, ovary, and mammary gland and converted back to active steroid hormones within these tissues, and it is tempting to speculate that Ostα-Ostβ may be involved in these processes. Steroid uptake by these tissues has also been associated with the induction and maintenance of endocrine-dependent cancers, namely prostate cancer and breast cancer (Billich et al. 2000
; Falany and Falany 1997
; Purohit et al. 1999
), and it will be of interest to examine whether this transporter may be involved.
Of significance, although direct quantitative comparisons of mRNA levels between human and mouse tissues are not possible from the present data, OST
α and OST
β mRNA expression in human brain and steroidogenic tissues appears to be much higher than that in the corresponding mouse tissues (), and these species differences parallel the differences in serum DHEAS levels. As noted above, human serum DHEAS concentrations are in the range of 1–5 µM (Dharia and Parker 2004
; Rainey and Nakamura 2008
), whereas mouse serum DHEAS levels are about 2 orders of magnitude lower, or 0.01–0.05 µM (Kobayashi et al. 2003
; and ). In Ost
mice, serum DHEA and DHEAS levels and the distribution of administered [3
H]DHEAS were altered, providing indirect evidence for a role of the transporter in neurosteroid disposition.
Although the mechanism for the altered DHEA/DHEAS levels in Ost
mice is presently undefined, some factors that may be involved include: a) diminished efflux of DHEAS from steroidogenic cells, as supported by the present findings showing that DHEAS is a relatively high affinity substrate for Ostα-Ostβ and that this transporter is localized to these cells; b) impaired biosynthesis of DHEA due to an imbalance of cholesterol and bile acid homeostasis, as previously reported in Ost
mice (Ballatori et al. 2008
; Rao et al. 2008
); c) decreased elimination of DHEA from the serum compartment; or d) decreased activity of the enzyme(s) that convert DHEA to DHEAS. However, the present findings demonstrate that hepatic expression of Sult2a1
, an important enzyme for the conversion of DHEA to DHEAS, was similar in both genotypes.
In summary, the present findings demonstrate that OSTα and OSTβ are expressed in steroidogenic cells in the brain and adrenal gland, and that this transporter can transport DHEAS and PREGS with high affinity. Thus, OSTα-OSTβ may contribute to neurosteroid transport in the brain and sterol conjugate transport in the adrenal gland and other steroidogenic tissues.