FXR expression was initially identified in the liver, gut, adrenal gland and kidney in rodents (Forman et al.,1995a
). Subsequent studies confirmed the same expression pattern in human tissues (Huber et al.,2002
; Zhang et al.,2003
). To date, most research has focused on FXR function in liver, and FXR has been shown to regulate a number of target genes involved in bile acid, lipid and glucose metabolism (Repa et al.,2000
; Song et al.,2001
; Claudel et al.,2003
; Francis et al.,2003
; Pircher et al.,2003
; Sirvent et al.,2004
; Claudel et al.,2005
; Cai and Boyer,2006b
; Ma et al.,2006
). In contrast to the intense interest in liver, little has been done to define the role of FXR in the adrenal gland. In this study, we have identified HSD3B2 as an FXR target gene in human adrenal gland.
FXR is expressed in both adult and fetal adrenal glands at relatively high levels (Forman et al.,1995a
; Lee et al.,2006b
; Houten et al.,2007
). The recent study by Auwerx and colleagues showed that FXR expression in mouse adrenal glands can be stimulated by oral feeding of the agonist GW4064 (Houten et al.,2007
). This provides another aspect of FXR regulation and further supports a role for FXR in the adrenal gland. In contrast to other organs that highly express FXR such as kidney, liver and intestine, the adrenal gland is however not commonly associated with any aspect of bile acid regulation, and calls for investigation of adrenal- specific functions of FXR.
The primary function of the adrenal gland is the production of steroid hormones; therefore we examined the effects of FXR on adrenal cell expression of transcripts encoding steroidogenic enzymes. This identified HSD3B2 as a potential FXR target in human adrenal cells. Further deletion and mutation experiments suggested a potential FXRE at -137 to -130 in the HSD3B2 promoter region. FXR binding to this element was confirmed by ChIP assay. When combined, these data strongly suggest that HSD3B2 is a direct FXR target gene in the human adrenal gland.
First identified from a human adrenal cDNA library (Rheaume et al.,1991
), HSD3B2 encodes a protein of 371 amino acids that is primarily expressed in the adrenal, ovary, and testis (Lachance et al.,1992
; Thomas et al.,2001
). It catalyzes the sequential 3ß-hydroxysteroid dehydrogenation and delta-5-4 isomerization of delta-5 steroid precursors that include pregnenolone, 17-hydroxypregnenolone, DHEA, and androst-5-ene-3ß, 17ß-diol into their respective 4-ketosteroids, namely progesterone, 17-hydroxyprogesterone, androstenedione, and testosterone. Thus HSD3B2 is required for the biosynthesis of all classes of steroid hormones, including glucocorticoids, mineralocorticoids, progestins, androgens, and estrogens (Simard et al.,2005
One intriguing result from the FXR immunohistochemistry experiments was the observation of high nuclear expression of FXR in ZF cells, but primarily cytoplasmic localization in ZR. Although there has been no report regarding FXR trafficking between cytoplasm and nuclei, it is well established that some nuclear receptors are effective only when present in nuclei and that receptors can effectively move between the nuclei and cytoplasm (Guiochon-Mantel et al.,1994
; Czar et al.,1995
; Hager et al.,2000
). This suggested that FXR transcriptional activity may differ between ZF and ZR. In contrast, similar mRNA expression levels of FXR were observed in both ZF and ZR (data not shown). HSD3B2 mRNA and protein are present at high levels in zona fasciculata but almost absent from zona reticularis (Sasano,1990
; Endoh et al.,1996
; Gell et al.,1998
). The regulatory mechanism for the HSD3B2 expression pattern in the adrenal has not been fully elucidated. The zone-specific distribution of HSD3B2 is in part responsible for DHEA production in the reticularis and cortisol synthesis in the fasciculata (Hornsby,1995
; Rainey and Nakamura,2008
). Therefore, by increasing HSD3B2 expression, FXR-mediated stimulation would increase the synthesis of cortisol while decreasing DHEA/DHEA-S production, effectively changing the ratio of cortisol to DHEA-S in adrenal, leading to the fasciculata phenotype. The significance of FXR in the regulation of zonation needs further evidence, but our results suggest that FXR is in the adrenal and can regulate HSD3B2 expression.
Since FXR has been reported to work with RXR as heterodimer for its transcriptional activity, we co-transfected the FXR and RXR expression vectors together with HSD3B2 promoter reporters, but no significant addictive effect of RXR under basal- or ligand-stimulated states was detected (data not shown). This suggests that RXR is not limiting in our transfection studies. Also, we tested NGFI-B – the nuclear receptor whose response element overlaps with that of FXR and which is known to regulate HSD3B2 (Bassett et al.,2004
). NGFI-B and FXR appeared to act independently as there was no addictive effect on promoter activity. Furthermore, in agreement with Houten and colleagues (Houten et al.,2007
), we did not find the classic FXR target SHP to be regulated by ligand treatment in human adrenal (data not shown).
The generation of a FXR knockout mouse model (Sinal et al.,2000
) offered a very powerful tool for the FXR study, but unfortunately, due to species differences, it may not be an appropriate model for the study of mouse Hsd3b1 (the ortholog of the human HSD3B2 gene). Using ECR browser (DCODE.org, Comparative Genomics Center), we compared the -1 kb 5’ promoter sequence of both human HSD3B2 and mouse Hsd3b1. There is only 74 % similarity between these two sequences. Not surprisingly, the FXRE defined for the human gene was not conserved in mouse Hsd3b1 promoter. This may explain why incubation of wild mouse adrenal glands with GW4064 did not induce Hsd3b1 mRNA expression and there was no difference in Hsd3b1 levels between wild and FXR-/-
mice (Lee et al.,2006b
). In support of this premise, transfection of the mouse Y-1 adrenal cell line with FXR expression vectors in a manner that increased FXR mRNA levels by more than 500-fold did not alter mRNA levels of Hsd3b1. Furthermore, in contrast to human adrenals, Hsd3b1 is expressed through-out the mouse adrenal cortex including zona reticularis (Schulte et al.,2007
). Thus HSD3B2 is regulated differently between human and mouse adrenal glands and it appears that FXR plays a more important role in human adrenal glands than in mouse adrenals.
There are at least two potential sources of ligands available for FXR activation within the adrenal gland. First, bile acids could be transported from the plasma into adrenal cells through the OSTα / OSTβ transport system that is present the adrenal cells (Seward et al.,2003
). This hypothesis is supported by the ability of FXR to increase adrenocortical cell expression of the OSTα gene. Second, adrenal-derived steroids or steroid intermediates that are found at very high levels within adrenal cells, may act to inhibit or activate FXR transcriptional activity. Indeed, an adrenal and gonad steroid metabolite was recently shown to bind and activate FXR transcriptional activity (Wang et al.,2006
). A thorough screening of all adrenocortical steroid metabolites for potential FXR ligands may provide new mechanisms for regulating FXR transcriptional activity.
In summary, we established that FXR is expressed at high levels in the human adrenal gland, and that it stimulates HSD3B2 under the control of both the natural bile acid ligand CDCA and the synthetic agonist GW4064. The identification of HSD3B2 as a novel FXR target in the adrenal gland will give new direction to the study of FXR and help expand the understanding of FXR function in human tissues.