The Steroid Metabolome and Transcriptome of the Human Adrenal Gland

Abstract

The adrenal glands serve as multifunctional endocrine organs which are anatomically and functionally divided into three concentric zones, namely the zona glomerulosa (ZG), zona fasciculata (ZF) and zona reticularis (ZR). The human ZR is considered the primary source for the C19 steroids like dehydroepiandrostenedione (DHEA) and DHEA sulfate (DHEAS), which have little direct activity on the androgen receptor but can act as precursors for more potent androgens. Adrenal-derived C19 steroids are significant contributors to circulating androgens in women and in pre-pubertal children. The ZR remains dormant until around 6 years of age, after which it expands and produces increasing amounts of DHEA and DHEAS. This phenomenon is known as adrenarche. ACTH has been the accepted primary (but not the sole) mediator of adrenal C19 steroid biosynthesis, and thus adrenarche. We also speculate that other fine-tuning mechanisms might also play important roles in the physiologic production of adrenal C19 steroids. The goal of our ongoing studies was to determine the ability of the human adrenal to synthesize active androgens, as well as to further define the mechanisms regulating adrenal C19 steroid biosynthesis. In this dissertation, I will summarize my studies aimed at defining the adrenal C19 steroid metabolome and the transcriptome of the two adrenocortical zones, ZF and ZR. In addition, I will describe the novel findings that demonstrate bone morphogenetic protein-4 (BMP4) as a paracrine negative regulator of adrenal C19 steroid secretion. Although there is overall agreement that the adrenal is a key provider of androgens in women, the overall adrenal C19 steroid metabolome and its responsiveness to ACTH has not been well studied. We used LC-MS/MS to quantify nine unconjugated C19 steroids and two estrogens in human adrenal vein (AV) samples, pre and post ACTH stimulation. DHEAS was quantified by RIA. LC-MS/MS profiling of AV samples from women demonstrated that AV levels of DHEAS, before and after ACTH infusion, are the highest among the steroids measured. Of the unconjugated steroids, 11OHA, DHEA and A4 are the most abundant in AV samples, pre and post ACTH stimulation. The study also demonstrated that the adrenal gland can synthesize potent C19 steroids like testosterone and11β-hydroxytestosterone (11OHT). We propose that 11OHT could represent a novel adrenal androgen biomarker because its synthesis requires the enzyme CYP11B1, which is expressed solely in the adrenal cortex. Based on the current study involving LC-MS/MS, measurements of various C19 steroids and a microarray profile of the different adrenal steroidogenic enzymes, we provided evidence of a novel pathway in the human adrenal leading to the production of several androgens and precursor steroids. Beyond steroidogenic enzymes and cofactor proteins, little is known about the differences in phenotypes of ZF and ZR. Microarray analysis was recruited to compare the expression patterns of around 48000 probe sets between human adrenal ZF and ZR in a non-biased approach. Our microarray analysis showed that a transcription factor involved in the Wnt signaling pathway, Lymphoid Enhancer-Binding Factor 1 (LEF1), was the most up-regulated ZF transcript when compared to ZR. LEF1 is a component of the canonical and non-canonical Wnt signaling mechanism, thereby hinting that along with ZG, cell proliferation may also occur in ZF to a limited extent. The speculation was also supported by our pathway analysis which shows that other components of the canonical and non-canonical Wnt signaling mechanism were also up-regulated in ZF. While cell proliferation was shown to be up-regulated in the ZF, our pathway analysis also showed that the cell death machinery was more dominant in the ZR. Another interesting point that was highlighted by our microarray study was the presence of a functional BMP system equipped with the ligand (BMP4), receptors (BMP type-II receptor, activin receptor-like kinase receptors 2, 3 and 4; also called as ALK2, ALK3 and ALK4 respectively) and the signaling proteins (SMADs 4 and 5) in the human adrenal gland, as well as the human adrenocortical cell line H295R. Our microarray, quantitative RT-PCR (qPCR) and immunohistochemistry studies confirmed that BMP4 expression was highest in the zona glomerulosa (ZG), followed by the ZF, and lowest in the ZR. Using the H295R cell culture model, we investigated the effect of BMP4 on the biochemical and molecular mechanisms defining C19 steroid synthesis. Our studies revealed that DHEA and A4 synthesis was significantly decreased by BMP4 treatment. This effect was largely caused by the inhibitory effects of BMP4 on the expression of CYP17. We also observed that BMP4 does not affect the mRNA levels of CYP11A1 and HSD3B2. Collectively, the present data suggest that BMP4 is a paracrine negative regulator of adrenal C19 steroid synthesis. In summary, our studies have identified a new series of active androgens produced by the human adrenal and also given us a differentiating transcriptomic profile between ZF and ZR. We have also defined a novel signaling cascade in the adrenal gland that regulates adrenal C19 steroid production.