Endocrine Abstracts

The adrenal cortex synthesizes glucocorticoids that are essential for metabolic homeostasis and modulation of immune responsiveness. Glucocorticoid-producing cells, located in the zona fasciculata (zF), constitute the critical effector of the HPA axis that serves to mediate the mammalian stress response. Dysregulation of glucocorticoid function results in significant morbidity with complete deficiency being incompatible with life. While the zF is composed of heterogenous cell populations exhibiting proliferative, remodeling, and differentiation capabilities, the mechanisms and physiological importance of such diversity remain unexplored. To define the functional significance of the cellular diversity of the zF, we first performed single-cell RNAseq of the steroidogenic lineage in the adult mouse adrenal. We identified the homeodomain protein, HHEX, as the top enriched transcription factor in zF cells. To determine its role in adrenal homeostasis, we generated adrenal-specific Hhex knockout mouse models. Importantly, HHEX KO mice exhibited a profound glucocorticoid deficiency at baseline. We then performed bulk RNAseq and identified Abcb1b as the top gene dysregulated in HHEX KO of both sexes. ABCB1 (also P-glycoprotein, multidrug resistance protein 1) is a steroid efflux pump that is enriched in an inner-most zF cluster of cells and centrifugally expands during chronic stress. In humans, ABCB1 polymorphisms are associated with altered HPA axis regulation suggesting a critical role in chronic stress adaptation. Using CUT&Tag technology to profile in vivo genomic binding, we provide evidence that HHEX directly occupies Abcb1b regulatory regions. By combining RNAscope for in situ localization, and chronic ACTH treatment, we observed that HHEX KO fail to increase Abcb1b expression. Taken together these results suggest a critical role for HHEX in Abcb1b expression and chronic stress adaptation in both sexes. We then proceeded to determine adrenocortical cholesterol content (steroid precursor), and a profound decrease was evident specifically in the inner zF of KO males. Temporal analyses of HHEX expression revealed an 8-fold higher level in males at puberty compared to female counterparts, and genomic binding studies confirmed AR binding to HHEX promoter region in vivo. These observations suggest a sexually dimorphic role of HHEX in lipid (cholesterol ester) homeostasis. To explore the possibility that the dimorphism was mediated in part by androgens, we gonadectomized males and observed a complete rescue of the lipid loss in HHEX KO adrenals. Taken together, our results provide evidence that HHEX orchestrates the function and identity of the zF to promote chronic stress adaptation and protect lipid droplet integrity in males.