The transcription factor HHEX maintains glucocorticoid levels and protects adrenals from androgen-induced lipid depletion.

Glucocorticoid-producing cells of the adrenal cortex (. zona fasciculata, zF) constitute the critical effectors of the hypothalamic-pituitary-adrenal axis, mediating the mammalian stress response. With glucocorticoids being essential for life, it is not surprising that zF dysfunction perturbs multiple organs that participate in optimizing cardiometabolic fitness. The zF forms a dynamic and heterogenous cell population endowed with the capacity to remodel through the engagement of both proliferative and differentiation programs that enable the adrenal to adapt and respond to diverse stressors. However, the mechanisms that sustain such differential responsiveness remain poorly understood. In this study, we resolved the transcriptome of the steroidogenic lineage by scRNA-seq using reporter mice. We identified HHEX, a homeodomain protein, as the most enriched transcription factor in glucocorticoid-producing cells. We developed new genetic mouse models to demonstrate that HHEX deletion causes glucocorticoid insufficiency in male animals. Molecularly, we demonstrated that HHEX is an androgen receptor (AR) target gene, shaping the sexual dimorphism of the adrenal gland by repressing the female transcriptional program at puberty, while also maintaining zF cholesterol ester content by protecting lipid droplets from androgen-induced-lipophagy. Moreover, our study revealed that, in both sexes, HHEX is crucial for maintaining the identity of the innermost adrenocortical cell subpopulation. Specifically, loss of HHEX impairs the expression of (P-glycoprotein/MDR1), an efflux pump regulating steroid export and cellular levels of xenobiotics. Together, these data demonstrate that HHEX serves as a multi-functional regulator of post-natal adrenal maturation that is potentiated by androgens.