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

## TL;DR

This study shows that the HHEX protein is essential for maintaining proper adrenal function, especially in males, by regulating glucocorticoid levels and protecting against androgen-induced lipid loss.

## Contribution

The study identifies HHEX as a novel transcription factor that regulates adrenal sexual dimorphism and lipid homeostasis through androgen signaling.

## Key findings

- HHEX deletion in male mice causes glucocorticoid insufficiency.
- HHEX protects lipid droplets from androgen-induced lipophagy in adrenal cells.
- HHEX maintains the identity of inner adrenocortical cells by regulating Abcb1b expression.

## Abstract

Glucocorticoid-producing cells of the adrenal cortex (i.e. 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 Sf1-Cre; RosamT/mG 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 Abcb1b (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.

In this study, we defined HHEX as a key regulator of adrenocortical cell function. By using multiple transgenic mouse models, we demonstrate that HHEX shapes AR-dependent sexual dimorphism in the zF following the onset of puberty. HHEX protects lipid droplets integrity from lipophagy induced by AR signaling and its absence results in glucocorticoid insufficiency in males. Additionally, in both sexes, HHEX supports the inner zF transcriptome, including the expression of Abcb1b, a critical regulator of intracellular levels of steroids and xenobiotics.