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, 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 resolve the transcriptome of the steroidogenic lineage by scRNA-seq using Sf1-Crehigh; RosamT/mG reporter mice. We identify HHEX, a homeodomain protein, as the most enriched transcription factor in glucocorticoid-producing cells. We utilize genetic mouse models to demonstrate that Hhex deletion causes glucocorticoid deficiency in male animals. Molecularly, we demonstrate 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 reveals 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.
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Data availability
All sequencing datasets generated in this study have been deposited in the Gene Expression Omnibus (GEO) database under the accession code GSE291343, GSE291472, and GSE291344. Processed data are provided in Supplementary Data 1. The CLOUPE file related to the scRNAseq dataset presented in this manuscript is accessible at the following address [https://doi.org/10.6084/m9.figshare.28612406]. Source data are provided with this paper.
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Acknowledgements
Research reported in this publication was supported by the International Fund for Congenital Adrenal Hyperplasia to G.D.H., F.B., and T.D., the Center for Cell Plasticity and Organ Design to T.D., the National Institutes of Health/National Institute of Diabetes and Digestive and Kidney Diseases of the National Institutes of Health under Award Number R01DK043140, R01DK062027 to G.D.H. and Dr. William Rainey, the National Heart, Lung, and Blood Institute under Award Number 1R01HL15583401 to A.F.T., the Swiss National Science Foundation (310030L_182700/1) to F.B., the Swiss NCCR “Kidney.CH” and from the University Research Priority Program of the University of Zurich ITINERARE–Innovative Therapies in Rare Disease to D.P. and F.B. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. We thank all the members of the Hammer Lab for helpful discussion and feedback on the manuscript. We especially thank Dr. Rainey (University of Michigan) for providing Human adrenal paraffin samples and sharing ARKO RNAseq. We thank Dr. Pierre Val (University of Clermont-Ferrand) for sharing reagents. We thank Dr. Gregg Myers (University of Michigan) for technical assistance with hematopoietic cells. We thank Dr. Leonard Cheung (Stony Brook University) for helpful discussion regarding HHEX expression in the mouse pituitary gland. We thank Dr. Klionsky (University of Michigan), autophagy expert for helpful discussion on lipophagy process. We thank Dr. Frank Claessens and Dr. Johan Swinnen (KU Leuven) for sharing the AR flox mice60. The research reported in this publication used the Advanced Genomic Core, the Microscopy Core, the Flow Cytometry Core, and the Orthopaedic Research Laboratories (ORL) Histology Core at the University of Michigan. We especially thank Emma Snyder-White and Carol Whitinger for technical assistance with histology experiments and Michael Pihalja with FACS sorting.
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We followed guidelines for authorship at University of Michigan Office of Research including 4 criteria. (1) significant contribution to the conception or design of the work; or the acquisition, analysis, or interpretation of data for the work; (2) drafting the work or revising it critically for important intellectual content; (3) final approval of the version to be published; (4) agreement to be accountable for all aspects of the work in ensuring that questions related to accuracy or integrity of any part of the work are appropriately investigated. T.D., K.J.B., and G.D.H. design the experiments, T.D. and A.M.L. analyzed the data, T.D., K.J.B., M.C.F., C.R.L., D.J, E.S., and K.A.H. genotyped mice, performed the experiments, or acquired data. C.L. and A.F.T. performed LCMSMS. S.W.P. and A.M.L. analyzed the ARKO RNAseq data. D.P. and F.B. provided Cyp11b1-CreERT2 mice. D.T.B. provided Cyp11b2-Cre (AS-Cre) mice. T.D. wrote the original manuscript, T.D., K.J.B., C.R.L., and G.D.H. edited the manuscript. All coauthors provided expertise and feedback.
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G.D.H. Founder and Board of Directors—Sling Therapeutics, Advisor—Orphagen Pharmaceuticals. The remaining authors declare no competing interests.
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Dumontet, T., Basham, K.J., Foster, M.C. et al. The transcription factor HHEX maintains glucocorticoid levels and protects adrenals from androgen-induced lipid depletion. Nat Commun (2026). https://doi.org/10.1038/s41467-025-68257-4
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DOI: https://doi.org/10.1038/s41467-025-68257-4
