HIF1α is a direct regulator of steroidogenesis in the adrenal gland

Abstract

Endogenous steroid hormones, especially glucocorticoids and mineralocorticoids, derive from the adrenal cortex, and drastic or sustained changes in their circulatory levels affect multiple organ systems. Although hypoxia signaling in steroidogenesis has been suggested, knowledge on the true impact of the HIFs (Hypoxia-Inducible Factors) in the adrenocortical cells of vertebrates is scant. By creating a unique set of transgenic mouse lines, we reveal a prominent role for HIF1α in the synthesis of virtually all steroids in vivo. Specifically, mice deficient in HIF1α in adrenocortical cells displayed enhanced levels of enzymes responsible for steroidogenesis and a cognate increase in circulatory steroid levels. These changes resulted in cytokine alterations and changes in the profile of circulatory mature hematopoietic cells. Conversely, HIF1α overexpression resulted in the opposite phenotype of insufficient steroid production due to impaired transcription of necessary enzymes. Based on these results, we propose HIF1α to be a vital regulator of steroidogenesis as its modulation in adrenocortical cells dramatically impacts hormone synthesis with systemic consequences. In addition, these mice can have potential clinical significances as they may serve as essential tools to understand the pathophysiology of hormone modulations in a number of diseases associated with metabolic syndrome, auto-immunity or even cancer.

Keywords: Adrenocortical steroids; Cytokines; Hypoxia-inducible factor; Oxygen sensors.

Fig. 1

Characterization of the Akr1b7:cre-P2H1^ff/ff mouse line with cortex-specific targeting of hypoxia pathway proteins. a Representative immunofluorescent image of anti-GFP stained (GFP +) area in the adrenal cortex of the Akr1b7:cre-mTmG mouse line. Region enclosed within the white dotted line represents the medulla and it demarcates the medulla from the cortex (scale bar, 100 μm). b qPCR-based mRNA expression analysis of Hif1α, Phd2 and Hif2α in entire adrenal tissue from P2H1 mice and WT littermates (n = 10–13). Relative gene expression was calculated using the 2^(⁻ddCt) method. The graphs represent data from 2 independent experiments. c Relative gene expression analysis using mRNA from the entire adrenal tissue in P2H1 mice and their WT counterparts (n = 10–13). All graphs represent data from 2 independent experiments. d Representative images (magnification 20x) of paraffin sections of adrenal glands (H&E) from 8-week-old WT and P2H1 mice (scale bars represent 100 μm). e Representative immunofluorescent images of CD31⁺ endothelial cell staining in adrenal gland sections from WT and P2H1 mice (scale bars represent 50 μm). Graph in the right-side panel represents quantification of CD31⁺ area as a fraction of total tissue area. Each data point represents a single measurement of the cortical area in the adrenal gland (collection of n= 6 vs 11 individual mice). f Representative images of cryosections of WT and P2H1 adrenal glands (H&E) (scale bars represent 50 μm). Graph in the right-side panel represents the normalized average size of an individual lipid droplet per section of adrenal gland tissue in WT versus P2H1 mice. Measurements were made from 6 sections per mouse. (n = 8 individual adrenals per genotype). The graphs in e and f are representative of 2 independent experiments

Fig. 2

Adrenal cortex-specific loss of PHD2 and HIF1 leads to enhanced steroidogenesis in P2H1 mice. a Box and whisker plots showing steroid hormone measurements in adrenal glands from WT mice and compared to littermate P2H1 mice (n = 20–31 individual adrenal glands). b Box and whisker plots showing steroid hormone measurements in the plasma of individual mice (n = 5–17). All data were normalized to the average value of WT mice and graphs represent results of pooled data of at least 3 independent experiments

Fig. 3

Gene expression analysis of P2H1 adrenocortical cells. a Gene expression analysis of enzymes involved in the steroidogenesis pathway using mRNA from whole adrenals from P2H1 mice and WT counterparts (n = 10–13). All graphs are the result of 2 independent experiments. b Schematic overview of the RNAseq approach which compared sorted GFP⁺ cells from WT controls and P2H1 mice (n = 3). c Gene signature analysis using Enrichr. d Gene set enrichment analyses (GSEA) showed positive signatures for steroidogenesis-related pathways. e Prominent HIF-related pathways. f The β-catenin nuclear pathway

Fig. 4

Immune system changes in P2H1 mice. a Box and whisker plots representing levels of pro/anti-inflammatory cytokines measured in the plasma of P2H1 mice and WT littermate controls (n = 7–12). All data were normalized to the average value seen in WT mice. Each dot represents data from one animal. b Box and whisker plots showing percentage lymphocytes and eosinophils in circulation which revealed reduced fractions in P2H1 mice compared to WT controls. c Greater numbers of circulating neutrophils in P2H1 mice compared to WT littermates. All graphs represent pooled results of 2 independent experiments

Fig. 5

Adrenal cortex-specific loss of PHD2 and PHD3 leads to reduced steroidogenesis in mice. a Box and whisker plots showing steroid hormone levels in the adrenal glands of WT mice and compared to that of littermate P2H1 mice (n = 14–16 individual adrenal glands). b Box and whisker plots showing steroid hormone measurements in the plasma of individual mice (n = 10–12). All data were normalized to the average value of WT mice and graphs represent results of pooled data of at least 3 independent experiments

Fig. 6

Inverse regulation of steroidogenesis in P2P3 mice compared to P2H1 mice. a Gene expression analysis of enzymes involved in the steroidogenesis pathway in P2P3 mice and their WT counterparts (n = 12–13) was performed on mRNA from entire adrenal glands. All graphs represent pooled data from at least 3 independent experiments. b Relative expression profile of all genes analyzed from the adrenal glands of P2H1 and P2P3 mice and compared to their respective WT littermates. Statistical significance was defined using an unpaired multiple t test (n = 13; Benjamini, Krieger and Yekutieli method; *p < 0.0001 for all individual genes)

Fig. 7

Immune system changes in P2P3 mice. a Box and whisker plots representing levels of pro/anti-inflammatory cytokines measured in the plasma of P2P3 mice and WT littermate controls (n = 6–13). Each dot represents data from one animal. All data were normalized to the average value seen in WT mice and one-tailed hypothesis tests were performed. All graphs represent samples of different litters. b Schematic overview of all changes in adrenocortical enzymes and their corresponding hormones and intermediates reported here in P2H1 (red) and P2P3 (yellow) mice