Integrative genomic analysis reveals a conserved role for prolactin signalling in the regulation of adrenal function

Abstract

We report here that the pituitary hormone prolactin (PRL) has an important, conserved role in regulating adrenal gland function. The adrenal plays a pivotal role in endocrine homeostasis and stress response, which vary across lifespan and have different features in the two sexes.[1] Furthermore, most adrenal disorders have a higher prevalence in women.[2] To get deeper insight into the mechanisms of age- and sex-dependent adrenal function, we performed an integrative analysis of the mouse adrenal transcriptome by RNA-seq and active enhancer (as defined by H3K27ac ChIP-seq) usage at different ages (from E18.5 to P12 weeks) in both sexes. Multivariate analysis showed that age, but not sex, had a significant effect on adrenal global gene expression profiles (Figure and Supporting information S1; Tables S1, S2). Expression of genes related to lipid metabolism and immunity was progressively enriched with age (Figure 1C and Supporting information Table S3), with the proportion of macrophages increasing and neutrophils/mast cells decreasing at older ages (Figure 1D). The expression of different classes of secreted proteins, ion channels and enzymes showed a lower level of sex-dependent enrichment (Figure 1E and Supporting information Table S4). Age-and sex-dependent long non-coding RNA differentially expressed genes (DEG) followed the same pattern as coding DEG, being mostly specific for each age and sex (Supporting information Figure S2 and Table S5). Remarkably, P12 weeks adrenals of both sexes showed increased percentages of novel gene transcripts compared to previous ages (Supporting information Figure S2E). Sexually dimorphic expression pervades all cell populations of the mouse adrenal gland (Supporting information Figure S3 and Table S6). We identified a few thousand enhancers active in the adrenal gland at each temporal stage and in each sex (Supporting information Table S7). A subset among those belong to the super-enhancer class (SEC), genomic regions highly enriched in active chromatin, which regulate developmental and tissue-specific programs and often encompass disease-associated genomic loci.[3] At all ages and in both sexes, adrenal SEC have a much higher level of overlap than typical-enhancer class (TEC) elements (Figure 2A), while adrenal TEC are significantly more conserved than SEC (Figure 2B). Adrenal SEC-associated genes are enriched in genes involved in transcriptional regulation, cell-cell adhesion, protein phosphorylation, biological rhythms and steroid hormone receptor function at all ages and in both sexes (Figure 2C and Supporting information Table S8). Mouse adrenal SEC-associated genes are expressed at significantly higher levels in the adrenal gland (Supporting information Figure S4A) and include a higher percentage of tissue-specific genes (Supporting information Figure S4B) than TEC-associated genes. Human adrenal SEC are also preferentially associated to genes encoding proteins with adrenal-enriched expression than to genes with higher expression in other tissues (Supporting information Figure S4C) and SEC-associated genes are enriched with age-dependent DEG at most times and sex-dependent DEG at P12 weeks compared to TEC (Supporting information Figure S4D,E). Examples of SEC associated to genes important in adrenal physiology are shown in Supporting information Figure S5. Of note, some adrenal SEC-associated genes are expressed preferentially in the adrenal and are involved in blood pressure regulation (Supporting information Table S9). In particular, non-coding SNPs associated with blood pressure traits with very high significance are located inside a conserved SEC encompassing the KCNK3 gene (Supporting information Figure S6 and Table S10). By comparing our list of adrenal sexually dimorphic DEG with data in mouse[4, 5] and rat,[6] we could highlight a core conserved sexually dimorphic gene expression program (Figure 3A and Supporting information Table S11). We focused on the role of the PRL receptor (PRLR), because of its conserved sexually dimorphic expression in the human adrenal (Figure 3B) and the well-known role of PRL signalling in physiological adaptations and response to stress.[7] All Prlr isoforms were upregulated in the female adrenal compared to male at P12, but not P2 weeks of age (Figure 3C). Adrenal gland weight was significantly reduced in adult female Prlr -/- mice compared to WT (Figure 3D), while total body weight was normal in all Prlr -/- animals (Supporting information Figure S7). Signalling pathway impact analysis revealed that the JAK-STAT pathway is significantly activated in female adrenals compared to male at P12 weeks of age (Supporting information Figure S8). The mean area of adrenocortical cells in female Prlr -/- adrenals was significantly decreased (Figure 3E,F). This is consistent with significantly reduced circulating corticosterone levels and a trend toward an increased ACTH/corticosterone ratio in female Prlr -/- mice compared to WT (Figure 3