Regulation of stem and progenitor cells in the adrenal cortex

Abstract

The adrenal cortex is an endocrine organ comprised of three histological zones, the outermost zona glomerulosa, the intermediate zona fasciculata, and the innermost zona reticularis. High plasticity of the adrenal gland is supported by pools of stem and progenitor cells that are deployed to sustain physiological and homeostatic demands. In recent decades, exciting new discoveries elucidating the identity, function, and fate of these cell populations have emerged. In this review, we describe paracrine and endocrine signaling loops that are crucial for adrenal biology, focusing on recent studies unpacking the enigmatic nature of adrenal stem and progenitor cell populations.

Introduction

The adrenal glands are paired endocrine organs that lie above each kidney and produce steroid hormones and catecholamines critical for life. The glands are comprised two embryonically distinct parts: an outer layer, the cortex, and a central zone, the medulla. The cortex, the subject of this review, produces three major types of steroids: mineralocorticoids, glucocorticoids, and androgens. The production of each hormone is compartmentalized and regulated by independent endocrine feedback loops: the renin-angiotensin-aldosterone system and the hypothalamus-pituitary-adrenal (HPA) systems, which control the production of mineralocorticoids and glucocorticoids/androgens, respectively. This functional compartmentalization is mirrored histologically as the cortex is structured into three distinct concentric histological zones. Accordingly, from the outmost to the innermost, the zona glomerulosa (zG), zona fasciculata (zF), and zona reticularis synthetize mineralocorticoids (e.g. aldosterone), glucocorticoids (e.g. cortisol/cortisone), and androgens (e.g. dehydroepiandrosterone-sulfate (DHEAS)), respectively. In addition, paracrine signaling pathways maintained by stem/progenitor populations and involving heterotypic cellular interactions are major determinants of adrenocortical anatomic and functional zonation. In this review, we will summarize the most recent findings on the paracrine regulatory loops that govern stem/progenitor cell function in the adrenal cortex and their importance for homeostasis throughout life.

Adrenal development

The fetal adrenal cortex is derived from the adrenogonadal primordium (AGP), which originates from the coelomic epithelium and underlying intermediate (mesonephric) mesoderm during weeks 4–6 of gestation in humans and at embryonic day 9.5 (E9.5) in mice. At this stage, expression of the transcription factor steroidogenic factor 1 (SF1, encoded by NR5A1 in humans or Nr5a1 in mice) is initiated, enabling the development of the AGP and subsequent bipotential gonad and adrenal cortex together with the unique steroidogenic programs of both organs [1]. The fetal adrenal-specific enhancer (FAdE), which is active exclusively during late AGP development, is essential for initiating adrenocortical Nr5a1 expression [2]. The AGP divides into adrenal and gonadal primordia (week 8 of gestation; E10.5), and by the 9th week of gestation (E12.5), cells of the neural crest infiltrate the adrenal primordium to form the central adrenal medulla. Mesenchymal cells coalesce around the adrenal primordium forming the adrenal capsule. In humans, the fetal adrenal cortex regresses during the weeks after birth and is eventually replaced by the definitive adult cortex [3]. Although the mechanisms supporting the transition between the fetal and definitive adult adrenal cortex are still poorly understood, studies suggest that DAX1 (DSS/AHC/X-chromosome gene-1; encoded by Nr0b1), an orphan nuclear receptor and SF1 target gene, acts as a corepressor of SF1 [4], repressing FAdE during the transition from the fetal to definitive cortex [2].

Stem/progenitor cell populations in the adrenal gland

Different populations of progenitor cells have been described in the adult adrenal gland, residing in the adrenal capsule and cortex. These capsular and cortical progenitor cell populations establish reciprocal signaling networks by unknown mechanisms to coordinate centripetal replenishment and differentiation of cortical cells in response to paracrine and endocrine signaling, thus maintaining homeostasis of the adrenal cortex. In the following sections, we will briefly summarize the current knowledge of these key progenitor populations and their involvement in paracrine signaling networks crucial for adrenocortical homeostasis.

Capsule

The capsule is comprised of thin layers of SF1-negative mesenchymal cells. Several long-term retained populations of distinct developmental origins have been characterized in the capsule. These include populations expressing Wilms tumor protein homolog 1 (WT1), transcription factor 21 (TCF21), GLI family zinc finger 1 (GLI1), R-spondin 3 (RSPO3), Yes-associated protein (YAP) and transcriptional coactivator with the PDZ-binding motif (TAZ), and Nestin (Figure 1). Furthermore, some of these cell types are essential for maintenance of the subcapsular SF1+ progenitor cell compartment. Here, we present a brief overview of these capsular populations.

Figure 1. Molecules and signaling in adrenal homeostasis.

Schematic of spatial location of molecules and signaling pathways discussed in this review. Nestin, YAP/TAZ, Tcf21, Wt1, Gli1, and Rspo3 are expressed in the adrenal capsule. YAP/TAZ are also found expressed throughout the adrenal cortex. EZH2 expression is particularly concentrated in the transition zone from zG to zF. Shh- and Wnt4-expressing cells, along with WNT-responsive cells, primarily populate the zG. Finally, the adrenocortical WNT gradient is stronger in the outer cortex and weaker in the innermost cortex. PKA signaling, active in the zF, inhibits a zG phenotype. Androgens suppress cell recruitment from capsule.

Wilms tumor protein homolog 1

WT1 is a transcriptional regulator of SF1 that is essential for proper adrenogonadal development [5]. Capsular cells expressing Wt1 may give rise to few SF1+ adrenocortical cells with little contribution to cortical homeostasis [5–7]. During development, cells expressing Wt1 partially overlap with progenitor cells expressing Gli1. Interestingly, WT1 binds the promoters of both Tcf21 and Gli1 [6], suggesting WT1 may be critical for regulating Gli1 and Tcf21 expression in these cell populations. Recently, investigators demonstrated that Sf1-driven deletion of the histone methyltransferase enhancer of zeste homolog 2 (Ezh2) leads to zF aplasia, coincident with a large accumulation of adrenocortical WT1+ cells [8]. These observations suggest that in the context of increased demand, WT1+ cells may be recruited as supraphysiological progenitors [8].

Transcription factor 21

During mouse adrenal development, Tcf21 is expressed starting at E9.5, and TCF21+ cells give rise to both SF1-capsular and SF1+ cortical lineages before capsular formation. After capsular formation, TCF21+ cells contribute to adrenal development by giving rise to SF1-negative stromal cells. By E14.5, Tcf21 expression is restricted only to a subset of capsular cells. Over time, capsular Tcf21 expression declines and is restricted to a small compartment of capsular cells in adult adrenals [9].

GLI family zinc finger 1

GLI1 is a transcriptional effector of the canonical Hedgehog (HH) signaling pathway. In the adrenal gland, Sonic hedgehog (SHH) is secreted by cells of the zG and signals to Gli1+ capsular cells, leading to Gli1-dependent transcription of canonical SHH target genes [10–12]. Recent studies suggest Gli1-dependent transcription may be activated through SHH-independent mechanisms [13]. During development, Gli1-expressing cells are derived from FAdE+ cells [9] and constitute the largest population of cells residing in the capsule. Their descendants migrate centripetally into the cortex, differentiating into SF1+ cells, a subset of which also expresses SHH [12]. Remarkably, in male mice during adulthood, the contribution of Gli1+ progenitor cells to cortical homeostasis is substantially reduced but may be exacerbated during zF regeneration [12,14]. While it has been observed that the turnover of the adrenals of female mice is faster than that of males [15], a recent study by Grabek et al [16] reported that this sexual dimorphism may be partially attributed to androgens restricting the contribution of Gli1+ cells to the male adrenal cortex.

R-spondin 3

RSPO3 is a paracrine factor released from the adrenal capsule that positively regulates the canonical WNT pathway [17]. Rspo3 is expressed in the mouse adrenal capsule starting at E12.5, colocalizing with Gli1+ cells and Nr2f2+ cells. RSPO3 loss leads to impairment of adrenal development, affecting the zG morphologically and functionally, evidenced by loss of progenitor markers Shh and Wnt4 and loss of zonation markers DAB2 and CYP11B2. Capsular Gli1 expression is also affected, consistent with loss of SHH signaling [18].

Yes-associated protein/transcriptional coactivator with the PDZ-binding motif

The Hippo signaling pathway has been implicated in control of organ size, renewal, and regeneration [19]. The two effectors of Hippo signaling, YAP and TAZ, are expressed in both the mouse adrenal capsule and throughout the cortex [20]. Sf1-driven loss of one copy of YAP and two copies of TAZ led to adrenal defects in male mice [20]. Notably, mice exhibited a minor decrease in adrenocorticotropic hormone (ACTH)–induced corticosterone levels, an increase in lipid accumulation, diminished expression of Shh, Nr0b1, and Gli1 mRNA at 10 weeks of age, suggesting partial depletion of adrenal progenitors.

Nestin

Nestin, a type VI intermediate filament protein, marks a small, distinct population of neural crest–derived cells scattered in the capsule and disseminated throughout the mouse adrenal cortex [21]. During homeostasis, Nestin+ cell descendants migrate centripetally toward the medulla and can express steroidogenic markers [21].

Cortex

Recent studies have also implicated the subcapsular outer cortex as the location of adrenocortical progenitors recruited in response to endocrine and paracrine factors (Figure 1). Under physiologic conditions, these progenitors are likely the major contributors to cortical homeostasis. The current model suggests that descendants of peripheral adrenocortical stem/progenitor cells differentiate and migrate centripetally, undergoing apoptosis at the corticomedullary boundary, thus giving rise to CYP11B2+ cells of the zG and CYP11B1+ cells of the zF (Figure 2). These peripheral progenitors are characterized by nuclear β-catenin, SF1+, SHH+, CYP11B2- expression [10–12,14,15,22]. Here, we present a brief overview of these cortical populations.

Figure 2. Cell lineages in adult mouse adrenal.

Schematic exemplifying cell lineages of the adrenal gland. Shh-expressing cells give rise to cells of the zG that can differentiate into CYP11B2+ cells, which in turn originate zF CYP11B1+ cells. In the capsule, SF1-/Nestin+ cells can contribute to the steroidogenic cortex, particularly under stress conditions. Capsular Gli1+ cells are recruited to produce differentiated steroidogenic cells that delaminate into the cortex. This process is hindered by androgens in male mice.

SHH signaling pathway

SHH is the only HH ligand expressed in the adrenal gland, secreted by a subset of SF1+ zG cells starting at E12.5. Lineage-tracing experiments in mice have revealed that SHH+ cells residing in the zG give rise to virtually all cortical cells, during development and adult homeostasis. It is now known that SHH+ cells differentiate into CYP11B2+ cells, which migrate and differentiate into zF CYP11B1+ cells via repression of WNT signaling and activation of ACTH-dependent protein kinase A (PKA) signaling [11,12,15,23]. In male mice, SHH signaling is particularly important during zF regeneration, in which capsular Gli1+ and subcapsular Shh+ are progenitors recruited to repopulate the zF and restore steroidogenesis. Indeed, pharmacological inhibition of the HH pathway restricts adrenocortical regeneration [14].

WNT signaling pathway

The WNT signaling pathway is involved in organogenesis, homeostasis, and progenitor cell control in many tissues including the adrenal cortex. In the canonical WNT signaling pathway, WNT ligands bind to cell surface receptors [24], leading to (TCF/LEF) translocation of β-catenin into the nucleus, where it complexes with T-cell factor/lymphocyte enhancer factor (TCF/LEF) transcription factors to initiate target gene transcription [25]. In the adrenal cortex, WNT signaling is zonally distributed, with prominent nuclear β-catenin staining in the zG and a fading gradient in the upper zF [23,26–28].

In mice, canonical WNT signaling is established after AGP formation (E9.5) and encapsulation (E12.5), coincident with the establishment of the definitive adrenal cortex [28]. Genetic impairment of β-catenin dosage in Sf1+ cells during organogenesis (after E12.5) leads to reduction in cortical cell proliferation and complete regression of the adrenal gland by E18.5 [28]. Loss of β-catenin signaling in 50% of the Sf1+ cortex results in adrenal failure in mice after 15 weeks, suggesting that canonical WNT signaling is essential for cortical progenitor activity during homeostasis [28]. Consistent with this observation, a majority of SHH+ cells bear active WNT signaling [29] and are depleted in a model of capsular RSPO3 deficiency [18]. Complete zF regeneration is also reliant on intact canonical WNT signaling [14].

Recent studies have focused on the role of WNT ligands in maintaining canonical WNT signaling in the adrenal cortex, with a specific focus on Wnt4. In humans, inactivating germline mutations in WNT4 causes SEx Reversion, Kidneys, Adrenal, and Lung dysgenesis (SERKAL) syndrome, a condition characterized by female-to-male sex reversal and adrenal insufficiency [30]. In mice, Wnt4 is expressed in the developing adrenal at E11.5 [27], and by E14.5, the expression is restricted to the outer cortex; in adults, WNT4 is produced by cells in the zG [26,27]. Global Wnt4 deficiency leads to a decrease in adrenal Cyp11b2 expression and aldosterone production, implicating WNT4 in sustaining canonical WNT signaling and steroidogenesis in the zG [23]. This was also supported by a mouse model in which Sf1-driven Wnt4 deletion led to diminished expression of canonical WNT target genes [23].

DAX1

Nr0b1 (encoding DAX1) is also a target of β-catenin, expressed primarily in subcapsular adrenocortical cells [31]. Intriguingly, aged mice lacking DAX1 display a phenotype partially resembling loss of β-catenin. DAX1-deficient aging mice bear hypofunctional, dysplastic adrenals accompanied by loss of proliferation, suggesting the importance of DAX1 in maintaining the adrenocortical progenitor pool [32].

ACTH/PKA signaling and the zF

The HPA axis regulates production of glucocorticoids and androgens from the adrenal cortex. In addition to promoting steroid production, activation of the HPA axis has mitogenic effects on the adrenal cortex. Pituitary ACTH is an essential component of this axis, stimulating release of cortisol (or corticosterone in mice) from the adrenal zF through binding the melanocortin 2 receptor and melanocortin accessory protein (MRAP) and activating PKA signaling [33]. In mice, MRAP deficiency results in neonatal lethality, rescued by administration of exogenous glucocorticoids. Survivors lacking MRAP exhibit postnatal impairment of steroidogenesis, a thickened and hyperplastic capsule, increased Shh expression, and an accumulation of progenitor cells with active WNT signaling. These findings suggest a critical role for ACTH in deploying adrenocortical stem/progenitor cells to differentiate into the steroidogenic cells of the zF. This finding also corroborates the recent demonstration that constitutive activation of PKA signaling in the adrenal cortex inhibits the canonical WNT pathway, favoring zF differentiation [23,34].

A deeper understanding of the role of PKA in zF fate determination comes from recent studies. Mathieu et al [8] developed a mouse model of Sf1-driven deletion of Ezh2, a member of the polycomb repressive complex 2 responsible for writing the repressive H3K27me3 mark. EZH2 expression in the adult cortex is largely confined to proliferating cells in the zG/zF boundary. These investigators observed that adult mice with Sf1-driven Ezh2 deletion developed primary glucocorticoid insufficiency with severe zonation defects, notably zF aplasia concomitant with zG disorganization. Interestingly, WNTsignaling was unchanged, suggesting the actions of EZH2 are primarily on cells of the zF. Indeed, these investigators observed that adrenocortical EZH2 deposits H3K27me3 on the promoters of genes encoding negative regulators of PKA signaling. These findings suggest EZH2 is crucial for priming cells of the zF to respond to ACTH and are consistent with recent observations that Ezh2 expression rises dramatically during the proliferative burst that accompanies zF regeneration [14]. Taken together, these data support an essential role of EZH2 in enabling cell cycle-avid zF cells to respond to ACTH during development and cortical renewal.

Final remarks

Numerous advances in the study of the adrenal gland biology have been made in recent years. Although the field is still evolving, new studies elucidating the signaling pathways controlling stem and progenitor cell fate illustrate that the adrenal gland is a multifaceted system where endocrine, autocrine, and paracrine factors converge to achieve physiological demands [22,35]. Many studies have taken advantage of mouse model systems and provided thought-provoking evidence that mouse adrenals exhibit a high degree of sexual dimorphism, which warrants future study [16,36]. In the future, we hope new studies will better characterize stem/progenitor cells in human tissues, perhaps using organoid models and single-cell approaches.