Never Underestimate the Complexity of Remodeling

As every experienced homeowner knows, one should never underestimate the complexity of remodeling. Three recent articles, including one in this issue of Endocrinology, highlight the remarkable complexity of tissue remodeling in the adrenal cortex (1–3). The adrenal cortex, a major site of steroid biosynthesis, is composed of anatomically and functionally distinct layers. The zona glomerulosa (zG) contains cells that produce mineralocorticoids under the control of the renin-angiotensin system. The zona fasciculata (zF) contains cells that secrete glucocorticoids as part of the hypothalamic-pituitary-adrenal axis. Adrenocortical cells undergo constant turnover, allowing rapid organ remodeling in response to physiological demands for mineralocorticoids or glucocorticoids. Stem/progenitor cells in the periphery of the adrenal gland differentiate to centripetally repopulate the cortex (4). The signaling pathways that control the maintenance and differentiation of adrenocortical stem/progenitor cells have been the subject of intense investigation (5, 6). Steroidogenic cells in the adrenal glands and gonads arise from a common pool of progenitors in the adrenogonadal primordia (AGP), specialized coelomic epithelial cells that coexpress the transcription factors steroidogenic factor-1 (Sf1) and Wilms tumor suppressor-1 (Wt1) (6). At about embryonic day 10.5 in the mouse, AGP cells delaminate, invade adjacent mesenchyme, and down-regulate expression of Wt1 while maintaining Sf1 expression (3). Some of these Sf1+ cells migrate dorsolaterally to form the nascent gonad; the remainder migrate dorsomedially to form the adrenal anlagen (6). Shortly thereafter, the adrenal anlagen are invaded by neural crest cells that ultimately give rise to neuroendocrine cells of the medulla. At embryonic day 14.5, the nascent adrenal glands become encapsulated by mesenchymal cells (6). Traditionally, the adrenal capsule has been viewed as merely a support structure, but it is now clear that it houses and maintains stem/progenitor cells that give rise to differentiated steroidogenic cells in the cortex (5). It is not until after birth that the adrenal cortex partitions into discrete zones (2). Developmental signaling pathways, including the sonic hedgehog (Shh) and Wnt/β-catenin pathways, are involved in the formation and homeostasis of the adrenal gland (6, 7). Shh is secreted by subcapsular cells that express Sf1 but not the terminal enzymes required for mineralocorticoid or glucocorticoid biosynthesis (8–10). Capsular cells, which do not express Sf1, transduce the Shh signal and up-regulate its downstream effector Gli1 (8, 10). Some of these Gli1+ cells migrate into the cortex, lose responsiveness to Shh, and become steroidogenic, as evidenced by expression of Sf1 and differentiation markers characteristic of the zG (Cyp11b2) or zF (Cyp11b1). Conditional deletion of Shh from steroidogenic cells results in adrenocortical hypoplasia and capsular thinning (8–10). Like Shh, β-catenin is expressed in the subcapsule (11), and Wnt/β-catenin signaling maintains the undifferentiated state of adrenocortical stem/progenitor cells in this region (5, 12). Targeted mutagenesis of β-catenin in Sf1+ cells causes late onset adrenal hypoplasia, thought to be the result of gradual depletion of a stem/progenitor cell pool (11). In this edition of the journal, Guasti et al (1) investigate the role of another developmental signaling molecule, δ-like homologue 1 (Dlk1), in adrenocortical homeostasis and remodeling. Dlk1, also known as preadipocyte factor-1, is a transmembrane protein related to the Notch family of signaling molecules. It was originally identified as a key regulator of the undifferentiated state in preadipocytes (13). Cleavage of the Dlk1 extracellular domain by TNF-α-converting enzyme produces a biologically active soluble peptide that inhibits the differentiation of preadipocytes into mature adipocytes by binding to fibronectin and activating the MAPK pathway, resulting in the phosphorylation of Erk1–2 (13). Besides its role in preadipocyte regulation, Dlk1is now thought to control the quiescence of stem/progenitor cells in other tissue types (13). Guasti et al (1) show that Dlk1 is coexpressed with Shh in the outer undifferentiated zone (zU), a distinctive layer located between the zG and zF in the rat adrenal cortex (Figure 1A). Soluble Dlk1, like Shh, activates Gli1 expression in nearby capsule cells. Using cultured cell models, Guasti et al provide evidence that Dlk1 activates Gli1 transcription through a β1-integrin and Erk1–2-dependent mechanism. Interestingly, Dlk1 can inhibit Shh activation of Gli1 in vitro, suggesting that Dlk1 may function to fine tune Shh signaling in common signal receiving cells in the adrenal capsule. In addition to being coexpressed, Dlk1 and Shh are coordinately regulated. Both genes are down-regulated in the adrenals of mice fed a low-sodium diet, which activates the renin-angiotensin system and increases the size of the zG. Conversely, Dlk1 and Shh are up-regulated in the adrenals of mice treated with captopril, which inhibits the renin-angiotensin system and decreases the size of the zG. Collectively, these findings suggest that Dlk1 and Shh may act together to regulate adrenal remodeling and homeostasis. New insights into adrenocortical homeostasis and remodeling. A, Shh and Dlk1 are coexpressed in the zU of the rat adrenal cortex and may act in concert to regulate stem/progenitor cells in the adrenal capsule (1). B, Adrenocortical zonation during postnatal mouse development results from lineage conversion of zG cells into zF cells, as evidenced by fate mapping using Cyp11b2-Cre and a GFP reporter (2). C, Wt1 marks a population of AGP-like progenitors within the adrenal capsule of the mouse. Under basal conditions, these AGP-like cells give rise to normal steroidogenic cells in the cortex, as evidenced by lineage tracing analysis with a GFP reporter. GDX triggers the differentiation of AGP-like cells into wedges of gonadal-like steroidogenic tissue (3). Secretion of sex steroids and other hormones by the ectopic gonadal tissue causes regression of the subjacent X-zone, a phenomenon first described over 70 years ago (17). Guasti et al make another interesting observation relevant to adrenocortical zonation. They show that cells in the inner zU stain intensely for Cyp11a1 (side chain cleavage enzyme), which catalyzes the first reaction in steroidogenesis, but lack expression of markers of the outer zU (Shh, Dlk1), zG (Cyp11b2), or zF (Cyp11b1). The authors suggest that the inner zU represents a transitional population of cells that is more committed to the steroidogenic phenotype than cells in the outer zU. Supporting this premise, the ACTH receptor (melanocortin 2 receptor, Mc2r) and its accessory protein Mrap have been shown to be expressed in the inner zU (14). A recent article by Freedman et al (2) provides further insight into the dynamic mechanisms underlying adrenocortical zonation and remodeling. They used Cyp11b2-Cre to indelibly mark zG cells and all their descendants with green fluorescent protein (GFP). By tracing the fate of GFP+ cells during postnatal development, these investigators concluded that adrenocortical zonation results from lineage conversion of “terminally” differentiated zG cells into zF cells (Figure 1B). These findings provide formal proof of the longstanding hypothesis of centripetal migration of adrenocortical cells, wherein stem/progenitor cells in the periphery of the gland give rise to zG cells that migrate centripetally and convert to zF before undergoing apoptosis at the corticomedullary junction (15). Thus, steroidogenic cell fate in the adrenal cortex is not preordained but rather determined by the cellular environment and hormonal milieu; the functional identity of any given adrenocortical cell may change during its lifetime in response to external cues. To show that zG-to-zF conversion occurs during adrenocortical remodeling, Freedman et al (2) treated older mice with dexamethasone to suppress the zF via negative feedback of the hypothalamic-pituitary-adrenal axis. Dexamethasone treatment caused a reduction in the size of the zF and loss of GFP+ cells in the zone, without altering GFP+ cells in the zG. After dexamethasone withdrawal, the zG-to-zF conversion resumed and the zF expanded. Remarkably, when zG-to-zF conversion was disrupted through conditional mutagenesis of Sf1 in Cyp11b2+ cells, a fully functional zF still formed, implying the existence of zG-independent pathways of zF formation and maintenance. Relatively undifferentiated progenitors, such as the transitional population of cells characterized by Guasti et al (1), are one possible source of cells for this zG-independent pathway of zF formation. Another potential source of progenitors is the adrenal X-zone, a juxtamedullary zone found in rodents (Figure 1, A–C) (16). The adrenal cortex of the mouse can undergo remodeling in response to physiological demand for not only mineralocorticoids and glucocorticoids but also sex steroids. In classic studies, Woolley et al (17) first demonstrated that gonadectomy (GDX) and the ensuing changes in plasma hormone levels induce sex steroid-producing adrenocortical neoplasms in certain inbred mouse strains. This phenomenon is thought to reflect gonadotropin-induced metaplasia of stem/progenitor cells in the adrenal capsule or adjacent cortex, although the term “neoplasia” is used more often than “metaplasia” to describe the process, because with time, the metaplastic lesions can evolve into frank tumors (18, 19). The neoplastic tissue expresses gonadal-like differentiation markers, including the LH receptor (Lhcgr), transcription factor Gata4, and enzymes required for sex steroid biosynthesis (Cyp17a1, Cyp19a1) (18). Typical markers of adrenocortical cell differentiation, such as Mc2r, Cyp11b1, and Cyp11b2, are not expressed in the neoplastic tissue (18). The formation of ectopic gonadal-like tissue from stem/progenitor cells in the adrenal gland can be viewed as an extreme example of adrenocortical remodeling in response to GDX. The molecular basis for GDX-induced adrenocortical neoplasia is not well understood. Linkage analysis of crosses between susceptible and nonsusceptible inbred strains has established that post-GDX adrenocortical neoplasia is a complex trait influenced by multiple genetic loci (20). Targeted mutagenesis of Gata4 attenuates post-GDX adrenocortical tumor formation and ectopic sex steroid production in susceptible strains (21), and transgenic expression of Gata4 induces adrenocortical neoplasia in a nonsusceptible strain (22). Epigenetic modifications may also play a role in the pathogenesis of GDX-induced adrenocortical neoplasia (23). A recent article by Bandiera et al (3) implicates Wt1 as a key player in GDX-induced adrenocortical neoplasia in the mouse and sheds light on a distinctive pool of stem/progenitor cells in the adrenal gland. Lineage tracing analysis of Wt1+ cells identified a long-lived progenitor population in the adrenal capsule characterized by expression of Wt1 and Gata4, markers of the AGP. Under basal conditions, these AGP-like cells in the capsule can give rise to normal steroidogenic cells in the adrenal cortex (Figure 1C, left). GDX activates these Wt1+ capsular progenitors and triggers their differentiation into gonadal-like steroidogenic tissue (Figure 1C, right). The authors conclude that Wt1+ adrenal capsular cells represent a reserve stem/progenitor cell population with AGP-like features that can be mobilized in response to extreme physiological demand. The 3 articles discussed here underscore the complexity of tissue remodeling in the adrenal cortex. The process entails: 1) a wide array of developmental signaling factors that interact with one another; 2) distinct pools of multipotential stem/progenitor cells in the capsule, subcapsule, and other sites; 3) direct lineage conversion in response to environmental cues; 4) functional redundancy with alternative pathways for differentiation; and 5) extraordinary plasticity. When deranged, the process can result in neoplasia. Lessons gleaned from studying remodeling in the adrenal cortex are relevant to other organ systems. For example, steroidogenic Leydig cells in the adult testis can arise from different populations of stem/progenitor cells, including from undifferentiated mesenchymal cells in the testicular interstitium and vascular progenitors (24, 25). Patients with disrupted adrenocortical function due to 21-hydroxylase deficiency develop neoplastic nodules of hormonally active adrenocortical tissue in the testis (testicular adrenal rest tumors), possibly arising from a distinctive pool of multipotential stem/progenitor cells (26, 27). The mechanisms governing remodeling may be complicated, but this degree of complexity is necessary to achieve the dual goals of tissue homeostasis and stress adaptation. We thank Joshua Rubin, Monita Wilson, Paul Hruz, and Maximiliaan Schillebeeckx for helpful comments. This work was supported by the American Heart Association Grant 13GRNT16850031 and by the Sigrid Jusélius Foundation. Disclosure Summary: The authors have nothing to disclose. adrenogonadal primordia δ-like homologue 1 gonadectomy green fluorescent protein steroidogenic factor-1 sonic hedgehog Wilms tumor suppressor-1 zona fasciculata zona glomerulosa undifferentiated zone.