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Dysregulation of cholesterol homeostasis is associated with many diseases such as cardiovascular disease and cancer. Liver X receptors (LXRs) are major upstream regulators of cholesterol homeostasis and are activated by endogenous cholesterol metabolites such as 27-hydroxycholesterol (27HC). LXRs and various LXR ligands such as 27HC have been described to influence several extra-hepatic biological systems. However, disparate reports of LXR function have emerged, especially with respect to immunology and cancer biology. This would suggest that, similar to steroid nuclear receptors, the LXRs can be selectively modulated by different ligands. Here, we use RNA-sequencing of macrophages and single-cell RNA-sequencing of immune cells from metastasis-bearing murine lungs to provide evidence that LXR satisfies the 2 principles of selective nuclear receptor modulation: (1) different LXR ligands result in overlapping but distinct gene expression profiles within the same cell type, and (2) the same LXR ligands differentially regulate gene expression in a highly context-specific manner, depending on the cell or tissue type. The concept that the LXRs can be selectively modulated provides the foundation for developing precision pharmacology LXR ligands that are tailored to promote those activities that are desirable (proimmune), but at the same time minimizing harmful side effects (such as elevated triglyceride levels).

TLX (NR2E1), an orphan member of the nuclear receptor superfamily, is a transcription factor that has been described to be generally repressive in nature. It has been implicated in several aspects of physiology and disease. TLX is best known for its ability to regulate the proliferation of neural stem cells and retinal progenitor cells. Dysregulation, overexpression, or loss of TLX expression has been characterized in numerous studies focused on a diverse range of pathological conditions, including abnormal brain development, psychiatric disorders, retinopathies, metabolic disease, and malignant neoplasm. Despite the lack of an identified endogenous ligand, several studies have described putative synthetic and natural TLX ligands, suggesting that this receptor may serve as a therapeutic target. Therefore, this article aims to briefly review what is known about TLX structure and function in normal physiology, and provide an overview of TLX in regard to pathological conditions. Particular emphasis is placed on TLX and cancer, and the potential utility of this receptor as a therapeutic target.

The involvement of small heterodimer partner (SHP) in the inhibition of hepatic bile acid synthesis from cholesterol has been established. However, extrahepatic expression of SHP implies that SHP may have regulatory functions other than those in the liver. Here, we find that SHP mRNA expression is high in murine bone marrow cells, suggesting a physiological role within macrophages. Indeed, expression of SHP in macrophages decreases the transcriptional activity and nuclear localization of nuclear factor κB, whereas downregulation of SHP has the opposite effects. Expression of genes associated with macrophage–T cell crosstalk were altered by overexpression or downregulation of SHP. Intriguingly, increasing SHP expression in macrophages resulted in decreased T cell expansion, a hallmark of T cell activation, whereas knockdown of SHP resulted in increased expansion. Analyses of the expanded T cells revealed a dichotomous skewing between effector T cells and regulatory T cells (Tregs), with SHP overexpression reducing Tregs and downregulation of SHP increasing their expansion. The expanded Tregs were confirmed to be suppressive via adoptive transfers. IL-2 and TGF-β, known inducers of Treg differentiation, were found to be regulated by SHP. Furthermore, SHP occupancy at the promoter region of IL-2 was increased after macrophages were challenged with lipopolysaccharide. Neutralizing antibodies to IL-2 and TGF-β inhibited the expansion of Tregs mediated by downregulation of SHP. This study demonstrates that expression and activity of SHP within macrophages can alter T cell fate and identifies SHP as a potential therapeutic target for autoimmune diseases or solid cancers.

Extracellular vesicles (EVs), including exosomes, are emerging as important carriers of signals in normal and pathological physiology. As EVs are a long-range communication or signaling modality—just like hormones are—the field of endocrinology is uniquely poised to offer insight into their functional biology and regulation. EVs are membrane-bound particles secreted by many different cell types and can have local or systemic effects, being transported in body fluids. They express transmembrane proteins, some of which are shared between EVs and some being specific to the tissue of origin, that can interact with target cells directly (much like hormones can). They also contain cargo within them that includes DNA, RNA, miRNA, and various metabolites. They can fuse with target cells to empty their cargo and alter their target cell physiology in this way also. Similar to the endocrine system, the EV system is likely to be under homeostatic control, making the regulation of their biogenesis and secretion important aspects to study. In this review, we briefly highlight select examples of how EVs are implicated in normal physiology and disease states. We also discuss what is known about their biogenesis and regulation of secretion. We hope that this paper inspires the endocrinology field to use our collective expertise to explore these new multimodal “hormones.”

Cholesterol has been implicated in the clinical progression of breast cancer, a disease that continues to be the most commonly diagnosed cancer in women. Previous work has identified the cholesterol metabolite 27-hydroxycholesterol (27HC) as a major mediator of the effects of cholesterol on breast tumor growth and progression. 27HC can act as an estrogen receptor (ER) modulator to promote the growth of ERα+ tumors, and as a liver X receptor (LXR) ligand in myeloid immune cells to establish an immune-suppressive program. In fact, the metastatic properties of 27HC require the presence of myeloid cells with neutrophils (polymorphonuclear neutrophils; PMNs) being essential for the increase in lung metastasis in murine models. In an effort to further elucidate the mechanisms by which 27HC alters breast cancer progression, we made the striking finding that 27HC promoted the secretion of extracellular vesicles (EVs), a diverse assortment of membrane bound particles that includes exosomes. The resulting EVs had a size distribution that was skewed slightly larger than EVs generated by treating cells with vehicle. The increase in EV secretion and size was consistent across 3 different subtypes: primary murine PMNs, RAW264.7 monocytic cells, and 4T1 murine mammary cancer cells. Label-free analysis of 27HC-EVs indicated that they had a different metabolite composition to those from vehicle-treated cells. Importantly, 27HC-EVs from primary PMNs promoted tumor growth and metastasis in 2 different syngeneic models, demonstrating the potential role of 27HC-induced EVs in the progression of breast cancer. EVs from PMNs were taken up by cancer cells, macrophages, and PMNs, but not T cells. Since EVs did not alter proliferation of cancer cells, it is likely that their protumor effects are mediated through interactions with myeloid cells. Interestingly, RNA-seq analysis of tumors from 27HC-EV-treated mice do not display significantly altered transcriptomes, suggesting that the effects of 27HC-EVs occur early on in tumor establishment and growth. Future work will be required to elucidate the mechanisms by which 27HC increases EV secretion, and how these EVs promote breast cancer progression. Collectively, however, our data indicate that EV secretion and content can be regulated by a cholesterol metabolite, which may have detrimental effects in terms of disease progression, important findings given the prevalence of both breast cancer and hypercholesterolemia.

Osteoporosis and age-related bone loss are important public health concerns. Therefore, there is a high level of interest in the development of medical interventions and lifestyle changes that reduce the incidence of osteoporosis and age-related bone loss. Decreased bone mineral density is associated with high cholesterol, and patients on statins have increased bone mineral densities, strongly implicating cholesterol as a negative regulator of bone homeostasis. In this study, using both molecular and pharmacological approaches, we have been able to demonstrate that the primary cholesterol metabolite, 27-hydroxycholesterol, through its actions on both estrogen receptors and liver X receptors, decreases osteoblast differentiation and enhances osteoclastogenesis, resulting in increased bone resorbtion in mice. Induction of the short heterodimer partner protein by estrogens in osteoblasts can attenuate the liver X receptor-mediated actions of 27-hydroxycholesterol in bone. These data establish a mechanistic link between cholesterol and bone quality, highlight an unexpected target of estrogens in osteoblasts, and define a signaling axis, the therapeutic exploitation of which is likely to yield novel antiosteoporotic drugs.

Estrogens work by binding to and activating specific estrogen receptors (ERs). Although mammals have two major nuclear ER subtypes (ERalpha and ERbeta), three subtypes have been shown in teleost fish (ERalpha, ERbeta-I, and ERbeta-II). 17beta-Estradiol stimulates the production of an egg yolk precursor protein (vitellogenin) in the liver of oviparous species, including the goldfish. However, the functional involvement of the ER subtypes in this process is not fully understood. Here, using primary goldfish hepatocytes, we test the hypothesis that all three ER subtypes are functionally involved in the liver of goldfish by using RNA interference to specifically knock-down the different ER subtypes. The results suggest that ERalpha is induced by estradiol through activation of the ERbeta subtypes. This induction serves to sensitize the liver to further stimulation by estradiol. The knock-down results were supported by use of ER subtype specific antagonists. Sensitization by up-regulation of ERalpha is likely to be important for seasonal spawners such as goldfish, to bring about a change from somatic growth to reproductive development, and vitellogenesis. The novel data presented in this study provide strong support for the hypothesis that the goldfish ER subtypes play functional roles in the regulation of vitellogenin and ERalpha and provide a framework for the better understanding of ER signaling in fish and other vertebrates.

Osteoporosis is an important clinical problem, affecting more than 50% of people over age 50 yr. Estrogen signaling is critical for maintaining proper bone density, and the identification of an endogenous selective estrogen receptor (ER) modulator, 27-hydroxycholesterol (27HC), suggests a mechanism by which nutritional/metabolic status can influence bone biology. With its levels directly correlated with cholesterol, a new possibility emerges wherein 27HC links estrogen and cholesterol signaling to bone homeostasis. In these studies, we found that increasing concentrations of 27HC, both by genetic and pharmacological means, led to decreased bone mineral density that was associated with decreased bone formation and increased bone resorption. Upon manipulation of endogenous estrogen levels, many of the responses to elevated 27HC were altered in such a way as to implicate ER as a likely mediator. In a model of postmenopausal bone loss, some pathologies associated with elevated 27HC were exacerbated by the absence of endogenous estrogens, suggesting that 27HC may act both in concert with and independently from classic ER signaling. These data provide evidence for interactions between estrogen signaling, cholesterol and metabolic disease, and osteoporosis. Patients with high cholesterol likely also have higher than average 27HC, perhaps putting them at a higher risk for bone loss and fracture. More studies are warranted to fully elucidate the mechanism of action of 27HC in bone and to identify ways to modulate this pathway therapeutically.

Thyroid hormones are important mediators of growth and development in vertebrates and act by binding to a specific family of thyroid receptors (TRs). The TRs belong to the nuclear receptor superfamily, with two conserved regions, a DNA binding domain and a ligand binding domain (LBD). We recently demonstrated the presence of four TR subtypes in goldfish, two with complete DNA binding domains and LBDs (TRalpha-1 and TRbeta) and two novel forms including a transcript resembling TRalpha with variation in the LBD as well as a TRalpha-truncated (TRalpha-t) form lacking a LBD. To study the functional significance of TR subtypes, we first investigated the regulation of hepatic goldfish deiodinase type 3 (D3) by T3 and validated a bioassay in which D3 gene expression is up-regulated significantly in vivo and in vitro. Using short interfering RNA, TRalpha-1, TRbeta, or TRalpha-t was specifically knocked down and thyroid hormone-induced D3 gene expression was measured. Short interfering RNA against TRalpha-1 or TRbeta reduced the T3 induction of deiodinase gene expression to 50% or less than 25% of control (T3 treated) cells, respectively. Knocking down TRalpha-t alone, however, increased D3 expression 500-fold supporting the hypothesis that TRalpha-t plays a modulatory role in thyroid hormone-induced gene expression. Our results provide important insight into thyroid receptor biology in goldfish and a framework for the better understanding of thyroid receptor function in all vertebrates.