Keith R. Stayrook

Identification of heme as the ligand for the orphan nuclear receptors REV-ERBα and REV-ERBβ

The nuclear receptors REV-ERBα (encoded by NR1D1) and REV-ERBβ (NR1D2) have remained orphans owing to the lack of identified physiological ligands. Here we show that heme is a physiological ligand of both receptors. Heme associates with the ligand-binding domains of the REV-ERB receptors with a 1:1 stoichiometry and enhances the thermal stability of the proteins. Results from experiments of heme depletion in mammalian cells indicate that heme binding to REV-ERB causes the recruitment of the co-repressor NCoR, leading to repression of target genes including BMAL1 (official symbol ARNTL), an essential component of the circadian oscillator. Heme extends the known types of ligands used by the human nuclear receptor family beyond the endocrine hormones and dietary lipids described so far. Our results further indicate that heme regulation of REV-ERBs may link the control of metabolism and the mammalian clock.

Iron deficiency drives an autosomal dominant hypophosphatemic rickets (ADHR) phenotype in fibroblast growth factor-23 (Fgf23) knock-in mice

Autosomal dominant hypophosphatemic rickets (ADHR) is unique among the disorders involving Fibroblast growth factor 23 (FGF23) because individuals with R176Q/W and R179Q/W mutations in the FGF23 176RXXR179/S180 proteolytic cleavage motif can cycle from unaffected status to delayed onset of disease. This onset may occur in physiological states associated with iron deficiency, including puberty and pregnancy. To test the role of iron status in development of the ADHR phenotype, WT and R176Q-Fgf23 knock-in (ADHR) mice were placed on control or low-iron diets. Both the WT and ADHR mice receiving low-iron diet had significantly elevated bone Fgf23 mRNA. WT mice on a low-iron diet maintained normal serum intact Fgf23 and phosphate metabolism, with elevated serum C-terminal Fgf23 fragments. In contrast, the ADHR mice on the low-iron diet had elevated intact and C-terminal Fgf23 with hypophosphatemic osteomalacia. We used in vitro iron chelation to isolate the effects of iron deficiency on Fgf23 expression. We found that iron chelation in vitro resulted in a significant increase in Fgf23 mRNA that was dependent upon Mapk. Thus, unlike other syndromes of elevated FGF23, our findings support the concept that late-onset ADHR is the product of gene–environment interactions whereby the combined presence of an Fgf23-stabilizing mutation and iron deficiency can lead to ADHR.

Modulation of Retinoic Acid Receptor-related Orphan Receptor α and γ Activity by 7-Oxygenated Sterol Ligands

The retinoic acid receptor-related orphan receptors α and γ (RORα (NR1F1) and RORγ (NR1F3)) are orphan nuclear receptors and perform critical roles in regulation of development, metabolism, and immune function. Cholesterol and cholesterol sulfate have been suggested to be RORα ligands, but the physiological significance is unclear. To date, no endogenous RORγ ligands have been described. Here, we demonstrate that 7-oxygenated sterols function as high affinity ligands for both RORα and RORγ by directly binding to their ligand-binding domains (Ki ∼20 nm), modulating coactivator binding, and suppressing the transcriptional activity of the receptors. One of the 7-oxygenated sterols, 7α-hydroxycholesterol (7α-OHC), serves as a key intermediate in bile acid metabolism, and we show that 7α-OHC modulates the expression of ROR target genes, including Glc-6-Pase and phosphoenolpyruvate carboxykinase, in an ROR-dependent manner. Furthermore, glucose output from hepatocytes is suppressed by 7α-OHC functioning as an RORα/γ ligand. Thus, RORα and RORγ are ligand-regulated members of the NR superfamily and may serve as sensors for 7-oxygenated sterols.

DNA binding alters coactivator interaction surfaces of the intact VDR-RXR complex.

The vitamin D receptor (VDR) functions as an obligate heterodimer in complex with the retinoid X receptor (RXR). These nuclear receptors are multidomain proteins, and it is unclear how various domains interact with one another within the nuclear receptor heterodimer. Here, we show that binding of intact heterodimer to DNA alters the receptor dynamics in regions remote from the DNA-binding domains (DBDs), including the coactivator binding surfaces of both co-receptors, and that the sequence of the DNA response element can determine these dynamics. Furthermore, agonist binding to the heterodimer results in changes in the stability of the VDR DBD, indicating that the ligand itself may play a role in DNA recognition. These data suggest a mechanism by which nuclear receptors show promoter specificity and have differential effects on various target genes, providing insight into the function of selective nuclear receptor modulators.

Identification and characterization of noncalcemic, tissue-selective, nonsecosteroidal vitamin D receptor modulators

Vitamin D receptor (VDR) ligands are therapeutic agents for the treatment of psoriasis, osteoporosis, and secondary hyperparathyroidism. VDR ligands also show immense potential as therapeutic agents for autoimmune diseases and cancers of skin, prostate, colon, and breast as well as leukemia. However, the major side effect of VDR ligands that limits their expanded use and clinical development is hypercalcemia that develops as a result of the action of these compounds mainly on intestine. In order to discover VDR ligands with less hypercalcemia liability, we sought to identify tissue-selective VDR modulators (VDRMs) that act as agonists in some cell types and lack activity in others. Here, we describe LY2108491 and LY2109866 as nonsecosteroidal VDRMs that function as potent agonists in keratinocytes, osteoblasts, and peripheral blood mononuclear cells but show poor activity in intestinal cells. Finally, these nonsecosteroidal VDRMs were less calcemic in vivo, and LY2108491 exhibited more than 270-fold improved therapeutic index over the naturally occurring VDR ligand 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] in an in vivo preclinical surrogate model of psoriasis.

Regulation of Cholesterologenesis by the Oxysterol Receptor, LXRα

Cholesterol is required for normal cellular and physiological function, yet dysregulation of cholesterol metabolism is associated with diseases such as atherosclerosis. Cholesterol biosynthesis is regulated by end product negative feedback inhibition where the levels of sterols and oxysterols regulate the expression of cholesterologenic enzymes. Sterol regulatory element-binding protein-2 is responsive to both sterols and oxysterols and has been shown to mediate the transcriptional response of the cholesterologenic enzymes to these lipids. Here, we show that the nuclear hormone receptor for oxysterols, the liver X receptor α (LXRα), regulates cholesterol biosynthesis by directly silencing the expression of two key cholesterologenic enzymes (lanosterol 14α-demethylase (CYP51A1), and squalene synthase (farnesyl diphosphate farnesyl transferase 1)) via novel negative LXR DNA response elements (nLXREs) located in each of these genes. Examination of the CYP51A1 gene revealed that both the SRE and nLXRE are required for normal oxysterol-dependent repression of this gene. Thus, these data suggest that LXRα plays an important role in the regulation of cholesterol biosynthesis.

Regulation of Adipogenesis by Natural and Synthetic REV-ERB Ligands

The nuclear hormone receptor, REV-ERB, plays an essential role in adipogenesis. Rev-erbalpha expression is induced in 3T3-L1 cells during adipogenesis, and overexpression of this receptor leads to expression of adipogenic genes. We recently demonstrated that the porphyrin heme functions as a ligand for REV-ERB, and binding of heme is required for the receptors activity. We therefore hypothesized that REV-ERB ligands may play a role in regulation of adipogenesis. We detected an increase intracellular heme levels during 3T3-L1 adipogenesis that correlated with induction of aminolevulinic acid synthase 1 (Alas1) expression, the rate-limiting enzyme in heme biosynthesis. If the increase in Alas1 expression was blocked, adipogenesis was severely attenuated, indicating that induction of expression of Alas1 and the increase in heme synthesis is critical for differentiation. Inhibition of heme synthesis during adipogenesis leads to decreased recruitment of nuclear receptor corepressor to the promoter of a REV-ERB target gene, suggesting alteration of REV-ERB activity. Treatment of 3T3-L1 cells with a synthetic REV-ERB ligand, SR6452, resulted in induction of adipocyte differentiation to a similar extent as treatment with the peroxisomal proliferator-activated receptor-gamma agonist, rosiglitazone. Combination of SR6452 and rosiglitazone had an additive effect on stimulation of adipocyte differentiation. These results suggest that heme, functioning as a REV-ERB ligand, is an important signaling molecule for induction of adipogenesis. Moreover, synthetic small molecule ligands for REV-ERB are effective modulators of adipogenesis and may be useful for treatment of metabolic diseases.

Hydrogen/deuterium exchange reveals distinct agonist/partial agonist receptor dynamics within vitamin D receptor/retinoid X receptor heterodimer.

Regulation of nuclear receptor (NR) activity is driven by alterations in the conformational dynamics of the receptor upon ligand binding. Previously, we demonstrated that hydrogen/deuterium exchange (HDX) can be applied to determine novel mechanism of action of PPARγ ligands and in predicting tissue specificity of selective estrogen receptor modulators. Here, we applied HDX to probe the conformational dynamics of the ligand binding domain (LBD) of the vitamin D receptor (VDR) upon binding its natural ligand 1α,25-dihydroxyvitamin D3 (1,25D3), and two analogs, alfacalcidol and ED-71. Comparison of HDX profiles from ligands in complex with the LBD with full-length receptor bound to its cognate receptor retinoid X receptor (RXR) revealed unique receptor dynamics that could not be inferred from static crystal structures. These results demonstrate that ligands modulate the dynamics of the heterodimer interface as well as provide insight into the role of AF-2 dynamics in the action of VDR partial agonists.

TGF-β in the Bone Microenvironment: Role in Breast Cancer Metastases.

Breast cancer is the most prevalent cancer among females worldwide. It has long been known that cancers preferentially metastasize to particular organs, and bone metastases occur in ∼70% of patients with advanced breast cancer. Breast cancer bone metastases are predominantly osteolytic and accompanied by bone destruction, bone fractures, pain, and hypercalcemia, causing severe morbidity and hospitalization. In the bone matrix, transforming growth factor-β (TGF-β) is one of the most abundant growth factors, which is released in active form upon tumor-induced osteoclastic bone resorption. TGF-β, in turn, stimulates bone metastatic cells to secrete factors that further drive osteolytic destruction of the bone adjacent to the tumor, categorizing TGF-β as a crucial factor responsible for driving the feed-forward vicious cycle of cancer growth in bone. Moreover, TGF-β activates epithelial-to-mesenchymal transition, increases tumor cell invasiveness and angiogenesis and induces immunosuppression. Blocking the TGF-β signaling pathway to interrupt this vicious cycle between breast cancer and bone offers a promising target for therapeutic intervention to decrease skeletal metastasis. This review will describe the role of TGF-β in breast cancer and bone metastasis, and pre-clinical and clinical data will be evaluated for the potential use of TGF-β inhibitors in clinical practice to treat breast cancer bone metastases.

The role of TGF-β in bone metastasis: novel therapeutic perspectives.

The skeleton is a preferred site for cancer metastasis. These bone metastases cause dysregulated bone remodeling and the associated morbidity of fractures, pain, hypercalcemia and catastrophic nerve compression syndromes. Transforming growth factor-β (TGF-β) is stored in mineralized bone matrix, and released and activated by osteoclastic bone resorption. Once activated, TGF-β stimulates nearby metastatic tumor cells within the bone microenvironment to secrete factors that further drive osteolytic destruction of the bone. Therefore, TGF-β and its signaling constitute a critical component driving the feed-forward vicious cycle of cancer growth in bone. Moreover, additional pro-tumorigenic activities attributed to TGF-β include activation of epithelial-to-mesenchymal transition, increased tumor cell invasion, enhanced angiogenesis and various immunomodulatory properties. Blocking the TGF-β signaling pathway to interrupt this vicious cycle and manipulate the bone microenvironment offers a promising area for therapeutic intervention to decrease skeletal metastasis and normalize bone homeostatic mechanisms. In this review, preclinical and clinical data are evaluated for the potential use of TGF-β pathway inhibitors in clinical practice to treat bone metastases and its associated comorbidities.