Jian-ping He

The orphan nuclear receptor Nur77 regulates LKB1 localization and activates AMPK

Liver kinase B1 (LKB1) has important roles in governing energy homeostasis by regulating the activity of the energy sensor kinase AMP-activated protein kinase (AMPK). The regulation of LKB1 function, however, is still poorly understood. Here we demonstrate that the orphan nuclear receptor Nur77 binds and sequesters LKB1 in the nucleus, thereby attenuating AMPK activation. This Nur77 function is antagonized by the chemical compound ethyl 2-[2,3,4-trimethoxy-6-(1-octanoyl)phenyl]acetate (TMPA), which interacts with Nur77 with high affinity and at specific sites. TMPA binding of Nur77 results in the release and shuttling of LKB1 to the cytoplasm to phosphorylate AMPKα. Moreover, TMPA effectively reduces blood glucose and alleviates insulin resistance in type II db/db and high-fat diet- and streptozotocin-induced diabetic mice but not in diabetic littermates with the Nur77 gene knocked out. This study attains a mechanistic understanding of the regulation of LKB1-AMPK axis and implicates Nur77 as a new and amenable target for the design and development of therapeutics to treat metabolic diseases.

Impeding the interaction between Nur77 and p38 reduces LPS-induced inflammation

Sepsis, a hyperinflammatory response that can result in multiple organ dysfunctions, is a leading cause of mortality from infection. Here, we show that orphan nuclear receptor Nur77 (also known as TR3) can enhance resistance to lipopolysaccharide (LPS)-induced sepsis in mice by inhibiting NF-κB activity and suppressing aberrant cytokine production. Nur77 directly associates with p65 to block its binding to the κB element. However, this function of Nur77 is countered by the LPS-activated p38α phosphorylation of Nur77. Dampening the interaction between Nur77 and p38α would favor Nur77 suppression of the hyperinflammatory response. A compound, n-pentyl 2-[3,5-dihydroxy-2-(1-nonanoyl) phenyl]acetate, screened from a Nur77-biased library, blocked the Nur77-p38α interaction by targeting the ligand-binding domain of Nur77 and restored the suppression of the hyperinflammatory response through Nur77 inhibition of NF-κB. This study associates the nuclear receptor with immune homeostasis and implicates a new therapeutic strategy to treat hyperinflammatory responses by targeting a p38α substrate to modulate p38α-regulated functions.

The orphan receptor TR3 participates in angiotensin II-induced cardiac hypertrophy by controlling mTOR signalling

Angiotensin II (AngII) induces cardiac hypertrophy and increases the expression of TR3. To determine whether TR3 is involved in the regulation of the pathological cardiac hypertrophy induced by AngII, we established mouse and rat hypertrophy models using chronic AngII administration. Our results reveal that a deficiency of TR3 in mice or the knockdown of TR3 in the left ventricle of rats attenuated AngII‐induced cardiac hypertrophy compared with the respective controls. A mechanistic analysis demonstrates that the TR3‐mediated activation of mTORC1 is associated with AngII‐induced cardiac hypertrophy. TR3 was shown to form a trimer with the TSC1/TSC2 complex that specifically promoted TSC2 degradation via a proteasome/ubiquitination pathway. As a result, mTORC1, but not mTORC2, was activated; this was accompanied by increased protein synthesis, enhanced production of reactive oxygen species and enlarged cell size, thereby resulting in cardiac hypertrophy. This study demonstrates that TR3 positively regulates cardiac hypertrophy by influencing the effect of AngII on the mTOR pathway. The elimination or reduction of TR3 may reduce cardiac hypertrophy; therefore, TR3 is a potential target for clinical therapy.

Prolyl isomerase Pin1 stabilizes and activates orphan nuclear receptor TR3 to promote mitogenesis

Pin1 regulates a subset of phosphoproteins by isomerizing phospho-Ser/Thr-Pro motifs via a ‘post-phosphorylation’ mechanism. Here, we characterize TR3 as a novel Pin1 substrate, and the mitogenic function of TR3 depends on Pin1-induced isomerization. There are at least three phospho-Ser-Pro motifs on TR3 that bind to Pin1. The Ser95-Pro motif of TR3 is the key site through which Pin1 enhances TR3 stability by retarding its degradation. Pin1 can also catalyze TR3 through phospho-Ser431-Pro motif, which is phosphorylated by extracellular signal-regulated kinase 2 (ERK2), resulting in enhanced TR3 transactivation. Furthermore, Pin1 not only facilitates TR3 targeting to the promoter of cyclin D2, a novel downstream target of TR3, but also promotes TR3 to recruit p300, thereby inducing cell proliferation. Importantly, we found that Pin1 is indispensable for TR3 to promote tumor growth both in vitro and in vivo. Our study thus suggests that Pin1 has an important role in cell proliferation by isomerizing TR3.

Orphan receptor TR3 is essential for the maintenance of stem-like properties in gastric cancer cells

The orphan receptor TR3 is an important regulator of cell proliferation and apoptosis. However, whether TR3 is involved in regulating the stem-like properties of cancer cells remains unknown. The present study shows that TR3 expression is increased in gastric tumorsphere cells and is positively correlated with cancer stem cell (CSC) characteristics. Knocking down TR3 leads to the suppression of its stem-like properties in both gastric cancer cells and tumorsphere cells. This process involves the decreased expression of the stemness-related genes Oct-4 and Nanog and the invasion-related gene MMP-9. We further identify Nanog as a new target for the transcription factor TR3. Together, these data demonstrate for the first time that TR3 is essential for the maintenance of stem-like properties in human gastric cancer cells and implicate TR3 as a new therapeutic target for gastric cancer.

Orphan Receptor TR3 Enhances p53 Transactivation and Represses DNA Double-Strand Break Repair in Hepatoma Cells under Ionizing Radiation

In response to ionizing radiation (IR)-induced DNA double-strand breaks (DSB), cells elicit an evolutionarily conserved checkpoint response that induces cell cycle arrest and either DNA repair or apoptosis, thereby maintaining genomic stability. DNA-dependent protein kinase (DNA-PK) is a central enzyme involved in DSB repair for mammalian cells that comprises a DNA-PK catalytic subunit and the Ku protein, which act as regulatory elements. DNA-PK also functions as a signaling molecule to selectively regulate p53-dependent apoptosis in response to IR. Herein, we demonstrate that the orphan nuclear receptor TR3 suppresses DSB repair by blocking Ku80 DNA-end binding activity and promoting DNA-PK-induced p53 activity in hepatoma cells. We find that TR3 interacts with Ku80 and inhibits its binding to DNA ends, which then suppresses DSB repair. Furthermore, TR3 is a phosphorylation substrate for DNA-PK and interacts with DNA-PK catalytic subunit in a Ku80-independent manner. Phosphorylated TR3, in turn, enhances DNA-PK-induced phosphorylation and p53 transcription activity, thereby enhancing IR-induced apoptosis in hepatoma cells. Together, our findings reveal novel functions for TR3, not only in DSB repair regulation but also in IR-induced hepatoma cell apoptosis, and they suggest that TR3 is a potential target for cancer radiotherapy.

Enhancement of hypothalamic STAT3 acetylation by nuclear receptor Nur77 dictates leptin sensitivity

Leptin, an anorexigenic hormone in the hypothalamus, suppresses food intake and increases energy expenditure. Failure to respond to leptin will lead to obesity. Here, we discovered that nuclear receptor Nur77 expression is lower in the hypothalamus of obese mice compared with normal mice. Injection of leptin results in significant reduction in body weight in wild-type mice but not in Nur77 knockout (KO) littermates or mice with specific Nur77 knockdown in the hypothalamus. Hypothalamic Nur77 not only participates in leptin central control of food intake but also expands leptin’s reach to liver and adipose tissues to regulate lipid metabolism. Nur77 facilitates signal transducer and activator of transcription 3 (STAT3) acetylation by recruiting acetylase p300 and disassociating deacetylase histone deacetylase 1 (HDAC1) to enhance the transcriptional activity of STAT3 and consequently modulates the expression of downstream gene Pomc in the hypothalamus. Nur77 deficiency compromises response to leptin in mice fed a high-fat diet. Severe leptin resistance in Nur77 KO mice with increased appetite, lower energy expenditure, and hyperleptinemia contributes to aging-induced obesity. Our study opens a new avenue for regulating metabolism with Nur77 as the positive modulator in the leptin-driven antiobesity in the hypothalamus.

Orphan receptor TR3 participates in cisplatin-induced apoptosis via Chk2 phosphorylation to repress intestinal tumorigenesis

Cisplatin is a widely used antitumor agent that induces aggressive cancer cell death via triggering cellular proteins involved in apoptosis. Here, we demonstrate that cisplatin effectively induces orphan nuclear receptor TR3 phosphorylation by activating Chk2 kinase activity and promoting cross talk between these two proteins, thereby contributing to the repression of intestinal tumorigenesis via apoptosis. Mechanistic analysis has demonstrated that Chk2-induced phosphorylation enables TR3 to bind to its response elements on the promoters of the BRE and RNF-7 genes, leading to the negative regulation of these two anti-apoptotic genes. Furthermore, the induction of apoptosis by cisplatin is mediated by TR3, and knockdown of TR3 reduces cisplatin-induced apoptosis in colon cancer cells by 27%. The role of TR3 in cisplatin chemotherapy is further clarified in mouse models. In Apc(min/+) mice, cisplatin inhibits intestinal tumorigenesis by 70% in a TR3 phosphorylation-dependent manner; however, the loss of TR3 function in Apc(min/+)/TR3(-/-) mice leads to the failure of cisplatin-induced repression of tumorigenesis. Consistently, xenografts derived from TR3 knockdown colon cancer cells are insensitive to cisplatin treatment, whereas a significant curative effect (50% inhibition) is observed in xenografts with functional TR3. Taken together, our study reveals a novel cross talk between Chk2 and TR3 and sheds light on the mechanism of cisplatin-induced apoptosis through TR3. Therefore, TR3 may be a new target of cisplatin for colon cancer therapy.

The orphan nuclear receptor TR3/Nur77 regulates ER stress and induces apoptosis via interaction with TRAPγ.

The orphan nuclear receptor TR3 (also known as Nur77) belongs to the steroid/thyroid/retinoid nuclear receptor superfamily and plays important roles in regulating cell proliferation, differentiation and apoptosis. No physiological ligand for TR3 has been found thus far; the determination of its binding partners is therefore important to clarify the biological functions of TR3. Here, we identified translocon-associated protein subunit γ (TRAPγ) as a novel TR3 binding partner using a tandem affinity purification method. This interaction between TR3 and TRAPγ was further confirmed, and the interacting regions were mapped. The ligand-binding domain of TR3 was required for TRAPγ binding, and the C terminus of TRAPγ was responsible for its interaction with TR3. When stimulated with 12-O-tetradecanoylphorbol 13-acetate (TPA) or CD437, this TR3-TRAPγ interaction not only induced Ca(2+) depletion in the endoplasmic reticulum (ER) but also promoted the expression of the proapoptotic transcriptional regulator CHOP. Notably, both TR3 and TRAPγ were required for ER stress-induced apoptosis in HepG2 cells. Overall, this study demonstrated a novel, TR3-initiated signaling pathway in which TR3 regulates ER stress and induces apoptosis of hepatoma cells through its interaction with TRAPγ.

Induction of Autophagic Death in Cancer Cells by Agonizing TR3 and Attenuating Akt2 Activity.

Apoptotic resistance is becoming a significant obstacle for cancer therapy as the majority of treatment takes the route of apoptotic induction. It is of great importance to develop an alternative strategy to induce cancer cell death. We previously reported that autophagic cell death mediated by nuclear receptor TR3 and driven by a chemical agonist, 1-(3,4,5-trihydroxyphenyl)nonan-1-one (THPN), is highly effective in the therapy of melanoma but not any other cancer types. Here, we discovered that the insensitivity of cancer cells to THPN originated from a high cellular Akt2 activity. Akt2 phosphorylation interferes with TR3 export to cytoplasm and targeting to mitochondria, which lead to the autophagic induction. Therefore, the TR3-mediated autophagy could be effectively induced in the otherwise insensitive cells by downregulating Akt2 activity. Highly effective antineoplastic compounds are developed through optimizing the structure of THPN. This study implicates a general strategy for cancer therapy by the induction of autophagic cell death.