LRH-1 mitigates intestinal inflammatory disease by maintaining epithelial homeostasis and cell survival.

Epithelial dysfunction and crypt destruction are defining features of inflammatory bowel disease (IBD). However, current IBD therapies targeting epithelial dysfunction are lacking. The nuclear receptor LRH-1 (NR5A2) is expressed in intestinal epithelium and thought to contribute to epithelial renewal. Here we show that LRH-1 maintains intestinal epithelial health and protects against inflammatory damage. Knocking out LRH-1 in murine intestinal organoids reduces Notch signaling, increases crypt cell death, distorts the cellular composition of the epithelium, and weakens the epithelial barrier. Human LRH-1 (hLRH-1) rescues epithelial integrity and when overexpressed, mitigates inflammatory damage in murine and human intestinal organoids, including those derived from IBD patients. Finally, hLRH-1 greatly reduces disease severity in T-cell-mediated murine colitis. Together with the failure of a ligand-incompetent hLRH-1 mutant to protect against TNFα-damage, these findings provide compelling evidence that hLRH-1 mediates epithelial homeostasis and is an attractive target for intestinal disease.

Disulfide-Trapping Identifies a New, Effective Chemical Probe for Activating the Nuclear Receptor Human LRH-1 (NR5A2).

Conventional efforts relying on high-throughput physical and virtual screening of large compound libraries have failed to yield high-efficiency chemical probes for many of the 48 human nuclear receptors. Here, we investigated whether disulfide-trapping, an approach new to nuclear receptors, would provide effective lead compounds targeting human liver receptor homolog 1 (hLRH-1, NR5A2). Despite the fact that hLRH-1 contains a large ligand binding pocket and binds phospholipids with high affinity, existing synthetic hLRH-1 ligands are of limited utility due to poor solubility, low efficacy or significant off-target effects. Using disulfide-trapping, we identified a lead compound that conjugates with remarkably high-efficiency to a native cysteine residue (Cys346) lining the hydrophobic cavity in the ligand binding domain of hLRH-1. Guided by computational modeling and cellular assays, the lead compound was elaborated into ligands PME8 and PME9 that bind hLRH-1 reversibly (no cysteine reactivity) and increase hLRH-1 activity in cells. When compared with the existing hLRH-1 synthetic agonist RJW100, both PME8 and PME9 showed comparable induction of the LRH-1 dependent target gene CYP24A1 in human HepG2 cells, beginning as early as 3 h after drug treatment. The induction is specific as siRNA-mediated knock-down of hLRH-1 renders both PME8 and PME9 ineffective. These data show that PME8 and PME9 are potent activators of hLRH-1 and suggest that with further development this lead series may yield useful chemical probes for manipulating LRH-1 activity in vivo.

A gene-expression screen identifies a non-toxic sumoylation inhibitor that mimics SUMO-less human LRH-1 in liver.

SUMO-modification of nuclear proteins has profound effects on gene expression. However, non-toxic chemical tools that modulate sumoylation in cells are lacking. Here, to identify small molecule sumoylation inhibitors we developed a cell-based screen that focused on the well-sumoylated substrate, human Liver Receptor Homolog-1 (hLRH-1, NR5A2). Our primary gene-expression screen assayed two SUMO-sensitive transcripts, APOC3 and MUC1, that are upregulated by SUMO-less hLRH-1 or by siUBC9 knockdown, respectively. A polyphenol, tannic acid (TA) emerged as a potent sumoylation inhibitor in vitro (IC50 = 12.8 µM) and in cells. TA also increased hLRH-1 occupancy on SUMO-sensitive transcripts. Most significantly, when tested in humanized mouse primary hepatocytes, TA inhibits hLRH-1 sumoylation and induces SUMO-sensitive genes, thereby recapitulating the effects of expressing SUMO-less hLRH-1 in mouse liver. Our findings underscore the benefits of phenotypic screening for targeting post-translational modifications, and illustrate the potential utility of TA for probing the cellular consequences of sumoylation.

Direct modification and activation of a nuclear receptor-PIP2 complex by the inositol lipid kinase IPMK.

Phosphatidylinositol 4,5-bisphosphate (PIP2) is best known as a plasma membrane-bound regulatory lipid. Although PIP2 and phosphoinositide-modifying enzymes coexist in the nucleus, their nuclear roles remain unclear. We showed that inositol polyphosphate multikinase (IPMK), which functions both as an inositol kinase and as a phosphoinositide 3-kinase (PI3K), interacts with the nuclear receptor steroidogenic factor 1 (SF-1) and phosphorylates its bound ligand, PIP2. In vitro studies showed that PIP2 was not phosphorylated by IPMK if PIP2 was displaced or blocked from binding to the large hydrophobic pocket of SF-1 and that the ability to phosphorylate PIP2 bound to SF-1 was specific to IPMK and did not occur with type 1 p110 PI3Ks. IPMK-generated SF-1-PIP3 (phosphatidylinositol 3,4,5-trisphosphate) was dephosphorylated by the lipid phosphatase PTEN. Consistent with the in vitro activities of IPMK and PTEN on SF-1-PIP(n), SF-1 transcriptional activity was reduced by silencing IPMK or overexpressing PTEN. This ability of lipid kinases and phosphatases to directly remodel and alter the activity of a non-membrane protein-lipid complex establishes a previously unappreciated pathway for promoting lipid-mediated signaling in the nucleus.

Eliminating SF-1 (NR5A1) sumoylation in vivo results in ectopic hedgehog signaling and disruption of endocrine development.

Sumoylation is generally considered a repressive mark for many transcription factors. However, the in vivo importance of sumoylation for any given substrate remains unclear and is questionable because the extent of sumoylation appears exceedingly low for most substrates. Here, we permanently eliminated SF-1/NR5A1 sumoylation in mice (Sf-1(K119R, K194R, or 2KR)) and found that Sf-1(2KR/2KR) mice failed to phenocopy a simple gain of SF-1 function or show elevated levels of well-established SF-1 target genes. Instead, mutant mice exhibited marked endocrine abnormalities and changes in cell fate that reflected an inappropriate activation of hedgehog signaling and other potential SUMO-sensitive targets. Furthermore, unsumoylatable SF-1 mutants activated Shh and exhibited preferential recruitment to Shh genomic elements in cells. We conclude that the sumoylation cycle greatly expands the functional capacity of transcription factors such as SF-1 and is leveraged during development to achieve cell-type-specific gene expression in multicellular organisms.

Demasculinization and feminization of male gonads by atrazine: consistent effects across vertebrate classes.

Atrazine is the most commonly detected pesticide contaminant of ground water, surface water, and precipitation. Atrazine is also an endocrine disruptor that, among other effects, alters male reproductive tissues when animals are exposed during development. Here, we apply the nine so-called "Hill criteria" (Strength, Consistency, Specificity, Temporality, Biological Gradient, Plausibility, Coherence, Experiment, and Analogy) for establishing cause-effect relationships to examine the evidence for atrazine as an endocrine disruptor that demasculinizes and feminizes the gonads of male vertebrates. We present experimental evidence that the effects of atrazine on male development are consistent across all vertebrate classes examined and we present a state of the art summary of the mechanisms by which atrazine acts as an endocrine disruptor to produce these effects. Atrazine demasculinizes male gonads producing testicular lesions associated with reduced germ cell numbers in teleost fish, amphibians, reptiles, and mammals, and induces partial and/or complete feminization in fish, amphibians, and reptiles. These effects are strong (statistically significant), consistent across vertebrate classes, and specific. Reductions in androgen levels and the induction of estrogen synthesis - demonstrated in fish, amphibians, reptiles, and mammals - represent plausible and coherent mechanisms that explain these effects. Biological gradients are observed in several of the cited studies, although threshold doses and patterns vary among species. Given that the effects on the male gonads described in all of these experimental studies occurred only after atrazine exposure, temporality is also met here. Thus the case for atrazine as an endocrine disruptor that demasculinizes and feminizes male vertebrates meets all nine of the "Hill criteria".

Stimulating the GPR30 estrogen receptor with a novel tamoxifen analogue activates SF-1 and promotes endometrial cell proliferation.

Estrogens and selective estrogen receptor (ER) modulators such as tamoxifen are known to increase uterine cell proliferation. Mounting evidence suggests that estrogen signaling is mediated not only by ERalpha and ERbeta nuclear receptors, but also by GPR30 (GPER), a seven transmembrane (7TM) receptor. Here, we report that primary human endometriotic H-38 cells express high levels of GPR30 with no detectable ERalpha or ERbeta. Using a novel tamoxifen analogue, STX, which activates GPR30 but not ERs, significant stimulation of the phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) pathways was observed in H-38 cells and in Ishikawa endometrial cancer cells expressing GPR30; a similar effect was observed in JEG3 choriocarcinoma cells. STX treatment also increased cellular pools of phosphatidylinositol (3,4,5) triphosphate, a proposed ligand for the nuclear hormone receptor SF-1 (NR5A1). Consistent with these findings, STX, tamoxifen, and the phytoestrogen genistein were able to increase SF-1 transcription, promote Ishikawa cell proliferation, and induce the SF-1 target gene aromatase in a GPR30-dependent manner. Our findings suggest a novel signaling paradigm that is initiated by estrogen activation of the 7TM receptor GPR30, with signal transduction cascades (PI3K and MAPK) converging on nuclear hormone receptors (SF-1/LRH-1) to modulate their transcriptional output. We propose that this novel GPR30/SF-1 pathway increases local concentrations of estrogen, and together with classic ER signaling, mediate the proliferative effects of synthetic estrogens such as tamoxifen, in promoting endometriosis and endometrial cancers.

The herbicide atrazine activates endocrine gene networks via non-steroidal NR5A nuclear receptors in fish and mammalian cells.

Atrazine (ATR) remains a widely used broadleaf herbicide in the United States despite the fact that this s-chlorotriazine has been linked to reproductive abnormalities in fish and amphibians. Here, using zebrafish we report that environmentally relevant ATR concentrations elevated zcyp19a1 expression encoding aromatase (2.2 microg/L), and increased the ratio of female to male fish (22 microg/L). ATR selectively increased zcyp19a1, a known gene target of the nuclear receptor SF-1 (NR5A1), whereas zcyp19a2, which is estrogen responsive, remained unchanged. Remarkably, in mammalian cells ATR functions in a cell-specific manner to upregulate SF-1 targets and other genes critical for steroid synthesis and reproduction, including Cyp19A1, StAR, Cyp11A1, hCG, FSTL3, LHss, INHalpha, alphaGSU, and 11ss-HSD2. Our data appear to eliminate the possibility that ATR directly affects SF-1 DNA- or ligand-binding. Instead, we suggest that the stimulatory effects of ATR on the NR5A receptor subfamily (SF-1, LRH-1, and zff1d) are likely mediated by receptor phosphorylation, amplification of cAMP and PI3K signaling, and possibly an increase in the cAMP-responsive cellular kinase SGK-1, which is known to be upregulated in infertile women. Taken together, we propose that this pervasive and persistent environmental chemical alters hormone networks via convergence of NR5A activity and cAMP signaling, to potentially disrupt normal endocrine development and function in lower and higher vertebrates.

A histone lysine methyltransferase activated by non-canonical Wnt signalling suppresses PPAR-gamma transactivation.

Histone modifications induced by activated signalling cascades are crucial to cell-lineage decisions. Osteoblast and adipocyte differentiation from common mesenchymal stem cells is under transcriptional control by numerous factors. Although PPAR-gamma (peroxisome proliferator activated receptor-gamma) has been established as a prime inducer of adipogenesis, cellular signalling factors that determine cell lineage in bone marrow remain generally unknown. Here, we show that the non-canonical Wnt pathway through CaMKII-TAK1-TAB2-NLK transcriptionally represses PPAR-gamma transactivation and induces Runx2 expression, promoting osteoblastogenesis in preference to adipogenesis in bone marrow mesenchymal progenitors. Wnt-5a activates NLK (Nemo-like kinase), which in turn phosphorylates a histone methyltransferase, SETDB1 (SET domain bifurcated 1), leading to the formation of a co-repressor complex that inactivates PPAR-gamma function through histone H3-K9 methylation. These findings suggest that the non-canonical Wnt signalling pathway suppresses PPAR-gamma function through chromatin inactivation triggered by recruitment of a repressing histone methyltransferase, thus leading to an osteoblastic cell lineage from mesenchymal stem cells.

Suppression of PPAR transactivation switches cell fate of bone marrow stem cells from adipocytes into osteoblasts.

Osteoblasts and adipocytes differentiate from common pleiotropic mesenchymal stem cells under transcriptional controls by numerous factors and multiple intracellular signalings. However, cellular signaling factors that determine cell fates of mensenchymal stem cells in bone marrow remain to be largely uncovered, though peroxisome proliferator-activated receptor-gamma (PPAR-gamma) is well established as a prime inducer of adipogenesis. Here, we describe two signaling pathways that induce the cell fate decision into osteoblasts from adipocytes. One signaling is a TAK1/TAB1/NIK cascade activated by TNF-alpha and IL-1, and the activated NF-kappaB blocked the DNA binding of PPAR-gamma, attenuating the activated PPAR-mediated adipogenesis. The second signaling is the noncanonical Wnt pathway through CaMKII-TAK1/TAB2-NLK. Activated NLK by a noncanonical Wnt ligand (Wnt-5a) transrepresses PPAR transactivation through a histone methyltransferase, SETDB1. Wnt-5a induces phosphorylation of NLK, leading to the formation of a corepressor complex that inactivates PPAR function through histone H3-K9 methylation. Thus, two signaling pathways lead to an osteoblastic cell lineage decision from mesenchymal stem cells through two distinct modes of PPAR transrepression.

Structural analyses reveal phosphatidyl inositols as ligands for the NR5 orphan receptors SF-1 and LRH-1.

Vertebrate members of the nuclear receptor NR5A subfamily, which includes steroidogenic factor 1 (SF-1) and liver receptor homolog 1 (LRH-1), regulate crucial aspects of development, endocrine homeostasis, and metabolism. Mouse LRH-1 is believed to be a ligand-independent transcription factor with a large and empty hydrophobic pocket. Here we present structural and biochemical data for three other NR5A members-mouse and human SF-1 and human LRH-1-which reveal that these receptors bind phosphatidyl inositol second messengers and that ligand binding is required for maximal activity. Evolutionary analysis of structure-function relationships across the SF-1/LRH-1 subfamily indicates that ligand binding is the ancestral state of NR5A receptors and was uniquely diminished or altered in the rodent LRH-1 lineage. We propose that phospholipids regulate gene expression by directly binding to NR5A nuclear receptors.

Nuclear receptors as targets for drug development: crosstalk between peroxisome proliferator-activated receptor gamma and cytokines in bone marrow-derived mesenchymal stem cells.

Peroxisome proliferator-activated receptor gamma (PPARgamma) is a ligand-dependent nuclear receptor and regulates adipogenesis and fat metabolism. PPARgamma is activated by fatty acid derivatives and some synthetic compounds such as the thiazolidinediones. In addition, certain cytokines were known to affect the transactivation function of PPARgamma. However, the molecular mechanism of the functional interaction between PPARgamma and cytokine signaling remains unclear. We found that combined treatment of PPARgamma and cytokines (IL-1 or TNF-alpha) inhibited adipogenesis and induced osteoblastgenesis in bone marrow-derived mesenchymal stem cells. Furthermore, we showed that the ligand dependent transactivation function of PPARgamma was suppressed by IL-1 and TNF-alpha. This suppression was mediated through NF-kappaB activated by the TAK1/TAB1-NIK cascade, a downstream cascade triggered with IL-1 or TNF-alpha signaling. Thus, we have identified a molecular mechanism of functional cross-talk between PPARgamma and cytokine signaling that may provide a theoretical basis for development of novel therapeutical strategies and design of novel compounds for treatment of obesity, diabetes, and some other chronic diseases.

Receptor tyrosine kinases inhibit bone morphogenetic protein-Smad responsive promoter activity and differentiation of murine MC3T3-E1 osteoblast-like cells.

Growth factors such as fibroblast growth factor-2 (FGF-2) and epidermal growth factor (EGF) that activate extracellular signal-regulated kinases (ERKs) through receptor tyrosine kinases (RTKs) stimulate proliferation but suppress differentiation of osteoblasts. To study the mechanism of this inhibitory action of these growth factors on osteoblastic differentiation, we evaluated Smad1 transactivity in MC3T3-E1 osteoblast-like cells by reporters of promoter activity of mouse Smad6, an early response gene to bone morphogenetic proteins (BMPs). FGF-2 and EGF inhibited alkaline phosphatase activity and Smad6 promoter activity stimulated by BMP-2. Overexpression of constitutively active MEK by adenovirus mimicked, but that of dominant negative Ras or treatment with a MEK1 inhibitor, PD098059, reversed, the inhibitory effects of these growth factors on both activities. These effects are mediated by BMP-responsive elements (BMPREs) on Smad6 promoter, because an artificial reporter driven by three tandem BMPREs gave similar results, and these effects were all abolished when the BMPREs were mutated. RTK-ERK activation inhibited the promoter activity even when BMP signal was mediated by a mutant Smad1, which lacks phosphorylation sites by ERKs, or by a Smad1 fused to Gal4 DNA binding domain, which constitutively localizes in the nucleus. These results show that the RTK-Ras-ERK pathway suppresses BMP signal by interfering with Smad1 transactivity. Because direct phosphorylation of Smad1 by ERKs is not required for the inhibition, other transcriptional factors that are phosphorylated by ERKs might be involved in the regulation of osteoblastic differentiation by ERKs.

Cytokines suppress adipogenesis and PPAR-gamma function through the TAK1/TAB1/NIK cascade.

Pluripotent mesenchymal stem cells in bone marrow differentiate into adipocytes, osteoblasts and other cells. Balanced cytodifferentiation of stem cells is essential for the formation and maintenance of bone marrow; however, the mechanisms that control this balance remain largely unknown. Whereas cytokines such as interleukin-1 (IL-1) and tumour-necrosis factor-alpha (TNF-alpha) inhibit adipogenesis, the ligand-induced transcription factor peroxisome proliferator-activated receptor-gamma (PPAR-gamma), is a key inducer of adipogenesis. Therefore, regulatory coupling between cytokine- and PPAR-gamma-mediated signals might occur during adipogenesis. Here we show that the ligand-induced transactivation function of PPAR-gamma is suppressed by IL-1 and TNF-alpha, and that this suppression is mediated through NF-kappaB activated by the TAK1/TAB1/NF-kappaB-inducing kinase (NIK) cascade, a downstream cascade associated with IL-1 and TNF-alpha signalling. Unlike suppression of the PPAR-gamma transactivation function by mitogen-activated protein kinase-induced growth factor signalling through phosphorylation of the A/B domain, NF-kappaB blocks PPAR-gamma binding to DNA by forming a complex with PPAR-gamma and its AF-1-specific co-activator PGC-2. Our results suggest that expression of IL-1 and TNF-alpha in bone marrow may alter the fate of pluripotent mesenchymal stem cells, directing cellular differentiation towards osteoblasts rather than adipocytes by suppressing PPAR-gamma function through NF-kappaB activated by the TAK1/TAB1/NIK cascade.