Constitutive Receptor Activity Research Articles

Mouse Hepatomas with Ha-ras and B-raf Mutations Differ in Mitogen-Activated Protein Kinase Signaling and Response to Constitutive Androstane Receptor Activation.

Nuclear receptors mediate the hepatic induction of drug-metabolizing enzymes by xenobiotics. Not much is known about enzyme induction in liver tumors. Here, we treated tumor-bearing mice with phenobarbital, an activator of the constitutive androstane receptor (CAR), to analyze the response of chemically induced Ha-ras- and B-raf-mutated mouse liver adenoma to CAR activation in vivo. Both tumor subpopulations possess almost identical gene expression profiles. CAR target gene induction in the tumors was studied at the mRNA and protein levels, and a reverse-phase protein microarray approach was chosen to characterize important signaling cascades. CAR target gene induction was pronounced in B-raf-mutated but not in Ha-ras-mutated tumors. Phosphoproteomic profiling revealed that phosphorylation-activated extracellular signal-regulated kinase (ERK) 1/2 was more abundant in Ha-ras-mutated than in B-raf-mutated tumors. ERK activation in tumor tissue was negatively correlated with CAR target induction. ERK activation is known to inhibit CAR-dependent transcription. In summary, profound differences exist between the two closely related tumor subpopulations with respect to the activation of mitogenic signaling cascades, and these dissimilarities might explain the differences in xenobiotic induction of CAR target genes.

PAC1 Receptors: Shapeshifters in Motion.

Shapeshifters, in common mythology, are entities that can undergo multiple physical transformations. As our understanding of G protein-coupled receptors (GPCRs) has accelerated and been refined over the last two decades, we now understand that GPCRs are not static proteins, but rather dynamic structures capable of moving from one posture to the next, and adopting unique functional characteristics at each transition. This model of GPCR dynamics underlies our current understanding of biased agonism-how different ligands to the same receptor can generate different intracellular signals-and constitutive receptor activity, or the level of unbound basal receptor signaling that can be attenuated by inverse agonists. From information derived from related class B receptors, we have recently modeled the structure and molecular dynamics of the full-length pituitary adenylate cyclase activating polypeptide (PACAP, Adcyap1)-selective PAC1 receptor (PAC1R, Adcyap1r1). The class B receptors are different from the class A GPCRs in part from the presence of a large extracellular domain (ECD); the transitions of the ECD along with the dynamics of the transmembrane domains (TMD or 7TM) of the PAC1R describes a series of open- and closed-state conformations that appear to identify the mechanisms for receptor activation. The PAC1R shapeshifts also have the ability of delineating the mechanisms and the design of reagents that may direct biased agonism (or antagonism) for potential therapeutics.

Structural Alterations in Human Fibroblast Growth Factor Receptors in Carcinogenesis.

Fibroblast growth factor (FGF) plays an important role in human embryogenesis, angiogenesis, cell proliferation, and differentiation. Carcinogenesis is accompanied by aberrant constitutive activation of FGF receptors (FGFRs) resulting from missense mutation in the FGFR1-4 genes, generation of chimeric oncogenes, FGFR1-4 gene amplification, alternative splicing shift toward formation of mesenchymal FGFR isoforms, and FGFR overexpression. Altogether, these alterations contribute to auto- and paracrine stimulation of cancer cells and neoangiogenesis. Certain missense mutations are found at a high rate in urinary bladder cancer and can be used for non-invasive cancer recurrence diagnostics by analyzing urine cell pellet DNA. Chimeric FGFR1/3 and amplified FGFR1/2 genes can predict cell response to the targeted therapy in various oncological diseases. In recent years, high-throughput sequencing has been used to analyze exomes of virtually all human tumors, which allowed to construct phylogenetic trees of clonal cancer evolution with special emphasis on driver mutations in FGFR1-4 genes. At present, FGFR blockers, such as multi-kinase inhibitors, specific FGFR inhibitors, and FGF ligand traps are being tested in clinical trials. In this review, we discuss current data on the functioning of the FGFR family proteins in both normal and cancer cells, mutations in the FGFR1-4 genes, and mechanisms underlying their oncogenic potential, which might be interesting to a broad range of scientists searching for specific tumor markers and targeted anti-cancer drugs.

Abstract 946: Suppression of breast cancer metastasis and extension of host animal survival by a new adamantyl antiestrogen, K-07, in a preclinical breast cancer metastasis model driven by constitutively active mutant estrogen receptors

Abstract Over one-third of human breast tumors that become resistant to endocrine therapies have acquired mutations in the estrogen receptor (ER alpha) that result in constitutive receptor activity in the absence of estrogen, and these tumors are much less sensitive to suppression by current standard-of-care antiestrogens such as tamoxifen and fulvestrant. We reported recently that the novel adamantyl antiestrogen, K-07, was effective in inhibiting the proliferation of breast tumor xenografts containing the two most frequent constitutively active mutant forms of the ER, Y537S ER and D538G ER, and that this antiestrogen reduced ER levels and the expression of ER-regulated genes (Zhao et al., Cancer Research 77(20):5602-5613, 2017). Because mutant ERs are usually enriched in recurrent, metastatic breast cancers compared to the primary breast cancer, we have now examined the effectiveness of compound K-07 in preventing the growth of breast cancer metastases and in extending survival in a metastatic tumor model. MCF-7 breast cancer cells expressing luciferase and Y537S ER or D538G ER (ca. 50% mutant ER and 50% wild type ER) were injected i.v. by the tail vein into NOD-SCID-gamma (NSG) female mice. The constitutively active ER-containing breast cancer cells established metastases in liver, bone and brain that increased in number and size over time (day zero – 80 days) as monitored by IVIS imaging and immunohistochemical (IHC) analysis. Daily oral treatment with K-07 (80 mg/kg orally for 30 days and then 40 mg/kg) versus oral vehicle greatly reduced the metastasis of mutant ER-containing breast cancer cells. Notably, mice with mutant ER-containing metastases treated with K-07 survived much longer than mice given daily control vehicle. In fact, by day 60, only 25% of vehicle treated mice with mutant ER breast cancers were alive whereas all K-07 treated D538G mice and 80% of Y537S mice were still alive. Hence, the findings indicate that this antiestrogen can reduce the in vivo metastasis of breast cancers driven by constitutively active mutant ERs and extend host survival. The findings suggest that the antiestrogen K-07 may be suitable for further translational and clinical examination of its efficacy in suppression of metastasis in patients with breast cancers containing constitutively active mutant ERs. Citation Format: Mary J. Laws, Sung Hoon Kim, Jian Min, Yuechao Zhao, Yvonne Ziegler, David Chu, Ben H. Park, John A. Katzenellenbogen, Benita S. Katzenellenbogen. Suppression of breast cancer metastasis and extension of host animal survival by a new adamantyl antiestrogen, K-07, in a preclinical breast cancer metastasis model driven by constitutively active mutant estrogen receptors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 946.

Abstract 5493: Targeted inhibition of glutaminase in GRM1-expressing melanoma cells inhibits cell proliferation by reducing glutamate bioavailability

Abstract Aberrant glutamatergic signaling has been implicated in many cancer types. Our laboratory has previously illustrated the role of metabotropic glutamate receptor 1 (GRM1) in neoplastic transformation of melanocytes in vitro and spontaneous metastatic melanoma in vivo. Glutamate, the natural ligand of GRM1 is also the predominant excitatory neurotransmitter in the central nervous system. We have demonstrated significant upregulation of glutaminase (GLS) expression in GRM1 expressing melanoma cells, resulting in excess glutamate production and the establishment of autocrine loops in vitro. GLS catalyzes the first step in glutamine metabolism–the conversion of glutamine to glutamate. Comparison of glutamate levels in circulating blood plasma between melanoma prone and wild type mice showed elevated glutamate levels in melanoma prone GRM1-transgenic mice, suggesting that aberrant GRM1 expression also promotes an increase in circulating glutamate, to ensure constitutive activation of the GRM1 receptor and its downstream signaling affecters in vivo. Inclusion of CB-839, a potent, selective, and orally bioavailable inhibitor of GLS, resulted in significant inhibition of GRM1+ melanoma cell proliferation compared to GRM1- ones. We also demonstrated that treatment with Riluzole, an inhibitor of glutamatergic signaling by decreasing the available ligand, glutamate, led to significantly reduced melanoma cell proliferation in vitro and tumor progression in vivo. We found that simultaneous targeting of glutamate production (CB-839) and release (Riluzole) in GRM1+ melanoma cells led to synergistic suppression of cell proliferation in vitro. Furthermore, we found that CB-839 altered the function not the expression of GLS in melanoma cells. Additionally, we also discovered that manipulation of GRM1 expression by genetic means led to robust parallel changes in gene expression levels and intracellular glutamate concentrations by mass spectrometry analysis, as well as the endogenous levels of GLS in melanoma cells, suggesting association between GRM1 and GLS. The sensitivity of GRM1+ melanoma cells to modulation of GLS point to the dependency of these cells to glutamate amounts via GLS activity. We are continuing to investigate the mechanisms and regulation of GLS in our experimental system, with the goal of developing a rational approach for the treatment of GRM1 expressing tumors. Citation Format: Raj Shah, Mengying Zhu, Andrew Boreland, Fabian Filipp, Suzie Chen. Targeted inhibition of glutaminase in GRM1-expressing melanoma cells inhibits cell proliferation by reducing glutamate bioavailability [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5493.

Combined deletion of the fibronectin-type III domains and the stalk region results in ligand-independent, constitutive activation of the Interleukin 6 signal-transducing receptor gp130

Gp130 is the common receptor within the Interleukin 6 cytokine family. Gp130 consists of 6 extracellular domains followed by a small stalk region connecting the last extracellular domain with the trans-membrane domain. Whereas the first three extracellular domains bind to IL-6-type cytokines, the domains 4–6 are needed for correct positioning of the intracellular domains to facilitate Janus kinase activation after cytokine binding. Interestingly, deletion within the cytokine-binding domain resulted in cytokine-independent constitutive activation of mutant gp130 receptors. Here, we tested the hypothesis, if deletions of the stalk region and/or domains 4–6 of gp130 might also result in constitutive receptor activation. Shortening of the stalk region of gp130 alone did, however, not result in constitutive receptor activation, whereas a gp130 receptor deletion variant only consisting of the three N-terminal cytokine binding domains but lacking all FNIII domains was biologically inactive. Importantly, combined deletion of the three FNIII domains plus shortening of the stalk region of gp130 resulted in ligand-independent, constitutive receptor activation of gp130.

Betulinic Acid Exerts Cytotoxic Activity Against Multidrug-Resistant Tumor Cells via Targeting Autocrine Motility Factor Receptor (AMFR).

Betulinic acid (BetA) is a naturally occurring pentacyclic triterpene isolated from the outer bark of white-barked birch trees and many other medicinal plants. Here, we studied betulinic acid's cytotoxic activity against drug-resistant tumor cell lines. P-glycoprotein (MDR1/ABCB1) and BCRP (ABCG2) are known ATP-binding cassette (ABC) drug transporters that mediating MDR. ABCB5 is a close relative to ABCB1, which also mediates MDR. Constitutive activation of the EGF receptor is tightly linked to the development of chemotherapeutic resistance. BetA inhibited P-gp, BCRP, ABCB5 and mutation activated EGFR overexpressing cells with similar efficacy as their drug-sensitive parental counterparts. Furthermore, the mRNA expressions of ABCB1, BCRP, ABCB5 and EGFR were not related to the 50% inhibition concentrations (IC50) for BetA in a panel of 60 cell lines of the National Cancer Institute (NCI), USA. In addition to well-established MDR mechanisms, we attempted to identify other molecular mechanisms that play a role in mediating BetA's cytotoxic activity. For this reason, we performed COMPARE and hierarchical cluster analyses of the transcriptome-wide microarray-based mRNA expression of the NCI cell lines panel. Various genes significantly correlating to BetA's activity were involved in different biological processes, e.g., cell cycle regulation, microtubule formation, signal transduction, transcriptional regulation, chromatin remodeling, cell adhesion, tumor suppression, ubiquitination and proteasome degradation. Immunoblotting and in silico analyses revealed that the inhibition of AMFR activity might be one of the mechanisms for BetA to overcome MDR phenotypes. In conclusion, BetA may have therapeutic potential for the treatment of refractory tumors.

Mini G protein probes for active G protein–coupled receptors (GPCRs) in live cells

G protein–coupled receptors (GPCRs) are key signaling proteins that regulate nearly every aspect of cell function. Studies of GPCRs have benefited greatly from the development of molecular tools to monitor receptor activation and downstream signaling. Here, we show that mini G proteins are robust probes that can be used in a variety of assay formats to report GPCR activity in living cells. Mini G (mG) proteins are engineered GTPase domains of Gα subunits that were developed for structural studies of active-state GPCRs. Confocal imaging revealed that mG proteins fused to fluorescent proteins were located diffusely in the cytoplasm and translocated to sites of receptor activation at the cell surface and at intracellular organelles. Bioluminescence resonance energy transfer (BRET) assays with mG proteins fused to either a fluorescent protein or luciferase reported agonist, superagonist, and inverse agonist activities. Variants of mG proteins (mGs, mGsi, mGsq, and mG12) corresponding to the four families of Gα subunits displayed appropriate coupling to their cognate GPCRs, allowing quantitative profiling of subtype-specific coupling to individual receptors. BRET between luciferase–mG fusion proteins and fluorescent markers indicated the presence of active GPCRs at the plasma membrane, Golgi apparatus, and endosomes. Complementation assays with fragments of NanoLuc luciferase fused to GPCRs and mG proteins reported constitutive receptor activity and agonist-induced activation with up to 20-fold increases in luminescence. We conclude that mG proteins are versatile tools for studying GPCR activation and coupling specificity in cells and should be useful for discovering and characterizing G protein subtype–biased ligands.

A method for the quantification of biased signalling at constitutively active receptors.

Biased agonism, the ability of an agonist to differentially activate one of several signal transduction pathways when acting at a given receptor, is an increasingly recognized phenomenon at many receptors. The Black and Leff operational model lacks a way to describe constitutive receptor activity and hence inverse agonism. Thus, it is impossible to analyse the biased signalling of inverse agonists using this model. In this theoretical work, we develop and illustrate methods for the analysis of biased inverse agonism. Methods were derived for quantifying biased signalling in systems that demonstrate constitutive activity using the modified operational model proposed by Slack and Hall. The methods were illustrated using Monte Carlo simulations. The Monte Carlo simulations demonstrated that, with an appropriate experimental design, the model parameters are 'identifiable'. The method is consistent with methods based on the measurement of intrinsic relative activity (RAi ) (ΔΔlogR or ΔΔlog(τ/Ka )) proposed by Ehlert and Kenakin and their co-workers but has some advantages. In particular, it allows the quantification of ligand bias independently of 'system bias' removing the requirement to normalize to a standard ligand. In systems with constitutive activity, the Slack and Hall model provides methods for quantifying the absolute bias of agonists and inverse agonists. This provides an alternative to methods based on RAi and is complementary to the ΔΔlog(τ/Ka ) method of Kenakin et al. in systems where use of that method is inappropriate due to the presence of constitutive activity.

Synaptic adhesion protein ELFN1 is a selective allosteric modulator of group III metabotropic glutamate receptors in trans.

Functional characterization of the GPCR interactome has been focused predominantly on intracellular interactions, yet GPCRs are increasingly found in complex with extracellular proteins. Extracellular leucine-rich repeat fibronectin type III domain containing 1 (ELFN1) was recently reported to physically anchor mGluR6 and mGluR7 across retinal and hippocampal synapses, respectively; however, the consequence of transsynaptic interactions on properties and pharmacology of these receptors are unknown. In the current study, we explore the effects of ELFN1 on mGluR signaling and pharmacology. First, we established the binding specificity of ELFN1 and found it to be recruited selectively to all group III mGluRs (mGluR4, mGluR6, mGluR7, and mGluR8), but not other mGluR species. Using site-directed mutagenesis we mapped binding determinants of this interaction to two distinct sites on the ELFN1 ectodomain. To evaluate functional aspects of the interaction, we developed a transcellular signaling assay in reconstituted HEK293 cells which monitors changes in mGluR activity in one cell following its exposure to separate ELFN1-containing cells. Using this platform, we found that ELFN1 acts as an allosteric modulator of class III mGluR activity in suppressing cAMP accumulation: altering both agonist-induced and constitutive receptor activity. Using bioluminescence resonance energy transfer-based real-time kinetic assays, we established that ELFN1 alters the ability of mGluRs to activate G proteins. Our findings demonstrate that core properties of class III mGluRs can be altered via extracellular interactions with ELFN1 which serves as a transsynaptic allosteric modulator for these receptors. Furthermore, our unique assay platform opens avenues for exploring transcellular/transsynaptic pharmacology of other GPCR transcomplexes.

Minimum datasets to establish a CAR-mediated mode of action for rodent liver tumors

Methods for investigating the Mode of Action (MoA) for rodent liver tumors via constitutive androstane receptor (CAR) activation are outlined here, based on current scientific knowledge about CAR and feedback from regulatory agencies globally. The key events (i.e., CAR activation, altered gene expression, cell proliferation, altered foci and increased adenomas/carcinomas) can be demonstrated by measuring a combination of key events and associative events that are markers for the key events. For crop protection products, a primary dataset typically should include a short-term study in the species/strain that showed the tumor response at dose levels that bracket the tumorigenic and non-tumorigenic dose levels. The dataset may vary depending on the species and the test compound. As examples, Case Studies with nitrapyrin (in mice) and metofluthrin (in rats) are described. Based on qualitative differences between the species, the key events leading to tumors in mice or rats by this MoA are not operative in humans. In the future, newer approaches such as a CAR biomarker signature approach and/or in vitro CAR3 reporter assays for mouse, rat and human CAR may eventually be used to demonstrate a CAR MoA is operative, without the need for extensive additional studies in laboratory animals.

An operational model for GPCR homodimers and its application in the analysis of biased signaling

G-protein-coupled receptors are one of the most important protein superfamilies as drug targets in drug discovery programs. Their interactions with ligands are influenced by their homomerization. In this study, we propose an operational model for receptor homodimers, which includes constitutive receptor activity. Distinct functional response curves can be obtained from this model, which can satisfactorily depict typical complex experimental data as biphasic and bell-shaped curves. Operational parameters in the model can provide mechanistic explanations for observed functional complexity associated with the cooperativity and intrinsic efficacy of ligands. Because the model presented here is derived from the conceptual framework of operational models, it takes advantage of the body of knowledge coming from the widespread use of this type of modeling. The operational homodimer model can also explain the biased signaling dependent on ligand concentration. In conclusion, this operational homodimer model has a wide range of applications in pharmacological research.

Long non-coding RNA metallothionein 1 pseudogene 3 promotes p2y12 expression by sponging miR-126 to activate platelet in diabetic animal model

Platelet hyperaggregation and hypercoagulation are associated with increase of thrombogenic risk, especially in patients with type 2 diabetes (T2D). High activity of P2Y12 receptor is found in T2D patients, exposing such patients to a prothrombotic condition. P2Y12 is a promising target for antiplatelet, but due to P2Y12 receptor constitutive activation, the clinical practical phenomena such as “clopidogrel resistance” are commonly occurring. In this study, we investigate the role of lncRNA on platelet activation. By lncRNA array, we screened thousands of differentially expressed lncRNA in megakaryocytes from T2D patients and confirmed that lncRNA metallothionein 1 pseudogene 3 (MT1P3) was significantly upregulated in megakaryocytes from T2D patients than in healthy controls. And we further investigate the biofunction of MT1P3 on platelet activation and the regulatory mechanism on p2y12. MT1P3 was positively correlated with p2y12 mRNA levels and promoted p2y12 expression by sponging miR-126. Knockdown of MT1P3 by siRNA reduced p2y12 expression, inhibiting platelet activation and aggregation in diabetes animal model. In conclusion, our findings identify MT1P3 as a key regulator in platelet activation by increasing p2y12 expression through sponging miR-126 under T2D condition. These findings may provide a new insight for managing platelet hyperactivity-related diseases.

Constitutive activity of the metabotropic glutamate receptor 2 explored with a whole-cell label-free biosensor

Label-free cellular assays using a biosensor provide new opportunities for studying G protein-coupled receptor (GPCR) signaling. As opposed to conventional in vitro assays, integrated receptor-mediated cellular responses are determined in real-time rather than a single downstream signaling pathway. In this study, we examined the potential of a label-free whole cell impedance-based biosensor system (i.e. xCELLigence) to study the pharmacology of one GPCR in particular, the mGlu2 receptor. This receptor is a target for the treatment of several psychiatric diseases such as schizophrenia and depression.After optimization of assay conditions to prevent interference of endogenous glutamate in the culture medium, detailed pharmacological assessments were performed. Concentration-response curves showed a concentration-dependent increase in impedance for agonists and positive allosteric modulators, whereas receptor inhibition by an antagonist or negative allosteric modulator resulted in a concentration-dependent decrease in cellular impedance. Interestingly, constitutive receptor activity was observed that was decreased by LY341495, which therefore behaved as an inverse agonist here, a property that was heretofore unappreciated. This was confirmed by concentration-dependent modulation of LY341495 potency and efficacy by a allosteric modulators.In summary, the use of the xCELLigence system to study mGlu2 receptor pharmacology was validated. This is the first class C GPCR to be characterized extensively by such method, opening new avenues to study receptor pharmacology including inverse agonism and demonstrating its value for future drug discovery efforts of mGlu receptors as well as other GPCRs.

Balancing tonic and phasic inhibition in hypothalamic corticotropin-releasing hormone neurons.

GABA transporter (GAT) blockade recruits extrasynaptic GABAA receptors (GABAA Rs) and amplifies constitutive presynaptic GABAB R activity. Extrasynaptic GABAA Rs contribute to a tonic current. Corticosteroids increase the tonic current mediated by extrasynaptic GABAA Rs. Corticotropin-releasing hormone (CRH) neurons in the paraventricular nucleus of the hypothalamus (PVN) are integratory hubs that regulate the endocrine response to stress. GABA inputs provide a basal inhibitory tone that constrains this system and circulating glucocorticoids (CORT) are important feedback controllers of CRH output. Surprisingly little is known about the direct effects of CORT on GABA synapses in PVN. Here we used whole-cell patch clamp recordings from CRH neurons in mouse hypothalamic brain slices to examine the effects of CORT on synaptic and extrasynaptic GABA signalling. We show that GABA transporters (GATs) limit constitutive activation of presynaptic GABAB receptors and ensure high release probability at GABA synapses. GATs in combination with GABAB receptors also curtail extrasynaptic GABAA R signalling. CORT has no effect on synaptic GABA signalling, but increases extrasynaptic GABA tone through upregulation of postsynaptic GABAA receptors. These data show that efficient GABA clearance and autoinhibition control the balance between synaptic (phasic) and extrasynaptic (tonic) inhibition in PVN CRH neurons. This balance is shifted towards increased extrasynaptic inhibition by CORT.

Case examples of an evaluation of the human relevance of the pyrethroids/pyrethrins-induced liver tumours in rodents based on the mode of action.

Rodent carcinogenicity studies are useful for screening for human carcinogens but they are not perfect. Some modes of action (MOAs) lead to cancers in both experimental rodents and humans, but others that lead to cancers in rodents do not do so in humans. Therefore, analysing the MOAs by which chemicals produce tumours in rodents and determining the relevance of such tumour data for human risk are critical. Recently, experimental data were obtained as case examples of an evaluation of the human relevance of pyrethroid (metofluthrin and momfluorothrin)- and pyrethrins-induced liver tumours in rats based on MOA. The MOA analysis, based on the International Programme on Chemical Safety (IPCS) framework, concluded that experimental data strongly support that the postulated MOA for metofluthrin-, momfluorothrin- and pyrethrins-produced rat hepatocellular tumours is mediated by constitutive androstane receptor (CAR) activation. Since metofluthrin and momfluorothrin are close structural analogues, reproducible outcomes for both chemicals provide confidence in the MOA findings. Furthermore, cultured human hepatocyte studies and humanized chimeric mouse liver studies demonstrated species difference between human hepatocytes (refractory to the mitogenic effects of these compounds) and rat hepatocytes (sensitive to their mitogenic effects). These data strongly support the hypothesis that the CAR-mediated MOA for liver tumorigenesis is of low carcinogenic risk for humans. In this research, in addition to cultured human hepatocyte studies, the usefulness of the humanized chimeric liver mouse models was clearly demonstrated. These data substantially influenced decisions in regulatory toxicology. In this review I comprehensively discuss the human relevance of the CAR-mediated MOA for rodent liver tumorigenesis based on published information, including our recent molecular research on CAR-mediated MOA.

MMP14 is a novel target of PTH signaling in osteocytes that controls resorption by regulating soluble RANKL production.

Parathyroid hormone (PTH) affects the skeleton by acting on osteocytes (Ots) in bone through yet unclear mechanisms. We report that matrix metalloproteinase 14 (MMP14) expression/activity are increased in bones from mice with genetic constitutive activation (ca) of the PTH receptor 1 (PTH1R) in Ots (caPTH1ROt) and in bones from mice exposed to elevated PTH levels but not in mice lacking [conditional knockout (cKO)] the PTH1R in Ots (cKOPTH1ROt). Furthermore, PTH upregulates MMP14 in human bone cultures and in Ot-enriched bones from floxed control mice but not from cKOPTH1ROt mice. MMP14 activity increases soluble receptor activator of NF-κΒ ligand production, which in turn, stimulates osteoclast differentiation and resorption. Pharmacologic inhibition of MMP14 activity reduced the high bone remodeling exhibited by caPTH1ROt mice or induced by chronic PTH elevation and decreased bone resorption but allowed full stimulation of bone formation induced by PTH injections, thereby potentiating bone gain. Thus, MMP14 is a new member of the intricate gene network activated in Ots by PTH1R signaling that can be targeted to adjust the skeletal responses to PTH in favor of bone preservation.-Delgado-Calle, J., Hancock, B., Likine, E. F., Sato, A. Y., McAndrews, K., Sanudo, C., Bruzzaniti, A., Riancho, J. A., Tonra, J. R., Bellido, T. MMP14 is a novel target of PTH signaling in osteocytes that controls resorption by regulating soluble RANKL production.

P125 TLR4 ACTIVATION UPREGULATES EXPRESSION OF OXIDATIVE ENZYMES: IMPLICATIONS FOR INTESTINAL TUMORIGENESIS AND LUMINAL MICROBIOTA

Overexpression of Dual oxidase 2 (Duox2) occurs as an early event in both human inflammatory bowel disease and mouse models of colitis, and has been associated with dysbiosis. We have previously shown that mice bearing constitutive activation of toll-like receptor 4 (TLR4) in intestinal epithelial cells (IECs) (villin-TLR4) have altered microbial populations, increased cytokine expression, and increased Nox1 and Duox2 expression. Villin-TLR4 mice develop spontaneous duodenal tumors. Here, we test whether TLR4-mediated induction of Nox1 and Duox2 is dependent on luminal microbiota. villin-TLR4 mice were treated or not with a combination of antibiotics for 4 weeks to deplete their microflora. IECs from duodenum and colon segments were isolated, as well as spontaneous tumors occurring in duodenum. Duodenum and colon organoids from wild type (WT) and TLR4-/- mice were stimulated with lipopolysaccharide (LPS) and/or TNF and IFN-γ. Nox1 and Duox2 expression was evaluated in all samples by quantitative PCR. Antibiotic treatment caused a marked depletion of bacterial populations in stool. Depletion of microflora decreased expression of Nox1 and Duox2 in villin-TLR4 duodenum but not in tumors or colon tissue. Even after antibiotic treatment, villin-TLR4 mice continued to have significantly elevated Nox-1 and Duox-2 expression in the duodenum and colon when compared to their untreated littermates. To further investigate whether TLR4 signaling is responsible for induction of Nox-1 and Duox-2, WT or TLR4-/- duodenum and colon organoids were treated with ultrapure LPS. This resulted in significant upregulation of Nox-1 and Duox-2 in WT but not TLR4-/- organoids. No increased expression was seen with TNF+IFN-g. TLR4 drives expression of oxidative enzymes in the intestine. LPS and luminal microbiota increase expression of Nox-1/Duox-2. We speculate that these oxidative pathways participate in TLR4-dependent neoplasia of the intestine.

Tyrosine kinase inhibitor sensitive PDGFRΑ mutations in GIST: Two cases and review of the literature.

Gastrointestinal stromal tumors (GISTs) are rare mesenchymal malignancies of the gastrointestinal tract. Most GISTs harbor a c-KIT (80%) or a PDGFRα (10%) mutation that leads to constitutive activation of the tyrosine kinase receptor. Response to treatment with tyrosine kinase inhibitors (TKIs) is dependent on mutational status of the tumor. The most common mutation in PDGFRα, D842V, is known to be imatinib resistant. Almost all other PDGFRα mutations are imatinib sensitive. We describe two patients with a PDGFRα exon 18 mutated GIST responding to treatment with TKIs. One of these patients has a p.M844_S847 deletion, not previously described in relation with TKI treatment response. Mutations in circulating tumor DNA were detectable with digital droplet PCR in serial plasma samples taken during treatment and correlated with treatment response of both patients. Computer 3D-modeling of the PDGFRα kinase domain of these two variants revealed no direct interference in imatinib or sunitinib binding and no effect in its activity in contrast to the reported structure of the imatinib resistant D842V mutation.An overview is given of the literature regarding the evidence of patients with different PDGFRα mutated GISTs on response to TKIs. The findings emphasize the use of mutational analysis in GIST to provide patients personalized treatment. Detection of mutations in plasma is feasible and can provide real-time information concerning treatment response. We suggest to register GIST patients with these uncommon mutations in a prospective international database to understand the tumor biology and obtain more evidence of such mutations to predict treatment response.

Modulation of pregnane X receptor (PXR) and constitutive androstane receptor (CAR) activation by ursolic acid (UA) attenuates rifampin-isoniazid cytotoxicity

BackgroundInteractions between transcriptional inducers of cytochrome P450 (CYP450) enzymes and therapeutic drugs may be prevented by antagonizing the activation of a nuclear receptor (NR), pregnane X receptor (PXR, NR1I2), thus improving therapeutic efficacy. PurposeIn the present study, we aim to identify that ursolic acid (UA), a widely distributed pentacyclic triterpene, may act as an effective antagonist of PXR and its sister NR receptor, constitutive androstane receptor (CAR, NR1I3). MethodsThe hepatocellular carcinoma cell line, HepG2, was used to evaluate the promoter activity of PXR and CAR target genes, CYP3A4 and CYP2B6, respectively. Catalytic activities, mRNA, and protein expression of CYP3A4 and CYP2B6 were evaluated in a differentiated HepaRG cell line. Coregulation of PXR with coregulators on CYP3A4 promoter response elements was also been characterized. ResultsTransient transfection assays showed that UA effectively attenuated CYP3A4 and CYP2B6 promoter activities mediated by rifampin (RIF, human PXR agonist) and CITCO (human CAR agonist). These inhibitory effects were well correlated with the expression and catalytic activities of CYP3A4 and CYP2B6. Furthermore, the interaction of co-regulators with PXR and the transcriptional complexes in the CYP3A4 promoter activity and CYP3A4 promoter xenobiotic response element (everted repeat 6, ER6), respectively, were disrupted in the presence of UA. UA showed an antagonistic effect against PXR, and reversed the cytotoxic effects of isoniazid (INH) induced by RIF. Taken together, these results show that UA inhibits the transactivation effects of PXR and CAR, and reduces the expression and function of CYP3A4 and CYP2B6. ConclusionThe present study suggests that UA could be a powerful agent for reducing potentially dangerous interactions between transcriptional inducers of CYP enzymes and therapeutic drugs.