Phase 2 Trial of Iberdomide in Systemic Lupus Erythematosus.

BACKGROUND: Iberdomide, a cereblon modulator promoting degradation of the transcription factors Ikaros and Aiolos, which affect leukocyte development and autoimmunity, is being evaluated for the treatment of systemic lupus erythematosus (SLE). METHODS: In this phase 2 trial, we randomly assigned patients in a 2:2:1:2 ratio to receive oral iberdomide (at a dose of 0.45, 0.30, or 0.15 mg) or placebo once daily for 24 weeks, in addition to standard medications. The primary end point at week 24 was a response on the SLE Responder Index (SRI-4), which was defined as a reduction of at least 4 points in the Systemic Lupus Erythematosus Disease Activity Index 2000 score (a 24-item weighted score of lupus activity that ranges from 0 to 105, with higher scores indicating greater disease activity), no new disease activity as measured on the British Isles Lupus Assessment Group 2004 index, and no increase of 0.3 points or more in the Physician's Global Assessment score (on a visual-analogue scale ranging from 0 [no disease activity] to 3 [maximal disease]). RESULTS: A total of 288 patients received the assigned intervention: 81 received iberdomide at a dose of 0.45 mg, 82 received iberdomide at a dose of 0.30 mg, 42 received iberdomide at a dose of 0.15 mg, and 83 received placebo. At week 24, the percentages of patients with an SRI-4 response were 54% in the iberdomide 0.45-mg group, 40% in the iberdomide 0.30-mg group, 48% in the iberdomide 0.15-mg group, and 35% in the placebo group (adjusted difference between the iberdomide 0.45-mg group and the placebo group, 19.4 percentage points; 95% confidence interval, 4.1 to 33.4; P = 0.01), with no significant differences between the groups that received the lower doses of iberdomide and the group that received placebo. Iberdomide-associated adverse events included urinary tract and upper respiratory tract infections and neutropenia. CONCLUSIONS: In this 24-week, phase 2 trial involving patients with SLE, iberdomide at a dose of 0.45 mg resulted in a higher percentage of patients with an SRI-4 response than did placebo. Data from larger, longer trials are needed to determine the efficacy and safety of iberdomide in SLE. (Funded by Bristol Myers Squibb; ClinicalTrials.gov number, NCT03161483; EudraCT number, 2016-004574-17.).

Influence of injection technique, drug formulation and tumor microenvironment on intratumoral immunotherapy delivery and efficacy.

BACKGROUND: Intratumoral delivery of immunotherapeutics represents a compelling solution to directly address local barriers to tumor immunity. However, we have previously shown that off-target delivery is a substantial problem during intratumoral injections; this can lead to diminished drug efficacy and systemic toxicities. We have identified three variables that influence intratumoral drug delivery: injection technique, drug formulation and tumor microenvironment. The purpose of this study was to characterize the impact of modifications in each variable on intratumoral drug delivery and immunotherapy efficacy. METHODS: Intratumoral injections were performed in a hybrid image-guided intervention suite with ultrasound, fluoroscopy and CT scanning capabilities in both rat and mouse syngeneic tumor models. Intratumoral drug distribution was quantified by CT volumetric imaging. The influence of varying needle design and hydrogel-based drug delivery on the immune response to a stimulator of interferon genes (STING) agonist was evaluated using flow cytometry and single cell RNA sequencing. We also evaluated the influence of tumor stiffness on drug injection distribution. RESULTS: Variations in needle design, specifically with the use of a multiside hole needle, led to approximately threefold improvements in intratumoral drug deposition relative to conventional end-hole needles. Likewise, delivery of a STING agonist through a multiside hole needle led to significantly increased expression of type I interferon-associated genes and 'inflammatory' dendritic cell gene signatures relative to end-hole STING agonist delivery. A multidomain peptide-based hydrogel embedded with a STING agonist led to substantial improvements in intratumoral deposition; however, the hydrogel was noted to generate a strong immune response against itself within the target tumor. Evaluation of tumor stroma on intratumoral drug delivery revealed that there was a greater than twofold improvement in intratumoral distribution in soft tumors (B16 melanoma) compared with firm tumors (MC38 colorectal). CONCLUSIONS: Injection technique, drug formulation and tumor stiffness play key roles in the accurate delivery of intratumoral immunotherapeutics.

A Role for Protease Activated Receptor Type 3 (PAR3) in Nociception Demonstrated Through Development of a Novel Peptide Agonist.

The protease activated receptor (PAR) family is a group of G-protein coupled receptors (GPCRs) activated by proteolytic cleavage of the extracellular domain. PARs are expressed in a variety of cell types with crucial roles in homeostasis, immune responses, inflammation, and pain. PAR3 is the least researched of the four PARs, with little known about its expression and function. We sought to better understand its potential function in the peripheral sensory nervous system. Mouse single-cell RNA sequencing data demonstrates that PAR3 is widely expressed in dorsal root ganglion (DRG) neurons. Co-expression of PAR3 mRNA with other PARs was identified in various DRG neuron subpopulations, consistent with its proposed role as a coreceptor of other PARs. We developed a lipid tethered PAR3 agonist, C660, that selectively activates PAR3 by eliciting a Ca2+ response in DRG and trigeminal neurons. In vivo, C660 induces mechanical hypersensitivity and facial grimacing in WT but not PAR3-/- mice. We characterized other nociceptive phenotypes in PAR3-/- mice and found a loss of hyperalgesic priming in response to IL-6, carrageenan, and a PAR2 agonist, suggesting that PAR3 contributes to long-lasting nociceptor plasticity in some contexts. To examine the potential role of PAR3 in regulating the activity of other PARs in sensory neurons, we administered PAR1, PAR2, and PAR4 agonists and assessed mechanical and affective pain behaviors in WT and PAR3-/- mice. We observed that the nociceptive effects of PAR1 agonists were potentiated in the absence of PAR3. Our findings suggest a complex role of PAR3 in the physiology and plasticity of nociceptors. PERSPECTIVE: We evaluated the role of PAR3, a G-protein coupled receptor, in nociception by developing a selective peptide agonist. Our findings suggest that PAR3 contributes to nociception in various contexts and plays a role in modulating the activity of other PARs.

Long non-coding RNA SNHG5 suppresses the development of acute respiratory distress syndrome by targeting miR-205/COMMD1 axis.

Previous studies have reported the important roles of long non-coding RNAs (lncRNAs) in acute respiratory distress syndrome (ARDS). Here, we focus on the role and regulatory mechanism of lncRNA SNHG5 in ARDS. LPS was used to induce mice to establish ARDS model in vivo and to induce A549 cells to establish ARDS model in vitro. qRT-PCR was performed to determine the expressions of SNHG5, miR-205, and inflammatory cytokines. MTT assay was applied to detect cell viability. Dual-luciferase reporter (DLR) assay was performed to test the interactions among SNHG5, miR-205 and COMMD1. Western blot was used to detect the protein expression of COMMD1. Lung injury was evaluated by evaluating the score of lung injury, lung wet/dry weight ratio, and myeloperoxidase (MPO) activity. SNHG5 was downregulated, while miR-205 was upregulated in the serum of ARDS patients and lung tissues of LPS-induced mice. Upregulation of SNHG5 or down-regulation of miR-205 inhibited inflammation and promoted the viability of LPS-induced A549 cells. SNHG5 alleviated the lung injury of ARDS mice. MiR-205 was a target of SNHG5 and inversely correlated with SNHG5. COMMD1 was targeted by miR-205, and was positively regulated by SNHG5. MiR-205 mimics or sh-COMMD1 reversed the promoting effect of SNHG5 on cell viability and the suppressing effect of SNHG5 on inflammation in cellular model of ARDS. Meantime, miR-205 mimics reversed the relieving effect of SNHG5 on lung injury in mouse model of ARDS. SNHG5 acted as a sponge for miR-205 to ameliorate LPS-induced ARDS by regulating COMMD1.

Downregulated miR­29a promotes B cell overactivation by upregulating Crk­like protein in systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is an autoimmune disorder; however, the pathogenesis is not fully understood. Accumulating evidence suggested an important role of microRNAs (miRNA/miR) in autoimmunity. The present study aimed therefore to determine the miRNA expression patterns in the B cells from the peripheral blood of 66 patients with SLE and 10 healthy controls (HCs) by using an Affymetrix GeneChip® miRNA 2.0 array. In addition, next­generation sequencing was used to obtain the peripheral blood mononuclear cell (PBMC) miRNA profiles from three patients with SLE and three HCs. Candidate miRNAs that were considered to contribute to the pathogenesis of SLE were obtained based on the intersection of miRNA profiles. The analysis revealed a significant downregulation in miR­29a expression levels in B cells from patients with SLE, which was subsequently verified using reverse transcription­quantitative PCR. Based on these results, the expression pattern of miR­29a in SLE was further investigated and its role in the hyperactivity of B cells was determined. miR­29a inhibitors and mimics were transfected into PBMCs obtained from HCs and patients with SLE, and an ELISA was used to demonstrate that miR­29a inhibition increased the production of IgG. Bioinformatics analysis predicted Crk­like protein (CRKL) as a target gene of miR­29a in patients with SLE. Therefore, CRKL expression levels were compared between patients with SLE and HCs by using western blotting, and its direct transcriptional regulation by miR­29a was determined using a dual­luciferase reporter assay. Low expression levels of miR­29a were revealed to upregulate the expression levels of CRKL in B cells, and the protein expression levels of CRKL in patients with SLE were significantly upregulated compared with the HCs. In conclusion, the results from the present study suggested that miR­29a may affect IgG antibody secretion in B cells by regulating CRKL, thereby contributing to the development and progression of SLE, which offers a novel candidate target for treatment.

Liprin alfa 2 gene expression is increased by cannabis use and associated with neuropsychological function.

The relation of heavy cannabis use with decreased neuropsychological function has frequently been described but the underlying biological mechanisms are still largely unknown. This study investigates the relation of cannabis use with genome wide gene expression and subsequently examines the relations with neuropsychological function. Genome-wide gene expression in whole blood was compared between heavy cannabis users (N = 90) and cannabis naïve participants (N = 100) that were matched for psychotic like experiences. The results were validated using quantitative real-time PCR. Psychotic like experiences were assessed using the Comprehensive Assessment of Psychotic Experiences (CAPE). Neuropsychological function was estimated using four subtasks of the Wechsler Adult Intelligence Scale (WAIS). Subsequent in vitro studies in monocytes and a neuroblastoma cell line investigated expression changes in response to two major psychotropic components of cannabis; tetrahydrocannabinol (THC) and cannabidiol (CBD). mRNA expression of Protein Tyrosine Phosphatase Receptor Type F Polypeptide-Interacting-Protein Alpha-2 (PPFIA2) was significantly higher in cannabis users (LogFold Change 0.17) and confirmed by qPCR analysis. PPFIA2 expression level was negatively correlated with estimated intelligence (B=-22.9, p = 0.002) also in the 100 non-users (B=-28.5, p = 0.037). In vitro exposure of monocytes to CBD led to significant increase in PPFIA2 expression. However, exposure of monocytes to THC and neuroblastoma cells to THC or CBD did not change PPFIA2 expression. Change in PPFIA2 gene expression in response to cannabinoids is a putative mechanism by which cannabis could influence neuropsychological functions. The findings warrant further exploration of the role of PPFIA2 in cannabis induced changes of neuropsychological function, particularly in relation to CBD.

Up-regulation of receptor interaction protein 140 promotes glucolipotoxicity-induced damage in MIN6 cells.

The receptor interaction protein 140 (RIP140) cofactor is a key regulator of metabolic balance, but its function in glucose- and lipid-mediated damage in islet ß cells is unknown and was investigated in this study. RIP140 expression and distribution was evaluated in MIN6 cells under high glucose and lipid conditions using real-time Polymerase Chain Reaction (PCR), western blotting and confocal laser scanning microscopy. Cells were separately treated with 500 µM palmitic acid and 25 mM glucose when RIP140 expression was upregulated or downregulated, and cell viability, apoptosis rate, the level of oxidative stress and insulin secretion was assessed, as was the expression of related genes. Increased glucose and palmitic acid elevated RIP140 expression and distribution in nuclei. Overexpression of RIP140 promoted apoptosis but inhibited cell viability in MIN6 cells, and basal insulin secretion and glucose-stimulated insulin secretion levels were altered following treatment with glucose and palmitic acid. In addition, oxidative stress was elevated, phosphorylated extracellular signal-regulated kinases 1/2 and uncoupling protein 2 messenger RNA (mRNA) abundance were increased, B-cell lymphoma-2 protein levels were decreased, and peroxisome proliferators activated receptor gamma co-activator 1 alpha, phosphoenolpyruvate carboxykinase , and pancreatic and duodenal homeobox-1 mRNA levels were downregulated. Furthermore, glucolipotoxicity-induced damage was reversed when RIP140 expression was downregulated by small interfering RNA (SiRNA). RIP140 promotes islet ß cells damage caused by glucolipotoxicity.

Targeting IRF3 as a YAP agonist therapy against gastric cancer.

The Hippo pathway plays a vital role in tissue homeostasis and tumorigenesis. The transcription factor IRF3 is essential for innate antiviral immunity. In this study, we discovered IRF3 as an agonist of Yes-associated protein (YAP). The expression of IRF3 is positively correlated with that of YAP and its target genes in gastric cancer; the expression of both IRF3 and YAP is up-regulated and prognosticates patient survival. IRF3 interacts with both YAP and TEAD4 in the nucleus to enhance their interaction, promoting nuclear translocation and activation of YAP. IRF3 and YAP-TEAD4 are associated genome-wide to cobind and coregulate many target genes of the Hippo pathway. Overexpression of active IRF3 increased, but depletion of IRF3 reduced, the occupancy of YAP on the target genes. Knockdown or pharmacological targeting of IRF3 by Amlexanox, a drug used clinically for antiinflammatory treatment, inhibits gastric tumor growth in a YAP-dependent manner. Collectively, our study identifies IRF3 as a positive regulator for YAP, highlighting a new therapeutic target against YAP-driven cancers.

Neuropeptide Y promotes adipogenic differentiation in primary cultured human adipose-derived stem cells.

Neuropeptide Y (NPY) is an important neurotransmitter in the control of energy metabolism. Several studies have shown that obesity is associated with increased levels of NPY in the hypothalamus. We hypothesized that the release of NPY has coordinated and integrated effects on energy metabolism in different tissues, such as adipocyte tissue, resulting in increased energy storage and decreased energy expenditure. Whether NPY has role in the molecular mechanism of human adipocyte tissue remains unclear. We established the model of human adipose derived stem cells (hADSCs) from human adipose tissue and differentiated it into adipocytes in the presence of NPY at different concentrations (10-15-10-6 mmol/L). We then assessed hADSCs proliferation and differentiation by quantifying lipid accumulation and examining the expression levels of related adipocyte markers after differentiation. Furthermore, the specific markers of white adipocyte tissue (WAT) in hADSCs were also analyzed. The results showed that low doses of NPY stimulated hADSCs proliferation (p < 0.05), while high doses of NPY inhibited hADSCs proliferation (p < 0.05). NPY significantly promoted lipid accumulation and increased the size of lipid droplets during human adipogenic differentiation; the levels of adipocyte markers PPAR-γ and C/EBPα were also increased. At the same time, NPY also increased the levels of WAT markers Cidec and RIP140 after adipocyte differentiation. The results suggested high dose NPY inhibits the proliferation of hADSCs while promotes adipocyte differentiation and increases the expression of WAT markers. This may be the reason why increased levels of NPY can lead to a rise in body weight.

Upregulation of BAG3 with apoptotic and autophagic activities in maggot extract­promoted rat skin wound healing.

Maggot extract (ME) accelerates rat skin wound healing, however its effect on cell maintenance in wound tissues remains unclear. B­cell lymphoma (Bcl) 2­associated athanogene (BAG)3 inhibits apoptosis and promotes autophagy by associating with Bcl­2 or Beclin 1. Bcl­2, the downstream effector of signal transducer and activator of transcription 3 signaling, is enhanced in ME­treated wound tissues, which may reinforce the Bcl­2 anti­apoptotic activity and/or cooperate with Beclin 1 to regulate autophagy during wound healing. The present study investigated expression levels of BAG3, Bcl­2, Beclin 1 and light chain (LC)3 levels in rat skin wound tissues in the presence and absence of ME treatment. The results revealed frequent TUNEL­negative cell death in the wound tissues in the early three days following injury, irrespective to ME treatment. TUNEL­positive cells appeared in the wound tissues following 4 days of injury and 150 µg/ml ME efficiently reduced apoptotic rate and enhanced BAG3 and Bcl­2 expression. Elevated Beclin 1 and LC3 levels and an increased LC3 II ratio were revealed in the ME­treated tissues during the wound healing. The results of the present study demonstrate the anti­apoptotic effects of BAG3 and Bcl­2 in ME­promoted wound healing. Beclin 1/LC3 mediated autophagy may be favorable in maintaining cell survival in the damaged tissues and ME­upregulated BAG3 may enhance its activity.

The Hippo kinases LATS1 and 2 control human breast cell fate via crosstalk with ERα.

Cell fate perturbations underlie many human diseases, including breast cancer. Unfortunately, the mechanisms by which breast cell fate are regulated are largely unknown. The mammary gland epithelium consists of differentiated luminal epithelial and basal myoepithelial cells, as well as undifferentiated stem cells and more restricted progenitors. Breast cancer originates from this epithelium, but the molecular mechanisms that underlie breast epithelial hierarchy remain ill-defined. Here, we use a high-content confocal image-based short hairpin RNA screen to identify tumour suppressors that regulate breast cell fate in primary human breast epithelial cells. We show that ablation of the large tumour suppressor kinases (LATS) 1 and 2 (refs 5, 6), which are part of the Hippo pathway, promotes the luminal phenotype and increases the number of bipotent and luminal progenitors, the proposed cells-of-origin of most human breast cancers. Mechanistically, we have identified a direct interaction between Hippo and oestrogen receptor-α (ERα) signalling. In the presence of LATS, ERα was targeted for ubiquitination and Ddb1-cullin4-associated-factor 1 (DCAF1)-dependent proteasomal degradation. Absence of LATS stabilized ERα and the Hippo effectors YAP and TAZ (hereafter YAP/TAZ), which together control breast cell fate through intrinsic and paracrine mechanisms. Our findings reveal a non-canonical (that is, YAP/TAZ-independent) effect of LATS in the regulation of human breast cell fate.

Incretin treatment and atherosclerotic plaque stability: Role of adiponectin/APPL1 signaling pathway.

AIMS: Glucagon like peptide 1 (GLP-1) analogues and dipeptidyl peptidase IV (DPP-4) inhibitors reduce atherosclerosis progression in type 2 diabetes mellitus (T2DM) patients and are associated with morphological and compositional characteristics of stable plaque phenotype. GLP-1 promotes the secretion of adiponectin which exerts anti-inflammatory effects through the adaptor protein PH domain and leucine zipper containing 1 (APPL1). The potential role of APPL1 expression in the evolution of atherosclerotic plaque in TDM2 patients has not previously evaluated. METHODS: The effect of incretin therapy in the regulation of adiponectin/APPL1 signaling was evaluated both on carotid plaques of asymptomatic diabetic (n=71) and non-diabetic patients (n=52), and through in vitro experiments on endothelial cell (EC). RESULTS: Atherosclerotic plaques of T2DM patients showed lower adiponectin and APPL1 levels compared with non-diabetic patients, along with higher oxidative stress, tumor necrosis factor-α (TNF-α), vimentin, and matrix metalloproteinase-9 (MMP-9) levels. Among T2DM subjects, current incretin-users presented higher APPL1 and adiponectin content compared with never incretin-users. Similarly, in vitro observations on endothelial cells co-treated with high-glucose (25mM) and GLP-1 (100nM) showed a greater APPL1 protein expression compared with high-glucose treatment alone. CONCLUSIONS: Our findings suggest a potential role of adiponectin/APPL1 signaling in mediating the effect of incretin in the prevention of atherosclerosis progression or plaque vulnerability in T2DM.

A High-Throughput Screening Platform Targeting PDLIM5 for Pulmonary Hypertension.

Pulmonary arterial hypertension is a complex disease with multiple etiologic factors. PDLIM5, a member of the Enigma subfamily of PDZ and LIM domain protein family, contains an N-terminal PDZ domain and three LIM domains at its C-terminus. We have previously shown that overexpression of PDLIM5 prevents hypoxia-induced pulmonary hypertension (PH), and deletion of PDLIM5 in smooth muscle cells enhances hypoxia-induced PH in vivo. These results suggest that PDLIM5 may be a novel therapeutic target of PH. In this study, we aim to establish a high-throughput screening platform for PDLIM5-targeted drug discovery. We generated a stable mink lung epithelial cell line (MLEC) containing a transforming growth factor-ß/Smad luciferase reporter with lentivirus-mediated suppression of PDLIM5 (MLEC-shPDLIM5) and measured levels of Smad2/3 and pSmad2/3. We found that in MLEC, suppression of PDLIM5 decreased Smad-dependent luciferase activity, Smad3, and pSmad3. We used MLEC-shPDLIM5 and a control cell line (MLEC-shCTL) to screen the Prestwick library (1200 compounds) and identified and validated paclitaxel as a PDLIM5 inhibitor in MLEC. Furthermore, we showed that paclitaxel inhibited Smad2 expression and Smad3 phosphorylation in A549 cells. Our study suggests that this system is robust and suitable for PDLIM5-targeted drug discovery.

Small-molecule agonists of mammalian Diaphanous-related (mDia) formins reveal an effective glioblastoma anti-invasion strategy.

The extensive invasive capacity of glioblastoma (GBM) makes it resistant to surgery, radiotherapy, and chemotherapy and thus makes it lethal. In vivo, GBM invasion is mediated by Rho GTPases through unidentified downstream effectors. Mammalian Diaphanous (mDia) family formins are Rho-directed effectors that regulate the F-actin cytoskeleton to support tumor cell motility. Historically, anti-invasion strategies focused upon mDia inhibition, whereas activation remained unexplored. The recent development of small molecules directly inhibiting or activating mDia-driven F-actin assembly that supports motility allows for exploration of their role in GBM. We used the formin inhibitor SMIFH2 and mDia agonists IMM-01/-02 and mDia2-DAD peptides, which disrupt autoinhibition, to examine the roles of mDia inactivation versus activation in GBM cell migration and invasion in vitro and in an ex vivo brain slice invasion model. Inhibiting mDia suppressed directional migration and spheroid invasion while preserving intrinsic random migration. mDia agonism abrogated both random intrinsic and directional migration and halted U87 spheroid invasion in ex vivo brain slices. Thus mDia agonism is a superior GBM anti-invasion strategy. We conclude that formin agonism impedes the most dangerous GBM component-tumor spread into surrounding healthy tissue. Formin activation impairs novel aspects of transformed cells and informs the development of anti-GBM invasion strategies.

The AhR and NF-κB/Rel Proteins Mediate the Inhibitory Effect of 2,3,7,8-Tetrachlorodibenzo-p-Dioxin on the 3' Immunoglobulin Heavy Chain Regulatory Region.

Transcriptional regulation of the murine immunoglobulin (Ig) heavy chain gene (Igh) involves several regulatory elements including the 3'Igh regulatory region (3'IghRR), which is composed of at least 4 enhancers (hs3A, hs1.2, hs3B, and hs4). The hs1.2 and hs4 enhancers exhibit the greatest transcriptional activity and contain binding sites for several transcription factors including nuclear factor kappaB/Rel (NF-κB/Rel) proteins and the aryl hydrocarbon receptor (AhR). Interestingly, the environmental immunosuppressant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), which potently inhibits antibody secretion, also profoundly inhibits 3'IghRR and hs1.2 enhancer activation induced by the B-lymphocyte activator lipopolysaccharide (LPS), but enhances LPS-induced activation of the hs4 enhancer. Within the hs1.2 and hs4 enhancers, the AhR binding site is in close proximity or overlaps an NF-κB/Rel binding site suggesting a potential reciprocal modulation of the 3'IghRR by AhR and NF-κB/Rel. The objective of the current study was to evaluate the role of NF-κB/Rel and the AhR on the 3'IghRR and its enhancers using the AhR ligand TCDD, the AhR antagonist CH223191, and toll-like receptor agonists LPS, Resiquimod (R848), or cytosine-phosphate-guanine-oligodeoxynucleotides (CpG). Utilizing the CH12.LX B-lymphocyte cell line and variants expressing either a 3'IghRR-regulated transgene reporter or an inducible IκBα (inhibitor kappa B-alpha protein) superrepressor (IκBαAA), we demonstrate an AhR- and NF-κB/Rel-dependent modulation of 3'IghRR and hs4 activity. Additionally, in mouse splenocytes or CH12.LX cells, binding within the hs1.2 and hs4 enhancer of the AhR and the NF-κB/Rel proteins RelA and RelB was differentially altered by the cotreatment of LPS and TCDD. These results suggest that the AhR and NF-κB/Rel protein binding profile within the 3'IghRR mediates the inhibitory effects of TCDD on Ig expression and therefore antibody levels.

A cell-based screening for TAZ activators identifies ethacridine, a widely used antiseptic and abortifacient, as a compound that promotes dephosphorylation of TAZ and inhibits adipogenesis in C3H10T1/2 cells.

Transcriptional co-activator with PSD-95/Dlg-A/ZO-1 (PDZ)-binding motif (TAZ) regulates in cell proliferation and differentiation. In mesenchymal stem cells it promotes osteogenesis and myogenesis, and suppresses adipogenesis. TAZ activators are expected to prevent osteoporosis, obesity and muscle atrophy. TAZ activation induces epithelial-mesenchymal transition, confers stemness to cancer cells and leads to poor clinical prognosis in cancer patients. In this point of view, TAZ inhibitors should contribute to cancer therapy. Thus, TAZ attracts attention as a two-faced drug target. We screened for TAZ modulators by using human lung cancer A549 cells expressing the fluorescent reporter. Through this assay, we obtained TAZ activator candidates. We unexpectedly found that ethacridine, a widely used antiseptic and abortifacient, enhances the interaction of TAZ and protein phosphatases and increases unphosphorylated and nuclear TAZ. Ethacridine inhibits adipogenesis in mesenchymal C3H10T1/2 cells through the activation of TAZ. This finding suggests that ethacridine is a bona fide TAZ activator and supports that our assay is useful to discover TAZ activators.

Differential in vivo genotoxicity of arsenic trioxide in glutathione depleted mouse bone marrow cells: expressions of Nrf2/Keap1/P62.

Generation of reactive oxygen species is one of the major contributors in arsenic-induced genotoxicity where reduced glutathione (GSH) could be an important determining factor. To understand the role of endogenous GSH, arsenic trioxide (As2O3) was administered in buthionine sulfoximine (BSO)- and N-acetyl-L-cysteine (NAC)-treated mice. As2O3-induced significant chromosome aberrations (CAs) in all treatment groups compared with the control. BSO-treated mouse bone marrow cells showed significant CAs at a dose of 2 mg As2O3 kg(-1) b.w. Similar induction was not evident at 4 mg As2O3 kg(-1) b.w. and exhibited antagonistic effect at 8 mg As2O3 kg(-1) b.w. To understand this differential effect, expression pattern of Nrf2 was observed. Nrf2 expression increased following As2O3 treatment in a dose-dependent manner up to 4 mg As2O3 kg(-1) b.w after which no further increase was noticed. NAC pre-treatment significantly reduced the extent of As2O3-induced CAs suggesting the protective role of endogenous GSH against arsenic-induced genotoxicity.

Changes in vitamin D target gene expression in adipose tissue monitor the vitamin D response of human individuals.

SCOPE: Vitamin D3, its biologically most active metabolite 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3), and the vitamin D receptor (VDR) are important for adipose tissue biology. METHODS AND RESULTS: We extrapolated genomic VDR association loci in adipocytes from 55 conserved genome-wide VDR-binding sites in nonfat tissues. Taking the genes DUSP10, TRAK1, NRIP1, and THBD as examples, we confirmed the predicted VDR binding sites upstream of their transcription start sites and showed rapid mRNA up-regulation of all four genes in SGBS human pre-adipocytes. Using adipose tissue biopsy samples from 47 participants of a 5-month vitamin D3 intervention study, we demonstrated that all four primary VDR target genes can serve as biomarkers for the vitamin D3 responsiveness of human individuals. Changes in DUSP10 gene expression appear to be the most comprehensive marker, while THBD mRNA changes characterized a rather different group of study participants. CONCLUSION: We present a new approach to predict vitamin D target genes based on conserved genomic VDR-binding sites. Using human adipocytes as examples, we show that such ubiquitous VDR target genes can be used as markers for the individual's response to a supplementation with vitamin D3.

Synthesis and biological evaluation of biotin conjugates of (±)-(4bS,8aR,10aS)-10a-ethynyl-4b,8,8-trimethyl-3,7-dioxo-3,4b,7,8,8a,9,10,10a-octahydro-phenanthrene-2,6-dicarbonitrile, an activator of the Keap1/Nrf2/ARE pathway, for the isolation of its protein targets.

The tricycle 1 ((±)-(4bS,8aR,10aS))-10a-ethynyl-4b,8,8-trimethyl-3,7-dioxo-3,4b,7,8,8a,9,10,10a-octahydrophenanthrene-2,6-dicarbonitrile), a potent activator of the Keap1/Nrf2/ARE pathway, has the potential to be a first in class drug for the treatment of diabetic nephropathy. To identify the protein targets for the development of 1, the (1:1)-diasteromeric mixture of biotinylated tricycles 3a and 3b were designed and synthesized. For the synthesis of 3a and 3b, a new important precursor, hydroxylated tricycle (±)-16 was synthesized from 4 by a C1 α-methyl group oxidation protocol, which involves cyclopalladation of the C1 α-methyl group from a C2-oxime. For the induction of the phase 2 cytoprotective enzyme NQO1 in Hepa1c1c7 murine hepatoma cells, the diasteromeric mixture 3a and 3b shows high potency (CD, 75 nM) although this potency is lower than that of 1 and 16. Thus, biotinylated tricycles 3a and 3b may be promising tools for the isolation of the protein targets of 1.