Induction Of Luciferase Activity Research Articles

YIV-818-A: a novel therapeutic agent in prostate cancer management through androgen receptor downregulation, glucocorticoid receptor inhibition, epigenetic regulation, and enhancement of apalutamide, darolutamide, and enzalutamide efficacy.

Introduction: Prostate cancer is the second leading cause of cancer death among men in the United States. Castration-Resistant Prostate Cancer (CRPC) often develops resistance to androgen deprivation therapy. Resistance in CRPC is often driven by AR variants and glucocorticoid receptor (GR). Thus, drugs that target both could be vital in overcoming resistance. Methods: Utilizing the STAR Drug Discovery Platform, three hundred medicinal plant extracts were examined across 25 signaling pathways to identify potential drug candidates. Effects of the botanical drug YIV-818-A, derived from optimized water extracts of Rubia cordifolia (R.C.), on Dihydrotestosterone (DHT) or Dexamethasone (DEX) induced luciferase activity were assessed in 22RV1 cells harboring the ARE luciferase reporter. Furthermore, the key active compounds in YIV-818-A were identified through activity guided purification. The inhibitory effects of YIV-818-A, RA-V, and RA-VII on AR and GR activities, their impact on AR target genes, and their roles in modifying epigenetic status were investigated. Finally, the synergistic effects of these compounds with established CRPC drugs were evaluated both in vitro and in vivo. Results: YIV-818-A was found to effectively inhibit DHT or DEX induced luciferase activity in 22RV1 cells. Deoxybouvardin (RA-V) was identified as the key active compound responsible for inhibiting AR and GR activities. Both YIV-818-A and RA-V, along with RA-VII, effectively downregulated AR and AR-V proteins through inhibiting protein synthesis, impacted the expression of AR target genes, and modified the epigenetic status by reducing levels of Bromodomain and Extra-Terminal proteins (Brd2/Brd4) and H3K27Ac. Furthermore, these compounds exhibited synergistic effects with apalutamide, darolutamide, or enzalutamide, and suppressed AR mediated luciferase activity of 22RV1 cells. Co-administration of YIV-818-A and enzalutamide led to a significant reduction of 22RV1 tumor growth in vivo. Different sources of R.C. had variable levels of RA-V, correlating with their potency in AR inhibition. Discussion: YIV-818-A, RA-V, and RA-VII show considerable promise in addressing drug resistance in CRPC by targeting both AR protein and GR function, along with modulation of vital epigenetic markers. Given the established safety profile of YIV-818-A, these findings suggest its potential as a chemopreventive agent and a robust anti-prostate cancer drug.

The Multi-CDK Inhibitor Dinaciclib Shows Synergistic Activity with the BET Inhibitor PLX51107 and Reverses BET Resistance through Inhibition of Canonical Wnt Signaling in Acute Myeloid Leukemia (AML)

PLX51107 is a novel BET inhibitor (BETi) with antineoplastic activity in pre-clinical models of both aggressive B-cell malignancies and AML. Differentiating it from other BET inhibitors currently under investigation, PLX51107 has a unique binding site in the acetylated lysine binding pocket of BRD4. Through interaction with pTEFb, which is comprised of CDK9 and several cyclin T1 heterodimers, BRD4 promotes phosphorylation of RNA polymerase II, ultimately influencing gene transcription. Dinaciclib is a potent inhibitor of multiple cyclin dependent kinases including CDK9 and has been shown to have pre-clinical activity in AML. Therefore, we hypothesized that PLX51107 would have synergistic activity when combined with dinaciclib. We found synergistic effects on proliferation in primary AML cells when PLX51107 was combined with dinaciclib in stromal co-cultures across a range of dose combinations below the IC50 of either compound alone. Synergistic activity was also present in cytokine support culture conditions, colony forming assays, and synergy assays utilizing COMbenefit software. Interestingly, the highly conserved canonical β-catenin dependent Wnt signaling pathway is a known BETi resistance mechanism in AML. Therefore, destruction of β-catenin and inhibition of Wnt signaling could represent a novel therapeutic target to circumvent development of BET-mediated resistance. We found dinaciclib treatment reduced protein levels of total β-catenin in primary AML cells and AML cell lines (Figure 1A). Phosphorylated β-catenin and non-phosphorylated "active" β-catenin protein levels were also reduced following dinaciclib treatment (Figure 1A). In addition, mRNA expression of multiple components of the Wnt pathway, including the Wnt co-receptor LRP6, AXIN2, FZD1, and many Wnt target genes were significantly decreased following dinaciclib and combination treatment. Moreover, in a luciferase based Wnt reporter cell line, we found dinaciclib significantly reduced Wnt3a induced luciferase activity, suggesting a direct effect on β-catenin mediated transcription of Wnt target genes. Collectively, this data suggests dinaciclib has multiple inhibitory effects on canonical β-catenin dependent Wnt signaling. We found combination treatment of PLX51107 and dinaciclib produced synergistic reduction in expression of both Wnt signaling components and Wnt target genes. Conversely, β-catenin and Wnt target gene expression levels were either not significantly changed or even increased when treated with PLX51107 alone. In order to examine the effects of dinaciclib in the setting of BET resistance, we developed PLX51107 resistant MOLM-13 and MV4-11 AML cell lines via serial drugging. These resistant cell lines remained sensitive to dinaciclib treatment and to cytotoxic chemotherapy drugs including cytarabine and etoposide while demonstrating resistance to PLX51107 via Annexin PI and MTS assays. We found sensitivity to dinaciclib treatment in these resistant cell lines remained unaffected by BETi resistance and continued to demonstrate inhibition of Wnt signaling, similar to the effects seen in parental cells. Therefore, dinaciclib represents a novel strategy to overcome potential therapy emergent BETi resistance. We next examined the efficacy of PLX51107 in combination with dinaciclib in vivo, utilizing luciferase labeled MOLM-13 AML cells xenotransplanted into NOD / SCID / IL2rgnull (NSG) immunodeficient mice. Daily oral dosing with 20 mg/kg PLX51107 resulted in prolonged survival (median 42 days) compared to vehicle treated control animals (median 28 days, p< 0.001). Weekly intraperitoneal 10 mg/kg dinaciclib did not significantly improve survival (median 30 days, p=0.09) in MOLM-13 xenograft mice. However, mice receiving combination therapy survived a median of 56.5 days, significantly longer than with either drug alone (p<0.05) (Figure 1B). We also found decreased disease burden by bioluminescence and by histopathology in combination treated animals, compared to both vehicle and single agent controls. In conclusion, we have demonstrated in vitro and in vivo synergistic activity of the BET inhibitor PLX51107 in combination with dinaciclib in pre-clinical models of AML and have identified novel inhibition of Wnt / β-catenin signaling by dinaciclib, thereby providing a strategy to circumvent resistance to BET inhibitors in AML. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Signaling Pathway of Taurine-Induced Upregulation of TXNIP

Taurine, a sulfur-containing β-amino acid, is present at high concentrations in mammalian tissues and plays an important role in several essential biological processes. However, the genetic mechanisms involved in these physiological processes associated with taurine remain unclear. In this study, we investigated the regulatory mechanism underlying the taurine-induced transcriptional enhancement of the thioredoxin-interacting protein (TXNIP). The results showed that taurine significantly increased the luciferase activity of the human TXNIP promoter. Further, deletion analysis of the TXNIP promoter showed that taurine induced luciferase activity only in the TXNIP promoter region (+200 to +218). Furthermore, by employing a bioinformatic analysis using the TRANSFAC database, we focused on Tst-1 and Ets-1 as candidates involved in taurine-induced transcription and found that the mutation in the Ets-1 sequence did not enhance transcriptional activity by taurine. Additionally, chromatin immunoprecipitation assays indicated that the binding of Ets-1 to the TXNIP promoter region was enhanced by taurine. Taurine also increased the levels of phosphorylated Ets-1, indicating activation of Ets-1 pathway by taurine. Moreover, an ERK cascade inhibitor significantly suppressed the taurine-induced increase in TXNIP mRNA levels and transcriptional enhancement of TXNIP. These results suggest that taurine enhances TXNIP expression by activating transcription factor Ets-1 via the ERK cascade.

Transcriptional regulation of human odontogenic ameloblast-associated protein gene by tumor necrosis factor-α.

Odontogenic ameloblast-associated protein (ODAM) is produced by maturation stage ameloblasts and junctional epithelium (JE). The function of ODAM is thought to be involved in the attachment of teeth and JE. To elucidate transcriptional regulation of human ODAM gene in inflamed gingiva, we have analyzed the effects of TNF-α on the expression of ODAM gene in Ca9-22 and Sa3 gingival epithelial cells. Total RNAs were extracted from Ca9-22 and Sa3 cells after stimulation by TNF-α (10ng/ml). ODAM mRNA and protein levels were analyzed by qPCR and Western blotting. Luciferase (LUC) analyses were performed using LUC constructs inserted in various lengths of ODAM gene promoter. Gel shift and chromatin immunoprecipitation (ChIP) assays were carried out. TNF-α increased ODAM mRNA and protein levels at 3 to 24h. TNF-α induced LUC activities of the ODAM gene promoter constructs, and the activities were inhibited by protein kinase A, tyrosine kinase, MEK1/2, PI3-kinase and NF-κB inhibitors. Gel shift and ChIP assays revealed that TNF-α increased CCAAT/enhancer-binding protein (C/EBP) β and Yin Yang1 (YY1) binding to three kinds of C/EBPs and YY1 elements. These results demonstrate that TNF-α stimulates ODAM gene transcription via C/EBPs and YY1 elements in the human ODAM gene promoter.

Merkel cell polyomavirus T-antigens regulate DICER1 mRNA stability and translation through HSC70

SummaryMerkel cell carcinoma is an aggressive skin malignancy, mostly caused by Merkel cell polyomavirus (MCPyV). MCPyV T-antigens can induce mature microRNA expressions through the DnaJ domain, but its underlying mechanism is still unknown. Here, we report that the T-antigens induce protein expression and mRNA stability of DICER1, a key factor in microRNA biogenesis, through heat shock cognate 70 (HSC70). HSC70 directly interacts with the AU-rich elements (ARE) of DICER1 mRNA in both coding and 3′ untranslated region in the presence of MCPyV T-antigen. The T-antigen/HSC70 interaction could induce luciferase activity of synthetic ARE-containing reporter, as well as the stability of ARE-containing mRNAs, suggesting a broader role of MCPyV T-antigens in regulating multiple mRNAs via HSC70. These findings highlight a new role for the interaction of HSC70 and MCPyV T-antigens in mRNA regulation and an undescribed regulatory mechanism of DICER1 mRNA stability and translation through its direct interaction with HSC70.

Development of a Sensitive Detection Method for Alphaviruses and Its Use as a Virus Neutralization Assay.

Alphaviruses have a single-stranded, positive-sense RNA genome that contains two open reading frames encoding either the non-structural or the structural genes. Upon infection, the genomic RNA is translated into the non-structural proteins (nsPs). NsPs are required for viral RNA replication and transcription driven from the subgenomic promoter (sgP). Transfection of an RNA encoding the luciferase gene under the control of the sgP into cells enabled the detection of replication-competent chikungunya virus (CHIKV) or Mayaro virus (MAYV) with high sensitivity as a function of the induced luciferase activity. This assay principle was additionally used to analyze virus-neutralizing antibodies in sera and might be an alternative to standard virus neutralization assays based on virus titration or the use of genetically modified tagged viruses.

Lactoferrin Is a Potential Activator of the Vitamin D Receptor in Its Regulation of Osteogenic Activities in C57BL/6J Mice and MC3T3-E1 Cells.

Lactoferrin (LF) has been shown to promote bone anabolism, and the vitamin D receptor (VDR) mediates the effects of vitamin D on bone. We hypothesized that LF improves bone health by increasing VDR expression. We sought to determine the role of VDR activation in LF-induced osteogenic activity in vivo and in vitro and the underlying molecular mechanisms. Sixty male C57BL/6J mice (aged 4 wk) were randomly assigned into 6 groups and fed vitamin D-deficient (VDD; 0 IU/kg) or vitamin D-normal diet (VDN; 1000 IU cholecalciferol/kg) and administered placebo or LF (100 or 1000 mg/kg body weight) by gavage for 24 wk. Trabecular bone structure was analyzed using micro-CT, and VDR expression was assessed by immunohistochemistry. In vitro, MC3T3-E1 cells were treated with 100 μg LF/mL to evaluate its effect on VDR expression. Finally, the direct recruitment of LF to the Vdr promoter was confirmed by chromatin immunoprecipitation assay. In addition, cells were transfected with pGL3-basic Vdr vector for monitoring Vdr promoter activation using luciferase assays. LF supplementation at 100 and 1000 mg/kg revealed an ∼6.5% (P<0.05) increase in bone mineral density in mice on VDD diet and exhibited an enhanced expression of VDR in bone compared with control. This increased expression of VDR was also observed in the bone of mice on the VDN diet, but the effect was more pronounced in VDD diet. In vitro, compared with the control group, Vdr mRNA expression was 18 times greater (P<0.05) and peaked at 2 h posttreatment of LF. By cotransfection of the pGL3-basic Vdr vector, LF induced luciferase activity by 30% (P<0.05) in MC3T3-E1 cells. In vivo and in vitro, LF, a potential activator of VDR, promotes osteogenesis. This suggests that dairy products, which are rich in LF, may serve as a functional food to improve bone health.

A high-throughput cell-based gaussia luciferase reporter assay for measurement of CYP1A1, CYP2B6, and CYP3A4 induction

The induction of cytochrome P450s can result in reduced drug efficacy and lead to potential drug–drug interactions. The xenoreceptors—aryl hydrocarbon receptor (AhR), constitutive androstane receptor (CAR), and pregnane X receptor (PXR)—play key roles in CYP induction by xenobiotics. In order to be able to rapidly screen for the induction of three enzymes (CYP1A1, CYP2B6, and CYP3A4), we generated a stable AhR-responsive HepG2 cell line, a stable CAR-responsive HepG2 cell line, and a stable PXR-responsive HepG2 cell line. To validate these stable xenoreceptor-responsive HepG2 cell lines, we evaluated the induction of the different Gaussia reporter activities, as well as the mRNA and protein expression levels of endogenous CYPs in response to different inducers. The induction of luciferase activity in the stable xenoreceptor-responsive HepG2 cell lines by specific inducers occurred in a concentration dependent manner. There was a positive correlation between the induction of luciferase activities and the induction endogenous CYP mRNA expression levels. These xenoreceptor-responsive HepG2 cell lines were further validated with known CYP1A1, CYP2B6, and CYP3A4 inducers. These stable xenoreceptor-responsive HepG2 cell lines may be used in preclinical research for the rapid and sensitive detection of AhR, CAR, and PXR ligands that induce CYP450 isoforms.

An in vitro investigation of endocrine disrupting potentials of ten bisphenol analogues

The endocrine disruption potency of BPA was reported elsewhere, but the mechanisms of its analogues have not been fully resolved. In this study, endocrine disruption potentials of nine alternative bisphenol analogues, namely 2,2-bis(4-hydroxyphenyl)butane (BPB), 2,2-Bis(4-hydroxy-3-methylphenyl)propane (BPC), 4,4′-dihydroxydiphenylmethane (BPF), 4,4′-(1,3-Phenylene diisopropylidene)bisphenol (BPM), 4,4′-(1,4-phenylenediisopropylidene)bisphenol (BPP), 4,4′- sulfonyldiphenol (BPS), 4,4′ cyclohexylidenebisphenol (BPZ), 4,4′ (hexafluoroisopropylidene)-diphenol (BPAF) and 4,4′-(1-phenylethylidene)bisphenol (BPAP), plus 2,2-bis(4-hydroxyphenyl)propane (BPA) were investigated by H295R cell and MVLN cell bioassays. In the H295R cell assay, the endpoints included hormone production and key genes for steroidogenesis (CYP11A, CYP17, CYP19 and 3βHSD2) or metabolism sulfotransferase (SULT1A1, SULT2A1 and SULT2B1) at the molecular level. The results indicated that except for BPP or BPAF, the eight other bisphenols significantly increased the E2/T ratio. In addition, BPB, BPF and BPS significantly up-regulate CYP19 gene expression, and only BPB significantly reduced sulfotransferase gene expression. In the MVLN luciferase gene reporter assay, seven bisphenols induced luciferase activity alone, and are 104 to 108-fold less potent than E2. Their nuclear ERα binding activity is in the order of BPAF > BPZ > BPP > BPB > BPA > BPF > BPS. In summary, all nine tested bisphenols showed endocrine toxicity through different mechanisms. Some had similar potency as BPA, but some had even higher potency. Further research is necessary to evaluate the toxicity of these potential BPA substitutes.

Drug Repurposing to Treat Glucocorticoid Resistance in Asthma.

Corticosteroid resistance causes significant morbidity in asthma, and drug repurposing may identify timely and cost-effective adjunctive treatments for corticosteroid resistance. In 95 subjects from the Childhood Asthma Management Program (CAMP) and 19 subjects from the Severe Asthma Research Program (SARP), corticosteroid response was measured by the change in percent predicted forced expiratory volume in one second (FEV1). In each cohort, differential gene expression analysis was performed comparing poor (resistant) responders, defined as those with zero to negative change in FEV1, to good responders, followed by Connectivity Map (CMap) analysis to identify inversely associated (i.e., negatively connected) drugs that reversed the gene expression profile of poor responders to resemble that of good responders. Mean connectivity scores weighted by sample size were calculated. The top five drug compound candidates underwent in vitro validation in NF-κB-based luciferase reporter A549 cells stimulated by IL-1β ± dexamethasone. In CAMP and SARP, 134 and 178 respective genes were differentially expressed in poor responders. CMap analysis identified 46 compounds in common across both cohorts with connectivity scores < −50. γ-linolenic acid, ampicillin, exemestane, brinzolamide, and INCA-6 were selected for functional validation. γ-linolenic acid, brinzolamide, and INCA-6 significantly reduced IL-1β induced luciferase activity and potentiated the anti-inflammatory effect of dexamethasone in A549/NF-κB-luc reporter cells. These results demonstrate how existing drugs, including γ-linolenic acid, brinzolamide, and INCA-6, may be repurposed to improve corticosteroid response in asthmatics.

MicroRNA-145 transcriptionally regulates Semaphorin 3A expression in prostate cancer cells.

Prostate cancer (PCa) is one of the most prevalent cancer types among males. Differential expression of microRNAs is associated with various cancers including PCa. Although mature microRNAs are preferentially located in the cytoplasm, several studies identified mature human microRNAs in purified nuclei and miR-145 has been found to be predominantly expressed in the nuclei of benign tissues compared to tumor lesions. However, the nuclear functions of miR-145 are yet limited. Here, we aimed at investigating the inductive role of miR-145 on the expression of Semaphorin 3A(SEMA3A) in PCa cell lines. To study the regulatory potential of miR-145 in the transcriptional level in PCa, we overexpressed miR-145 in PC3 and DU145 cells, and confirmed its upregulation by quantitative-real-time-PCR. Then we investigated the tumor suppressor potential of miR-145 upon inducing SEMA3A expression using cell viability assay, western blot analysis, Chromatin Immunoprecipitation assay and luciferase reporter assay. Our results revealed that p53, miR-145, and SEMA3A expressions are significantly downregulated in PC3 and DU145 cells compared to nontumorigenic prostate epithelial PNT1a cells. miR-145 overexpression in PCa cells induced the expression of SEMA3A at both messenger RNA and protein levels. Furthermore, increased miR-145 expression enriched RNA Pol-II antibody on the promoter of SEMA3A and induced luciferase activity controlled by SEMA3A promoter. In this study, we showed that the functions of miR-145 are not limited to gene silencing, and found that it may lead to changes in gene expression in the transcriptional level.

Abstract 3587: NRF2-driven KEAP1 transcription in human lung cancers

Abstract Introduction: NRF2/KEAP1 copy number variations and somatic mutations lead to NRF2 activation in a variety of human cancers. Though NRF2 plays a role in activating KEAP1 expression in turn, exact mechanisms and clinical implications remain unknown. Methods: We examined expression correlation between NRF2 and KEAP1 in RNA profiling databases (cBioportal, GEO) with focus on lung cancer. We performed qPCR and western blotting to measure KEAP1 mRNA and protein alterations during NRF2 activator or knockdown treatments. We also queried ENCODE and SRA for ChIP-seq datasets to identify NRF2 binding within KEAP1 promoters in human cells. To demonstrate the regulatory role of NRF2 binding on KEAP1 expression, we applied luciferase reporter assay and CRISPR editing to assess the ARE-related KEAP1 promoter activity and endogenous mRNA abundance change. To determine specimen-level implication of the feedback pattern, we tested gene expression ratio for predicting NRF2/KEAP1 copy number variations and somatic mutations. Results: RNA profiling datasets analysis showed that mRNA expression of KEAP1 was consistently in positive correlation with NRF2 in multiple primary squamous cell cancers, including LUSC (Pearson r=0.50, p&amp;lt;1e-15), ESCA: squamous (r=0.49, p=7.1e-7), HNSC (r=0.33, p=5e-15), and CESC: squamous (r=0.2, p=0.0014). The positive correlations were consistent across all squamous cell lung cancer datasets (Pooled r=0.5, p&amp;lt;1e-15, n=1409), but not in lung adenocarcinoma (Pooled r=0.06, p=0.11, n=2428). To determine regulatory role of NRF2 on KEAP1, we first treated multiple lung cell lines with NRF2 activator and observed significantly increased KEAP1 mRNA and protein levels. Correspondingly, we observed KEAP1 reduction in NRF2 knockdown A549 cells. ChIP-seq dataset analysis revealed highly consistent and variable NRF2 occupancy in KEAP1 promoter regions. This occupancy was drastically increased in lymphocytes and Bease2B cells after NRF2 activation. We then deleted NRF2 binding site in KEAP1 promoter and observed significant reduction of baseline and inducible luciferase activity. The ARE deletion in H292 genome reduced KEAP1 mRNA expression and increased NRF2 downstream gene expression. In one ARE homozygous deletion clone, we observed an elimination of inducible KEAP1 mRNA expression. To see if the feedback pattern can be applied to predict per-sample NRF2 signaling disruption, we calculated gene expression ratios of NRF2/KEAP1 and TXN (NRF2 target gene) /KEAP1. We found significant associations of increased TXN/KEAP1 ratio with KEAP1 deletion, and decreased NRF2/KEAP1 ratio with somatic mutation. From this result, we further developed a linear regression model that can reliably predict functional mutations in NRF2 pathway. Conclusion: NRF2-driven KEAP1 transcription suggested a calibrated NRF2 signaling transduction. Novel cancer prevention and therapy targeting NRF2 signaling will benefit from understanding this unique pattern. Citation Format: Yijun Tian, Qian Liu, Shengnan Yu, Qian Chu, Yuan Chen, Kongming Wu, Liang Wang. NRF2-driven KEAP1 transcription in human lung cancers [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3587.

Human and Zebrafish Nuclear Progesterone Receptors Are Differently Activated by Manifold Progestins.

The environmental risk of natural and synthetic ligands of the nuclear progesterone receptor (nPR) has been pointed out, however there is still a lack of mechanistic information regarding their ability to interact with nuclear PR in aquatic species. To identify possible interspecies differences, we assessed in vitro the ability of manifold progestins to transactivate zebrafish (zf) and human (h) PRs, using two established reporter cell lines, U2OS-zfPR and HELN-hPR, respectively. Reference ligands highlighted some differences between the two receptors. The reference human agonist ligands promegestone and progesterone induced luciferase activity in both cell lines in a concentration-dependent manner, whereas the natural zebrafish progestin 17α,20β-dihydroxy-4-pregnen-3-one activated zfPR but not hPR. The potent human PR antagonist mifepristone (RU486) blocked PR-induced luciferase in both cell models but with different potencies. In addition, a set of 22 synthetic progestins were screened on the two cell lines. Interestingly, all of the tested compounds activated hPR in the HELN-hPR cell line, whereas the majority of them acted as zfPR antagonists in U2OS-zfPR. Such zfPR-specific response was further confirmed in zebrafish liver cells. This study provides novel information regarding the activity of a large set of progestins on human and zebrafish PR and highlights major interspecies differences in their activity, which may result in differential effects of progestins between fish and humans.

Neutraceutical Targeting of the Bile Acid Receptor, Farnesoid X Receptor, for Intestinal Disease

INTRODUCTIONBile acids, classically known for their roles in fat digestion, are now recognised as important regulators of many aspects of intestinal physiology, including epithelial proliferation/apoptosis, transport and barrier function. Thus, the nuclear bile acid receptor, farnesoid X receptor (FXR), represents a promising therapeutic target for intestinal disorders, such as inflammatory bowel disease and colorectal cancer. Previous studies suggest dietary plant‐derived phytochemicals may modulate FXR activity.AIMSTo investigate the effects of a phytochemical, denoted KFS1, and a KFS1 rich plant extract, denoted as QE1, on FXR signalling in colonic epithelial cells.METHODST84 colonic epithelial cells, grown as polarised monolayers, were treated bilaterally with the FXR agonist, GW4064 (5 μM), KFS1 (1 – 100 μM) or QE1 (30 – 100 μg/ml) for 24 hr. Expression of FXR and FGF‐19, an index of FXR activation, were measured by qRT‐PCR, western blotting and ELISA. Transepithelial electrical resistance (TEER) was measured as an index of epithelial barrier function. Lactate dehydrogenase (LDH) release and PARP cleavage were used as measurements of necrosis and apoptosis, respectively. FXR activity was also studied using an FXR/luciferase reporter HepG2 cell line. Data are expressed as the mean ± SEM for a series of n experiments.RESULTSGW4064 (5 μM) induced a 719.8 ± 183.3 fold increase in FGF‐19 mRNA expression (n = 20; p&lt;0.001) and a 10.4 ± 4.9 fold increase in FGF‐19 protein release (n = 5; p&lt;0.01). KFS1 (5 – 100 μM) did not alter FGF‐19 protein expression and at higher concentrations (50 – 100 μM) reduced TEER. Similarly, GW4064 (5 μM) significantly induced luciferase activity in FXR HepG2 reporter cells by 1580.7 ± 195.7 counts per second (p&lt;0.001; n = 3), whereas treatment with KFS1 (1 – 100 μM) was without effect. Lower concentrations of KFS1 (5 μM), which alone did not alter TEER, increased FXR mRNA and protein expression by 4.2 ± 0.4 (p&lt;0.05; n = 4) and 1.5 ± 0.04 fold (p&lt;0.05; n = 4), respectively. Moreover, pre‐treatment of T84 cells with KFS1 (1, 5, or 10 μM) for 1 hr prior to treatment with GW4064 (5 μM; 24 hr) potentiated GW4064‐induced FGF‐19 mRNA expression by 2.5 ± 0.8, 2.9 ± 0.2, and 4.6 ± 0.5 fold, respectively (p&lt;0.05; n = 6) and FGF‐19 protein expression by 2.5 ± 0.4, 4.1 ± 0.5, and 4.6 ± 0.5 fold, respectively (p&lt;0.05, p&lt;0.001; n = 7). Combined treatment with KFS1 (1, 5, 10 μM) and GW4064 (5 μM) also significantly reduced TEER to 49.8 ± 7.4%, 50.6 ± 8.3%, and 40.6 ± 6.6% of control values, respectively (n = 7). TEER reductions in response to such co‐treatments were not associated with significant LDH release or PARP cleavage (n = 3). Finally, a methanolic extract of plant material known to be rich in KFS1, QE1 (100 μg/ml), increased GW4064‐induced FGF‐19 release in T84 cells by 2.1 ± 0.3 fold (n = 6; p&lt;0.01).CONCLUSIONThese data demonstrate that KFS1, a common dietary phytochemical, induces FXR expression in colonic epithelial cells and prime the cells for agonist‐induced FXR activation. These findings suggest that foods, or food supplements, rich in such plant‐derived phytochemicals have potential for development as FXR‐targeted neutraceuticals.Support or Funding InformationStAR PhD Scholarship from RCSI &amp; Science Foundation Ireland (SFI)

Generation of a Quantitative Luciferase Reporter for Sox9 SUMOylation.

Sox9 is a master transcription factor for chondrogenesis, which is essential for chondrocyte proliferation, differentiation, and maintenance. Sox9 activity is regulated by multiple layers, including post-translational modifications, such as SUMOylation. A detection method for visualizing the SUMOylation in live cells is required to fully understand the role of Sox9 SUMOylation. In this study, we generated a quantitative reporter for Sox9 SUMOylation that is based on the NanoBiT system. The simultaneous expression of Sox9 and SUMO1 constructs that are conjugated with NanoBiT fragments in HEK293T cells induced luciferase activity in SUMOylation target residue of Sox9-dependent manner. Furthermore, the reporter signal could be detected from both cell lysates and live cells. The signal level of our reporter responded to the co-expression of SUMOylation or deSUMOylation enzymes by several fold, showing dynamic potency of the reporter. The reporter was active in multiple cell types, including ATDC5 cells, which have chondrogenic potential. Finally, using this reporter, we revealed a extracellular signal conditions that can increase the amount of SUMOylated Sox9. In summary, we generated a novel reporter that was capable of quantitatively visualizing the Sox9-SUMOylation level in live cells. This reporter will be useful for understanding the dynamism of Sox9 regulation during chondrogenesis.

Transcriptional Regulation of NAMPT Gene by Glycogen Synthase Kinase 3β in Goat Adipocytes.

Nicotinamide phosphoribosyl transferase (NAMPT) is a cytokine which is secreted by adipose tissue and involved in the process of immunoreactions, inflammation, and lipid metabolism through the endocrine or paracrine. However, the information regarding NAMPT and its transcriptional regulation in goats remains unknown. In this study, the inactivation of glycogen synthase kinase 3β (GSK3β) decreased the supernatant NAMPT productions in goat adipocytes. The luciferase activities were kept in high levels in pGL3 (-735/+34) and the deletion from -735 bp to -486 bp significantly decreased the luciferase activity (p < 0.01). In addition, a peroxisome proliferator-activated receptor gamma (PPARγ) element in this region was responsible for NAMPT promoter activity. The transcription activity of PPARγ was activated by SB216763 stimulation and failed to induce luciferase activity of the pGL3 (Mut-PPARγ-735/+34). In contrast, the GSK3β overexpression repressed luciferase activity of the NAMPT promoter. Besides, chromatin immunoprecipitation assay indicated that PPARγ was capable of binding to the NAMPT promoter, and inhibition of GSK3β increased the binding capacity of PPARγ to the NAMPT promoter. These results confirm the role of GSK3β as a negative regulator of NAMPT transcription in goat adipocytes, and GSK3β-regulated expression of NAMPT is mediated by PPARγ.

Transcriptional regulation of human amelotin gene by interleukin-1β.

One of the major causes of tooth loss is chronic inflammation of the periodontium, the tissues surrounding the tooth. Amelotin (AMTN) is a tooth enamel protein which is expressed in maturation‐stage ameloblasts and also in the internal basal lamina of junctional epithelium, a unique epithelial structure attached to the tooth surface which protects against the constant microbiological challenge to the periodontium. Localization of AMTN suggests that its function could be involved in the dentogingival attachment. The purpose of this study was to investigate the effect of interleukin‐1β (IL‐1β) on AMTN gene transcription in human gingival epithelial Ca9‐22 cells. IL‐1β increased AMTN mRNA and protein levels at 3 h, and the levels reached maximum at 6 and 12 h. IL‐1β induced luciferase activities of human AMTN gene promoter constructs (−211, −353, −501, −769, and −950AMTN), but these activities were partially inhibited in −353AMTN constructs that included 3‐bp mutations in CCAAT/enhancer binding protein 1 (C/EBP1), C/EBP2, and Ying Yang 1 (YY1) elements. Transcriptional activities induced by IL‐1β were abrogated by protein kinase A (PKA), tyrosine kinase, mitogen‐activated protein kinase kinase (MEK1/2), and phosphatidylinositol 3‐kinase (PI3K) inhibitors. Gel shift and ChIP assays showed that IL‐1β increased C/EBPβ binding to C/EBP1 and C/EBP2, and YY1 binding to YY1 elements after 3 h, and that these DNA–protein interactions were inhibited by PKA, tyrosine kinase, MEK1/2, and PI3K inhibitors. These results demonstrated that IL‐1β increases AMTN gene transcription in human gingival epithelial cells mediated through C/EBP1, C/EBP2, and YY1 elements in the human AMTN gene promoter.

Pharmacologically relevant concentrations of berberine transiently stimulate dihydrotestosterone-inducible androgen receptor-mediated luciferase activity in human prostate cancer cells

Background: Herbal medicines containing berberine have a wide spectrum of usage. However, the effect on androgen receptor (AR) activity at plasmatic relevant concentrations is omitted. Therefore, the effect of berberine on AR activity in prostate cancer cell lines was investigated. Methods: We used the reporter gene assay (RGA) and polymerase chain reaction (qPCR) for monitoring the activity of AR. Moreover, we monitored the proliferation of two prostate cancer cell lines treated with Berberine. Results: Berberine significantly potentiated dihydrotestosterone (DHT)-inducible AR-dependent luciferase activity in RGA. It also significantly decreased basal KLK3 mRNA level in 22Rv1 cells, but DHT-inducible KLK3 mRNA was not affected in androgen-independent (22Rv1) or androgen-dependent (LNCaP) cells. Proliferation of both cell lines was significantly inhibited by 100 and 1,000 nM concentrations in similar manner and Berberine increased Annexin V staining, suggesting an apoptotic event. Conclusions: Berberine inhibits proliferation of prostatic cell lines at pharmacologically reachable concentrations but with no apparent link to androgen receptor-driven target gene expression.

In vitro nuclear receptor inhibition and cytotoxicity of hydraulic fracturing chemicals and their binary mixtures

The widespread use of hydraulic fracturing (HF) in oil and gas extraction operations has led to concern over environmental risks posed by chemicals used in HF fluids. Here we employed a suite of stable luciferase reporter gene assays to investigate the potential for selected HF chemicals or geogenics to activate or antagonise nuclear receptor signalling. We screened three biocides (bronopol [BP], glutaraldehyde [GA], and tetrakis(hydroxymethyl)phosphonium sulfate [THPS]), a surfactant (2-butoxyethanol), a friction reducer (polyacrylamide), and a coal seam geogenic (o-cresol) for their potential to act as agonists or antagonists of the estrogen receptor, androgen receptor, progesterone receptor (PR), glucocorticoid receptor or peroxisome proliferator-activated receptor gamma (PPARγ). None of the chemicals induced luciferase activity in any of assays used in the study. In antagonistic mode, BP, GA and THPS caused reductions in luciferase activity in the reporter assays at higher concentrations (50–100 μM), while at low concentrations (2–10 μM) GA and THPS enhanced luciferase activity in some assays relative to controls. None of the other tested chemicals exhibited antagonism in the selected assays. In most cases, altered receptor signalling only occurred at concentrations exhibiting cytotoxicity. However, PPARγ activity, and to a lesser extent PR activity, were inhibited by THPS at sub-cytotoxic concentrations. The majority of binary combinations tested exhibited significantly less-than-additive cytotoxicity, and none of the combinations exhibited synergistic cytotoxicity. In summary, the results of the present study indicate that the selected chemicals are not likely to function as direct agonists of the nuclear receptors tested, and only one chemical, THPS was an apparent partial antagonist of two nuclear receptors.

Mycophenolate Mofetil induces c-Jun-N-terminal kinase expression in 22Rv1 cells: an impact on androgen receptor signaling.

Mycophenolate Mofetil (MYC) is a transplant drug used to prevent rejection in heart and kidneys transplant patients. Inosine monophosphate dehydrogenase (IMPDH), an enzyme involved in de novo synthesis of guanosine nucleotides, was considered as a primary target for MYC. Recently, we described that MYC was activates aryl hydrocarbon receptor and it antagonizes glucocorticoid receptor. Here we describe an androgen receptor (AR) as another off-target for MYC.We found that MYC increased basal and dihydrotestosterone (DHT)-inducible AR-dependent luciferase activity in AIZ-AR cells. In the same manner it induced or augmented mRNA level of KLK3 (prostate specific antigen; PSA) in 22Rv1 cells. Herein it displayed a hormetic effect on proliferation activity, since it significantly stimulated proliferation in lower concentrations but inhibited in higher (>1 µg/ml) concentrations in the presence of DHT. In contrast, MYC suppressed DHT-inducible KLK3 mRNA expression and cell proliferation in androgen-dependent LNCaP cells. MYC augmented DHT-inducible nuclear translocation of AR and increased the expression of MAPK8/9 (JNK46/54) resulting in the drop of their phosphorylation status. Moreover, MYC sensitized DHT-treated 22Rv1 cells to JNK-IN-8 mediated growth inhibition with the drop of IC50 from 1425 nM to 84 nM within 24 hrs. In conclusion, we suggest that, castrate-resistant prostate cancers progression might be retarded with the combination of MYC and chemical JNK inhibitors, involving AR-dependent mechanism.