Response Element Transcription Research Articles

Discovery of Highly Potent and Efficient CBP/p300 Degraders with Strong In Vivo Antitumor Activity.

The transcriptional coactivator cAMP response element binding protein (CREB)-binding protein (CBP) and its homologue p300 have emerged as attractive therapeutic targets for human cancers such as acute myeloid leukemia (AML). Herein, we report the design, synthesis, and biological evaluation of a series of cereblon (CRBN)-recruiting CBP/p300 proteolysis targeting chimeras (PROTACs) based on the inhibitor CCS1477. The representative compounds 14g (XYD190) and 14h (XYD198) potently inhibited the growth of AML cells with low nanomolar IC50 values and effectively degraded CBP and p300 proteins in a concentration- and time-dependent manner. Mechanistic studies confirmed that 14g and 14h can selectively bind to CBP/p300 bromodomains and induce CBP and p300 degradation in bromodomain family proteins in a CRBN- and proteasome-dependent manner. 14g and 14h displayed remarkable antitumor efficacy in the MV4;11 xenograft model (TGI = 88% and 93%, respectively). Our findings demonstrated that 14g and 14h are useful lead compounds and deserve further optimization and activity evaluation for the treatment of human cancers.

SUMOylation of the ubiquitin ligase component KEAP1 at K39 upregulates NRF2 and its target function in lung cancer cell proliferation

Nuclear Factor Erythroid 2-Related Factor 2 (NRF2) is important for the expression of genes associated with oxidative stress. The levels of NRF2 are controlled by Kelch-like ECH-associated protein 1 (KEAP1)-dependent degradation. Although oxidative stress is known to suppress KEAP1 activity to stabilize the levels of NRF2, the mechanism for this control is unclear. Here, we identify that KEAP1 is modified by SUMO1 at the lysine residue position 39 (K39). Arginine replacement of this lysine (K39R) in KEAP1 did not affect its stability, subcellular localization, or dimerization but promoted the formation of the Cullin 3 ubiquitin ligase and increased NRF2 ubiquitination. This was accompanied by decreased NRF2 expression. Gene reporter assays showed that the transcription of antioxidant response elements was heightened in KEAP1-WT cells compared to cells expressing the KEAP1-K39R SUMO1 substrate mutant. Consistent with this, chromatin immunoprecipitation assays revealed higher NRF2 binding to the promoter regions of antioxidant genes in cells expressing the KEAP1-WT compared to the KEAP1-K39R mutant protein in H1299 lung cancer cell. The significance of this suppression of KEAP1 activity by its SUMOylation was tested in a subcutaneous tumor model of H1299 lung cancer cell lines that differentially expressed the WT and K39R KEAP1 constructs. This model showed that mutating the SUMOylation site on KEAP1 altered the production of reactive oxygen species and suppressed tumor growth. Taken together, our study recognizes that NRF2-dependent redox control is regulated by the SUMOylation of KEAP1. These findings identify a potential new therapeutic option to counteract oxidative stress.

Genomic Redistribution of Metal-Response Transcription Factor-1 (MTF-1) in Cadmium Resistant Cells.

(1) Background: Metal homeostasis is an important part of cellular programs and is disrupted when cells are exposed to carcinogenic heavy metals. Metal response is mediated by the metal response element transcription factor MTF-1. However, where MTF-1 binds and how that binding changes in response to heavy metals, such as cadmium, remains unknown. (2) Methods: To investigate the effects of prolonged cadmium exposure on the genomic distribution of MTF-1, we performed MTF-1 CUT&RUN, RNA-seq and ATAC-seq on control and cadmium-resistant cells. (3) Results: Changes in MTF-1 binding primarily occur distal to the transcription start sight. Newly occupied MTF-1 sites are enriched for FOS/JUN DNA binding motifs, while regions that lose MTF-1 binding in cadmium are enriched for the FOX transcription factor family member DNA binding sites. (4) Conclusions: Relocalization of MTF-1 to new genomic loci does not alter the accessibility of these locations. Our results support a model whereby MTF-1 is relocalized to accessible FOS/JUN-bound genomic locations in response to cadmium.

Quercetin and resveratrol inhibit ferroptosis independently of Nrf2-ARE activation in mouse hippocampal HT22cells.

Oxidative stress is the central pathomechanism in multiple cell death pathways, including ferroptosis, a form of iron-dependent programmed cell death. Various phytochemicals, which include the inducers of the nuclear factor erythroid-2-related factor 2-antioxidant response element (Nrf2-ARE) transcription pathway, prevent ferroptosis. We recently reported that several compounds, such as the potent Nrf2-ARE inducer curcumin, protect mouse hippocampus-derived HT22cells against ferroptosis independently of Nrf2-ARE activity. The present study characterized the anti-ferroptotic mechanisms of two additional Nrf2-ARE inducers, quercetin and resveratrol. Both compounds prevented erastin- and RSL3-induced ferroptosis of wild-type HT22cells, and also blocked the exacerbated erastin- and RSL3-induced ferroptosis of Nrf2-knockdown HT22cells. In both HT22cells, quercetin and resveratrol blocked erastin- and RSL3-induced elevation in reactive oxygen species. These results suggest that the Nrf2-ARE pathway does protect against ferroptosis, but quercetin and resveratrol act by reducing oxidative stress independently of Nrf2-ARE induction. Quercetin and resveratrol also reduced Fe2+ concentrations in HT22cells and in cell-free reactions. Thus, quercetin and resveratrol likely protect against erastin- and RSL3-induced ferroptosis by inhibiting the iron-catalyzed generation of hydroxyl radicals. Unlike quercetin, resveratrol cannot form a chelate structure with Fe2+ but the density functional theory computation demonstrates that resveratrol can form stable monodentate complexes with the alkene moiety and the electron-rich A ring.

Improving Reporter Gene Assay Methodology for Evaluating the Ability of Compounds to Restore P53 Activity.

Tumor suppressor protein P53 induces cycle arrest and apoptosis by mediating the transcriptional expression of its target genes. Mutations causing conformational abnormalities and post-translational modifications that promote degradation are the main reasons for the loss of P53 function in tumor cells. Reporter gene assays that can scientifically reflect the biological function can help discover the mechanism and therapeutic strategies that restore P53 function. In the reporter gene system of this work, tetracycline-inducible expression of wild-type P53 was used to provide a fully activated state as a 100% activity reference for the objective measurement of biological function. It was confirmed by RT-qPCR, cell viability assay, immunofluorescence, and Western blot analysis that the above-mentioned reporter gene system could correctly reflect the differences in biological activity between the wild-type and mutants. After that, the system was tentatively used for related mechanism research and compound activity evaluation. Through the tetracycline-induced co-expression of wild-type P53 and mutant P53 in exact proportion, it was observed that the response modes of typical transcriptional response elements (TREs) to dominant negative P53 mutation effect were not exactly the same. Compared to the relative multiple-to-solvent control, the activity percentage relative to the 100% activity reference of wild-type P53 can better reflect the actual influence of the so-called P53 mutant reactivator. Similarly, relative to the 100% activity reference, it can objectively reflect the biological effects caused by the inhibitor of P53 negative factors, such as MDM2. In conclusion, this study provides a 100% activity reference and a reliable calculation model for relevant basic research and drug development.

Abstract A03: Discovery and evaluation of GT19630, a c-Myc/n-Myc degrader, for targeting c-Myc-driven B-cell malignancies, acute myeloid leukemia (AML) and n-Myc driven cancers

Abstract Myc family members (MYC, MYCN and MYCL) are oncogenic transcriptional factors, which form dimers with Max to activate transcription activities to drive tumor initiation and progression. c-Myc deregulation has been identified in 80% of all tumor types, including B-cell malignancies, AML and a variety of solid tumors. N-Myc genetic alterations have been identified in small cell lung cancer, neuroblastoma, neuroendocrine prostate cancers and sarcoma. Myc-deregulation has been directly linked to the poor clinical outcome in these cancers, which makes Myc a therapeutic target for pharmacological inhibition. Here we described GT19630, a c-Myc/n-Myc degrader. GT19630 selectively degraded c-Myc proteins in c-Myc dependent blood cancer cells (IC50=1.5 nM) as compared to growth-factor regulated c-Myc in hematopoietic progenitor cells (TF-1) (IC50=52.5 nM). Similar selectivity of GT19630 has been demonstrated in cell proliferation and granulocyte–macrophage progenitor colony forming unit (GM-CFU) assays with the IC50s of 26.2 and 39.0 nM, respectively. GT19630 was shown to degrade c-Myc via proteasome degradation system and degraded CRBN-dependent endogenous neo-substrates of GSPT1, CK1α and IKZF1. GT19630 reduced transcriptional factors including c-Myc, Max, MXI1, SWF/SNF family members/associated proteins; ARID1A, SMARCE1, and SMARCC1 in transcriptional factor response element (TFRE) assays. Moreover, GT19630 inhibited the cell proliferation with IC50<10 nM in 74% of B-cell malignant cell lines (20/27) bearing deregulated c-Myc. Importantly, GT19630 was demonstrated to degrade Myc proteins completely and induced tumor regression or tumor eradication in AML, lymphoma and multiple myeloma (MM) animal xenograft tumor at lowest dose of 0.3 mpk/qd. In addition, GT19630 demonstrated an even-driven pharmacology in vivo and induced complete AML tumor regression with an intermittent dosing regimen of 3d on/7d off. Furthermore, GT19630 degraded n-Myc in SCLC, and neuroblastoma cells and demonstrated target-engaged efficacy in SCLC tumor models. Finally, GT19630 demonstrated favorable PK and safety profiles in rat after Rx for 14 days. In conclusion, GT19630 is a potent c-Myc/n-Myc degrader, which induced complete tumor regression and eradicated lymphoma cells in c-Myc/n-Myc dependent animal models (lymphoma, MM and AML and SCLC) without heme toxicity in vivo. GT19630 has achieved favorable PK profile and therapeutic index, which help advance this compound to IND-enabling stage. Citation Format: Liandong Ma, Yuzhi Tong, Zhaohui Yang, Qianxiang Zhou, Honghua Yan, Ye Chen, Dong Chen, Ru Xu, Yini Wang, Jun Qin. Discovery and evaluation of GT19630, a c-Myc/n-Myc degrader, for targeting c-Myc-driven B-cell malignancies, acute myeloid leukemia (AML) and n-Myc driven cancers [abstract]. In: Proceedings of the Third AACR International Meeting: Advances in Malignant Lymphoma: Maximizing the Basic-Translational Interface for Clinical Application; 2022 Jun 23-26; Boston, MA. Philadelphia (PA): AACR; Blood Cancer Discov 2022;3(5_Suppl):Abstract nr A03.

Genetic and Functional Identifying of Novel STAT1 Loss-of-Function Mutations in Patients with Diverse Clinical Phenotypes.

Mutations in signal transducer and activator of transcription 1 (STAT1) cause a broad spectrum of disease phenotypes. Heterozygous STAT1 loss-of-function (LOF) mutations cause Mendelian susceptibility to mycobacterial diseases (MSMD) infection, which is attributable to impaired IFN-γ signaling. The identification of novel mutations may extend the phenotypes associated with autosomal dominant (AD) STAT1 deficiency. Five patients with heterozygous STAT1 variations were recruited and their clinical and immunologic phenotypes were analyzed, with particular reference to JAK-STAT1 signaling pathways. Four, heterozygous STAT1 deficiency mutations were identified, three of which were novel mutations. Two of the mutations were previously unreported mRNA splicing mutations in AD STAT1-deficient patients. Patients with heterozygous STAT1 deficiency suffered not only mycobacterial infection, but also intracellular non-mycobacterial bacterial infection and congenital multiple malformations. AD-LOF mutation impaired IFN-γ-mediated STAT1 phosphorylation, gamma-activated sequence (GAS), and IFN-stimulated response element (ISRE) transcription activity and IFN-induced gene expression to different extents, which might account for the diverse clinical manifestations observed in these patients. The infectious disease susceptibility and phenotypic spectrum of patients with AD STAT1-LOF are broader than simply MSMD. The susceptibility to infections and immunological deficiency phenotypes, observed in AD-LOF patients, confirms the importance of STAT1 in host-pathogen interaction and immunity. However, variability in the nature and extent of these phenotypes suggests that functional analysis is required to identify accurately novel, heterozygous STAT1 mutations, associated with pathogenicity. Aberrant splice of STAT1 RNA could result in AD-LOF for STAT1 signaling which need more cases for confirmation.

Withaphysalins from Medicinal and Edible Physalis minima and Their Anti-inflammatory Activities.

Physalis minima is a medicinal and edible plant in China. In this study, 22 new withaphysalins, including a novel 1(10 → 6)abeo-14β-hydroxy one (1) and other 15 unusual 14β-hydroxy ones (3-4, 6-17, 19), were isolated from the whole herbs of P. minima together with two known analogues (23-24). Their structures were established by extensive analysis of high-resolution electrospray ionization mass spectrometry, IR, and 1D and 2D NMR spectroscopic data. Their absolute configurations were determined by electronic circular dichroism (ECD) spectra and single-crystal X-ray crystallographic analyses, together with DFT NMR calculations. All isolated compounds were evaluated for their anti-inflammatory activity via measuring the colorimetric reporter of the secreted embryonic alkaline phosphatase gene driven by an IFN-β minimal promoter fused to five copies of the NF-κB consensus transcriptional response element and three copies of the c-Rel binding site in LPS-stimulated human THP1-Dual cells. Compounds 2, 5, 6, 9, 10, 11, and 20 showed significant anti-inflammatory effects with IC50 values in the range of 3.01-13.39 μM. Among them, compounds 2 and 10 showed better anti-inflammatory effects to inhibit the secretion of IL-6, IL-1β, and TNF-α in LPS-stimulated THP1-Dual cells.

CREM Is Correlated With Immune-Suppressive Microenvironment and Predicts Poor Prognosis in Gastric Adenocarcinoma.

The transcriptional repressor cAMP response element modulator (CREM) has an important role in T-cell development. In this study, we used the integrated Bioinformatics Methods to explore the role of CREM in gastric adenocarcinoma (GAC). Our results showed that high CREM expression was closely related with poorer overall survival in GAC. By GSEA cluster analysis, we found that the high expression of CREM was associated with the cancer-associated pathway in GAC. Moreover, single-cell sequencing data showed that CREM is mainly localized in exhausted CD8+ T cells. Its prognostic value and the potential function lead to T-cell exhaustion in the tumor microenvironment (TME). Similar results were also obtained in glioma and lung cancer. High expression of CREM, correlated with clinical relevance of GAC, was associated with T-cell exhaustion and M2 polarization in GAC. These findings suggest that CREM can be used as a prognostic biomarker in GAC, which might provide a novel direction to explore the pathogenesis of GAC.

Computational resources to define alleles and altered regulatory motifs at genomically edited candidate response elements.

Unequivocal functional assessment of candidate genomic regulatory regions, such as transcriptional response elements, requires genetic alteration at their native chromosomal loci. Targeted DNA cleavage by Cas9 or other programmable nucleases enables analysis at virtually any genomic region, and diverse alleles generated by editing can be defined by deep sequencing for functional analysis. Interpretation of disrupted response elements, however, presents a special challenge, as these regions typically comprise clustered DNA binding motifs for multiple transcriptional regulatory factors (TFs); DNA sequence differences, natural or engineered, that affect binding by one TF can confer loss or gain of binding sites for other TFs. To address these and other analytical complexities, we created three computational tools that together integrate, in a single experiment, allele definition and TF binding motif evaluation for up to 9216 clones isolated, sequenced and propagated from Cas9-treated cell populations. We demonstrate 1) the capacity to functionally assess edited TF binding sites to query response element function, and 2) the efficacy and utility of these tools, by analyzing cell populations targeted by Cas9 for disruption of example glucocorticoid receptor (GR) binding motifs near FKBP5, a GR-regulated gene in the human adenocarcinoma cell line A549.

Abstract LB213: ACTL6A (Actin-like Protein 6A) enhances the cancer phenotype

Abstract Epidermal squamous cell carcinoma (SCC) is an extremely common, invasive and metastatic cancer. ACTL6A, an important protein subunit of the SWI/SNF epigenetic regulatory complex, is frequently overexpressed in cancer cells. In the present study, we show that ACTL6A drives SCC cell proliferation, invasion, migration and tumor formation, and that this aggressive phenotype is markedly reduced in ACTL6A knockdown cells. Expression of ACTL6A is associated with reduced p21Cip1 level and treatment with ACTL6A-siRNA results in a marked increase in p21Cip1 and attenuation of the cancer phenotype. Molecular studies reveal that ACTL6A suppresses p21Cip1 promoter activity to reduce mRNA and p21Cip1 protein and that loss of p21Cip1 expression appears to be biologically meaningful, as p21Cip1 knockdown enhances the aggressive phenotype. ACTL6A suppression of p21Cip1 expression requires the p53 and Sp1 transcription response elements in the p21Cip1 gene promoter. In addition, ACTL6A knockdown results in increased p53 tumor suppressor expression. These studies show that ACTL6A facilitates an aggressive cancer phenotype by reducing p53 and p21Cip1 mRNA and protein and suggests that ACTL6A contributes to an aggressive cancer phenotype in epidermal squamous cell carcinoma. Citation Format: Suruchi Shrestha, Gautam Adhikary, Wen Xu, Sivaveera Kandaysamy, Richard L. Eckert. ACTL6A (Actin-like Protein 6A) enhances the cancer phenotype [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB213.

Hemocyanin and hexamerinsexpression in response to hypoxia in stoneflies (Plecoptera, Insecta).

Many freshwater ecosystems worldwide undergo hypoxia events that can trigger physiological, behavioral, and molecular responses in many organisms. Among such molecular responses, the regulation of the hemocyanin (Hc) protein expression which plays a major role in oxygen transportation within aquatic insects remains poorly understood. The stoneflies (Plecoptera) are aquatic insects that possess a functional Hc in the hemolymph similar to crustacean that co-occurs with a nonfunctional Hc protein, hexamerins (Hx). However, the role of both proteins during hypoxia remains undetermined. Here, we evaluated the effect of hypoxia on the expression of Hc and Hx proteins via a comparison between hypoxia and normoxia amino acid sequence variation and protein expression pattern within 23 stonefly species. We induced short-term hypoxia in wild-caught stoneflies species, sequenced the target region of Hc and Hx by complementary DNA synthesis, characterized the protein biochemistry using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, ultrafiltration, and polarographic fluorometric method, and amplified the genome region of the hypoxia-inducible factor (HIF) transcriptional response element that regulated Hc using genome walking library approach. We found a lack of Hc expression in all examined species during hypoxia conditions, despite recognition of the HIF gene region as a possible regulatory factor of Hc, suggesting that compensatory responses as metabolic changes or behavioral tracheal movements to enhance respiratory efficiency could be possible mechanics to compensate for hypoxia. A short Hc-like novel isoform was detected instead in these 23 species, possibly due to either protein degradation or alternative splicing mechanisms, suggesting that the protein could be performing a different function other than oxygen transportation. Hx during hypoxia was expressed and exhibited species-level amino acid changes, highlighting a possible role during hypoxia. Our results demonstrate that hypoxia could enable a similar potential adaptive response of multiple species regarding specific physiological requirements, thereby shedding light on community behavior in stress environments that may help us to improve conservation practices and biomonitoring.

Simultaneous Examination of Cellular Pathways using Multiplex Hextuple Luciferase Assaying.

Multiplex experimentation that can assay multiple cellular signaling pathways in the same cells requires orthogonal genetically encoded reporters that report over large dynamic ranges. Luciferases are cost-effective, versatile candidates whose output signals can be sensitively detected in a multiplex fashion. Commonly used dual luciferase reporter assays detect one luciferase that is coupled to a single cellular pathway and a second that is coupled to a control pathway for normalization purposes. We have expanded this approach to multiplex hextuple luciferase assays that can report on five cellular signaling pathways and one control, each of which is encoded by a unique luciferase. Light emission by the six luciferases can be distinguished by the use of two distinct substrates, each specific for three luciferases, followed by spectral decomposition of the light emitted by each of the three luciferase enzymes with bandpass filters. Here, we present detailed protocols on how to perform multiplex hextuple luciferase assaying to monitor pathway fluxes through transcriptional response elements for five specific signaling pathways (i.e., c-Myc, NF-κβ, TGF-β, p53, and MAPK/JNK) using the constitutive CMV promoter as normalization control. Protocols are provided for preparing reporter vector plasmids for multiplex reporter assaying, performing cell culture and multiplex luciferase reporter vector plasmid transfection, executing multiplex luciferase assays, and analyzing and interpreting data obtained by a plate reader appropriately equipped to detect the different luminescences. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Preparation of vectors for multiplex hextuple luciferase assaying Basic Protocol 2: Cell culture work for multiplex hextuple luciferase assays Basic Protocol 3: Transfection of luciferase reporter plasmids followed by drug and recombinant protein treatments Basic Protocol 4: Performing the multiplex hextuple luciferase assay.

Estrogenic activity of benzotriazole UV stabilizers evaluated through in vitro assays and computational studies

Benzotriazole UV stabilizers (BUVs) are used in a variety of products to prevent yellowing and degradation. However, knowledge of the estrogenic activity of BUVs is still lacking. In the present study, a strategy combining in vitro assays and computational studies was adopted to evaluate the estrogenic activity of BUVs. 2-(2-Hydroxy-5-methlphenyl) benzotriazole (UV-P), 2-(5-tert-butyl-2-hydroxyphenyl)benzotriazole (UV-PS), and 2-(3-Allyl-2-hydroxy-5-methylphenyl)-2H-benzotriazole (UV-9) induced partial estrogenic activity while 2-(2-hydroxy-5-tert-octyl-phenyl)benzotriazole (UV-329), 2-(3-s-butyl-5-tert-butyl-2-hydroxyphenyl)benzotriazole (UV-350), and 3-(2H-benzotriazolyl)-5- (1,1-di-methylethyl)-4-hydroxy-benzene-propanoic acid octyl esters (UV-384) showed no estrogenic activity in MVLN assays. The results of in vitro assays were in accord with the results of computational studies. Moreover, ICI 182,780 suppressed the estrogenic activity of BUVs both in the absence and presence of E2, demonstrating that the estrogen responsive element (ERE) transcription activities of BUVs are generated through an estrogen receptor (ER) mediated pathway. Our findings suggest that the endocrine disruption effects of BUVs are a cause for concern.

Ozone at low concentrations does not affect motility and proliferation of cancer cells in vitro.

Exposure to low ozone concentrations is used in medicine as an adjuvant/complementary treatment for a variety of diseases. The therapeutic potential of low ozone concentrations relies on their capability to increase the nuclear translocation of the Nuclear factor erythroid 2-related factor 2 (Nrf2), thus inducing the transcription of Antioxidant Response Elements (ARE)-driven genes and, through a cascade of events, a general cytoprotective response. However, based on the controversial role of Nrf2 in cancer initiation, progression and resistance to therapies, possible negative effects of ozone therapy may be hypothesised in oncological patients. With the aim to elucidate the possible changes in morphology, migration capability and proliferation of cancer cells following mild ozone exposure, we performed wound healing experiments in vitro on HeLa cells treated with low ozone concentrations currently used in the clinical practice. By combining a multimodal microscopy approach (light and fluorescence microscopy, scanning electron microscopy, atomic force microscopy) with morphometric analyses, we demonstrated that, under our experimental conditions, exposure to low ozone concentrations does not alter cytomorphology, motility and proliferation features, thus supporting the notion that ozone therapy should not positively affect tumour cell growth and metastasis.

IL‐6 Increases ROS via Rac1, Leading to Mineralocorticoid Receptor and ENaC Activation

HTN is an inflammatory disease characterized by excessive sodium (Na+) reabsorption and increased pro‐inflammatory cytokines, such as interleukin 6 (IL‐6). Aldosterone (Aldo) is the primary ligand for the mineralocorticoid receptor (MR), which is expressed in the aldosterone‐sensitive portion of the distal nephron (ASDN). Although studies show benefits of MR inhibition in HTN, Aldo levels are not always increased, implying an alternate MR activation pathway. The epithelial sodium channel (ENaC) is in both the late distal convoluted tubule (DCT2) and cortical collecting duct (CCD), and is a major Na+ transport protein. We have shown that IL‐6 activates the MR in vitro, and that pharmacological Rac inhibition reduces mineralocorticoid response element (MRE) transcription. We have also shown that IL‐6 increases reactive oxygen species (ROS). Therefore, we hypothesized that IL‐6 activates the MR through increases in ROS generation and Rac1 activity, leading to increased ENaC activity.Since our data show that IL‐6 induces nuclear MR translocation, we tested if Rac1 affects IL‐6 mediated MR translocation. We co‐transfected MR‐eGFP tagged constructs and Rac1 expression vectors into murine distal convoluted tubule cells (mDCT15). We found IL‐6 mediated MR translocation decreased after Rac1 inhibition, or inhibition of MR activation; therefore, we investigated the role of Rac1 in IL‐6 mediated ENaC activity in the CCD and DCT2. We measured transepithelial voltage and resistance using a voltohmmeter, and calculated current in both mDCT15 and cortical collecting duct cells (mpkCCD) (n=4–15/group). Cells were transfected with vectors containing wild‐type Rac1 (WT), Rac1 dominant‐negative (DN Rac1) or constitutively active Rac1 (CA) and then treated with vehicle, Aldo [100nM], or IL‐6 [100ng/mL]. Data are shown as a fold change of current before and after treatments (1hr). We observed a significant increase in amiloride‐sensitive current in both mDCT2 and mpkCCD cells following both Aldo and IL‐6 treatments. Interestingly, the current was higher in the DCT2 than in the CCD (10.04 vs.1.98‐fold change/baseline). We also found that DN Rac1 completely inhibited IL‐6‐induced Na+ current (−1.16‐fold change/baseline) and that IL‐6 did not further increase current in cells with Rac1 CA (See Figure 1). Our data are the first to investigate ENaC activity in DCT2. Additionally, we show that IL‐6 is a strong stimulus for ENaC activity in both the DCT2 and CCD.Since IL‐6 increases ROS production in the DCT2, we investigated the role of Rac1 in this mechanism. mDCT15 cells were transfected with Rac1 inhibition and treated with IL6 [100ng/mL]. Cells were stained with dihydroethidium (DHE, ROS stain) and with confocal microscopy, we visualized ROS production. Our preliminary data show that Rac1 knockdown inhibits IL‐6 mediated ROS production (832.5±18 vs. 1073±29 mean DHE fluorescence, p=0.001).Together, our data show that IL‐6 may play an important role in MR activation, coupled with Rac1 and ROS‐generation in the distal nephron. Our data reveal a potential mechanism of MR activation during HTN, without increased Aldo levels.Support or Funding InformationDK115660, ASN Gottschalk Award to BMW; DK110409 to DCERac1 Inhibition Reduced IL‐6‐Mediated ENaC Current in mDCT15 cell line.Data are shown as fold‐changes in current from baseline (n=4–15, *p<0.05 ANOVA, Kruskal‐Wallis).Figure 1

Fenretinide attenuates lipopolysaccharide (LPS)-induced blood-brain barrier (BBB) and depressive-like behavior in mice by targeting Nrf-2 signaling

The transcription factor nuclear factor erythroid-2 related factor 2 (Nrf2) is a dominant manager to inhibit oxidative and inflammatory damage. Fenretinide (Fen) is a novel agent, showing significant role in regulating oxidative stress and inflammatory response. However, its effects on lipopolysaccharide (LPS)-induced brain injury are still unclear. In the present study, we explored the regulatory role of Fen in LPS-triggered neuroinflammation, and the underlying molecular mechanisms. Results here indicated that Fen treatment markedly improved Nrf2 expression and nuclear translocation in mouse brain endothelial cell line bEnd.3 cells, and promoted Nrf2-antioxidant responsive element (ARE) transcription activity, as well as its down-streaming signals, which was Nrf2-dependent. Fen also exhibited cytoprotective role in LPS-stimulated bEnd.3 cells through improving anti-oxidant capacity and inhibiting inflammation by the blockage of nuclear factor-kappa B (NF-κB) signaling. Mouse model with brain injury induced by LPS, Fen administration markedly attenuated the behavior impairments, blood-brain-barrier (BBB) and the histological changes in hippocampus samples. Additionally, Fen attenuated oxidative stress and blunted inflammation in hippocampus of LPS-challenged mice. Therefore, results in the study highlighted the protective role of Fen against LPS-elicited brain injury.

Examining multiple cellular pathways at once using multiplex hextuple luciferase assaying

Sensitive simultaneous assessment of multiple signaling pathways within the same cells requires orthogonal reporters that can assay over large dynamic ranges. Luciferases are such genetically encoded candidates due to their sensitivity, versatility, and cost-effectiveness. We expand luciferase multiplexing in post-lysis endpoint luciferase assays from two to six. Light emissions are distinguished by a combination of distinct substrates and emission spectra deconvolution. All six luciferase reporter units are stitched together into one plasmid facilitating delivery of all reporter units through a process we termed solotransfection, minimizing experimental errors. We engineer a multiplex hextuple luciferase assay to probe pathway fluxes through five transcriptional response elements against a control constitutive promoter. We can monitor effects of siRNA, ligand, and chemical compound treatments on their target pathways along with the four other probed cellular pathways. We demonstrate the effectiveness and adaptiveness of multiplex luciferase assaying, and its broad application across different research fields.

Janthinobacterium CG23_2: Comparative Genome Analysis Reveals Enhanced Environmental Sensing and Transcriptional Regulation for Adaptation to Life in an Antarctic Supraglacial Stream.

As many bacteria detected in Antarctic environments are neither true psychrophiles nor endemic species, their proliferation in spite of environmental extremes gives rise to genome adaptations. Janthinobacterium sp. CG23_2 is a bacterial isolate from the Cotton Glacier stream, Antarctica. To understand how Janthinobacterium sp. CG23_2 has adapted to its environment, we investigated its genomic traits in comparison to genomes of 35 published Janthinobacterium species. While we hypothesized that genome shrinkage and specialization to narrow ecological niches would be energetically favorable for dwelling in an ephemeral Antarctic stream, the genome of Janthinobacterium sp. CG23_2 was on average 1.7 ± 0.6 Mb larger and predicted 1411 ± 499 more coding sequences compared to the other Janthinobacterium spp. Putatively identified horizontal gene transfer events contributed 0.92 Mb to the genome size expansion of Janthinobacterium sp. CG23_2. Genes with high copy numbers in the species-specific accessory genome of Janthinobacterium sp. CG23_2 were associated with environmental sensing, locomotion, response and transcriptional regulation, stress response, and mobile elements—functional categories which also showed molecular adaptation to cold. Our data suggest that genome plasticity and the abundant complementary genes for sensing and responding to the extracellular environment supported the adaptation of Janthinobacterium sp. CG23_2 to this extreme environment.

Interplay Between Mitochondrial Peroxiredoxins and ROS in Cancer Development and Progression.

Mitochondria are multifunctional cellular organelles that are major producers of reactive oxygen species (ROS) in eukaryotes; to maintain the redox balance, they are supplemented with different ROS scavengers, including mitochondrial peroxiredoxins (Prdxs). Mitochondrial Prdxs have physiological and pathological significance and are associated with the initiation and progression of various cancer types. In this review, we have focused on signaling involving ROS and mitochondrial Prdxs that is associated with cancer development and progression. An upregulated expression of Prdx3 and Prdx5 has been reported in different cancer types, such as breast, ovarian, endometrial, and lung cancers, as well as in Hodgkin’s lymphoma and hepatocellular carcinoma. The expression of Prdx3 and Prdx5 in different types of malignancies involves their association with different factors, such as transcription factors, micro RNAs, tumor suppressors, response elements, and oncogenic genes. The microenvironment of mitochondrial Prdxs plays an important role in cancer development, as cancerous cells are equipped with a high level of antioxidants to overcome excessive ROS production. However, an increased production of Prdx3 and Prdx5 is associated with the development of chemoresistance in certain types of cancers and it leads to further complications in cancer treatment. Understanding the interplay between mitochondrial Prdxs and ROS in carcinogenesis can be useful in the development of anticancer drugs with better proficiency and decreased resistance. However, more targeted studies are required for exploring the tumor microenvironment in association with mitochondrial Prdxs to improve the existing cancer therapies and drug development.