Increased DNA Binding Activity Research Articles

The p38 MAPK/NF-κB pathway mediates GLT-1 up-regulation during cerebral ischemic preconditioning-induced brain ischemic tolerance in rats

Our previous finding suggests that p38 MAPK contributes to the GLT-1 upregulation during induction of brain ischemic tolerance by cerebral ischemic preconditioning (CIP), however, the underlying mechanism is still unclear. Here, we investigated the molecular mechanisms underlying the CIP-induced GLT-1 upregulation by using Western blotting, Co-immunoprecipitation (Co-IP), electrophoretic mobility shift assay (EMSA) and thionin staining in rat hippocampus CA1 subset. We found that application of BAY11-7082 (an inhibitor of NF-κB), or dihydrokainate (an inhibitor of GLT-1), or SB203580 (an inhibitor of p38 MAPK) could attenuate the CIP-induced neuronal protection in hippocampus CA1 region of rats. Moreover, CIP caused rapid activation of NF-κB, as evidenced by nuclear translocation of NF-κB p50 protein, which led to active p50/p65 dimer formation and increased DNA binding activity. GLT-1 was also increased after CIP. Pretreatment with BAY11-7082 blocked the CIP-induced GLT-1 upregulation. The above results suggest that NF-κB participates in GLT-1 up-regulation during the induction of brain ischemic tolerance by CIP. We also found that pretreatment with SB203580 caused significant reduction of NF-κB p50 protein in nucleus, NF-κB p50/p65 dimer nuclear translocation and DNA binding activity of NF-κB. Together, we conclude that p38 MAPK/NF-κB pathway participates in the mediation of GLT-1 up-regulation during the induction of brain ischemic tolerance induced by CIP.

Effect of semaphorin-3A on the cellular stability of CD4+CD25+ regulatory T cells induced by lipopolysaccharide

To investigate the effect and mechanism of semaphorin-3A (Sema3A) in maintaining the cellular stability of CD4+CD25+ regulatory T cells (Tregs) induced by lipopolysaccharide (LPS). In vitro, using immunomagnetic beads, splenic CD4+CD25+ Tregs of C57BL/6J mice were isolated and cultured. According to the random number table, the isolated cells were divided into control group (treated with anti-CD3e and anti-CD28 for polyclonal activation), LPS group (on the basis of control group, treated with LPS at the dose of 100 μg/L), LPS + nuclear factor kappa B (NF-κB) inhibitor pyrrolidine dithiocarbamate (PDTC) group (treated with LPS at the dose of 100 μg/L and PDTC at the dose of 25 mg/L), LPS + phosphate buffer solution (PBS) group (treated with LPS at the dose of 100 μg/L and PBS at the volume of 10 μL), LPS + PDTC + recombinant Sema3A (rSema3A) group (treated with LPS at the dose of 100 μg/L, PDTC at the dose of 25 mg/L and rSema3A at the dose of 300 μg/L), and LPS + PBS + rSema3A group (treated with LPS at the dose of 100 μg/L, PBS at the volume of 10 μL and rSema3A at the dose of 300 μg/L). mRNA and protein expressions of the specific markers of CD4+CD25+ Tregs, including forkhead box protein P-3 (Foxp-3), cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) and membrane-associated transforming growth factor-β1 (TGF-β1m+) were detected by reverse transcription-polymerase chain reaction (RT-PCR) and immunofluorescence methods after 24 hours. The supernatant interleukin-10 (IL-10) and secretory type TGF-β1 (sTGF-β1) were detected by enzyme-linked immunosorbent assay (ELISA). The apoptotic level was detected by immunofluorescence. The demethylation of Foxp3-Tregs-specific demethylated region (Foxp-3-TSDR) was detected by methylation specific PCR (MSP) in order to reflect the cellular stability of CD4+CD25+ Tregs. DNA binding activity of NF-κB signaling pathway was determined by electrophoretic mobility shift assay (EMSA), and activity of NF-κB signaling pathway was determined by Western blotting. Compared with control group, LPS could increase the cellular stability, including an increase in the mRNA and protein expressions of Foxp-3, CTLA-4 and TGF-β1m+ and secretion of IL-10 and sTGF-β1, decrease in the level of apoptosis and increase in the methylation of Foxp-3-TSDR. At the same time, LPS increased DNA binding activity of NF-κB signaling pathway and phosphorylation levels of the major molecules of NF-κB, including inhibitory protein kinaseβ (IKKβ) and p65, suggesting that the mechanism of enhancing cellular stability by LPS was related to the NF-κB signaling pathway. Compared with LPS group, PBS had no effect on cellular stability and NF-κB signaling pathway. However, administration of rSema3A further promoted cellular stability and activated NF-κB signaling pathway. Compared with LPS + PBS + rSema3A group, PDTC inhibited the function of rSema3A to increase cellular stability, as the mRNA and protein expressions of Foxp-3, CTLA-4 and TGF-β1m+ were significantly down-regulated in the LPS + PDTC + rSema3A group [Foxp-3 mRNA (2-ΔΔCt): 8.092±1.117 vs. 18.509±1.068, Foxp-3 protein (relative fluorescence intensity): 1.224±0.033 vs. 1.826±0.181; CTLA-4 mRNA (2-ΔΔCt): 3.254±0.760 vs. 11.840±0.827, CTLA-4 protein (relative fluorescence intensity): 1.305±0.058 vs. 1.842±0.111; TGF-β1m+ mRNA (2-ΔΔCt): 3.589±1.180 vs. 8.509±0.472, TGF-β1m+ protein (relative fluorescence intensity): 1.319±0.033 vs. 1.822±0.063, all P < 0.01], the secretion of IL-10 and sTGF-β1 were significantly decreased [IL-10 (ng/L): 445.33±54.08 vs. 992.67±83.10, sTGF-β1 (ng/L): 1 116.67±65.25 vs. 1 494.67±94.45, both P < 0.01], the apoptosis was significantly increased (fluorescence intensity: 0.398±0.031 vs. 0.268±0.046, P < 0.01), the methylation of Foxp-3-TSDR was significantly decreased (grey value: 0.467±0.048 vs. 1.780±0.119, P < 0.01), the DNA binding activity of NF-κB signaling pathway was significantly inhibited (grey value: 1.23±0.02 vs. 3.95±0.06, P < 0.01), and the phosphorylation levels of IKKβ and p65 were significantly decreased [p-IKKβ (p-IKKβ/IKKβ): 0.97±0.07 vs. 1.97±0.04, p-p65 (p-p65/p65): 0.95±0.08 vs. 1.93±0.06, both P < 0.01]. LPS increases the cellular stability of CD4+CD25+ Tregs through the NF-κB signaling pathway, and Sema3A further increases the cellular stability of CD4+CD25+ Tregs, and is related to the NF-κB signaling pathway.

HSB-1 Inhibition and HSF-1 Overexpression Trigger Overlapping Transcriptional Changes To Promote Longevity in Caenorhabditis elegans.

Heat shock factor 1 (HSF-1) is a component of the heat shock response pathway that is induced by cytoplasmic proteotoxic stress. In addition to its role in stress response, HSF-1 also acts as a key regulator of the rate of organismal aging. Overexpression of HSF-1 promotes longevity in C. elegans via mechanisms that remain less understood. Moreover, genetic ablation of a negative regulator of HSF-1, termed as heat shock factor binding protein 1 (HSB-1), results in hsf-1-dependent life span extension in animals. Here we show that in the absence of HSB-1, HSF-1 acquires increased DNA binding activity to its genomic target sequence. Using RNA-Seq to compare the gene expression profiles of the hsb-1 mutant and hsf-1 overexpression strains, we found that while more than 1,500 transcripts show ≥1.5-fold upregulation due to HSF-1 overexpression, HSB-1 inhibition alters the expression of less than 500 genes in C. elegans. Roughly half of the differentially regulated transcripts in the hsb-1 mutant have altered expression also in hsf-1 overexpressing animals, with a strongly correlated fold-expression pattern between the two strains. In addition, genes that are upregulated via both HSB-1 inhibition and HSF-1 overexpression include numerous DAF-16 targets that have known functions in longevity regulation. This study identifies how HSB-1 acts as a specific regulator of the transactivation potential of HSF-1 in non-stressed conditions, thus providing a detailed understanding of the role of HSB-1/HSF-1 signaling pathway in transcriptional regulation and longevity in C. elegans.

Constitutive activation and overexpression of NF-κB/c-Rel in conjunction with p50 contribute to aggressive tongue tumorigenesis.

Tongue squamous cell carcinoma (TSCC) is a most aggressive head and neck cancer often associated with a poor survival rate. Yet, it always shows better prognosis in presence of HPV16 infection. NF-κB plays a pivotal role in carcinogenesis and chemo-radio resistance of cancer but its role in tongue cancer is not yet explored. In this study, a total of hundred tongue tissue biopsies comprising precancer, cancer and adjacent normal controls including two tongue cancer cell lines (HPV+/−ve) were employed to examine expression and transactivation of NF-κB proteins, their silencing by siRNA and invasion assays to understand their contributions in tongue carcinogenesis. An exclusive prevalence (28%) of HR-HPV type 16 was observed mainly in well differentiated tumors (78.5%). Increased DNA binding activity and differential expression of NF-κB proteins was observed with p50 and c-Rel being the two major DNA binding partners forming the functional NF-κB complex that increased as a function of severity of lesions in both HPV+/−ve tumors but selective participation of p65 in HPV16+ve TSCCs induced well differentiation of tumors resulting in better prognosis. siRNA treatment against c-Rel or Fra-2 led to upregulation of p27 but strong inhibition of c-Rel, c-Jun, c-myc, HPVE6/E7 and Fra-2 which is exclusively overexpressed in HPV−ve aggressive tumors. In conclusion, selective participation of c-Rel with p50 that in cross-talk with AP-1/Fra-2 induced poor differentiation and aggressive tumorigenesis mainly in HPV−ve smokers while HPV infection induced expression of p65 and p27 leading to well differentiation and better prognosis preferably in non-smoking TSCC patients.

A feed-forward regulation of endothelin receptors by c-Jun in human non-pigmented ciliary epithelial cells and retinal ganglion cells.

c-Jun, c-Jun N-terminal kinase(JNK) and endothelin B (ETB) receptor have been shown to contribute to the pathogenesis of glaucoma. Previously, we reported that an increase of c-Jun and CCAAT/enhancer binding protein β (C/EBPβ) immunohistostaining is associated with upregulation of the ETB receptor within the ganglion cell layer of rats with elevated intraocular pressure (IOP). In addition, both transcription factors regulate the expression of the ETB receptor in human non-pigmented ciliary epithelial cells (HNPE). The current study addressed the mechanisms by which ET-1 produced upregulation of ET receptors in primary rat retinal ganglion cells (RGCs) and HNPE cells. Treatment of ET-1 and ET-3 increased the immunocytochemical staining of c-Jun and C/EBPβ in primary rat RGCs and co-localization of both transcription factors was observed. A marked increase in DNA binding activity of AP-1 and C/EBPβ as well as elevated protein levels of c-Jun and c-Jun-N-terminal kinase (JNK) were detected following ET-1 treatment in HNPE cells. Overexpression of ETA or ETB receptor promoted the upregulation of c-Jun and also elevated its promoter activity. In addition, upregulation of C/EBPβ augmented DNA binding and mRNA expression of c-Jun, and furthermore, the interaction of c-Jun and C/EBPβ was confirmed using co-immunoprecipitation. Apoptosis of HNPE cells was identified following ET-1 treatment, and overexpression of the ETA or ETB receptor produced enhanced apoptosis. ET-1 mediated upregulation of c-Jun and C/EBPβ and their interaction may represent a novel mechanism contributing to the regulation of endothelin receptor expression. Reciprocally, c-Jun was also found to regulate the ET receptors and C/EBPβ appeared to play a regulatory role in promoting expression of c-Jun. Taken together, the data suggests that ET-1 triggers the upregulation of c-Jun through both ETA and ETB receptors, and conversely c-Jun also upregulates endothelin receptor expression, thereby generating a positive feed-forward loop of endothelin receptor activation and expression. This feed-forward regulation may contribute to RGC death and astrocyte proliferation following ET-1 treatment.

Abstract 5856: Molecular mechanism underlying breast cancer cell radioresistance: role of radiation-induced NF-KB self-sustenance

Abstract Understanding the mechanism of breast cancer cell radioresistance and the associated selection pressure, tumor recurrence, would help us to develop molecular interventions and achieve disease/progression-free survival in patients with cancer. In the present study, we investigated the molecular mechanisms prompted in breast cancer cells in response to clinical radiation and the associated coordination of intra- and inter-cellular signaling that orchestrate radioresistance and tumor relapse/recurrence. Our findings showed that clinical doses of radiation increase NF-κB transcription and DNA-binding activity. Further, radiation exposure resulted in de novo TNF-α synthesis and secretion. An NF-κB gene manipulation approach revealed that NF-κB triggered NF-κB-mediated TNF-α transcriptional activity. NF-κB DNA binding kinetic analysis demonstrated both sustained and dual phase NF-κB activation with radiation. Radiation prompted NF-κB-dependent synthesis/secretion of TNF-α, a requisite for the second phase of NF-κB activation and maintenance. Radiation-associated NF-κB-dependent secretion of TNF-α from irradiated cells also activated NF-κB in the non-irradiated, non-targeted bystander cells. Together, these findings demonstrated that radiation-triggered NF-κB-dependent TNFα secretion is critical for self-sustenance of NF-κB (through autocrine positive feedback signaling) and for coordinating bystander response (through intercellular signaling: de novo activation of NF-κB in bystander cells) after radiation exposure. Further, the data suggest that this NF-κB→TNF-α→NF-κB feedback-dependent self-sustenance of NF-κB could drive the selection pressure (proliferation, clonal expansion) of surviving breast cancer cells and subsequent tumor relapse/recurrence. Note: This abstract was not presented at the meeting. Citation Format: Hui Yu, Natarajan Aravindan, Ji Xu, Mohan Natarajan. Molecular mechanism underlying breast cancer cell radioresistance: role of radiation-induced NF-KB self-sustenance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5856. doi:10.1158/1538-7445.AM2017-5856

Uric Acid Induces Endothelial Dysfunction by Activating the HMGB1/RAGE Signaling Pathway.

Uric acid (UA) is a risk factor for endothelial dysfunction, a process in which inflammation may play an important role. UA increases high mobility group box chromosomal protein 1 (HMGB1) expression and extracellular release in endothelial cells. HMGB1 is an inflammatory cytokine that interacts with the receptor for advanced glycation end products (RAGE), inducing an oxidative stress and inflammatory response, which leads to endothelial dysfunction. In this study, human umbilical vein endothelial cells (HUVECs) were incubated with a high concentration of UA (20 mg/dL) after which endothelial function and the expression of HMGB1, RAGE, nuclear factor kappa B (NF-κB), inflammatory cytokines, and adhesion molecules were evaluated. UA inhibited endothelial nitric oxide synthase (eNOS) expression and nitric oxide (NO) production in HUVECs, increased intracellular HMGB1 expression and extracellular HMGB1 secretion, and upregulated RAGE expression. UA also activated NF-κB and increased the level of inflammatory cytokines. Blocking RAGE significantly suppressed the upregulation of RAGE and HMGB1 and prevented the increase in DNA binding activity of NF-κB and the levels of inflammatory cytokines. It also blocked the decrease in eNOS expression and NO production induced by UA. Our results suggest that high concentrations of UA cause endothelial dysfunction via the HMGB1/RAGE signaling pathway.

Abstract 12745: Interleukin-4 Promotes Procollagen Type-I Alpha 1 Gene Expression via Activator Protein 1 Activation in Cardiac Fibroblasts

Interleukin-4 (IL-4) activates the signal transducer and activator of transcription 6 signaling pathway, resulting in the upregulation of procollagen genes and promotion of collagen production in cardiac fibroblasts (CFs). In addition, transcription factors may participate in the transcriptional activation of collagen genes. We hypothesize that IL-4 activates activator protein 1 (AP-1) via c-Jun phosphorylation, promoting transcriptional activation of the procollagen type-I alpha 1 (Col1α1) gene and collagen type-I expression in CFs. Primary mouse CFs were used. Western blot analyses revealed that IL-4 (10 ng/ml) increased phosphorylation of c-Jun, a subunit of AP-1. Additionally, our experiments utilizing electrophoretic mobility shift assay (EMSA) revealed a significant increase in DNA binding activity for AP-1 in IL-4-treated CFs compared to control cells (1.00±0.07 to 2.14±0.38 fold change versus control, p &lt;0.05). Here we aimed to determine whether IL-4-activated AP-1 participates in transcriptional activation of the Col1α1 gene leading to increased collagen type-I expression in CFs. To that end, we blocked AP-1 with shRNA lentiviral particles specific for c-Jun in CFs, detected Col1α1 mRNA by qRT-PCR, and measured collagen type-I by Western blot. We found that IL-4 significantly induced Col1α1 mRNA expression after incubation with control shRNA-transduced cells for 6 hours (1.01±0.01 to 1.52±0.07 fold change versus control, p &lt;0.005), and it increased collagen type-I expression after 24 hours of incubation (1.00±0.01 to 1.95±0.12 fold change versus control, p &lt;0.005). In contrast, IL-4 did not increase mRNA levels of Col1α1 (1.09±0.02 to 0.98±0.03, p &gt;0.05) and the collagen type-I (0.95±0.05 to 1.01±0.10, p &gt;0.05) in c-Jun shRNA-transfected cells. Our data demonstrate that IL-4-activated AP-1, an ubiquitous transcription factor containing phosphorylated c-Jun, enhanced Col1α1 gene expression and collagen type-I expression in CFs. Data generated from this study provides a novel molecular basis for potential treatment of cardiac fibrosis by targeting IL-4-induced signaling in CFs.

Novel Roles of Epoxyeicosanoids in Regulating Cardiac Mitochondria.

Maintenance of a healthy pool of mitochondria is important for the function and survival of terminally differentiated cells such as cardiomyocytes. Epoxyeicosatrienoic acids (EETs) are epoxy lipids derived from metabolism of arachidonic acid by cytochrome P450 epoxygenases. We have previously shown that EETs trigger a protective response limiting mitochondrial dysfunction and reducing cellular death. The aim of this study was to investigate whether EET-mediated effects influence mitochondrial quality in HL-1 cardiac cells during starvation. HL-1 cells were subjected to serum- and amino acid free conditions for 24h. We employed a dual-acting synthetic analog UA-8 (13-(3-propylureido)tridec-8-enoic acid), possessing both EET-mimetic and soluble epoxide hydrolase (sEH) inhibitory properties, or 14,15-EET as model EET molecules. We demonstrated that EET-mediated events significantly improved mitochondrial function as assessed by preservation of the ADP/ATP ratio and oxidative respiratory capacity. Starvation induced mitochondrial hyperfusion observed in control cells was attenuated by UA-8. However, EET-mediated events did not affect the expression of mitochondrial dynamic proteins Fis1, DRP-1 or Mfn2. Rather we observed increased levels of OPA-1 oligomers and increased mitochondrial cristae density, which correlated with the preserved mitochondrial function. Increased DNA binding activity of pCREB and Nrf1/2 and increased SIRT1 activity together with elevated mitochondrial proteins suggest EET-mediated events led to preserved mitobiogenesis. Thus, we provide new evidence for EET-mediated events that preserve a healthier pool of mitochondria in cardiac cells following starvation-induced stress.

Abstract 740: Silica promotes anchorage-independent growth of human bronchial cells via activation of NF-êB1 p50

Abstract Although crystalline silica (hereafter silica) was judged as carcinogen by IARC, the mechanism of silica-induced carcinogenesis remains elusive. As limited studies have been reported to support the direct carcinogenic effect of silica on human lung epithelial cells, silica-induced inflammatory microenvironment may be postulated to facilitate lung neoplastic transformation. To test this hypothesis, we investigate the growth of immortalized human lung bronchial epithelial cell lines NL20, BEAS-2B and 16HBE14o-, following incubation with conditioned media collected 48h after exposed to silica (Silica CM) in each cell line. All cell lines promoted anchorage-independent growth in each Silica CM, compared with those in CM without silica exposure. All the bronchial cell lines in Silica CM increased DNA binding activity of NF-êB1 p50. After incubated with Silica CM, culture supernatants of NL20 and BEAS-2B kept higher concentration of EGF, and those of 16HBE14o- contained higher concentration of TNFá and IGF1, respectively. Recombinant EGF, TNFá and IGF1 are all sufficient to promote anchorage-independent growth of each bronchial cell line, and to increase DNA binding activity of NF-êB1 p50. Neutralizing human EGF and TNFá antibodies dampened the growth of each bronchial cell line. NF-êB inhibitor Bay11-7082 suppressed the growth of all bronchial cell lines incubated with Silica CM. Taken together, silica promoted anchorage-independent growth of human lung epithelial cells via silica-induced secretion of soluble factors, followed by activation of NF-êB1 p50 in an autocrine manner. The present study supports the hypothesis that silica-induced inflammatory microenvironment may be actively involved in the potential carcinogenic effect of silica. Citation Format: Motoo Katabami, Ichiro Kinoshita, Yasushi Shimizu, Satoshi Takeuchi, Hirotoshi Dosaka-Akita. Silica promotes anchorage-independent growth of human bronchial cells via activation of NF-êB1 p50. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 740.

Retraction Note to: Platelet-derived growth factor-D contributes to aggressiveness of breast cancer cells by up-regulating Notch and NF-κB signaling pathways.

Platelet-derived growth factor-D (PDGF-D) has been linked with several human malignancies; however, its role in breast cancer progression is not known. We found that PDGF-D expressing breast cancer cell lines MDA-MB-231 and SUM-149 are more invasive compared to cell lines with little or no expression of PDGF-D such as MDA-MB-468 and MCF-7 cells. Over-expression of PDGF-D in PDGF-D low expressing MDA-MB-468 and MCF-7 cells by cDNA transfection showed increased cell proliferation while silencing the expression of PDGF-D by siRNA in PDGF-D high expressing MDA-MB-231 and SUM-149 cells showed decreased cell proliferation and increased apoptosis. Moreover, PDGF-D over-expression was positively correlated with the expression of Notch-1 and Jagged-1, and the expression of mesenchymal markers (Vimentin and ZEB-2) with concomitant decreased expression of epithelial marker E-cadherin. Since NF-κB activation plays a crucial role in Notch signaling as well as in epithelial–mesenchymal transition and tumor aggressiveness, we determined the DNA binding activity of NF-κB and our findings are consistent showing that PDGF-D over-expression led to increased DNA binding activity of NF-κB while it was found to be decreased by inactivation of PDGF-D. These results were also consistent with the expression and activity of MMP-9 and VEGF, as well as invasive characteristics. Further, forced expression of Notch-1/Jagged-1 by cDNA transfection de-repressed the effects of PDGF-D silencing on NF-κB activity and invasion. From these results, we conclude that PDGF-D plays an important role in breast tumor aggressiveness and this process is mechanistically linked with the activation of Notch and NF-κB signaling.

A Surfactant-Induced Functional Modulation of a Global Virulence Regulator from Staphylococcus aureus

Triton X-100 (TX-100), a useful non-ionic surfactant, reduced the methicillin resistance in Staphylococcus aureus significantly. Many S. aureus proteins were expressed in the presence of TX-100. SarA, one of the TX-100-induced proteins, acts as a global virulence regulator in S. aureus. To understand the effects of TX-100 on the structure, and function of SarA, a recombinant S. aureus SarA (rSarA) and its derivative (C9W) have been investigated in the presence of varying concentrations of this surfactant using various probes. Our data have revealed that both rSarA and C9W bind to the cognate DNA with nearly similar affinity in the absence of TX-100. Interestingly, their DNA binding activities have been significantly increased in the presence of pre-micellar concentration of TX-100. The increase of TX-100 concentrations to micellar or post-micellar concentration did not greatly enhance their activities further. TX-100 molecules have altered the secondary and tertiary structures of both proteins to some extents. Size of the rSarA-TX-100 complex appears to be intermediate to those of rSarA and TX-100. Additional analyses show a relatively moderate interaction between C9W and TX-100. Binding of TX-100 to C9W has, however, occurred by a cooperative pathway particularly at micellar and higher concentrations of this surfactant. Taken together, TX-100-induced structural alteration of rSarA and C9W might be responsible for their increased DNA binding activity. As TX-100 has stabilized the somewhat weaker SarA-DNA complex effectively, it could be used to study its structure in the future.

Corticotropin Releasing Hormone and Urocortin 3 Stimulate Vascular Endothelial Growth Factor Expression through the cAMP/CREB Pathway

Colonic epithelium is the first line of defense against various pathological offenses in the gut. Previous studies have shown that the peptides of the corticotropin-releasing hormone (CRH) family modulate vascular endothelial growth factor (VEGF)-A production in other cells. Here we sought to investigate whether CRH and urocortin (Ucn) 3 regulate VEGF-A secretion in colonocytes through CRH receptors and to elucidate the underlying mechanism of action. CRH and Ucn 3 significantly increased the expression levels of VEGF-A mRNA and protein through CRH receptor 1 and 2, respectively, in human colonic epithelial cells and primary mouse intestinal epithelial cells. Underlying mechanisms involve activation of adenylyl cyclase with subsequent increase of intracellular cAMP level and increased DNA binding activity of transcription factor CREB on VEGF-A promoter region. Finally, genetic deficiency of CREB decreased intestinal inflammation and VEGF-A expression in a dextran sodium sulfate-induced colitis model. These results show that activation of CRH receptors by CRH ligands stimulates VEGF-A expression in intestinal epithelial cells through the cAMP/CREB pathway. Since VEGF-A boosts inflammatory responses through angiogenesis, these data suggest that CREB may be a key effector of CRH and Ucn 3-dependent inflammatory angiogenesis.

Abstract 42: The NF-κB/MDM2 pathway is engaged in activin/ PI3 kinase induced degradation of p21 in colon cancer cells

Abstract Introduction: The signaling pathways for activin and TGFβ are frequently disrupted in colorectal cancers (CRC) and these mutations are associated with metastatic disease. Understanding the cellular consequences wrought by mutations to these signaling pathways may provide opportunities for the development of new therapeutic approaches. Our lab has previously shown opposing effects on a downstream target of TGFβ and activin, namely the cyclin-dependent kinase (CDK) inhibitor p21. TGFβ induces SMAD4-dependent upregulation of p21, while activin downregulates p21 in a SMAD4-independent mechanism via ubiquitination and degradation allowing for enhanced cell migration. However, the mode of regulation leading to p21 degradation is not completely known. We hypothesize that regulation of the E3 ubiquitin ligase MDM2 by NF-κB is involved in p21 degradation. Here we explore the role of MDM2 in the regulation of p21 in response to activin and TGFβ treatment in FET CRC cells. Methods: Colon cancer cell lines were assessed for ligand-induced PI3K/NF-κB/MDM2/p21 pathway engagement. FET cells were transfected with increasing concentrations of either wild type p65 or a dominant-negative mutant of p65 and treated with activin or TGFβ prior to immunoblot analysis for IκB-alpha, MDM2 and p21. DNA-binding activity of NF-κB was assessed by electrophoretic mobility shift assay (EMSA). The requirement for NF-κB activation was determined through use of the selective inhibitor peptide NBD (NEMO-binding domain), and PI3 kinase activity was confirmed by inhibition with LY294002. Results: Because activin induces ubiquitination of p21, we investigated the potential involvement of MDM2 and further the regulation of MDM2 by NF-κB. NF-κB has been reported to regulate MDM2 through direct binding to an NF-κB binding site in the promoter of MDM2. Activin induced activation of NF-κB as measured by increased DNA binding activity to an NF-κB consensus-binding site as well as by a reduction in IκB-alpha protein. Furthermore, we found this regulation to be dependent on activation of PI3 kinase. Overexpression of wild type p65 plasmid but not dominant negative p65 was found to increase MDM2 expression while the NF-κB specific inhibitor NBD blocked the activin-induced increase in MDM2. By contrast, TGFβ treatment did not contribute to an increase in MDM2 levels via NF-κB pathway. Conclusions: Here, we provide evidence to indicate that in colon cancer cells, activin may be working through NF-κB to promote MDM2 activity leading to the degradation of p21 in a PI3 kinase dependent mechanism. Future experiments will explore if pharmaceutical intervention in this pathway can inhibit the metastatic activity of activin as a therapeutic approach for advanced colon cancer. Citation Format: Arundhati Jana, Seung Hyua Baik, Timothy Carroll, Ozkan Ozden, Nancy L. Krett, Barbara Jung. The NF-κB/MDM2 pathway is engaged in activin/ PI3 kinase induced degradation of p21 in colon cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 42. doi:10.1158/1538-7445.AM2015-42

Activation of the carbohydrate response element binding protein (ChREBP) in response to anoxia in the turtle Trachemys scripta elegans

BackgroundChREBP (carbohydrate response element binding protein) is a glucose-responsive transcription factor that is known to be an important regulator of glycolytic and lipogenic genes in response to glucose. We hypothesized that activation of ChREBP could be relevant to anoxia survival by the anoxia-tolerant turtle, Trachemys scripta elegans. MethodsExpression of ChREBP in response to 5 and 20h of anoxia was examined using RT-PCR and Western immunoblotting. In addition, subcellular localization and DNA-binding activity of ChREBP protein were assessed and transcript levels of liver pyruvate kinase (LPK), a downstream gene under ChREBP control were quantified using RT-PCR. ResultsChREBP was anoxia-responsive in kidney and liver, with transcript levels increasing by 1.2–1.8 fold in response to anoxia and protein levels increasing by 1.8–1.9 fold. Enhanced nuclear presence under anoxia was also observed in both tissues by 2.2–2.8 fold. A 4.2 fold increase in DNA binding activity of ChREBP was also observed in liver in response to 5h of anoxia. In addition, transcript levels of LPK increased by 2.1 fold in response to 5h of anoxia in the liver. ConclusionsThe results suggest that activation of ChREBP in response to anoxia might be a crucial factor for anoxia survival in turtle liver by contributing to elevated glycolytic flux in the initial phases of oxygen limitation. General significanceThis study provides the first demonstration of activation of ChREBP in response to anoxia in a natural model of anoxia tolerance, further improving our understanding of the molecular nature of anoxia tolerance.

Targeted Gene Deletion of Prolyl Hydroxylase Domain Protein 3 Triggers Angiogenesis and Preserves Cardiac Function by Stabilizing Hypoxia Inducible Factor 1 Alpha Following Myocardial Infarction

The key oxygen sensing molecules, Prolyl-hydroxylase domain 1-3 enzymes (PHD1-3), regulate hypoxia-inducible factor (HIF) under hypoxia. In the settings of cardiomyopathy and ischemia-reperfusion injury, PHD3 expression is elevated, resulting in decreased HIF activation. The role of PHD3 in myocardial injury is poorly understood. Hence, we aimed to determine the effects of PHD3 deletion in mice on HIF-1α and other related pathways following myocardial infarction (MI). Left coronary artery (LAD) in both wild type and prolyl hydroxylase 3 knock out (PHD3⁻/⁻) mice was ligated to induce myocardial infarction. Electrophoretic mobility shift analysis showed significant increase in DNA-binding activity of HIF-1α in PHD3⁻/⁻ mice as compared to wild type (WT) mice post MI. The PHD3⁻/⁻MI group also showed decreased fibrosis. Seven days after MI, enhanced capillary/arteriolar density was observed compared to WTMI group. PHD3⁻/⁻ mice subjected to MI also showed improved cardiac functions (Ejection fraction and Fractional shortening), as assessed by echocardiogram, compared to WT. Western blot analysis showed increased VEGF, Ang-1 & Bcl-2 expression in PHD3⁻/⁻MI group. In conclusion, ablation of the PHD3 gene resulted in increased angiogenesis and cardiac function after infarction thereby offering a potential target for pharmacological management of ischemic myocardial disease.

Nuclear Interaction between ADR-Induced p65 and p53 Mediates Cardiac Injury in iNOS (−/−) Mice

Adriamycin (ADR) treatment causes an imbalance in the levels of nitric oxide (•NO) and superoxide (O2 •−) production leading to cardiac injury. Previously we demonstrated that mice lacking inducible nitric oxide synthase (iNOS) have increased oxidative stress and mitochondrial injury. The molecular events leading to increased mitochondrial injury in iNOS deficient mice is unknown. ADR in the absence of iNOS preferentially activates a proapoptotic pathway without a concurrent increase in prosurvival pathways. Treatment with ADR leads to an increase in DNA binding activity of nuclear factor kappa B (NFκB) and p53 in wildtype mice. Following ADR treatment, p53, but not NFκB DNA binding activity, as well as the level of Bax, a p53 target gene, was increased in iNOS (−/−) mice. This apoptotic signaling effect in iNOS (−/−) is alleviated by overexpression of manganese superoxide dismutase (MnSOD). Increases in NFκB and p53 in ADR-treated wildtype mice did not lead to increases in target genes such as MnSOD, bcl-xL, or Bax. Moreover, co-immunoprecipitation analysis revealed that p65, a prominent member of the NFκB family, interacts with p53 in the nucleus. These results suggest that NFκB and p53 may counter act one another's actions in ADR-treated wildtype (WT) mice. Further, these results identify a novel mechanism by which oxidative stress may regulate transcription of proapoptotic genes.

Transcriptional activation of p21WAF1/CIP1 is mediated by increased DNA binding activity and increased interaction between p53 and Sp1 via phosphorylation during replicative senescence of human embryonic fibroblasts

Although p21(WAF1/CIP1) is known to be elevated during replicative senescence of human embryonic fibroblasts (HEFs), the mechanism for p21 up-regulation has not been elucidated clearly. In order to explore the mechanism, we analyzed expression of p21 mRNA and protein and luciferase activity of full-length p21 promoter. The result demonstrated that p21 up-regulation was accomplished largely at transcription level. The promoter assay using serially-deleted p21 promoter constructs revealed that p53 binding site was the most important site and Sp1 binding sites were necessary but not sufficient for transcriptional activation of p21. In addition, p53 protein was shown to interact with Sp1 protein. The interaction was increased in aged fibroblasts and was regulated by phosphorylation of p53 and Sp1. DNA binding activity of p53 was significantly elevated in aged fibroblasts but that of Sp1 was not. DNA binding activities of p53 and Sp1 were also regulated by phosphorylation. Phosphorylation of p53 at serine-15 and of Sp1 at serines appears to be involved. Taken together, the result demonstrated that p21 transcription during replicative senescence of HEFs is up-regulated by increase in DNA binding activity and interaction between p53 and Sp1 via phosphorylation.

APE1/Ref-1 enhances DNA binding activity of mutant p53 in a redox-dependent manner

Apurinic/apyrimidinic endonuclease 1/redox factor-1 (APE1/Ref-1) is a dual function protein; in addition to its DNA repair activity, it can stimulate DNA binding activity of numerous transcription factors as a reduction-oxidation (redox) factor. APE1/Ref-1 has been found to be a potent activator of wild-type p53 (wtp53) DNA binding in vitro and in vivo. Although p53 is mutated in most types of human cancer including hepatocellular carcinoma (HCC), little is known about whether APE1/Ref-1 can regulate mutant p53 (mutp53). Herein, we reported the increased APE1/Ref-1 protein and accumulation of mutp53 in HCC by immunohistochemistry. Of note, it was observed that APE1/Ref-1 high-expression and mutp53 expression were associated with carcinogenesis and progression of HCC. To determine whether APE1/Ref-1 regulates DNA binding of mutp53, we performed electromobility shift assays (EMSAs) and quantitative chromatin immunoprecipitation (ChIP) assays in HCC cell lines. In contrast to sequence-specific and DNA structure-dependent binding of wtp53, reduced mutp53 efficiently bound to nonlinear DNA, but not to linear DNA. Notably, overexpression of APE1/Ref-1 resulted in increased DNA binding activity of mutp53, while downregulation of APE1/Ref-1 caused a marked decrease of mutp53 DNA binding. In addition, APE1/Ref-1 could not potentiate the accumulation of p21 mRNA and protein in mutp53 cells. These data indicate that APE1/Ref-1 can stimulate mutp53 DNA binding in a redox-dependent manner.

Farnesoid X receptor directly regulates xenobiotic detoxification genes in the long-lived Little mice

Activation of xenobiotic metabolism pathways has been linked to lifespan extension in different models of aging. However, the mechanisms underlying activation of xenobiotic genes remain largely unknown. Here we showed that although farnesoid X receptor (FXR, Nr1h4) mRNA levels do not change significantly, FXR protein levels are elevated in the livers of the long-lived Little mice, leading to increased DNA binding activity of FXR. Hepatic FXR expression is sex-dependent in wild-type mice but not in Little mice, implying that up-regulation of FXR might be dependent on the reduction of growth hormone in Little mice. Growth hormone treatment decreased hepatic expression of FXR and xenobiotic genes Abcb1a, Fmo3 and Gsta2 in both wild-type and Little mice, suggesting an association between FXR and xenobiotic gene expression. We found that Abcb1a is transactivated by FXR via direct binding of FXR/retinoid X receptor α (RXRα) heterodimer to a response element at the proximal promoter. FXR also positively controls Fmo3 and Gsta2 expression through direct interaction with the response elements in these genes. Our study demonstrates that xenobiotic genes are direct transcriptional targets of FXR and suggests that FXR signaling may play a critical role in the lifespan extension observed in Little mice.