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Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

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At least 217 records · Page 12
All sourcesNASA NTRSDOE OSTI
DOE OSTI2013

MicroRNA-106b-25 cluster targets β-TRCP2, increases the expression of Snail and enhances cell migration and invasion in H1299 (non small cell lung cancer) cells

Highlights: •miR-106b-25 cluster directly targets the 3′UTR of the β-TRCP2 transcript. •β-TRCP2 mRNA was lower in H1299 cells stably expressing miR-106b-25 cluster. •miR-106b-25 cluster increased the expression of Snail. •miR-106b-25 cluster promoted the migration, colony formation and invasion. •miR-106b-25 cluster enhanced endothelial tube formation. -- Abstract: Lung cancer causes high mortality without a declining trend and non small cell lung cancer represents 85% of all pulmonary carcinomas. MicroRNAs (miRNAs) serve as fine regulators of proliferation, migration, invasion/metastasis and angiogenesis of normal and cancer cells. Using TargetScan6.2, we predicted that the ubiquitin ligase, β-TRCP2, could be a target for two of the constituent miRNAs of the miR-106b-25 cluster (miR-106b and miR-93). We generated a stable clone of miR-106b-25 cluster (CL) or the empty vector (EV) in H1299 (non small cell lung cancer) cells. The expression of β-TRCP2 mRNA was significantly lower in CL than that in EV cells. Transient expression of miR-93 but not antimiR-93 decreased the expression of β-TRCP2 mRNA in H1299 cells. β-TRCP2-3′UTR reporter assay revealed that its activity in CL cells was only 60% of that in EV cells. Snail protein expression was higher in CL than that in EV cells and H1299 cells exhibited an increase in the expression of Snail upon transient transfection with miR-93. miR-106b-25 cluster-induced migration of CL measured by scratch assay was more than that in EV cells and no significant difference in migration was observed between antimiR-93-transfected H1299 cells and the corresponding control-oligo-transfected cells. miR-106b-25 cluster-induced migration of CL cells was again confirmed in a Boyden chamber assay without the matrigel. CL cells were more invasive than EV cells when assessed using Boyden chambers with matrigel but there were no significant changes in the cell viabilities between EV and CL cells. Colony formation assay revealed that the CL cells formed more number of colonies than EV cells but they were smaller in size than those formed by EV cells. The supernatant from CL cells was more effective than that from EV cells in inducing tube formation in endothelial cells. Taken together, our data indicate that miR-106b-25 cluster may play an important role in the metastasis of human non-small cell lung cancer cells by directly suppressing the β-TRCP2 gene expression with a consequent increase in the expression of Snail.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2014

TGF-β promotes glioma cell growth via activating Nodal expression through Smad and ERK1/2 pathways

Highlights: •TGF-β promoted Nodal expression in glioma cells. •TGF-β promoted Nodal expression via activating Smad and ERK1/2 pathways. •TGF-β promotes glioma cell growth via activating Nodal expression. -- Abstract: While there were certain studies focusing on the mechanism of TGF-β promoting the growth of glioma cells, the present work revealed another novel mechanism that TGF-β may promote glioma cell growth via enhancing Nodal expression. Our results showed that Nodal expression was significantly upregulated in glioma cells when TGF-β was added, whereas the TGF-β-induced Nodal expression was evidently inhibited by transfection Smad2 or Smad3 siRNAs, and the suppression was especially significant when the Smad3 was downregulated. Another, the attenuation of TGF-β-induced Nodal expression was observed with blockade of the ERK1/2 pathway also. Further detection of the proliferation, apoptosis, and invasion of glioma cells indicated that Nodal overexpression promoted the proliferation and invasion of tumor cells and inhibited their apoptosis, resembling the effect of TGF-β addition. Downregulation of Nodal expression via transfection Nodal-specific siRNA in the presence of TGF-β weakened the promoting effect of the latter on glioma cells growth, and transfecting Nodal siRNA alone in the absence of exogenous TGF-β more profoundly inhibited the growth of glioma cells. These results demonstrated that while both TGF-β and Nodal promoted glioma cells growth, the former might exert such effect by enhancing Nodal expression, which may form a new target for glioma therapy.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2014

MiR-29b inhibits collagen maturation in hepatic stellate cells through down-regulating the expression of HSP47 and lysyl oxidase

Highlights: • Enhanced HSP47 and LOX expression is associated with decreased miR-29b level in liver fibrosis. • miR-29b down-regulates HSP47 and LOX expression. • The suppression of HSP47 and LOX by miR-29b is mediated by putative sites at their 3′-UTRs. • miR-29b inhibits extracellular LOX activity and collagen maturation. - Abstract: Altered expression of miR-29b is implicated in the pathogenesis and progression of liver fibrosis. We and others previously demonstrated that miR-29b down-regulates the expression of several extracellular-matrix (ECM) genes including Col 1A1, Col 3A1 and Elastin via directly targeting their 3′-UTRs. However, whether or not miR-29b plays a role in the post-translational regulation of ECM biosynthesis has not been reported. Heat shock protein 47 (HSP47) and lysyl oxidase (LOX) are known to be essential for ECM maturation. In this study we have demonstrated that expression of HSP47 and LOX was significantly up-regulated in culture-activated primary rat hepatic stellate cells (HSCs), TGF-β stimulated LX-2 cells and liver tissue of CCl{sub 4}-treated mice, which was accompanied by a decrease of miR-29b level. In addition, over-expression of miR-29b in LX-2 cells resulted in significant inhibition on HSP47 and LOX expression. Mechanistically, miR-29b inhibited the expression of a reporter gene that contains the respective full-length 3′-UTR from HSP47 and LOX gene, and this inhibitory effect was abolished by the deletion of a putative miR-29b targeting sequence from the 3′-UTRs. Transfection of LX-2 cells with miR-29b led to abnormal collagen structure as shown by electron-microscopy, presumably through down-regulation of the expression of molecules involved in ECM maturation including HSP47 and LOX. These results demonstrated that miR-29b is involved in regulating the post-translational processing of ECM and fibril formation.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2014

Regulation of hemeoxygenase-1 gene expression by Nrf2 and c-Jun in tertiary butylhydroquinone-stimulated rat primary astrocytes

Highlights: • tBHQ increased HO-1 mRNA and protein levels in rat primary astrocytes. • tBHQ enhanced HO-1 gene transcription in an ARE-dependent manner. • tBHQ increased the nuclear translocation and DNA binding of Nrf2 and c-Jun to ARE. • Nrf2 and c-Jun are involved in the differential modulation of HO-1 expression. • Nrf2 and c-Jun regulate HO-1 expression via their coordinated interaction. - Abstract: Hemeoxygenase-1 (HO-1) is a phase II antioxidant enzyme that is primarily involved in detoxification and cytoprotection in a variety of tissues. However, the mechanism underlying HO-1 gene expression remains unclear. In the present study, we investigated the regulation of HO-1 expression in primary cultured astrocytes by using the natural antioxidant compound tertiary butylhydroquinone (tBHQ). We found that tBHQ increased HO-1 mRNA and protein levels. Promoter analysis revealed that tBHQ enhanced HO-1 gene transcription in an antioxidant response element (ARE)-dependent manner. In addition, tBHQ increased the nuclear translocation and DNA binding of Nrf2 and c-Jun to ARE. Small interfering RNA (siRNA) experiments demonstrated that Nrf2 and c-Jun are involved in the differential modulation of HO-1 expression. Thus, Nrf2 knockdown reduced the basal level of HO-1 expression but did not affect the fold induction by tBHQ. On the other hand, knockdown of c-Jun diminished tBHQ-mediated induction of HO-1 without affecting basal expression. The data suggest that Nrf2 generally modulates the basal expression of HO-1, while c-Jun mediates HO-1 induction in response to tBHQ. The results of co-immunoprecipitation assays demonstrated a physical interaction between Nrf2 and c-Jun in tBHQ-treated astrocytes. The results suggest that Nrf2 and c-Jun regulate HO-1 expression via their coordinated interaction in tBHQ-treated rat primary astrocytes.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2014

Nuclear factor-κB is a common upstream signal for growth differentiation factor-5 expression in brown adipocytes exposed to pro-inflammatory cytokines and palmitate

Highlights: • GDF5 expression is up-regulated by IL-1β, TNF-α and palmitate in brown pre-adipocytes. • NF-κB stimulates promoter activity and expression of GDF5 in brown pre-adipocytes. • Recruitment of NF-κB to the GDF5 promoter is facilitated in BAT from ob/ob mice. • An NF-κB inhibitor prevents upregulation of GDF5 expression in brown pre-adipocytes. - Abstract: We have previously demonstrated that genetic and acquired obesity similarly led to drastic upregulation in brown adipose tissue (BAT), rather than white adipose tissue, of expression of both mRNA and corresponding protein for the bone morphogenic protein/growth differentiation factor (GDF) member GDF5 capable of promoting brown adipogenesis. In this study, we evaluated expression profiles of GDF5 in cultured murine brown pre-adipocytes exposed to pro-inflammatory cytokines and free fatty acids (FFAs), which are all shown to play a role in the pathogenesis of obesity. Both interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) were effective in up-regulating GDF5 expression in a concentration-dependent manner, while similar upregulation was seen in cells exposed to the saturated FFA palmitate, but not to the unsaturated FFA oleate. In silico analysis revealed existence of the putative nuclear factor-κB (NF-κB) binding site in the 5′-flanking region of mouse GDF5, whereas introduction of NF-κB subunits drastically facilitated both promoter activity and expression of GDF5 in brown pre-adipocytes. Chromatin immunoprecipitation analysis confirmed significant facilitation of the recruitment of NF-κB to the GDF5 promoter in lysed extracts of BAT from leptin-deficient ob/ob obese mice. Upregulation o GDF5 expression was invariably inhibited by an NF-κB inhibitor in cultured brown pre-adipocytes exposed to IL-1β, TNF-α and palmitate. These results suggest that obesity leads to upregulation of GDF5 expression responsible for the promotion of brown adipogenesis through a mechanism relevant to activation of the NF-κB pathway in response to particular pro-inflammatory cytokines and/or saturated FFAs in BAT.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2015

Transforming growth factor β-activated kinase 1 negatively regulates interleukin-1α-induced stromal-derived factor-1 expression in vascular smooth muscle cells

Stromal-derived Factor-1 (SDF-1) derived from vascular smooth muscle cells (VSMCs) contributes to vascular repair and remodeling in various vascular diseases. In this study, the mechanism underlying regulation of SDF-1 expression by interleukin-1α (IL-1α) was investigated in primary rat VSMCs. We found IL-1α promotes SDF-1 expression by up-regulating CCAAT-enhancer-binding protein β (C/EBPβ) in an IκB kinase β (IKKβ) signaling-dependent manner. Moreover, IL-1α-induced expression of C/EBPβ and SDF-1 was significantly potentiated by knockdown of transforming growth factor β-activated kinase 1 (TAK1), an upstream activator of IKKβ signaling. In addition, we also demonstrated that TAK1/p38 mitogen-activated protein kinase (p38 MAPK) signaling exerted negative effect on IL-1α-induced expression of C/EBPβ and SDF-1 through counteracting ROS-dependent up-regulation of nuclear factor erythroid 2-related factor 2 (NRF2). In conclusion, TAK1 acts as an important regulator of IL-1α-induced SDF-1 expression in VSMCs, and modulating activity of TAK1 may serve as a potential strategy for modulating vascular repair and remodeling. - Highlights: • IL-1α induces IKKβ signaling-dependent SDF-1 expression by up-regulating C/EBPβ. • Activation of TAK1 by IL-1α negatively regulates C/EBPβ-dependent SDF-1 expression. • IL-1α-induced TAK1/p38 MAPK signaling counteracts ROS-dependent SDF-1 expression. • TAK1 counteracts IL-1α-induced SDF-1 expression by attenuating NRF2 up-regulation.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2016

Role of protein kinase A and class II phosphatidylinositol 3-kinase C2β in the downregulation of KCa3.1 channel synthesis and membrane surface expression by lyso-globotriaosylceramide

The intermediate conductance calcium-activated potassium channel (KCa3.1) mediates proliferation of many cell types including fibroblasts, and is a molecular target for intervention in various cell proliferative diseases. Our previous study showed that reduction of KCa3.1 channel expression by lyso-globotriaosylceramide (lyso-Gb3) inhibits differentiation into myofibroblasts and collagen synthesis, which might lead to development of ascending thoracic aortic aneurysm secondary to Fabry disease. However, how lyso-Gb3 downregulates KCa3.1 channel expression is unknown. Therefore, we aimed to investigate the underlying mechanisms of lyso-Gb3-mediated KCa3.1 channel downregulation, focusing on the cAMP signaling pathway. We found that lyso-Gb3 increased the intracellular cAMP concentration by upregulation of adenylyl cyclase 6 and inhibited ERK 1/2 phosphorylation through the protein kinase A (PKA) pathway, leading to the inhibition of KCa3.1 channel synthesis, not the exchange protein directly activated by cAMP (Epac) pathway. Moreover, lyso-Gb3 suppressed expression of class II phosphatidylinositol 3-kinase C2β (PI3KC2β) by PKA activation, which reduces the production of phosphatidylinositol 3-phosphate [PI(3)P], and the reduced membrane surface expression of KCa3.1 channel was recovered by increasing the intracellular levels of PI(3)P. Consequently, our findings that lyso-Gb3 inhibited both KCa3.1 channel synthesis and surface expression by increasing intracellular cAMP, and controlled surface expression through changes in PI3KC2β-mediated PI(3)P production, suggest that modulation of PKA and PI3KC2β activity to control of KCa3.1 channel expression can be an alternative important target to attenuate ascending thoracic aortic aneurysms in Fabry disease. - Highlights: • Lyso-Gb3 causes elevation of intracellular cAMP. • Lyso-Gb3 inhibits the ERK 1/2 phosphorylation through PKA, thereby reducing KCa3.1 channel synthesis. • Lyso-Gb3 reduces PI3KC2β-mediated intracellular PI(3)P production. • Lyso-Gb3 reduces both surface and total expression of the KCa3.1 channel. • Increasing intracellular levels of PI(3)P only recovers the reduced surface expression.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2016

Sustained expression of GLP-1 receptor differentially modulates β-cell functions in diabetic and nondiabetic mice

Glucagon-like peptide 1 (GLP-1) has been shown to play important roles in maintaining β-cell functions, such as insulin secretion and proliferation. While expression levels of GLP-1 receptor (Glp1r) are compromised in the islets of diabetic rodents, it remains unclear when and to what degree Glp1r mRNA levels are decreased during the progression of diabetes. In this study, we performed real-time PCR with the islets of db/db diabetic mice at different ages, and found that the expression levels of Glp1r were comparable to those of the islets of nondiabetic db/misty controls at the age of four weeks, and were significantly decreased at the age of eight and 12 weeks. To investigate whether restored expression of Glp1r affects the diabetic phenotypes, we generated the transgenic mouse model Pdx1{sup PB}-CreER{sup TM}; CAG-CAT-Glp1r (βGlp1r) that allows for induction of Glp1r expression specifically in β cells. Whereas the expression of exogenous Glp1r had no measurable effect on glucose tolerance in nondiabetic βGlp1r;db/misty mice, βGlp1r;db/db mice exhibited higher glucose and lower insulin levels in blood on glucose challenge test than control db/db littermates. In contrast, four weeks of treatment with exendin-4 improved the glucose profiles and increased serum insulin levels in βGlp1r;db/db mice, to significantly higher levels than those in control db/db mice. These differential effects of exogenous Glp1r in nondiabetic and diabetic mice suggest that downregulation of Glp1r might be required to slow the progression of β-cell failure under diabetic conditions. - Highlights: • Expression levels of incretin receptors were significantly decreased in diabetic db/db islets after the age of eight weeks. • A transgenic mouse model expressing Glp1r specifically in β cells was generated. • Exogenous expression of Glp1r in β cells did not affect metabolic profiles in nondiabetic mice. • Sustained expression of Glp1r in diabetic db/db β cells deteriorated glucose profiles, but significantly improved insulin secretion after treatment with a GLP-1 analog.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2016

LncRNA FER1L4 suppresses cancer cell proliferation and cycle by regulating PTEN expression in endometrial carcinoma

Background: Dysregulated long non-coding RNAs (lncRNAs) might exert key roles in pathways associated with endometrial carcinoma (EC) development. This study aims to investigate the new role of lncRNA FER1L4 in EC pathogenesis due to its correlation with phosphatase and tensin homolog (PTEN), one important indicator of EC progression. Methods: Real time PCR was performed to detect the expression of FER1L4 in thirty paired EC samples and two EC cell lines. Plasmid containing FER1L4 was transfected into HEC-50 cells with a relative lower level of FER1L4 expression, followed which PTEN expression and Akt phosphorylation were measured by western blotting. Cell proliferation was analyzed through MTT and colony-formation assays, while cell cycle and apoptosis were determined by flow cytometry. Results: FER1L4 showed significantly downregulation in EC tissues compared to control, which was positively correlated with decreased PTEN expression. Moreover, FER1L4 could promote PTEN expression and inhibit Akt phosphorylation. Additionally, a significant decrease of cell proliferation was observed in FER1L4 overexpressing cells, along with cell cycle arrest at G0/G1 phase and increased proportion of apoptotic cells. Conclusion: FER1L4 not only showed downregulation in EC tissues and cells, but also regulated PTEN expression and Akt signaling, which might contribute to its inhibition on cell proliferation. This study might provide a new potential therapeutic target for EC treatment. - Highlights: •FER1L4 expression was markedly downregulated in EC tissues compared to control. •FER1L4 downregulation was positively correlated with decreased PTEN expression. •FER1L4 could promote PTEN expression and inhibit Akt phosphorylation. • FER1L4 showed significant inhibition on EC cell proliferation. • FER1L4 also induced cell cycle arrest and apoptosis promotion in EC cells.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2016

ID4 promotes AR expression and blocks tumorigenicity of PC3 prostate cancer cells

Deregulation of tumor suppressor genes is associated with tumorigenesis and the development of cancer. In prostate cancer, ID4 is epigenetically silenced and acts as a tumor suppressor. In normal prostate epithelial cells, ID4 collaborates with androgen receptor (AR) and p53 to exert its tumor suppressor activity. Previous studies have shown that ID4 promotes tumor suppressive function of AR whereas loss of ID4 results in tumor promoter activity of AR. Previous study from our lab showed that ectopic ID4 expression in DU145 attenuates proliferation and promotes AR expression suggesting that ID4 dependent AR activity is tumor suppressive. In this study, we examined the effect of ectopic expression of ID4 on highly malignant prostate cancer cell, PC3. Here we show that stable overexpression of ID4 in PC3 cells leads to increased apoptosis and decreased cell proliferation and migration. In addition, in vivo studies showed a decrease in tumor size and volume of ID4 overexpressing PC3 cells, in nude mice. At the molecular level, these changes were associated with increased androgen receptor (AR), p21, and AR dependent FKBP51 expression. At the mechanistic level, ID4 may regulate the expression or function of AR through specific but yet unknown AR co-regulators that may determine the final outcome of AR function. - Highlights: • ID4 expression induces AR expression in PC3 cells, which generally lack AR. • ID4 expression increased apoptosis and decreased cell proliferation and invasion. • Overexpression of ID4 reduces tumor growth of subcutaneous xenografts in vivo. • ID4 induces p21 and FKBP51 expression- co-factors of AR tumor suppressor activity.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2017

lncRNA-Map2k4 sequesters miR-199a to promote FGF1 expression and spinal cord neuron growth

Spinal cord injury (SCI) is a common critical illness in clinical practice. SCI prevention, treatment and rehabilitation have become important topics in today's medical profession. Studies have shown that long noncoding RNAs (lncRNAs) also play an important role in the pathology of SCI. The biology software analysis identified miR-199a binding sites in the lncRNA-Map2k4 and FGF1 sequences, which were confirmed by the subsequent dual luciferase reporter assay. When lncRNA-Map2k4 expression was down-regulated by siRNA, miR-199a expression in neurons was up-regulated and FGF1 expression was down-regulated. In turn, miR-199a up-regulation inhibited lncRNA-Map2k4 and FGF1 expression. But when lncRNA-Map2k4-m (a lncRNA-Map2k4 overexpression vector with mutated miR-199a binding sites) was co-transfected into neuronal cells with miR-199a mimics, lncRNA-Map2k4-m over-expression did not block the inhibition of FGF1 expression by miR-199a. Moreover, lncRNA-Map2k4 and FGF1 promoted the proliferation and inhibited the apoptosis of neuronal cells, whereas miR-199a down-regulated the aforementioned functions of lncRNA-Map2k4 and FGF1; however, lncRNA-Map2k4-m could not block the inhibitory action of miR-199a on proliferation. Thus, lncRNA-Map2k4 regulates neuronal proliferation and apoptosis through a miR-199a/FGF1 pathway. This finding provides more evidence for the role of lncRNAs in SCI. - Highlights: • lncRNA-Map2k4 is the target gene of miR-199a, and its down-regulation promotes miR-199a expression in neurons. • miR-199a targeted regulation of FGF1 expression in neurons. • Knockdown of lncRNA-Map2k4 inhibited the FGF1 expression by up-regulating miR-199a. • lncRNA-Map2k4 regulates neuron proliferation and apoptosis through an miR-199a/FGF1 pathway.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2017

Shear stress-mediated changes in the expression of complement regulatory protein CD59 on human endothelial progenitor cells by ECM-integrinαVβ3-F-actin pathway in vitro

Highlights: • Shear stress increases the expression of complement regulatory protein CD59 in human EPCs. • Shear stress-induced CD59 expression is closely related to extracellular matrix components. • Shear stress augments the expression of complement regulatory protein CD59 by ECM-integrin-F-actin pathway. Membrane regulatory proteins, such as CD46, CD55, and CD59, prevent excess complement activation and to protect cells from damage. Previous investigations confirmed that shear stress in the physiological range was more favorable for endothelial progenitor cells (EPCs) to repair injured vascular endothelial cells and operates mainly in atheroprotective actions. However, detailed events that contribute to shear stress-induced protection in EPCs, particularly the mechanisms of signal transduction, remain poorly understood. In this study, we observed shear stress-mediated changes in the expression of complement regulatory proteins CD46, CD55, and CD59 on human EPCs and focused on the mechanical transmission mechanism in transformed cells in response to the ECM-F-actin pathway in vitro. Shear stress was observed to promote the expression of complement regulatory protein CD59, but not CD46 or CD55, on EPCs. In addition, the shear stress-induced CD59 expression was confirmed to be associated with the ECM components and was alleviated in EPCs pretreated with GRGDSP, which inhibits ECM components-integrin interaction. Furthermore, shear stress also promotes the rearrangement and polymerization of F-actin. However, shear stress-induced CD59 expression was reduced when the F-actin stress fiber formation process was delayed by Gly-Arg-Gly-Asp-Ser-Pro (GRGDSP) or destroyed by cytochalasin D (Cyto D), while Jasplakinolide (JAS) reversed the expression of CD59 through promotion of F-actin polymerization and its stabilizing capacities. Our results indicates that shear stress is an important mediator in EPC expression of CD59 regulated by the ECM-F-actin pathway, which is a key factor in preventing membrane attack complex (MAC) -mediated cell autolysis.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Tyrosine kinase Fyn regulates iNOS expression in LPS-stimulated astrocytes via modulation of ERK phosphorylation

Highlights: • Fyn regulates LPS-induced iNOS expression in the cortices. • Fyn modulates iNOS expression and nitrite levels in LPS-stimulated astrocytes. • Fyn regulates iNOS expression in LPS-stimulated astrocytes via ERK phosphorylation. The high concentrations of nitric oxide (NO) produced by inducible nitric oxide synthase (iNOS) in activated glial cells in response to neuroinflammatory stimuli have neurotoxic effects on the brain. At basal levels, iNOS expression is low, and proinflammatory stimuli induce iNOS expression in astrocytes, microglia, and oligodendrocytes. Fyn, a non-receptor tyrosine kinase, regulates iNOS expression in several types of immune cells. However, its role in stimulated astrocytes is less clear. In this study, we investigated the role of Fyn in the regulation of lipopolysaccharide (LPS)-induced iNOS expression in astrocytes from mice and rats. Intracerebroventricular LPS injections in cortical regions enhanced iNOS mRNA and protein levels, which were increased in Fyn-deficient mice. Accordingly, LPS-induced nitrite production was enhanced in primary astrocytes cultured from Fyn-deficient mice or rats. Similar results were observed in cultured astrocytes after the siRNA-induced knockdown of Fyn expression. Finally, we observed increased LPS-induced extracellular signal-regulated protein kinase (ERK) activation in Fyn-deficient astrocytes. These results suggested that Fyn has a regulatory role in iNOS expression in astrocytes during neuroinflammatory responses.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

IL-13 regulates IL-17C expression by suppressing NF-κB-mediated transcriptional activation in airway epithelial cells

Highlights: • IL-1β markedly elevated IL-17C expression viathe NF-κB-signaling pathway in normal human bronchial epithelial cells. • IL-13 attenuated IL-1β-mediated IL-17C expression via a STAT6-signaling pathway. • IL-1β enhanced binding of the NF-κB p65 sub unit to two IL-17C promoter regions (−130/−120 and −157/−147). • IL-13 attenuated IL-1β-mediated p65 binding to IL-17C promoter regions, resulting in lower IL-17C expression. The cytokine interleukin (IL)-17C is highly expressed in epithelial tissues and involved in innate immune responses; however, the regulation of IL-17C expression in the airways remains poorly understood. Here, we show that IL-1β strongly induces both IL-17C mRNA and protein expression in primary normal human bronchial epithelial cells. Conversely, IL-13 significantly reduced the IL-1β-induced IL-17C expression. Attenuation of the nuclear factor (NF)-κB-signaling pathway using an NF-κB-subunit p65-specific small-interfering RNA (siRNA), reduced IL-1β-induced IL-17C expression, demonstrating the importance of NF-κB signaling in IL-17C regulation. The inhibitory effects of IL-13 on IL-17C expression were abolished when the Janus kinase (JAK)/signal transducer and activator of transcription 6 (STAT6)-signaling pathway was impaired, using either the JAK inhibitor ruxolitinib or a STAT6-specific siRNA. Western blot analysis demonstrated that IL-1β promoted both IκB-α phosphorylation and degradation, and p65 nuclear translocation. Although IL-13 induced STAT6 phosphorylation and nuclear translocation, it did not affect the activation of the IL-1β-mediated NF-κB-pathway. Using chromatin immunoprecipitation, we confirmed that IL-1β enhanced p65 binding to regions within the IL-17C promoter that flank putative NF-κB-binding sites (−130/−120 and −157/−147). Interestingly, IL-13 treatment reduced the IL-1β-mediated p65 binding to these regions. These findings demonstrate that NF-κB-mediated transcriptional mechanisms are critically involved in the IL-1β-mediated IL-17C induction, and that IL-13 negatively regulates this induction by suppressing NF-κB-based transcriptional activation.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

KSHV vIRF4 enhances BCL6 transcription via downregulation of IRF4 expression

Highlights: • KSHV vIRF4 enhances the BCL6 expression by inhibiting IRF4 expression. • vIRF4 binds to the promoter region of IRF4 and suppresses IRF4 expression. • vIRF4-mediated deregulation of BCL6-IRF4 axis requires efficient KSHV lytic gene expression. Primary effusion lymphoma (PEL), strongly linked with latent infection of Kaposi's sarcoma-associated herpesvirus (KSHV), constitutively expresses cellular interferon regulatory factor 4 (IRF4) while suppressing the expression of B cell lymphoma 6 (BCL6). Recently, it was shown that IRF4, a key transcriptional repressor of BCL6, might be a pivotal regulator of KSHV for balancing between latency and its reactivation in PEL cells. However, the action of the BCL6-IRF4 transcription factor axis during KSHV's life cycle is not clear. Herein we found that the KSHV lytic protein viral interferon regulatory factor 4 (vIRF4) dramatically enhanced the transcriptional activity of the BCL6 through the inhibition of its negative regulator IRF4. Using a chromatin immunoprecipitation (ChIP) assay, we further showed that vIRF4 bound to the specific promoter region of IRF4, contributing to a dramatic suppression of IRF4 gene expression. Correspondingly, we also found BCL6 expression to be positively and inversely correlated with vIRF4 and IRF4 expression, respectively, during KSHV reactivation. Finally, we observed that these processes require efficient KSHV lytic replication. Thus, our findings suggest a crucial role of the BCL6-IRF4 axis in triggering the transition between KSHV latency and lytic reactivation.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Corin is highly expressed in atherosclerosis models

Highlights: • Tested the efficacy of different commercial corin antibodies. • Corin was basally expressed in aorta and induced in atherosclerotic mouse model. • Corin was expressed in vascular endothelial cells and induced by ox-LDL stimulation. • Demonstrated corin involvement in atherosclerosis. Corin is a serine protease mainly expressed in the heart, where it regulates blood pressure and cardiac function through activating pro-atrial natriuretic peptide (pro-ANP) to ANP. Its expression has also been detected in non-cardiac tissues. However, there is no report so far about the distribution and function of corin in aorta and in related diseases such as atherosclerosis. This study was the first to explore corin expression in aorta both under normal conditions and in atherosclerosis models. In vivo, we found corin had a basal level of expression in aortas, mainly in intimal endothelial cells and was significantly elevated in mouse atherosclerosis model. Moreover, we observed pro-ANP, the specific substrate of corin, was also expressed in mice aortas and increased in mouse atherosclerosis model. In vitro, we further demonstrated corin expression in cultured vascular endothelial cells and its induced expression after ox-LDL stimulation. Our results suggested that corin may play important roles in aorta physiology and in the pathophysiological process of atherosclerosis in an autocrine manner and has potential clinical value for the treatment of atherosclerosis.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Molecularly targeted anti-cancer drugs inhibit the invasion and metastasis of hepatocellular carcinoma by regulating the expression of MMP and TIMP gene families

Highlights: • The multi-target drugs of liver cancer bringing new hope for advance HCC patients. • Sorafenib, regorafenib, and lenvatinib have inhibitory effect on the metastasis and invasion. • They can down-regulate the expression of MMPs and up-regulate the TIMPs level. To investigate the effect of multi-kinase kinase inhibitors (sorafenib; regorafenib; lenvatinib) on the invasion and metastasis of human hepatocellular carcinoma (HCC) cells, and the outcome of this effect on the expression of matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs), yet unclarified. Cells were subjected to four different treatments: blank control group, sorafenib (10 μmol/L) treatment group, regorafenib (20 mmol/L) treatment group, and lenvatinib (4 μmol/L) treatment group. Anti-invasion and anti-metastasis effects were tested using the wound-healing assay and transwell invasion assay. Real-time PCR and Western blot analyses were used to determine the impact of sorafenib, regorafenib, and lenvatinib on the gene expression of MMPs and TIMPs in the two HCC lines (Hep3B and SMMC-7721). Results from the wound-healing and transwell invasion assays showed the three tested anti-cancer drugs to have a significant inhibitory effect on the metastasis and invasion of HCC cells. Real-time PCR and western blot analyses revealed that sorafenib down-regulated the expressions of MMP-7,10,16 and up-regulated those of TIMP-1,3,4, regorafenib down-regulated the expression of MMP-1 and up-regulated TIMP-3 gene expression, and lenvatinib down-regulated the expressions of MMP-1,2,7,9,10,16 and up-regulated those of TIMP-1,3,4. However, these three targeted anti-cancer drugs seem to have no significant regulatory effect on the expressions of other MMPs and TIMPs family genes. In conclusion, sorafenib, regorafenib, and lenvatinib inhibit the invasion and metastasis of HCC cells by regulating MMPs/TIMPs expression levels.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

δ-Tocopherol promotes thermogenic gene expression via PGC-1α upregulation in 3T3-L1 cells

Highlights: • PGC-1a and UCP1 gene expressions were significantly increased by d-tocopherol treatment in 3T3 L1 cells. • PGC-1a and UCP1 protein expressions were increased in d-tocopherol-treated cells. • Nuclear translocation of PGC-1a was enhanced in d-tocopherol-treated cells. • PGC-1a knockdown canceled the induction of UCP1 gene expression by d-tocopherol treatment. • Expression and activation of p38 MAPK was enhanced by d-tocopherol treatment. Activation of thermogenic adipocytes (brown and beige) has been considered an attractive target for weight loss and treatment of metabolic disease. Peroxisome proliferator-activated receptor γ co-activator-1 α (PGC1-α) is a master regulator of thermogenic gene expression in thermogenic adipocytes. We previously reported that α-tocopherol upregulated PGC-1α gene expression and promoted thermogenic adipocyte differentiation in mammalian adipocytes. In this study, we investigated the effects of the vitamin E analogs (α-, γ- and δ-tocopherol) on PGC-1α and uncoupling protein 1 (UCP1) gene expression in 3T3-L1 cells. The expression of PGC-1α and UCP1 increased significantly with the addition of δ-tocopherol. In δ-tocopherol-treated cells, nuclear translocation of PGC-1α increased, as did p38 mitogen-activated protein kinase (MAPK) expression and phosphorylation. Our results suggest that p38 MAPK activation by δ-tocopherol contributes to PGC-1α activation and UCP1 induction.

60 APPLIED LIFE SCIENCES↗