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At least 955 records · Page 53
All sourcesNASA NTRSDOE OSTI
DOE OSTI2016

mTORC1 is a critical mediator of oncogenic Semaphorin3A signaling

Aberration of signaling pathways by genetic mutations or alterations in the surrounding tissue environments can result in tumor development or metastasis. However, signaling molecules responsible for these processes have not been completely elucidated. Here, we used mouse Lewis lung carcinoma cells (LLC) to explore the mechanism by which the oncogenic activity of Semaphorin3A (Sema3A) signaling is regulated. Sema3A knockdown by shRNA did not affect apoptosis, but decreased cell proliferation in LLCs; both the mammalian target of rapamycin complex 1 (mTORC1) level and glycolytic activity were also decreased. In addition, Sema3A knockdown sensitized cells to inhibition of oxidative phosphorylation by oligomycin, but conferred resistance to decreased cell viability induced by glucose starvation. Furthermore, recombinant SEMA3A rescued the attenuation of cell proliferation and glycolytic activity in LLCs after Sema3A knockdown, whereas mTORC1 inhibition by rapamycin completely counteracted this effect. These results demonstrate that Sema3A signaling exerts its oncogenic effect by promoting an mTORC1-mediated metabolic shift from oxidative phosphorylation to aerobic glycolysis. -- Highlights: •Sema3A knockdown decreased proliferation of Lewis lung carcinoma cells (LLCs). •Sema3A knockdown decreased mTORC1 levels and glycolytic activity in LLCs. •Sema3A knockdown sensitized cells to inhibition of oxidative phosphorylation. •Sema3A promotes shift from oxidative phosphorylation to aerobic glycolysis via mTORC1.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2016

Matrine suppresses airway inflammation by downregulating SOCS3 expression via inhibition of NF-κB signaling in airway epithelial cells and asthmatic mice

Matrine has been demonstrated to attenuate allergic airway inflammation. Elevated suppressor of cytokine signaling 3 (SOCS3) was correlated with the severity of asthma. The aim of this study was to investigate the effect of matrine on SOCS3 expression in airway inflammation. In this study, we found that matrine significantly inhibited OVA-induced AHR, inflammatory cell infiltration, goblet cell differentiation, and mucous production in a dose-dependent manner in mice. Matrine also abrogated the level of interleukin (IL)-4 and IL-13, but enhanced interferon (IFN)-γ expression, both in BALF and in lung homogenates. Furthermore, matrine impeded TNF-α-induced the expression of IL-6 and adhesion molecules in airway epithelial cells (BEAS-2B and MLE-12). Additionally, we found that matrine inhibited SOCS3 expression, both in asthmatic mice and TNF-α-stimulated epithelial cells via suppression of the NF-κB signaling pathway by using pcDNA3.1-SOCS3 plasmid, SOCS3 siRNA, or nuclear factor kappa-B (NF-κB) inhibitor PDTC. Conclusions: Matrine suppresses airway inflammation by downregulating SOCS3 expression via inhibition of NF-κB signaling in airway epithelial cells and asthmatic mice. - Highlights: • Matrine attenuates asthmatic symptoms and regulates Th1/Th2 balance in vivo. • Matrine suppresses inflammation responses in vitro. • Matrine decreases SOCS3 expression both in vivo and in vitro. • Matrine inhibits SOCS3 expression by suppressing NF-κB signaling.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Shaping mitochondrial dynamics: The role of cAMP signalling

Highlights: • Mitochondrial dynamics are controlled by mitochondrial and cellular signalling pathways. • Mitochondria host cAMP/PKA signalling microdomains. • cAMP is a key player in the orchestration of mitochondrial dynamics. • PKA phosphorylation of mitochondrial targets regulates fission, mitophagy & motility. In recent years, our idea of mitochondria evolved from “mere” energy and metabolite producers to key regulators of many cellular functions. In order to preserve and protect their functional status, these organelles engage a number of dynamic processes that allow them to decrease accumulated burden and maintain their homeostasis. Indeed, mitochondria can unite (fusion), divide (fission), position themselves strategically in the cell (motility/trafficking) and if irreversibly damaged or dysfunctional eliminated (mitophagy). These dynamic processes can be controlled both by mitochondrial and cellular signalling pathways, hence allowing mitochondria to tune their function to the cellular needs. Among the regulatory mechanisms, reversible phosphorylation downstream the cyclic AMP (cAMP) signalling cascade was shown to deeply influence mitochondrial dynamics. This review explores the emerging evidence suggesting that cAMP is a key player in the orchestration of mitochondrial fusion/fission, motility and mitophagy, extending the repertoire of this second messenger, which is now recognised as a major regulator of mitochondrial homeostasis.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2017

Stearic acid suppresses mammary gland development by inhibiting PI3K/Akt signaling pathway through GPR120 in pubertal mice

It has been demonstrated that dietary high fat diet negatively affects the pubertal mammary gland development. The aim of the present study was to investigate the effects of stearic acid (SA), an 18-carbon chain saturated fatty acid, on mammary gland development in pubertal mice and to explore the underlying mechanism. Our results demonstrated that dietary supplementation of 2% SA suppressed mammary duct development, with significant reduction of terminal end bud (TEB) number and ductal branch. In accord, the expression of proliferative marker Cyclin D1 was markedly decreased by dietary SA. Furthermore, dietary SA led to increase of G protein-coupled receptor 120 (GPR120) expression and inhibition of PI3K/Akt signaling pathway in mammary gland of pubertal mice. In good agreement with the in vivo findings, the in vitro results showed that 40 μM SA significantly suppressed proliferation of mouse mammary epithelial cell HC11 by regulating mRNA and/or protein expression of proliferative markers such as Cyclin D1/3, p21, and PCNA. Meanwhile, SA activated GPR120 and inhibited PI3K/Akt signaling pathway in a GPR120-dependent manner. In addition, SA-induced inhibition of PI3K/Akt signaling pathway, suppression of HC11 proliferation, and alteration of proliferative markers expression were abolished by knockdown of GPR120 with siRNA. Collectively, these findings showed that SA suppressed mammary gland development of pubertal mice, which was coincident with the SA-inhibited HC11 proliferation, and was associated with inhibition of PI3K/Akt signaling pathway through activation of GPR120. These data provided new insights into the regulation of mammary gland development by dietary fatty acids. - Highlights: • Dietary supplementation of 2% SA suppressed mammary duct growth in pubertal mice. • SA inhibited HC11 proliferation by regulating expression of proliferative markers. • SA enhanced GPR120 and inhibited PI3K/Akt in mice mammary gland and HC11 cells. • Knockdown of GPR120 eliminated SA-suppressed PI3K/Akt and HC11 proliferation.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2017

MKK4 activates non-canonical NFκB signaling by promoting NFκB2-p100 processing

The NFκB family of transcription factors is crucial for innate or adaptive immunity, inflammation, and diseases including cancer. The two NFκB signaling pathways (canonical and non-canonical) differ from each other in extracellular signals, membrane receptors, signaling adaptors, and dimer subunits. The p52 (NFκB2) subunit, which participates in the non-canonical pathway, is generated by ubiquitin-mediated processing of the p100 precursor. Here, we found that NFκB2 processing and activation were mediated by mitogen-activated protein kinase kinase-4 (MKK4) and its substrate c-Jun N-terminal kinase (JNK). In MKK4-null mouse embryonic fibroblasts (MEFs), serum- and lymphotoxin β receptor (LTβR) antibody-induced processing of p100 and nuclear translocation of p52 were found to be defective. Serum and LTβR antibody activated the MKK4-JNK signaling pathway, and SP600125, a JNK inhibitor, blocked p100 processing. Cellular senescence, one of the responses regulated by the non-canonical NFκB pathway, was observed more frequently in MKK4-null MEFs than in wildtype cells. These results suggest that the MKK4/JNK-dependent pathway regulates NFκB2 processing/activation and, through this mechanism, MKK4 and NFκB2 control cellular growth and senescence. - Highlights: • MKK4 is required for processing and activation of non-canonical NFκB2-p100. • Nuclear translocation of NFκB2-p52 is dependent on ERK activity. • MKK4 depletion and NFκB2 inactivation induces cellular senescence.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Edaravone attenuates lipopolysaccharide-induced acute respiratory distress syndrome associated early pulmonary fibrosis via amelioration of oxidative stress and transforming growth factor-β1/Smad3 signaling

Highlights: • The ARDS associated early pulmonary fibrosis in rat can be induced by injecting LPS. • Edaravone has anti-fibrosis activities against ARDS associated lung fibrosis. • The mechanism relies on amelioration of oxidative stress and TGF-β1/Smad3 signaling. Pulmonary fibrosis is responsible for the both short-term and long-term outcomes in patients with acute respiratory distress syndrome (ARDS). There is still no effective cure to improve prognosis. The purpose of this study was to investigate whether edaravone, a free radical scavenger, have anti-fibrosis effects in the rat model of ARDS associated early pulmonary fibrosis by lipopolysaccharide (LPS) administration. Rats were subjected to intravenous injection of LPS, and edaravone was given intraperitoneally after LPS administration daily for 7 consecutive days. LPS treatment rapidly increased lung histopathology abnormalities, coefficient of lung, hydroxyproline and collagen I levels, stimulated myofibroblast differentiation and induced expression of TGF-β1 and activation of TGF-β1/Smad3 signaling as early as day 7 after LPS injection. Moreover, LPS intoxication significantly increased the contents of malondialdehyde (MDA), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α), whereas it dramatically decreased superoxide dismutase (SOD) and glutathione peroxidase (GSH-PX) activities from day 1 after LPS treatment. On the contrary, edaravone treatment ameliorated LPS-induced myofibroblast differentiation and pulmonary fibrosis, simultaneously, and attenuated LPS-stimulated oxidative stress and activation of TGF-β1/Smad3 signaling. Collectively, edaravone may attenuate ARDS associated early pulmonary fibrosis through amelioration of oxidative stress and TGF-β1/Smad3 signaling pathway. Edaravone may be a promising drug candidate for the treatment of ARDS-related pulmonary fibrosis in early period.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Impaired vagus function in rats suppresses bile acid synthesis in the liver by disrupting tight junctions and activating Fxr-Fgf15 signaling in the intestine

Highlights: • Unconjugated and glycine-conjugated bile acids were increased in vagotomized rats. • The bile acid synthetic enzyme Cyp7a1 was suppressed in vagotomized rats. • Intestinal Fxr-Fgf15 signaling was activated in vagotomized rats. • Tight junction of the intestine was impaired in vagotomized rats. Bile acids (BAs) circulate between the liver and intestine, and regulate the homeostasis of glucose, lipid, and energy. Recent studies demonstrated an essential role of BAs in neurological diseases, suggesting an interaction between BAs and the nervous system. In the present study, we showed that impaired vagus function in rats induced by vagotomy resulted in an increase in bile flow without causing liver injury. The concentrations of unconjugated and glycine-conjugated BAs were increased in both serum and bile of rats after vagotomy, which was due to impaired tight junctions and thus increased passive absorption of BAs in the intestine. Vagotomy markedly suppressed the expression of the rate-limiting BA synthetic enzyme Cyp7a1, which was not due to activation of Fxr-Shp signaling in the liver, but due to activation of Fxr-Fgf15 signaling in the intestine. Furthermore, vagotomy produced a BA profile in the bile favorable for Fxr activation by decreasing tauro-β-muricholic acid, a natural Fxr antagonist, and increasing glyco-chenodeoxycholic acid, a natural Fxr agonist. In summary, the present study provides the first comprehensive analysis of the critical role of the vagus nerve in regulating BA metabolism and signaling pathway.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

CtBP represses Dpp signaling as a dimer

Highlights: • Ectopic expression of CtBP causes a disrupted wing vein pattern. • CtBP causes a decreased pMad level. • CtBP requires dimerization to repress Dpp signaling pathway. C-terminal binding protein (CtBP) is a highly conserved transcriptional co-repressor in animal development and human diseases. In Drosophila, CtBP is critical for fly development and is thought to exert its repressive roles in many signaling pathways including Dpp/BMP pathway. Here we provide evidence that although wild type CtBP negatively and dominantly influences Dpp signaling in fly presumptive wings, mutant CtBP unable to form dimer does not, indicating that dimerization is required for the repression role of CtBP in Dpp signaling in vivo.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Inhibition of Notch signaling pathway using γ-secretase inhibitor delivered by a low dose of Triton-X100 in cultured oral cancer cells

Highlights: • A novel method is developed to deliver γ-secretase inhibitor DAPT using a low dose of Triton-X100. • This approach effectively blocks the cleavage of Notch1 receptor. • The elimination of Notch1 intracellular domain from cell nuclei is confirmed. • Notch1 signaling blockade in mRNA level is demonstrated upon applying this delivery system. How to effectively delivering therapeutic agents, including γ-secretase inhibitors (GSIs), into live cells, remains a significant challenge. This study assessed the effect of Notch signaling inhibition by examining levels of the Notch1 intracellular domain (N1ICD) in cultured oral cancer cells analyzed with random stitched images (2D) and 3D visualizations using confocal microscopy and quantitative gene analysis. Substantially, we have developed a novel method to assist the delivery of γ-secretase inhibitor, DAPT, into live cells in the presence of an effective minimum concentration of Triton-X100 (0.001%) without damaging cell activity and membrane integrity assessed with cell proliferation assays. The images obtained in this study showed that DAPT alone could not block the γ-secretase inhibitor despite inhibiting cell growth. Further analysis of quantitative gene expressions of Notch signaling canonical pathway to verify the effectiveness of the novel method for delivering inhibitor into live cells, displayed deregulation of Notch1, Delta-like ligand 1 (DLL1) and hairy and enhancer of split 1 (Hes1). Our data suggest that Notch1/Hes1 signaling pathway is deactivated using DAPT with a low dose of Triton-X100 in this cancer cells. And the finding also suggests that Notch1 could be engaged by DLL1 to promote differentiation in oral cancer cells. Using this approach, we demonstrate that Triton-X100 is a promising and effective permeabilization agent to deliver γ-secretase inhibitor DAPT into live oral epithelial cells. This strategy has the potential to implicate in the treatment of cancer diseases.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

BMP4 signaling in NPCs upregulates Bcl-xL to promote their survival in the presence of FGF-2

Highlights: • BMP4 promoted survival of neural stem/progenitor cells (NPCs) in the presence of FGF-2. • BMP4 activated Smad1/5/8 signaling and promoted Id1 and Bcl-xL expression. • BMP4 inhibited cytochrome c release from mitochondria via Bcl-xL expression. We previously reported that BMP4 does not promote proliferation or differentiation of CD44-positive astrocyte precursor cells (APCs) but greatly promotes their survival in the presence of fibroblast growth factor-2 (FGF-2). In this study, we examined if BMP4 acts as a survival factor also for neural stem/progenitor cells (NPCs) isolated from ganglionic eminence of neonatal mouse brain. We found BMP4 promotes survival but not proliferation or differentiation of these cells, just as in the case for CD44-positive APCs. Microarray analysis revealed some candidate molecules in the signaling pathway downstream of BMP4. Among them, we focused on Id1 (inhibitor of DNA-binding 1) and Bcl-xL in this study. Expression of both genes was promoted in the presence of BMP4, and this promotion was reduced by dorsomorphin, an inhibitor of BMP4 signaling. Furthermore, cytochrome c release from mitochondria was significantly reduced in the presence of BMP4, suggesting up-regulation of Bcl-xL activity by BMP4. Id1 siRNA reduced the expression of Bcl-xL, and negated survival promoting effect of BMP4. These data suggest that BMP4 promotes survival of NPCs by enhancing the anti-apoptotic function of Bcl-xL via BMP4-Smad1/5/8-Id1 signaling.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Perturbations in carotenoid and porphyrin status result in differential photooxidative stress signaling and antioxidant responses

Highlights: • Two modes of photooxidation by inhibition of carotenoid and porphyrin biosynthesis. • We examine differential photooxidative stress signaling and antioxidant responses. • OF results in greater oxidative damage with respect to membrane integrity than NF. • Free SA and redox state of Asc and GSH have roles in protection from photooxidation. • Differential regulation of ROS-scavenging enzymes between NF and OF plants. We examined differential photooxidative stress signaling and antioxidant responses in rice plants treated with norflurazon (NF) and oxyfluorfen (OF), which are inhibitors of carotenoid and porphyrin biosynthesis, respectively. Plants treated with OF markedly increased levels of cellular leakage and malondialdehyde, compared with NF-treated plants, showing that OF plants suffered greater oxidative damage with respect to membrane integrity. The enhanced production of H{sub 2}O{sub 2} in response to OF, but not NF, indicates the important role of H{sub 2}O{sub 2} in activation of photooxidative stress signaling in OF plants. In response to NF and OF, the increased levels of free salicylic acid as well as maintenance of the redox ratio of ascorbate and glutathione pools to a certain level are considered to be crucial factors in the protection against photooxidation. Plants treated with OF greatly up-regulated catalase (CAT) activity and Cat transcript levels, compared with NF-treated plants. Interestingly, NF plants showed no noticeable increase in oxidative metabolism, although they did show considerable increases in ascorbate peroxidase (APX) and peroxidase activities and transcript levels of APX, as in OF plants. Our results suggest that perturbations in carotenoid and porphyrin status by NF and OF can be sensed by differential photooxidative stress signaling, such as that involving H{sub 2}O{sub 2}, redox state of ascorbate and glutathione, and salicylic acid, which may be responsible for at least part of the induction of ROS-scavenging enzymes.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

FTO is involved in Alzheimer's disease by targeting TSC1-mTOR-Tau signaling

Diabetes and obesity are commonly associated with Alzheimer's disease (AD). Accumulating evidence show that insulin signaling defects are protentional upstream driver of AD. However, the mechanism by which diabetes and insulin signaling defects contribute to AD remains unknown. Here we show that Fat mass and obesity-associated protein (FTO) is involved the insulin defects-associated AD. Defective insulin signaling in diabetes and obesity in human and mice activated Fto in the brain tissues. Lentivirus-mediated knockdown of Fto reduced the phosphorylation of Tau protein whereas overexpression of FTO promoted the level of phosphorylated Tau in neurons. Mechanism study demonstrated that FTO activated the phosphorylation of Tau in a mTOR-dependent manner because FTO activated mTOR and its downstream signaling and rapamycin blocked FTO-mediated phosphorylation of Tau. FTO promoted the activation of mTOR by increasing the mRNA level of TSC1 but not TSC2, the upstream inhibitor of mTOR. Finally, we found that conditional knockout of Fto in the neurons reduced the cognitive deficits in 3xTg AD mice. Collectively, our evidence demonstrated that FTO is critically involved in insulin defects-related AD.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Molecular identification of BrHAB2a, one of the two AtHAB2-like proteins in Brassica rapa, is an important component of ABA signaling

Highlights: • Fourteen putative PP2C-A-like phosphatases were identified in Brassica rapa. • BrHAB2a was verified as a putative negative regulator of ABA signaling. • The inhibition of BrHAB2a by ABA and its receptors was successfully recapitulated. Abscisic acid (ABA) signaling is a vital physiological step that is used by many land plants to fight against environmental stress. As components of the linear ABA signaling pathway, clade A protein phosphatases type 2C (PP2C-As) are mainly inhibited by PYRABACTIN RESISTANCE1/PYR1-LIKE/REGULATORY COMPONENTS OF ABA RECEPTORS (PYLs)-type receptors upon their binding to ABA. Here, we show that the genome of Brassica rapa encodes 14 putative clade A PP2C-like proteins (BrPP2C-As). Two of these BrPP2C-As, Bra025964 and Bra016595, show high similarity to the HAB2 (Homology to ABI2) protein in Arabidopsis. RNAseq data reveal that nearly all BrPP2C-As, like BrHAB2a (Bra025964) and BrHAB2b (Bra016595), were highly expressed in at least one tissue. Overexpression of BrHAB2a conferred ABA insensitivity to Arabidopsis thaliana seedlings. Furthermore, the phosphatase activity of BrHAB2a could be inhibited by AtPYL1 or BrPYL1 in the presence of ABA. Overall, these results suggest that BrHAB2a is a functional PP2C-A like protein phosphotase and a key component of ABA signaling in Brassica rapa.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

MicroRNA-139-5p suppresses myosin heavy chain I and IIa expression via inhibition of the calcineurin/NFAT signaling pathway

Highlights: • MiR-139-5p was higher in mouse slow-twitch than in fast-twitch muscle. • MiR-139-5p overexpression downregulated the expression of MyHC I and MyHC IIa. • CaN/NFAT signaling was notably inhibited by miR-139-5p overexpression. • MiR-139-5p suppressed MyHC I and IIa expression via inhibiting CaN/NFAT signaling. MicroRNAs (miRNAs) are a class of small non-coding RNAs that are widely involved in a variety of biological processes. Different skeletal muscle fiber type composition exhibits characteristic differences in functional properties and energy metabolism of skeletal muscle. However, the molecular mechanism by which miRNAs control the different type of muscle fiber formation is still not fully understood. In the present study, we characterized the role of microRNA-139-5p (miR-139-5p) in the regulation of myosin heavy chain (MyHC) isoform expression and its underlying mechanisms. Here we found that the expression of miR-139-5p was significantly higher in mouse slow-twitch muscle than in fast-twitch muscle. Overexpression of miR-139-5p downregulated the expression of MyHC I and MyHC IIa, whereas inhibition of miR-139-5p upregulated them. We also found that the levels of calcineurin (CaN), NFATc1, MEF2C and MCIP1.4, which are the components of CaN/NFAT signaling pathway that has shown to positively regulate slow fiber-selective gene expression, were notably inhibited by miR-139-5p overexpression. Furthermore, treatment of phenylephrine (PE), a α1-adrenoceptor agonist, abolished the inhibitory effect of miR-139-5p on MyHC I and MyHC IIa expression. Together, our findings indicated that the role of miR-139-5p in regulating the MyHC isoforms, especially MyHC I and MyHC IIa, may be achieved through inhibiting CaN/NFAT signaling pathway.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Keratinocyte growth factor protects endometrial cells from oxygen glucose deprivation/re-oxygenation via activating Nrf2 signaling

Highlights: • Keratinocyte growth factor (KGF) receptor (KGFR) is functionally expressed in endometrial cells. • KGF protects endometrial cells from oxygen glucose deprivation/re-oxygenation (OGDR). • KGF inhibits OGDR-induced programmed necrosis in endometrial cells. • KGF activates Nrf2 signaling to protect endometrial cells from OGDR. • Akt-mTOR activation mediates KGF-induced Nrf2 activation and endometrial cell protection. Oxygen and glucose deprivation (OGD)-re-oxygenation (OGDR) exposure to endometrial cells mimics ischemia-reperfusion injury. The present study tests the potential effect of keratinocyte growth factor (KGF) on the process. We show that KGF receptor KGFR is expressed in human endometrial T-HESC cells and primary murine endometrial cells. KGF pre-treatment protected endometrial cells from OGDR, inhibiting cell viability reduction and cell death. KGF attenuated OGDR-induced programmed necrosis in endometrial cells. Significantly, KGF activated Nrf2 signaling, causing Nrf2 Ser-40 phosphorylation, protein stabilization, nuclear translocation to promote anti-oxidant gene (HO1, NOQ1 and GCLC) expression. Nrf2 silencing (by targeted shRNAs) or CRISPR/Cas9 knockout almost abolished KGF-induced endometrial cell protection against OGDR. Furthermore, KGF activated Akt-mTOR signaling in endometrial cells. Whereas Akt-mTOR inhibitors (LY294002, AZD2014 and RAD001) abolished KGF-induced Nrf2 activation and anti-OGDR cytoprotection. Together, KGF protects endometrial cells from OGDR via activating Akt-mTOR-Nrf2 signaling.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

PF-06409577 activates AMPK signaling to protect retinal pigment epithelium cells from UV radiation

Highlights: • PF-06409577 protects retinal pigment epithelium (RPE) cells from ultra-violet radiation (UVR). • PF-06409577 activates AMPK signaling in RPE cells. • AMPK activation mediates PF-06409577-induced RPE cytoprotection against UVR. • PF-06409577 activates NADPH and Nrf2 signaling in RPE cells. • PF-06409577 inhibits UVR-induced oxidative injury in RPE cells. Ultra-violet (UV) radiation (UVR) to human retinas induces oxidative injury to the resident retinal pigment epithelium (RPE) cells. PF-06409577 a novel, potent and direct AMP-activated protein kinase (AMPK) activator. In ARPE-19 cells and primary murine RPE cells, PF-06409577 significantly inhibited UVR-induced viability reduction, cell death and apoptosis. PF-06409577 activated AMPK signaling in RPE cells by increasing AMPKα1-acetyl-CoA carboxylase phosphorylation and AMPK activity. AMPK inhibition, by AMPKα1-shRNA, -CRISPR/Cas9 knockout or -T172A dominant negative mutation, almost abolished PF-06409577-induced RPE cytoprotection against UVR. PF-06409577 enhanced nicotinamide adenine dinucleotide phosphate (NADPH) activity and expression levels of Nrf2-dependent genes in RPE cells. Furthermore, UVR-induced reactive oxygen species (ROS) production, lipid peroxidation and DNA damage were largely inhibited by the AMPK activator. In summary, PF-06409577 inhibits UVR-induced oxidative stress and RPE cell death by activating AMPK signaling.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

TGF-β1 induced fascin1 expression facilitates the migration and invasion of kidney carcinoma cells through ERK and JNK signaling pathways

Highlights: • In 769-P and OSRC cell lines, TGF-β1 can increase the expression of fascin1. • TGF-β1 promoted the invasive and migratory capabilities of the two cell lines via increased the expression of fascin1. • TGF-β1 induced fascin1 expression through ERK and JNK signal pathways. Transforming growth factor-β1 (TGF-β1) plays a crucial role in the signaling network that controls cellular invasion and motility capability during tumor development. To investigate whether fascin1 plays a crucial role in TGF-β1-facilitated invasion and migration of kidney cancer cells (KCC), real-time PCR and western blotting were used to test the fascin1 expression after TGF-β1 treatment (10 ng/ml) in 769-P and OSRC cells. Fascin1 was silenced using the small interfering RNA (siRNA) technique. Cytoskeleton staining was used to test the change of Cytoskeleton. Cell migration and invasion changes were measured by wound-healing and Transwell assay. The results indicate that mRNA and protein levels of fascin1 were dramatically increased after treatment with 10 ng/ml TGF-β1 in 769-P and OSRC cells. TGF-β1 promoted the occurrence of EMT (Epithelial-Mesenchymal Transition) and the invasive and migratory capabilities of the two cell lines after treatment with 10 ng/ml TGF-β1. In addition, fascin1 siRNA dramatically attenuated the invasiveness and migration induced by TGF-β1. Furthermore, we identified that specific inhibitors of ERK and JNK signaling pathways, FR180204 and SP600125, can suppress TGF-β1-induced fascin1 expression. In conclusion, these results reveal that fascin1 is an important mediator of TGF-β1-induced invasion and migration of KCC through ERK and JNK signal pathways.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Neuroligin-3 protects retinal cells from H2O2-induced cell death via activation of Nrf2 signaling

Highlights: • NLGN3 protects retinal pigment epithelium (RPE) cells and retinal ganglion cells (RGCs) from H{sub 2}O{sub 2}. • NLGN3 inhibits H{sub 2}O{sub 2}-induced ROS production, lipid peroxidation and DNA damage in retinal cells. • NLGN3 efficiently activates Nrf2 signaling in human RPE cells and RGCs. • Nrf2 silencing abolishes NLGN3-induced anti-H{sub 2}O{sub 2} actions in human retinal cells. Intensified oxidative stress can cause severe damage to human retinal pigment epithelium (RPE) cells and retinal ganglion cells (RGCs). The potential effect of neuroligin-3 (NLGN3) against the process is studied here. Our results show that NLGN3 efficiently inhibited hydrogen peroxide (H{sub 2}O{sub 2})-induced death and apoptosis in human RPE cells and RGCs. H{sub 2}O{sub 2}-induced reactive oxygen species (ROS) production, lipid peroxidation and DNA damage in retinal cells were alleviated by NLGN3. NLGN3 activated nuclear-factor-E2-related factor 2 (Nrf2) signaling, enabling Nrf2 protein stabilization, nuclear translocation and expression of key anti-oxidant enzymes (HO1, NOQ1 and GCLC) in RPE cells and RGCs. Further results demonstrate that NLGN3 activated Akt-mTORC1 signaling in retinal cells. Conversely, Akt-mTORC1 inhibitors (RAD001 and LY294002) reduced NLGN3-induced HO1, NOQ1 and GCLC mRNA expression. Significantly, Nrf2 silencing by targeted shRNAs reversed NLGN3-induced retinal cytoprotection against H{sub 2}O{sub 2}. We conclude that NLGN3 activates Nrf2 signaling to protect human retinal cells from H{sub 2}O{sub 2}. NLGN3 could be further tested as a valuable retinal protection agent.

60 APPLIED LIFE SCIENCES↗