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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 757 records · Page 42
All sourcesNASA NTRSDOE OSTI
DOE OSTI2018

MicroRNA-613 promotes colon cancer cell proliferation, invasion and migration by targeting ATOH1

The aim of the present study is to investigate the expression and function of miR-613 in colon cancer (CC) and illuminate the molecular mechanisms underlying miR-613-regulated CC progression. Our data demonstrated that miR-613 was upregulated in CC tissue samples (P = 0.009) and human CC cell lines (HCT-116 and Lovo; P = 0.001 and P = 0.003, respectively), which also promoted the proliferation, invasion and migration of CC cells (P < 0.05). The dual-luciferase reporter assay confirmed that Atonal homolog1 (ATOH1) was the target mRNA of miR-613. Rescue experiments showed that ATOH1 overexpression vector significantly reversed the stimulative effects of miR-613 mimic on the progression of HCT-116 and Lovo cells (P < 0.001). Positive ATOH1 expression in CC tissues was significantly associated with lower grade (χ2 = 3.592, P = 0.043), lower TNM stage (χ{sup 2} = 3.537, P = 0.048) and better overall survival (P=0.041). Jun N-terminal kinase 1 (JNK1) pathway and Mucin 2 (MUC2) were the potential downstream proteins of miR-613/ATOH1. miR-613 is an oncogene in CC and promotes the proliferation, invasion and migration of CC cells by targeting ATOH1 likely via activating JNK1 pathway and upregulating MUC2.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

RBM17 controls apoptosis and proliferation to promote Glioma progression

Highlights: • RBM17 overexpressed in glioma patients and resulted in the poor prognosis. • Downregulation of RBM17 suppressed proliferation and induced apoptosis in glioma cells. • The silencing of RBM17 in U87 cells suppressed the tumor growth in vivo. • RBM17 on main signaling pathways in human glioma cells. The splicing factor SPF45 (RBM17) is a well-known component of the spliceosome that is involved in alternative splicing. RBM17 is frequently overexpressed in many tumors and plays a crucial role in cancer progression and drug resistance. However, the role of RBM17 in the development of glioma has not been thoroughly elucidated to date. In the present study, we found that RBM17 was overexpressed in glioma and that a high level of expression of RBM17 was closely associated with a poor prognosis in glioma patients. We investigated the effect of RBM17 on apoptosis, cell growth and cell cycle indexes and the activation of apoptosis signaling by shRNA in human U87 and U251 glioma cells. The downregulated expression of RBM17 mRNA was accompanied by the induction of cell cycle arrest, and apoptosis, reduced cell proliferation in the two cell lines, and reduced cell survival, as measured by the increased activation of caspase-3, caspase-9, and PARP (poly ADP-ribose polymerase). Furthermore, in subcutaneous U87 cell xenograft tumors in nude mice, intradermal administration of an shRNA targeting RBM17 significantly downregulated RBM17 expression in vivo and was accompanied by the suppressed growth of glioma. To the best of our knowledge, our results are the first to confirm that RBM17 functions in promoting cell proliferation, affecting the cell cycle, and inducing apoptosis in human glioma cells both in vitro and in vivo. These results indicate that RBM17 may be a therapeutic target in the clinical management of glioma.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Administration of ubiquitin-activating enzyme UBA1 inhibitor PYR-41 attenuates angiotensin II-induced cardiac remodeling in mice

Highlights: • Ang II induces UBA1 expression. • PYR-41 inhibits cardiac remodeling. • PYR-41 stabilizes PTEN. Pathological cardiac hypertrophy is the main risk factor for heart diseases. The ubiquitin-proteasome system (UPS) is the major intracellular protein degradation system involved in the development of cardiac hypertrophic remodeling. Ubiquitin-activating enzyme E1, a key component of the UPS, catalyzes the first step in ubiquitin conjugation to mark cellular proteins for degradation via proteasome. However, the functional role of E1 (UBA1) in regulation of hypertrophic remodeling in angiotensin II (Ang II)-infused mice remains unknown. In this study, male wild-type mice were treated with UBA1 inhibitor PYR-41 at two doses of 5 and 10 mg and infused with Ang II (1000 ng/kg/min) for 14 days. Systolic blood pressure was detected by using tail-cuff system. Cardiac function was assessed by echocardiography. Hypertrophic remodeling was analyzed examined by histological examinations. The expressions of genes and proteins were detected by quantitative real-time PCR and immunoblotting analysis. After 14 days, Ang II infusion significantly increased UBA1 expression at both mRNA and protein levels in the hearts. Furthermore, Ang II-infused mice showed a significant increase in systolic blood pressure compensatory cardiac function, hypertrophy, interstitial fibrosis, inflammation and oxidative stress compared with saline-treated controls, whereas these effects were dose-dependently attenuated in PYR-41-treated mice. These beneficial actions were associated mainly with inhibition of PTEN degradation and multiple downstream mediators (AKT, ERK1/2, STAT3, TGF-β/Smad2/3 and NF-kB(p65)). In conclusion, these results indicate that inhibition of UBA1 suppresses Ang II-induced hypertrophic remodeling, and suggest that administration of low dose PYR-41 may be a new potential therapeutic approach for treating hypertensive heart diseases.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

microRNA-9 selectively targets LMX1A to promote gastric cancer cell progression

Highlights: • miR-9 overexpression downregulates LMX1A, promoting gastric cancer (GC) cell proliferation. • miR-9 inhibition upregulates LMX1A to induce GC cell apoptosis. • LMX1A is the direct and primary target of miR-9 in GC cells. • miR-9 upregulation in human GC tissues correlates with LMX1A downregulation. LIM homeobox transcription factor 1, alpha (LMX1A) is downregulated in human gastric cancer (GC), functioning as a tumor suppressor. The current study aims to identify specific microRNA that can regulate LMX1A expression. By sequence analysis of LMX1A mRNA 3′-untranslated region (3′-UTR), we show that microRNA-9 (miR-9) putatively targets human LMX1A. In established (AGS cells) and primary human GC cells, ectopic overexpression of miR-9 by a lentiviral construct decreased LMX1A 3′-UTR activity, causing LMX1A mRNA and protein downregulation. Functional analyses show that miR-9 overexpression enhanced GC cell survival and proliferation. On the contrary, miR-9 inhibition by antagomir-9 lentivirus increased LMX1A 3′-UTR activity to upregulate LMX1A mRNA and protein expression, causing GC cell apoptosis. CRISPR/Cas9-mediated LMX1A knockout promoted AGS cell survival and proliferation. Importantly, miR-9 and antagomiR-9 were ineffective to the function of LMX1A-knockout AGS cells. In human GC tissues miR-9 is upregulated, which is negatively correlated with LMX1A downregulation. Together, we conclude that miR-9 selectively targets LMX1A to promote GC cell progression.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Efficient mRNA delivery system utilizing chimeric VSVG-L7Ae virus-like particles

Highlights: • Generation of chimeric virus-like particles by fusion of VSV-G and L7Ae proteins. • VSVG-L7Ae VLPs efficiently incorporate and deliver mRNA into various human cell lines. • Inclusion of mRNA is mediated by RNA binding domain of L7Ae. The delivery of mRNA is advantageous over DNA delivery as it is transient and does not carry the risk of genomic DNA integration. However, there are currently few efficient mRNA delivery options available, especially for hard-to-transfect cell types, and thus new delivery methods are needed. To this end, we have established a novel mRNA delivery system utilizing chimeric virus-like particles (VLPs). We generated a novel VLP by fusing protein G of Vesicular stomatitis virus (VSV-G) with a ribosomal protein L7Ae of Archeoglobus fulgidus. This system allowed the efficient delivery of EGFP mRNA which was independent from the presence of BoxC/D motif in the mRNA sequence. Our VSVG-L7Ae VLP system demonstrated high transduction efficacy in hard-to-transfect cell lines, such as human induced pluripotent stem cells (iPS cells) and monocytes. In summary, this platform may serve as an efficient and transient transgene delivery tool for an mRNA of interest.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Conversion of dietary trans-vaccenic acid to trans11,cis13-conjugated linoleic acid in the rat lactating mammary gland by Fatty Acid Desaturase 3-catalyzed methyl-end Δ13-desaturation

Highlights: • FADS3 was shown in vitro to catalyze the Δ13-desaturation of trans-vaccenic acid. • The product is the trans11,cis13-18:2 conjugated linoleic acid isomer. • The proof of this concept is so far lacking in vivo. • Lactating female rats fed with trans-vaccenic acid produced the trans11,cis13-18:2. • This study demonstrates this new pathway in the lactating mammary gland. In vitro, the rat Fatty Acid Desaturase 3 (FADS3) gene was shown to code for an enzyme able to catalyze the unexpected Δ13-desaturation of trans-vaccenic acid, producing the trans11,cis13-conjugated linoleic acid (CLA) isomer. FADS3 may therefore be the first methyl-end trans-vaccenate Δ13-desaturase functionally characterized in mammals, but the proof of this concept is so far lacking in vivo. The present study therefore aimed at investigating further the putative in vivo synthesis of trans11,cis13-CLA from dietary trans-vaccenic acid in rodents. During one week of pregnancy and two weeks post-partum, Sprague-Dawley female rats were fed two diets either high (10.0% of fatty acids and 3.8% of energy intake) or low (0.4% of fatty acids and 0.2% of energy intake) in trans-vaccenic acid. The trans11,cis13-CLA was specifically detected, formally identified and reproducibly quantified (0.06% of total fatty acids) in the mammary gland phospholipids of lactating female rats fed the high trans-vaccenic acid-enriched diet. This result was consistent with FADS3 mRNA expression being significantly higher in the lactating mammary gland than in the liver. Although the apparent metabolic conversion is low, this physiological evidence demonstrates the existence of this new pathway described in the lactating mammary gland and establishes the FADS3 enzyme as a reliable mammalian trans-vaccenate Δ13-desaturase in vivo.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Gene expression profiling in adipose tissue of Sprague Dawley rats identifies olfactory receptor 984 as a potential obesity treatment target

Highlights: • Olr984 is identified as candidate gene for obesity resistance in rats. • Olr984 is highly expressed in human adipose tissue. • Olr984mRNA expression in human adipose tissue correlates with obesity parameters. The aim of the study was to identify and functionally characterize novel candidate gene/s involved in the development of resistance to diet-induced obesity in rats. In a high-fat-diet (HFD) study of rats, we found subgroups which either developed resistance to HFD-induced obesity (DR) or showed an obesity-prone phenotype (DIO). Gene expression analysis in 10 samples (5 DIO vs 5 DR) was performed. The most promising gene, OR6C3 (orthologous with rat Olr984 and mouse Olfr788) was measured by qRT-PCR in paired samples of human visceral (Vis) and subcutaneous (SC) adipose tissue (AT) (n = 225) and in sub-fractions of adipocytes and cells of stromal vascular fraction. Gene expression analyses showed Olr984 with significantly reduced mRNA expression in DR rats. In the Vis AT of human samples we found an up-regulation of OR6C3 compared to SC AT, independent of gender, glucose tolerance or type 2 diabetes. We observed significantly lower levels of SC AT OR6C3 mRNA in subjects with obesity compared to those with normal-weight or overweight. OR6C3 is more expressed in SVF than in adipocytes. Olr984 could be a novel candidate gene related to diet-induced obesity in rats. Variation in human AT mRNA expression is related to obesity parameters and glucose homeostasis and linked to the regulatory role of insulin on the Olr984.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Maternal supplementation with calcium varying with feeding time daily during late pregnancy affects lipid metabolism and transport of placenta in pigs

To investigate effects of Ca level varying with feeding time daily in sows during late pregnancy on placental lipid metabolism and transport in pigs, sixty pregnant sows were assigned to 3 groups: the CON group was fed low-Ca diet with 11.25 g CaCO3 at 0600 h and 1500 h, H-L group was fed low-Ca diet with 22.5 g CaCO3 at 0600 h and low-Ca diet at 1500 h, and L-H group was fed low-Ca diet at 0600 h and low-Ca diet with 22.5 g CaCO3 at 1500 h, respectively. Serum from sows and umbilical cord and placenta were collected during delivery. Results showed that, compared with the CON group, H-L feeding significantly increased maternal serum total triglyceride (TG) and umbilical serum high-density lipoprotein (HDL) (P P P < 0.05). It is concluded that maternal supplementation with Ca varying with feeding time daily during late pregnancy affects placental lipid metabolism and transport in pigs by regulating the mRNA expression related to lipid metabolism and the circadian clock.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

MiR-429 suppresses neurotrophin-3 to alleviate perineural invasion of pancreatic cancer

Highlights: • MiR-429 is downregulated in pancreatic cancer tissues and cells. • MiR-429 functions to inhibit cancer cell proliferation and invasion. • MiR-429 directly targets to NT-3 mRNA. • MiR-429 suppress NT-3 in cancer cells to resist the attraction TrkC in PC12 cells. • NT-3-TrkC signaling promotes PC12 neurite outgrowth and neuropathic pain. Perineural invasion (PNI) potentially increases the risk of relapse and abdominal pain in patients with pancreatic ductal adenocarcinoma (PDAC). However, the underlying mechanisms of PNI of PDAC is incompletely revealed. Our study aimed to investigate roles of miR-429 in modulating PNI in PDAC. We found that miR-429 was downregulated in PDAC cancer tissues and was profoundly decreased in tissues with PNI. It was reduced in nine of the ten examined pancreatic cancer cell lines. MiR-429 mimics restored its cellular expressions in MIA PaCa-2 and BxCP3 cells and significantly suppressed cell viability and invasion of the cancer cells. The online bioinformatic software predicted that neurotrophin-3 (NT-3) was a potential target gene of miR-429. It was showed that NT-3 mRNA elevated in PC cancer tissues, especially in patients presenting PNI. MiR-429 upregulation substantially suppressed the NT-3 mRNA and secretion in cancer cells. Also, the dual luciferase reporter assays confirmed the interaction between miR-429 and NT-3. When co-culturing the two PDAC cells with PC-12 cells, the invaded cell counts significantly increased comparing with the sole culture of cancer cells. However, miR-429 mimic transfection or NT-3 blocking retarded the cancer invasion in the co-culture system. Besides, we found that cancer cells conditioned medium (CM) treatment significantly increased the neurite outgrowth percentage in PC-12 cells, which was suppressed by culturing with CM from miR-429 mimics-transfected cells. In the CM cultured PC-12 cells, NT-3 receptor TrkC as well as pain-related proteins TRPV1 and TRPV2 significantly elevated. Collectively, miR-429 potentially suppressed neurotrophin-3 to alleviate PNI of PDAC.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Sterol regulatory element binding protein-1: Molecular cloning, tissue distribution and gene expression level in response to nutritional regulation in mud crab, Scylla paramamosain

Highlights: • The complete SREBP-1 cDNA sequence was cloned from the hepatopancreas of mud crab based on the RACE technology. • Tissue distribution analysis revealed that SREBP-1 transcripts were widely distributed in various organs, with higher mRNA levels in the eyestalk and cranial ganglia. • The expression level of SREBP-1 mRNA were up-regulated in proportion to the replacement of dietary fish oil with soybean oil. In the present study, SREBP-1 cDNA was cloned from the hepatopancreas of mud crab (Scylla paramamosain) and characterized by performing rapid-amplification of cDNA ends. The 3361bp long full-length cDNA encodes a polypeptide with 1039 amino acids. Tissue distribution analysis revealed that SREBP-1 transcripts were widely distributed in various organs, with higher mRNA levels in the eyestalk and cranial ganglia. Further, expression level of SREBP-1 mRNA were up-regulated in proportion to the replacement of dietary fish oil (FO) with soybean oil (SO). These results may contribute to better understanding of the long-chain polyunsaturated fatty acids (LC-PUFA) biosynthetic pathway and regulation mechanism in mud crab.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Aspergillus fumigatus phospholipase D may enhance reactive oxygen species production by accumulation of histone deacetylase 6

Highlights: • Expression of A. fumigatus PLD in human cells was able to increase the production of ROS. • A. fumigatus PLD was found to interact with HDAC6 and significantly increased its expression level. • A. fumigatus PLD may enhance the production of ROS via the accumulation of HDAC6. Aspergillus fumigatus, an airborne pathogen, causes many diseases, including aspergilloma, invasive aspergillosis, and allergic bronchopulmonary aspergillosis. Phospholipase D (PLD) is an important virulence factor for A. fumigatus infection, but the manner by which PLD contributes to the virulence of this pathogen is not clear. Our results show that expression of A. fumigatus PLD in human cells was able to increase the production of reactive oxygen species (ROS), which play an important role in several signaling pathways as well as in lung infection. Meanwhile, A. fumigatus PLD was found to interact with human endogenous histone deacetylase 6 (HDAC6), a known regulator of ROS production and inflammatory responses; PLD significantly increased the expression level of HDAC6 protein without altering its mRNA level. These results suggest that A. fumigatus PLD may enhance the production of ROS via the accumulation of HDAC6, which may be involved in host immunomodulation during A. fumigatus infection.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Von Hippel-Lindau (VHL) protein antagonist, VH298, promotes functional activities of tendon-derived stem cells and accelerates healing of entheses in rats by inhibiting ubiquitination of hydroxy-HIF-1α

Enthesis is the region where a tendon attaches to a bone. It is a relatively vulnerable position, and in most cases surgical treatment is required upon rupture. The reconstructed enthesis is usually weaker compared to the original, and is prone to rupture again. Hypoxia-inducible factor-1 α (HIF-1α) is known to be involved in extensive activities in cells. It is inhibited under normoxic conditions, and undergoes two essential processes, hydroxylation and ubiquitination, the latter of which has been largely unexplored. Herein, we measured the levels of HIF-1α and hydroxy-HIF-1α in VH298-treated rat tendon-derived stem cells (TDSCs) by immunoblotting. We also detected the proliferation of TDSCs using CCK-8 assay and the mRNA levels of related genes by quantitative RT-PCR. The TDSCs were observed to be induced and the chondrogenic differentiation related genes were found to be enhanced. We also simulated in-vitro wounding in a scratch test and reconstructed the enthesis in a rat model of Achilles tendon by classical surgery followed by administration of phosphate buffer saline (PBS) injection or VH298 injection. We observed that HIF-1α and hydroxy-HIF-1α levels were increased in VH298-treated TDSCs in a dose- and time-dependent manner. Thirty micromolar VH298 could significantly increase cell proliferation, migration, and expression of collagen-1α, collagen-3α, decorin, tenomodulin, tenascin C genes, and chondrogenic differentiation-related genes, collagen-2α, SRY-box9, aggrecan. VH298-treated enthesis could tolerate more load-to-failure, had a better healing pattern, and activation of HIF signaling pathway. VH298 can thus enhance the functional activities of TDSCs, enhance their chondrogenic differentiation potential, and accelerate enthesis healing by inhibiting the ubiquitination of hydroxy-HIF-1α.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

The role of UNC5b in ox-LDL inhibiting migration of RAW264.7 macrophages and the involvement of CCR7

Highlights: ∙The netrin-1, and its receptor UNC5b, are expressed by macrophage foam cells. ∙The up-regulation of UNC5b and netrin-1 was in respond to ox-LDL in Raw264.7 macrophage, and was associated with decreasing cell migration. ∙In ox-LDL inducement, UNC5b down-regulated CCR7, and then inhibited macrophage migration. The formation of macrophage foam cells by ingesting ox-LDL and focal retention in the subendothelial space are the hallmarks of the early atherosclerotic lesion. The C-C chemokine receptor type 7 (CCR7) is positively correlated with the macrophage migration. But the mechanism of CCR7 regulation is not fully clearness. In the present study, we demonstrates that expression in UNC5b and netrin-1 was enhanced in respond to ox-LDL in Raw264.7 macrophage and associated with decreasing cell migration. Interestingly, it was found that ox-LDL significantly downregulate CCR7 gene expression. The expression of CCR7 in mRNA and protein levels were decreased in ox-LDL treated Raw264.7 macrophage when we over expression of UNC5b with pcDNA3.1-UNC5b plasmid. We got the inverse results after silence UNC5b gene with siUNC5b. Meanwhile, the data show that in ox-LDL inducement, UNC5b down-regulated CCR7, and then inhibited macrophage migration. This novel phenomenon is of a crucial highlights to understand deeply the pathogenesis of atherosclerosis. The molecular mechanism of CCR7 regulation deserves intensive study.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Targeting ninjurin 2 by miR-764 regulates hydrogen peroxide (H2O2)-induced neuronal cell death

Highlights: • MicroRNA-764 (miR-764) directly regulates ninjurin2 (NINJ2) expression. • Forced overexpression of miR-764 downregulates NINJ2 in human neuronal cells. • miR-764 inhibitor upregulates NINJ2 in human neuronal cells. • NINJ2 downregulation by miR-764 is cytotoxic to human neuronal cells. • NINJ2 overexpression by lentivirus or mir-764 inhibitor protects neuronal cells from H{sub 2}O{sub 2}. Ninjurin 2 (NINJ2) is a novel adhesion molecule expressed in neurons and glial cells. The current study determined if specific microRNA (miRNA) can regulate NINJ2 expression in human neuronal cells. Sequence analysis of NINJ2 mRNA 3′-untranslated region (3′-UTR) revealed that microRNA-764 (miR-764) putatively targets NINJ2. In SH-SY5Y/SK-N-BE neuronal cells and primary human neurons, lentivirus-mediated overexpression of miR-764 conferred significant repression on NINJ2 3′-UTR luciferase activity and downregulation of NINJ2 mRNA/protein. Conversely, transfection of the miR-764 inhibitor increased NINJ2 mRNA and protein expression in the neuronal cells. Function studies show that NINJ2 downregulation by miR-764 induced significant viability reduction and apoptosis in the neuronal cells. Further, CRISPR/Cas9-mediated NINJ2 knockout mimicked and abolished miR-764-induced actions in SH-SY5Y cells. Conversely, lentivirus-mediated NINJ2 overexpression or transfection of miR-764 inhibitor protected neuronal cells from hydrogen peroxide (H{sub 2}O{sub 2})-induced cell death and apoptosis. Collectively, these results show that NINJ2 is a pro-survival factor in human neuronal cells. miR-764 regulates NINJ2 expression and neuron functions.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

FTY720 attenuates intestinal injury and suppresses inflammation in experimental necrotizing enterocolitis via modulating CXCL5/CXCR2 axis

Highlights: • FTY720 reduces weight loss and improves survival in experimental NEC. • FTY720 ameliorates intestinal injury in experimental NEC. • FTY720 attenuates inflammatory response in experimental NEC. • FTY720 down-regulates CXCL5/CXCR2 axis in experimental NEC. Necrotizing enterocolitis (NEC) remains one of the leading causes of death in neonatal infants and new therapeutic strategies for NEC are urgently required. The immunomodulatory agent FTY720 has been shown to have protective effects in various inflammatory diseases. In this study, we hypothesized that treatment with FTY720 confers protection against experimental NEC. Experimental NEC was induced in five-day-old C57BL/6 neonatal mice by hyperosmolar formula feeding plus hypoxia and lipopolysaccharide (LPS) challenges. Induction of NEC resulted in substantial weight loss and high mortality compared to the control group, whereas FTY720 treatment significantly attenuated weight loss and improved survival in NEC-challenged neonatal mice. FTY720 treatment strongly ameliorated NEC-induced intestinal injury with reduced apoptosis and up-regulation of intestinal barrier proteins in the ileal tissues. Furthermore, FTY720 treatment abrogated NEC-initiated intestinal and systemic inflammation with markedly diminished inflammatory cytokines and chemokines. Moreover, FTY720 treatment suppressed NEC-activated CXCL5/CXCR2 axis with down-regulated expression of CXCL5 and CXCR2 at both mRNA and protein levels. Thus, we demonstrate that FTY720 protects neonatal mice against NEC-associated lethality by ameliorating intestinal injury and attenuating inflammation, possibly via its down-regulation of NEC-induced activation of intestinal CXCL5/CXCR2 axis.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Knockout of Raptor destabilizes ornithine decarboxylase mRNA and decreases binding of HuR to the ODC transcript in cells exposed to ultraviolet-B irradiation

Non-melanoma skin cancer (NMSC) is the most commonly diagnosed cancer in the United States. Ultraviolet-B (UVB) irradiation is the primary carcinogen responsible for stimulating NMSC development. Ornithine Decarboxylase (ODC), the first rate-limiting enzyme in the synthesis of polyamines, is upregulated in response to a variety of proliferation stimuli, including UVB exposure. Our previous studies have demonstrated regulation of ODC synthesis by the mammalian target of rapamycin complex 1 (mTORC1) in cells transformed by oncogenic Ras. The goal of these studies was to better understand the link between mTORC1 and ODC in nontransformed cells treated with UVB. We show that the ablation of mTORC1 activity by conditional knockout of its essential component Raptor led to decreased levels of ODC protein both before and after exposure to 10 mJ/cm{sup 2} UVB. Moreover, ODC mRNA was destabilized in the absence of Raptor, suggesting post-transcriptional regulation. We have previously shown that the ODC transcript is stabilized by the RNA binding protein (RBP) human antigen R (HuR), and the intracellular localization of HuR responds to changes in mTORC1 activity. To expand these studies, we investigated whether HuR functions to regulate ODC mRNA stability after UVB exposure. Our results show an increased localization of HuR to the cytoplasm after UVB exposure in wild-type cells compared to Raptor knockout cells, and this is accompanied by greater association of HuR with the ODC transcript. These data suggest that the localization of HuR in response to UVB is influenced, at least in part, by mTORC1 and that HuR can bind to and stabilize ODC mRNA after UVB exposure in an mTORC1-dependent manner.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

NRF2 and HSF1 coordinately regulate heme oxygenase-1 expression

Heme oxygenase-1 (HO-1) is an inducible enzyme responding to various stresses and has cytoprotective activities. Although HO-1 has been referred to as heat shock protein (HSP) 32, the heat-mediated induction of HO-1 varies among different species and cell lines. We examined the effects of heat shock on HO-1 expression in mouse embryonic fibroblast (MEF) cells deficient in heat shock factor 1 (HSF1) or nuclear factor-erythroid-2-related factor 2 (NRF2). Heme-induced expression of HO-1 was 2-fold higher in Hsf1{sup −/−} cells than in the wild-type cells at both mRNA and protein levels. In Nrf2{sup −/−} cells, heme-induced expression of HO-1 was not detected. In contrast, HO-1 expression was markedly induced by heat shock at 40–42 °C in Nrf2{sup −/−} cells while the wild-type cells were not responsive. The heat-induced expression of HO-1 in Nrf2{sup −/−} cells were almost completely diminished by transfection of siRNA against Hsf1 gene. These results suggest that HSF1 and NRF2 suppress heme-induced and heat-induced HO-1 expression, respectively.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Intestinal epithelial cell-specific Raptor is essential for high fat diet-induced weight gain in mice

Mammalian target of rapamycin complex 1 (mTORC1) is a major regulator of cell growth and proliferation through fuel sensing. Systemic inhibition of mTOR as well as manipulation of its downstream products prevent diet-induced obesity. The purpose of this study was to determine the consequences of intestine-targeted mTORC1 inhibition. To attenuate intestinal mTORC1 activity, Villin-Cre{sup ER} mice were crossed with Raptor{sup flox/flox} mice, creating an intestinal-specific Raptor null line (i-Raptor −/−). Mice were fed a high fat diet (HFD) and compositional changes as well as food intake levels were assessed. Over a five-week time course, i-Raptor −/− mice consistently gained less body weight on a HFD compared to wildtype (WT) mice secondary to significantly reduced food intake. Importantly, the i-Raptor −/− mice did not appear to be malnourished, demonstrated by their preservation of lean body mass. i-Raptor −/− mice also maintained a normal metabolic profile without significant changes in triglyceride or fasting glucose levels. Further investigation revealed that GDF-15 mRNA expression was significantly enhanced in i-Raptor −/− enterocytes when refed with HFD after overnight starvation. In summary, our study establishes that loss of intestinal specific-mTORC1 is protective of the development of diet-induced obesity by reducing food intake without altering the metabolic profile.

60 APPLIED LIFE SCIENCES↗