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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 505 records · Page 28
All sourcesNASA NTRSDOE OSTI
DOE OSTI2016

Dioscin protects against ANIT–induced cholestasis via regulating Oatps, Mrp2 and Bsep expression in rats

Alpha-naphthylisothiocyanate (ANIT) is a toxicant that is widely used in rodents to model human intrahepatic cholestasis. The aim of the study is to investigate whether effects of dioscin on ANIT-induced cholestasis are related to changes in expression of hepatic transporters in rats. Effects of dioscin on cholestasis were examined by histology and biochemical marker levels. The functional changes of hepatic transporters were determined by in vitro, in situ and in vivo. qRT-PCR and western blot were used to assess the expression of hepatic transporters in cholestatic rats. Dioscin administration could ameliorate cholestasis, as evidenced by reduced biochemical markers as well as improved liver pathology. The uptakes of organic anion transporting polypeptide (Oatp) substrates were altered in liver uptake index in vivo, perfused rat liver in situ and isolated rat hepatocytes in vitro in cholestasis rats. qRT-PCR and western blot analysis indicated co-treatment of ANIT with dioscin prevented the adaptive down-regulation of Oatp1a1, 1b2, and prompted the up-regulation of Oatp1a4, multidrug resistance-associated protein (Mrp) 2 and bile salt export pump (Bsep). In addition, concerted effects on Mrp2 and Bsep occurred through up-regulation of small heterodimer partner by activating farnesoid X receptor. Dioscin might prevent impairment of hepatic function by restoring hepatic transporter expression. - Highlights: • Cholestasis was improved by dioscin via up-regulating the expression of Oatps, Mrp2 and Bsep. • Dioscin regulated Mrp2 and Bsep via activating FXR. • Dioscin may be a candidate drug for the prevention of intrahepatic cholestasis.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2016

Up-regulation of granzyme B and perforin by staphylococcal enterotoxin C2 mutant induces enhanced cytotoxicity in Hepa1–6 cells

Staphylococcal enterotoxin C2 (SEC2), a member of bacterial superantigen, is one of the most potent known activators of T lymphocytes. With this property, SEC2 has already been used in clinic as a tumor immunotherapy agent in China. To increase the antitumor activity, a SEC2 mutant named ST-4 (GKVTG102-106WWH) with amino acid substitutions in T cell receptor (TCR)-binding domain was generated by site-directed mutagenesis, and the molecular mechanism of the enhanced antitumor activity was investigated. Results showed that ST-4 could activate much more Vβ 8.2 and 8.3 T cells and NK cells compared with SEC2, and exhibited significantly enhanced immunocyte stimulation and antitumor activity in vitro. The synthetic peptide sequencing the residues of mutant TCR-binding domain could competitively inhibit the immunocyte stimulation activity of ST-4. Most importantly, ST-4 up-regulated granzyme B and perforin at both mRNA and protein levels. We also found that expression of proapoptotic proteins cytochrome c, BAX and activation of caspase-3, 9 was up-regulated, and antiapoptotic protein Bcl-xL was down-regulated in the treatment with either ST-4 or SEC2. When granzyme B inhibitor or perforin inhibitor is presented, tumor cell viability was significantly rescued. Taken together, we demonstrate that increased ST-4-TCR recognition contributed to massive T cells and NK cells activation. These activated cells released up-regulated granzyme B and perforin, which induced the enhanced tumor cells apoptosis by mitochondrial apoptotic pathway, and ultimately led to enhanced tumor cell growth inhibition. ST-4 may be a promising candidate for antitumor clinic usage in future. - Highlights: • We obtained a SEC2 mutant ST-4 with enhanced superantigen and antitumor activity. • Increased ST-4-TCR recognition contributed to massive T cells and NK cells activation. • Up-regulated GzmB and PRF1 in T cell by ST-4 induced enhanced tumor cells apoptosis. • Enhanced tumor cell apoptosis induced by ST-4 via mitochondrial apoptotic pathway.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2016

Progressive effects of silver nanoparticles on hormonal regulation of reproduction in male rats

The growing use of silver nanoparticles (AgNPs) in various applications, including consumer, agriculture and medicine products, has raised many concerns about the potential risks of nanoparticles (NPs) to human health and the environment. An increasing body of evidence suggests that AgNPs may have adverse effects of humans, thus the aim of this study was to investigate the effects of AgNPs on the male reproductive system. Silver particles (20 nm AgNPs (groups Ag I and Ag II) and 200 nm Ag sub-micron particles (SPs) (group Ag III)) were administered intravenously to male Wistar rats at a dose of 5 (groups Ag I and Ag III) or 10 (group Ag II) mg/kg of body weight. The biological material was sampled 24 h, 7 days and 28 days after injection. The obtained results revealed that the AgNPs had altered the luteinising hormone concentration in the plasma and the sex hormone concentration in the plasma and testes. Plasma and intratesticular levels of testosterone and dihydrotestosterone were significantly decreased both 7 and 28 days after treatment. No change in the prolactin and sex hormone-binding globulin concentration was observed. Exposure of the animals to AgNPs resulted in a considerable decrease in 5α-reductase type 1 and the aromatase protein level in the testis. Additionally, expression analysis of genes involved in steroidogenesis and the steroids metabolism revealed significant down-regulation of Star, Cyp11a1, Hsd3b1, Hsd17b3 and Srd5a1 mRNAs in AgNPs/AgSPs-exposed animals. The present study demonstrates the potential adverse effect on the hormonal regulation of the male reproductive function following AgNP/AgSP administration, in particular alterations of the sex steroid balance and expression of genes involved in steroidogenesis and the steroids metabolism. - Highlights: • Assessment of the toxic effects of AgNPs/AgSPs on the regulation of male reproductive function • AgNP −/AgSP-induced alterations of sex steroid status in male Wistar rats. • Regulation of male reproductive function is differently modulated by AgNPs and AgSPs. • Endocrine-mediated toxicity of AgNPs/AgSPs increased over time. • AgNPs/AgSPs alter male reproductive function regulation at the transcriptional level.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2012

Autoimmune regulator is acetylated by transcription coactivator CBP/p300

The Autoimmune Regulator (AIRE) is a regulator of transcription in the thymic medulla, where it controls the expression of a large set of peripheral-tissue specific genes. AIRE interacts with the transcriptional coactivator and acetyltransferase CBP and synergistically cooperates with it in transcriptional activation. Here, we aimed to study a possible role of AIRE acetylation in the modulation of its activity. We found that AIRE is acetylated in tissue culture cells and this acetylation is enhanced by overexpression of CBP and the CBP paralog p300. The acetylated lysines were located within nuclear localization signal and SAND domain. AIRE with mutations that mimicked acetylated K243 and K253 in the SAND domain had reduced transactivation activity and accumulated into fewer and larger nuclear bodies, whereas mutations that mimicked the unacetylated lysines were functionally similar to wild-type AIRE. Analogously to CBP, p300 localized to AIRE-containing nuclear bodies, however, the overexpression of p300 did not enhance the transcriptional activation of AIRE-regulated genes. Further studies showed that overexpression of p300 stabilized the AIRE protein. Interestingly, gene expression profiling revealed that AIRE, with mutations mimicking K243/K253 acetylation in SAND, was able to activate gene expression, although the affected genes were different and the activation level was lower from those regulated by wild-type AIRE. Our results suggest that the AIRE acetylation can influence the selection of AIRE activated genes. -- Highlights: Black-Right-Pointing-Pointer AIRE is acetylated by the acetyltransferases p300 and CBP. Black-Right-Pointing-Pointer Acetylation occurs between CARD and SAND domains and within the SAND domain. Black-Right-Pointing-Pointer Acetylation increases the size of AIRE nuclear dots. Black-Right-Pointing-Pointer Acetylation increases AIRE protein stability. Black-Right-Pointing-Pointer AIRE acetylation mimic regulates a different set of AIRE target genes.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2012

A biphasic endothelial stress-survival mechanism regulates the cellular response to vascular endothelial growth factor A

Vascular endothelial growth factor A (VEGF-A) is an essential cytokine that regulates endothelial function and angiogenesis. VEGF-A binding to endothelial receptor tyrosine kinases such as VEGFR1 and VEGFR2 triggers cellular responses including survival, proliferation and new blood vessel sprouting. Increased levels of a soluble VEGFR1 splice variant (sFlt-1) correlate with endothelial dysfunction in pathologies such as pre-eclampsia; however the cellular mechanism(s) underlying the regulation and function of sFlt-1 are unclear. Here, we demonstrate the existence of a biphasic stress response in endothelial cells, using serum deprivation as a model of endothelial dysfunction. The early phase is characterized by a high VEGFR2:sFlt-1 ratio, which is reversed in the late phase. A functional consequence is a short-term increase in VEGF-A-stimulated intracellular signaling. In the late phase, sFlt-1 is secreted and deposited at the extracellular matrix. We hypothesized that under stress, increased endothelial sFlt-1 levels reduce VEGF-A bioavailability: VEGF-A treatment induces sFlt-1 expression at the cell surface and VEGF-A silencing inhibits sFlt-1 anchorage to the extracellular matrix. Treatment with recombinant sFlt-1 inhibits VEGF-A-stimulated in vitro angiogenesis and sFlt-1 silencing enhances this process. In this response, increased VEGFR2 levels are regulated by the phosphatidylinositol-3-kinase and PKB/Akt signaling pathways and increased sFlt-1 levels by the ERK1/2 signaling pathway. We conclude that during serum withdrawal, cellular sensing of environmental stress modulates sFlt-1 and VEGFR2 levels, regulating VEGF-A bioavailability and ensuring cell survival takes precedence over cell proliferation and migration. These findings may underpin an important mechanism contributing to endothelial dysfunction in pathological states. -- Highlights: Black-Right-Pointing-Pointer Endothelial cells mount a stress response under conditions of low serum. Black-Right-Pointing-Pointer Endothelial VEGFR levels are modulated during this response. Black-Right-Pointing-Pointer The cell regulates VEGF-A bioavailability and cell survival. Black-Right-Pointing-Pointer This may partly underlie endothelial dysfunction seen in many pathologies.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2013

Protein kinase B/Akt1 inhibits autophagy by down-regulating UVRAG expression

Autophagy, or autophagocytosis, is a selective intracellular degradative process involving the cell's own lysosomal apparatus. An essential component in cell development, homeostasis, repair and resistance to stress, autophagy may result in either cell death or survival. The targeted region of the cell is sequestered within a membrane structure, the autophagosome, for regulation of the catabolic process. A key factor in both autophagosome formation and autophagosome maturation is a protein encoded by the ultraviolet irradiation resistance-associated gene (UVRAG). Conversely, the serine/threonine-specific protein kinase B (PKB, also known as Akt), which regulates survival in various cancers, inhibits autophagy through mTOR activation. We found that Akt1 may also directly inhibit autophagy by down-regulating UVRAG both in a 293T transient transfection system and breast cancer cells stably expressing Akt1. The UVRAG with mutations at putative Akt1-phosphorylation sites were still inhibited by Akt1, and dominant-negative Akt1 also inhibited UVRAG expression, suggesting that Akt1 down-regulates UVRAG by a kinase activity-independent mechanism. We showed that Akt1 overexpression in MDA-MB-231 breast cancer cells down-regulated UVRAG transcription. Cells over-expressing Akt1 were more resistant than control cells to ultraviolet light-induced autophagy and exhibited the associated reduction in cell viability. Levels of the autophagosome indicator protein LC3B-II and mRFP-GFP-LC3 were reduced in cells that over-expressing Akt1. Inhibiting Akt1 by siRNA or reintroducing UVRAG gene rescued the level of LC3B-II in UV-irradiation. Altogether, these data suggest that Akt1 may inhibit autophagy by decreasing UVRAG expression, which also sensitizes cancer cells to UV irradiation.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2014

Defining the role of mesenchymal stromal cells on the regulation of matrix metalloproteinases in skeletal muscle cells

Recent studies indicate that mesenchymal stromal cell (MSC) transplantation improves healing of injured and diseased skeletal muscle, although the mechanisms of benefit are poorly understood. In the present study, we investigated whether MSCs and/or their trophic factors were able to regulate matrix metalloproteinase (MMP) expression and activity in different cells of the muscle tissue. MSCs in co-culture with C2C12 cells or their conditioned medium (MSC-CM) up-regulated MMP-2 and MMP-9 expression and function in the myoblastic cells; these effects were concomitant with the down-regulation of the tissue inhibitor of metalloproteinases (TIMP)-1 and -2 and with increased cell motility. In the single muscle fiber experiments, MSC-CM administration increased MMP-2/9 expression in Pax-7{sup +} satellite cells and stimulated their mobilization, differentiation and fusion. The anti-fibrotic properties of MSC-CM involved also the regulation of MMPs by skeletal fibroblasts and the inhibition of their differentiation into myofibroblasts. The treatment with SB-3CT, a potent MMP inhibitor, prevented in these cells, the decrease of α-smooth actin and type-I collagen expression induced by MSC-CM, suggesting that MSC-CM could attenuate the fibrogenic response through mechanisms mediated by MMPs. Our results indicate that growth factors and cytokines released by these cells may modulate the fibrotic response and improve the endogenous mechanisms of muscle repair/regeneration. - Highlights: • MSC-CM contains paracrine factors that up-regulate MMP expression and function in different skeletal muscle cells. • MSC-CM promotes myoblast and satellite cell migration, proliferation and differentiation. • MSC-CM negatively interferes with fibroblast-myoblast transition in primary skeletal fibroblasts. • Paracrine factors from MSCs modulate the fibrotic response and improve the endogenous mechanisms of muscle regeneration.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2015

Hypoxia induced E-cadherin involving regulators of Hippo pathway due to HIF-1α stabilization/nuclear translocation in bone metastasis from breast carcinoma

The present study deals with the molecular mechanisms involved in the regulation of E-cadherin expression under hypoxia, because the adjustment of the amount of E-cadherin due to physical stimuli of the microenvironment might influence the colonization of metastasis to skeleton. We analyzed the effect of 1% oxygen tension, that is similar to that encountered in the bone marrow by metastatic cells spreading from breast carcinoma. The purpose was to evaluate the hypoxia-orchestrated control of E-cadherin transactivation via hypoxia inducible factor-1 (HIF-1) and peroxisome proliferator activated receptor-γ (PPARγ), and the involvement of Hippo pathway members, as regulators of transcription factors. To give a translational significance to the study, we took into consideration human pair-matched ductal breast carcinoma and bone metastasis: E-cadherin and Wwox were expressed in bone metastasis but not in breast carcinoma, while HIF-1α and TAZ seemed localized principally in nuclei of metastasis and were found in all cell compartments of breast carcinoma. A close examination of the regulatory mechanisms underlying E-cadherin expression in bone metastasis was done in 1833 clone derived from MDA-MB231 cells. Hypoxia induced E-cadherin only in 1833 clone, but not in parental cells, through HIF-1 and PPARγ activities, while Wwox decreased. Since Wwox was highly expressed in bone metastasis, the effect of ectopic Wwox was evaluated, and we showed E-cadherin transactivation and enhanced invasiveness in WWOX transfected 1833 cells. Also, hypoxia was additive with ectopic Wwox remarkably enhancing HIF-1α nuclear shuttle and accumulation due to the lengthening of the half-life of HIF-1α protein; under this experimental condition HIF-1α appeared as a slower migrated band compared with control, in agreement with the phosphorylation state. The in vitro data strongly supported the almost exclusive presence of HIF-1α in nuclei of human-bone metastasis. Thus, we identified Wwox as a novel molecule in the HIF-1α-HDM2 regulatory loop, necessary for the dynamic regulation of the HIF-1α amount, and we suggested that the reduction of endogenous Wwox free pool under hypoxia might also be due to the interaction with HDM2, sequestering the E3 ubiquitin ligase. We highlighted the importance of nuclear HIF-1α in the biology of metastasis for the mesenchymal-epithelial transition: this phenotype was regulated by Wwox plus hypoxia through E-cadherin target gene, playing a pivotal role in bone metastasis colonization. - Highlights: • E-cadherin accumulates in hypoxic bone metastasis opposite to primary carcinoma. • HIF-1 and PPARγ cooperate in inducing E-cadherin under hypoxia in metastatic cells. • Wwox regulates HIF-1α phosphorylation and nuclear translocation. • Hypoxia plus Wwox prevent HIF-1α degradation via HDM2 forming a regulatory loop.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2017

Deubiquitinase USP33 is negatively regulated by β-TrCP through ubiquitin-dependent proteolysis

Ubiquitin-mediated proteolysis regulates cellular levels of various proteins, and therefore plays important roles in controlling cell signaling and disease progression. The Skp1-Cul1-F-box ubiquitin ligase β-TrCP is recognized as an important negative regulator for numerous key signaling proteins. Recently, the deubiquitinases (DUBs) have turned out to be essential to regulate signaling pathways related to human diseases. However, whether β-TrCP is able to regulate the deubiquitinase family members remains largely unexplored. Here, we found that β-TrCP downregulated cellular levels of endogenous USP33. We also revealed that β-TrCP interacted with USP33 independently of the classic binding motif for β-TrCP, and mediated USP33 degradation via the ubiquitin proteasome pathway. Furthermore, we found that the WD40 motif of β-TrCP and 201–400 amino acid motif of USP33 are required for the interaction between β-TrCP and USP33. Consequently, β-TrCP attenuated USP33-mediated inhibition of cell proliferation and cell invasion. Taken together, our study clarified that the E3 ligase β-TrCP regulates cellular USP33 levels by the ubiquitin-proteasomal proteolysis. - Highlights: •β-TrCP downregulates protein levels of USP33. •β-TrCP promotes USP33 ubiquitination and degradation. • The effect of β-TrCP on USP33 is independent of DSG motif. • β-TrCP attenuates USP33-mediated suppression of cell proliferation and invasion.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2017

VANGL2 interacts with integrin αv to regulate matrix metalloproteinase activity and cell adhesion to the extracellular matrix

Highlights: • VANGL2 regulates cell adhesion to ECM proteins. • VANGL2 function influences cell surface integrin αvβ3 expression. • Inhibition of MMP activity rescues the adhesion defect in VANGL2 knockdown cells. • Integrin αv is required for VANGL2-dependent MMP2 activation. • VANGL2 binds integrin αvβ3. Planar cell polarity (PCP) proteins are implicated in a variety of morphogenetic processes including embryonic cell migration and potentially cancer progression. During zebrafish gastrulation, the transmembrane protein Vang-like 2 (VANGL2) is required for PCP and directed cell migration. These cell behaviors occur in the context of a fibrillar extracellular matrix (ECM). While it is thought that interactions with the ECM regulate cell migration, it is unclear how PCP proteins such as VANGL2 influence these events. Using an in vitro cell culture model system, we previously showed that human VANGL2 negatively regulates membrane type-1 matrix metalloproteinase (MMP14) and activation of secreted matrix metalloproteinase 2 (MMP2). Here, we investigated the functional relationship between VANGL2, integrin αvβ3, and MMP2 activation. We provide evidence that VANGL2 regulates cell surface integrin αvβ3 expression and adhesion to fibronectin, laminin, and vitronectin. Inhibition of MMP14/MMP2 activity suppressed the cell adhesion defect in VANGL2 knockdown cells. Furthermore, our data show that MMP14 and integrin αv are required for increased proteolysis by VANGL2 knockdown cells. Lastly, we have identified integrin αvβ3 as a novel VANGL2 binding partner. Together, these findings begin to dissect the molecular underpinnings of how VANGL2 regulates MMP activity and cell adhesion to the ECM.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Potential role of MG53 in the regulation of transforming-growth-factor-β1-induced atrial fibrosis and vulnerability to atrial fibrillation

Highlights: • MG53 is expressed in human atrium and associated with AF. • MG53 could regulate TGF-β1/Smad pathway to induce atrial fibrosis. • Exogenous TGF-β1 suppresses expression of MG53. • MG53 could serve as a promising target agent for atrial fibrosis induced by the TGF-β1/Smad pathway in patients with AF. Atrial fibrosis plays a critical role in atrial fibrillation (AF) by the transforming growth factor (TGF)-β1/Smad pathway. The disordered differentiation, proliferation, migration and collagen deposition of atrial fibroblasts play significant roles in atrial fibrosis. Mitsugumin (MG)53 is predominantly expressed in myocardium of rodents and has multiple biological functions. However, the role of MG53 in cardiac fibrosis remains unclear. This study provided clinical and experimental evidence for the involvement of MG53 in atrial fibrosis in humans and atrial fibrosis phenotype in cultured rat atrial fibroblasts. In atrial tissue from patients we demonstrated that MG53 was expressed in human atrium. Expression of MG53 increased with the extent of atrial fibrosis, which could induce AF. In cultured atrial fibroblasts, depletion of MG53 by siRNA caused down-regulation of the TGF-β1/Smad pathway, while overexpression of MG53 by adenovirus up-regulated the pathway. MG53 regulated the proliferation and migration of atrial fibroblasts. Besides, exogenous TGF-β1 suppressed expression of MG53. In conclusion, we demonstrated that MG53 was expressed in human atrium, and may be a potential upstream of the TGF-β1/Smad pathway in human atrium and rat atrial fibroblasts. This suggests that MG53 is a potential regulator of atrial fibrosis induced by the TGF-β1/Smad pathway in patients with AF.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Artemin regulates CXCR4 expression to induce migration and invasion in pancreatic cancer cells through activation of NF-κB signaling

Highlights: • Artemin regulates CXCR4 expression to induce migration and invasion in pancreatic cancer cells through activation of NF-κB signaling. • Artemin activates Akt and ERK, which cooperatively mediates activation of NF-κB and subsequent CXCR4 up-regulation. • Artemin induces an increase in migration and invasion potential in pancreatic cancer cell by regulating Akt and ERK1/2/NF-κB/CXCR4 signaling. Pancreatic ductal adenocarcinoma (PDAC) is the most lethal human malignant tumor because of the early onset of local invasion and distant metastasis. Perineural invasion is a prominent characteristic of pancreatic adenocarcinoma, which is a multifactorial process that involves various signaling molecules from different signaling pathways. The glial cell line-derived neurotrophic factor family of ligands was reported to be involved in perineural invasion in pancreatic cancer. Artemin is one member of the glial cell line-derived neurotrophic factor family of ligands. Although Artemin has previously been demonstrated to promote invasiveness of pancreatic cancer, the mechanisms remain poorly understood. In this study, we performed an analysis to determine the effects of Artemin on modulating tumor cell metastatic potential and invasion activity and explored its mechanisms in pancreatic cancer. We indicated that Artemin and CXCR4 were overexpressed in cancer tissues and widely expressed in pancreatic cancer cell lines. We observed that activation of ERK1/2 and Akt in Artemin-treated cells led to enhanced nuclear accumulation of NF-κB, which then induced CXCR4 expression. Through regulation of the expression of CXCR4, Artemin functionally promoted the migration and invasion in pancreatic cancer cells. The present study indicated that Artemin induced CXCR4 expression by activating Akt and ERK 1/2/NF-κB signaling, thereby modulating tumor cell metastatic potential and invasion activity in pancreatic cancer by regulating SDF-1α/CXCR4 axis. Artemin might be an effective and potent therapeutic target for pancreatic cancer metastasis, especially in perineural invasion.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Leptin up-regulates microRNA-27a/b-3p level in hepatic stellate cells

Highlights: • Leptin up-regulates miR-27a/b-3p levels in hepatic stellate cells (HSCs). • Multiple signaling pathways mediate the effect of leptin on miR-27a/b-3p. • miR-27a/b-3p reduce SREBP1c and LXRα levels, and inhibit HSC activation. • miR-27a/b-3p involved in leptin-associated liver fibrosis. Obese patients, often accompanied by hyperleptinemia, are prone to liver fibrogenesis. Leptin is an adipocyte-derived hormone and plays a promotion role in liver fibrosis. Sterol regulatory element binding protein-1c (SREBP1c) exerts a crucial role in inhibiting hepatic stellate cell (HSC) activation, a key step in liver fibrogenesis. Our previous studies indicated that leptin inhibited SREBP1c expression, contributing to leptin-induced HSC activation and liver fibrosis. microRNAs (miR) have emerged as important layers of regulatory control and regulate gene expression, and are implicated in numerous diseases. The present study revealed leptin up-regulation of miR-27a/b-3p levels in HSCs in vitro and in vivo. Three signaling pathways were required for leptin regulation of miR-27a/b-3p levels. miR-27a/b-3p could reduce SREBP1c and liver x receptor α (LXRα) levels, increased α-smooth muscle actin (α-SMA, a marker for HSC activation) and α1(I)collagen levels in cultured HSCs. miR-27a/b-3p regulation of SREBP1c and LXRα were independent of 3′-untranslated region of SREBP1c and LXRα mRNA. In vivo experiments further demonstrated the miR-27a/b-3p involved in leptin-associated decrease in SREBP1 level in HSCs, HSC activation, and liver fibrosis. These data might have potential implications for our understanding of molecular mechanisms underlying leptin roles in liver fibrogenesis of obese patients with hyperleptinaemia.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

miR-675 promotes odontogenic differentiation of human dental pulp cells by epigenetic regulation of DLX3

Highlights: • miR-675 promotes the odontogenic differentiation of human dental pulp cells (hDPCs). • miR-675 regulates the odontogenic differentiation of hDPCs by epigenetic regulating DLX3. • Our findings shed light on the regulatory mechanism of dentin hypoplasia in TDO patients. In a previous study, we showed that microRNA-675 (miR-675) was significantly down-regulated in patients with tricho-dento-osseous (TDO) syndrome. One of the main features of TDO syndrome is dentin hypoplasia. Thus, we hypothesize that miR-675 plays a role in dentin development. In this study, we determined the role of miR-675 in the odontogenic differentiation of human dental pulp cells (hDPCs). Stable overexpression and knockdown of miR-675 in hDPCs were performed using recombinant lentiviruses containing U6 promoter-driven miR-675 and short hairpin-miR675 expression cassettes, respectively. Alkaline phosphatase (ALP) assay, Alizarin red staining assay, quantitative polymerase chain reaction (qPCR), Western blot analysis, and immunofluorescent staining revealed the promotive effects of miR-675 on the odontogenic differentiation of hDPCs. Further, we found that miR-675 facilitates the odontogenic differentiation process of hDPCs by epigenetic regulation of distal-less homeobox (DLX3). Thus, for the first time, we determined that miR-675 regulates the odontogenic differentiation of hDPCs by inhibiting the DNA methyltransferase 3 beta (DNMT3B)-mediated methylation of DLX3. Our findings uncover an unanticipated regulatory role for miR-675 in the odontogenic differentiation of hDPCs by epigenetic changes in DLX3 and provide novel insight into dentin hypoplasia feature in TDO patients.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Withaferin A and sulforaphane regulate breast cancer cell cycle progression through epigenetic mechanisms

Highlights: • Tumor suppressor p21protein is reactivated via withaferin A (WA) and sulforaphane (SFN). • Combinatorial nutritive compounds have the ability to impede cell cycle progression. • Mutant p53 is down-regulated via the studied compounds where wild type p53 is not. • Epigenetic mechanisms are regulated by WA and SFN. • The H3K4Me3 marker is increased at the p21 promoter after WA and SFN treatment. Little is known about the effects of combinatorial dietary compounds on the regulation of epigenetic mechanisms involved in breast cancer prevention. The human diet consists of a multitude of components, and there is a need to elucidate how certain compounds interact in collaboration. Withaferin A (WA), found in the Indian winter cherry and documented as a DNA methyltransferase (DNMT) inhibitor, and sulforaphane (SFN), a well-known histone deacetylase (HDAC) inhibitor found in cruciferous vegetables, are two epigenetic modifying compounds that have only recently been studied in conjunction. The use of DNMT and HDAC inhibitors to reverse the malignant expression of certain genes in breast cancer has shown considerable promise. Previously, we found that SFN + WA synergistically promote breast cancer cell death. Herein, we determined that these compounds inhibit cell cycle progression from S to G2 phase in MDA-MB-231 and MCF-7 breast cancer. Furthermore, we demonstrate that this unique combination of epigenetic modifying compounds down-regulates the levels of Cyclin D1 and CDK4, and pRB; conversely, the levels of E2F mRNA and tumor suppressor p21 are increased independently of p53. We find these events coincide with an increase in unrestricted histone methylation. We propose SFN + WA-induced breast cancer cell death is attributed, in part, to epigenetic modifications that result in the modulated expression of key genes responsible for the regulation of cancer cell senescence.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Differentiation in stem/progenitor cells along fetal or adult hepatic stages requires transcriptional regulators independently of oscillations in microRNA expression

Highlights: • The role of microRNAs in differentiation of stem cells is not well understood. • This study found microRNA levels oscillated during differentiation of hepatocytes. • Many functions but not differentiation were regulated by microRNAs in hepatocytes. • Regulators of gene networks independently maintained hepatic differentiation. • These insights will advance how stem cells may contribute in disease mechanisms and therapies. Understanding mechanisms in lineage differentiation is critical for organ development, pathophysiology and oncogenesis. To determine whether microRNAs (miRNA) may serve as drivers or adjuncts in hepatic differentiation, we studied human embryonic stem cell-derived hepatocytes and primary hepatocytes representing fetal or adult stages. Model systems were used for hepatic lineage advancement or regression under culture conditions with molecular assays. Profiles of miRNA in primary fetal and adult hepatocytes shared similarities and distinctions from pluripotent stem cells or stem cell-derived early fetal-like hepatocytes. During phenotypic regression in fetal or adult hepatocytes, miRNA profiles oscillated to regain stemness-associated features that had not been extinguished in stem cell-derived fetal-like hepatocytes. These oscillations in stemness-associated features were not altered in fetal-like hepatocytes by inhibitory mimics for dominantly-expressed miRNA, such as hsa-miR-99b, -100, -214 and -221/222. The stem cell-derived fetal-like hepatocytes were permissive for miRNA characterizing mature hepatocytes, including mimics for hsa-miR-122, -126, -192, -194 and -26b, although transfections of the latter did not advance hepatic differentiation. Examination of genome-wide mRNA expression profiles in stem cell-derived or primary fetal hepatocytes indicated targets of highly abundant miRNA regulated general processes, e.g., cell survival, growth and proliferation, functional maintenance, etc., without directing cell differentiation. Among upstream regulators of gene networks in stem cell-derived hepatocytes included HNF4A, SNAI1, and others, which affect transcriptional circuits directing lineage development or maintenance. Therefore, miRNA expression oscillated in response to microenvironmental conditions, whereas lineage-specific transcriptional regulators, such as HNF4A, were necessary for directing hepatic differentiation. This knowledge will be helpful for understanding the contribution of stem cells in pathophysiological states and oncogenesis, as well as for applications of stem cell-derived hepatocytes.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

Human adenylate kinase 6 regulates WNK1 (with no lysine kinase-1) phosphorylation states and affects ion homeostasis in NT2 cells

Highlights: • Depletion of AK6 inhibits cell proliferation and promotes apoptosis in NT2 cells. • AK6 interacts with N terminal of WNK1 and regulates its phosphorylation states. • AK6 mediates Akt/WNK1 signaling pathway and regulates ion homeostasis in NT2 cells. • Chloride channel CLC-3 is responsible for ion transport in NT2 cells. Adenylate kinase 6 (AK6), a nucleus localized phosphotransferase in mammalians, shows ubiquitously expression and broad substrate activity in different tissues and cell types. Although the function of AK6 has been extensively studied in different cancer cell lines, its role in mammalian germline is still unknown. Here we showed that knockdown of AK6 inhibits cell proliferation and promotes cell apoptosis in human testicular carcinoma (NT2 cells). Co-immunoprecipitation experiment and in vitro pull down assay identified WNK1 (with no lysine kinase-1) as one of the AK6 interacting proteins in NT2 cells. Moreover, we found that AK6 regulates the phosphorylation states of WNK1 (Thr60) and affects phosphorylation level of Akt (Ser473) upon hypotonic condition, probably affecting chloride channel and regulating ion transport and homeostasis in NT2 cells and consequently contributing to the decreased cell proliferation rate. In conclusion, AK6 regulates WNK1 phosphorylation states and affects ion homeostasis in NT2 cells. These findings provide new insights into the function of AK6 and WNK1 in human testicular carcinoma. This work also provides foundation for further mechanism study of AK6 in spermatogenesis.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2016

Cooperative Electronic and Structural Regulation in a Bioinspired Allosteric Photoredox Catalyst

Here in this article, we report the first allosteric photoredox catalyst regulated via constructively coupled structural and electronic control. While often synergistically exploited in nature, these two types of control mechanisms have only been applied independently in the vast majority of allosteric enzyme mimics and receptors in the literature. By embedding a model of photosystem II in a supramolecular coordination complex that responds to chloride as an allosteric effector, we show that distance and electronic control of light harvesting can be married to maximize allosteric regulation of catalytic activity. This biomimetic system is composed of a Bodipy photoantenna, which is capable of transferring excited-state energy to a photoredox pair, wherein the excitation energy is used to generate a catalytically active charge-separated state. The structural aspect of allosteric regulation is achieved by toggling the coordination chemistry of an antenna-functionalized hemilabile ligand via partial displacement from a Rh I structual node using chloride. In doing so, the distance between the antenna and the central photoredox catalyst is increased, lowering the inherent efficiency of through-space energy transfer. At the same time, coordination of chloride lowers both the charge of the Rh I node and the reduction potential of the Rh II/I couple, to the extent that electronic quenching of the antenna excited state is possible via photoinduced electron transfer from the metal center. Compared to a previously developed system that operates solely via electronic regulation, the present system demonstrates that coupling electronic and structural approaches to allosteric regulation gives rise to improved switching ratios between catalytically active and inactive states. Contributions from both structural and electronic control mechanisms are probed via nuclear magnetic resonance, X-ray diffraction, electrochemical, spectroelectrochemical, and transient absorption studies. Overall, this work establishes that intertwined electronic and structural regulatory mechanisms can be borrowed from nature to build stimuli-responsive inorganic materials with potential applications in sensing, catalysis, and photonic devices.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗