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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 343 records · Page 19
All sourcesNASA NTRSDOE OSTI
DOE OSTI2018

Egress of murine regulatory T cells from the thymus requires TIPE2

Highlights: • TIPE2 deficiency leads to the accumulation of Tregs in the thymus. • TIPE2 deficiency doesn't affect the development and apoptosis of tTregs. • TIPE2 promotes the chemotaxis of tTregs in vitro. • TIPE2 promotes the polarization of pAKT and F-actin in tTregs undergoing directed migration. Regulatory T cells (Tregs) can be divided into thymus-derived Treg (tTregs) and peripheral induced Tregs (pTregs) in vivo according to their origins and are essential for the maintenance of immune hemostasis and immune tolerance. Tumor necrosis factor-α-induced protein 8 like 2 (TIPE2) is expressed primarily by immune cells and is a negative regulator of the innate and adaptive immune response. Previous studies indicate that TIPE2 is required for the expression of Treg signature genes and promotes leading-edge formation in neutrophils through cytoskeleton remodeling. In the current study, we showed that TIPE2 deficient mice accumulate more Treg cells in the thymus. Further studies revealed that TIPE2 deficiency doesn't affect the development and apoptosis of tTregs. Instead, TIPE2 promotes the chemotaxis of tTregs in vitro, which may account for the accumulation of Tregs in the thymus of TIPE2 deficient mice. Mechanistic study revealed that TIPE2 promotes the polarization of pAKT and F-actin in tTregs undergoing directed migration. Taken together, these results demonstrated that TIPE2 enhances the cytoskeleton remodeling and promotes the thymus egress of tTregs, which may play an important role in the maintenance of self-tolerance.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Citrullinated histone 3 causes endothelial barrier dysfunction

Highlights: • We report a new function of citrullinated histones. • H3Cit mediates microvascular leakage and endothelial barrier dysfunction. • H3Cit causes cell-cell junction disorganization and actin stress fiber formation. • H3Cit-mediated barrier dysfunction is not due to cell death or Rho/MLCK signaling. • Adenylyl cyclase activator forskolin blocks H3Cit-induced barrier dysfunction. Circulating components of neutrophil extracellular traps (NETs), especially histones, are associated with tissue injury during inflammatory conditions like sepsis. Commonly used as a NET biomarker, citrullinated histone 3 (H3Cit) may also functionally contribute to the NET-associated inflammatory response. To this end, we sought to examine the role of H3Cit in mediating microvascular endothelial barrier dysfunction. Here we show that H3Cit can directly contribute to inflammatory injury by disrupting the microvascular endothelial barrier. We found that endothelial responses to H3Cit are characterized by cell-cell adherens junction opening and cytoskeleton reorganization with increased F-actin stress fibers. Several signaling pathways often implicated in the transduction of hyperpermeability, such as Rho and MLCK, did not appear to play a major role; however, the adenylyl cyclase activator forskolin blocked the endothelial barrier effect of H3Cit. Taken together, the data suggest that H3Cit-induced endothelial barrier dysfunction may hold promise to treat inflammatory injury.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Diacylglycerol kinase β induces filopodium formation via its C1, catalytic and carboxy-terminal domains and interacts with the Rac1-GTPase-activating protein, β2-chimaerin

Highlights: • Overexpression of DGKβ in COS-7 cells strongly induced filopodium-formation. • The C1, catalytic and C-terminal domains are essential for the filopodium-formation. • Filopodium formation correlates with plasma membrane localization and F-actin colocalization. • DGKβ strongly interacts with β2-chimaerin, a Rac1-GAP. The β-isoform of diacylglycerol kinase (DGK) localizes predominantly to neurons and induces neurite outgrowth and spine formation. However, the detailed molecular mechanisms underlying the functions of DGKβ remain elusive. During the course of studies on other DGK isozymes, we unexpectedly found that the overexpression of wild-type DGKβ in COS-7 cells markedly induced filopodium formation. Because filopodium formation is closely related to neurite outgrowth and spine formation, we constructed various DGKβ mutants and compared their abilities to induce filopodium formation in order to elucidate the structure-function relationships of DGKβ. We found that the C-terminal, C1 and catalytic domains and catalytic activity were indispensable for filopodium formation, but the recoverin homology domain and EF-hand motifs were not. Moreover, the extent of plasma membrane localization and F-actin colocalization were positively correlated with filopodium formation. Intriguingly, DGKβ selectively interacted and colocalized at the plasma membrane with a Rac1-GTPase-activating protein, β2-chimaerin, which is an inducer of filopodia; it also interacted, to lesser extent, with α2-chimaerin, but not with α1- or β1-chimaerin. Moreover, DGKβ enhanced the plasma membrane localization of β2-chimaerin. These results suggest that DGKβ plays an important role in neurite outgrowth and spine formation in neurons via its ability to induce filopodium formation.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

The C-terminus of Ubl4A is critical for pro-death activity and association with the Arp2/3 complex

Highlights: • The Ubl4A C-terminus has a pro-death function. • Ubl4A interacts with the Arp2/3 complex through its C-terminus. • A single mutation in Ubl4A C-terminus abolishes its association with Arp2/3. • The D122A mutation also diminishes Ubl4A cytotoxicity in cancer cells. Ubl4A is a small ubiquitin-like protein involved in diverse cellular functions. We have shown that Ubl4A is critical for survival of the starvation-mediated cell death in vivo. The underlying mechanism for this is through interaction with the actin-related protein Arp2/3 complex and promotion of actin branching. Interestingly, “put-back” of Ubl4A to Ubl4A-deficient cells also results in cell death. Removal of the Ubl4A N-terminus significantly enhances its cytotoxicity, indicating that the pro-death activity of Ubl4A is mainly from its C-terminal region. In vitro protein pull-down assays show that the C-terminal region of Ubl4A can directly interact with the Arp2/3 complex. The single point mutation of an aspartic acid to alanine (D122A) in the Ubl4A C-terminus abolishes its ability to bind the Arp2/3 complex. This mutation also destabilizes Ubl4A proteins susceptible to protease degradation. Importantly, ectopic expression of wild-type Ubl4A can induce cell death in colon cancer cells, but such pro-death activity is diminished in the D122A mutant. These data suggest that Ubl4A C-terminus, especially D122, is critical for Ubl4A-Arp2/3 interaction and its pro-death function.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

A role for myosin VI in retinal pigment epithelium phagocytosis

Highlights: • Quashing myosin VI activity reduces rates of phagosome trafficking in RPE cells. • Trafficked phagosomes in RPE cells undergo two modes of motion. • The more randomly directed mode of phagosome trafficking is myosin VI dependent. • The more directed mode of phagosome trafficking is myosin VI independent. The retinal pigment epithelium (RPE) is a monolayer of pigmented cells adjacent to the choroid coat, the vascular layer of the eye. Among other functions, these cells are responsible for the phagocytosis of rod and cone cell waste shed from the photoreceptor outer segments. We describe here studies to understand the involvement of the motor protein myosin VI in the trafficking of internalized microspheres by a human retinal pigment epithelium primary cell line (ARPE-19). We perturbed the myosin VI-actin interaction by overexpressing a dominant negative myosin VI construct in these cells. We used single particle tracking to characterize the trajectories of internalized fluorescent microspheres. Analysis of the speed of the microspheres' motions revealed that the magnitude of the average speed over short time scales is reduced in the presence of the dominant negative motor. Analysis of the mean-squared displacement of these trajectories demonstrated that trafficking of phagosomes involves two modes of motion, a rapid, more randomly directed motion that is myosin VI dependent and a slower, more directed motion that is independent of the motor. From these data, we posit that this trafficking process involves an interplay between myosin VI motors, which are involved in motion through the actin periphery of the cell, and microtubule motors.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Planar compression of extracellular substrates induces S phase arrest via ATM-independent CHK2 activation

Highlights: • Planar compression of the extracellular substrate induces S phase arrest. • Cancer cells do not exhibit compression-induced S phase arrest. • The CHK2-p53 pathway mediates compression-induced S phase arrest. • Compression-induced activation of CHK2 is independent of ATM. • Actin disassembly upon substrate compression is required for S phase arrest. Cell proliferation is regulated not only by soluble chemical factors but also by mechanical cues surrounding cells. Mechanical stretch of extracellular substrates is known to promote cell proliferation by driving exit from the G{sub 0} phase and entry into the S phase. Here, we report that planer compression of extracellular substrates induces cell cycle arrest in the S phase. The compression-induced S phase arrest is mediated by the checkpoint kinase 2 (CHK2)-p53 pathway. In contrast to the canonical S phase checkpoint pathway activated by DNA damage, CHK2 activation by the substrate compression is independent of ataxia telangiectasia mutated (ATM). We further find that disassembly of the actin cytoskeleton is required for the compression-induced S phase arrest. Notably, cancer cells do not exhibit S phase arrest upon the substrate compression. Our results suggest a novel mechanism for homeostatic control of cell growth under mechanical perturbations.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2011

Regulation of gene expression and subcellular protein distribution in MLO-Y4 osteocytic cells by lysophosphatidic acid: Relevance to dendrite outgrowth.

Osteoblastic and osteocytic cells are highly responsive to the lipid growth factor lysophosphatidic acid (LPA) but the mechanisms by which LPA alters bone cell functions are largely unknown. A major effect of LPA on osteocytic cells is the stimulation of dendrite membrane outgrowth, a process that we predicted to require changes in gene expression and protein distribution. We employed DNA microarrays for global transcriptional profiling of MLO-Y4 osteocytic cells grown for 6 and 24h in the presence or absence of LPA. We identified 932 transcripts that displayed statistically significant changes in abundance of at least 1.25-fold in response to LPA treatment. Gene ontology (GO) analysis revealed that the regulated gene products were linked to diverse cellular processes, including DNA repair, response to unfolded protein, ossification, protein-RNA complex assembly, and amine biosynthesis. Gene products associated with the regulation of actin microfilament dynamics displayed the most robust expression changes, and LPA-induced dendritogenesis in vitro was blocked by the stress fiber inhibitor cytochalasin D. Mass spectrometry-based proteomic analysis of MLO-Y4 cells revealed significant LPA-induced changes in the abundance of 284 proteins at 6h and 844 proteins at 24h. GO analysis of the proteomic data linked the effects of LPA to cell processes that control of protein distribution and membrane outgrowth, including protein localization, protein complex assembly, Golgi vesicle transport, cytoskeleton-dependent transport, and membrane invagination/endocytosis. Dendrites were isolated from LPA-treated MLO-Y4 cells and subjected to proteomic analysis to quantitatively assess the subcellular distribution of proteins. Sets of 129 and 36 proteins were enriched in the dendrite fraction as compared to whole cells after 6h and 24h of LPA exposure, respectively. Protein markers indicated that membranous organelles were largely excluded from the dendrites. Highly represented among the proteins with elevated abundances in dendrites were molecules that regulate cytoskeletal function, cell motility and membrane adhesion. Our combined transcriptomic/proteomic analysis of the response of MLO-Y4 osteocytic cells to LPA indicates that dendritogenesis is a membrane- and cytoskeleton-driven process with actin dynamics playing a particularly critical role.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2015

Effect of prolonged exposure to sublethal concentrations of DDT and DDE on protein expression in human pancreatic beta cells

Pollution of the environment represents one of less explored potential reasons for the worldwide epidemic of type 2 diabetes. One of the most prevalent organochlorine pollutants remains the pesticide DDT and its degradation product DDE. Despite some epidemiologic correlations between levels of DDT and DDE in human organism and the prevalence of diabetes, there is almost no information about the exact targets of these compounds inside pancreatic beta cells. To detect functional areas of pancreatic beta cells that could be affected by exposure to DDT and DDE, we analyzed changes in protein expression in the NES2Y human pancreatic beta cell line exposed to three sublethal concentrations (0.1 μM, 1 μM, 10 μM) of DDT and DDE for 1 month. Protein separation and identification was achieved using high-resolution 2D-electrophoresis, computer analysis and mass spectrometry. With these techniques, four proteins were found downregulated after exposure to 10 μM DDT: three cytoskeletal proteins (cytokeratin 8, cytokeratin 18 and actin) and one protein involved in glycolysis (alpha-enolase). Two proteins were downregulated after exposure to 10 μM DDE: cytokeratin 18 and heterogenous nuclear ribonucleoprotein H1 (HNRH1). These changes correlate with previously described effects of other stress conditions (e.g. exposure to palmitate, hyperglycemia, imidazoline derivative, and cytokines) on protein expression in pancreatic beta cells. We conclude that cytoskeletal proteins and their processing, glucose metabolism, and mRNA processing may represent targets affected by exposure to conditions hostile to pancreatic beta cells, including exposure to DDT and DDE. - Highlights: • Epidemiologic studies connect pollution with incidence of diabetes mellitus. • We explored the effect of DDT and DDE on protein expression in the NES2Y pancreatic beta cell line. • One month exposure to three sublethal concentrations of DDT and DDE was employed. • Expression of alpha-enolase, actin, cytokeratin 8 and 18 was reduced in NES2Y/DDT. • Expression of HNRH1 and cytokeratin 18 was reduced in NES2Y/DDE.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2018

Irradiation of rainbow trout at early life stages results in a proteomic legacy in adult gills. Part A; proteomic responses in the irradiated fish and in non-irradiated bystander fish

Highlights: • The effects of early life irradiation on the trout gill proteome was investigated. • The bystander effect proteomic responses in non-irradiated fish were investigated. • Proteomic responses of embryonic irradiation were anti-tumorigenic or anti-oxidant. • Proteomic responses of larval or first feeder irradiation were “dual function”. • Bystander effect proteomic responses were anti-tumorigenic or protective. Exposure to a single 0.5 Gy X-ray dose of eggs at 48 h after fertilisation (48 h egg), eyed eggs, yolk sac larvae (YSL) and first feeders induces a legacy effect in adult rainbow trout. This includes the transmission of a bystander effect to non-irradiated adult trout which had swam with the irradiated fish. The aim of this study was to investigate this legacy by analysing the gill proteome of these irradiated and bystander fish. Irradiation at all of the early life stages resulted in changes to proteins which play a key role in development but are also known to be anti-tumorigenic and anti-oxidant: upregulation of haemoglobin subunit beta (48 h egg), haemoglobin, serum albumin 1 precursor (eyed eggs), clathrin heavy chain 1 isoform X10 (eyed eggs and first feeders), and actin-related protein 2/3 complex subunit 4 (first feeders), downregulation of pyruvate dehydrogenase, histone 1 (48 h egg), triosephosphate isomerase (TPI), collagen alpha-1(1) chain like proteins (YSL), pyruvate kinase PKM-like protein (YSL and first feeders), ubiquitin-40S ribosomal proteins S27 and eukaryotic translation initiation factor 4 A isoform 1B (first feeders). However irradiation of YSL and first feeders (post hatching early life stages) also induced proteomic changes which have a complex relationship with tumorigenesis or cancer progression; downregulation of alpha-1-antiprotease-like protein precursor, vigilin isoform X2 and nucleoside diphosphate kinase (YSL) and upregulation of hyperosmotic glycine rich protein (first feeders). In bystander fish some proteomic changes were similar to those induced by irradiation: upregulation of haemoglobin subunit beta (48 h egg), haemoglobin (eyed eggs), actin-related protein 2/3 complex subunit 4, hyperosmotic glycine rich protein (first feeders), and downregulation of alpha-1-antiprotease-like protein, vigilin isoform X2, nucleoside diphosphate kinase (YSL), pyruvate kinase PKM-like protein and ubiquitin-40S ribosomal protein S27a-like (first feeders). Other proteomic changes were unique to bystander fish; downregulation of TPI, ubiquitin-40S ribosomal protein S2 (eyed egg), cofilin-2, cold-inducible RNA-binding protein B-like isoform X3 (YSL) and superoxide dismutase (first feeder), and upregulation of haemoglobin subunit alpha, collagen 1a1 precursor, apolipoprotein A-1-1 and A-1–2 precursor (first feeders). These bystander effect proteomic changes have been shown to be overwhelmingly anti-tumorigenic or protective of the fish gill.

63 RADIATION, THERMAL, AND OTHER ENVIRON. POLLUTAN↗
DOE OSTI2014

Exploring the potential role of tungsten carbide cobalt (WC-Co) nanoparticle internalization in observed toxicity toward lung epithelial cells in vitro

Tungsten carbide cobalt (WC-Co) has been recognized as a workplace inhalation hazard in the manufacturing, mining and drilling industries by the National Institute of Occupational Safety and Health. Exposure to WC-Co is known to cause “hard metal lung disease” but the relationship between exposure, toxicity and development of disease remain poorly understood. To better understand this relationship, the present study examined the role of WC-Co particle size and internalization on toxicity using lung epithelial cells. We demonstrated that nano- and micro-WC-Co particles exerted toxicity in a dose- and time-dependent manner and that nano-WC-Co particles caused significantly greater toxicity at lower concentrations and shorter exposure times compared to micro-WC-Co particles. WC-Co particles in the nano-size range (not micron-sized) were internalized by lung epithelial cells, which suggested that internalization may play a key role in the enhanced toxicity of nano-WC-Co particles over micro-WC-Co particles. Further exploration of the internalization process indicated that there may be multiple mechanisms involved in WC-Co internalization such as actin and microtubule based cytoskeletal rearrangements. These findings support our hypothesis that WC-Co particle internalization contributes to cellular toxicity and suggest that therapeutic treatments inhibiting particle internalization may serve as prophylactic approaches for those at risk of WC-Co particle exposure. - Highlights: • Hard metal (WC-Co) particle toxicity was established in lung epithelial cells. • Nano-WC-Co particles caused greater toxicity than micro-WC-Co particles. • Nano- and micro-WC-Co particles were capable of inducing cellular apoptosis. • Nano-WC-Co particles were internalized by lung epithelial cells. • WC-Co particle internalization was mediated by actin dynamics.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2013

HES6 enhances the motility of alveolar rhabdomyosarcoma cells

Absract: HES6, a member of the hairy-enhancer-of-split family of transcription factors, plays multiple roles in myogenesis. It is a direct target of the myogenic transcription factor MyoD and has been shown to regulate the formation of the myotome in development, myoblast cell cycle exit and the organization of the actin cytoskeleton during terminal differentiation. Here we investigate the expression and function of HES6 in rhabdomyosarcoma, a soft tissue tumor which expresses myogenic genes but fails to differentiate into muscle. We show that HES6 is expressed at high levels in the subset of alveolar rhabdomyosarcomas expressing PAX/FOXO1 fusion genes (ARMSp). Knockdown of HES6 mRNA in the ARMSp cell line RH30 reduces proliferation and cell motility. This phenotype is rescued by expression of mouse Hes6 which is insensitive to HES6 siRNA. Furthermore, expression microarray analysis indicates that the HES6 knockdown is associated with a decrease in the levels of Transgelin, (TAGLN), a regulator of the actin cytoskeleton. Knockdown of TAGLN decreases cell motility, whilst TAGLN overexpression rescues the motility defect resulting from HES6 knockdown. These findings indicate HES6 contributes to the pathogenesis of ARMSp by enhancing both proliferation and cell motility.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2013

Myosin heavy chain-like localizes at cell contact sites during Drosophila myoblast fusion and interacts in vitro with Rolling pebbles 7

Besides representing the sarcomeric thick filaments, myosins are involved in many cellular transport and motility processes. Myosin heavy chains are grouped into 18 classes. Here we show that in Drosophila, the unconventional group XVIII myosin heavy chain-like (Mhcl) is transcribed in the mesoderm of embryos, most prominently in founder cells (FCs). An ectopically expressed GFP-tagged Mhcl localizes in the growing muscle at cell–cell contacts towards the attached fusion competent myoblast (FCM). We further show that Mhcl interacts in vitro with the essential fusion protein Rolling pebbles 7 (Rols7), which is part of a protein complex established at cell contact sites (Fusion-restricted Myogenic-Adhesive Structure or FuRMAS). Here, branched F-actin is likely needed to widen the fusion pore and to integrate the myoblast into the growing muscle. We show that the localization of Mhcl is dependent on the presence of Rols7, and we postulate that Mhcl acts at the FuRMAS as an actin motor protein. We further show that Mhcl deficient embryos develop a wild-type musculature. We thus propose that Mhcl functions redundantly to other myosin heavy chains in myoblasts. Lastly, we found that the protein is detectable adjacent to the sarcomeric Z-discs, suggesting an additional function in mature muscles. - Highlights: ► The class XVIII myosin encoding gene Mhcl is transcribed in the mesoderm. ► Mhcl localization at contact sites of fusing myoblasts depends on Rols7. ► Mhcl interacts in vitro with Rols7 which is essential for myogenesis. ► Functional redundancy with other myosins is likely as mutants show no muscle defects. ► Mhcl localizes adjacent to Z-discs of sarcomeres and might support muscle integrity.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2013

One isoform of Arg/Abl2 tyrosine kinase is nuclear and the other seven cytosolic isoforms differently modulate cell morphology, motility and the cytoskeleton

The non-receptor tyrosine kinase Abelson related gene (Arg/Abl2) regulates cell migration and morphogenesis by modulating the cytoskeleton. Arg promotes actin-based cell protrusions and spreading, and inhibits cell migration by attenuating stress fiber formation and contractility via activation of the RhoA inhibitor, p190RhoGAP, and by regulating focal adhesion dynamics also via CrkII phosphorylation. Eight full-length Arg isoforms with different N- and C-termini are endogenously expressed in human cells. In this paper, the eight Arg isoforms, subcloned in the pFLAG-CMV2 vector, were transfected in COS-7 cells in order to study their subcellular distribution and role in cell morphology, migration and cytoskeletal modulation. The transfected 1BSCTS Arg isoform has a nuclear distribution and phosphorylates CrkII in the nucleus, whilst the other isoforms are detected in the cytoplasm. The 1BLCTL, 1BSCTL, 1ASCTS isoforms were able to significantly decrease stress fibers, induce cell shrinkage and filopodia-like protrusions with a significant increase in p190RhoGAP phosphorylation. In contrast, 1ALCTL, 1ALCTS, 1ASCTL and 1BLCTS isoforms do not significantly decrease stress fibers and induce the formation of retraction tail-like protrusions. The 1BLCTL and 1ALCTL isoforms have different effects on cell migration and focal adhesions. All these data may open new perspectives to study the mechanisms of cell invasiveness. -Highlights: • Each of the eight Arg isoforms was transfected in COS-7 cells. • Only the 1BSCTS Arg isoform has a nuclear distribution in transfected cells. • The cytoplasmic isoforms and F-actin colocalize cortically and in cell protrusions. • Arg isoforms differently phosphorylate p190RhoGAP and CrkII. • Arg isoforms differently modulate stress fibers, cell protrusions and motility.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2015

Studying p53 family proteins in yeast: Induction of autophagic cell death and modulation by interactors and small molecules

In this work, the yeast Saccharomyces cerevisiae was used to individually study human p53, p63 (full length and truncated forms) and p73. Using this cell system, the effect of these proteins on cell proliferation and death, and the influence of MDM2 and MDMX on their activities were analyzed. When expressed in yeast, wild-type p53, TAp63, ΔNp63 and TAp73 induced growth inhibition associated with S-phase cell cycle arrest. This growth inhibition was accompanied by reactive oxygen species production and autophagic cell death. Furthermore, they stimulated rapamycin-induced autophagy. On the contrary, none of the tested p53 family members induced apoptosis either per se or after apoptotic stimuli. As previously reported for p53, also TAp63, ΔNp63 and TAp73 increased actin expression levels and its depolarization, suggesting that ACT1 is also a p63 and p73 putative yeast target gene. Additionally, MDM2 and MDMX inhibited the activity of all tested p53 family members in yeast, although the effect was weaker on TAp63. Moreover, Nutlin-3a and SJ-172550 were identified as potential inhibitors of the p73 interaction with MDM2 and MDMX, respectively. Altogether, the yeast-based assays herein developed can be envisaged as a simplified cell system to study the involvement of p53 family members in autophagy, the modulation of their activities by specific interactors (MDM2 and MDMX), and the potential of new small molecules to modulate these interactions. - Highlights: • p53, p63 and p73 are individually studied in the yeast S. cerevisiae. • p53 family members induce ROS production, cell cycle arrest and autophagy in yeast. • p53 family members increase actin depolarization and expression levels in yeast. • MDM2 and MDMX inhibit the activity of p53 family members in yeast. • Yeast can be a useful tool to study the biology and drugability of p53, p63 and p73.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2015

Experimental evidence of Migfilin as a new therapeutic target of hepatocellular carcinoma metastasis

Migfilin is a novel cell–matrix adhesion protein known to interact with Vasodilator Stimulated Phosphoprotein (VASP) and be localized both at cell–matrix and cell–cell adhesions. To date there is nothing known about its role in hepatocellular carcinoma (HCC). As matrix is important in metastasis, we aimed to investigate the Migfilin's role in HCC metastasis using two human HCC cell lines that differ in their metastatic potential; non-invasive Alexander cells and the highly invasive HepG2 cells. We silenced Migfilin by siRNA and studied its effect on signaling and metastasis-related cellular properties. We show that Migfilin's expression is elevated in HepG2 cells and its silencing leads to upregulation of actin reorganization-related proteins, namely phosphor-VASP (Ser157 and Ser239), Fascin-1 and Rho-kinase-1, promoting actin polymerization and inhibiting cell invasion. Phosphor-Akt (Ser473) is decreased contributing to the upregulation of free and phosphor-β-catenin (Ser33/37Thr41) and inducing proliferation. Migfilin elimination upregulates Extracellular Signal–regulated kinase, which increases cell adhesion in HepG2 and reduces invasiveness. This is the first study to reveal that Migfilin inhibition can halt HCC metastasis in vitro, providing the molecular mechanism involved and presenting Migfilin as potential therapeutic target against HCC metastasis. - Highlights: • Migfilin is a cell–matrix and cell–cell adhesion protein known to interact with VASP. • Nothing is known about Migfilin's role in hepatocellular carcinoma (HCC). • We eliminated Migfilin from 2 HCC cell lines and studied in vitro metastasis. • Its silencing inhibits cell invasion and promotes adhesion in HepG2 invasive cells. • We provide molecular mechanism by which Migfilin elimination halts HCC metastasis.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2015

Wip1 knockout inhibits the proliferation and enhances the migration of bone marrow mesenchymal stem cells

Mesenchymal stem cells (MSCs), a unique population of multipotent adult progenitor cells originally found in bone marrow (BM), are extremely useful for multifunctional therapeutic approaches. However, the growth arrest and premature senescence of MSCs in vitro prevent the in-depth characterization of these cells. In addition, the regulatory factors involved in MSCs migration remain largely unknown. Given that protein phosphorylation is associated with the processes of MSCs proliferation and migration, we focused on wild-type p53-inducible phosphatase-1 (Wip1), a well-studied modulator of phosphorylation, in this study. Our results showed that Wip1 knockout significantly inhibited MSCs proliferation and induced G2-phase cell-cycle arrest by reducing cyclinB1 expression. Compared with WT-MSCs, Wip1{sup −/−} MSCs displayed premature growth arrest after six passages in culture. Transwell and scratch assays revealed that Wip1{sup −/−} MSCs migrate more effectively than WT-MSCs. Moreover, the enhanced migratory response of Wip1{sup −/−} MSCs may be attributed to increases in the induction of Rac1-GTP activity, the pAKT/AKT ratio, the rearrangement of filamentous-actin (f-actin), and filopodia formation. Based on these results, we then examined the effect of treatment with a PI3K/AKT and Rac1 inhibitor, both of which impaired the migratory activity of MSCs. Therefore, we propose that the PI3K/AKT/Rac1 signaling axis mediates the Wip1 knockout-induced migration of MSCs. Our findings indicate that the principal function of Wip1 in MSCs transformation is the maintenance of proliferative capacity. Nevertheless, knocking out Wip1 increases the migratory capacity of MSCs. This dual effect of Wip1 provides the potential for purposeful routing of MSCs. - Highlights: • Wip1 knockout inhibited MSCs proliferation through reducing cyclinB1 expression. • Wip1{sup −/−} MSCs displayed premature growth arrest in vitro after six passages. • Knocking out Wip1 increases the migratory capacity of MSCs. • The PI3K/AKT/Rac1 pathway mediates the Wip1-knockout-induced migration of MSCs. • Overexpression of Wip1 reversed premature senescence and migration of Wip1{sup −/−} MSCs.

60 APPLIED LIFE SCIENCES↗