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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 397 records · Page 22
All sourcesNASA NTRSDOE OSTI
DOE OSTI2014

Adenoviral overexpression of Lhx2 attenuates cell viability but does not preserve the stem cell like phenotype of hepatic stellate cells

Hepatic stellate cells (HSC) are well known initiators of hepatic fibrosis. After liver cell damage, HSC transdifferentiate into proliferative myofibroblasts, representing the major source of extracellular matrix in the fibrotic organ. Recent studies also demonstrate a role of HSC as progenitor or stem cell like cells in liver regeneration. Lhx2 is described as stem cell maintaining factor in different organs and as an inhibitory transcription factor in HSC activation. Here we examined whether a continuous expression of Lhx2 in HSC could attenuate their activation and whether Lhx2 could serve as a potential target for antifibrotic gene therapy. Therefore, we evaluated an adenoviral mediated overexpression of Lhx2 in primary HSC and investigated mRNA expression patterns by qRT-PCR as well as the activation status by different in vitro assays. HSC revealed a marked increase in activation markers like smooth muscle actin alpha (αSMA) and collagen 1α independent from adenoviral transduction. Lhx2 overexpression resulted in attenuated cell viability as shown by a slightly hampered migratory and contractile phenotype of HSC. Expression of stem cell factors or signaling components was also unaffected by Lhx2. Summarizing these results, we found no antifibrotic or stem cell maintaining effect of Lhx2 overexpression in primary HSC. - Highlights: • We performed adenoviral overexpression of Lhx2 in primary hepatic stellate cells. • Hepatic stellate cells expressed stem cell markers during cultivation. • Cell migration and contractility was slightly hampered upon Lhx2 overexpression. • Lhx2 overexpression did not affect stem cell character of hepatic stellate cells.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2015

The cytoskeletal inhibitors latrunculin A and blebbistatin exert antitumorigenic properties in human hepatocellular carcinoma cells by interfering with intracellular HuR trafficking

The impact of the RNA-binding protein HuR for the post-transcriptional deregulation of tumor-relevant genes is well established. Despite of elevations in HuR expression levels, an increase in cytoplasmic HuR abundance in many cases correlates with a high grade of malignancy. Here, we demonstrated that administration of the actin-depolymerizing macrolide latrunculin A, or blebbistatin, an inhibitor of myosin II ATPase activity, caused a dose- and time-dependent reduction in the high cytoplasmic HuR content of HepG2 and Huh7 hepatocellular carcinoma (HCC) cells. Subcellular fractionation revealed that in addition, both inhibitors strongly attenuated cytoskeletal and membrane-bound HuR abundance and conversely increased the HuR amount in nuclear cell fractions. Concomitant with changes in intracellular HuR localization, both cytoskeletal inhibitors markedly decreased the half-lives of cyclooxygenase-2 (COX-2), cyclin A and cyclin D{sub 1} encoding mRNAs resulting in a significant reduction in their expression levels in HepG2 cells. Importantly, a similar reduction in the expression of these HuR targets was achieved by a RNA interference (RNAi)-mediated knockdown of either HuR or nonmuscle myoin IIA. Using polysomal fractionation, we further demonstrate that the decrease in cytoplasmic HuR by latrunculin A or blebbistatin is accompanied by a marked change in the allocation of HuR and its mRNA cargo from polysomes to ribonucleoprotein (RNP) particles. Functionally, the basal migration and prostaglandin E{sub 2} synthesis are similarly impaired in inhibitor-treated and stable HuR-knockdown HepG2 cells. Our data demonstrate that interfering with the actomyosin-dependent HuR trafficking may comprise a valid therapeutic option for antagonizing pathologic posttranscriptional gene expression by HuR and furthermore emphasize the potential benefit of HuR inhibitory strategies for treatment of HCC. - Highlights: • We tested the effects of latrunculin A and blebbistatin on different HuR activities. • Both cytoskeletal inhibitors disturbed the RNP-polysome HuR allocation in HepG2 cells. • Accordingly, both inhibitors reduced the stability and levels of HuR controlled mRNAs. • Functionally, important HuR-triggered cell functions were impaired by both compounds. • Targeting of HuR trafficking implies a useful approach for novel antitumor therapy.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2016

MicroRNA-29b regulates TGF-β1-mediated epithelial–mesenchymal transition of retinal pigment epithelial cells by targeting AKT2

The role of microRNA (miRNA) in proliferative vitreoretinopathy (PVR) progression has not been studied extensively, especially in retinal pigment epithelial–mesenchymal transition (EMT) which is the main reason for formation of PVR. In this study, we first investigated the miRNA expression profile in transforming growth factor beta 1 (TGF-β1) mediated EMT of ARPE-19 cells. Among the five changed miRNAs, miR-29b showed the most significant downregulation. Enhanced expression of miR-29b could reverse TGF-β1 induced EMT through targeting Akt2. Akt2 downregulation could inhibit TGF-β1-induced EMT. Furthermore, inhibition of miR-29b in ARPE-19 cells directly triggered EMT process, which characterized by the phenotypic transition and the upregulation of α-smooth muscle actin (α-SMA) and downregulation of E-cadherin and zona occludin-1 (ZO-1) with increased cell migration. Akt2-shRNA also inhibited miR-29 inhibitor-induced EMT process. These data indicate that miR-29b plays an important role in TGF-β1-mediated EMT in ARPE-19 cells by targeting Akt2. - Highlights: • MiR-29b expression is decreased in TGF-β1-induced EMT of ARPE-19 cells. • MiR-29b inhibits TGF-β1-induced EMT in ARPE-19 cells. • MiR-29b inhibitor induces EMT in ARPE-19 cells. • Akt2 is the target for miR-29b. • Downregulation of Akt2 prevents TGF-β1-induced EMT of ARPE-19 cells.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2015

Targeted inhibition of disheveled PDZ domain via NSC668036 depresses fibrotic process

In this study, we determined the effects of transforming growth factor-beta (TGF-β) and Wnt/β-catenin signaling on myofibroblast differentiation of NIH/3T3 fibroblasts in vitro and evaluated the therapeutic efficacy of NSC668036 in bleomycin-induced pulmonary fibrosis murine model. In vitro study, NSC668036, a small organic inhibitor of the PDZ domain in Dvl, suppressed β-catenin-driven gene transcription and abolished TGF-β1-induced migration, expression of collagen I and α-smooth muscle actin (α-SMA) in fibroblasts. In vivo study, we found that NSC668036 significantly suppressed accumulation of collagen I, α-SMA, and TGF-β1 but increased the expression of CK19, Occludin and E-cadherin that can inhibit pulmonary fibrogenesis. Because fibrotic lung exhibit aberrant activation of Wnt/β-catenin signaling, these data collectively suggest that inhibition of Wnt/β-catenin signaling at the Dvl level may be an effective approach to the treatment of fibrotic lung diseases. - Highlights: • NSC668036 inhibited the proliferation and migration of NIH/3T3 fibroblasts. • NSC668036 suppressed the Wnt/β-catenin signaling pathway. • TGF-β-induced stimulation of profibrotic responses were inhibited by NSC668036. • NSC668036 can inhibit the development of bleomycin-induced pulmonary fibrosis.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2015

Stabilization of gene expression and cell morphology after explant recycling during fin explant culture in goldfish

The development of fin primary cell cultures for in vitro cellular and physiological studies is hampered by slow cell outgrowth, low proliferation rate, poor viability, and sparse cell characterization. Here, we investigated whether the recycling of fresh explants after a first conventional culture could improve physiological stability and sustainability of the culture. The recycled explants were able to give a supplementary cell culture showing faster outgrowth, cleaner cell layers and higher net cell production. The cells exhibited a highly stabilized profile for marker gene expression including a low cytokeratin 49 (epithelial marker) and a high collagen 1a1 (mesenchymal marker) expression. Added to the cell spindle-shaped morphology, motility behavior, and actin organization, this suggests that the cells bore stable mesenchymal characteristics. This contrast with the time-evolving expression pattern observed in the control fresh explants during the first 2 weeks of culture: a sharp decrease in cytokeratin 49 expression was concomitant with a gradual increase in col1a1. We surmise that such loss of epithelial features for the benefit of mesenchymal ones was triggered by an epithelial to mesenchymal transition (EMT) process or by way of a progressive population replacement process. Overall, our findings provide a comprehensive characterization of this new primary culture model bearing mesenchymal features and whose stability over culture time makes those cells good candidates for cell reprogramming prior to nuclear transfer, in a context of fish genome preservation. - Highlights: • Recycled fin explants outgrow cells bearing stable mesenchymal traits. • Cell production and quality is enhanced in the recycled explant culture system. • Fresh fin primary culture is highly variable and loose epithelial traits over time.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2016

Overexpression of transcription factor FOXC2 in cultured human podocytes upregulates injury markers and increases motility

Obesity and diabetes-related kidney diseases associate with renal failure and cardiovascular morbidity, and represent a major health issue worldwide. However, the molecular mechanisms leading to their development remain poorly understood. We observed increased expression of transcription factor FoxC2 in the podocytes of obese Zucker rats that are insulin resistant and albuminuric. We also found that depletion of adiponectin, an adipocyte-derived hormone whose secretion is decreased in obesity, upregulated FOXC2 in differentiated human podocytes in vitro. Overexpression of FOXC2 in cultured human podocytes led to increased nuclear expression of FOXC2 associated with a change of cellular morphology. This was accompanied by upregulation of vimentin, a key mesenchymal marker, and active beta-catenin, associated with podocyte injury. We also observed re-organization of the actin cytoskeleton, disrupted localization of the tight junction protein ZO-1, and increased motility of podocytes overexpressing FOXC2. These data indicate that the expression of FOXC2 in podocytes needs to be tightly regulated, and that its overexpression induces a chain of cellular events leading to podocyte dysfunction. These changes may lead to podocyte detachment and depletion ultimately contributing to albuminuria. We also suggest a novel molecular mechanism linking obesity-induced decrease in adiponectin to podocyte dysfunction via upregulation of FOXC2. - Highlights: • Obese Zucker rats show an increase in nuclear FoxC2 in glomerular podocytes. • Adiponectin depletion upregulates FOXC2 in cultured human podocytes. • Overexpression of FOXC2 in podocytes leads to upregulation of injury markers. • FOXC2 overexpressing podocytes have altered morphology and increased motility. • Overexpression of FOXC2 could ultimately contribute to albuminuria.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2016

Isolation and characterization of human islet stellate cells

Background and aims: We have previously demonstrated that islet stellate cells (ISCs) exhibiting a similar phenotype to classical pancreatic stellate cells (PSCs) could be isolated from rat islets, where they may contribute to islet fibrosis in type 2 diabetes mellitus (T2DM). This study was designed to determine whether human islets also contain ISC. Materials and methods: Using standard explants techniques, human ISCs were enriched from freshly isolated human islets. Immunofluorescence visualization of markers for PSCs(α-smooth muscle actin;α-SMA), desmin, vimentin, glial fibrillary acidic protein (GFAP) was used to characterize the human ISC. Cell counting kit-8 (CCK-8) was used to assess the proliferation of ISC. The wound-healing assay and the transwell migration were used to assess the migration capacity of ISC. Immunofluorescence against collagen typesI (col-I), collagen typesIII (col-III) and fibronectin (FN) was performed to identify extracellular matrix (ECM) component synthesized by ISC. Adipogenic and osteogenic differentiation were tried to detected stem cell potential. Results: In culture, ISC with triangular shape grow out from human islets. The passaged ISC expressed α-SMA, desmin, vimentin, GFAP and was positive for col-I, col-III and FN. The proliferation and migration ability of ISC was significantly slower than those of PSC. And both the human PSC and ISC were able to differentiate in vitro into adipocyte- and osteoblast-like cells. Conclusion: Similar to our previous rat experiment, the current study shows that human islets also contain ISC which is phenotypically similar but not identical to human PSC. - Highlights: • ISCs grow out from human islets. ISC expressed α-SMA, desmin, vimentin, GFAP and was positive for col-I, col-III and FN. The proliferation and migration ability of human islets contain ISC. • ISC is phenotypically similar but not identical to PSC. • ISC may play an important role in the islet fibrosis in T2DM.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2016

The journey of integrins and partners in a complex interactions landscape studied by super-resolution microscopy and single protein tracking

Cells adjust their adhesive and cytoskeletal organizations according to changes in the biochemical and physical nature of their surroundings. In return, by adhering and generating forces on the extracellular matrix (ECM) cells organize their microenvironment. Integrin-dependent focal adhesions (FAs) are the converging zones integrating biochemical and biomechanical signals arising from the ECM and the actin cytoskeleton. Thus, integrin-mediated adhesion and mechanotransduction, the conversion of mechanical forces into biochemical signals, are involved in critical cellular functions such as migration, proliferation and differentiation, and their deregulation contributes to pathologies including cancer. A challenging problem is to decipher how stochastic protein movements and interactions lead to formation of dynamic architecture such as integrin-dependent adhesive structures. In this review, we will describe recent advances made possible by super-resolution microscopies and single molecule tracking approaches that provided new understanding on the organization and the dynamics of integrins and intracellular regulators at the nanoscale in living cells.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2016

Vitamin A and insulin are required for the maintenance of hepatic stellate cell quiescence

Transdifferentiation of vitamin A-storing hepatic stellate cells (HSCs) to vitamin A-depleted myofibroblastic cells leads to liver fibrosis. Vitamin A regulates lipid accumulation and gene transcription, suggesting that vitamin A is involved in the maintenance of HSC quiescence under a physiological condition. However, the precise mechanism remains elusive because there is no appropriate in vitro culture system for quiescent HSCs. Here, we show that treatment of quiescent HSCs with vitamin A partially maintained the accumulation of lipid droplets and expression of quiescent HSC markers (glial fibrillary acidic protein, peroxisome proliferator-activator receptor-γ and CCAAT/enhancer-binding protein-α) and also the expression of myofibroblastic markers (α-smooth muscle actin, heat shock protein 47 and collagen type I). On the other hand, combined treatment with vitamin A and insulin sustained the characteristic of HSC quiescence and completely suppressed the expression of myofibroblastic markers through activation of the JAK2/STAT5 signaling pathway and increased expression of sterol regulatory element binding protein-1. These treated HSCs transdifferentiated to myofibroblastic cells under a culture condition with fetal bovine serum. The results suggest an important role of vitamin A and insulin in the maintenance of HSC quiescence under a physiological condition. - Highlights: • Vitamin A partially maintained the quiescent phenotypes of HSCs. • Combined treatment with vitamin A and insulin sustained HSC quiescence. • These treated HSCs activated under a culture condition with FBS. • HSC quiescence is maintained by activation of JAK2/STAT5 and SREBP-1 expression.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2016

Nitric Oxide and ERK mediates regulation of cellular processes by Ecdysterone

The complex process of wound healing is a major problem associated with diabetes, venous or arterial disease, old age and infection. A wide range of pharmacological effects including anabolic, anti-diabetic and hepato-protective activities have been attributed to Ecdysterone. In earlier studies, Ecdysterone has been shown to modulate eNOS and iNOS expression in diabetic animals and activate osteogenic differentiation through the Extracellular-signal-Regulated Kinase (ERK) pathway in periodontal ligament stem cells. However, in the wound healing process, Ecdysterone has only been shown to enhance granulation tissue formation in rabbits. There have been no studies to date, which elucidate the molecular mechanism underlying the complex cellular process involved in wound healing. The present study, demonstrates a novel interaction between the phytosteroid Ecdysterone and Nitric Oxide Synthase (NOS), in an Epidermal Growth Factor Receptor (EGFR)-dependent manner, thereby promoting cell proliferation, cell spreading and cell migration. These observations were further supported by the 4-amino-5-methylamino- 2′ ,7′ -difluorofluorescein diacetate (DAF FM) fluorescence assay which indicated that Ecdysterone activates NOS resulting in increased Nitric Oxide (NO) production. Additionally, studies with inhibitors of both the EGFR and ERK, demonstrated that Ecdysterone activates NOS through modulation of EGFR and ERK. These results clearly demonstrate, for the first time, that Ecdysterone enhances Nitric Oxide production and modulates complex cellular processes by activating ERK1/2 through the EGF pathway. - Highlights: • Ecdysterone significantly enhances cell migration in a dose dependent manner. • Ecdysterone augments cell spreading during the initial phase of cell migration through actin cytoskeletal rearrangement. • Ecdysterone enhances cell proliferation in a nitric oxide dependent manner. • Ecdysterone enhances nitric oxide production via activation of EGFR and phosphorylation of ERK.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2016

Inhibition of hydrogen sulfide on the proliferation of vascular smooth muscle cells involved in the modulation of calcium sensing receptor in high homocysteine

Hyperhomocysteinemia induces the proliferation of vascular smooth muscle cells (VSMCs). Hydrogen sulfide (H{sub 2}S) inhibits the phenotype switch of VSMCs and calcium-sensing receptor (CaSR) regulated the production of endogenous H{sub 2}S. However, whether CaSR inhibits the proliferation of VSMCs by regulating the endogenous cystathionine-gamma-lyase (CSE, a major enzyme that produces H{sub 2}S) pathway in high homocysteine (HHcy) has not been previously investigated. The intracellular calcium concentration, the concentration of H{sub 2}S, the cell viability, the proliferation and the expression of proteins of cultured VSMCs from rat thoracic aortas were measured, respectively. The results showed that the [Ca{sup 2+}]{sub i} and the expression of p-CaMK and CSE increased upon treatment with CaSR agonist. In HHcy, the H{sub 2}S concentration decrease, the proliferation and migration rate increased, the expression of Cyclin D1, PCNA, Osteopontin and p-Erk1/2 increased while the α-SM actin, P21{sup Cip/WAK−1} and Calponin decreased. The CaSR agonist or exogenous H{sub 2}S significantly reversed the changes of VSMCs caused by HHcy. In conclusion, our results demonstrated that CaSR regulate the endogenous CSE/H{sub 2}S is related to the PLC-IP{sub 3} receptor and CaM signal pathways which inhibit the proliferation of VSMCs, and the latter is involved in the Erk1/2 dependent signal pathway in high homocysteine. - Highlights: • CaSR activation increased the production of endogenous H{sub 2}S in high homocysteine VSMCs. • CaSR modulated the CSE/H{sub 2}S are related to the PLC-IP{sub 3}R and Ca{sup 2+}-CaM signal pathways. • Inhibition of H{sub 2}S on the proliferation of VSMCs is involved in the Erk1/2 pathway. • Explore the potential roles of CaSR in regulating VSMCs proliferation in high homocysteine.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2016

Nucleus and nucleus-cytoskeleton connections in 3D cell migration

Cell migration plays an important role in many physiological and pathological settings, ranging from embryonic development to cancer metastasis. Currently, accumulating data suggest that cells migrating in three-dimensional (3D) environments show well-defined differences compared to their well-established two-dimensional (2D) counterparts. During 3D migration, the cell body and nucleus must deform to allow cellular passage through the available spaces, and the deformability of the relatively rigid nucleus may constitute a limiting step. Here, we highlight the key evidence regarding the role of the nuclear mechanics in 3D migration, including the molecular components that govern the stiffness of the nucleus and review how the nuclear dynamics are connected to and controlled by cytoskeleton-based migration machinery. Intriguingly, nuclear movement must be coordinated with the cytoskeletal dynamics at the leading and trailing edges, which in turn impact the cytoplasmic dynamics that affect the migration efficiency. Thus, we suggest that alterations in the nuclear structure may facilitate cellular reorganizations that are necessary for efficient migration. - Graphical abstract: Schematic representations of a cell migrating on a 2D substrate and a cell migrating in a 3D extracellular matrix environment. (A) Nucleus-cytoskeleton connections are essential to 3D migration. Mechanical signals are transduced by integrins at the cell surface and channeled to cytoskeletal proteins, which generates prestress. The nucleus-cytoskeleton connections can either act as a stable skeleton to anchor the nuclei or provide active force to move the nuclei. The LINC complex is responsible for the nucleo-cytoskeletal coupling. Nesprins connect the cytoskeletal proteins to the inner nuclear membrane proteins SUN1 and SUN2. The SUN proteins connect to the lamins that form the lamina, which attaches to the chromatin. This physical connectivity transmits the mechanical signals from receptors at the cell membrane through the cytoskeletal architecture to the nucleus and into the chromosomes. On a 2D substrate (B), the nucleus can be subjected to tensional forces emanating from the stress fibers and compressive forces due to the actin cap structures and the resistance of the surface. In a 3D environment (C), the migration process requires reshaping of the nucleus and squeezing it through narrow openings in the ECM. During this process the cells may also experience both tension generated by the actomyosin filaments and compression resulting from the high pressure of the anterior compartment. - Highlights: • The influence of nuclear size and stiffness in cell migration is discussed. • We describe molecular components that govern the mechanical properties of the nucleus. • We discuss the roles of chromatin, lamin A/C in nuclear mechanical properties and cell migration. • We review how nuclear dynamics are connected to cytoskeleton. • We discuss the role of nucleo-cytoskeletal coupling in cell migration.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2017

ERα inhibited myocardin-induced differentiation in uterine fibroids

Uterine fibroids, also known as uterine leiomyomas, are a benign tumor of the human uterus and the commonest estrogen-dependent benign tumor found in women. Myocardin is an important transcriptional regulator in smooth and cardiac muscle development. The role of myocardin and its relationship with ERα in uterine fibroids have barely been addressed. We noticed that the expression of myocardin was markedly reduced in human uterine fibroid tissue compared with corresponding normal or adjacent myometrium tissue. Here we reported that myocardin induced the transcription and expression of differentiation markers SM22α and alpha smooth muscle actin (α-SMA) in rat primary uterine smooth muscle cells (USMCs) and this effect was inhibited by ERα. Notably, we showed that, ERα induced expression of proliferation markers PCNA and ki-67 in rat primary USMCs. We also found ERα interacted with myocardin and formed complex to bind to CArG box and inhibit the SM22α promoter activity. Furthermore, ERα inhibited the transcription and expression of myocardin, and reduced the levels of transcription and expression of downstream target SM22α, a SMC differentiation marker. Our data thus provided important and novel insights into how ERα and myocardin interact to control the cell differentiation and proliferation of USMCs. Thus, it may provide potential therapeutic target for uterine fibroids.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2017

The Drosophila DOCK family protein Sponge is required for development of the air sac primordium

Dedicator of cytokinesis (DOCK) family genes are known as DOCK1-DOCK11 in mammals. DOCK family proteins mainly regulate actin filament polymerization and/or depolymerization and are GEF proteins, which contribute to cellular signaling events by activating small G proteins. Sponge (Spg) is a Drosophila counterpart to mammalian DOCK3/DOCK4, and plays a role in embryonic central nervous system development, R7 photoreceptor cell differentiation, and adult thorax development. In order to conduct further functional analyses on Spg in vivo, we examined its localization in third instar larval wing imaginal discs. Immunostaining with purified anti-Spg IgG revealed that Spg mainly localized in the air sac primordium (ASP) in wing imaginal discs. Spg is therefore predicted to play an important role in the ASP. The specific knockdown of Spg by the breathless-GAL4 driver in tracheal cells induced lethality accompanied with a defect in ASP development and the induction of apoptosis. The monitoring of ERK signaling activity in wing imaginal discs by immunostaining with anti-diphospho-ERK IgG revealed reductions in the ERK signal cascade in Spg knockdown clones. Furthermore, the overexpression of D-raf suppressed defects in survival and the proliferation of cells in the ASP induced by the knockdown of Spg. Collectively, these results indicate that Spg plays a critical role in ASP development and tracheal cell viability that is mediated by the ERK signaling pathway. - Highlights: • Spg mainly localizes in the air sac primordium in wing imaginal discs. • Spg plays a critical role in air sac primordium development. • Spg positively regulates the ERK signal cascade.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

1,25(OH)2D3 attenuates pulmonary arterial hypertension via microRNA-204 mediated Tgfbr2/Smad signaling

Highlights: • miRNA-204 is significantly lower in hypoxic pulmonary hypertension. • miRNA-204 is involved in the development of pulmonary hypertension. • 1,25(OH){sub 2}D{sub 3} significantly prevented the development of PAH by blocking the Tgfbr2/Smad signaling. • 1,25(OH){sub 2}D{sub 3} reduced pulmonary hypertension mediated by increased the level of miRNA-204. • miRNA-204 strengthened the anti-PAH function of 1,25(OH){sub 2}D{sub 3} by targeting Tgfbr2. Pulmonary arterial hypertension (PAH) is a devastating disease characterized by high pulmonary artery pressure. It is reported that microRNA-204 (miR-204) plays an important role in the development of PAH. Calcitriol [1,25-dihydroxyvitamin D3, 1,25(OH){sub 2}D{sub 3}] mediates multiple pathophysiological processes. The aim of the current study was to explore the role of 1,25(OH){sub 2}D{sub 3} in PAH. PAH was induced in rats and rat pulmonary arterial endothelial cells (PAECs) were isolated as in vitro PAH model. The mean pulmonary artery pressure, morphologic changes, and expressions of transforming growth factor-beta1 (Tgfbr2), Smad2/7, alpha smooth muscle actin (α-SMA), and p21 were then measured. Furthermore, the effect of 1,25(OH){sub 2}D{sub 3} on rat PAECs with or without hypoxia treatment was also assessed by measuring the proliferation, migration, and cell cycle distribution of PAECs. The potential targets of miR-204 were also predicted and validated with a dual-luciferase reporter system. Then the role of miR-204 and Tgfbr2 in the anti-PAH effect of 1,25(OH){sub 2}D{sub 3} was further explored by modulating the expression of the two genes. The overall pulmonary hypertension and hypoxia-induced proliferation and migration of PAECs were attenuated by administration of 1,25(OH){sub 2}D{sub 3}, which was associated with the suppressed expressions of Tgfbr2, α-SMA, and Smad7 and induced expressions of miR-204, p21 and Smad2 both in vitro and in vivo. Moreover, the luciferase reporter assay identified Tgfbr2 as a novel direct target of miR-204. Both overexpression of miR-204 and inhibition of Tgfbr2 would strengthen the effect of 1,25(OH){sub 2}D{sub 3} administration. Findings outlined in the current study demonstrated that 1,25(OH){sub 2}D{sub 3} was a promising therapeutic modality for treatment of PAH, function of which was exerted through miR-204 mediated Tgfbr2 signaling.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

RalGPS2 is involved in tunneling nanotubes formation in 5637 bladder cancer cells

Highlights: • RalGPS2 is expressed in 5637 cancer cells and is essential for their growth. • RalGPS2 is required for TNT formation. • The overexpression of RalGPS2 PH domain markedly increased the number and length of TNT. • Interaction between RalA and Sec5 is required for TNT formation in 5637 cells. • RalGPS2 interacts with LST1 and RalA leading to TNT formation. RalGPS2 is a Ras-independent Guanine Nucleotide Exchange Factor (GEF) for RalA containing a PH domain and an SH3-binding region and it is involved in several cellular processes, such as cytokinesis, control of cell cycle progression, differentiation, cytoskeleton organization and rearrangement. Up to now, few data have been published regarding RalGPS2 role in cancer cells, and its involvement in bladder cancer is yet to be established. In this paper we demonstrated that RalGPS2 is expressed in urothelial carcinoma-derived 5637 cancer cells and is essential for cellular growth. These cells produces thin membrane protrusions that displayed the characteristics of actin rich tunneling nanotubes (TNTs) and here we show that RalGPS2 is involved in the formation of these cellular protrusions. In fact the overexpression of RalGPS2 or of its PH-domain increased markedly the number and the length of nanotubes, while the knock-down of RalGPS2 caused a strong reduction of these structures. Moreover, using a series of RalA mutants impaired in the interaction with different downstream components (Sec5, Exo84, RalBP1) we demonstrated that the interaction of RalA with Sec5 is required for TNTs formation. Furthermore, we found that RalGPS2 interacts with the transmembrane MHC class III protein leukocyte specific transcript 1 (LST1) and RalA, leading to the formation of a complex which promotes TNTs generation. These findings allow us to add novel elements to molecular models that have been previously proposed regarding TNTs formation.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Interaction between oral squamous cell carcinoma cells and fibroblasts through TGF-β1 mediated by podoplanin

Highlights: • Paracrine TGF-β1 from PDPN-positive OSCC cells activates NFs. • PDPN-positive OSCC cells promote fibroblast invasion in direct co-culture. • The fibroblast invasion is promoted through MMP-2, MMP-14, and ITGA5 signaling. • MMP-2 and MMP-14 upregulation by TGF-β1 from CAFs and cancer cells promote OSCC cell invasion. Podoplanin is upregulated in the invasive front of oral squamous cell carcinoma (OSCC). Carcinoma-associated fibroblasts (CAFs) may mediate podoplanin expression. However, the role of podoplanin in OSCC cell and fibroblast interaction remains elusive. In the present study, we found that positive podoplanin expression in OSCC cells correlated with smooth muscle actin (α-SMA) expression in CAFs. Using CAFs and normal mucosal fibroblasts (NFs), we established indirect and direct co-culture systems mimicking the structure of OSCC. Podoplanin-overexpressing OSCC cells promoted NF activation; in direct co-culture, but not in indirect co-culture, podoplanin-overexpressing OSCC cells increased fibroblast invasion via matrix metalloproteinase 2 (MMP-2), MMP-14, and αv/β6 integrin receptor (ITGA5/ITGB6) signaling. CAFs also induced podoplanin expression through the transforming growth factor-β1 (TGF-β1)/Smad pathway. TGF-β1 increased the podoplanin-dependent activation of epidermal growth factor receptor (EGFR), AKT, and extracellular signal–regulated kinase (ERK) signaling. Additionally, CAFs promoted OSCC cell invasion by upregulating MMP-2 and MMP-14 expression in both indirect and direct co-culture. Taken together, our findings indicate that podoplanin regulates the interaction between OSCC cells and CAFs via the mutual paracrine effects of TGF-β1.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Calcium influx differentially regulates migration velocity and directedness in response to electric field application

Neural precursor cells (NPCs) respond to externally applied direct current electrical fields (DCEFs) by undergoing rapid and directed migration toward the cathode in a process known as galvanotaxis. It is unknown if the underlying mechanisms of galvanotactic migration is common to non-electrosensitive cells and if so, how NPCs and other galvanotactic cells sense and transduce electrical fields into cellular motility. In this study, we show that distinct aspects of NPC galvanotactic migration: motility (quantified through |velocity|) and directedness, are differentially regulated by calcium. We use low-Ca{sup 2+} culture conditions; an intracellular Ca{sup 2+} chelator; and voltage gated calcium channel (VGCC) inhibitors to specific channels expressed on NPCs, to demonstrate the role of Ca{sup 2+} influx in DCEF-induced NPC migration. Consistent with existing literature, we show Ca{sup 2+} is involved in F-actin polymerization that lengthens NPC membrane protrusions necessary for cellular motility. However, inhibiting Ca{sup 2+} results in reduced velocity but has no effect on DCEF-induced directedness. This dissociation between velocity and directedness reveal that these migration parameters can be independently regulated, thus suggesting a parallel process of sensing DCEFs by NPCs.

60 APPLIED LIFE SCIENCES↗