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Sample records for 3t3 mouse fibroblasts

  1. Lysophosphatidic acid receptor-5 negatively regulates cellular responses in mouse fibroblast 3T3 cells.

    PubMed

    Dong, Yan; Hirane, Miku; Araki, Mutsumi; Fukushima, Nobuyuki; Tsujiuchi, Toshifumi

    2014-04-04

    Lysophosphatidic acid (LPA) signaling via G protein-coupled LPA receptors (LPA1-LPA6) mediates a variety of biological functions, including cell migration. Recently, we have reported that LPA1 inhibited the cell motile activities of mouse fibroblast 3T3 cells. In the present study, to evaluate a role of LPA5 in cellular responses, Lpar5 knockdown (3T3-L5) cells were generated from 3T3 cells. In cell proliferation assays, LPA markedly stimulated the cell proliferation activities of 3T3-L5 cells, compared with control cells. In cell motility assays with Cell Culture Inserts, the cell motile activities of 3T3-L5 cells were significantly higher than those of control cells. The activity levels of matrix metalloproteinases (MMPs) were measured by gelatin zymography. 3T3-L5 cells stimulated the activation of Mmp-2, correlating with the expression levels of Mmp-2 gene. Moreover, to assess the co-effects of LPA1 and LPA5 on cell motile activities, Lpar5 knockdown (3T3a1-L5) cells were also established from Lpar1 over-expressing (3T3a1) cells. 3T3a1-L5 cells increased the cell motile activities of 3T3a1 cells, while the cell motile activities of 3T3a1 cells were significantly lower than those of control cells. These results suggest that LPA5 may act as a negative regulator of cellular responses in mouse fibroblast 3T3 cells, similar to the case for LPA1.

  2. Lysophosphatidic acid receptor-5 negatively regulates cellular responses in mouse fibroblast 3T3 cells

    SciTech Connect

    Dong, Yan; Hirane, Miku; Araki, Mutsumi; Fukushima, Nobuyuki; Tsujiuchi, Toshifumi

    2014-04-04

    Highlights: • LPA{sub 5} inhibits the cell growth and motile activities of 3T3 cells. • LPA{sub 5} suppresses the cell motile activities stimulated by hydrogen peroxide in 3T3 cells. • Enhancement of LPA{sub 5} on the cell motile activities inhibited by LPA{sub 1} in 3T3 cells. • The expression and activation of Mmp-9 were inhibited by LPA{sub 5} in 3T3 cells. • LPA signaling via LPA{sub 5} acts as a negative regulator of cellular responses in 3T3 cells. - Abstract: Lysophosphatidic acid (LPA) signaling via G protein-coupled LPA receptors (LPA{sub 1}–LPA{sub 6}) mediates a variety of biological functions, including cell migration. Recently, we have reported that LPA{sub 1} inhibited the cell motile activities of mouse fibroblast 3T3 cells. In the present study, to evaluate a role of LPA{sub 5} in cellular responses, Lpar5 knockdown (3T3-L5) cells were generated from 3T3 cells. In cell proliferation assays, LPA markedly stimulated the cell proliferation activities of 3T3-L5 cells, compared with control cells. In cell motility assays with Cell Culture Inserts, the cell motile activities of 3T3-L5 cells were significantly higher than those of control cells. The activity levels of matrix metalloproteinases (MMPs) were measured by gelatin zymography. 3T3-L5 cells stimulated the activation of Mmp-2, correlating with the expression levels of Mmp-2 gene. Moreover, to assess the co-effects of LPA{sub 1} and LPA{sub 5} on cell motile activities, Lpar5 knockdown (3T3a1-L5) cells were also established from Lpar1 over-expressing (3T3a1) cells. 3T3a1-L5 cells increased the cell motile activities of 3T3a1 cells, while the cell motile activities of 3T3a1 cells were significantly lower than those of control cells. These results suggest that LPA{sub 5} may act as a negative regulator of cellular responses in mouse fibroblast 3T3 cells, similar to the case for LPA{sub 1}.

  3. Interferon inhibits the conversion of 3T3-L1 mouse fibroblasts into adipocytes.

    PubMed Central

    Keay, S; Grossberg, S E

    1980-01-01

    Confluent Swiss mouse 3T3-L1 fibroblasts slowly differentiate functionally and morphologically into adipocytes, a conversion hastened by insulin. The cells are sensitive (although less than L929 cells) to the antiviral action of mouse fibroblast interferons but not to interferons from heterologous species (human and chicken). Cultures stimulated with insulin in the presence of partially purified or electrophoretically pure mouse interferons have a much lower percentage of cells accumulating lipid than do insulin-treated control cultures. Interferon-treated cell cultures also contain much less triglyceride, cholesterol, and cholesterol esters than do replicate control cultures stimulated by insulin to differentiate. Increased de novo lipid biosynthesis that occurs during differentiation is inhibited, as determined by incorporation of [14C]acetate into lipids extractable by the Folch method. This incorporation is a sensitive bioassay of the antidifferentiation effect of interferon; less than 1 antiviral unit is inhibitory. Variously inactivated or mock interferon preparations as well as interferons from several heterologous species fail to inhibit 3T3-L1 adipocyte conversion. Interferon is inhibitory even when applied as long as 3 days after insulin stimulation. The effect of interferon does not appear to depend upon its competition with insulin for cell surface receptors. Because interferon can alter the program of events involved in conversion of 3T3-L1 fibroblasts into adipose cells, it may be able to affect the regulation of eukaryotic cell differentiation. Images PMID:6159626

  4. Regulation of p53 in NIH3T3 mouse fibroblasts following hyperosmotic stress

    PubMed Central

    Lambert, Ian Henry; Enghoff, Maria Stine; Brandi, Marie-Luise; Hoffmann, Else Kay

    2015-01-01

    The aim of this project was to analyze the regulation of p53 expression in NIH3T3 fibroblasts under the influence of increasing hyperosmotic stress. Expression of p53 showed a biphasic response pattern in NIH3T3 cells under increasing osmotic stress (337 mOsm to 737 mOsm) with a maximum at 587 mOsm. Under isotonic conditions p53 expression increased after addition of the proteasome inhibitor MG132 indicating that cellular p53 levels in unperturbed cells is kept low by proteasomal degradation. However, under hypertonic conditions p53 synthesis as well as p53 degradation were significantly reduced and it is demonstrated that the increase in p53 expression observed when tonicity is increased from 337 to 587 mOsm reflects that degradation is more inhibited than synthesis, whereas the decrease in p53 expression at higher tonicities reflects that synthesis is more inhibited than degradation. The activity of the p53 regulating proteins p38 MAP kinase and the ubiquitin ligase MDM2 were studied as a function of increasing osmolarity. MDM2 protein expression was unchanged at all osmolarities, whereas MDM2 phosphorylation (Ser166) increased at osmolarities up to 537 mOsm and remained constant at higher osmolarities. Phosphorylation of p38 increased at osmolarities up to 687 mOsm which correlated with an increased phosphorylation of p53 (Ser15) and the decreased p53 degradation. Caspase-3 activity increased gradually with hypertonicity and at 737 mOsm both Caspase-3 activity and annexin V binding are high even though p53 expression and activity are low, indicating that initiation of apoptosis under severe hypertonic conditions is not strictly controlled by p53. PMID:26056062

  5. MUC4, a multifunctional transmembrane glycoprotein, induces oncogenic transformation of NIH3T3 mouse fibroblast cells

    PubMed Central

    Bafna, Sangeeta; Singh, Ajay P; Moniaux, Nicolas; Eudy, James D; Meza, Jane L; Batra, Surinder K.

    2008-01-01

    Numerous studies have established the association of MUC4 with the progression of cancer and metastasis. An aberrant expression of MUC4 is reported in precancerous lesions indicating its early involvement in the disease process; however, its precise role in cellular transformation has not been explored. MUC4 contains many unique domains and is proposed to impact on cell signaling pathways and behavior of the tumor cells. In the present study, to decipher its oncogenic potential of MUC4, we stably expressed the MUC4 mucin in NIH3T3 mouse fibroblast cells. Stable ectopic expression of MUC4 resulted in increased growth, colony formation and motility of NIH3T3 cells in vitro and tumor formation in nude mice, when cells were injected subcutaneously. Microarray analysis demonstrated increased expression of several growth- and mitochondrial energy production-associated genes in MUC4-expressing NIH3T3 cells. In addition, expression of MUC4 in NIH3T3 cells resulted in enhanced levels of oncoprotein ErbB2 and its phosphorylated form (pY1248-ErbB2). In conclusion, our studies provide the first evidence that MUC4 alone induces cellular transformation and indicates a novel role of MUC4 in cancer biology. PMID:19010895

  6. Comparative Culturing of 3T3 Swiss J2 Mouse Embryo Fibroblasts on Modified Chitosan Matrices.

    PubMed

    Alekhin, A I; Gaenko, G P

    2016-07-01

    Comparative culturing of mouse embryo fibroblasts on chitosan matrices and culture plastic was carried out. During the first 2 h of culturing (lag phase), cell adhesion to chitosan and chitosan-gelatin matrices was 20-30% higher than adhesion to culture plastic (control). During the stationary phase, 80% cells adhered to chitosan matrices (vs. 60% in the control). Cell culturing on chitosan matrices was carried out without medium replacement with fresh portions. The cells remained viable within 5 days of culturing. Cell death phase was observed on day 6 of culturing on chitosan matrices: cell adhesion dropped to 50%. Culturing on culture plastic was carried out with daily medium replacement with a fresh portion. Cell death phase (50% decrease in the number of adherent cell) under these condition was observed on day 5. It seems that the observed effect was a result of contact interactions of cell integrins and chitosan ligands, modulation of transmembrane signal, eventually modifying the expression of cell genes. This effect can be required in regenerative medicine for production of primary cell culture.

  7. Human lung-derived mature mast cells cultured alone or with mouse 3T3 fibroblasts maintain an ultrastructural phenotype different from that of human mast cells that develop from human cord blood cells cultured with 3T3 fibroblasts.

    PubMed Central

    Dvorak, A. M.; Furitsu, T.; Estrella, P.; Ishizaka, T.

    1991-01-01

    Culture systems designed to maintain or develop human mast cells have proved difficult, yet these systems would provide valuable resources for future investigations of human mast cell biology. Cocultures of either isolated mature human lung mast cells (Levi-Schaffer et al., J Immunol 1987, 139:494-500) or human cord blood mononuclear cells (Furitsu, Proc Natl Acad Sci USA 1989, 86:10039-10043) with 3T3 embryonic mouse skin fibroblasts have implicated fibroblasts as an important factor in the successful maintenance and development of human mast cells in vitro. The authors cultured isolated, mature human lung mast cells either with or without 3T3 cells for 1 month and examined their ultrastructural phenotype. Mast cell viability in each circumstance was equivalent, but mast cell yield was improved in the presence of 3T3 cells. The ultrastructural phenotype was identical in both culture systems. Mast cells were shown to maintain the phenotype of their in vivo lung counterparts (ie, scroll granules predominanted, and numerous lipid bodies were present). This ultrastructural phenotype differs from that of mast cells that develop in cocultures of human cord blood cells and 3T3 cells, where developing mast cells with crystalline granules and few lipid bodies prevail, a phenotype much like that of human skin mast cells in vivo (Furitsu, Proc Natl Acad Sci USA 1989, 86:10039-10043). Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 PMID:1750506

  8. Malignant transformation induced by incorporated radionuclides in BALB/3T3 mouse embryo fibroblasts.

    PubMed Central

    LeMotte, P K; Adelstein, S J; Little, J B

    1982-01-01

    The induction of lethality and malignant transformation by 5-[125I]iododeoxyuridine and [3H]thymidine incorporated into cellular DNA and by x-irradiation was studied in vitro in BALB/3T3 cells. Under these conditions, 125I radiation is highly localized to small regions of the DNA at the site of each decay and produces DNA double-strand breaks with high efficiency. Incorporated 125I was found to be 12-16 times as lethal per decay as incorporated 3H. For the induction of malignant transformation, however, 125I was approximately 25 times as effective per decay as 3H. When the frequencies of transformation induced at various levels of survival by 125I, 3H, and x-rays were compared, lethality was found to correlate closely with transformation at doses that yielded significant cell killing. An exception occurred at low doses, where 125I appeared much more efficient than x-irradiation in inducing transformation; transformation frequencies equal to those induced by 3-5 Gy of x-rays resulted from 125I exposures that yielded little or no cell killing. PMID:6961448

  9. Mechanisms of vitamin K transport and metabolism in Swiss 3T3 mouse fibroblasts

    SciTech Connect

    Canfield, L.M.; Townsend, A.F.; Hibbs, D.B.

    1986-03-01

    Transport of vitamin K into isolated fibroblasts was followed using /sup 3/H vitamin K/sub 1/. The initial rate is saturable by 5 min. at 25..mu..M vitamin K with a Km(app) of 10..mu..M and V/sub max/ of 50 pmols/min/10/sup 6/ cells. Kinetics of uptake are biphasic with a second slower rate ensuing after 10 minutes. Insensitivity of the initial rate of uptake to FCCP or ouabain indicates an ATP-independent transport mechanism. Specificity of transport is shown by competition of uptake of /sup 3/H vitamin K by unlabelled vitamin and strong (>90%) inhibition of the initial rate by equimolar concentrations of the vitamin K analog, Chloro-K. In addition, following uptake, both vitamins K/sub 1/ and K/sub 2/ are metabolized to their respective epoxides. Vitamin K/sub 1/ epoxide is also transported into fibroblasts and metabolized to the parent quinone in a Warfarin-sensitive reaction. Following alkaline hydrolysis of isolated intracellular protein, the vitamin K-dependent amino acid, gamma carboxyglutamic acid (gla) was detected. It is concluded that vitamin K is specifically transported into fibroblasts and metabolized via the classical pathway described in liver with the concomitant production of vitamin K-dependent proteins.

  10. Roughness threshold for cell attachment and proliferation on plasma micro-nanotextured polymeric surfaces: the case of primary human skin fibroblasts and mouse immortalized 3T3 fibroblasts

    NASA Astrophysics Data System (ADS)

    Bourkoula, A.; Constantoudis, V.; Kontziampasis, D.; Petrou, P. S.; Kakabakos, S. E.; Tserepi, A.; Gogolides, E.

    2016-08-01

    Poly(methyl methacrylate) surfaces have been micro-nanotextured in oxygen plasmas with increasing ion energy, leading to micro-nanotopography characterized by increased root mean square roughness, correlation length and fractal dimension. Primary human skin fibroblasts and mouse immortalized 3T3 fibroblasts were cultured on these surfaces and the number of adhering cells, their proliferation rate and morphology (cytoplasm and nucleus area) were evaluated as a function of roughness height, correlation length, and fractal dimension. A roughness threshold behavior was observed for both types of cells leading to dramatic cell number decrease above this threshold, which is almost similar for the two types of cells, despite their differences in size and stiffness. The results are discussed based on two theoretical models, which are reconciled and unified when the elastic moduli and the size of the cells are taken into account.

  11. Role of the crystalline form of titanium dioxide nanoparticles: Rutile, and not anatase, induces toxic effects in Balb/3T3 mouse fibroblasts.

    PubMed

    Uboldi, Chiara; Urbán, Patricia; Gilliland, Douglas; Bajak, Edyta; Valsami-Jones, Eugenia; Ponti, Jessica; Rossi, François

    2016-03-01

    The wide use of titanium dioxide nanoparticles (TiO2 NPs) in industrial applications requires the investigation of their effects on human health. In this context, we investigated the effects of nanosized and bulk titania in two different crystalline forms (anatase and rutile) in vitro. By colony forming efficiency assay, a dose-dependent reduction of the clonogenic activity of Balb/3T3 mouse fibroblasts was detected in the presence of rutile, but not in the case of anatase NPs. Similarly, the cell transformation assay and the micronucleus test showed that rutile TiO2 NPs were able to induce type-III foci formation in Balb/3T3 cells and appeared to be slightly genotoxic, whereas anatase TiO2 NPs did not induce any significant neoplastic or genotoxic effect. Additionally, we investigated the interaction of TiO2 NPs with Balb/3T3 cells and quantified the in vitro uptake of titania using mass spectrometry. Results showed that the internalization was independent of the crystalline form of TiO2 NPs but size-dependent, as nano-titania were taken up more than their respective bulk materials. In conclusion, we demonstrated that the cytotoxic, neoplastic and genotoxic effects triggered in Balb/3T3 cells by TiO2 NPs depend on the crystalline form of the nanomaterial, whereas the internalization is regulated by the particle size.

  12. 3T3 fibroblasts induce cloned interleukin 3-dependent mouse mast cells to resemble connective tissue mast cells in granular constituency

    SciTech Connect

    Dayton, E.T.; Pharr, P.; Ogawa, M.; Serafin, W.E.; Austen, K.F.; Levi-Schaffer, F.; Stevens, R.L.

    1988-01-01

    As assessed by ultrastructure, histochemical staining, and T-cell dependency, in vitro-differentiated interleukin 3-dependent mouse mast cells are comparable to the mast cells that reside in the gastrointestinal mucosa but not in the skin or the serosal cavity of the mouse. The authors now demonstrate that when cloned interleukin 3-dependent mast cells are cocultured with mouse skin-derived 3T3 fibroblasts in the presence of WEHI-3 conditioned medium for 28 days, the mast cells acquire the ability to stain with safranin, increase their histamine content approx. 50-fold and their carboxypeptidase. A content approx. 100-fold, and augment approx. their biosynthesis of proteoglycans bearing /sup 35/S-labeled haparin relative to /sup 35/S-labeled chondroitin sulfate glycosaminoglycans. Thus, fibroblasts induce interleukin 3-dependent mouse mast cells to change phenotype from mucosal-like to connective tissue-like, indicating that the biochemical and functional characteristics of this mast cell type are strongly influenced by the connective tissue microenvironment.

  13. A quantitative description of the extension and retraction of surface protrusions in spreading 3T3 mouse fibroblasts.

    PubMed

    Albrecht-Buehler, G; Lancaster, R M

    1976-11-01

    We suggest a method of quantitating the motile actions of surface protrusions in spreading animal cells in culture. Its basis is the determination of the percentage of freshly plated cells which produce particle-free areas around them on a gold particle-coated glass cover slip within 50 min. Studying 3T3 cells with this assay, we found that the presence of Na+, K+, Cl-, and Mg++ or Ca++ in a neutral or slightly alkaline phosphate or bicarbonate buffered solution is sufficient to support the optimal particle removal by the cells for at least 50 min. Two metabolic inhibitors, 2,4-dinitrophenol and Na-azide, inhibit the particle removal. If D-glucose is added along with the inhibitors, particle removal can be restored, whereas the addition of three glucose analogues which are generally believed to be nonmetabolizable cannot restore the activity. Serum is not required for the mechanism(s) of the motile actions of surface protrusions in spreading 3T3 cells. However, it contains components which can neutralize the inhibitory actions of bovine serum albumin and several amino acids, particularly L-cystine or L-cystein and L-methionine. Furthermore, serum codetermines which of the major surface extension, filopodia, lamellipodia, or lobopodia, is predominantly active. We found three distinct classes of extracellular conditions under which the active surface projections are predominantly either lamellipodia, (sheetlike projections), lobopodia (blebs), or filopodia (microspikes). The quantitated dependencies on temperature, pH and the inhibition by cytochalasin B or the particle removal are very similar in all three cases. Preventing the cells from anchoring themselves for 15-20 min before plating in serum-free medium seems to stimulate particle removal threefold.

  14. Study of smart antibacterial PCL-xFe3 O4 thin films using mouse NIH-3T3 fibroblast cells in vitro.

    PubMed

    Pai B, Ganesh; Kulkarni, Ajay V; Jain, Shilpee

    2016-01-13

    Surface energy plays a major role in prokaryotic and eukaryotic cell interactions with biomedical devices. In the present study, poly(ε-caprolactone)-xFe3 O4 nanoparticles (PCL-xFO NPs; x = 0, 10, 20, 30, 40, 60 wt% FO concentration in PCL) composite thin films were developed for skin tissue regeneration. The surface properties in terms of roughness, surface energy, wettability of the thin films were altered with the incorporation of Fe3 O4 NPs. These thin films show antimicrobial properties and cyto-compatibility with NIH 3T3 mouse embryonic fibroblast cells. The porosity and thickness of the films were controlled by varying RPM of the spin coater. Interestingly, at 1000 RPM the roughness of the film decreased with increasing concentrations of FO NPs in PCL, whereas the surface energy increased with increasing FO NPs concentrations. Furthermore, the spreading of NIH-3T3 cells grown on PCL-xFO thin films was less as compared to control (TCPS), however cells overcame this effect after 48 h of seeding and cells spread similarly to those grown on TCPS after 48 h. Also, the incorporation of FO NPs in thin films induced inner membrane permeabilization in E. coli bacteria leading to bacterial cell death. The viability of E. coli bacteria decreased with increasing concentration of FO NPs in PCL. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2016.

  15. Dehydrodiconiferyl alcohol isolated from Cucurbita moschata shows anti-adipogenic and anti-lipogenic effects in 3T3-L1 cells and primary mouse embryonic fibroblasts.

    PubMed

    Lee, Junghun; Kim, Donghyun; Choi, Jonghyun; Choi, Hyounjeong; Ryu, Jae-Ha; Jeong, Jinhyun; Park, Eun-Jin; Kim, Seon-Hee; Kim, Sunyoung

    2012-03-16

    A water-soluble extract from the stems of Cucurbita moschata, code named PG105, was previously found to contain strong anti-obesity activities in a high fat diet-induced obesity mouse model. One of its biological characteristics is that it inhibits 3T3-L1 adipocyte differentiation. To isolate the biologically active compound(s), conventional solvent fractionation was performed, and the various fractions were tested for anti-adipogenic activity using Oil Red O staining method. A single spot on thin layer chromatography of the chloroform fraction showed a potent anti-adipogenic activity. When purified, the structure of its major component was resolved as dehydrodiconiferyl alcohol (DHCA), a lignan, by NMR and mass spectrometry analysis. In 3T3-L1 cells, synthesized DHCA significantly reduced the expression of several adipocyte marker genes, including peroxisome proliferator-activated receptor γ (Pparg), CCAAT/enhancer-binding protein α (Cebpa), fatty acid-binding protein 4 (Fabp4), sterol response element-binding protein-1c (Srebp1c), and stearoyl-coenzyme A desaturase-1 (Scd), and decreased lipid accumulation without affecting cell viability. DHCA also suppressed the mitotic clonal expansion of preadipocytes (an early event of adipogenesis), probably by suppressing the DNA binding activity of C/EBPβ, and lowered the production level of cyclinA and cyclin-dependent kinase 2 (Cdk2), coinciding with the decrease in DNA synthesis and cell division. In addition, DHCA directly inhibited the expression of SREBP-1c and SCD-1. Similar observations were made, using primary mouse embryonic fibroblasts. Taken together, our data indicate that DHCA may contain dual activities, affecting both adipogenesis and lipogenesis.

  16. Poly(2-hydroxyethyl methacrylate)-b-poly(L-Lysine) cationic hybrid materials for non-viral gene delivery in NIH 3T3 mouse embryonic fibroblasts.

    PubMed

    Johnson, Renjith P; Uthaman, Saji; John, Johnson V; Heo, Min Seon; Park, In Kyu; Suh, Hongsuk; Kim, Il

    2014-09-01

    In order to develop efficient and nontoxic gene delivery vectors, a series of biocompatible block copolymers, poly[(2-hydroxyethyl methacrylate)40 -block-(L-lysine)n ] (n = 40, 80, 120, 150), are prepared by combining an atom transfer radical polymerization of 2-hydroxyethyl methacrylate with a ring-opening polymerization of N(ϵ) -(carbobenzoxy)-L-lysine N-carboxyanhydride. The block copolymers are successfully condensed with plasmid DNA (pDNA) into nanosized (<200 nm) polyplexes. As a representative sample, p(HEMA)40 -b-p(lys)150 is utilized to confirm the effective cellular and nuclear uptake of pDNA. The polymer/pDNA polyplexes exhibit very low cytotoxicity and enhanced transfection activity by being easily taken up into mouse embryonic fibroblast cell line (NIH 3T3). Thus, the chimeric block copolymers provide a means for developing versatile nonviral gene vectors harboring the ideal requirements of low cytotoxicity, good stability, and high transfection efficiency for gene therapy.

  17. Pharmaco-Phylogenetic Investigation of Methyl Gallate Isolated from Acacia nilotica (L.) Delile and Its Cytotoxic Effect on NIH3T3 Mouse Fibroblast.

    PubMed

    Mishra, Rohit K; Ramakrishna, M; Mishra, Vani; Pathak, Ashutosh; Rajesh, S; Sharma, Shivesh; Pandey, Avinash C; Nageswara Rao, G; Dikshit, Anupam

    2016-01-01

    Present exploration deals with the therapeutic perspective of methyl gallate isolated from the leaf extract of Acacia nilotica (L.) Delile in contrast to food-borne bacterial pathogen's viz., Escherichia coli, Klebsiella pneumoniae, Salmonella typhimurium, Pseudomonas aeruginosa and Staphylococcus aureus with their evolutionary succession. The extract was subjected to phytochemical analysis and isolated compound was identified as methyl gallate using UV-vis, IR and NMR spectra. It was found most potent against K. pneumoniae with its minimum inhibition concentration (MIC) of 0.32 mg/ml and minimum bactericidal concentration (MBC) at 0.62 mg/ml. The correlation of MIC values with an evolutionary succession assists the relationship between their genetic and toxic properties. The cytotoxic pursuit of methyl gallate was additionally assessed over NIH3T3 mouse fibroblast by Neutral red (NR) uptake, MTT cell proliferation assay and did not disclose any relevant influence on cell viability as well as cell proliferation. As such, the methyl gallate extracted from the leaf of A. nilotica holds massive antibacterial aptitude and hands out towards a new paradigm for food and pharmaceutical industries.

  18. Neoplastic transformation and tumorigenesis associated with overexpression of imup-1 and imup-2 genes in cultured NIH/3T3 mouse fibroblasts

    SciTech Connect

    Ryoo, Zae Young . E-mail: jaewoong64@hanmail.net; Jung, Boo Kyoung; Lee, Sang Ryeul; Kim, Myoung Ok; Kim, Sung Hyun; Kim, Hyo Jin; Ahn, Jung Yong; Lee, Tae-Hoon; Cho, Youl Hee; Park, Jae Hak; Kim, Jin Kyeoung

    2006-10-27

    Immortalization-upregulated protein 1 (IMUP-1) and immortalization-upregulated protein 2 (IMUP-2) genes have been recently cloned and are known to be involved in SV40-mediated immortalization. IMUP-1 and IMUP-2 genes were strongly expressed in various cancer cell lines and tumors, suggesting the possibility that they might be involved in tumorigenicity. To directly elucidate the functional role of IMUP-1 and IMUP-2 on neoplastic transformation and tumorigenicity, we stably transfected IMUP-1 and IMUP-2 into NIH/3T3 mouse fibroblast cells. Cellular characteristics of the neoplastic transformation were assessed by transformation foci, growth in soft agar, and tumor development in nude mice. We found that IMUP-1 and IMUP-2 overexpressing cells showed altered growth properties, anchorage-independent growth in soft agar and inducing tumor in nude mice. Furthermore, IMUP-1 and IMUP-2 transformants proliferated in reduced serum and shortened cell cycle. These results suggest that ectopic overexpression of IMUP-1 and IMUP-2 may play an important role in acquiring a transformed phenotype, tumorigenicity in vivo, and be related to cellular proliferation.

  19. A comparative evaluation of photo-toxic effect of fractionated melanin and chlorpromazine hydrochloride on human (dermal fibroblasts and epidermal keratinocytes) and mouse cell line/s (fibroblast Balb/c 3T3).

    PubMed

    Rai, V; Dayan, N; Michniak-Kohn, B

    2011-03-01

    Fractionated melanin (Mel-HEV), a bleached version of natural melanin, offers protection against the high energy visible (HEV/UVA) and ultraviolet (specifically UVA) irradiation making it a potential compound to be added to skin care and sunscreen formulations and other cosmetic and personal care products. Chlorpromazine (CPZ) has been shown to exhibit photosensitivity and phototoxicity reaction in vitro and in vivo. Comparative evaluation of chemotoxicity and phototoxicity using Mel-HEV and CPZ (as positive control) was performed on mouse fibroblast cell line 'Balb/c 3T3'. This is the recommended method for evaluating the phototoxic potential of compounds under the European Center of Validation of Alternative Methods (ECVAM) guidelines (OECD, 2004). This study was expanded from a mouse cell line - Balb 3T3/c to two human cell lines - HDF and HEKn for two reasons: to compare the difference between the sensitivity and behavior of two fibroblast cell lines (Balb/c 3T3 vs. HDF) and to compare the differences between two fibroblast cell lines with the keratinocyte cell line (HDF & Balb/c 3T3 vs. HEKn). It was found that Balb/c 3T3 and HEKn were both sensitive to the phototoxic potential of CPZ. However, HDF showed insensitivity to phototoxic evaluation. The test compound, Mel-HEV, was found to be non-phototoxic. The mean toxic concentration (MTC) for CPZ during HEV and UVA exposure conditions was found to be similar using Balb/c 3T3 (36.25 μg/ml) and HEKn (39.99 μg/ml) showing that cells exhibit similar responses at HEV/UVA- conditions. However, Balb/c 3T3 showed more sensitivity to CPZ at HEV/UVA+ condition (MTC=0.87 μg/ml; mean PIF=55.33; MPE=0.395) than HEKn (MTC=5.35 μg/ml; PIF=7.61; MPE=0.276) making it the preferred cell line for phototoxicity evaluations.

  20. Cytotoxicity and morphological transforming potential of cobalt nanoparticles, microparticles and ions in Balb/3T3 mouse fibroblasts: an in vitro model.

    PubMed

    Sabbioni, Enrico; Fortaner, Salvador; Farina, Massimo; Del Torchio, Riccardo; Olivato, Iolanda; Petrarca, Claudia; Bernardini, Giovanni; Mariani-Costantini, Renato; Perconti, Silvia; Di Giampaolo, Luca; Gornati, Rosalba; Di Gioacchino, Mario

    2014-06-01

    We previously described the behaviour of different cobalt forms, i.e., cobalt nanoparticles (CoNP), cobalt microparticles (CoMP) and cobalt ions (Co(2+)), in culture medium (dissolution, interaction with medium components, bioavailability) as well as their uptake and intracellular distribution in Balb/3T3 mouse fibroblasts (Sabbioni, Nanotoxicology, 2012). Here, we assess the cytotoxicity and morphological transformation of CoNP compared not only to Co(2+), but also to CoMP and to released Co products. Cytotoxicity reached maximum at 4-h exposure, with ranking CoMP > CoNP > Co(2+). However, if we consider toxicity as a function of intracellular Co, toxicity of the ionic forms seems to prevail over the particles. Co forms other than Co(2+) released from particles had toxicity intermediate between particles and ions. Alterations in concentrations of essential elements (Cu, Mg, Zn) in cells exposed to Co particles may contribute to toxicity. Both CoMP and CoNP (but not Co(2+) and other released Co forms) induced morphological transformation (CoMP > CoNP). This was dependent on reactive oxygen species production and lipid peroxidation, as indicated by inhibition of type III foci with ascorbic acid. The present results suggest that the previously demonstrated massive mitochondrial and nuclear Co internalisation and DNA adduct formation by CoMP and CoNP (Sabbioni, Nanotoxicology, 2012) induce toxicity and transformation. On the contrary, the role of ions released by particles in culture medium is negligible. Thus, both the chemical and the physical properties of Co particles contribute to cytotoxicity and morphological transformation.

  1. Study of lactoferrin gene expression in human and mouse adipose tissue, human preadipocytes and mouse 3T3-L1 fibroblasts. Association with adipogenic and inflammatory markers.

    PubMed

    Moreno-Navarrete, José María; Serrano, Marta; Sabater, Mònica; Ortega, Francisco; Serino, Matteo; Pueyo, Neus; Luche, Elodie; Waget, Aurelie; Rodriguez-Hermosa, José Ignacio; Ricart, Wifredo; Burcelin, Remy; Fernández-Real, José Manuel

    2013-07-01

    Lactoferrin is considered an epithelial protein present in different gland secretions. Administration of exogenous lactoferrin is also known to modulate adipogenesis and insulin action in human adipocytes. Here, we aimed to investigate lactoferrin gene expression (real-time polymerase chain reaction) and protein (enzyme-linked immunosorbent assay) levels in human (n=143) and mice adipose tissue samples, in adipose tissue fractions and during human preadipocyte and 3T3-L1 cell line differentiation, evaluating the effects of inducers (rosiglitazone) and disruptors (inflammatory factors) of adipocyte differentiation. Lactoferrin (LTF) gene and protein were detectable at relatively high levels in whole adipose tissue and isolated adipocytes in direct association with low-density lipoprotein-related protein 1 (LRP1, its putative receptor). Obese subjects with type 2 diabetes and increased triglycerides had the lowest levels of LTF gene expression in subcutaneous adipose tissue. Specifically, LTF gene expression was significantly increased in adipocytes, mainly from lean subjects, increasing during differentiation in parallel to adipogenic genes and gene markers of lipid droplets. The induction or disruption of adipogenesis led to concomitant changes (increase and decrease, respectively) of lactoferrin levels during adipocyte differentiation also in parallel to gene markers of adipogenesis and lipid droplet development. The administration of lactoferrin led to autopotentiated increased expression of the LTF gene. The decreased lactoferrin mRNA levels in association with obesity and diabetes were replicated in mice adipose tissue. In conclusion, this is the first observation, to our knowledge, of lactoferrin gene expression in whole adipose tissue and isolated adipocytes, increasing during adipogenesis and suggesting a possible contribution in adipose tissue physiology through LRP1.

  2. Peanut sprout ethanol extract inhibits the adipocyte proliferation, differentiation, and matrix metalloproteinases activities in mouse fibroblast 3T3-L1 preadipocytes.

    PubMed

    Kim, Woo Kyoung; Kang, Nam E; Kim, Myung Hwan; Ha, Ae Wha

    2013-06-01

    3T3-L1 preadipocyte were differentiated to adipocytes, and then treated with 0, 10, 20, and 40 µg/mL of peanut sprout ethanol extract (PSEE). The main component of PSEE is resveratrol which contained 5.55 mg/mL of resveratrol. The MTT assay, Oil-Red O staining, glycerol-3-phosphate dehydrogenase (GPDH) activity, and the triglyceride concentration were determined in 3T3-L1 cells. MMP-2 and MMP-9 activities as well as mRNA expressions of C/EBP β and C/EBP α were also investigated. As the concentration of PSEE in adipocytes increased, the cell proliferation was decreased in a dose-dependent manner from 4 days of incubation (P < 0.05). The GDPH activity (P < 0.05) and the triglyceride concentration (P < 0.05) were decreased as the PSEE treatment concentration increased. The mRNA expression of C/EBPβ in 3T3-L1 cells was significantly low in groups of PSEE-treated, compared with control group (P < 0.05). The MMP-9 (P < 0.05) and MMP-2 (P < 0.05) activities were decreased in a dose-dependent manner as the PSEE concentration increased from 20 µg/mL. In conclusion, it was found that PSEE has an effect on restricting proliferation and differentiation of adipocytes.

  3. Genotoxicity and morphological transformation induced by cobalt nanoparticles and cobalt chloride: an in vitro study in Balb/3T3 mouse fibroblasts.

    PubMed

    Ponti, Jessica; Sabbioni, Enrico; Munaro, Barbara; Broggi, Francesca; Marmorato, Patrick; Franchini, Fabio; Colognato, Renato; Rossi, François

    2009-09-01

    Nanotechnology is an emerging field that involves the development, manufacture and measurement of materials and systems in the submicron to nanometer range. Its development is expected to have a large socio-economical impact in practically all fields of industrial activity. However, there is still a lack of information about the potential risks of manufactured nanoparticles for the environment and for human health. In this work, we studied the cytotoxicity, genotoxicity and morphological transforming activity of cobalt nanoparticles (Co-nano) and cobalt ions (Co(2+)) in Balb/3T3 cells. We also evaluated Co-nano dissolution in culture medium and cellular uptake of both Co-nano and Co(2+). Our results indicated dose-dependent cytotoxicity, assessed by colony-forming efficiency test, for both compounds. The toxicity was higher for Co-nano than for Co(2) after 2 and 24 h of exposure, while dose-effect relationships were overlapping after 72 h. Statistically significant results were observed for Co-nano with the micronucleus test and the comet assay, while for Co(2+) positive results were observed only with the latter. In addition, even when Co-nano was genotoxic (at >1 microM), no evident dose-dependent effect was observed. Concerning morphological transformation, we found a statistically significant increase in the formation of type III foci (morphologically transformed colonies) only for Co-nano. Furthermore, we observed a higher cellular uptake of Co-nano compared with Co(2+).

  4. Chemical composition of the essential oil from basil (Ocimum basilicum Linn.) and its in vitro cytotoxicity against HeLa and HEp-2 human cancer cell lines and NIH 3T3 mouse embryonic fibroblasts.

    PubMed

    Kathirvel, Poonkodi; Ravi, Subban

    2012-01-01

    This study examines the chemical composition and in vitro anticancer activity of the essential oil from Ocimum basilicum Linn. (Lamiaceae), cultivated in the Western Ghats of South India. The chemical compositions of basil fresh leaves were identified by GC-MS: 11 components were identified. The major constituents were found to be methyl cinnamate (70.1%), linalool (17.5%), β-elemene (2.6%) and camphor (1.52%). The results revealed that this plant may belong to the methyl cinnamate and linalool chemotype. A methyl thiazol tetrazolium assay was used for in vitro cytotoxicity screening against the human cervical cancer cell line (HeLa), human laryngeal epithelial carcinoma cell line (HEp-2) and NIH 3T3 mouse embryonic fibroblasts. The IC(50) values obtained were 90.5 and 96.3 µg mL(-1), respectively, and the results revealed that basil oil has potent cytotoxicity.

  5. PDGF-induced receptor phosphorylation and phosphoinositide hydrolysis are unaffected by protein kinase C activation in mouse swiss 3T3 and human skin fibroblasts

    SciTech Connect

    Sturani, E.; Vicentini, L.M.; Zippel, R.; Toschi, L.; Pandiella-Alonso, A.; Comoglio, P.M.; Meldolesi, J.

    1986-05-29

    Short (1-10 min) pretreatment of intact cells with activators of protein kinase C (e.g. phorbol-12 myristate, 13-acetate, PMA) affects the activity of a variety of surface receptors (for growth factors, hormones and neurotransmitters), with inhibition of transmembrane signal generation. In two types of fibroblasts it is demonstrated that the PDGF receptor is unaffected by PMA. Exposure to PMA at concentrations up to 100 nM for 10 min failed to inhibit either one of the agonist-induced, receptor-coupled responses of PDGF: the autophosphorylation of receptor molecules at tyrosine residues, and the hydrolysis of membrane polyphosphoinositides. In contrast, the EGF receptor autophosphorylation (in A 431 cells) and the bombesin-induced phosphoinositide hydrolysis were readily inhibited by PMA.

  6. Coculture with BJ fibroblast cells inhibits the adipogenesis and lipogenesis in 3T3-L1 cells

    SciTech Connect

    Jeong, Hyun Jeong; Park, Sahng Wook; Kim, Hojeong; Park, Sang-Kyu; Yoon, Dojun

    2010-02-19

    Mouse or human fibroblasts are commonly used as feeder cells to prevent differentiation in stem or primary cell culture. In the present study, we addressed whether fibroblasts can affect the differentiation of adipocytes. We found that the differentiation of 3T3-L1 preadipocytes was strongly suppressed when the cells were cocultured with human fibroblast (BJ) cells. BrdU incorporation analysis indicated that mitotic clonal expansion, an early event required for 3T3-L1 cell adipogenesis, was not affected by BJ cells. The 3T3-L1 cell expression levels of peroxisome proliferator-activated receptor {gamma}2, CCAAT/enhancer-binding protein alpha (C/EBP{alpha}), sterol regulatory element binding protein-1c, and Krueppel-like factor 15, but not those of C/EBP{beta} or C/EBP{delta}, were decreased by coculture with BJ cells. When mature 3T3-L1 adipocytes were cocultured with BJ cells, their lipid contents were significantly reduced, with decreased fatty acid synthase expression and increased phosphorylated form of acetyl-CoA carboxylase 1. Our data indicate that coculture with BJ fibroblast cells inhibits the adipogenesis of 3T3-L1 preadipocytes and decreases the lipogenesis of mature 3T3-L1 adipocytes.

  7. Effect of in vitro administered 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on DNA-binding activities of nuclear transcription factors in NIH-3T3 mouse fibroblasts.

    PubMed

    Hwang, Seong Gu; Sasagawa, Hiromi; Matsumura, Fumio

    2007-01-01

    TCDD is a very toxic environmental contaminant which is known to cause a variety of toxic symptoms in many species. Because of a myriad of biochemical changes TCDD is known to induce in many test animals, it has been difficult to pinpoint the causative event common to all those symptoms in different species. One of the research avenues we have been following is identification of the pattern of TCDD-induced changes in DNA binding characteristics of nuclear transcription factors (NTFs), each of which has the property to trigger a set of coordinated changes in many gene expressions. Since in our previous work we studied animals affected by TCDD in vivo using gel mobility shift assay approached with32P labeled oligonucleotide probes, we examined in the current study the possibility whether we could establish an equivalent in vitro system in NIH-3T3 mouse fibroblast cells so as to be able to learn the similarities and the dissimilarities of TCDD-induced responses of NTFs between in vitro and in vivo. The results indicated that, for a large part, this in vitro test system could reasonably reproduce the pattern of changes occurring in vivo at the early stages of TCDD's action in terms of induced changes in binding of thes NTSs to DNA. The key features were TCDD induced upregulation of NTFs binding to the response elements for AP-1, dioxin (DRE) and T3 (thyroid hormone) and down-regulation of those to response elements (REs) for c-Myc, Sp-1 and retinoic acid receptor (RARE). However, the time course required the changes in DNA binding activity was much shorter in vitro. To study the basic cause for such changes in NTF binding, we studied the effects of exogenously added EGF, forskolin, TPA (12-0-tetradecanoylphorbol acetate) and TNFalpha on the expression of TCDD's action on some of these NTFs. The results showed that these agents indeed greatly influence the outcome. The most influential agents were TNFalpha, forskolin and EGF. These results indicate that this in vitro

  8. Definition of metabolism-dependent xenobiotic toxicity with co-cultures of human hepatocytes and mouse 3T3 fibroblasts in the novel integrated discrete multiple organ co-culture (IdMOC) experimental system: results with model toxicants aflatoxin B1, cyclophosphamide and tamoxifen.

    PubMed

    Li, Albert P; Uzgare, Aarti; LaForge, Yumiko S

    2012-07-30

    The integrated discrete multiple organ co-culture system (IdMOC) allows the co-culturing of multiple cell types as physically separated cells interconnected by a common overlying medium. We report here the application of IdMOC with two cell types: the metabolically competent primary human hepatocytes, and a metabolically incompetent cell line, mouse 3T3 fibroblasts, in the definition of the role of hepatic metabolism on the cytotoxicity of three model toxicants: cyclophosphamide (CPA), aflatoxin B1 (AFB) and tamoxifen (TMX). The presence of hepatic metabolism in IdMOC with human hepatocytes was demonstrated by the metabolism of the P450 isoform 3A4 substrate, luciferin-IPA. The three model toxicants showed three distinct patterns of cytotoxic profile: TMX was cytotoxic to 3T3 cells in the absence of hepatocytes, with slightly lower cytotoxicity towards both 3T3 cells and hepatocytes in the IdMOC. AFB was selective toxic towards the human hepatocytes and relatively noncytotoxic towards 3T3 cells both in the presence and absence of the hepatocytes. CPA cytotoxicity to the 3T3 cells was found to be significantly enhanced by the presence of the hepatocytes, with the cytotoxicity dependent of the number of hepatocytes, and with the cytotoxicity attenuated by the presence of a non-specific P450 inhibitor, 1-aminobenzotriazole. We propose here the following classification of toxicants based on the role of hepatic metabolism as defined by the human hepatocyte-3T3 cell IdMOC assay: type I: direct-acting cytotoxicants represented by TMX as indicated by cytotoxicity in 3T3 cells in the absence of hepatocytes; type II: metabolism-dependent cytotoxicity represented by AFB1 with effects localized within the site of metabolic activation (i. e. hepatocytes); and type III: metabolism-dependent cytotoxicity with metabolites that can diffuse out of the hepatocytes to cause toxicity in cells distal from the site of metabolism, as exemplified by CPA.

  9. Inhibitory effects of LPA1 on cell motile activities stimulated by hydrogen peroxide and 2,3-dimethoxy-1,4-naphthoquinone in fibroblast 3T3 cells.

    PubMed

    Hirane, Miku; Araki, Mutsumi; Dong, Yan; Honoki, Kanya; Fukushima, Nobuyuki; Tsujiuchi, Toshifumi

    2013-11-08

    Reactive oxygen species (ROS) are known to mediate a variety of biological responses, including cell motility. Recently, we indicated that lysophosphatidic acid (LPA) receptor-3 (LPA3) increased cell motile activity stimulated by hydrogen peroxide. In the present study, we assessed the role of LPA1 in the cell motile activity mediated by ROS in mouse fibroblast 3T3 cells. 3T3 cells were treated with hydrogen peroxide and 2,3-dimethoxy-1,4-naphthoquinone (DMNQ) at concentrations of 0.1 and 1 μM for 48 h. In cell motility assays with Cell Culture Inserts, the cell motile activities of 3T3 cells treated with hydrogen peroxide and DMNQ were significantly higher than those of untreated cells. 3T3 cells treated with hydrogen peroxide and DMNQ showed elevated expression levels of the Lpar3 gene, but not the Lpar1 and Lpar2 genes. To investigate the effects of LPA1 on the cell motile activity induced by hydrogen peroxide and DMNQ, Lpar1-overexpressing (3T3-a1) cells were generated from 3T3 cells and treated with hydrogen peroxide and DMNQ. The cell motile activities stimulated by hydrogen peroxide and DMNQ were markedly suppressed in 3T3-a1 cells. These results suggest that LPA signaling via LPA1 inhibits the cell motile activities stimulated by hydrogen peroxide and DMNQ in 3T3 cells.

  10. Synthesis, Characterization, and Study of In Vitro Cytotoxicity of ZnO-Fe3O4 Magnetic Composite Nanoparticles in Human Breast Cancer Cell Line (MDA-MB-231) and Mouse Fibroblast (NIH 3T3).

    PubMed

    Bisht, Gunjan; Rayamajhi, Sagar; Kc, Biplab; Paudel, Siddhi Nath; Karna, Deepak; Shrestha, Bhupal G

    2016-12-01

    Novel magnetic composite nanoparticles (MCPs) were successfully synthesized by ex situ conjugation of synthesized ZnO nanoparticles (ZnO NPs) and Fe3O4 NPs using trisodium citrate as linker with an aim to retain key properties of both NPs viz. inherent selectivity towards cancerous cell and superparamagnetic nature, respectively, on a single system. Successful characterization of synthesized nanoparticles was done by XRD, TEM, FTIR, and VSM analyses. VSM analysis showed similar magnetic profile of thus obtained MCPs as that of naked Fe3O4 NPs with reduction in saturation magnetization to 16.63 emu/g. Also, cell viability inferred from MTT assay showed that MCPs have no significant toxicity towards noncancerous NIH 3T3 cells but impart significant toxicity at similar concentration to breast cancer cell MDA-MB-231. The EC50 value of MCPs on MDA-MB-231 is less than that of naked ZnO NPs on MDA-MB-231, but its toxicity on NIH 3T3 was significantly reduced compared to ZnO NPs. Our hypothesis for this prominent difference in cytotoxicity imparted by MCPs is the synergy of selective cytotoxicity of ZnO nanoparticles via reactive oxygen species (ROS) and exhausting scavenging activity of cancerous cells, which further enhance the cytotoxicity of Fe3O4 NPs on cancer cells. This dramatic difference in cytotoxicity shown by the conjugation of magnetic Fe3O4 NPs with ZnO NPs should be further studied that might hold great promise for the development of selective and site-specific nanoparticles. Schematic representation of the conjugation, characterization and cytotoxicity analysis of Fe3O4-ZnO magnetic composite particles (MCPs).

  11. Synthesis, Characterization, and Study of In Vitro Cytotoxicity of ZnO-Fe3O4 Magnetic Composite Nanoparticles in Human Breast Cancer Cell Line (MDA-MB-231) and Mouse Fibroblast (NIH 3T3)

    NASA Astrophysics Data System (ADS)

    Bisht, Gunjan; Rayamajhi, Sagar; KC, Biplab; Paudel, Siddhi Nath; Karna, Deepak; Shrestha, Bhupal G.

    2016-12-01

    Novel magnetic composite nanoparticles (MCPs) were successfully synthesized by ex situ conjugation of synthesized ZnO nanoparticles (ZnO NPs) and Fe3O4 NPs using trisodium citrate as linker with an aim to retain key properties of both NPs viz. inherent selectivity towards cancerous cell and superparamagnetic nature, respectively, on a single system. Successful characterization of synthesized nanoparticles was done by XRD, TEM, FTIR, and VSM analyses. VSM analysis showed similar magnetic profile of thus obtained MCPs as that of naked Fe3O4 NPs with reduction in saturation magnetization to 16.63 emu/g. Also, cell viability inferred from MTT assay showed that MCPs have no significant toxicity towards noncancerous NIH 3T3 cells but impart significant toxicity at similar concentration to breast cancer cell MDA-MB-231. The EC50 value of MCPs on MDA-MB-231 is less than that of naked ZnO NPs on MDA-MB-231, but its toxicity on NIH 3T3 was significantly reduced compared to ZnO NPs. Our hypothesis for this prominent difference in cytotoxicity imparted by MCPs is the synergy of selective cytotoxicity of ZnO nanoparticles via reactive oxygen species (ROS) and exhausting scavenging activity of cancerous cells, which further enhance the cytotoxicity of Fe3O4 NPs on cancer cells. This dramatic difference in cytotoxicity shown by the conjugation of magnetic Fe3O4 NPs with ZnO NPs should be further studied that might hold great promise for the development of selective and site-specific nanoparticles.

  12. Lipid droplet changes in proliferating and quiescent 3T3 fibroblasts.

    PubMed

    Diaz, Giacomo; Batetta, Barbara; Sanna, Francesca; Uda, Sabrina; Reali, Camilla; Angius, Fabrizio; Melis, Marta; Falchi, Angela Maria

    2008-05-01

    Lipid droplets (LDs) are fat-storing organelles present in virtually all eukaryotic cells and involved in many aspects of cell biology related to lipid metabolism and cholesterol homeostasis. In this study, we investigated the presence of LDs in proliferating and quiescent (contact-inhibited) 3T3 fibroblasts to verify a correlation with cell growth. LDs were characterized by Nile red staining, positivity to adipophilin and negativity to perilipin. LDs were numerous in proliferating cells, but very few in quiescent cells. However, the fraction of quiescent cells, which resumed proliferation after scratch-wound assay, also resumed the formation of LDs. In proliferating cells, the number of LDs correlated with the DNA content, suggesting a continuous accumulation of LDs during cell growth. These findings were supported by biochemical data showing much higher rates of cholesterol esterification and triglyceride synthesis in proliferating cells. Both filipin staining and the fluorescent cholesterol analog dehydroergosterol revealed the presence of an intense traffic of free cholesterol, mediated by acidic vesicles, in proliferating cells. Nile red ratiometric measurements revealed a different lipid composition of LDs in proliferating and quiescent cells. Changes in the number and composition of LDs were also found in growing cells treated with inhibitors of cholesterol esterification (Sandoz 58-035), endosomal cholesterol efflux (U18666A) and V-ATPase (bafilomycin-A1).

  13. Mitogenic stimuli and phosphatidylinositol (PI) turnover in cultured 3T3 fibroblasts

    SciTech Connect

    Kohler, C.; Petersen, R.

    1986-03-01

    The hydrolysis of PI and polyphosphoinositides by phopholipase C is an early and rapid response to cell activation by a variety of neurotransmitters, hormones, growth factors and pharmacological agonists. The authors have examined the role of PI turnover and the generation of second messengers (diacylglycerol and inositol trisphosphate) in the mitogenic response of cultured Balb/c and Swiss 3T3 cells to polypeptide growth factors. Cells were prelabelled with /sup 3/H inositol for 18-20 hours, washed and suspended in Herpes + Li/sup +/ buffer, and stimulated with platelet-derived growth factor (PDGF), vasopressin, insulin, and other growth factors. PI turnover was measured as the increase in total inositol phosphate (IP) production. IP1, IP2, and IP3 were characterized by sequential elution from a Dowex column. Partially purified PDGF produced a 2-4 fold stimulation of total IP production. This was seen as early as 30 seconds after stimulation and increased for up to 1-2 hours. Balb/c cells were more sensitive than Swiss cells to the mitogenic and PI effects of PDGF. Other mitogenic stimuli had differential effects on PI turnover. Vasopressin (4-400 ng/ml) markedly stimulated PI turnover (3-6 fold) in Swiss, but not Balb/c cells. Insulin (100 ng/ml - 10 ..mu..g/ml) increased total IP to a greater degree in Balb/c cells. Epidermal growth factor (10 ng/ml - 10 ..mu..g/ml) had no effect on PI turnover and fibroblast growth factor (10 ng/ml - 10 ..mu..g/ml) only stimulated at the higher concentrations in Swiss cells. Thrombin (1U/ml - 10 U/ml) produced a 1.5 - 2 fold stimulation in Balb/c cells. Thus, various polypeptide growth factors have differential effects on PI turnover depending on their mitogenic potential and the effector cell type.

  14. Effect of botulinum neurotoxin type A (BoNTA) on the morphology and viability of 3T3 murine fibroblasts

    PubMed Central

    Bandala, Cindy; Terán-Melo, Juan Luis; Anaya-Ruiz, Maricruz; Mejía-Barradas, Cesar Miguel; Domínguez-Rubio, Rene; la Garza-Montano, Paloma De; Alfaro-Rodríguez, Alfonso; Lara-Padilla, Eleazar

    2015-01-01

    Aim: BoNTA is used in the treatment of ophthalmological disorders, muscular hyperactivity and pain. In recent years it has been described that BoNTA reduces cellular viability and induces apoptosis in prostate cells lines. Studies about the effect of BoNTA are no well known. There have been studies about the effect of BoNTA on the expression levels of collagenase in fibroblasts, but not on its morphological impact on these cells. The aim of this study was to determine the effect of BoNTA on the morphology and viability of the 3T3 fibroblast cell line. Material and methods: The 3T3 fibroblast cell line was cultured and the experimental group received 10 U BoNTA added to a 0.9% sterile saline solution in a reconstituted vial. The control group received saline solution only. Cultured cells were observed and photographed at 5, 10, 15 and 20 h. Cell viability was evaluated by means of the trypan blue test, and cell proliferation with the Proliferation Assay kit (PROMEGA). Results: The application of BoNTA to 3T3 fibroblast cells induced morphological changes, such as a loss of normal fibroblast morphology. Additionally, we observed the cytoplasmic retraction and spread phenomena. The nuclei showed other important changes with Giemsa staining. Conclusion: The results indicate that BoNTA induced a loss of spindle form, increase in cytoplasmic vesicles, and the presence of nuclear vesicles (compacted chromatin surrounded by a nuclear envelope). This suggests an apoptotic process and decreased cell viability. Further studies are needed to explore the mechanisms of these alterations. PMID:26464704

  15. Response of MC3T3-E1 osteoblasts, L929 fibroblasts, and J774 macrophages to fluoride surface-modified AZ31 magnesium alloy.

    PubMed

    Lozano, Rosa María; Pérez-Maceda, Blanca Teresa; Carboneras, Mónica; Onofre-Bustamante, Edgar; García-Alonso, María Cristina; Escudero, María Lorenza

    2013-10-01

    The present work evaluates the biocompatibility of a fluoride surface-modified AZ31 magnesium alloy (AZ31HF) with different cell lines that coexist in the implant environment to test its potential use as a biodegradable and absorbable biomaterial for bone repair. A clear stimulation of cell proliferation and an enhancement of the mitochondrial respiratory activity were observed when mouse osteoblasts (MC3T3-E1), fibroblasts (L929), and macrophages (J774) cell lines were cultured with the modified alloy. No significant change in apoptosis or viability rates was observed when osteoblasts and fibroblasts cultures were grown in the presence of this alloy. A proteomic analysis of the MC3T3-E1 cell extracts cultured in the presence of AZ31HF showed an overexpression of proteins related with the mineralization process, which is a necessary step for bone repair. An increase in the lactate dehydrogenase activity was observed in the MC3T3-E1 and J774 cell cultures that could be a response of the oxidative stress produced by the presence of the material. This stress could be related to the increase observed in the respiratory mitochondrial activity or respiratory burst measured in theses cultures that indicate damage in the cell membranes and subsequently some cell death. Results reported here, for and against AZ31HF, should be taken into account when considering the potential use of this modified alloy in bone repair applications.

  16. Strategies of NF-κB signaling modulation by ectromelia virus in BALB/3T3 murine fibroblasts.

    PubMed

    Struzik, Justyna; Szulc-Dąbrowska, Lidia; Winnicka, Anna; Niemiałtowski, Marek

    2015-10-01

    Nuclear factor κB (NF-κB) is a pleiotropic transcription factor that regulates the expression of immune response genes. NF-κB signaling can be disrupted by pathogens that prevent host immune response. In this work, we examined the influence of ectromelia (mousepox) virus (ECTV) on NF-κB signaling in murine BALB/3T3 fibroblasts. Activation of NF-κB via tumor necrosis factor (TNF) receptor 1 (TNFR1) in these cells induces proinflammatory cytokine secretion. We show that ECTV does not recruit NF-κB to viral factories or induce NF-κB nuclear translocation in BALB/3T3 cells. Additionally, ECTV counteracts TNF-α-induced p65 NF-κB nuclear translocation during the course of infection. Inhibition of TNF-α-induced p65 nuclear translocation was also observed in neighboring cells that underwent fusion with ECTV-infected cells. ECTV inhibits the key step of NF-κB activation, i.e. Ser32 phosphorylation and degradation of inhibitor κBα (IκBα) induced by TNF-α. We also observed that ECTV prevents TNF-α-induced Ser536 of p65 phosphorylation in BALB/3T3 cells. Studying TNFR1 signaling provides information about regulation of inflammatory response and cell survival. Unraveling poxviral immunomodulatory strategies may be helpful in drug target identification as well as in vaccine development.

  17. Immunohistochemical evidence for an association of ribosomes with microfilaments in 3T3 fibroblasts.

    PubMed

    Hesketh, J E; Horne, Z; Campbell, G P

    1991-02-01

    Ribosome distribution in cultured fibroblasts was investigated immunohistochemically using antibodies which recognize the 60S ribosomal subunit. After treatment of cells with buffer containing 25mM KCl and 0.05% Nonidet-P40 immunostained material was present in punctate patterns and linear arrays consistent with some ribosomes being associated with the cytoskeleton. Treatment of the cells with 130mM KCl caused loss of both the beaded lines of immunostaining and micro-filaments. Double immunostaining showed ribosomes to be closely associated with microfilaments.

  18. Erythropoietin and interleukin-1beta modulate nitrite production in a Swiss 3T3 cell model of rheumatoid synovial fibroblasts.

    PubMed

    Baig, S; Patel, Y; Coussons, P; Grant, R

    2002-11-01

    Erythropoietin (EPO), a haemopoietic growth factor and a primary regulator of erythropoiesis, is widely used to treat anaemia in various chronic complications of rheumatoid arthritis (RA). Fibroblast-like cells, found in the pannus tissue of joints, are thought to contribute to the inflammatory pathology of RA. Thus for the current study we investigated the effects of recombinant human EPO (rHuEPO) on NO metabolism, using an interleukin-1beta (IL-1beta)-stimulated Swiss 3T3 fibroblast monolayer as a model for fibroblast activity in RA. The results show that, over 3 days, both alone and in combination with the pro-inflammatory cytokine IL-1beta (10 ng/ml), rHuEPO (25 micro-units/ml) induced significant production of nitrite in cell culture supernatants. This is an indicator of NO production by nitric oxide synthase (NOS), which is a well-documented mediator of metalloproteinase-mediated tissue remodelling in RA. It therefore appears that, through modulation of NOS-dependent NO production, rHuEPO may influence remodelling of connective tissue in RA, independently of its established erythropoietic role.

  19. Effects of salvianolic acid-A on NIH/3T3 fibroblast proliferation, collagen synthesis and gene expression

    PubMed Central

    Liu, Cheng-Hai; Hu, Yi-Yang; Wang, Xiao-Ling; Xu, Lie-Ming; Liu, Ping

    2000-01-01

    AIM: To investigate the mechanisms of salvianolic acid A (SA-A) against liver fibrosis in vitro. METHODS: NIH/3T3 fibroblasts were cultured routinely, and incubated with 10-4 mol/L-10-7 mol/L SA-A for 22 h. The cell viability was assayed by [3H]proline incorporation, cell proliferation by [3H]TdR incorporation, cell collagen synthetic rate was measured with [3H]proline impulse and collagenase digestion method. The total RNA was prepared from the control cells and the drug treated cells respectively, and α (1) I pro-collagen mRNA expression was semi-quantitatively analyzed with RT-PCR. RESULTS: 10-4 mol/L SA-A decreased cell viability and exerted some cytotoxiciy, while 10-5 mol/L-10-7 mol/L SA-A did not affect cell viability, but inhibited cell proliferation significantly, and 10-6 mol/L SA-A had the best effect on cell viability among these concentrations of drugs. 10-5 mol/L-10-6 mol/L SA-A inhibited intracellular collagen synthetic rate, but no significant influence on extracellular collagen secretion. Both 10-5 mol/L and 10-6 mol/L SA-A could decrease α (1) I pro-collagen mRNA expression remarkably. CONCLUSION: SA-A had potent action against liver fibrosis. It inhibited NIH/3T3 fibroblast proliferation, intracellular collagen synthetic rate and type I pro-collagen gene expression, which may be one of the main mechanisms of the drug. PMID:11819598

  20. Bone marrow-derived cultured mast cells and peritoneal mast cells as targets of a growth activity secreted by BALB/3T3 fibroblasts

    SciTech Connect

    Jozaki, K.; Kuriu, A.; Hirota, S.; Onoue, H.; Ebi, Y.; Adachi, S.; Ma, J.Y.; Tarui, S.; Kitamura, Y. )

    1991-03-01

    When fibroblast cell lines were cultured in contact with bone marrow-derived cultured mast cells (CMC), both NIH/3T3 and BALB/3T3 cell lines supported the proliferation of CMC. In contrast, when contact between fibroblasts and CMC was prohibited by Biopore membranes or soft agar, only BALB/3T3 fibroblasts supported CMC proliferation, suggesting that BALB/3T3 but not NIH/3T3 cells secreted a significant amount of a mast cell growth activity. Moreover, the BALB/3T3-derived growth activity induced the incorporation of (3H)thymidine by CMC and the clonal growth of peritoneal mast cells in methylcellulose. The mast cell growth activity appeared to be different from interleukin 3 (IL-3) and interleukin 4 (IL-4), because mRNAs for these interleukins were not detectable in BALB/3T3 fibroblasts. Although mast cells are genetically deficient in tissues of W/Wv mice, CMC did develop when bone marrow cells of W/Wv mice were cultured with pokeweed mitogen-stimulated spleen cell-conditioned medium. Because BALB/3T3 fibroblast-conditioned medium (BALB-FCM) did not induce the incorporation of (3H)thymidine by W/Wv CMC, the growth activity in BALB-FCM appeared to be a ligand for the receptor encoded by the W (c-kit) locus. Because CMC and peritoneal mast cells are obtained as homogeneous suspensions rather easily, these cells may be potentially useful as targets for the fibroblast-derived mast cell growth activity.

  1. High-density lipoprotein contribute to G0-G1/S transition in Swiss NIH/3T3 fibroblasts

    PubMed Central

    Angius, Fabrizio; Spolitu, Stefano; Uda, Sabrina; Deligia, Stefania; Frau, Alessandra; Banni, Sebastiano; Collu, Maria; Accossu, Simonetta; Madeddu, Clelia; Serpe, Roberto; Batetta, Barbara

    2015-01-01

    High density lipoproteins (HDLs) play a crucial role in removing excess cholesterol from peripheral tissues. Although their concentration is lower during conditions of high cell growth rate (cancer and infections), their involvement during cell proliferation is not known. To this aim, we investigated the replicative cycles in synchronised Swiss 3T3 fibroblasts in different experimental conditions: i) contact-inhibited fibroblasts re-entering cell cycle after dilution; ii) scratch-wound assay; iii) serum-deprived cells induced to re-enter G1 by FCS, HDL or PDGF. Analyses were performed during each cell cycle up to quiescence. Cholesterol synthesis increased remarkably during the replicative cycles, decreasing only after cells reached confluence. In contrast, cholesteryl ester (CE) synthesis and content were high at 24 h after dilution and then decreased steeply in the successive cycles. Flow cytometry analysis of DiO-HDL, as well as radiolabeled HDL pulse, demonstrated a significant uptake of CE-HDL in 24 h. DiI-HDL uptake, lipid droplets (LDs) and SR-BI immunostaining and expression followed the same trend. Addition of HDL or PDGF partially restore the proliferation rate and significantly increase SR-BI and pAKT expression in serum-deprived cells. In conclusion, cell transition from G0 to G1/S requires CE-HDL uptake, leading to CE-HDL/SR-BI pathway activation and CEs increase into LDs. PMID:26640042

  2. Regulation of the beta-adrenergic receptor-adenylate cyclase complex of 3T3-L1 fibroblasts by sodium butyrate

    SciTech Connect

    Stadel, J.M.; Poksay, K.S.; Nakada, M.T.; Crooke, S.T.

    1986-05-01

    Mouse 3T3-L1 fibroblasts contain beta-adrenergic receptors (BAR), predominantly of the B/sub 1/ subtype. Incubation of these cells with 2-10 mM sodium butyrate (SB) for 24-48 hr results in a switch in the BAR subtype from B/sub 1/ to B/sub 2/ and promotes a 1.5 to 2.5 fold increase in total BAR number. Other short chain acids were not as effective as SB in promoting changes in BAR. BAR were assayed in membranes prepared from the 3T3-L1 cells using the radiolabeled antagonist (/sup 125/I)-cyanopindolol and the B/sub 2/ selective antagonist ICI 118.551. BAR subtype switch was confirmed functionally by measuring cellular cAMP accumulation in response to agonists. The structure and amount of the alpha subunits of the guanine nucleotide regulatory proteins N/sub s/ and N/sub i/ were determined by ADP-ribosylation using /sup 32/P-NAD and either cholera toxin or pertussis toxin for labeling of the respective subunits. Preincubation of cells with 5 mM SB for 48 hr resulted in a 2-3 fold increase in the labeling of the alpha subunits of both N/sub s/ and N/sub i/. A protein of M/sub r/ = 44,000 showed enhanced labeling by cholera toxin following SB treatment of the cells. These data indicate SB concomitantly regulates expression of BAR subtype and components of the adenylate cyclase in 3T3-L1 cells.

  3. TGF beta induces a sustained c-fos expression associated with stimulation or inhibition of cell growth in EL2 or NIH 3T3 fibroblasts.

    PubMed

    Liboi, E; Di Francesco, P; Gallinari, P; Testa, U; Rossi, G B; Peschle, C

    1988-02-29

    We have previously indicated that epidermal growth factor (EGF) plays a fundamental role in the proliferation control of EL2 rat fibroblast line. It is shown here that transforming growth factor beta (TGF beta) stimulates both DNA synthesis and proliferation of EL2 cells, while exerting an inhibitory effect on the growth of murine NIH-3T3 fibroblasts. We also report the effect of TGF beta and EGF on c-fos expression in EL2 cells, as compared to that of TGF beta in NIH-3T3 fibroblasts. In EL2 cells EGF induces a transient c-fos expression at both mRNA and protein level, as previously observed in NIH-3T3 fibroblasts treated with platelet-derived or fibroblast growth factor (PDGF, FGF). Conversely, TGF beta induces in EL2 cells a sustained expression of fos mRNA and protein, which are still detectable at least 24 and 7 hr after treatment respectively. In NIH-3T3 fibroblasts TGF beta causes a sustained fos RNA expression, which is not associated, however, with detectable fos protein. We conclude that in fibroblasts stimulated by mitogens c-fos expression may be differentially modulated, depending of the growth factor and the cell line. This is seemingly due to differential regulation of fos gene expression, not only at the transcriptional and/or post-transcriptional level (transient or sustained fos RNA induction by EGF or TGF beta in EL2 cells), but also at the translational level (fos protein(s) induction by TGF beta in EL2 but not NIH-3T3 fibroblasts, possibly related to the stimulatory vs inhibitory effect of this factor on the growth of the former vs the latter line).

  4. Neurite outgrowth stimulatory effects of culinary-medicinal mushrooms and their toxicity assessment using differentiating Neuro-2a and embryonic fibroblast BALB/3T3

    PubMed Central

    2013-01-01

    Background Mushrooms are not only regarded as gourmet cuisine but also as therapeutic agent to promote cognition health. However, little toxicological information is available regarding their safety. Therefore, the aim of this study was to screen selected ethno-pharmacologically important mushrooms for stimulatory effects on neurite outgrowth and to test for any cytotoxicity. Methods The stimulatory effect of mushrooms on neurite outgrowth was assessed in differentiating mouse neuroblastoma (N2a) cells. Neurite length was measured using Image-Pro Insight processor system. Neuritogenesis activity was further validated by fluorescence immunocytochemical staining of neurofilaments. In vitro cytotoxicity was investigated by using mouse embryonic fibroblast (BALB/3T3) and N2a cells for any embryo- and neuro-toxic effects; respectively. Results Aqueous extracts of Ganoderma lucidum, Lignosus rhinocerotis, Pleurotus giganteus and Grifola frondosa; as well as an ethanol extract of Cordyceps militaris significantly (p < 0.05) promoted the neurite outgrowth in N2a cells by 38.4 ± 4.2%, 38.1 ± 2.6%, 33.4 ± 4.6%, 33.7 ± 1.5%, and 35.8 ± 3.4%; respectively. The IC50 values obtained from tetrazolium (MTT), neutral red uptake (NRU) and lactate dehydrogenase (LDH) release assays showed no toxic effects following 24 h exposure of N2a and 3T3 cells to mushroom extracts. Conclusion Our results indicate that G. lucidum, L. rhinocerotis, P. giganteus, G. frondosa and C. militaris may be developed as safe and healthy dietary supplements for brain and cognitive health. PMID:24119256

  5. Mouse osteoblastic cell line (MC3T3-E1) expresses extracellular calcium (Ca2+o)-sensing receptor and its agonists stimulate chemotaxis and proliferation of MC3T3-E1 cells

    NASA Technical Reports Server (NTRS)

    Yamaguchi, T.; Chattopadhyay, N.; Kifor, O.; Butters, R. R. Jr; Sugimoto, T.; Brown, E. M.; O'Malley, B. W. (Principal Investigator)

    1998-01-01

    The calcium-sensing receptor (CaR) is a G protein-coupled receptor that plays key roles in extracellular calcium ion (Ca2+o) homeostasis in parathyroid gland and kidney. Osteoblasts appear at sites of osteoclastic bone resorption during bone remodeling in the "reversal" phase following osteoclastic resorption and preceding bone formation. Bone resorption produces substantial local increases in Ca2+o that could provide a signal for osteoblasts in the vicinity, leading us to determine whether such osteoblasts express the CaR. In this study, we used the mouse osteoblastic, clonal cell line MC3T3-E1. Both immunocytochemistry and Western blot analysis, using an antiserum specific for the CaR, detected CaR protein in MC3T3-E1 cells. We also identified CaR transcripts in MC3T3-E1 cells by Northern analysis using a CaR-specific riboprobe and by reverse transcription-polymerase chain reaction with CaR-specific primers, followed by nucleotide sequencing of the amplified products. Exposure of MC3T3-E1 cells to high Ca2+o (up to 4.8 mM) or the polycationic CaR agonists, neomycin and gadolinium (Gd3+), stimulated both chemotaxis and DNA synthesis in MC3T3-E1 cells. Therefore, taken together, our data strongly suggest that the osteoblastic cell line MC3T3-E1 possesses both CaR protein and mRNA very similar, if not identical, to those in parathyroid and kidney. Furthermore, the CaR in these osteoblasts could play a key role in regulating bone turnover by stimulating the proliferation and migration of such cells to sites of bone resorption as a result of local release of Ca2+o.

  6. Interactions between sub-10-nm iron and cerium oxide nanoparticles and 3T3 fibroblasts: the role of the coating and aggregation state

    NASA Astrophysics Data System (ADS)

    Safi, M.; Sarrouj, H.; Sandre, O.; Mignet, N.; Berret, J.-F.

    2010-04-01

    Recent nanotoxicity studies revealed that the physico-chemical characteristics of engineered nanomaterials play an important role in the interactions with living cells. Here, we report on the toxicity and uptake of cerium and iron oxide sub-10-nm nanoparticles by NIH/3T3 mouse fibroblasts. Coating strategies include low-molecular weight ligands (citric acid) and polymers (poly(acrylic acid), MW = 2000 g mol - 1). Electrostatically adsorbed on the surfaces, the organic moieties provide a negatively charged coating in physiological conditions. We find that most particles were biocompatible, as exposed cells remained 100% viable relative to controls. Only the bare and the citrate-coated nanoceria exhibit a slight decrease in mitochondrial activity at very high cerium concentrations (>1 g l - 1). We also observe that the citrate-coated particles are internalized/adsorbed by the cells in large amounts, typically 250 pg/cell after 24 h incubation for iron oxide. In contrast, the polymer-coated particles are taken up at much lower rates (<30 pg/cell). The strong uptake shown by the citrated particles is related to the destabilization of the dispersions in the cell culture medium and their sedimentation down to the cell membranes. In conclusion, we show that the uptake of nanomaterials by living cells depends on the coating of the particles and on its ability to preserve the colloidal nature of the dispersions.

  7. Receptor-mediated inhibition of adenylate cyclase and stimulation of arachidonic acid release in 3T3 fibroblasts. Selective susceptibility to islet-activating protein, pertussis toxin

    SciTech Connect

    Murayama, T.; Ui, M.

    1985-06-25

    Thrombin exhibited diverse effects on mouse 3T3 fibroblasts. It (a) decreased cAMP in the cell suspension, (b) inhibited adenylate cyclase in the Lubrol-permeabilized cell suspension in a GTP-dependent manner, increased releases of (c) arachidonic acid and (d) inositol from the cell monolayer prelabeled with these labeled compounds, (e) increased /sup 45/Ca/sup 2 +/ uptake into the cell monolayer, and (f) increased /sup 86/Rb/sup +/ uptake into the cell monolayer in a ouabain-sensitive manner. Most of the effects were reproduced by bradykinin, platelet-activating factor, and angiotensin II. The receptors for these agonists are thus likely to be linked to three separate effector systems: the adenylate cyclase inhibition, the phosphoinositide breakdown leading to Ca/sup 2 +/ mobilization and phospholipase A2 activation, and the Na,K-ATPase activation. Among the effects of these agonists, (a), (b), (c), and (e) were abolished, but (d) and (f) were not, by prior treatment of the cells with islet-activating protein (IAP), pertussis toxin, which ADP-ribosylates the Mr = 41,000 protein, the alpha-subunit of the inhibitory guanine nucleotide regulatory protein (Ni), thereby abolishing receptor-mediated inhibition of adenylate cyclase. The effects (a), (c), (d), and (e) of thrombin, but not (b), were mimicked by A23187, a calcium ionophore. The effects of A23187, in contrast to those of receptor agonists, were not affected by the treatment of cells with IAP. Thus, the IAP substrate, the alpha-subunit of Ni, or the protein alike, may play an additional role in signal transduction arising from the Ca/sup 2 +/-mobilizing receptors, probably mediating process(es) distal to phosphoinositide breakdown and proximal to Ca/sup 2 +/ gating.

  8. A commercial formulation of glyphosate inhibits proliferation and differentiation to adipocytes and induces apoptosis in 3T3-L1 fibroblasts.

    PubMed

    Martini, Claudia N; Gabrielli, Matías; Vila, María del C

    2012-09-01

    Glyphosate-based herbicides are extensively used for weed control all over the world. Therefore, it is important to investigate the putative toxic effects of these formulations which include not only glyphosate itself but also surfactants that may also be toxic. 3T3-L1 fibroblasts are a useful tool to study adipocyte differentiation, this cell line can be induced to differentiate by addition of a differentiation mixture containing insulin, dexamethasone and 3-isobutyl-1-methylxanthine. We used this cell line to investigate the effect of a commercial formulation of glyphosate (GF) on proliferation, survival and differentiation. It was found that treatment of exponentially growing cells with GF for 48h inhibited proliferation in a dose-dependent manner. In addition, treatment with GF dilution 1:2000 during 24 or 48h inhibited proliferation and increased cell death, as evaluated by trypan blue-exclusion, in a time-dependent manner. We showed that treatment of 3T3-L1 fibroblasts with GF increased caspase-3 like activity and annexin-V positive cells as evaluated by flow cytometric analysis, which are both indicative of induction of apoptosis. It was also found that after the removal of GF, remaining cells were able to restore proliferation. On the other hand, GF treatment severely inhibited the differentiation of 3T3-L1 fibroblasts to adipocytes. According to our results, a glyphosate-based herbicide inhibits proliferation and differentiation in this mammalian cell line and induces apoptosis suggesting GF-mediated cellular damage. Thus, GF is a potential risk factor for human health and the environment.

  9. Met promotes the formation of double minute chromosomes induced by Sei-1 in NIH-3T3 murine fibroblasts

    PubMed Central

    You, Jia; Wu, Di; Yu, Yang; Liu, Chang; Wang, Lei; Wang, Fei; Xu, Lu; Wang, Liqun; Wang, Nan; Tian, Xing; Wang, Falin; Liang, Hongbin; Gao, Yating; Cui, Xiaobo; Ji, Guohua; Bai, Jing; Yu, Jingcui; Meng, Xiangning; Jin, Yan; Sun, Wenjing; Guan, Xin-yuan; Zhang, Chunyu; Fu, Songbin

    2016-01-01

    Background Sei-1 is an oncogene capable of inducing double minute chromosomes (DMs) formation. DMs are hallmarks of amplification and contribute to oncogenesis. However, the mechanism of Sei-1 inducing DMs formation remains unelucidated. Results DMs formation significantly increased during serial passage in vivo and gradually decreased following culture in vitro. micro nuclei (MN) was found to be responsible for the reduction. Of the DMs-carrying genes, Met was found to be markedly amplified, overexpressed and highly correlated with DMs formation. Inhibition of Met signaling decreased the number of DMs and reduced the amplification of the DMs-carrying genes. We identified a 3.57Mb DMs representing the majority population, which consists of the 1.21 Mb AMP1 from locus 6qA2 and the 2.36 Mb AMP2 from locus 6qA2-3. Materials and Methods We employed NIH-3T3 cell line with Sei-1 overexpression to monitor and characterize DMs in vivo and in vitro. Array comparative genome hybridization (aCGH) and fluorescence in situ hybridization (FISH) were performed to reveal amplification regions and DMs-carrying genes. Metaphase spread was prepared to count the DMs. Western blot and Met inhibition rescue experiments were performed to examine for involvement of altered Met signaling in Sei-1 induced DMs. Genomic walking and PCR were adopted to reveal DMs structure. Conclusions Met is an important promotor of DMs formation. PMID:27494853

  10. Dynamics of Actin Stress Fibers and Focal Adhesions during Slow Migration in Swiss 3T3 Fibroblasts: Intracellular Mechanism of Cell Turning

    PubMed Central

    Miyoshi, Hiromi; Miura, Takuya; Tanaka, Hiroto; Tsubota, Ken-ichi; Liu, Hao

    2016-01-01

    To understand the mechanism regulating the spontaneous change in polarity that leads to cell turning, we quantitatively analyzed the dynamics of focal adhesions (FAs) coupling with the self-assembling actin cytoskeletal structure in Swiss 3T3 fibroblasts. Fluorescent images were acquired from cells expressing GFP-actin and RFP-zyxin by laser confocal microscopy. On the basis of the maximum area, duration, and relocation distance of FAs extracted from the RFP-zyxin images, the cells could be divided into 3 regions: the front region, intermediate lateral region, and rear region. In the intermediate lateral region, FAs appeared close to the leading edge and were stabilized gradually as its area increased. Simultaneously, bundled actin stress fibers (SFs) were observed vertically from the positions of these FAs, and they connected to the other SFs parallel to the leading edge. Finally, these connecting SFs fused to form a single SF with matured FAs at both ends. This change in SF organization with cell retraction in the first cycle of migration followed by a newly formed protrusion in the next cycle is assumed to lead to cell turning in migrating Swiss 3T3 fibroblasts. PMID:28119928

  11. Enhanced expression of haem oxygenase-1 by nitric oxide and antiinflammatory drugs in NIH 3T3 fibroblasts.

    PubMed

    Alcaraz, M J; Habib, A; Lebret, M; Créminon, C; Lévy-Toledano, S; Maclouf, J

    2000-05-01

    1. Haem oxygenase-1 (HO-1) can exert protective effects against oxidative stress and inflammation. Fibroblasts participate in inflammatory responses where they produce high levels of prostaglandins (PGs) and nitric oxide (NO). However, little is known of the presence of HO-1 in these cells and the possible interactions among these pathways. Incubation of cells with NO donors, spermine nonoate (SPNO) and S-nitroso-N-acetylpenicillamine (SNAP), induced a dose- and time-dependent expression of HO-1 protein. 2. NO donors increased basal PGE(2) release although they reduced PGE(2) accumulated in the medium and cyclo-oxygenase (COX) activity when cells were stimulated with lipopolysaccharide (LPS). COX-2 protein was weakly induced by SPNO in basal conditions and in the presence of LPS a synergy for HO-1 and COX-2 protein expression was observed. 3. Our results indicate that reactive oxygen species participate in the inductive effect of NO donors or LPS on HO-1 expression, whereas endogenous NO production may play a role in the mechanism of the synergy exhibited by SPNO and LPS on HO-1 and COX-2 expression. In this system, zinc protoporphyrin IX did not affect nitrite levels but reduced COX activity. 4. The selective COX-2 inhibitors SC58125 and NS398 as well as the non-selective COX inhibitor, indomethacin, strongly reduced PGE(2) synthesis and showed a synergy with NO donors in HO-1 and COX-2 induction. Addition of PGE(2) had no effect, suggesting a mechanism independent of PGs formation. 5. In inflammatory conditions a number of factors could cooperate to induce HO-1 and COX-2, with a positive regulation by COX inhibitors.

  12. Transformation by viral and cellular oncogenes of a mouse BALB/3T3 cell mutant resistant to transformation by chemical carcinogens

    SciTech Connect

    Ono, M.; Yakushinji, M.; Segawa, K.; Kuwano, M.

    1988-10-01

    The mouse cell line MO-5 is resistant to transformation by various chemical carcinogens and also by UV irradiation. Northern (RNA) blot analysis showed active expression of ras and myc genes in MO-5 and BALB/3T3 cells. The effect of transfection of various oncogenes on transformation was compared in MO-5 cells and parental BALB/3T3 cells. Activated c-H-ras, c-N-ras, and v-mos gene induced transformation foci of MO-5 and BALB/3T3. Introduction of the polyomavirus middle T-antigen (mTag) or the Rous sarcoma virus-related oncogene v-src, however, efficiently transformed BALB/3T3 but not MO-5 cells. Expression and phosphorylation of mTag and the associated c-src proteins were observed in mTag-transfected clones of MO-5 as in BALB/3T3 and phosphorylation of the src protein was observed in v-src-transfected BALB/3T3 and MO-5 clones. Hybrids between mTag- or v-src-induced transformants of BALB/3T3 and untransformed MO-5 maintained the transformation phenotype, suggesting that no dominant suppressor of transformation exists in MO-5. A hybrid clone between BALB/3T3 and MO-5 induced efficient transformation foci after transfection with the mTag gene, suggesting that the deficient transformation phenotype of MO-5 was recessive. Instead, some other alteration of MO-5, plausibly membrane function, might lead to abortive transformation by chemical carcinogens and also by mTag and the v-src gene product.

  13. Fibroblast growth factor-1 stimulation of quiescent NIH 3T3 cells increases G/T mismatch-binding protein expression.

    PubMed Central

    Donohue, P J; Feng, S L; Alberts, G F; Guo, Y; Peifley, K A; Hsu, D K; Winkles, J A

    1996-01-01

    Polypeptide growth factors promote cell-cycle progression in part by the transcriptional activation of a diverse group of specific genes. We have used an mRNA differential-display approach to identify several fibroblast growth factor (FGF)-1 (acidic FGF)-inducible genes in NIH 3T3 cells. Here we report that one of these genes, called FGF-regulated (FR)-3, is predicted to encode G/T mismatch-binding protein (GTBP), a component of the mammalian DNA mismatch correction system. The murine GTBP gene is transiently expressed after FGF-1 or calf serum treatment, with maximal mRNA levels detected at 12 and 18 h post-stimulation. FGF-1-stimulated NIH 3T3 cells also express an increased amount of GTBP as determined by immunoblot analysis. These results indicate that elevated levels of GTBP may be required during the DNA synthesis phase of the cell cycle for efficient G/T mismatch recognition and repair. PMID:8870641

  14. Tubby-like protein superfamily member PLSCR3 functions as a negative regulator of adipogenesis in mouse 3T3-L1 preadipocytes by suppressing induction of late differentiation stage transcription factors

    PubMed Central

    Inokawa, Akira; Inuzuka, Tatsutoshi; Takahara, Terunao; Shibata, Hideki; Maki, Masatoshi

    2015-01-01

    PLSCR3 (phospholipid scramblase 3, Scr3) belongs to the superfamily of membrane-associated transcription regulators named Tubby-like proteins (TULPs). Physiological phospholipid scrambling activities of PLSCRs in vivo have been skeptically argued, and knowledge of the biological functions of Scr3 is limited. We investigated the expression of Scr3 during differentiation of mouse 3T3-L1 preadipocytes by Western blotting (WB) and by reverse-transcription and real-time quantitative PCR (RT-qPCR). The Scr3 protein decreased during 3T3-L1 differentiation accompanied by a reduction in the mRNA level, and there was a significant increase in the amount of Scr3 protein secreted into the culture medium in the form of extracellular microvesicles (exosomes). On the other hand, Scr3 expression did not significantly decrease, and the secretion of Scr3 in 3T3 Swiss-albino fibroblasts (a parental cell-line of 3T3-L1) was not increased by differentiation treatment. Overexpression of human Scr3 during 3T3-L1 differentiation suppressed triacylglycerol accumulation and inhibited induction of the mRNAs of late stage pro-adipogenic transcription factors [CCAAT/enhancer-binding protein α (C/EBPα) and peroxisome proliferator-activated receptor γ (PPARγ)] and X-box-binding protein 1 (XBP1). Expression of early stage pro-adipogenic transcription factors (C/EBPβ and C/EBPδ) was not significantly affected. These results suggest that Scr3 functions as a negative regulator of adipogenesis in 3T3-L1 cells at a specific differentiation stage and that decrease in the intracellular amount of Scr3 protein caused by reduction in Scr3 mRNA expression and enhanced secretion of Scr3 protein appears to be important for appropriate adipocyte differentiation. PMID:26677203

  15. Exchange protein activated by cyclic AMP is involved in the regulation of adipogenic genes during 3T3-L1 fibroblasts differentiation.

    PubMed

    Gabrielli, Matías; Martini, Claudia N; Brandani, Javier N; Iustman, Laura J R; Romero, Damián G; del C Vila, María

    2014-02-01

    Adipogenesis is stimulated in 3T3-L1 fibroblasts by a combination of insulin, dexamethasone and isobutylmethylxanthine, IBMX, (I+D+M). Two transcription factors are important for the acquisition of the adipocyte phenotype, C/EBP beta (CCAT enhancer-binding protein beta) and PPAR gamma (peroxisome proliferator-activated receptor gamma). IBMX increases cAMP content, which can activate protein kinase A (PKA) and/or EPAC (exchange protein activated by cAMP). To investigate the importance of IBMX in the differentiation mixture, we first evaluated the effect of the addition of IBMX on the increase of C/EBP beta and PPAR gamma and found an enhancement of the amount of both proteins. IBMX addition (I+D+M) or its replacement with a cAMP analogue, dibutyryl-cAMP or 8-(4-chlorophenylthio)-2-O'-methyl-cAMP (8CPT-2-Me-cAMP), the latter activates EPAC and not PKA, remarkably increased PPAR gamma mRNA. However, neither I+D nor any of the inducers alone, increased PPAR gamma mRNA to a similar extent, suggesting the importance of the presence of both IBMX and I+D. It was also found that the addition of IBMX or 8CPT-2-Me-cAMP was able to increase the content of C/EBP beta with respect to I+D. In agreement with these findings, a microarray analysis showed that the presence of either 8CPT-2-Me-cAMP or IBMX in the differentiation mixture was able to upregulate PPAR gamma and PPAR gamma-activated genes as well as other genes involved in lipid metabolism. Our results prove the involvement of IBMX-cAMP-EPAC in the regulation of adipogenic genes during differentiation of 3T3-L1 fibroblasts and therfore contributes to elucidate the role of cyclic AMP in this process.

  16. A homeopathic remedy from arnica, marigold, St. John’s wort and comfrey accelerates in vitro wound scratch closure of NIH 3T3 fibroblasts

    PubMed Central

    2012-01-01

    Background Drugs of plant origin such as Arnica montana, Calendula officinalis or Hypericum perforatum have been frequently used to promote wound healing. While their effect on wound healing using preparations at pharmacological concentrations was supported by several in vitro and clinical studies, investigations of herbal homeopathic remedies on wound healing process are rare. The objective of this study was to investigate the effect of a commercial low potency homeopathic remedy Similasan® Arnica plus Spray on wound closure in a controlled, blind trial in vitro. Methods We investigated the effect of an ethanolic preparation composed of equal parts of Arnica montana 4x, Calendula officinalis 4x, Hypericum perforatum 4x and Symphytum officinale 6x (0712–2), its succussed hydroalcoholic solvent (0712–1) and unsuccussed solvent (0712–3) on NIH 3T3 fibroblasts. Cell viability was determined by WST-1 assay, cell growth using BrdU uptake, cell migration by chemotaxis assay and wound closure by CytoSelect ™Wound Healing Assay Kit which generated a defined “wound field”. All assays were performed in three independent controlled experiments. Results None of the three substances affected cell viability and none showed a stimulating effect on cell proliferation. Preparation (0712–2) exerted a stimulating effect on fibroblast migration (31.9%) vs 14.7% with succussed solvent (0712–1) at 1:100 dilutions (p < 0.001). Unsuccussed solvent (0712–3) had no influence on cell migration (6.3%; p > 0.05). Preparation (0712–2) at a dilution of 1:100 promoted in vitro wound closure by 59.5% and differed significantly (p < 0.001) from succussed solvent (0712–1), which caused 22.1% wound closure. Conclusion Results of this study showed that the low potency homeopathic remedy (0712–2) exerted in vitro wound closure potential in NIH 3T3 fibroblasts. This effect resulted from stimulation of fibroblasts motility rather than of their mitosis. PMID:22809174

  17. The Ras-related protein Cdc42Hs and bradykinin promote formation of peripheral actin microspikes and filopodia in Swiss 3T3 fibroblasts.

    PubMed Central

    Kozma, R; Ahmed, S; Best, A; Lim, L

    1995-01-01

    The Ras-related protein Cdc42 plays a role in yeast cell budding and polarity. Two related proteins, Rac1 and RhoA, promote formation in mammalian cells of membrane ruffles and stress fibers, respectively, which contain actin microfilaments. We now show that microinjection of the related human Cdc42Hs into Swiss 3T3 fibroblasts induced the formation of peripheral actin microspikes, determined by staining with phalloidin. A proportion of these microspikes was found to be components of filopodia, as analyzed by time-lapse phase-contrast microscopy. The formation of filopodia was also found to be promoted by Cdc42Hs microinjection. This was followed by activation of Rac1-mediated membrane ruffling. Treatment with bradykinin also promoted formation of microspikes and filopodia as well as subsequent effects similar to that seen upon Cdc42Hs microinjection. These effects of bradykinin were specifically inhibited by prior microinjection of dominant negative Cdc42HsT17N, suggesting that bradykinin acts by activating cellular Cdc42Hs. Since filopodia have been ascribed an important sensory function in fibroblasts and are required for guidance of neuronal growth cones, these results indicate that Cdc42Hs plays an important role in determining mammalian cell morphology. PMID:7891688

  18. Cocoa tea (Camellia ptilophylla) water extract inhibits adipocyte differentiation in mouse 3T3-L1 preadipocytes

    PubMed Central

    Li, Kai Kai; Liu, Chuek Lun; Shiu, Hoi Ting; Wong, Hing Lok; Siu, Wing Sum; Zhang, Cheng; Han, Xiao Qiang; Ye, Chuang Xing; Leung, Ping Chung; Ko, Chun Hay

    2016-01-01

    Cocoa tea (Camellia ptilophylla) is a naturally decaffeinated tea plant. Previously we found that cocoa tea demonstrated a beneficial effect against high-fat diet induced obesity, hepatic steatosis, and hyperlipidemia in mice. The present study aimed to investigate the anti-adipogenic effect of cocoa tea in vitro using preadipocytes 3T3-L1. Adipogenic differentiation was confirmed by Oil Red O stain, qPCR and Western blot. Our results demonstrated that cocoa tea significantly inhibited triglyceride accumulation in mature adipocytes in a dose-dependent manner. Cocoa tea was shown to suppress the expressions of key adipogenic transcription factors, including peroxisome proliferator-activated receptor gamma (PPAR γ) and CCAAT/enhancer binding protein (C/EBP α). The tea extract was subsequently found to reduce the expressions of adipocyte-specific genes such as sterol regulatory element binding transcription factor 1c (SREBP-1c), fatty acid synthase (FAS), Acetyl-CoA carboxylase (ACC), fatty acid translocase (FAT) and stearoylcoenzyme A desaturase-1 (SCD-1). In addition, JNK, ERK and p38 phosphorylation were inhibited during cocoa tea inhibition of 3T3-L1 adipogenic differentiation. Taken together, this is the first study that demonstrates cocoa tea has the capacity to suppress adipogenesis in pre-adipocyte 3T3-L1 similar to traditional green tea PMID:26833256

  19. Cocoa tea (Camellia ptilophylla) water extract inhibits adipocyte differentiation in mouse 3T3-L1 preadipocytes.

    PubMed

    Li, Kai Kai; Liu, Chuek Lun; Shiu, Hoi Ting; Wong, Hing Lok; Siu, Wing Sum; Zhang, Cheng; Han, Xiao Qiang; Ye, Chuang Xing; Leung, Ping Chung; Ko, Chun Hay

    2016-02-01

    Cocoa tea (Camellia ptilophylla) is a naturally decaffeinated tea plant. Previously we found that cocoa tea demonstrated a beneficial effect against high-fat diet induced obesity, hepatic steatosis, and hyperlipidemia in mice. The present study aimed to investigate the anti-adipogenic effect of cocoa tea in vitro using preadipocytes 3T3-L1. Adipogenic differentiation was confirmed by Oil Red O stain, qPCR and Western blot. Our results demonstrated that cocoa tea significantly inhibited triglyceride accumulation in mature adipocytes in a dose-dependent manner. Cocoa tea was shown to suppress the expressions of key adipogenic transcription factors, including peroxisome proliferator-activated receptor gamma (PPAR γ) and CCAAT/enhancer binding protein (C/EBP α). The tea extract was subsequently found to reduce the expressions of adipocyte-specific genes such as sterol regulatory element binding transcription factor 1c (SREBP-1c), fatty acid synthase (FAS), Acetyl-CoA carboxylase (ACC), fatty acid translocase (FAT) and stearoylcoenzyme A desaturase-1 (SCD-1). In addition, JNK, ERK and p38 phosphorylation were inhibited during cocoa tea inhibition of 3T3-L1 adipogenic differentiation. Taken together, this is the first study that demonstrates cocoa tea has the capacity to suppress adipogenesis in pre-adipocyte 3T3-L1 similar to traditional green tea.

  20. PPARgamma induces the insulin-dependent glucose transporter GLUT4 in the absence of C/EBPalpha during the conversion of 3T3 fibroblasts into adipocytes.

    PubMed Central

    Wu, Z; Xie, Y; Morrison, R F; Bucher, N L; Farmer, S R

    1998-01-01

    To define the molecular mechanisms that control GLUT4 expression during adipogenesis, NIH-3T3 fibroblasts ectopically expressing different adipogenic transcription factors (C/EBPbeta, C/EBPdelta, C/EBPalpha, and PPARgamma) under the control of a tetracycline-responsive inducible (C/EBPs) or a constitutive retroviral (PPARgamma) expression system were used. Enhanced production of C/EBPbeta (beta2 cell line), C/EBPbeta together with C/EBPdelta (beta/delta39 cell line), C/EBPalpha (alpha1 cell line), or PPARgamma (Pgamma2 cell line) in cells exposed to dexamethasone and the PPARgamma ligand ciglitazone (a thiazolidinedione) resulted in expression of GLUT4 mRNA as well as other members of the adipogenic gene program, including aP2 and adipsin. Focusing our studies on the beta/delta39 cells, we have demonstrated that C/EBPbeta along with C/EBPdelta in the presence of dexamethasone induces PPARgamma, adipsin, and aP2 mRNA production; however, GLUT4 mRNA is only expressed in cells exposed to ciglitazone. In addition, enhanced expression of a ligand-activated form of PPARgamma in the beta/delta39 fibroblasts stimulates synthesis of GLUT4 protein and gives rise to a population of adipocytic cells that take up glucose in direct response to insulin. C/EBPalpha is not expressed in the beta/delta39 cells under conditions that stimulate the adipogenic program. This observation suggests that PPARgamma alone or in combination with C/EBPbeta and C/EBPdelta is capable of activating GLUT4 gene expression. PMID:9421462

  1. The protective effects of guaraná extract (Paullinia cupana) on fibroblast NIH-3T3 cells exposed to sodium nitroprusside.

    PubMed

    Bittencourt, L S; Machado, D C; Machado, M M; Dos Santos, G F F; Algarve, T D; Marinowic, D R; Ribeiro, E E; Soares, F A A; Barbisan, F; Athayde, M L; Cruz, I B M

    2013-03-01

    The antioxidant effects of the hydro-alcoholic guaraná extract (Paullinia cupana var. sorbilis Mart.) on nitric oxide (NO) and other compounds generated from the degradation of sodium nitroprusside (SNP) in an embryonic fibroblast culture (NIH-3T3 cells) were evaluated. The guaraná bioactive compounds were initially determined by high-performance liquid chromatography: caffeine=12.240 mg/g, theobromine=6.733 mg/g and total catechins=4.336 mg/g. Cells were exposed to 10 μM SNP during a 6 h period because the cells exhibited >90% mortality at this concentration. Guaraná was added to the cultures in five concentrations (0.5, 1, 5, 10 and 20 mg/mL). The guaraná antioxidant effect was evaluated by viability assays, biochemical oxidation [lipid peroxidation, catalase and superoxide dismutase (SOD) activity] and genotoxicity (DNA Comet assay) analysis. Additionally, oxidative stress was evaluated by a 2,7-dihydrodichlorofluorescein diacetate fluorescence assay. Guaraná reverted the SNP toxicity mainly at lower concentrations (<5 mg), which decreased cell mortality, lipid peroxidation, DNA damage and cell oxidative stress as well as increased the SOD levels. These results demonstrate that guaraná has an antioxidant effect on NO metabolism in situations with higher cellular NO levels.

  2. Regulation of glucose transport by insulin, bombesin, and bradykinin in Swiss 3T3 fibroblasts: Involvement of protein kinase C-dependent and -independent mechanisms

    SciTech Connect

    Dettori, C.; Meldolesi, J. )

    1989-05-01

    Glucose transport stimulation by insulin, bombesin, and bradykinin in Swiss 3T3 fibroblasts was compared with the phosphoinositide hydrolysis effects of the same stimulants in a variety of experimental paradigms known to affect generation and/or functioning of intracellular second messengers: short- and long-term treatments with phorbol dibutyrate, that cause activation and down-regulation of protein kinase C, respectively; cell loading with high (quin2), that causes clamping of (Ca{sup 2+}){sub i} near the resting level; poisoning with pertussis toxin, that affects the GTP binding proteins of the Go/Gi class; treatment with Ca{sup 2+} ionophores. ({sup 14}C) glucose transport stimulation by maximal (insulin) was affected by neither pertussis toxin nor protein kinase C down-regulation. This result correlates with the lack of effect of insulin on phosphoinositide hydrolysis. In contrast, part of the glucose transport responses induced by bombesin and bradykinin appeared to be mediated by protein kinase C in proportion with the stimulation induced by these peptides on the phosphoinositide hydrolysis. The protein kinase C-independent portion of the response to bradykinin was found to be inhibitable by pertussis toxin. This latter result might suggest an interaction between the bradykinin receptor and a glucose transporter, mediated by a protein of the Go/Gi class.

  3. Characterization of a bombesin receptor on Swiss mouse 3T3 cells by affinity cross-linking

    SciTech Connect

    Sinnett-Smith, J.; Zachary, I.; Rozengurt, E.

    1988-12-01

    We have previously identified by chemical cross-linking a cell surface protein in Swiss 3T3 cells of apparent Mr 75,000-85,000, which may represent a major component of the receptor for peptides of the bombesin family in these cells. Because bombesin-like peptides may interact with other cell surface molecules, it was important to establish the correlation between receptor binding and functions of this complex and further characterize the Mr 75,000-85,000 cross-linked protein. Detailed time courses carried out at different temperatures demonstrated that the Mr 75,000-85,000 affinity-labelled band was the earliest cross-linked complex detected in Swiss 3T3 cells incubated with 125I-labelled gastrin-releasing peptide (125I-GRP). Furthermore, the ability of various nonradioactive bombesin agonists and antagonists to block the formation of the Mr 75,000-85,000 cross-linked complex correlated extremely well (r = 0.994) with the relative capacity of these peptides to inhibit 125I-GRP specific binding. Pretreatment with unlabelled GRP for up to 6 h caused only a slight decrease in both specific 125I-GRP binding and the affinity labelling of the Mr 75,000-85,000 protein. We also show that the cross-linked complex is a glycoprotein. First, solubilized affinity labelled Mr 75,000-85,000 complex applied to wheat germ lectin-sepharose columns was eluted by addition of 0.3 M N-acetyl-D-glucosamine. Second, treatment with endo-beta-N-acetylglucosaminidase F reduced the apparent molecular weight of the affinity-labelled band from 75,000-85,000 to 43,000, indicating the presence of N-linked oligosaccharide groups.

  4. 3T3 fibroblasts transfected with a cDNA for mitochondrial aspartate aminotransferase express plasma membrane fatty acid-binding protein and saturable fatty acid uptake.

    PubMed Central

    Isola, L M; Zhou, S L; Kiang, C L; Stump, D D; Bradbury, M W; Berk, P D

    1995-01-01

    To explore the relationship between mitochondrial aspartate aminotransferase (mAspAT; EC 2.6.1.1) and plasma membrane fatty acid-binding protein (FABPpm) and their role in cellular fatty acid uptake, 3T3 fibroblasts were cotransfected with plasmid pMAAT2, containing a full-length mAspAT cDNA downstream of a Zn(2+)-inducible metallothionein promoter, and pFR400, which conveys methotrexate resistance. Transfectants were selected in methotrexate, cloned, and exposed to increasing methotrexate concentrations to induce gene amplification. Stably transfected clones were characterized by Southern blotting; those with highest copy numbers of pFR400 alone (pFR400) or pFR400 and pMAAT2 (pFR400/pMAAT2) were expanded for further study. [3H]Oleate uptake was measured in medium containing 500 microM bovine serum albumin and 125-1000 microM total oleate (unbound oleate, 18-420 nM) and consisted of saturable and nonsaturable components. pFR400/pMAAT2 cells exhibited no increase in the rate constant for nonsaturable oleate uptake or in the uptake rate of [14C]octanoate under any conditions. By contrast, Vmax (fmol/sec per 50,000 cells) of the saturable oleate uptake component increased 3.5-fold in pFR400/pMAAT2 cells compared to pFR400, with a further 3.2-fold increase in the presence of Zn2+. Zn2+ had no effect in pFR400 controls (P > 0.5). The overall increase in Vmax between pFR400 and pFR400/pMAAT2 in the presence of Zn2+ was 10.4-fold (P < 0.01) and was highly correlated (r = 0.99) with expression of FABPpm in plasma membranes as determined by Western blotting. Neither untransfected 3T3 nor pFR400 cells expressed cell surface FABPpm detectable by immunofluorescence. By contrast, plasma membrane immunofluorescence was detected in pFR400/pMAAT2 cells, especially if cultured in 100 microM Zn2+. The data support the dual hypotheses that mAspAT and FABPpm are identical and mediate saturable long-chain free fatty acid uptake. Images Fig. 1 Fig. 2 Fig. 4 Fig. 5 PMID:7568234

  5. 13-Methylberberine, a berberine analogue with stronger anti-adipogenic effects on mouse 3T3-L1 cells

    PubMed Central

    Chow, Yit-Lai; Sogame, Mami; Sato, Fumihiko

    2016-01-01

    Lipid metabolism modulation is a main focus of metabolic syndrome research, an area in which many natural and synthetic chemicals are constantly being screened for in vitro and in vivo activity. Berberine, a benzylisoquinoline plant alkaloid, has been extensively investigated for its anti-obesity effects and as a potential cholesterol and triglyceride-lowering drug. We screened 11 protoberberine and 2 benzophenanthridine alkaloids for their anti-adipogenic effects on 3T3-L1 adipocytes and found that 13-methylberberine exhibited the most potent activity. 13-Methylberberine down-regulated the expression of the main adipocyte differentiation transcription factors, peroxisome proliferator-activated receptor gamma (PPARγ) and CCAAT enhancer binding protein alpha (C/EBPα), as well as their target genes. PPARγ, C/EBPα, and sterol regulatory element binding protein 1 (SREBP-1) protein levels were reduced, and this lipid-reducing effect was attenuated by an AMP-activated protein kinase (AMPK) inhibitor, indicating that the effect of this compound requires the AMPK signaling pathway. Decreased Akt phosphorylation suggested reduced de novo lipid synthesis. C-13 methyl substitution of berberine increased its accumulation in treated cells, suggesting that 13-methylberberine has improved absorption and higher accumulation compared to berberine. Our findings suggest that 13-methylberberine has potential as an anti-obesity drug. PMID:27917887

  6. 13-Methylberberine, a berberine analogue with stronger anti-adipogenic effects on mouse 3T3-L1 cells.

    PubMed

    Chow, Yit-Lai; Sogame, Mami; Sato, Fumihiko

    2016-12-05

    Lipid metabolism modulation is a main focus of metabolic syndrome research, an area in which many natural and synthetic chemicals are constantly being screened for in vitro and in vivo activity. Berberine, a benzylisoquinoline plant alkaloid, has been extensively investigated for its anti-obesity effects and as a potential cholesterol and triglyceride-lowering drug. We screened 11 protoberberine and 2 benzophenanthridine alkaloids for their anti-adipogenic effects on 3T3-L1 adipocytes and found that 13-methylberberine exhibited the most potent activity. 13-Methylberberine down-regulated the expression of the main adipocyte differentiation transcription factors, peroxisome proliferator-activated receptor gamma (PPARγ) and CCAAT enhancer binding protein alpha (C/EBPα), as well as their target genes. PPARγ, C/EBPα, and sterol regulatory element binding protein 1 (SREBP-1) protein levels were reduced, and this lipid-reducing effect was attenuated by an AMP-activated protein kinase (AMPK) inhibitor, indicating that the effect of this compound requires the AMPK signaling pathway. Decreased Akt phosphorylation suggested reduced de novo lipid synthesis. C-13 methyl substitution of berberine increased its accumulation in treated cells, suggesting that 13-methylberberine has improved absorption and higher accumulation compared to berberine. Our findings suggest that 13-methylberberine has potential as an anti-obesity drug.

  7. Dissociation of bradykinin-induced prostaglandin formation from phosphatidylinositol turnover in Swiss 3T3 fibroblasts: evidence for G protein regulation of phospholipase A/sub 2/

    SciTech Connect

    Burch, R.M.; Axelrod, J.

    1987-09-01

    In Swiss 3T3 fibroblasts bradykinin stimulated inositol phosphate (InsP) formation and prostaglandin E/sub 2/ (PGE/sub 2/) synthesis. The EC/sub 50/ values for stimulation of PGE/sub 2/ synthesis and InsP formation by bradykinin were similar, 200 pM and 275 pM, respectively. Guanosine-5'-(..gamma..-thio)triphosphate stimulated PGE/sub 2/ synthesis and InsP formation, and guanosine-5'-(..beta..-thio)diphosphate inhibited both PGE/sub 2/ synthesis and InsP formation stimulated by bradykinin. Neither bradykinin-stimulated PGE/sub 2/ synthesis nor InsP formation was sensitive to pertussis toxin. Phorbol ester, dexamethasone, and cycloheximide distinguished between bradykinin-stimulated PGE/sub 2/ synthesis and InsP formation. Phorbol 12-myristate 13-acetate enhanced bradykinin-stimulated PGE/sub 2/ synthesis but inhibited bradykinin-stimulated InsP formation. Pretreatment of cells with dexamethasone for 24 hr inhibited bradykinin-stimulated PGE/sub 2/ synthesis but was without effect on bradykinin-stimulated InsP formation. Cycloheximide inhibited on bradykinin-stimulated InsP formation. When bradykinin was added to cells prelabeled with (/sup 3/H) choline, the phospholipase A/sub 2/ products lysophosphatidylcholine and glycerophosphocholine were generated. The data suggest that bradykinin receptors are coupled by GTP-binding proteins to both phospholipase C and phospholipase A/sub 2/ and that phospholipase A/sub 2/ is the enzyme that catalyzes release of arachidonate for prostaglandin synthesis.

  8. Phenotypic and genotypic characteristics of novel mouse cell line (NIH/3T3)-adapted human enterovirus 71 strains (EV71:TLLm and EV71:TLLmv).

    PubMed

    Victorio, Carla Bianca Luena; Xu, Yishi; Ng, Qimei; Chow, Vincent T K; Chua, Kaw Bing

    2014-01-01

    Since its identification in 1969, Enterovirus 71 (EV71) has been causing periodic outbreaks of infection in children worldwide and most prominently in the Asia-Pacific Region. Understanding the pathogenesis of Enterovirus 71 (EV71) is hampered by the virus's inability to infect small animals and replicate in their derived in vitro cultured cells. This manuscript describes the phenotypic and genotypic characteristics of two selected EV71 strains (EV71:TLLm and EV71:TLLmv), which have been adapted to replicate in mouse-derived NIH/3T3 cells, in contrast to the original parental virus which is only able to replicate in primate cell lines. The EV71:TLLm strain exhibited productive infection in all primate and rodent cell lines tested, while EV71:TLLmv exhibited greater preference for mouse cell lines. EV71:TLLmv displayed higher degree of adaptation and temperature adaptability in NIH/3T3 cells than in Vero cells, suggesting much higher fitness in NIH/3T3 cells. In comparison with the parental EV71:BS strain, the adapted strains accumulated multiple adaptive mutations in the genome resulting in amino acid substitutions, most notably in the capsid-encoding region (P1) and viral RNA-dependent RNA polymerase (3D). Two mutations, E167D and L169F, were mapped to the VP1 canyon that binds the SCARB2 receptor on host cells. Another two mutations, S135T and K140I, were located in the VP2 neutralization epitope spanning amino acids 136-150. This is the first report of human EV71 with the ability to productively infect rodent cell lines in vitro.

  9. Effect of surface topography and bioactive properties on early adhesion and growth behavior of mouse preosteoblast MC3T3-E1 cells.

    PubMed

    Li, Na; Chen, Gang; Liu, Jue; Xia, Yang; Chen, Hanbang; Tang, Hui; Zhang, Feimin; Gu, Ning

    2014-10-08

    The effects of bioactive properties and surface topography of biomaterials on the adhesion and spreading properties of mouse preosteoblast MC3T3-E1 cells was investigated by preparation of different surfaces. Poly lactic-co-glycolic acid (PLGA) electrospun fibers (ES) were produced as a porous rough surface. In our study, coverslips were used as a substrate for the immobilization of 3,4-dihydroxyphenylalanine (DOPA) and collagen type I (COL I) in the preparation of bioactive surfaces. In addition, COL I was immobilized onto porous electrospun fibers surfaces (E-COL) to investigate the combined effects of bioactive molecules and topography. Untreated coverslips were used as controls. Early adhesion and growth behavior of MC3T3-E1 cells cultured on the different surfaces were studied at 6, 12, and 24 h. Evaluation of cell adhesion and morphological changes showed that the all the surfaces were favorable for promoting the adhesion and spreading of cells. CCK-8 assays and flow cytometry revealed that both topography and bioactive properties were favorable for cell growth. Analysis of β1, α1, α2, α5, α10 and α11 integrin expression levels by immunofluorescence, real-time RT-PCR, and Western blot and indicated that surface topography plays an important role in the early stage of cell adhesion. However, the influence of topography and bioactive properties of surfaces on integrins is variable. Compared with any of the topographic or bioactive properties in isolation, the combined effect of both types of properties provided an advantage for the growth and spreading of MC3T3-E1 cells. This study provides a new insight into the functions and effects of topographic and bioactive modifications of surfaces at the interface between cells and biomaterials for tissue engineering.

  10. Ca/sup 2 +/-mobilizing actions of platelet-derived growth factor differ from those of bombesin and vasopressin in Swiss 3T3 mouse cells

    SciTech Connect

    Lopez-Rivas, A.; Mendoza, S.A.; Nanberg, E.; Sinnett-Smith, J.; Rozengurt, E.

    1987-08-01

    Addition of the mitogenic peptides bombesin and vasopressin to quiescent Swiss 3T3 mouse cells increased the cytosolic Ca/sup 2 +/ concentration without any measurable delay. In contrast, there was a significant lag period (16 +/- 1.2 s) before platelet-derived growth factor (PDGF) increased cytosolic Ca/sup 2 +/ concentration. This lag was not diminished at high concentrations of either porcine or human PDGF. Similar results were obtained in 3T3 cells loaded with quin-2 or fura-2. The differences in the effects of bombesin, vasopressin, and PDGF on Ca/sup 2 +/ movements were also substantiated by measurements of /sup 45/Ca/sup 2 +/ efflux and of cellular /sup 45/Ca/sup 2 +/ content. Activation of protein kinase C by phorbol esters inhibited Ca/sup 2 +/ mobilization induced by either bombesin or vasopressin. In contrast, phorbol esters had no effect on PDGF-induced cytosolic Ca/sup 2 +/ concentration increase or acceleration of /sup 45/Ca/sup 2 +/ efflux. Finally, bombesin and vasopressin caused a rapid increase in the production of inositol 1,4,5-trisphosphate and inositol 1,3,4-trisphosphate, whereas PDGF, even at a saturating concentration, exerted only a small effect. These results indicate that the signal transduction pathway activated by PDGF that lead to Ca/sup 2 +/ mobilization can be distinguished form those utilized by bombesin and vasopressin.

  11. KGF and BMP-6 intervene in cellular reprogramming and in mesenchymal-epithelial transition (MET) of 3T3L1 mouse adipose cells.

    PubMed

    Reza, Abu M M T; Lee, Sungjin; Shiwani, Supriya; Singh, Naresh K

    2015-04-01

    Mesenchymal-epithelial transition (MET) is an inevitable process for cellular reprogramming. MET could be induced by suppressing epithelial-mesenchymal transition (EMT) signaling and activating an epithelial program within the cells. Aiming at MET, we investigated the potential of keratinocyte growth factor (KGF) and bone morphogenetic protein (BMP)-6 separately for the induction of MET in 3T3L1 mouse adipose cells and to trace the molecular events that probably upregulate during MET induction. KGF successfully induced MET through upregulation of epithelial related genes and transcript expression on 3T3L1 cells. In contrast, BMP-6 plays completely the reverse role through downregulation of all epithelial related genes and transcript expression. In KGF based treatment, seven genes (K8, K18, EpCAM, K5, K14, SMN1 and α-SMA) out of a total of eight genes were significantly (P < 0.05/P < 0.01) upregulated. Immunostaining and immunoblotting also revealed significant (P < 0.05/P < 0.01) expression of several epithelial-specific surface antigens and transcripts. Moreover, Ayoub Shaklar staining (specific to keratin) of KGF treated cells showed formation of keratin (reddish brown color) within cytoplasm of the cells, whereas control and BMP-6 treated cells did not. Conclusively, KGF was observed to have the potential to enhance MET and these clues could be used in future research into cellular reprogramming and regenerative medicine.

  12. Stable release of BDNF from the fibroblast cell line NIH3T3 grown on silicone elastomers enhances survival of spiral ganglion cells in vitro and in vivo.

    PubMed

    Warnecke, Athanasia; Sasse, Susanne; Wenzel, Gentiana I; Hoffmann, Andrea; Gross, Gerhard; Paasche, Gerrit; Scheper, Verena; Reich, Uta; Esser, Karl-Heinz; Lenarz, Thomas; Stöver, Timo; Wissel, Kirsten

    2012-07-01

    The treatment of choice for profound sensorineural hearing loss (SNHL) is direct electrical stimulation of spiral ganglion cells (SGC) via a cochlear implant (CI). The number and excitability of SGC seem to be critical for the success that can be achieved via CI treatment. However, SNHL is associated with degeneration of SGC. Long-term drug delivery to the inner ear for improving SGC survival may be achieved by functionalisation of CI electrodes with cells providing growth factors. Therefore, the capacity of brain-derived neurotrophic factor (BDNF)-secreting NIH3T3 cells grown on cylindrically shaped silicone elastomers (SE) to exert local and sustained neuroprotective effects was assessed in vitro and in vivo. An in vitro model to investigate adhesion and cell growth of lentivirally modified NIH3T3 cells synthesising BDNF on SE was established. The bioactivity of BDNF was characterised by co-cultivation of SGC with cell-coated SE. In addition, cell-coated SE were implanted into deafened guinea pigs. The recombinant NIH3T3 cells proliferated on silicone surfaces during 14 days of cultivation and expressed significantly increasing BDNF levels. Enhanced survival rates and neurite outgrowth of SGC demonstrated the bioactivity of BDNF in vitro. Implantation of SE with adhering BDNF-secreting NIH3T3 cells into the cochleae of systemically deafened guinea pigs induced a significant increase in SGC survival in comparison to SE without cell coating. Our data demonstrate a novel approach of cell-based long-term drug delivery to support SGC survival in vitro and in vivo. This therapeutic strategy--once transferred to cells suitable for clinical application--may improve CI performance.

  13. Ethanol extracts of chickpeas alter the total lipid content and expression levels of genes related to fatty acid metabolism in mouse 3T3-L1 adipocytes.

    PubMed

    Shinohara, Shigeo; Gu, Yuanjun; Yang, Ying; Furuta, Yasuo; Tanaka, Masahiko; Yue, Xiaohua; Wang, Weiqing; Kitano, Masaru; Kimura, Hiroshi

    2016-08-01

    Desi-type chickpeas, which have long been used as a natural treatment for diabetes, have been reported to lower visceral adiposity, dyslipidemia and insulin resistance induced by a chronic high-fat diet in rats. In this study, in order to examine the effects of chickpeas of this type in an in vitro system, we used the 3T3-L1 mouse cell line, a subclone of Swiss 3T3 cells, which can differentiate into cells with an adipocyte-like phenotype, and we used ethanol extracts of chickpeas (ECP) instead of chickpeas. Treatment of the 3T3-L1 cells with ECP led to a decrease in the lipid content in the cells. The desaturation index, defined as monounsaturated fatty acids (MUFAs)/saturated fatty acids (SFAs), was also decreased by ECP due to an increase in the cellular content of SFAs and a decrease in the content of MUFAs. The decrease in this index may reflect a decreased reaction from SFA to MUFA, which is essential for fat storage. To confirm this hypothesis, we conducted a western blot analysis, which revealed a reduction in the amount of stearoyl-CoA desaturase 1 (SCD1), a key enzyme catalyzing the reaction from SFA to MUFA. We observed simultaneous inactivations of enzymes participating in lipogenesis, i.e., liver kinase B1 (LKB1), acetyl-CoA carboxylase (ACC) and AMPK, by phosphorylation, which may lead to the suppression of reactions from acetyl-CoA to SFA via malonyl-CoA in lipogenesis. We also investigated whether lipolysis is affected by ECP. The amount of carnitine palmitoyltransferase 1 (CPT1), an enzyme important for the oxidation of fatty acids, was increased by ECP treatment. ECP also led to an increase in uncoupling protein 2 (UCP2), reported as a key protein for the oxidation of fatty acids. All of these results obtained regarding lipogenesis and fatty acid metabolism in our in vitro system are consistent with the results previously shown in rats. We also examined the effects on SCD1 and lipid contents of ethanol extracts of Kabuli

  14. Ethanol extracts of chickpeas alter the total lipid content and expression levels of genes related to fatty acid metabolism in mouse 3T3-L1 adipocytes

    PubMed Central

    Shinohara, Shigeo; Gu, Yuanjun; Yang, Ying; Furuta, Yasuo; Tanaka, Masahiko; Yue, Xiaohua; Wang, Weiqing; Kitano, Masaru; Kimura, Hiroshi

    2016-01-01

    Desi-type chickpeas, which have long been used as a natural treatment for diabetes, have been reported to lower visceral adiposity, dyslipidemia and insulin resistance induced by a chronic high-fat diet in rats. In this study, in order to examine the effects of chickpeas of this type in an in vitro system, we used the 3T3-L1 mouse cell line, a subclone of Swiss 3T3 cells, which can differentiate into cells with an adipocyte-like phenotype, and we used ethanol extracts of chickpeas (ECP) instead of chickpeas. Treatment of the 3T3-L1 cells with ECP led to a decrease in the lipid content in the cells. The desaturation index, defined as monounsaturated fatty acids (MUFAs)/saturated fatty acids (SFAs), was also decreased by ECP due to an increase in the cellular content of SFAs and a decrease in the content of MUFAs. The decrease in this index may reflect a decreased reaction from SFA to MUFA, which is essential for fat storage. To confirm this hypothesis, we conducted a western blot analysis, which revealed a reduction in the amount of stearoyl-CoA desaturase 1 (SCD1), a key enzyme catalyzing the reaction from SFA to MUFA. We observed simultaneous inactivations of enzymes participating in lipogenesis, i.e., liver kinase B1 (LKB1), acetyl-CoA carboxylase (ACC) and AMPK, by phosphorylation, which may lead to the suppression of reactions from acetyl-CoA to SFA via malonyl-CoA in lipogenesis. We also investigated whether lipolysis is affected by ECP. The amount of carnitine palmitoyltransferase 1 (CPT1), an enzyme important for the oxidation of fatty acids, was increased by ECP treatment. ECP also led to an increase in uncoupling protein 2 (UCP2), reported as a key protein for the oxidation of fatty acids. All of these results obtained regarding lipogenesis and fatty acid metabolism in our in vitro system are consistent with the results previously shown in rats. We also examined the effects on SCD1 and lipid contents of ethanol extracts of Kabuli-type chickpeas, which are

  15. Factors influencing the expression of endogenous reverse transcriptases and viral-like 30 elements in mouse NIH3T3 cells.

    PubMed

    Tzavaras, Theodore; Eftaxia, Sofia; Tavoulari, Sotiria; Hatzi, Paraskevi; Angelidis, Charalambos

    2003-10-01

    Retroviral reverse transcriptase (RT) plays a definite role in retroviral life cycle and is essential for the process of retrotransposition. We investigated the RNA expression of endogenous reverse transcriptases (enRTs) in the NIH3T3 mouse genome using, as a probe, a mixture of RT-PCR generated reverse transcriptase products potentially detecting a large number of RTs following treatment with different agents. We found that the expression of enRTs is induced approximately 500-fold following 5'-azacytidine-treatment. Amongst steroid hormones used such as estradiol, diethylstilbestrol, progesterone and dexamethasone only the latter was effective in inducing enRTs up to 4-fold at a concentration of 10(-7) M. Expression of a mouse dominant-negative form of p53 protein in cell clones resulted in induction of 20- to 50-fold, whereas C2-ceramide in a 4-fold induction at concentrations of 20-80 micro M. In a parallel analysis, the respective expression of the transposable viral-like 30 elements (VL30s) was also measured. Their expression was induced up to 50-fold by 5'-azacytidine, overexpression of the p53 gene and C2-ceramide at 80 micro M. It was also induced approximately 3- to 5-fold following estradiol, diethylstilbestrol or progesterone treatment and 30-fold by dexamethasone. Collectively, our results suggest that such stimuli inducing enRTs might play a role in the activation of transcription and retrotransposition of VL30.

  16. Expression of discoidin domain receptor 2 (DDR2) extracellular domain in pichia pastoris and functional analysis in synovial fibroblasts and NIT3T3 cells.

    PubMed

    Zhang, Wei; Ding, Tianbing; Zhang, Jian; Su, Jin; Li, Fuyang; Liu, Xinping; Ma, Wenyu; Yao, Libo

    2006-10-01

    Discoidin domain receptor 2 (DDR2) is a kind of protein tyrosine kinases associated with cell proliferation and tumor metastasis, and collagen, identified as a ligand for DDR2, up-regulates matrix metallloproteinase 1 (MMP-1) and MMP-2 expression in cellular matrix. To investigate the roles of DDR2 in destruction of cartilage in rheumatoid arthritis (RA) and tumor metastasis, we tried to express extracellular domain of DDR2 fused with a His tag to increase protein solubility and facilitate purification (without signal peptide and transmembrane domain, designated DR) in Pichia pastoris, purify the expressed protein, and characterize its function, for purpose of future application as a specific DDR2 antagonist. Two clones of relative high expression of His-DR were obtained. After purification by a Ni-NTA (nitric-tri-acetic acid) chromatographic column, soluble fused His-DR over 90% purity were obtained. Competitive binding inhibition assay demonstrated that expressed His-DR could block the binding of DDR2 and natural DDR2 receptors on NIT3T3 and synovial cell surfaces. Results of RT-PCR, Western blotting, and gelatinase zymography showed that His-DR was capable of inhibiting MMP-1 and MMP-2 secretion from NIT3T3 cells and RA synoviocytes stimulated by collagen II. For MMP-1, the inhibitory effect was displayed at the levels of mRNA and protein, whereas for MMP-2 it was demonstrated at the level of protein physiological activity. All these findings suggested that the fused expressed His-DR inhibited the activity of natural DDR2, and relevant MMP-1 and MMP-2 expression in synoviocytes and NIH3T3 cells provoked by collagen II.

  17. Enhancing effect of daidzein on the differentiation and mineralization in mouse osteoblast-like MC3T3-E1 cells.

    PubMed

    Ge, Yuebin; Chen, Dawei; Xie, Liping; Zhang, Rongqing

    2006-08-01

    The effect of daidzein, an important isoflavone, on the differentiation and mineralization in MC3T3-E1 cells, a mouse calvaria osteoblast-like cell line, was investigated. The MTT assay, the alizarin red S and von Kossa staining, the measurement of calcium (Ca) and phosphorus (P) concentrations by inductively coupled plasma-atomic emission spectrometry and the nitrophenol liberation method were used to determine the cell proliferation, mineralization and intracellular alkaline phosphatase (ALP) activity, respectively. Daidzein enhanced the cell proliferation after the culture for 2 days and the effect reached maximum on day 6. ALP activity and cellular Ca and P contents were increased time- and dose-dependently when the cells were treated with daidzein in the presence of disodium beta-glycerophosphate and L-ascorbic acid. Differentiation of the cells to the mature osteoblasts was prompted under incubation in the presence of daidzein for 21 days, by the time the mineralized nodules formed. The calcium depositions of the cells by alizarin red S staining were increased significantly after the culture with daidzein as long as 28 days. It has been demonstrated that daidzein may be able to enhance the bone differentiation and mineralization and prompt the bone formation in the early growing stage and the late growing stage of osteoblasts.

  18. Widening the mutation spectrum of EVC and EVC2: ectopic expression of Weyer variants in NIH 3T3 fibroblasts disrupts Hedgehog signaling.

    PubMed

    Valencia, Maria; Lapunzina, Pablo; Lim, Derek; Zannolli, Raffaella; Bartholdi, Deborah; Wollnik, Bernd; Al-Ajlouni, Othman; Eid, Suhair S; Cox, Helen; Buoni, Sabrina; Hayek, Joseph; Martinez-Frias, Maria L; Antonio, Perez-Aytes; Temtamy, Samia; Aglan, Mona; Goodship, Judith A; Ruiz-Perez, Victor L

    2009-12-01

    Autosomal recessive Ellis-van Creveld syndrome and autosomal dominant Weyer acrodental dysostosis are allelic conditions caused by mutations in EVC or EVC2. We performed a mutation screening study in 36 EvC cases and 3 cases of Weyer acrodental dysostosis, and identified pathogenic changes either in EVC or in EVC2 in all cases. We detected 40 independent EVC/EVC2 mutations of which 29 were novel changes in Ellis-van Creveld cases and 2 were novel mutations identified in Weyer pedigrees. Of interest one EvC patient had a T>G nucleotide substitution in intron 7 of EVC (c.940-150T>G), which creates a new donor splice site and results in the inclusion of a new exon. The T>G substitution is at nucleotide +5 of the novel 5' splice site. The three Weyer mutations occurred in the final exon of EVC2 (exon 22), suggesting that specific residues encoded by this exon are a key part of the protein. Using murine versions of EVC2 exon 22 mutations we demonstrate that the expression of a Weyer variant, but not the expression of a truncated protein that mimics an Ellis-van Creveld syndrome mutation, impairs Hedgehog signal transduction in NIH 3T3 cells in keeping with its dominant effect.

  19. Molecularly Characterized Solvent Extracts and Saponins from Polygonum hydropiper L. Show High Anti-Angiogenic, Anti-Tumor, Brine Shrimp, and Fibroblast NIH/3T3 Cell Line Cytotoxicity

    PubMed Central

    Ayaz, Muhammad; Junaid, Muhammad; Ullah, Farhat; Sadiq, Abdul; Subhan, Fazal; Khan, Mir Azam; Ahmad, Waqar; Ali, Gowhar; Imran, Muhammad; Ahmad, Sajjad

    2016-01-01

    Polygonum hydropiper is used as anti-cancer and anti-rheumatic agent in folk medicine. This study was designed to investigate the anti-angiogenic, anti-tumor, and cytotoxic potentials of different solvent extracts and isolated saponins. Samples were analyzed using GC, Gas Chromatography–Mass Spectrometry (GC–MS) to identify major and bioactive compounds. Quantitation of antiangiogenesis for the plant's samples including methanolic extract (Ph.Cr), its subsequent fractions; n-hexane (Ph.Hex), chloroform (Ph.Chf), ethyl acetate (Ph.EtAc), n-Butanol (Ph.Bt), aqueous (Ph.Aq), saponins (Ph.Sp) were performed using the chick embryo chorioallantoic membrane (CAM) assay. Potato disc anti-tumor assay was performed on Agrobacterium tumefaciens containing tumor inducing plasmid. Cytotoxicity was performed against Artemia salina and mouse embryonic fibroblast NIH/3T3 cell line following contact toxicity and MTT cells viability assays, respectively. The GC–MS analysis of Ph.Cr, Ph.Hex, Ph.Chf, Ph.Bt, and Ph.EtAc identified 126, 124, 153, 131, and 164 compounds, respectively. In anti-angiogenic assay, Ph.Chf, Ph.Sp, Ph.EtAc, and Ph.Cr exhibited highest activity with IC50 of 28.65, 19.21, 88.75, and 461.53 μg/ml, respectively. In anti-tumor assay, Ph.Sp, Ph.Chf, Ph.EtAc, and Ph.Cr were most potent with IC50 of 18.39, 73.81, 217.19, and 342.53 μg/ml, respectively. In MTT cells viability assay, Ph.Chf, Ph.EtAc, Ph.Sp were most active causing 79.00, 72.50, and 71.50% cytotoxicity, respectively, at 1000 μg/ml with the LD50 of 140, 160, and 175 μg/ml, respectively. In overall study, Ph.Chf and Ph.Sp have shown overwhelming results which signifies their potentials as sources of therapeutic agents against cancer. PMID:27065865

  20. Chinese herbal medicine Yi-Gan-San decreases the lipid accumulation in mouse 3T3-L1 adipocytes by modulating the activities of transcription factors SREBP-1c and FoxO1.

    PubMed

    Izumi, Masayuki; Seki, Takashi; Iwasaki, Koh; Sakamoto, Kazuichi

    2009-09-01

    Abnormal lipid metabolism in adipose tissue is closely related to the occurrence and progression of a wide variety of metabolic syndromes. We have analyzed the pharmacological effects of Chinese herbal medicines on cell differentiation and lipid metabolism in adipocytes. Yi-Gan-San (YGS) is a Chinese herbal medicine that is effective in treating the behavioral and psychological symptoms of dementia; however, its physiological mechanism remains unclear. We analyzed the effects of YGS on lipid accumulation in mouse 3T3-L1 adipocytes. Adipocyte differentiation was induced in mouse 3T3-L1 preadipocytes by treatment with the mixture of dexamethasone, 3-iso-butyl-1-methylxanthine, and insulin, and cells were cultured for 8 days with Chinese herbal medicines, including YGS. YGS effectively reduced the lipid accumulation in the differentiated 3T3-L1 cells in a dose-dependent manner, but had no effect on cell viability. YGS also reduced the activity of glycerol-3-phosphate dehydrogenase, an enzyme involved in lipid synthesis. In contrast, YGS gave no noticeable effect on glucose uptake and fatty acid uptake in the differentiated 3T3-L1 cells. Moreover, we established the stably transfected 3T3-L1 cell lines, each of which expresses the luciferase reporter gene under the control of sterol regulatory element-binding protein-1c (SREBP-1c) or FoxO1. SREBP-1c is a transcription factor involved in fatty acid synthesis, and FoxO1 is a forkhead-type transcription factor involved in adipocyte differentiation. Using these cell lines, we showed that YGS reduced the transcriptional activity of SREBP-1c, whereas YGS increased the activity of FoxO1. Thus, YGS may suppress lipid synthesis and fat accumulation in adipocytes through modulating the activities of SREBP-1c and FoxO1.

  1. T24 HRAS transformed NIH/3T3 mouse cells (GhrasT-NIH/3T3) in serial tumorigenic in vitro/in vivo passages give rise to increasingly aggressive tumorigenic cell lines T1-A and T2-A and metastatic cell lines T3-HA and T4-PA.

    PubMed

    Ray, Durwood B; Merrill, Gerald A; Brenner, Frederic J; Lytle, Laurie S; Lam, Tan; McElhinney, Aaron; Anders, Joel; Rock, Tara Tauber; Lyker, Jennifer Kier; Barcus, Scott; Leslie, Kara Hust; Kramer, Jill M; Rubenstein, Eric M; Pryor Schanz, Karen; Parkhurst, Amy J; Peck, Michelle; Good, Kimberly; Granath, Kristi Lemke; Cifra, Nicole; Detweiler, Jessalee Wantz; Stevens, Laura; Albertson, Richard; Deir, Rachael; Stewart, Elisabeth; Wingard, Katherine; Richardson, Micah Rose; Blizard, Sarah B; Gillespie, Lauren E; Kriley, Charles E; Rzewnicki, Daniel I; Jones, David H

    2016-01-01

    Cancer cells often arise progressively from "normal" to "pre-cancer" to "transformed" to "local metastasis" to "metastatic disease" to "aggressive metastatic disease". Recent whole genome sequencing (WGS) and spectral karyotyping (SKY) of cancer cells and tumorigenic models have shown this progression involves three major types of genome rearrangements: ordered small step-wise changes, more dramatic "punctuated evolution" (chromoplexy), and large catastrophic steps (chromothripsis) which all occur in random combinations to generate near infinite numbers of stochastically rearranged metastatic cancer cell genomes. This paper describes a series of mouse cell lines developed sequentially to mimic this type of progression. This starts with the new GhrasT-NIH/Swiss cell line that was produced from the NIH/3T3 cell line that had been transformed by transfection with HRAS oncogene DNA from the T24 human bladder carcinoma. These GhrasT-NIH/Swiss cells were injected s.c. into NIH/Swiss mice to produce primary tumors from which one was used to establish the T1-A cell line. T1-A cells injected i.v. into the tail vein of a NIH/Swiss mouse produced a local metastatic tumor near the base of the tail from which the T2-A cell line was established. T2-A cells injected i.v. into the tail vein of a nude NIH/Swiss mouse produced metastases in the liver and one lung from which the T3-HA (H=hepatic) and T3-PA (P=pulmonary) cell lines were developed, respectively. T3-HA cells injected i.v. into a nude mouse produced a metastasis in the lung from which the T4-PA cell line was established. PCR analysis indicated the human T24 HRAS oncogene was carried along with each in vitro/in vivo transfer step and found in the T2-A and T4-PA cell lines. Light photomicrographs indicate that all transformed cells are morphologically similar. GhrasT-NIH/Swiss cells injected s.c. produced tumors in 4% of NIH/Swiss mice in 6-10 weeks; T1-A cells injected s.c. produced tumors in 100% of NIH/Swiss mice in 7

  2. Differential regulation of S6 phosphorylation by insulin and epidermal growth factor in Swiss mouse 3T3 cells: insulin activation of type 1 phosphatase.

    PubMed Central

    Olivier, A R; Ballou, L M; Thomas, G

    1988-01-01

    Insulin and epidermal growth factor (EGF) induce distinct kinetics of S6 kinase activation and S6 phosphorylation in Swiss 3T3 cells. Both events are differentially regulated by specific phosphatases. The major S6 phosphatase in cell extracts was identified as a type 1 enzyme by its chromatographic properties, its sensitivity to inhibitor 2, and its substrate specificity. This enzyme is different from the major S6 kinase phosphatase, which is a type 2A enzyme. Insulin at physiological concentrations causes up to a 2-fold activation of a type 1 S6 phosphatase, whereas at higher concentrations this effect is significantly diminished. EGF alone has little effect on this enzyme, and with both agents together the total phosphatase activity remains basal. The results are consistent with the phosphorylation state of S6 observed in vivo and suggest a role of phosphatase type 1 in the regulation of protein synthesis. Images PMID:2838844

  3. Vasoactive intestinal peptide synergistically stimulates DNA synthesis in mouse 3T3 cells: Role of cAMP, Ca sup 2+ , and protein kinase C

    SciTech Connect

    Zurier, B.B.; Kozma, M.; Sinnett-Smith, J.; Rozengurt, E. )

    1988-05-01

    Vasoactive intestinal peptide synergistically stimulated initiation of DNA synthesis in Swiss 3T3 cells. The peptide stimulated ({sup 3}H)thymidine incorporation in the presence of insulin and either forskolin or an inhibitor of cAMP phosphodiesterase in a concentration-dependent manner. Half-maximal effect was obtained at 1 nM. At mitogenic concentrations, VIP stimulated a marked accumulation (eightfold) of cAMP. In contrast to other growth-promoting neuropeptides, VIP did not induce an increase in cytosolic free Ca{sup 2+} or the activation of protein kinase C. The authors conclude that neuropeptides can modulate long-term cell proliferation through multiple signaling pathways.

  4. Expression of Caveolin-1 reduces cellular responses to TGF-{beta}1 through down-regulating the expression of TGF-{beta} type II receptor gene in NIH3T3 fibroblast cells

    SciTech Connect

    Lee, Eun Kyung; Lee, Youn Sook; Han, In-Oc; Park, Seok Hee . E-mail: parks@skku.edu

    2007-07-27

    Transcriptional repression of Transforming Growth Factor-{beta} type II receptor (T{beta}RII) gene has been proposed to be one of the major mechanisms leading to TGF-{beta} resistance. In this study, we demonstrate that expression of Caveolin-1 (Cav-1) gene in NIH3T3 fibroblast cells down-regulates the expression of T{beta}RII gene in the transcriptional level, eventually resulting in the decreased responses to TGF-{beta}. The reduced expression of T{beta}RII gene by Cav-1 appeared to be due to the changes of the sequence-specific DNA binding proteins to either Positive Regulatory Element 1 (PRE1) or PRE2 of the T{beta}RII promoter. In addition, Cav-1 expression inhibited TGF-{beta}-mediated cellular proliferation and Plasminogen Activator Inhibitor (PAI)-1 gene expression as well as TGF-{beta}-induced luciferase activity. Furthermore, the inhibition of endogeneous Cav-1 by small interfering RNA increased the expression of T{beta}RII gene. These findings strongly suggest that expression of Cav-1 leads to the decreased cellular responsiveness to TGF-{beta} through down-regulating T{beta}RII gene expression.

  5. Design, synthesis and characterization of novel binary V(V)-Schiff base materials linked with insulin-mimetic vanadium-induced differentiation of 3T3-L1 fibroblasts to adipocytes. Structure-function correlations at the molecular level.

    PubMed

    Halevas, E; Tsave, O; Yavropoulou, M P; Hatzidimitriou, A; Yovos, J G; Psycharis, V; Gabriel, C; Salifoglou, A

    2015-06-01

    Among the various roles of vanadium in the regulation of intracellular signaling, energy metabolism and insulin mimesis, its exogenous activity stands as a contemporary challenge currently under investigation and a goal to pursue as a metallodrug against Diabetes mellitus II. In this regard, the lipogenic activity of vanadium linked to the development of well-defined anti-diabetic vanadodrugs has been investigated through: a) specifically designing and synthesizing Schiff base organic ligands L, bearing a variable number of terminal alcohols, b) a series of well-defined soluble binary V(V)-L compounds synthesized and physicochemically characterized, c) a study of their cytotoxic effect and establishment of adipogenic activity in 3T3-L1 fibroblasts toward mature adipocytes, and d) biomarker examination of a closely-linked molecular target involving or influenced by the specific V(V) forms, cumulatively delineating factors involved in potential pathways linked to V(V)-induced insulin-like activity. Collectively, the results a) project the importance of specific structural features in Schiff ligands bound to V(V), thereby influencing the emergence of its (a)toxicity and for the first time its insulin-like activity in pre-adipocyte differentiation, b) contribute to the discovery of molecular targets influenced by the specific vanadoforms seeking to induce glucose uptake, and c) indicate an interplay of V(V) structural speciation and cell-differentiation biological activity, thereby gaining insight into vanadium's potential as a future metallodrug in Diabetes mellitus.

  6. Capsaicin partially mimics heat in mouse fibroblast cells in vitro.

    PubMed

    Sugimoto, Naotoshi; Katakura, Masanori; Matsuzaki, Kentaro; Nakamura, Hiroyuki; Yachie, Akihiro; Shido, Osamu

    2017-03-01

    Capsaicin activates transient receptor potential vanilloid 1 (TRPV1), a cation channel in the transient receptor potential family, resulting in the transient entry of Ca(2+) and Mg(2+) and a warm sensation. However, the effects of capsaicin on cells have not fully elucidated in fibroblasts. In this study, we investigated whether capsaicin could induce signal transduction in mouse fibroblast cells and compared the effect with that of heat-induced signal transduction. The activation of the mitogen-activated protein kinases (MAPKs) ERK and p38 MAPK, expression levels of heat shock protein 70 (HSP70) and HSP90, actin assembly, and cell proliferation were analyzed in NIH3T3 mouse fibroblast cells. A 15-min stimulation with capsaicin (∼100 μM) phosphorylated ERK and p38 MAPK and induced actin assembly. A 2-day stimulation with capsaicin increased the level of HSP70, but not HSP90, and the 2-day stimulation with capsaicin (∼100 μM) did not affect cell proliferation. A 15-min exposure to moderate heat (39.5 °C) phosphorylated both ERK and p38 MAPK and induced actin assembly to similar degrees as stimulation with capsaicin. A 2-day exposure to moderate heat increased the levels of both HSP70 and HSP90 and prevented cell proliferation. However, the 2-day stimulation with capsaicin (100 μM) failed to prevent heat shock-induced cell death. Thus, our results suggest that the effects of capsaicin on fibroblast cells partially differ from those of heat. Notably, the 2-day stimulation with capsaicin was not sufficient to develop heat tolerance in fibroblast cells.

  7. 3T3 cells in adipocytic conversion.

    PubMed

    O'Shea Alvarez, M S

    1991-01-01

    3T3 are murine cells of an established heteroploid cellular line. Some clones of this cellular line, when cultured under adequate conditions differentiate into adipocytes. During the process of differentiation, the cells undergo a change from the elongated fibroblastic shape to a round or oval form and accumulate small drops of lipids within their cytoplasma. These lipid drops fuse into one large drop which displaces the nucleus towards the periphery, giving the cell the aspect of a mature adipocyte of white adipose tissue. The cells not only change their morphology, but they also present important biochemical changes. They show a simultaneous increase in triglyceride synthesis and activity of lipogenic enzymes. There is also an increase in the response of the activity of various hormones and the de novo synthesis of the receptors to such hormones, as insulin and ACTH. During the process of differentiation important changes occur in the synthesis of various proteins, such as actin, tubulin, and other proteins which also make up the cellular cytoskeleton, forming part of the lipid transportation within the adipose cell. The adipocytic differentiation of 3T3 cells depends on adipogenic serum factors used in the supplementary culture medium. These adipogenic factors seem to play an important role in the development of adipose tissue. There are hormones, chemical agents and serum factors which modulate adipocytic differentiation. The clone must be susceptible to adipocytic differentiation, it must reach a quiescent state and find itself in adipogenic conditions for the 3T3 cells to differentiate into adipocytes. It must also carry out an DNA synthesis which is an expression of the new phenotype. The differentiation of 3T3 cells in terminal. The fact that these cells present an adipocytic conversion under physiologic conditions and with adipogenic hormones which exist in the whole animal has been demonstrated. All of these characteristics show that the 3T3 cells may be

  8. Curcumin represses mouse 3T3-L1 cell adipogenic differentiation via inhibiting miR-17-5p and stimulating the Wnt signalling pathway effector Tcf7l2.

    PubMed

    Tian, Lili; Song, Zhuolun; Shao, Weijuan; Du, William W; Zhao, Lisa R; Zeng, Kejing; Yang, Burton B; Jin, Tianru

    2017-01-19

    Understanding mechanisms underlying adipogenic differentiation may lead to the discovery of novel therapeutic targets for obesity. Wnt signalling pathway activation leads to repressed adipogenic differentiation while certain microRNAs may regulate pre-adipocyte proliferation and differentiation. We show here that in mouse white adipose tissue, miR-17-5p level is elevated after high fat diet consumption. miR-17-5p upregulates adipogenic differentiation, as its over-expression increased while its inhibition repressed 3T3-L1 differentiation. The Tcf7l2 gene encodes a key Wnt signalling pathway effector, and its human homologue TCF7L2 is a highly regarded diabetes risk gene. We found that Tcf7l2 is an miR-17-5p target and confirmed the repressive effect of Tcf7l2 on 3T3-L1 adipogenic differentiation. The natural plant polyphenol compound curcumin possesses the body weight lowering effect. We observed that curcumin attenuated miR-17-5p expression and stimulated Tcf7l2 expression in 3T3-L1 cells. These, along with the elevation of miR-17-5p expression in mouse epididymal fat tissue in response to high fat diet consumption, allowed us to suggest that miR-17-5p is among central switches of adipogenic differentiation. It activates adipogenesis via repressing the Wnt signalling pathway effector Tcf7l2, and its own expression is likely nutritionally regulated in health and disease.

  9. Fisetin Suppresses Lipid Accumulation in Mouse Adipocytic 3T3-L1 Cells by Repressing GLUT4-Mediated Glucose Uptake through Inhibition of mTOR-C/EBPα Signaling.

    PubMed

    Watanabe, Marina; Hisatake, Mitsuhiro; Fujimori, Ko

    2015-05-27

    3,7,3',4'-Tetrahydroxyflavone (fisetin) is a flavonoid found in vegetables and fruits having broad biological activities. Here the effects of fisetin on adipogenesis and its regulatory mechanism in mouse adipocytic 3T3-L1 cells are studied. Fisetin inhibited the accumulation of intracellular lipids and lowered the expression of adipogenic genes such as peroxisome proliferator-activated receptor γ and CCAAT/enhancer-binding protein (C/EBP) α and fatty acid-binding protein 4 (aP2) during adipogenesis. Moreover, the mRNA levels of genes such as acetyl-CoA carboxylase, fatty acid synthase, and stearoyl-CoA desaturase involved in the fatty acid biosynthesis (lipogenesis) were reduced by the treatment with fisetin. The expression level of the glucose transporter 4 (GLUT4) gene was also decreased by fisetin, resulting in down-regulation of glucose uptake. Furthermore, fisetin inhibited the phosphorylation of the mammalian target of rapamycin (mTOR) and that of p70 ribosomal S6 kinase, a target of the mTOR complex, the inhibition of which was followed by a decreased mRNA level of the C/EBPα gene. The results obtained from a chromatin immunoprecipitation assay demonstrated that the ability of C/EBPα to bind to the GLUT4 gene promoter was reduced by the treatment with fisetin, which agreed well with those obtained when 3T3-L1 cells were allowed to differentiate into adipocytes in medium in the presence of rapamycin, an inhibitor for mTOR. These results indicate that fisetin suppressed the accumulation of intracellular lipids by inhibiting GLUT4-mediated glucose uptake through inhibition of the mTOR-C/EBPα signaling in 3T3-L1 cells.

  10. Analysis of propolis from Baccharis dracunculifolia DC. (Compositae) and its effects on mouse fibroblasts.

    PubMed

    de Funari, Cristiano Soleo; de Oliveira Ferro, Vicente; Mathor, Monica Beatriz

    2007-05-04

    This paper confirms Baccharis dracunculifolia DC. (Compositae) as the main botanical source of the propolis from southeastern Brazil (state of São Paulo) investigated to ascertain specific biological activity in relation to mouse NIH-3T3 fibroblasts, skin cells directly involved in the cicatrization processes. Flavonoid and total phenolic compounds were determined by spectrophotometry, and chemical composition by HPLC; the chromatographic profile, characterized largely by flavonoids and aromatic acids, was found to be qualitatively similar to that of Baccharis dracunculifolia DC. The adsorption of phenolic compounds in the propolis to skin powder was also investigated, and 68% of these compounds adsorbed to the skin powder. At concentrations from 0.12 to 7.81 microg/ml, the propolis revealed no statistical significant differences from its control solutions; however, at concentrations of 31.25 microg/ml or more, the propolis was toxic to NIH-3T3 cells. Thus, the propolis from Baccharis dracunculifolia DC. (Compositae) presents an in vitro concentration-dependent toxicity on mouse NIH-3T3 fibroblasts.

  11. Gene expression profile of mouse fibroblasts exposed to a biodegradable iron alloy for stents.

    PubMed

    Purnama, Agung; Hermawan, Hendra; Champetier, Serge; Mantovani, Diego; Couet, Jacques

    2013-11-01

    Iron-based materials could constitute an interesting option for cardiovascular biodegradable stent applications due to their superior ductility compared to their counterparts - magnesium alloys. Since the predicted degradation rate of pure iron is considered slow, manganese (35% w/w), an alloying element for iron, was explored to counteract this problem through the powder metallurgy process (Fe-35 Mn). However, manganese presents a high cytotoxic potential; thus its effect on cells must first be established. Here, we established the gene expression profile of mouse 3T3 fibroblasts exposed to Fe-35 Mn degradation products in order to better understand cell response to potentially cytotoxic degradable metallic material (DMM). Mouse 3T3 cells were exposed to degradation products eluting through tissue culture insert filter (3 μm pore size) containing cytostatic amounts of 3.25 mg ml(-1) of Fe-35 Mn powder, 0.25 mg ml(-1) of pure Mn powder or 5 mg ml(-1) of pure iron powder for 24 h. We then conducted a gene expression profiling study from these cells. Exposure of 3T3 cells to Fe-35 Mn was associated with the up-regulation of 75 genes and down-regulation of 59 genes, while 126 were up-regulated and 76 down-regulated genes in the presence of manganese. No genes were found regulated for the iron powder. When comparing the GEP of 3T3 fibroblasts in the presence of Fe-35 Mn and Mn, 68 up-regulated and 54 down-regulated genes were common. These results were confirmed by quantitative RT-PCR for a subset of these genes. This GEP study could provide clues about the mechanism behind degradation products effects on cells of the Fe-35 Mn alloy and may help in the appraisal of its potential for DMM applications.

  12. Combined Effects of Androgen and Growth Hormone on Osteoblast Marker Expression in Mouse C2C12 and MC3T3-E1 Cells Induced by Bone Morphogenetic Protein

    PubMed Central

    Kimura, Kosuke; Terasaka, Tomohiro; Iwata, Nahoko; Katsuyama, Takayuki; Komatsubara, Motoshi; Nagao, Ryota; Inagaki, Kenichi; Otsuka, Fumio

    2017-01-01

    Osteoblasts undergo differentiation in response to various factors, including growth factors and steroids. Bone mass is diminished in androgen- and/or growth hormone (GH)-deficient patients. However the functional relationship between androgen and GH, and their combined effects on bone metabolism, remains unclear. Here we investigated the mutual effects of androgen and GH on osteoblastic marker expression using mouse myoblastic C2C12 and osteoblast-like MC3T3-E1 cells. Combined treatment with dihydrotestosterone (DHT) and GH enhanced BMP-2-induced expression of Runx2, ALP, and osteocalcin mRNA, compared with the individual treatments in C2C12 cells. Co-treatment with DHT and GH activated Smad1/5/8 phosphorylation, Id-1 transcription, and ALP activity induced by BMP-2 in C2C12 cells but not in MC3T3-E1 cells. The insulin-like growth factor (IGF-I) mRNA level was amplified by GH and BMP-2 treatment and was restored by co-treatment with DHT in C2C12 cells. The mRNA level of the IGF-I receptor was not significantly altered by GH or DHT, while it was increased by IGF-I. In addition, IGF-I treatment increased collagen-1 mRNA expression, whereas blockage of endogenous IGF-I activity using an anti-IGF-I antibody failed to suppress the effect of GH and DHT on BMP-2-induced Runx2 expression in C2C12 cells, suggesting that endogenous IGF-I was not substantially involved in the underlying GH actions. On the other hand, androgen receptor and GH receptor mRNA expression was suppressed by BMP-2 in both cell lines, implying the existence of a feedback action. Collectively the results showed that the combined effects of androgen and GH facilitated BMP-2-induced osteoblast differentiation at an early stage by upregulating BMP receptor signaling. PMID:28067796

  13. Mouse white adipocytes and 3T3-L1 cells display an anomalous pattern of carnitine palmitoyltransferase (CPT) I isoform expression during differentiation. Inter-tissue and inter-species expression of CPT I and CPT II enzymes.

    PubMed Central

    Brown, N F; Hill, J K; Esser, V; Kirkland, J L; Corkey, B E; Foster, D W; McGarry, J D

    1997-01-01

    The outer mitochondrial membrane enzyme carnitine palmitoyltransferase I (CPT I) represents the initial and regulated step in the beta-oxidation of fatty acids. It exists in at least two isoforms, denoted L (liver) and M (muscle) types, with very different kinetic properties and sensitivities to malonyl-CoA. Here we have examined the relative expression of the CPT I isoforms in two different models of adipocyte differentiation and in a number of rat tissues. Adipocytes from mice, hamsters and humans were also evaluated. Primary monolayer cultures of undifferentiated rat preadipocytes expressed solely L-CPT I, but significant levels of M-CPT I emerged after only 3 days of differentiation in vitro; in the mature cell M-CPT I predominated. In sharp contrast, the murine 3T3-L1 preadipocyte expressed essentially exclusively L-CPT I, both in the undifferentiated state and throughout the differentiation process in vitro. This was also true of the mature mouse white fat cell. Fully developed adipocytes from the hamster and human behaved similarly to those of the rat. Thus the mouse white fat cell differs fundamentally from those of the other species examined in terms of tis choice of a key regulatory enzyme in fatty acid metabolism. In contrast, brown adipose tissue from all three rodents displayed the same isoform profiles, each expressing overwhelmingly M-CPT I. Northern blot analysis of other rat tissues established L-CPT I as the dominant isoform not only in liver but also in kidney, lung, ovary, spleen, brain, intestine and pancreatic islets. In addition to its primacy in skeletal muscle, heart and fat, M-CPT I was also found to dominate the testis. The same inter-tissue isoform pattern (with the exception of white fat) was found in the mouse. Taken together, the data bring to light an intriguing divergence between white adipocytes of the mouse and other mammalian species. They also raise a cautionary note that should be considered in the choice of animal model used

  14. A specific protein, p92, detected in flat revertants derived from NIH/3T3 transformed by human activated c-Ha-ras oncogene.

    PubMed

    Fujita, H; Suzuki, H; Kuzumaki, N; Müllauer, L; Ogiso, Y; Oda, A; Ebisawa, K; Sakurai, T; Nonomura, Y; Kijimoto-Ochiai, S

    1990-01-01

    Total proteins from a mouse embryo fibroblast cell line NIH/3T3, NIH/3T3 cells transformed by human activated c-Ha-ras (EJ-ras) oncogene (EJ-NIH/3T3), and the two flat revertant cell lines, R1 and R2, were analyzed by two-dimensional gel electrophoresis (IEF and NEPHGE). Several hundred polypeptides were resolved as seen by silver staining. Common alterations in four polypeptide spots were observed in the revertants when compared with NIH/3T3 and EJ-NIH/3T3 cells. In these alterations, a new polypeptide spot p92-5.7 (designated by molecular weight x 10(-3) and pI) was detected only in the revertants and not in NIH/3T3 and EJ-NIH/3T3 cells. Furthermore, the expression level of p92-5.7 seemed to be associated with the flat morphology and the reduced tumorigenicity of the revertants. Polypeptide p92-5.7 was also not detected in the total proteins extracted from BALB/3T3 cells, NIH Swiss mouse primary embryo fibroblasts, NRK (normal rat kidney) cells, and L6 (rat myoblast). Subcellular fractionation of total protein from R1 cells revealed that the p92-5.7 was present in the cytosol. Western blot analysis using an anti-gelsolin antibody demonstrated that the p92-5.7 might be a variant form of gelsolin which is thought to be an actin regulatory protein or a gelsolin-like polypeptide. These results may suggest that the expression of p92-5.7 detected only in the revertants is associated, at least in part, with the reversion. This may be the first demonstration of specific protein expression in the flat revertants.

  15. Actin dynamics in mouse fibroblasts in microgravity

    NASA Astrophysics Data System (ADS)

    Moes, Maarten J. A.; Bijvelt, Jose J.; Boonstra, Johannes

    2007-09-01

    After stimulating with the growth factor PDGF, cells exhibit abundant membrane ruffling and other morphological changes under normal gravity conditions. These morphological changes are largely determined by the actin microfilament system. Now these actin dynamics were studied under microgravity conditions in mouse fibroblasts during the DELTA mission. The aim of the present study was to describe the actin morphology in detail, to establish the effect of PDGF on actin morphology and to study the role of several actin-interacting proteins involved in introduced actin dynamics in microgravity. Identical experiments were conducted at 1G on earth as a reference. No results in microgravity were obtained due to a combination of malfunctioning hardware and unfulfilled temperature requirements.

  16. Mitomycin C modulates the circadian oscillation of clock gene period 2 expression through attenuating the glucocorticoid signaling in mouse fibroblasts.

    PubMed

    Kusunose, Naoki; Matsunaga, Naoya; Kimoto, Kenichi; Akamine, Takahiro; Hamamura, Kengo; Koyanagi, Satoru; Ohdo, Shigehiro; Kubota, Toshiaki

    2015-11-06

    Clock gene regulates the circadian rhythm of various physiological functions. The expression of clock gene has been shown to be attenuated by certain drugs, resulting in a rhythm disorder. Mitomycin C (MMC) is often used in combination with ophthalmic surgery, especially in trabeculectomy, a glaucoma surgical procedure. The purpose of this study was to investigate the influence of MMC on clock gene expression in fibroblasts, the target cells of MMC. Following MMC treatment, Bmal1 mRNA levels was significantly decreased, whereas Dbp, Per1, and Rev-erbα mRNA levels were significantly increased in the mouse fibroblast cell line NIH3T3 cells. Microarray analysis was performed to explore of the gene(s) responsible for MMC-induced alteration of clock gene expression, and identified Nr3c1 gene encoding glucocorticoid receptor (GR) as a candidate. MMC suppressed the induction of Per1 mRNA by dexamethasone (DEX), ligand of GR, in NIH3T3 cells. MMC also modulated the DEX-driven circadian oscillations of Per2::Luciferase bioluminescence in mouse-derived ocular fibroblasts. Our results demonstrate a previously unknown effect of MMC in GR signaling and the circadian clock system. The present findings suggest that MMC combined with trabeculectomy could increase the risk for a local circadian rhythm-disorder at the ocular surface.

  17. Mouse bone marrow-derived mast cells (BMMC) change their phenotype when cultured with fibroblasts

    SciTech Connect

    Levi-Schaffer, F.; Austen, K.F.; Stevens, R.L.

    1986-03-05

    The heparin-containing mast cells (HP-MC) that reside in the connective tissues of the mouse, but not the chondroitin sulfate containing mast cells in the gastrointestinal mucosa, stain with safranin when exposed to alcian blue/safranin. Mouse BMMC (the presumptive in vitro counterpart of the in vivo differentiated mucosal mast cell) were cultured for 2-14 days with confluent skin-derived 3T3 fibroblasts in RPMI-1640 containing 10% fetal calf serum and 50% WEHI-3 conditioned medium. Although the BMMC adhered to the fibroblast monolayer, they continued to divide, probably due to the presence of interleukin-3 in the conditioned medium. The mast cells remained viable throughout the period of co-culture, since they failed to release LDG and because they increased their histamine content per cell approx.15-fold. After 8-9 days of co-culture, >50% of the BMMC changed histochemically becoming safranin positive. At this time, 30-50% of the (/sup 35/S)glycosaminoglycans on the proteoglycans synthesized by these co-cultured mass cells were heparin, whereas the initial BMMC synthesized proteoglycans containing only chondroitin sulfate E. That interleukin 3-dependent mouse BMMC can be induced to undergo a phenotypic change so as to express characteristics of a HP-MC suggests that the tissue microenvironment determines the differentiated characteristics of these cells.

  18. Oxidative changes and apoptosis induced by 1800-MHz electromagnetic radiation in NIH/3T3 cells.

    PubMed

    Hou, Qingxia; Wang, Minglian; Wu, Shuicai; Ma, Xuemei; An, Guangzhou; Liu, Huan; Xie, Fei

    2015-03-01

    To investigate the potential adverse effects of mobile phone radiation, we studied reactive oxygen species (ROS), DNA damage and apoptosis in mouse embryonic fibroblasts (NIH/3T3) after intermittent exposure (5 min on/10 min off, for various durations from 0.5 to 8 h) to an 1800-MHz GSM-talk mode electromagnetic radiation (EMR) at an average specific absorption rate of 2 W/kg. A 2',7'-dichlorofluorescin diacetate fluorescence probe was used to detect intracellular ROS levels, immunofluorescence was used to detect γH2AX foci as a marker for DNA damage, and flow cytometry was used to measure apoptosis. Our results showed a significant increase in intracellular ROS levels after EMR exposure and it reached the highest level at an exposure time of 1 h (p < 0.05) followed by a slight decrease when the exposure continued for as long as 8 h. No significant effect on the number of γH2AX was detected after EMR exposure. The percentage of late-apoptotic cells in the EMR-exposed group was significantly higher than that in the sham-exposed groups (p < 0.05). These results indicate that an 1800-MHz EMR enhances ROS formation and promotes apoptosis in NIH/3T3 cells.

  19. miR-194 functions as a novel modulator of cellular senescence in mouse embryonic fibroblasts.

    PubMed

    Xu, Shun; Zhang, Bing; Zhu, Yanmei; Huang, Haijiao; Yang, Wenping; Huang, Haiyong; Zheng, Hui-Ling; Liu, Xinguang

    2017-03-01

    MicroRNA-194 (miR-194), a typical p53 responsive miRNA, serves as a tumor suppressor similar as p53, and has been demonstrated to play an anti-proliferation role in various human cancers. In spite of the pivotal role of p53 during aging process, the knowledge of miR-194's contribution to cellular senescence is limited. We herein sought to explore the role of miR-194 in the replicative senescence and stress-induced senescence of mouse embryonic fibroblasts. Our results unraveled that, compared to young cells, miR-194 is highly expressed in senescent cells, and extra expression of miR-194 significantly triggers the replicative senescence of MEFs and H2 O2 -induced senescence of NIH/3T3 cells, while inhibition of miR-194 exhibited the opposite effect. We further unveiled that DNMT3A was a direct and authentic target of miR-194, which has been reported to be closely associated with cellular senescence. Taken together, our data suggest that miR-194 may significantly promote the development of cellular senescence in mouse embryonic fibroblasts, which potentially occurs through inhibiting the DNMT3A expression.

  20. New mouse xenograft model modulated by tumor-associated fibroblasts for human multi-drug resistance in cancer

    PubMed Central

    MA, YAN; LIN, ZHIQIANG; FALLON, JOHN K.; ZHAO, QIANG; LIU, DAN; WANG, YONGJUN; LIU, FENG

    2015-01-01

    We developed an MDR tumor model that is modulated by tumor-associated fibroblasts. Studies on proliferation of tumor cell lines including paclitaxel-sensitive and resistant cell lines were performed. The expressions of P-gp and α-smooth muscle actin (α-SMA) antigen were evaluated by immunohistochemistry and western blot analysis. Quantitative P-gp analyses of different cell lines were accomplished by nanoUPLC-MS/MS. Tumor cell colony formation assay and established xenograft model was used to investigate the relationship between P-gp expression, fibroblast levels and tumorigenesis. The mouse xenograft model was developed after co-inoculation with MDR tumor cells and NIH/3T3 fibroblast cells. There was no correlation between tumorigenesis in vivo and the growth rate of cells in vitro. The proliferation among different cell lines had no significant differences, but the P-gp expression and tumor growth in the xenograft model were fairly different. P-gp determination and α-SMA immunofluorescence staining clarified the relationship between P-gp expression, fibroblast levels and tumorigenesis. It was more difficult for tumor cells with higher P-gp levels to recruit fibroblasts in vivo, resulting in lower tumorigenesis due to the lack of structural and chemical support during tumor progression. In the established paclitaxel-resistant mouse xenograft model, no obvious antitumor effect was observed after Taxol treatment, but a significant decrease in tumor size for the group treated with gemcitabine sensitive to the model. The results show that the added fibroblasts do not disturb the applicability of the model in MDR. Therefore, this mouse xenograft MDR model could serve as an effective tool for MDR research. PMID:26352907

  1. Cloning and Expression of CD19, a Human B-Cell Marker in NIH-3T3 Cell Line

    PubMed Central

    Abbasi-Kenarsari, Hajar; Shafaghat, Farzaneh; Baradaran, Behzad; Movassaghpour, Ali Akbar; Shanehbandi, Dariush; Kazemi, Tohid

    2015-01-01

    Background CD19 is a pan B cell marker that is recognized as an attractive target for antibody-based therapy of B-cell disorders including autoimmune disease and hematological malignancies. The object of this study was to stably express the human CD19 antigen in the murine NIH-3T3 cell line aimed to be used as an immunogen in our future study. Methods Total RNA was extracted from Raji cells in which high expression of CD19 was confirmed by flow cytometry. Synthesized cDNA was used for CD19 gene amplification by conventional PCR method using Pfu DNA polymerase. PCR product was ligated to pGEM-T Easy vector and ligation mixture was transformed to DH5α competent bacteria. After blue/white selection, one positive white colony was subjected to plasmid extraction and direct sequencing. Then, CD19 cDNA was sub-cloned into pCMV6-Neo expression vector by double digestion using KpnI and HindIII enzymes. NIH-3T3 mouse fibroblast cell line was subsequently transfected by the construct using Jet-PEI transfection reagent. After 48 hours, surface expression of CD19 was confirmed by flow cytometry and stably transfected cells were selected by G418 antibiotic. Results Amplification of CD19 cDNA gave rise to 1701 bp amplicon confirmed by alignment to reference sequence in NCBI database. Flow cytometric analysis showed successful transient and stable expression of CD19 on NIH-3T3 cells (29 and 93%, respectively). Conclusion Stable cell surface expression of human CD19 antigen in a murine NIH-3T3 cell line may develop a proper immunogene which raises specific anti-CD19 antibody production in the mice immunized sera. PMID:25926951

  2. Online monitoring of BALB/3T3 metabolism and adhesion with multiparametric chip-based system.

    PubMed

    Ceriotti, L; Kob, A; Drechsler, S; Ponti, J; Thedinga, E; Colpo, P; Ehret, R; Rossi, F

    2007-12-01

    A multiparametric chip-based system was employed to measure cell adhesion, metabolism, and response to metal compounds previously classified as cytotoxic in immortalized mouse fibroblasts (BALB/3T3 cell line). The system measures in parallel, online, and in label-free conditions the extracellular acidification rates (with pH-sensitive field effect transistors [ISFETs]), the cellular oxygen consumption (with amperometric electrode structures [Clark-type sensors]), and cell adhesion (with impedimetric interdigitated electrode structures [IDESs]). The experimental protocol was optimized to monitor metabolism and adhesion of the BALB/3T3 cell line. A total of 70,000 cells and a bicarbonate buffer-free running low-glucose Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal clone serum III and 1mM Hepes were selected to maintain cells in good conditions on the chip during the measurements performed under perfusion conditions. Cells were exposed to sodium arsenite, cadmium chloride, and cis-platinum at concentrations ranging from 1 to 100 microM. The kinetics of cell response to these compounds was analyzed and suggests that the Clark-type sensors can be more sensitive than IDESs and ISFETs in detecting the presence of high chemical concentration when short exposure times (i.e., 2h) are considered. The cytotoxicity data obtained from the online measurements of acidification, respiration, and adhesion at 24h compare well, in terms of half-inhibition concentration values (IC(50)), with the ones obtained using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test and colony-forming efficiency (CFE) assay. The results show a good sensitivity of the system combined with the advantages of the online and label-free detection methods that allow following cell status before, during, and after the treatment in the same experiment.

  3. Differentiation-specific decrease in heat shock protein synthesis in 3T3-L1 cells

    SciTech Connect

    Sorhage, F.; Kim, J.; Liu, A.Y.C.; Chen, K.Y.

    1986-05-01

    The regulation of synthesis of heat shock proteins (HSPs) in 3T3-L1 preadipocytes (fibroblasts) and adipocytes was examined using the techniques of pulse labeling with (/sup 35/S)methionine followed by analysis of the pattern and amount of radioactivity incorporated by SDS-polyacrylamide gel electrophoresis and autoradiography. Exposure of the 3T3-L1 preadipocyte cultures either to elevated temperature (42..mu..C) or to the amino acid analogue canavanine (400 ..mu..g/ml), markedly induced the synthesis of six major HSPs with apparent molecular weights of 105,000, 89,000, 74,000, 72,000, 50,000, and 42,000. The time course of induction of the HSPs by canavanine was significantly delayed as compared to that of heat shock; maximal increase in synthesis of the HSPs was observed at 3-7 hrs after incubation at 42..mu..c and at 22-24 hrs after incubation with 400 ..mu..g/ml canavanine. The magnitude of induction of HSP in the differentiated adipocytes was significantly reduced as compared to that of the undifferentiated fibroblast cells. The reduced expression of HSPs in 3T3-L1 adipocytes appears to be related to the terminal adipogenic differentiation process. The phenomenon was not observed in the control 3T3-C2 cells nor in a transformed variant of the 3T3-L1 cells.

  4. Induction of mutagenesis and transformation in BALB/c-3T3 clone A31-1 cells by diverse chemical carcinogens

    SciTech Connect

    Lubet, R.A. ); Kouri, R.E.; Curren, R.A.; Putman, D.L.; Schechtman, L.M. )

    1990-01-01

    BALB/c-3T3 cells were employed to examine the genotoxic potential of a variety of known chemical carcinogens. BALB/c-3T3 cells displayed a dose-dependent transformation response to a variety of carcinogens (polycyclic hydrocarbons, methylating agents, ethylating agents, aflatoxin B{sub 1} (AFT{sub 1}), and 4-nitroquinoline-N-oxide (4-NQO)). When the ability of these compounds to induce mutagenesis to resistance to the cardiac glycoside ouabain (OUA{sup R}) was examined, the authors found the short chain alkylating agents to be particularly effective mutagens, causing biologic effects at doses below those necessary to induce a transformation response. In contrast, the polycyclic hydrocarbons which were potent transforming agents were weaker, albeit significant, mutagens for the OUA{sup R} locus in this system, while AFB{sub 1} was quite weak. Further studies were performed with 5-azacytidine (5-AZA) and the nongenotoxic carcinogen cinnamyl anthranilate (CIN). 5-AZA was a potent transforming agent, but failed to cause mutagenesis. CIN similarly caused in vitro transformation. When a series of eight structurally diverse compounds were examined in both the BALB/c-3T3 and C3H10T1/2 mouse fibroblast transformation systems, the BALB/c-3T3 system was shown to be sensitive to a wide variety of potential carcinogens, whereas the C3H10T1/2 system proved routinely sensitive only to the polycyclic hydrocarbons.

  5. Combined effects of 60 Hz electromagnetic field exposure with various stress factors on cellular transformation in NIH3T3 cells.

    PubMed

    Lee, Hae-June; Jin, Yeung Bae; Lee, Jae Seon; Choi, Jong-Il; Lee, Ju-Woon; Myung, Sung Ho; Lee, Yun-Sil

    2012-04-01

    Epidemiological studies have suggested that extremely low-frequency magnetic fields (ELF-MF) are associated with an increased incidence of cancer. Studies using in vitro systems have reported mixed results for the effects of ELF-MF alone, and the World Health Organization (WHO) Research Agenda published in 2007 suggested that high priority research should include an evaluation of the co-carcinogenic effects of ELF-MF exposure using in vitro models. Here, the carcinogenic potential of ELF-MF exposure alone and in combination with various stress factors was investigated in NIH3T3 mouse fibroblasts using an in vitro cellular transformation assay. NIH3T3 cells were exposed to a 60 Hz ELF-MF (1 mT) alone or in combination with ionizing radiation (IR), hydrogen peroxide (H₂O₂), or c-Myc overexpression, and the resulting number of anchorage-independent colonies was counted. A 4 h exposure of NIH3T3 cells to ELF-MF alone produced no cell transformation. Moreover, ELF exposure did not influence the transformation activity of IR, H₂O₂, or activated c-Myc in our in vitro assay system, suggesting that 1 mT ELF-MF did not affect any additive or synergistic transformation activities in combination with stress factors such as IR, H₂O₂, or activated c-Myc in NIH3T3 cells.

  6. Adhesion, Proliferation and Migration of NIH/3T3 Cells on Modified Polyaniline Surfaces

    PubMed Central

    Rejmontová, Petra; Capáková, Zdenka; Mikušová, Nikola; Maráková, Nela; Kašpárková, Věra; Lehocký, Marián; Humpolíček, Petr

    2016-01-01

    Polyaniline shows great potential and promises wide application in the biomedical field thanks to its intrinsic conductivity and material properties, which closely resemble natural tissues. Surface properties are crucial, as these predetermine any interaction with biological fluids, proteins and cells. An advantage of polyaniline is the simple modification of its surface, e.g., by using various dopant acids. An investigation was made into the adhesion, proliferation and migration of mouse embryonic fibroblasts on pristine polyaniline films and films doped with sulfamic and phosphotungstic acids. In addition, polyaniline films supplemented with poly (2-acrylamido-2-methyl-1-propanesulfonic) acid at various ratios were tested. Results showed that the NIH/3T3 cell line was able to adhere, proliferate and migrate on the pristine polyaniline films as well as those films doped with sulfamic and phosphotungstic acids; thus, utilization of said forms in biomedicine appears promising. Nevertheless, incorporating poly (2-acrylamido-2-methyl-1-propanesulfonic) acid altered the surface properties of the polyaniline films and significantly affected cell behavior. In order to reveal the crucial factor influencing the surface/cell interaction, cell behavior is discussed in the context of the surface energy of individual samples. It was clearly demonstrated that the lesser the difference between the surface energy of the sample and cell, the more cyto-compatible the surface is. PMID:27649159

  7. [The comparison of biologic character between mouse embryonic fibroblast and human embryonic fibroblast].

    PubMed

    Zhang, Yi; Zhao, Liansan; Wang, Chengxiao; Lei, Binjun

    2003-06-01

    To evaluate the feasibility of using human embryonic fibroblast(HEF) as feeder layer in the culture of human embryonic stem(ES) cells in vitro, we investigated the morphology, the sensitivity to 0.25% trypsin, the growth curve and cell cycle of HEF with DMEM(low glucose) +10% FBS used as culture medium, and then we compared HEF with mouse embryonic fibroblast (MEF). The results showed that both HEF and MEF are adherent cells in vitro, and HEF has longer life span and better growth ability than MEF. In room temperature, HEF is more sensitive to 0.25% trypsin. Our research suggested that HEF can be used as feeder layer in culture of ES cells. HEF has longer service life than MEF and is worthy to be studied further.

  8. Cytotoxicity of folic acid conjugated hollow silica nanoparticles toward Caco2 and 3T3 cells, with and without encapsulated DOX.

    PubMed

    Patel, Kunal; Sundara Raj, Behin; Chen, Yan; Lou, Xia

    2016-04-01

    Hollow silica nanoparticles of two sizes with and without a folic acid targeting ligand were synthesized. Fickian diffusion of the antitumor drug doxorubicin hydrochloride (DOX) was demonstrated by the produced nanoparticles, achieving a cumulative release of 73% and 45% for 215 nm and 430 nm particles respectively over a period of 500 h. The hollow silica nanoparticles presented a time and dose dependent toxicity, selective to human epithelial colorectal adenocarcinoma (Caco2) cells, over mouse embryonic fibroblast (3T3) cells. At 24h Caco2 cell viability was reduced to 66% using pure hollow silica at a concentration of 50 μg mL(-1), while that of 3T3 cells remained at 94% under the same conditions. The selective cytotoxicity of hollow silica nanoparticles was further enhanced by conjugation of folic acid and incorporation of DOX: at 24h and an equivalent DOX concentration of 0.5 μg mL(-1), viable Caco2 cells were reduced to 45% while 3T3 cells were reduced to 83%. Interestingly the equivalent dose of free DOX was more toxic to 3T3 than to Caco2 cells, reducing the 3T3 viability to 72% and the Caco2 viability to 80%, which is likely due to the presence of the p-glycoprotein pumps in Caco2 cells. Folic acid conjugation served to enhance the viability of both cell lines in this work. Careful optimization of the folate content should further improve the cell specificity of the hollow silica nanoparticles, thus providing a viable targeting platform for cancer therapy.

  9. Butyrate modulates the expression of. beta. -adrenergic receptor subtype in 3T3-L1 cells

    SciTech Connect

    Poksay, K.S.; Nakada, M.T.; Crooke, S.T.; Stadel, J.M.

    1986-03-05

    In mouse 3T3-L1 fibroblasts, the glucocorticoid dexamethasone (dex) affects a switch in ..beta..-adrenergic receptor (..beta..AR) subtype expression from ..beta../sub 1/AR to ..beta../sub 2/AR and increases total ..beta..AR number. They now demonstrate a similar effect by sodium butyrate (B) and find that the combined effect of these two gene-activating agents is greater than additive suggesting different mechanisms of action on the ..beta..AR. ..beta..AR are assayed in membranes prepared from 3T3-L1 cells using the radiolabeled ..beta..AR-specific antagonist (/sup 125/I)-cyanopindolol. ..beta..AR subtype is determined by competition binding of the ..beta../sub 2/AR-selective antagonist ICI 118.551 for the radioligand. B (2-10mM) causes a dose-dependent increase in total ..beta..AR number (up to 2-fold over control) and the proportion of ..beta../sub 2/AR. B (5mM) causes a time-dependent increase in total ..beta..AR number (2-fold) and the proportion of ..beta../sub 2/AR up to 24 hr. Dex maximally increases total ..beta..AR number (2-fold) when treated for 48 hr at concentrations greater than or equal to 100nM. B (2 or 5mM) together with dex (250nM) have a greater than additive effect on total ..beta..AR number at 24 hr (1.7-fold) and at 48 hr (1.4-2.4-fold, using 5 or 10mM B and dex greater than or equal to 10nM). The proportion of ..beta../sub 2/AR is also greater when both compounds are added together. In comparison with proprionate and valerate, B increases total ..beta..AR number and the proportion of ..beta../sub 2/AR to a greater extent and at lower concentrations. To determine a functional correlate to these findings, cells were pre-treated for 48 hr with B and/or dex, intracellular ATP labeled with /sup 3/H-adenine, followed by treatment with forskolin (10..mu..M) and ..beta..AR agonists. B caused a dramatic increase in /sup 3/H-cAMP produced compared to control and dex treatments and a greater than additive effect was again achieved when B and dex were

  10. Antiproliferative activity of flower hexane extract obtained from Mentha spicata associated with Mentha rotundifolia against the MCF7, KB, and NIH/3T3 cell lines.

    PubMed

    Nedel, Fernanda; Begnini, Karine; Carvalho, Pedro Henrique de Azambuja; Lund, Rafael Guerra; Beira, Fátima T A; Del Pino, Francisco Augusto B

    2012-11-01

    This study assessed the antiproliferative effect in vitro of the flower hexane extract obtained from Mentha spicata associated with Mentha rotundifolia against the human breast adenocarcinoma (MCF-7), human mouth epidermal carcinoma (KB), and mouse embryonic fibroblast (NIH 3T3) cell lines, using sulforhodamine B (SRB) assay. A cell density of 2×10(4)/well was seeded in 96-well plates, and samples at different concentrations ranging from 10 to 500 mg/mL were tested. The optical density was determined in an ELISA multiplate reader (Thermo Plate TP-Reader). Results demonstrated that the hexane extract presented antiproliferative activity against both the tumor cell lines KB and MCF-7, presenting a GI(50) (MCF-7=13.09 mg/mL), TGI (KB=37.76 mg/mL), and IL(50) (KB=291.07 mg/mL). Also, the hexane extract presented antiproliferative activity toward NIH 3T3 cells GI(50) (183.65 mg/mL), TGI (280.54 mg/mL), and IL(50) (384.59 mg/mL). The results indicate that the flower hexane extract obtained from M. spicata associated with M. rotundifolia presents an antineoplastic activity against KB and MCF-7, although an antiproliferative effect at a high concentration of the extract was observed toward NIH 3T3.

  11. Copper Redistribution in Atox1-deficient Mouse Fibroblast Cells

    PubMed Central

    McRae, Reagan; Lai, Barry

    2010-01-01

    Quantitative SXRF imaging of adherent mouse fibroblast cells deficient in Atox1, a metallochaperone protein responsible for delivering copper (Cu) to cuproenzymes in the trans-Golgi network, revealed striking differences in the subcellular Cu distribution compared to wildtype cells. While the latter showed a pronounced perinculear localization of Cu, the Atox1-deficient cells displayed a mostly unstructured and diffuse distribution throughout the entire cell body. Comparison of the SXRF elemental maps for Zn and Fe of the same samples showed no marked differences between the two cell lines. The data underscore the importance of Atox1, not only as a metallochaperone for delivering Cu to cuproenzymes, but also as a key player in maintaining the proper distribution and organization of Cu at the cellular level. PMID:19865834

  12. Direct reprogramming of mouse fibroblasts into cardiomyocytes with chemical cocktails.

    PubMed

    Fu, Yanbin; Huang, Chenwen; Xu, Xinxiu; Gu, Haifeng; Ye, Youqiong; Jiang, Cizhong; Qiu, Zilong; Xie, Xin

    2015-09-01

    The direct conversion, or transdifferentiation, of non-cardiac cells into cardiomyocytes by forced expression of transcription factors and microRNAs provides promising approaches for cardiac regeneration. However, genetic manipulations raise safety concerns and are thus not desirable in most clinical applications. The discovery of full chemically induced pluripotent stem cells suggest the possibility of replacing transcription factors with chemical cocktails. Here, we report the generation of automatically beating cardiomyocyte-like cells from mouse fibroblasts using only chemical cocktails. These chemical-induced cardiomyocyte-like cells (CiCMs) express cardiomyocyte-specific markers, exhibit sarcomeric organization, and possess typical cardiac calcium flux and electrophysiological features. Genetic lineage tracing confirms the fibroblast origin of these CiCMs. Further studies show the generation of CiCMs passes through a cardiac progenitor stage instead of a pluripotent stage. Bypassing the use of viral-derived factors, this proof of concept study lays a foundation for in vivo cardiac transdifferentiation with pharmacological agents and possibly safer treatment of heart failure.

  13. Expression of the Saccharomyces cerevisiae glycoprotein invertase in mouse fibroblasts: glycosylation, secretion, and enzymatic activity

    SciTech Connect

    Bergh, M.L.E.; Cepko, C.L.; Wolf, D.; Robbins, P.W.

    1987-06-01

    Oligosaccharide processing is controlled by host- and protein-dependent factors. To increase our understanding of the relative contribution of those factors the authors studied the glycosylation of yeast invertase expressed in a heterologous system. Invertase synthesized in psi-2 cells (an NIH 3T3-derived packaging line) is secreted efficiently, enzymatically active, and heavily glycosylated. It was estimated that the protein contains 8 or 9 carbohydrate chains. Two classes can be observed, of an approximate size of 100-110 kDa and 115-130 kDa, respectively. The size differences are due to differences in glycosylation. The smaller class contains two high-mannose carbohydrate chains; the remainder is of the complex type, sialylated and most likely tri- or tetraantennary. This profile parallels the situation observed with invertase glycosylation in yeast, where 2 of 9 or 10 chains remain unprocessed. The larger size class of invertase expressed in mouse fibroblasts has a different profile, since it contains probably only complex-type glycans. There are no apparent differences, however, in the size of the protein backbone between the two size classes. When invertase is synthesized in the presence of the mannosidase inhibitor 1-deoxymannojirimycin, processing is blocked completely. The glucosidase inhibitor 1-deoxynojirimycin does not inhibit processing completely. The glycosylation inhibitor tunicamycin prevents secretion of invertase completely when cells are cultured at 37/sup 0/C. At 26/sup 0/C, however, nonglycosylated invertase can be detected in the medium. These data suggest that glycosylation of invertase seems to be essential for the early steps of the secretory pathway but is less critical for later events.

  14. Changes in gap junction organization and decreased coupling during induced apoptosis in lens epithelial and NIH-3T3 cells.

    PubMed

    Theiss, Carsten; Mazur, Antonina; Meller, Karl; Mannherz, Hans Georg

    2007-01-01

    We demonstrate that global induction of apoptosis in primary bovine lens epithelial (LEC) or fibroblastic mouse NIH-3T3 cells by staurosporine, puromycin, cycloheximide, or etoposide is accompanied by a decrease in coupling by gap junctions. Cell coupling as tested by neurobiotin spreading was maintained when the LEC or NIH-3T3 cells were pre-incubated with the pan-caspase inhibitor zVAD or the caspase-3 inhibiting tetrapeptide DEVD. Immunohistochemistry using anti-connexin-43 antibodies showed a reduction of plasma membrane integrated connexin-43 in both cell lines when undergoing apoptosis. Western blotting indicated degradation of connexin-43 that was inhibited by zVAD or DEVD. Cell coupling at single cell level was tested by direct microinjecting into LEC apoptosis-inducing agents of low molecular mass like staurosporine, etoposide and puromycin or the high molecular mass proteins caspase-3 and -8 in activated state. Microinjection of puromycin or etoposide induced apoptotic morphological changes of only the injected cell within 90 or 180 min, but did not affect adjacent cells. In contrast, microinjection of staurosporine led to a rapid induction of apoptosis of the injected and a number of adjacent cells suggesting spreading of staurosporine most probably through gap junction pores held open by dephosphorylation of connexin-43 as verified by immunoblotting and staining using a phospho-serine368-specific anti-connexin-43 antibody. Microinjection of active caspase-8 led after 3 h to morphological apoptotic alterations of only the injected cell, but did not inhibit spreading of co-injected neurobiotin to neighboring cells during the first hour. In contrast, microinjection of active caspase-3-induced apoptosis only of the injected cell after 60 min and rapidly and completely suppressed coupling to neighboring cells.

  15. Vaspin promotes 3T3-L1 preadipocyte differentiation

    PubMed Central

    Liu, Ping; Wu, Jine; Zhou, Xin; Wang, Liping; Han, Wenqi; Lv, Ying; Sun, Chaofeng

    2015-01-01

    Vaspin, a novel adipocyte factor secreted from visceral adipose tissues, is associated with obesity and insulin resistance and can regulate glucose and lipid metabolism, increase insulin sensitivity, and suppress inflammation; however, the underlying mechanisms remain unknown. Proliferation and maladaptive differentiation are important pathological mechanisms underlying obesity. This study aimed to evaluate the effects of vaspin on the proliferation and differentiation of preadipocyte 3T3-L1 cells and to explore the likely mechanisms responsible for 3T3-L1 differentiation. Vaspin was added to cultured 3T3-L1 cells, and the differentiation of adipocytes was evaluated using Oil Red O staining. The AKT signaling pathway and specific differentiation factors related to the differentiation of preadipocyte 3T3-L1 cells, peroxisome proliferator-activated γ and the CCAAT/enhancer-binding protein (C/EBP) family, were evaluated using reverse transcription polymerase chain reaction (RT-PCR) and western blot analyses during the early phase of differentiation. Additionally, adiponectin mRNA, interleukin-6 mRNA (IL-6 mRNA), and glucose transporter-4 (GLUT4) protein levels were measured in the differentiated adipocytes. The results indicated that vaspin promotes the intracellular accumulation of lipids and increases differentiation-related factors, including peroxisome proliferator-activated receptor γ, C/EBPα, and free fatty acid-binding protein 4 (FABP4), in a dose-dependent manner. Additionally, vaspin (200 ng/mL) increased the mRNA and protein levels of C/EBPβ, peroxisome proliferator-activated γ, C/EBPα, and FABP4. Moreover, compared with the control, significantly smaller eight-day differentiated adipocytes were observed, and these cells exhibited decreased IL-6 mRNA and increased GLUT4 mRNA levels; these results also indicated the potential of vaspin to promote the insulin-mediated AKT signaling pathway during the early phase of differentiation. In conclusion

  16. Curcumin Inhibits Transforming Growth Factor β Induced Differentiation of Mouse Lung Fibroblasts to Myofibroblasts

    PubMed Central

    Liu, Daishun; Gong, Ling; Zhu, Honglan; Pu, Shenglan; Wu, Yang; Zhang, Wei; Huang, Guichuan

    2016-01-01

    Transforming growth factor β (TGF-β) induced differentiation of lung fibroblasts to myofibroblasts is a key event in the pathogenesis of pulmonary fibrosis. This study aimed to evaluate the effect of curcumin on TGF-β induced differentiation of lung fibroblasts to myofibroblasts and explore the underlying mechanism. Mouse lung fibroblasts were cultured and treated with TGF-β2 and curcumin or rosiglitazone. Cell vitality was examined by MTT assay. The secretion of collagen-1 was assessed by ELISA. α smooth muscle actin (α-SMA) was visualized by immunofluorescence technique. The expression of peroxisome proliferator activated receptor γ (PPAR-γ) and platelet derived growth factor R β (PDGFR-β) was detected by PCR and Western blot analysis. We found that curcumin and rosiglitazone inhibited the proliferation and TGF-β induced differentiation of mouse lung fibroblasts. In addition, curcumin and rosiglitazone inhibited collagen-1 secretion and α-SMA expression in mouse lung fibroblasts. Furthermore, curcumin and rosiglitazone upregulated PPAR-γ and downregulated PDGFR-β expression in mouse lung fibroblasts. In conclusion, our study reveals novel mechanism by which curcumin inhibits TGF-β2 driven differentiation of lung fibroblasts to myofibroblasts. Curcumin could potentially be used for effective treatment of pulmonary fibrosis. PMID:27877129

  17. Hypoxia Enhances Direct Reprogramming of Mouse Fibroblasts to Cardiomyocyte-Like Cells.

    PubMed

    Wang, Yanyan; Shi, Shujun; Liu, Huiwen; Meng, Li

    2016-02-01

    Recent work has shown that mouse and human fibroblasts can be reprogrammed to cardiomyocyte-like cells with a combination of transcription factors. Current research has focused on improving the efficiency and mechanisms for fibroblast reprogramming. Previously, it has been reported that hypoxia enhances fibroblast cell reprogramming to pluripotent stem cells. In this study, we observed that 6 h of hypoxic conditions (2% oxygen) on newborn mouse dermal fibroblasts can improve the efficiency of reprogramming to cardiomyocyte-like cells. Expression of cardiac-related genes and proteins increased at 4 weeks after transfer of three transcription factors (Gata4/Mef2c/Tbx5 [GMT]). However, beating cardiomyocyte cells were not detected. The epigenetic mechanism of hypoxia-induced fibroblast reprogramming to cardiomyocyte cells requires further study.

  18. Aspartame downregulates 3T3-L1 differentiation.

    PubMed

    Pandurangan, Muthuraman; Park, Jeongeun; Kim, Eunjung

    2014-10-01

    Aspartame is an artificial sweetener used as an alternate for sugar in several foods and beverages. Since aspartame is 200 times sweeter than traditional sugar, it can give the same level of sweetness with less substance, which leads to lower-calorie food intake. There are reports that consumption of aspartame-containing products can help obese people lose weight. However, the potential role of aspartame in obesity is not clear. The present study investigated whether aspartame suppresses 3T3-L1 differentiation, by downregulating phosphorylated peroxisome proliferator-activated receptor γ (p-PPARγ), peroxisome proliferator-activated receptor γ (PPARγ), fatty acid-binding protein 4 (FABP4), CCAAT/enhancer-binding protein α (C/EBPα), and sterol regulatory element-binding protein 1 (SREBP1), which are critical for adipogenesis. The 3T3-L1 adipocytes were cultured and differentiated for 6 d in the absence and presence of 10 μg/ml of aspartame. Aspartame reduced lipid accumulation in differentiated adipocytes as evidenced by Oil Red O staining. qRT-PCR analysis showed that the PPARγ, FABP4, and C/EBPα mRNA expression was significantly reduced in the aspartame-treated adipocytes. Western blot analysis showed that the induction of p-PPARγ, PPARγ, SREBP1, and adipsin was markedly reduced in the aspartame-treated adipocytes. Taken together, these data suggest that aspartame may be a potent substance to alter adipocyte differentiation and control obesity.

  19. Cannabidiol promotes browning in 3T3-L1 adipocytes.

    PubMed

    Parray, Hilal Ahmad; Yun, Jong Won

    2016-05-01

    Recruitment of the brown-like phenotype in white adipocytes (browning) and activation of existing brown adipocytes are currently being investigated as a means to combat obesity. Thus, a wide variety of dietary agents that contribute to browning of white adipocytes have been identified. The present study was designed to investigate the effects of cannabidiol (CBD), a major nonpsychotropic phytocannabinoid of Cannabis sativa, on induction of browning in 3T3-L1 adipocytes. CBD enhanced expression of a core set of brown fat-specific marker genes (Ucp1, Cited1, Tmem26, Prdm16, Cidea, Tbx1, Fgf21, and Pgc-1α) and proteins (UCP1, PRDM16, and PGC-1α). Increased expression of UCP1 and other brown fat-specific markers contributed to the browning of 3T3-L1 adipocytes possibly via activation of PPARγ and PI3K. In addition, CBD increased protein expression levels of CPT1, ACSL, SIRT1, and PLIN while down-regulating JNK2, SREBP1, and LPL. These data suggest possible roles for CBD in browning of white adipocytes, augmentation of lipolysis, thermogenesis, and reduction of lipogenesis. In conclusion, the current data suggest that CBD plays dual modulatory roles in the form of inducing the brown-like phenotype as well as promoting lipid metabolism. Thus, CBD may be explored as a potentially promising therapeutic agent for the prevention of obesity.

  20. WEHI-3 cells inhibit adipocyte differentiation in 3T3-L1 cells

    SciTech Connect

    Lai, Jing; Liu, Gexiu; Yan, Guoyao; He, Dongmei; Zhou, Ying; Chen, Shengting

    2015-06-26

    By investigating the anti-adipogenic effects of WEHI-3 cells – a murine acute myelomonocytic leukemia cell line – we sought to improve the efficiency of hematopoietic stem cell transplantation (HSCT). Analysis of Oil Red O staining and the expression of adipogenic genes, including PPARγ, C/EBPα, FAS and LPL, indicated that WEHI-3 cells significantly inhibited 3T3-L1 mouse preadipocyte cells from differentiating into adipocytes. In vivo, fat vacuoles in mice injected with WEHI-3 cells were also remarkably reduced in the murine bone marrow pimelosis model. Moreover, the key gene in the Rho signaling pathway, ROCKII, and the key gene in the Wnt signaling pathway, β-catenin, were both upregulated compared with the control group. siRNA-mediated knockdown of ROCKII and β-catenin reversed these WEHI-3-mediated anti-adipogenic effects. Taken together, these data suggest that WEHI-3 cells exert anti-adipogenic effects and that both ROCKII and β-catenin are involved in this process. - Highlights: • WEHI-3, an acute myelomonocytic leukemia cell line, inhibited 3T3-L1 preadipocyte from differentiating into adipocyte. • WEHI-3 cells can arrest 3T3-L1 cells in G0/G1 phase by secreting soluble factors and thus inhibit their proliferation. • WEHI-3 cells reduced bone marrow pimelosis in the murine model. • Both ROCKII and β-catenin were involved in the WEHI-3-mediated anti-adipogenic effects.

  1. Alteration of glycolipids in ras-transfected NIH 3T3 cells

    SciTech Connect

    Matyas, G.R.; Aaronson, S.A.; Brady, R.O.; Fishman, P.H.

    1987-09-01

    Glycosphingolipid alterations upon viral transformation are well documented. Transformation of mouse 3T3 cells with murine sarcoma viruses results in marked decreases in the levels of gangliosides GM1 and GD1a and an increase in gangliotriaosylceramide. The transforming oncogenes of these viruses have been identified as members of the ras gene family. The authors analyzed NIH 3T3 cells transfected with human H-, K- and N-ras oncogenes for their glycolipid composition and expression of cell surface gangliosides. Using conventional thin-layer chromatographic analysis, they found that the level of GM3 was increased and that of GD1a was slightly decreased or unchanged, and GM1 was present but not in quantifiable levels. Cell surface levels of GM1 were determined by /sup 125/I-labeled cholera toxin binding to intact cells. GD1a was determined by cholera toxin binding to cells treated with sialidase prior to toxin binding. All ras-transfected cells had decreased levels of surface GM1 and GD1 as compared to logarithmically growing normal NIH 3T3 cells. Levels of GM1 and, to a lesser extent, GD1a increased as the latter cells became confluent. Using a monoclonal antibody assay, they found that gangliotriaosylceramide was present in all ras-transfected cells studied but not in logarithmically growing untransfected cells. These results indicated that ras oncogenes derived form human tumors are capable of inducing alterations in glycolipid composition.

  2. Characterization of hyaluronate binding proteins isolated from 3T3 and murine sarcoma virus transformed 3T3 cells

    SciTech Connect

    Turley, E.A.; Moore, D.; Hayden, L.J.

    1987-06-02

    A hyaluronic acid binding fraction was purified from the supernatant media of both 3T3 and murine sarcoma virus (MSV) transformed 3T3 cultures by hyaluronate and immunoaffinity chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis resolved the hyaluronate affinity-purified fraction into three major protein bands of estimated molecular weight (M/sub r,e/) 70K, 66K, and 56K which contained hyaluronate binding activity and which were termed hyaluronate binding proteins (HABP). Hyaluronate affinity chromatography combined with immunoaffinity chromatography, using antibody directed against the larger HABP, allowed a 20-fold purification of HABP. Fractions isolated from 3T3 supernatant medium also contained additional binding molecules in the molecular weight range of 20K. This material was present in vanishingly small amounts and was not detected with a silver stain or with (/sup 35/S)methionine label. The three protein species isolated by hyaluronate affinity chromatography (M/sub r,e/ 70K, 66K, and 56K) were related to one another since they shared antigenic determinants and exhibited similar pI values. In isocratic conditions, HABP occurred as aggregates of up to 580 kilodaltons. Their glycoprotein nature was indicated by their incorporation of /sup 3/H-sugars. Enzyme-linked immunoadsorbent assay showed they were antigenically distinct from other hyaluronate binding proteins such as fibronectin, cartilage link protein, and the hyaluronate binding region of chondroitin sulfate proteoglycan. The results are discussed with regard both to the functional significance of hyaluronate-cell surface interactions in transformed as well as normal cells and to the relationship of HABP to other reported hyaluronate binding proteins.

  3. Ultrasound associated uptake of chitosan nanoparticles in MC3T3-E1 cells

    NASA Astrophysics Data System (ADS)

    Wu, Junyi

    Chitosan is a natural linear polysaccharide that has been well known for its applications in drug delivery system due to its unique physicochemical and biological properties. However, challenges still remain for it to become a fully realized therapeutic agent. In this study, we investigated the uptake of chitosan nanoparticles (CNP) under the ultrasound stimulation, using a model cell culture system (MC3T3-E1 mouse pre-osteoblasts). The CNP were fabricated by an ionic gelation method and were lyophilized prior to characterization and delivery to cells. Particle size and zeta potential were measured using Dynamic Light Scattering (DLS); the efficiency of chitosan complexation was measured using the ninhydrin assay. Cytotoxicity was examined by neutral red assay within 48 hours after delivery. The effect of ultrasound (US) on the efficiency of nanoparticle delivery to the MC3T3-E1 cells was examined at 1MHz and at either 1 or 2 W/cm2. Fluorescein isothiocyanate (FITC)-conjugated-CNP were used to visualize the internalized particles within the cytosol. The uptake of FITC-CNP exhibits a dose and time dependent effect, a strong FITC fluorescence was detected at the concentration of 500microg/mL under fluorescence microscope. Ultrasound assisted uptake of FITC-CNP performed a significant positive effect at 2W/cm2 with 60s of ultrasound exposure time. CNP displayed a slightly decrease in cell viability from 25microg/mL to 100microg/mL, while higher concentration of CNP facilitates the proliferation of MC3T3-E1 cells. Less than 10% of reduction in cell viability was observed for US at 1W/cm2 and 2W/cm2 with 30s and 60s of exposure time, which suggest a mild effect of US to MC3T3-E1 cell line.

  4. The 3T3-L1 adipocyte glycogen proteome

    PubMed Central

    2013-01-01

    Background Glycogen is a branched polysaccharide of glucose residues, consisting of α-1-4 glycosidic linkages with α-1-6 branches that together form multi-layered particles ranging in size from 30 nm to 300 nm. Glycogen spatial conformation and intracellular organization are highly regulated processes. Glycogen particles interact with their metabolizing enzymes and are associated with a variety of proteins that intervene in its biology, controlling its structure, particle size and sub-cellular distribution. The function of glycogen in adipose tissue is not well understood but appears to have a pivotal role as a regulatory mechanism informing the cells on substrate availability for triacylglycerol synthesis. To provide new molecular insights into the role of adipocyte glycogen we analyzed the glycogen-associated proteome from differentiated 3T3-L1-adipocytes. Results Glycogen particles from 3T3-L1-adipocytes were purified using a series of centrifugation steps followed by specific elution of glycogen bound proteins using α-1,4 glucose oligosaccharides, or maltodextrins, and tandem mass spectrometry. We identified regulatory proteins, 14-3-3 proteins, RACK1 and protein phosphatase 1 glycogen targeting subunit 3D. Evidence was also obtained for a regulated subcellular distribution of the glycogen particle: metabolic and mitochondrial proteins were abundant. Unlike the recently analyzed hepatic glycogen proteome, no endoplasmic proteins were detected, along with the recently described starch-binding domain protein 1. Other regulatory proteins which have previously been described as glycogen-associated proteins were not detected, including laforin, the AMPK beta-subunit and protein targeting to glycogen (PTG). Conclusions These data provide new molecular insights into the regulation of glycogen-bound proteins that are associated with the maintenance, organization and localization of the adipocyte glycogen particle. PMID:23521774

  5. Anti-adipogenic effect of mulberry leaf ethanol extract in 3T3-L1 adipocytes

    PubMed Central

    Yang, Soo Jin; Park, Na-Young

    2014-01-01

    BACKGROUND/OBJECTIVES Adipogenesis is part of the cell differentiation process in which undifferentiated fibroblasts (pre-adipocytes) become mature adipocytes with the accumulation of lipid droplets and subsequent cell morphological changes. Several transcription factors and food components have been suggested to be involved in adipogenesis. The aim of this study was to determine whether mulberry leaf ethanol extract (MLEE) affects adipogenesis in 3T3-L1 adipocytes. MATERIALS/METHODS The 3T3-L1 adipocytes were treated with different doses of MLEE for 8 days starting 2 days post-confluence. Cell viability, fat accumulation, and adipogenesis-related factors including CCAAT-enhancer-binding protein alpha (C/EBPα), peroxisome proliferator-activated receptor gamma (PPARγ), PPARγ coactivator 1 alpha (PGC-1α), fatty acid synthase (FAS), and adiponectin were analyzed. RESULTS Results showed that MLEE treatments at 10, 25, 50, and 100 µg/ml had no effect on cell morphology and viability. Without evident toxicity, all MLEE treated cells had lower fat accumulation compared with control as shown by lower absorbances of Oil Red O stain. MLEE at 50 and 100 µg/ml significantly reduced protein levels of PPARγ, PGC-1α, FAS, and adiponectin in differentiated adipocytes. Furthermore, protein level of C/EBPα was significantly decreased by the treatment of 100 µg/ml MLEE. CONCLUSION These results demonstrate that MLEE treatment has an anti-adipogenic effect in differentiated adipocytes without toxicity, suggesting its potential as an anti-obesity therapeutic. PMID:25489399

  6. Characterization and cloning of a receptor for BMP-2 and BMP-4 from NIH 3T3 cells.

    PubMed Central

    Koenig, B B; Cook, J S; Wolsing, D H; Ting, J; Tiesman, J P; Correa, P E; Olson, C A; Pecquet, A L; Ventura, F; Grant, R A

    1994-01-01

    The bone morphogenetic proteins (BMPs) are a group of transforming growth factor beta (TGF-beta)-related factors whose only receptor identified to date is the product of the daf-4 gene from Caenorhabditis elegans. Mouse embryonic NIH 3T3 fibroblasts display high-affinity 125I-BMP-4 binding sites. Binding assays are not possible with the isoform 125I-BMP-2 unless the positively charged N-terminal sequence is removed to create a modified BMP-2, 125I-DR-BMP-2. Cross-competition experiments reveal that BMP-2 and BMP-4 interact with the same binding sites. Affinity cross-linking assays show that both BMPs interact with cell surface proteins corresponding in size to the type I (57- to 62-kDa) and type II (75- to 82-kDa) receptor components for TGF-beta and activin. Using a PCR approach, we have cloned a cDNA from NIH 3T3 cells which encodes a novel member of the transmembrane serine/threonine kinase family most closely resembling the cloned type I receptors for TGF-beta and activin. Transient expression of this receptor in COS-7 cells leads to an increase in specific 125I-BMP-4 binding and the appearance of a major affinity-labeled product of approximately 64 kDa that can be labeled by either tracer. This receptor has been named BRK-1 in recognition of its ability to bind BMP-2 and BMP-4 and its receptor kinase structure. Although BRK-1 does not require cotransfection of a type II receptor in order to bind ligand in COS cells, complex formation between BRK-1 and the BMP type II receptor DAF-4 can be demonstrated when the two receptors are coexpressed, affinity labeled, and immunoprecipitated with antibodies to either receptor subunit. We conclude that BRK-1 is a putative BMP type I receptor capable of interacting with a known type II receptor for BMPs. Images PMID:8065329

  7. In vitro cytotoxicity of hydrothermally synthesized ZnO nanoparticles on human periodontal ligament fibroblast and mouse dermal fibroblast cells.

    PubMed

    Seker, Sükran; Elçin, A Eser; Yumak, Tuğrul; Sınağ, Ali; Elçin, Y Murat

    2014-12-01

    The use of metal oxide nanoparticles (NPs) in industrial applications has been expanding, as a consequence, risk of human exposure increases. In this study, the potential toxic effects of zinc oxide (ZnO) NPs on human periodontal ligament fibroblast cells (hPDLFs) and on mouse dermal fibroblast cells (mDFs) were evaluated in vitro. We synthesized ZnO NPs (particle size; 7-8 nm) by the hydrothermal method. Characterization assays were performed with atomic force microscopy, Braun-Emmet-Teller analysis, and dynamic light scattering. The hPDLFs and mDFs were incubated with the NPs with concentrations of 0.1, 1, 10, 50 and 100 μg/mL for 6, 24 and 48h. Under the control and NP-exposed conditions, we have made different types of measurements for cell viability and morphology, membrane leakage and intracellular reactive oxygen species generation. Also, we monitored cell responses to ZnO NPs using an impedance measurement system in real-time. While the morphological changes were visualized using scanning electron microscopy, the subcellular localization of NPs was investigated by transmission electron microscopy. Results indicated that ZnO NPs have significant toxic effects on both of the primary fibroblastic cells at concentrations of ∼50-100 μg/mL. The cytotoxicity of ZnO NPs on fibroblasts depended on concentration and duration of exposure.

  8. Evaluation of chylomicron effect on ASP production in 3T3-L1 adipocytes.

    PubMed

    Gao, Ying; Gauvreau, Danny; Cui, Wei; Lapointe, Marc; Paglialunga, Sabina; Cianflone, Katherine

    2011-02-01

    In the past few years, there has been increasing interest in the production and physiological role of acylation-stimulating protein (ASP), identical to C3adesArg, a product of the alternative complement pathway generated through C3 cleavage. Recent studies in C3 (-/-) mice that are ASP deficient have demonstrated a role for ASP in postprandial triglyceride clearance and fat storage. The aim of the present study was to establish a cell model and sensitive ELISA assay for the evaluation of ASP production using 3T3-L1 adipocytes. 3T3-L1 preadipocytes were differentiated into adipocytes, then cultured in different media such as serum-free (SF), Dulbecco's modified Eagle's medium (DMEM)/F12 + 10% fetal calf serum (FBS), and at varying concentrations of chylomicrons and insulin + chylomicrons up to 48 h. ASP production in SF and DMEM/F12 + 10% FBS was compared. Chylomicrons stimulated ASP production in a concentration- and time-dependent manner. By contrast, chylomicron treatment had no effect on the production of C3, the precursor protein of ASP, which was constant over 48 h. Addition of insulin (100 nM) to a low-dose of chylomicrons (100 µg TG/ml) significantly increased ASP production compared with chylomicrons alone at 48 h (P < 0.001). Furthermore, addition of insulin significantly increased C3 secretion at both 18 and 48 h of incubation (P < 0.05, P < 0.001, respectively). Overall, the proportion of ASP to C3 remained constant, indicating no change in the ratio of C3 cleaved to generate ASP. This study demonstrated that 3T3-L1 adipocyte is a useful model for the evaluation of C3 secretion and ASP production by using a sensitive mouse-specific ELISA assay. The stimulation of ASP production with chylomicrons demonstrates a physiologically relevant response, and provides a strategy for further studies on ASP production and function.

  9. Molecular mechanism of 9-cis-retinoic acid inhibition of adipogenesis in 3T3-L1 cells

    SciTech Connect

    Sagara, Chiaki; Takahashi, Katsuhiko; Kagechika, Hiroyuki; Takahashi, Noriko

    2013-03-29

    Highlights: ► We examined the effects of 9-cis-RA on adipogenesis in mouse preadipocyte 3T3-L1. ► 9-cis-RA inhibited lipid accumulation in adipogenetically-induced 3T3-L1 cells. ► A RXR pan-antagonist suppressed the inhibitory effects of 9-cis-RA on adipogenesis. ► This antagonist had no effects on RXRα and PPARγ levels in 9-cis-RA-treated cells. ► 9-cis-RA-induced decrease in both RXRα and PPARγ was independent of RXR activation. -- Abstract: Retinoic acid (RA) signaling is mediated by specific nuclear hormone receptors. Here we examined the effects of 9-cis-RA on adipogenesis in mouse preadipocyte 3T3-L1 cells. 9-cis-RA inhibits the lipid accumulation of adipogenetically induced 3T3-L1 cells. The complex of retinoid X receptor α (RXRα) with peroxisome proliferator-activated receptor γ (PPARγ) is a major transcription factor in the process of adipogenesis, and the levels of these molecules were decreased by 9-cis-RA treatment. A RXR pan-antagonist suppressed 9-cis-RA’s inhibitory effects on adipogenesis, but not on the intracellular levels of both RXRα and PPARγ. These results suggest that 9-cis-RA could inhibit adipogenesis by activating RXR, and decrease both RXR and PPARγs levels in a RXR activation-independent manner.

  10. S-phase induction and transformation of quiescent NIH 3T3 cells by microinjection of phospholipase C

    SciTech Connect

    Smith, M.R.; Ryu, Sungho; Suh, Panghill; Rhee, Suegoo; Kung, Hsiangfu )

    1989-05-01

    Two inositol phospholipid-specific phospholipase C (PLC) isozymes (PLC-I and -II) have been purified from bovine brain. When PLC-I or PLC-II was microinjected into quiescent NIH 3T3 cells, a time- and dose-dependent induction of DNA synthesis occurred, as demonstrated by ({sup 3}H)thymidine incorporation into nuclear DNA. In addition, {approx} 8 hr after PLC injection, NIH 3T3 fibroblasts appeared spindle-shaped, refractile, and highly vacuolated, displaying a morphology similar to transformed cells. The morphologic transformation was apparent for 26-30 hr after which the injected cells reverted back to a normal phenotype. Microinjected PLC at a high concentration was cytotoxic, dissolving the cytoplasmic membrane and leaving behind cellular ghosts. PLC is a key regulatory enzyme involved in cellular membrane signal transduction. Introduction of exogenous PLC into NIH 3T3 cells by microinjection induced a growth and oncogenic potential, as demonstrated by the ability of microinjected PLC to override the cellular G{sub 0} block, inducing DNA synthesis and morphologic transformation of growth-arrested fibroblast cells.

  11. S-phase induction and transformation of quiescent NIH 3T3 cells by microinjection of phospholipase C.

    PubMed

    Smith, M R; Ryu, S H; Suh, P G; Rhee, S G; Kung, H F

    1989-05-01

    Two inositol phospholipid-specific phospholipase C (PLC) isozymes (PLC-I and -II) have been purified from bovine brain. When PLC-I or PLC-II was microinjected (100-700 micrograms/ml) into quiescent NIH 3T3 cells, a time- and dose-dependent induction of DNA synthesis occurred, as demonstrated by [3H]thymidine incorporation into nuclear DNA. In addition, approximately to 8 hr after PLC injection, NIH 3T3 fibroblasts appeared spindle-shaped, refractile, and highly vacuolated, displaying a morphology similar to transformed cells. The morphologic transformation was apparent for 26-30 hr after which the injected cells reverted back to a normal phenotype. Microinjected PLC at a high concentration (1 mg/ml) was cytotoxic, dissolving the cytoplasmic membrane and leaving behind cellular ghosts. PLC is a key regulatory enzyme involved in cellular membrane signal transduction. Introduction of exogenous PLC into NIH 3T3 cells by microinjection induced a growth and oncogenic potential, as demonstrated by the ability of microinjected PLC (approximately 10,000 molecules per cell) to override the cellular G0 block, inducing DNA synthesis and morphologic transformation of growth-arrested fibroblast cells.

  12. Adventitial fibroblasts are activated in the early stages of atherosclerosis in the apolipoprotein E knockout mouse

    SciTech Connect

    Xu Fang; Ji Jian; Li Li; Chen Rong; Hu Weicheng . E-mail: huweicheng@sdu.edu.cn

    2007-01-19

    The role of the adventitia in vascular function and vascular lesion formation has been largely ignored. This study observed the activation of the adventitia and specifically the fibroblasts in the development of atherosclerosis in the apoE(-/-) mouse. The results showed a gradual increase in expression of collagen types I and III after 2, 4, and 8 weeks of hyperlipidic diet. The earliest expression of monocyte chemoattractant protein-1 (MCP-1) protein and mRNA was detected in the adventitial fibroblast before the formation of intimal lesions. Proliferation, too, was first found in the adventitial fibroblasts. We hypothesize that the adventitial fibroblast is activated in the early stage of atherosclerosis. Adventitial inflammation may be an early event in the development of atherosclerotic lesions.

  13. Reprogramming mouse fibroblasts into engraftable myeloerythroid and lymphoid progenitors

    PubMed Central

    Cheng, Hui; Ang, Heather Yin-Kuan; A. EL Farran, Chadi; Li, Pin; Fang, Hai Tong; Liu, Tong Ming; Kong, Say Li; Chin, Michael Lingzi; Ling, Wei Yin; Lim, Edwin Kok Hao; Li, Hu; Huber, Tara; Loh, Kyle M.; Loh, Yuin-Han; Lim, Bing

    2016-01-01

    Recent efforts have attempted to convert non-blood cells into hematopoietic stem cells (HSCs) with the goal of generating blood lineages de novo. Here we show that hematopoietic transcription factors Scl, Lmo2, Runx1 and Bmi1 can convert a developmentally distant lineage (fibroblasts) into ‘induced hematopoietic progenitors' (iHPs). Functionally, iHPs generate acetylcholinesterase+ megakaryocytes and phagocytic myeloid cells in vitro and can also engraft immunodeficient mice, generating myeloerythoid and B-lymphoid cells for up to 4 months in vivo. Molecularly, iHPs transcriptionally resemble native Kit+ hematopoietic progenitors. Mechanistically, reprogramming factor Lmo2 implements a hematopoietic programme in fibroblasts by rapidly binding to and upregulating the Hhex and Gfi1 genes within days. Moreover the reprogramming transcription factors also require extracellular BMP and MEK signalling to cooperatively effectuate reprogramming. Thus, the transcription factors that orchestrate embryonic hematopoiesis can artificially reconstitute this programme in developmentally distant fibroblasts, converting them into engraftable blood progenitors. PMID:27869129

  14. Treatment with LPS plus INF-γ induces the expression and function of muscarinic acetylcholine receptors, modulating NIH3T3 cell proliferation: participation of NOS and COX

    PubMed Central

    Español, A J; Maddaleno, M O; Lombardi, M G; Cella, M; Martínez Pulido, P; Sales, M E

    2014-01-01

    Background and Purpose LPS and IFN-γ are potent stimuli of inflammation, a process in which fibroblasts are frequently involved. We analysed the effect of treatment with LPS plus IFN-γ on the expression and function of muscarinic acetylcholine receptors in NIH3T3 fibroblasts with regards to proliferation of these cells. We also investigated the participation of NOS and COX, and the role of NF-κB in this process. Experimental Approach NIH3T3 cells were treated with LPS (10 ng·mL−1) plus IFN-γ (0.5 ng·mL−1) for 72 h (iNIH3T3 cells). Cell proliferation was evaluated with MTT and protein expression by Western blot analysis. NOS and COX activities were measured by the Griess method and radioimmunoassay respectively. Key Results The cholinoceptor agonist carbachol was more effective at stimulating proliferation in iNIH3T3 than in NIH3T3 cells, probably due to the de novo induction of M3 and M5 muscarinic receptors independently of NF-κB activation. iNIH3T3 cells produced higher amounts of NO and PGE2 than NIH3T3 cells, concomitantly with an up-regulation of NOS1 and COX-2, and with the de novo induction of NOS2/3 in inflamed cells. We also found a positive feedback between NOS and COX that could potentiate inflammation. Conclusions and Implications Inflammation induced the expression of muscarinic receptors and, therefore,stimulated carbachol-induced proliferation of fibroblasts. Inflammation also up-regulated the expression of NOS and COX-2, thus potentiating the effect of carbachol on NO and PGE2 production. A positive crosstalk between NOS and COX triggered by carbachol in inflamed cells points to muscarinic receptors as potential therapeutic targets in inflammation. PMID:24990429

  15. Behavior of a fluorescent analogue of calmodulin in living 3T3 cells

    PubMed Central

    1985-01-01

    We have prepared and partially characterized a lissamine-rhodamine B fluorescent analogue of calmodulin, LRB-CM. The analogue had a dye/protein ratio of approximately 1.0 and contained no free dye or contaminating labeled proteins. LRB-CM was indistinguishable from native calmodulin upon SDS PAGE and in assays of phosphodiesterase and myosin light chain kinase. The emission spectrum of LRB-CM was insensitive to changes in pH, ionic strength, and temperature over the physiological range, but the apparent quantum yield was influenced somewhat by divalent cation concentration. LRB-CM injected into living Swiss 3T3 fibroblasts became associated with nitrobenzoxadiazole- phallacidin staining stress fibers in some interphase cells. LRB-CM and acetamidofluorescein-labeled actin co-injected into the same cell both became associated with fibers in some cells, but in most cases association of the two analogues with fibers was mutually exclusive. This suggests that calmodulin may differ from actin in the timing of incorporation into stress fibers or that we have distinguished distinct populations of stress fibers. We were able to detect no direct interaction of LRB-CM with actin by fluorescence photobleaching recovery (FRAP) of aqueous solutions. Interaction of LRB-CM with myosin light chain kinase also was not detected by FRAP. This suggests that the mean lifetime of the calmodulin-myosin light chain kinase complex is too short to affect the diffusion coefficient of calmodulin. We examined various fluorescent derivatives of proteins and dextrans as suitable control molecules for quantitative fluorescent analogue cytochemistry in living cells. Fluorescein isothiocyanate-dextrans were found to be preferable to all the proteins tested, since their mobilities in cytoplasm were inversely dependent on molecular size and there was no evidence of binding to intracellular components. In contrast, FRAP of LRB-CM in the cytoplasm of living 3T3 cells suggested that the analogue

  16. Iodothyronine Interactions with the System L1 Amino Acid Exchanger in 3T3-L1 Adipocytes.

    PubMed

    Mitchell, Fiona E; Roy, Lisa A; Taylor, Peter M

    2010-06-24

    Thyroid hormones enter isolated white adipocytes largely by a System L1-type amino acid transporter en route to exerting genomic actions. Differentiated 3T3-L1 mouse adipocytes in culture express mRNA for LAT1 (the catalytic subunit of high-affinity System L1). L-[(125)I]-T(3) uptake into 3T3-L1 adipocytes included a substantial saturable component inhibited by leucine. L-[(3)H]phenylalanine uptake into 3T3-L1 cells was saturable (K(m) of 31 μM), competitively inhibited by T(3) (K(i) of 1.2 μM) and blocked by leucine, BCH, and rT(3) as expected for substrate interactions of System L1. Efflux of preloaded L-[(3)H]phenylalanine from 3T3-L1 adipocytes was trans stimulated by external leucine, demonstrating the obligatory exchange mechanism of System L1 transport. T(3) (10 μM) did not significantly trans stimulate L-[(3)H]phenylalanine efflux, but did competitively inhibit the trans stimulatory effect of 10 μM leucine. The results highlight strong competitive interactions between iodothyronines (T(3), rT(3)) and amino acids for transport by System L1 in adipocytes, which may impact cellular iodothyronine exchanges during altered states of protein nutrition.

  17. Efficient introduction of specific TP53 mutations into mouse embryonic fibroblasts and embryonic stem cells.

    PubMed

    Wei, Quan-Xiang; van der Hoeven, Franciscus; Hollstein, Monica; Odell, Adam F

    2012-05-17

    This protocol describes a rapid, precise method for generating sets of embryonic stem (ES) cells or mouse embryonic fibroblasts (MEFs) harboring point mutations in the p53 tumor suppressor gene (officially known as Trp53). The strategy uses cells from the Trp53 (p53-null) 'platform' mouse, which allows site-specific integration of plasmid DNA into the Trp53 locus. Simple PCR protocols identify correctly targeted clones and immunoblots verify re-expression of the protein. We also present protocol modifications needed for efficient recovery of MEF clones expressing p53 constructs that retain wild-type function, including growth at low (3%) oxygen and transient downregulation of p53 regulators to forestall cell senescence of primary MEFs. A library of cell lines expressing various p53 mutants derived from the same population of primary fibroblasts or platform ES cells can be acquired and screened in less than 1 month.

  18. Effects of Berberine on Adipose Tissues and Kidney Function in 3T3-L1 Cells and Spontaneously Hypertensive Rats.

    PubMed

    Kishimoto, Aya; Dong, Shi-Fen; Negishi, Hiroko; Yasui, Naomi; Sun, Jian-Ning; Ikeda, Katsumi

    2015-09-01

    We aimed to investigate the effect of berberine on adipose tissues, as well as its effect on renal injury in 3T3-L1 cells and spontaneously hypertensive rats. 3T3-L1 cells were cultured and treated with berberine (5-20 pM) from days 3 to 8. Berberine added to the cultured medium could significantly down-regulate transcription factors, including CCAAT/enhancer binding protein β, CCAAT/enhancer binding protein a, and peroxisome pro liferator-activated receptor y, and suppress peroxisome proliferator-activated receptor target genes, such as adipocyte fatty acid binding protein and fatty acid synthase, and inhibit 3T3-Ll fibroblast differentiation to adipocytes. Male spontaneously hypertensive rats received either 150 mg/day of berberine or saline orally for 8 weeks. Compared with the control, berberine-treated rats exhibited significant reductions in body weight gain (p < 0.05), as well as retroperitoneal and mesenteric adipose tissues (p < 0.05). Berberine-treated rats significantly decreased urinary albumin excretion, a marker of renal injury (p < 0.05). Long-term treatment with berberine decreased the adipose tissues weight and attenuated renal injury in spontaneously hypertensive rats. Based on these results, berberine has an important role in regulating adipose tissues. These results suggest the protective effect of berberine on metabolic syndrome related diseases, such as renal injury.

  19. Bovine Collagen Peptides Compounds Promote the Proliferation and Differentiation of MC3T3-E1 Pre-Osteoblasts

    PubMed Central

    Liu, JunLi; Zhang, Bing; Song, ShuJun; Ma, Ming; Si, ShaoYan; Wang, YiHu; Xu, BingXin; Feng, Kai; Wu, JiGong; Guo, YanChuan

    2014-01-01

    Objective Collagen peptides (CP) compounds, as bone health supplements, are known to play a role in the treatment of osteoporosis. However, the molecular mechanisms of this process remain unclear. This study aimed to investigate the effects of bovine CP compounds on the proliferation and differentiation of MC3T3-E1 cells. Methods Mouse pre-osteoblast cell line MC3T3-E1 subclone 4 cells were treated with bovine CP compounds. Cell proliferation was analyzed by MTT assays and the cell cycle was evaluated by flow cytometry scanning. Furthermore, MC3T3-E1 cell differentiation was analyzed at the RNA level by real-time PCR and at the protein level by western blot analysis for runt-related transcription factor 2 (Runx2), a colorimetric p-nitrophenyl phosphate assay for alkaline phosphatase (ALP), and ELISA for osteocalcin (OC). Finally, alizarin red staining for mineralization was measured using Image Software Pro Plus 6.0. Results Cell proliferation was very efficient after treatment with different concentrations of bovine CP compounds, and the best concentration was 3 mg/mL. Bovine CP compounds significantly increased the percentage of MC3T3-E1 cells in G2/S phase. Runx2 expression, ALP activity, and OC production were significantly increased after treatment with bovine CP compounds for 7 or 14 days. Quantitative analyses with alizarin red staining showed significantly increased mineralization of MC3T3-E1 cells after treatment with bovine CP compounds for 14 or 21 days. Conclusions Bovine CP compounds increased osteoblast proliferation, and played positive roles in osteoblast differentiation and mineralized bone matrix formation. Taking all the experiments together, our study indicates a molecular mechanism for the potential treatment of osteoarthritis and osteoporosis. PMID:24926875

  20. Effect of blueberry polyphenols on 3T3-F442A preadipocyte differentiation.

    PubMed

    Moghe, Shiwani S; Juma, Shanil; Imrhan, Victorine; Vijayagopal, Parakat

    2012-05-01

    Today obesity is an epidemic, and its prevalence has increased significantly over the last few decades. To avoid excessive accumulation of fat, optimum energy intake along with regular exercise is mandatory. Polyphenols present in green tea, grape seeds, orange, and grapefruit combat adipogenesis at the molecular level and also induce lipolysis. However, very little is known regarding the role of blueberry polyphenols on adipocyte differentiation. Hence we tested the dose-dependent effects of blueberry polyphenols on mouse 3T3-F442A preadipocyte differentiation and lipolysis. 3T3-F442A preadipocytes were incubated with three doses of blueberry polyphenols (150, 200, and 250 μg/mL [BB-150, BB-200, and BB-250, respectively]), and intracellular lipid content, cell proliferation, and lipolysis were assayed. Blueberry polyphenols suppressed adipocyte differentiation determined by Oil Red-O staining and AdipoRed assay. Intracellular lipid content in control (11,385.51±1,169.6 relative fluorescence units) was significantly higher (P<.05) than with the three doses of blueberry polyphenols (8336.86±503.57, 4235.67±323.17, and 3027.97±346.61, respectively). This corresponds to a reduction of 27%, 63%, and 74%, respectively. Cell proliferation was observed to be significantly higher in the control (0.744±0.035 optical density units) than with BB-150 (0.517±0.031), BB-200 (0.491±0.023), and BB-250 (0.455±0.012). However, when tested for lipolysis, there was no significant difference observed among the groups. We conclude that blueberry polyphenols may play an effective role in inhibiting adipogenesis and cell proliferation.

  1. ATF3 inhibits adipocyte differentiation of 3T3-L1 cells

    SciTech Connect

    Jang, Min Kyung; Kim, Cho Hee; Seong, Je Kyung; Jung, Myeong Ho

    2012-04-27

    Highlights: Black-Right-Pointing-Pointer Overexpression of ATF3 inhibits adipocyte differentiation in 3T3-L1 cells. Black-Right-Pointing-Pointer Overexpression of ATF3 represses C/EBP{alpha} expression. Black-Right-Pointing-Pointer ATF3 directly binds to mouse C/EBP{alpha} promoter spanning from -1928 to -1907. Black-Right-Pointing-Pointer ATF3 may play a role in hypoxia-mediated inhibition of adipocyte differentiation. -- Abstract: ATF3 is a stress-adaptive gene that regulates proliferation or apoptosis under stress conditions. However, the role of ATF3 is unknown in adipocyte cells. Therefore, in this study, we investigated the functional role of ATF3 in adipocytes. Both lentivirus-mediated overexpression of ATF3 and stably-overexpressed ATF3 inhibited adipocyte differentiation in 3T3-L1 cells, as revealed by decreased lipid staining with oil red staining and reduction in adipogenic genes. Thapsigargin treatment and overexpression of ATF3 decreased C/EBP{alpha} transcript and repressed the activity of the 3.6-kb mouse C/EBP{alpha} promoter, demonstrating that ATF3 downregulates C/EBP{alpha} expression. Transfection studies using mutant constructs containing 5 Prime -deletions in the C/EBP{alpha} promoter revealed that a putative ATF/CRE element, GGATGTCA, is located between -1921 and -1914. Electrophoretic mobility shift assay and chromatin immunoprecipitation assay demonstrated that ATF3 directly binds to mouse C/EBP{alpha} promoter spanning from -1928 to -1907. Both chemical hypoxia-mimetics or physical hypoxia led to reduce the C/EBP{alpha} mRNA and repress the promoter activity of the C/EBP{alpha} gene, whereas increase ATF3 mRNA, suggesting that ATF3 may contribute to the inhibition of adipocyte differentiation in hypoxia through downregulation of C/EBP{alpha} expression. Collectively, these results demonstrate that ATF3 represses the C/EBP{alpha} gene, resulting in inhibition of adipocyte differentiation, and thus plays a role in hypoxia-mediated inhibition

  2. Electrophysiological and functional effects of sphingosine-1-phosphate in mouse ventricular fibroblasts

    SciTech Connect

    Benamer, Najate; Bois, Patrick

    2011-04-29

    Highlights: {yields} In cardiac fibroblasts, SUR2/Kir6.1 channel is activated by S1P via the S1P3R. {yields} S1P increases cell proliferation through SUR2/Kir6.1 activation. {yields} S1P decreases collagen and IL-6 secretion through SUR2/Kir6.1 activation. {yields} S1P stimulates fibroblast migration independently from SUR2/Kir6.1 channel. -- Abstract: The aim of this study was to characterize the effects of sphingosine-1-phosphate (S1P) on cardiac ventricular fibroblasts. Impacts of S1P on fibroblast excitability, cell migration, proliferation and secretion were characterized. The patch-clamp technique in the whole-cell configuration was used to study the S1P-induced current from mouse ventricular fibroblasts. The expression level of the S1P receptor during cell culture duration was evaluated by western-blot. Fibroblast proliferation and migration were quantified using the methylene blue assay and the Boyden chamber technique, respectively. Finally, fibroblast secretion properties were estimated by quantification of the IL-6 and collagen levels using ELISA and SIRCOL collagen assays, respectively. We found that S1P activated SUR2/Kir6.1 channel and that this effect was sensitive to specific inhibition of the S1P receptor of type 3 (S1P3R). In contrast, S1P1R receptor inhibition had no effect. Moreover, the S1P-induced current increased with cell culture duration whereas S1P3R expression level remained constant. The activation of SUR2/Kir6.1 channel by S1P via S1P3R stimulated cell proliferation and decreased IL-6 and collagen secretions. S1P also stimulated fibroblast migration via S1P3R but independently from SUR2/Kir6.1 channel activation. This study demonstrates that S1P, via S1P3R, affects cardiac ventricular fibroblasts function independently or through activation of SUR2/Kir6.1 channel. The latter effect occurs after fibroblasts differentiate into myofibroblasts, opening a new potential therapeutic strategy to modulate fibrosis after cardiac

  3. Phosphorylation of the growth arrest-specific protein Gas2 is coupled to actin rearrangements during Go-->G1 transition in NIH 3T3 cells

    PubMed Central

    1994-01-01

    Growth arrest-specific (Gas2) protein has been shown to be a component of the microfilament system, that is highly expressed in growth arrested mouse and human fibroblasts and is hyperphosphorylated upon serum stimulation of quiescent cells. (Brancolini, C., S. Bottega, and C. Schneider. 1992. J. Cell Biol. 117:1251-1261). In this study we demonstrate that the kinetics of Gas2 phosphorylation, during Go-->G1 transition, as induced by addition of 20% FCS to serum starved NIH 3T3 cells, is temporally coupled to the reorganization of actin cytoskeleton. To better dissect the relationship between Gas2 phosphorylation and the modification of the microfilament architecture we used specific stimuli for both membrane ruffling (PDGF and PMA) and stress fiber formation (L-alpha-lysophosphatidic acid LPA) (Ridley, A. J., and A. Hall. 1992. Cell. 70:389-399). All of them, similarly to 20% FCS, are able to downregulate Gas2 biosynthesis. PDGF and PMA induce Gas2 hyperphosphorylation that is temporally coupled with the appearance of membrane ruffling where Gas2 localizes. On the other hand LPA, a specific stimulus for stress fiber formation, fails to induce a detectable Gas2 hyperphosphorylation. Thus, Gas2 hyperphosphorylation is specifically correlated with the formation of membrane ruffling possibly implying a role of Gas2 in this process. PMID:8120096

  4. Nanofiber Alignment Regulates NIH3T3 Cell Orientation and Cytoskeletal Gene Expression on Electrospun PCL+Gelatin Nanofibers

    PubMed Central

    Fee, Timothy; Surianarayanan, Swetha; Downs, Crawford; Zhou, Yong; Berry, Joel

    2016-01-01

    To examine the influence of substrate topology on the behavior of fibroblasts, tissue engineering scaffolds were electrospun from polycaprolactone (PCL) and a blend of PCL and gelatin (PCL+Gel) to produce matrices with both random and aligned nanofibrous orientations. The addition of gelatin to the scaffold was shown to increase the hydrophilicity of the PCL matrix and to increase the proliferation of NIH3T3 cells compared to scaffolds of PCL alone. The orientation of nanofibers within the matrix did not have an effect on the proliferation of adherent cells, but cells on aligned substrates were shown to elongate and align parallel to the direction of substrate fiber alignment. A microarray of cyotoskeleton regulators was probed to examine differences in gene expression between cells grown on an aligned and randomly oriented substrates. It was found that transcriptional expression of eight genes was statistically different between the two conditions, with all of them being upregulated in the aligned condition. The proteins encoded by these genes are linked to production and polymerization of actin microfilaments, as well as focal adhesion assembly. Taken together, the data indicates NIH3T3 fibroblasts on aligned substrates align themselves parallel with their substrate and increase production of actin and focal adhesion related genes. PMID:27196306

  5. Proliferation of mouse fibroblast-like and osteoblast-like cells on pure titanium films manufactured by electron beam melting.

    PubMed

    Kawase, Mayu; Hayashi, Tatsuhide; Asakura, Masaki; Tomino, Masafumi; Mieki, Akimichi; Kawai, Tatsushi

    2016-10-01

    The physical characteristics and biological compatibility of surfaces produced by electron beam melting (EBM) are not well known. In particular, there are not many reports on biocompatibility qualities. In this study, pure Ti films were manufactured using EBM. While it is reported that moderately hydrophilic biomaterial surfaces display improved cell growth and biocompatibility, contact angle measurements on the EBM-produced pure Ti films showed slight hydrophobicity. Nonetheless, we found the cell count of both fibroblast-like cells (L929) and osteoblast-like cells (MC3T3-E1) increased on pure Ti films, especially the MC3T3-E1, which increased more than that of the control. In addition, the morphology of L929 and MC3T3-E1 was polygonal and spindle-shaped and the cytoskeleton was well developed in the pure Ti surface groups. Upon staining with Alizarin red S, a slight calcium deposition was observed and this level gradually rose to a remarkable level. These results indicate that pure Ti films manufactured by EBM have good biocompatibility and could be widely applied as biomedical materials in the near future.

  6. Dermal fibroblasts participate in the formation of new muscle fibres when implanted into regenerating normal mouse muscle.

    PubMed

    Pye, D; Watt, D J

    2001-02-01

    Both in vitro and in vivo studies have described the conversion of fibroblasts to myogenesis when in the presence of dysfunctional myogenic cells. Myogenic conversion of fibroblasts subjected to a normal, as opposed to a diseased muscle environment has only been reported in vitro. The primary aim of this work was to determine if fibroblasts can convert to a myogenic lineage and contribute to new fibre formation when implanted into the regenerating muscle of a normal mouse. Dermal fibroblasts were prepared from neonatal mouse skin and labelled prior to implantation with the fluorescent nuclear marker 4',6-diamidino-2-phenylindole (DAPI). Cells were implanted into muscles of host mice that had been subjected to either cold/crush or minced muscle injury. Some host muscles were x-irradiated to deplete the muscle of endogenous muscle precursor cells. Muscles were removed at 3 wk postimplantation and analysed both histologically and for the presence of DAPI labelled nuclei. Fibres containing DAPI labelled central nuclei indicated that the implanted cells had participated in the regenerative process. Mouse dermal fibroblasts therefore do contribute to muscle fibre formation in regenerating normal mouse muscle but the extent of their contribution is dependent on the nature of the trauma induced in the host muscle. The study also showed that regeneration was more successful in muscles which had not been irradiated, which is contrary to the previous studies where dermal fibroblasts were introduced into myopathic mouse muscle.

  7. Dermal fibroblasts participate in the formation of new muscle fibres when implanted into regenerating normal mouse muscle

    PubMed Central

    PYE, DEBORAH; WATT, DIANA J.

    2001-01-01

    Both in vitro and in vivo studies have described the conversion of fibroblasts to myogenesis when in the presence of dysfunctional myogenic cells. Myogenic conversion of fibroblasts subjected to a normal, as opposed to a diseased muscle environment has only been reported in vitro. The primary aim of this work was to determine if fibroblasts can convert to a myogenic lineage and contribute to new fibre formation when implanted into the regenerating muscle of a normal mouse. Dermal fibroblasts were prepared from neonatal mouse skin and labelled prior to implantation with the fluorescent nuclear marker 4′,6-diamidino-2-phenylindole (DAPI). Cells were implanted into muscles of host mice that had been subjected to either cold/crush or minced muscle injury. Some host muscles were x-irradiated to deplete the muscle of endogenous muscle precursor cells. Muscles were removed at 3 wk postimplantation and analysed both histologically and for the presence of DAPI labelled nuclei. Fibres containing DAPI labelled central nuclei indicated that the implanted cells had participated in the regenerative process. Mouse dermal fibroblasts therefore do contribute to muscle fibre formation in regenerating normal mouse muscle but the extent of their contribution is dependent on the nature of the trauma induced in the host muscle. The study also showed that regeneration was more successful in muscles which had not been irradiated, which is contrary to the previous studies where dermal fibroblasts were introduced into myopathic mouse muscle. PMID:11273041

  8. PIWI Proteins Are Dispensable for Mouse Somatic Development and Reprogramming of Fibroblasts into Pluripotent Stem Cells

    PubMed Central

    Cheng, Ee-Chun; Kang, Dongwan; Wang, Zhong; Lin, Haifan

    2014-01-01

    PIWI proteins play essential and conserved roles in germline development, including germline stem cell maintenance and meiosis. Because germline regulators such as OCT4, NANOG, and SOX2 are known to be potent factors that reprogram differentiated somatic cells into induced pluripotent stem cells (iPSCs), we investigated whether the PIWI protein family is involved in iPSC production. We find that all three mouse Piwi genes, Miwi, Mili, and Miwi2, are expressed in embryonic stem cells (ESCs) at higher levels than in fibroblasts, with Mili being the highest. However, mice lacking all three Piwi genes are viable and female fertile, and are only male sterile. Furthermore, embryonic fibroblasts derived from Miwi/Mili/Miwi2 triple knockout embryos can be efficiently reprogrammed into iPS cells. These iPS cells expressed pluripotency markers and were capable of differentiating into all three germ layers in teratoma assays. Genome-wide expression profiling reveals that the triple knockout iPS cells are very similar to littermate control iPS cells. These results indicate that PIWI proteins are dispensable for direct reprogramming of mouse fibroblasts. PMID:25238487

  9. Immunization of stromal cell targeting fibroblast activation protein providing immunotherapy to breast cancer mouse model.

    PubMed

    Meng, Mingyao; Wang, Wenju; Yan, Jun; Tan, Jing; Liao, Liwei; Shi, Jianlin; Wei, Chuanyu; Xie, Yanhua; Jin, Xingfang; Yang, Li; Jin, Qing; Zhu, Huirong; Tan, Weiwei; Yang, Fang; Hou, Zongliu

    2016-08-01

    Unlike heterogeneous tumor cells, cancer-associated fibroblasts (CAF) are genetically more stable which serve as a reliable target for tumor immunotherapy. Fibroblast activation protein (FAP) which is restrictively expressed in tumor cells and CAF in vivo and plays a prominent role in tumor initiation, progression, and metastasis can function as a tumor rejection antigen. In the current study, we have constructed artificial FAP(+) stromal cells which mimicked the FAP(+) CAF in vivo. We immunized a breast cancer mouse model with FAP(+) stromal cells to perform immunotherapy against FAP(+) cells in the tumor microenvironment. By forced expression of FAP, we have obtained FAP(+) stromal cells whose phenotype was CD11b(+)/CD34(+)/Sca-1(+)/FSP-1(+)/MHC class I(+). Interestingly, proliferation capacity of the fibroblasts was significantly enhanced by FAP. In the breast cancer-bearing mouse model, vaccination with FAP(+) stromal cells has significantly inhibited the growth of allograft tumor and reduced lung metastasis indeed. Depletion of T cell assays has suggested that both CD4(+) and CD8(+) T cells were involved in the tumor cytotoxic immune response. Furthermore, tumor tissue from FAP-immunized mice revealed that targeting FAP(+) CAF has induced apoptosis and decreased collagen type I and CD31 expression in the tumor microenvironment. These results implicated that immunization with FAP(+) stromal cells led to the disruption of the tumor microenvironment. Our study may provide a novel strategy for immunotherapy of a broad range of cancer.

  10. Regulation of Na+-H+ exchange in normal NIH-3T3 cells and in NIH-3T3 cells expressing the ras oncogene

    SciTech Connect

    Owen, N.E.; Knapik, J.; Strebel, F.; Tarpley, W.G.; Gorman, R.R.

    1989-04-01

    Our laboratory and others have demonstrated that Na+-H+ exchange can be regulated by two different pathways; one that is mediated by an inositol trisphosphate-stimulated increase in intracellular calcium activity, and one that is mediated by an increase in protein kinase C activity. To determine whether one of these pathways is more important than the other, or whether one pathway is physiologically relevant, we employed normal NIH-3T3 cells (3T3 cells) and NIH-3T3 cells expressing the EJ human bladder ras oncogene (EJ cells). The EJ cells were chosen because they provide a genetic model that does not exhibit serum- or platelet-derived growth factor (PDGF)-stimulated inositol trisphosphate release or Ca2+ mobilization. It was found that serum- or PDGF-stimulated Na+-H+ exchange was more pronounced in EJ cells than in control 3T3 cells. As expected, serum- or PDGF-stimulated Na+-H+ exchange in 3T3 cells was inhibited by chelating intracellular Ca2+ with the intracellular Ca2+ chelator quin2, by the intracellular Ca2+ antagonist 8-(N,N-diethylamino)octyl 3,4,5-trimethoxybenzoate (TMB-8), and by the calmodulin antagonist trifluoperazine. In contrast, these agents did not inhibit serum- or PDGF-stimulated Na+-H+ exchange in EJ cells. Activators of protein kinase C (e.g., 1-oleoyl-2-acetylglycerol or biologically active phorbol esters) were found to stimulate Na+-H+ exchange in EJ cells to the same extent as serum. However, these agents were considerably less effective than serum in control 3T3 cells. Despite these findings, PDGF did not stimulate diacylglycerol levels in EJ cells.

  11. Fibulin-6 regulates pro-fibrotic TGF-β responses in neonatal mouse ventricular cardiac fibroblasts

    PubMed Central

    Chowdhury, Arpita; Hasselbach, Lisa; Echtermeyer, Frank; Jyotsana, Nidhi; Theilmeier, Gregor; Herzog, Christine

    2017-01-01

    Fibulin-6, an essential component of extracellular matrix determines the architecture of cellular junctions in tissues undergoing strain. Increased expression and deposition of fibulin-6 facilitates fibroblast migration in response to TGF-β, following myocardial infarction in mouse heart. The underlying mechanism still remains elusive. In conjunction with our previous study, we have now demonstrated that in fibulin-6 knockdown (KD) fibroblasts, not only TGF-β dependent migration, but also stress fiber formation, cellular networking and subsequently fibroblast wound contraction is almost abrogated. SMAD dependent TGF-β pathway shows ~75% decreased translocation of R-SMAD and co-SMAD into the nucleus upon fibulin-6 KD. Consequently, SMAD dependent pro-fibrotic gene expression is considerably down regulated to basal levels both in mRNA and protein. Also, investigating the non-SMAD pathways we observed a constitutive increase in pERK-levels in fibulin-6 KD fibroblast compared to control, but no change was seen in pAKT. Immunoprecipitation studies revealed 60% reduced interaction of TGF-β receptor II and I (TGFRII and I) accompanied by diminished phosphorylation of TGFRI at serin165 in fibulin-6 KD cells. In conclusion, fibulin-6 plays an important role in regulating TGF-β mediated responses, by modulating TGF-β receptor dimerization and activation to further trigger downstream pathways. PMID:28209981

  12. BMPs functionally replace Klf4 and support efficient reprogramming of mouse fibroblasts by Oct4 alone

    PubMed Central

    Chen, Jiekai; Liu, Jing; Yang, Jiaqi; Chen, You; Chen, Jing; Ni, Su; Song, Hong; Zeng, Lingwen; Ding, Ke; Pei, Duanqing

    2011-01-01

    Generation of induced pluripotent stem cells by defined factors has become a useful model to investigate the mechanism of reprogramming and cell fate determination. However, the precise mechanism of factor-based reprogramming remains unclear. Here, we show that Klf4 mainly acts at the initial phase of reprogramming to initiate mesenchymal-to-epithelial transition and can be functionally replaced by bone morphogenetic proteins (BMPs). BMPs boosted the efficiency of Oct4/Sox2-mediated reprogramming of mouse embryonic fibroblasts (MEFs) to ∼1%. BMPs also promoted single-factor Oct4-based reprogramming of MEFs and tail tibial fibroblasts. Our studies clarify the contribution of Klf4 in reprogramming and establish Oct4 as a singular setter of pluripotency in differentiated cells. PMID:21135873

  13. Exendin-4 promotes proliferation and differentiation of MC3T3-E1 osteoblasts by MAPKs activation.

    PubMed

    Feng, Yingyu; Su, Lei; Zhong, Xing; Guohong, Wei; Xiao, Haipeng; Li, Yanbing; Xiu, Lingling

    2016-04-01

    Glucagon-like peptide-1 (GLP1) and its receptor agonist have been previously reported to play a positive role in bone metabolism in aged ovariectomized rats and insulin-resistant models. However, whether GLP1 has a direct effect on the proliferation and differentiation of osteoblasts or any cellular mechanism for this potential role is unknown. We examined the effects of the GLP1 receptor agonist exendin-4 on the proliferation, differentiation, and mineralization of mouse osteoblastic MC3T3-E1 cells. GLP1 receptor was detected in MC3T3-E1 cells by polymerase chain reaction (PCR) and Western blot assay. Cell proliferation was assessed using MTT assay, revealing that exendin-4 increased cell proliferation at effective concentrations between 10(-10) and 10(-5) M. Quantitative PCR analysis showed that exendin-4 increased the mRNA expression of the differentiation markers alkaline phosphatase (ALP), collagen-1 (COL1), osteocalcin (OC), and runt-related transcription factor 2 (RUNX2) under osteogenic conditions. Alizarin red staining confirmed that 10(-7) M exendin-4 increased osteoblast mineralization by 18.7%. Exendin-4 upregulated the phosphorylation of ERK1/2, p38, and JNK, with the peak effect at 1.5 h in the Western blot analysis. The use of selective MAPK inhibitors, namely PD98059, SB203580, and SP600125, blocked the effects of exendin-4 on kinase activation (ERK1/2, p38, and JNK), as well as cell proliferation and differentiation in MC3T3-E1 cells. These findings demonstrate that exendin-4 promotes both the proliferation and differentiation of preosteoblasts MC3T3-E1 via activation of the MAPK pathway.

  14. Functional expression of 5-HT{sub 2A} receptor in osteoblastic MC3T3-E1 cells

    SciTech Connect

    Hirai, Takao; Kaneshige, Kota; Kurosaki, Teruko; Nishio, Hiroaki

    2010-05-28

    In the previous study, we reported the gene expression for proteins related to the function of 5-hydroxytryptamine (5-HT, serotonin) and elucidated the expression patterns of 5-HT{sub 2} receptor subtypes in mouse osteoblasts. In the present study, we evaluated the possible involvement of 5-HT receptor subtypes and its inactivation system in MC3T3-E1 cells, an osteoblast cell line. DOI, a 5-HT{sub 2A} and 5-HT{sub 2C} receptor selective agonist, as well as 5-HT concentration-dependently increased proliferative activities of MC3T3-E1 cells in their premature period. This effect of 5-HT on cell proliferation were inhibited by ketanserin, a 5-HT{sub 2A} receptor specific antagonist. Moreover, both DOI-induced cell proliferation and phosphorylation of ERK1 and 2 proteins were inhibited by PD98059 and U0126, selective inhibitors of MEK in a concentration-dependent manner. Furthermore, treatment with fluoxetine, a 5-HT specific re-uptake inhibitor which inactivate the function of extracellular 5-HT, significantly increased the proliferative activities of MC3T3-E1 cells in a concentration-dependent manner. Our data indicate that 5-HT fill the role for proliferation of osteoblast cells in their premature period. Notably, 5-HT{sub 2A} receptor may be functionally expressed to regulate mechanisms underlying osteoblast cell proliferation, at least in part, through activation of ERK/MAPK pathways in MC3T3-E1 cells.

  15. Lambda-Chain Production in Human Lymphoblast-Mouse Fibroblast Hybrids

    PubMed Central

    Orkin, Stuart H.; Buchanan, Philip D.; Yount, William J.; Reisner, Howard; Littlefield, John W.

    1973-01-01

    Mutant human lymphoblast cells deficient in hypoxanthine phosphoribosyltransferase (EC 2.4.2.8) activity were hybridized with thymidine kinase (EC 2.7.1.21)-deficient mouse fibroblasts. Hybrid cells were readily selected, as both parental lines were nonreverting and eliminated by hypoxanthine-amethopterinthymidine medium. Human lambda (λ) chain was the only immunoglobulin chain produced by the lymphoblast parent, as determined by immunofluorescent techniques. Two independent hybrid clones chosen for detailed study synthesized human λ chain, and continued to do so after prolonged culture. As in both parental lines, no human immunoglobulin heavy chains, complements C3 or C4, or α1-antitrypsin, or mouse immunoglobulin chains or complement C5 were detectable in the hybrids. Selection against thymidine kinase-containing hybrid cells with 5-bromodeoxyuridine did not eliminate positive λ-chain reactivity, suggesting that the kinase and λ-chain loci are not linked. The continued production of an immunoglobulin chain by human lymphoblast-mouse fibroblast hybrids contrasts with the extinction of other differentiated functions in several hybrid systems, and indicates that gene localization and linkage analysis for human immunoglobulin chains should be feasible with this system. Images PMID:4599625

  16. Production of glycosylated physiologically "normal" human alpha 1-antitrypsin by mouse fibroblasts modified by insertion of a human alpha 1-antitrypsin cDNA using a retroviral vector.

    PubMed Central

    Garver, R I; Chytil, A; Karlsson, S; Fells, G A; Brantly, M L; Courtney, M; Kantoff, P W; Nienhuis, A W; Anderson, W F; Crystal, R G

    1987-01-01

    Alpha 1-Antitrypsin (alpha 1AT) deficiency is a hereditary disorder characterized by reduced serum levels of alpha 1AT, resulting in destruction of the lower respiratory tract by neutrophil elastase. As an approach to augment alpha 1AT levels in this disorder with physiologically normal human alpha 1AT, we have integrated a full-length normal human alpha 1AT cDNA into the genome of mouse fibroblasts. To accomplish this, the retroviral vector N2 was modified by inserting the simian virus 40 early promoter followed by the alpha 1AT cDNA. Southern analysis demonstrated that the intact cDNA was present in the genome of selected clones of the transfected murine fibroblasts psi 2 and infected NIH 3T3. The clones produced three mRNA transcripts (5.8, 4.8, and 2.4 kilobases) containing human alpha 1AT sequences, secreted an alpha 1AT molecule recognized by an anti-human alpha 1AT antibody, with the same molecular mass (52 kDa) as normal human alpha 1AT and that complexed with and inhibited human neutrophil elastase. The psi 2 produced alpha 1AT was glycosylated, and when infused intravenously into mice, it had a serum half-life similar to normal alpha 1AT purified from human plasma and markedly longer than that of nonglycosylated human alpha 1AT cDNA-directed yeast-produced alpha 1AT. These studies demonstrate the feasibility of using a retroviral vector to insert the normal human alpha 1AT cDNA into non-alpha 1AT-producing cells, resulting in the synthesis and secretion of physiologically "normal" human alpha 1AT. Images PMID:3029759

  17. Conditional knockout of N-WASP in mouse fibroblast caused keratinocyte hyper proliferation and enhanced wound closure

    PubMed Central

    Jain, Neeraj; Kalailingam, Pazhanichamy; Tan, Kai Wei; Tan, Hui Bing; Sng, Ming Keat; Chan, Jeremy Soon Kiat; Tan, Nguan Soon; Thanabalu, Thirumaran

    2016-01-01

    Neural-Wiskott Aldrich Syndrome Protein (N-WASP) is expressed ubiquitously, regulates actin polymerization and is essential during mouse development. We have previously shown that N-WASP is critical for cell-ECM adhesion in fibroblasts. To characterize the role of N-WASP in fibroblast for skin development, we generated a conditional knockout mouse model in which fibroblast N-WASP was ablated using the Cre recombinase driven by Fibroblast Specific Protein promoter (Fsp-Cre). N-WASPFKO (N-WASPfl/fl; Fsp-cre) were born following Mendelian genetics, survived without any visible abnormalities for more than 1 year and were sexually reproductive, suggesting that expression of N-WASP in fibroblast is not critical for survival under laboratory conditions. Histological sections of N-WASPFKO mice skin (13 weeks old) showed thicker epidermis with higher percentage of cells staining for proliferation marker (PCNA), suggesting that N-WASP deficient fibroblasts promote keratinocyte proliferation. N-WASPFKO mice skin had elevated collagen content, elevated expression of FGF7 (keratinocyte growth factor) and TGFβ signaling proteins. Wound healing was faster in N-WASPFKO mice compared to control mice and N-WASP deficient fibroblasts were found to have enhanced collagen gel contraction properties. These results suggest that N-WASP deficiency in fibroblasts improves wound healing by growth factor-mediated enhancement of keratinocyte proliferation and increased wound contraction in mice. PMID:27909303

  18. Increased Oxidative Stress in Cultured 3T3-L1 Cells was Attenuated by Berberine Treatment.

    PubMed

    Dong, Shi-Fen; Yasui, Naomi; Negishb, Hiroko; Kishimoto, Aya; Sun, Jian-Ning; Ikeda, Katsumi

    2015-06-01

    The 3T3-L1 cell line is one of the most well-characterized and reliable models for studying adipocytes. Increased oxidative stress in accumulated fat was found in 3T3-L1 cells. Berberine, an isoquinoline alkaloid, could suppress fat deposition in 3T3-L1 cells; however, whether berberine suppresses increased oxidative stress is not well known. In this study, we observed the effect of berberine on increased oxidative stress in 3T3-L1 cells. 3T3-L1 cells were cultured and treated with berberine (5-20 μM) from day 3 to day 8. We confirmed that berberine markedly inhibited fat accumulation and lipid droplets in 3T3-L1 adipocytes and decreased triglyceride content. Berberine inhibited increased oxidative stress in 3T3-L1 cells by suppressing reactive oxygen species (ROS) production, and increased glutathione peroxidase (GPx) gene expression and GPx activity. Berberine also markedly reduced adipokines secreted by adipocytes, including leptin and resistin.

  19. Active form Notch4 promotes the proliferation and differentiation of 3T3-L1 preadipocytes

    SciTech Connect

    Lai, Peng-Yeh; Tsai, Chong-Bin; Tseng, Min-Jen

    2013-01-18

    Highlights: ► Notch4IC modulates the ERK pathway and cell cycle to promote 3T3-L1 proliferation. ► Notch4IC facilitates 3T3-L1 differentiation by up-regulating proadipogenic genes. ► Notch4IC promotes proliferation during the early stage of 3T3-L1 adipogenesis. ► Notch4IC enhances differentiation during subsequent stages of 3T3-L1 adipogenesis. -- Abstract: Adipose tissue is composed of adipocytes, which differentiate from precursor cells in a process called adipogenesis. Many signal molecules are involved in the transcriptional control of adipogenesis, including the Notch pathway. Previous adipogenic studies of Notch have focused on Notch1 and HES1; however, the role of other Notch receptors in adipogenesis remains unclear. Q-RT-PCR analyses showed that the augmentation of Notch4 expression during the differentiation of 3T3-L1 preadipocytes was comparable to that of Notch1. To elucidate the role of Notch4 in adipogenesis, the human active form Notch4 (N4IC) was transiently transfected into 3T3-L1 cells. The expression of HES1, Hey1, C/EBPδ and PPARγ was up-regulated, and the expression of Pref-1, an adipogenic inhibitor, was down-regulated. To further characterize the effect of N4IC in adipogenesis, stable cells expressing human N4IC were established. The expression of N4IC promoted proliferation and enhanced differentiation of 3T3-L1 cells compared with those of control cells. These data suggest that N4IC promoted proliferation through modulating the ERK pathway and the cell cycle during the early stage of 3T3-L1 adipogenesis and facilitated differentiation through up-regulating adipogenic genes such as C/EBPα, PPARγ, aP2, LPL and HSL during the middle and late stages of 3T3-L1 adipogenesis.

  20. Imaging collagen remodeling and sensing transplanted autologous fibroblast metabolism in mouse dermis using multimode nonlinear optical imaging

    NASA Astrophysics Data System (ADS)

    Zhuo, Shuangmu; Chen, Jianxin; Cao, Ning; Jiang, Xingshan; Xie, Shusen; Xiong, Shuyuan

    2008-06-01

    Collagen remodeling and transplanted autologous fibroblast metabolic states in mouse dermis after cellular injection are investigated using multimode nonlinear optical imaging. Our findings show that the technique can image the progress of collagen remodeling in mouse dermis. It can also image transplanted autologous fibroblasts in their collagen matrix environment in the dermis, because of metabolic activity. It was also found that the approach can provide two-photon ratiometric redox fluorometry based on autologous fibroblast fluorescence from reduced nicotinamide adenine dinucleotide coenzyme and oxidized flavoproteins for sensing the autologous fibroblast metabolic state. These results show that the multimode nonlinear optical imaging technique may have potential in a clinical setting as an in vivo diagnostic and monitoring system for cellular therapy in plastic surgery.

  1. Exogenous fibroblast growth factor 8 rescues development of mouse diastemal vestigial tooth ex vivo.

    PubMed

    Li, Lu; Yuan, Guohua; Liu, Chao; Zhang, Lu; Zhang, Yanding; Chen, YiPing; Chen, Zhi

    2011-06-01

    Regression of vestigial tooth buds results in the formation of the toothless diastema, a unique feature of the mouse dentition. Revitalization of the diastemal vestigial tooth bud provides an excellent model for studying tooth regeneration and replacement. It has been previously shown that suppression of fibroblast growth factor (FGF) signaling in the diastema results in vestigial tooth bud regression. In this study, we report that application of exogenous FGF8 to the mouse embryonic diastemal region rescues diastemal tooth development. However, this rescue of diastemal tooth development occurs only in an isolated diastemal regions and not in the mandibular quadrant, which includes the incisor and molar germs. FGF8 promotes cell proliferation and inhibits apoptosis in diastemal tooth epithelium, and revitalizes the tooth developmental program, as evidenced by the expression of genes critical for normal tooth development. Our results also support the idea that the adjacent tooth germs contribute to the suppression of diastemal vestigial tooth buds by means of multiple signals.

  2. Assessment of the phototoxic hazard of some essential oils using modified 3T3 neutral red uptake assay.

    PubMed

    Dijoux, Nathalie; Guingand, Yannick; Bourgeois, Caroline; Durand, Sandrine; Fromageot, Claude; Combe, Corinne; Ferret, Pierre-Jacques

    2006-06-01

    When substances are developed in the aim to be a constituent of personal care products, and to be applied on the skin, it is necessary to carry out an assessment of potential phototoxic hazard. Phototoxicity is skin reaction caused by concurrent topical or systemic exposure to specific molecule and ultraviolet radiation. Most phototoxic compounds absorb energy particularly from UVA light leading to the generation of activated derivatives which can induce cellular damage. This type of adverse cutaneous response can be reproduced in vitro using different models of phototoxicity such as the validated 3T3 Neutral Red Uptake (NRU) phototoxicity assay. In the present study we utilised two different cell lines (the murine fibroblastic cell line 3T3 and the rabbit cornea derived cell line SIRC) to compare the photo-irritation potential of a strong phototoxic compound, chlorpromazine, to a weaker composite, such as 8-methoxypsoralen and Bergamot oil. After comparison of the different systems, five other essential oils were tested with both cell lines. Cellular damage was evaluated by the NRU cytotoxicity test or by MTT conversion test.

  3. EGF raises cytosolic Ca sup 2+ in A431 and Swiss 3T3 cells by a dual mechanism

    SciTech Connect

    Pandiella, A.; Malgaroli, A.; Meldolesi, J.; Vicentini, L.M. )

    1987-05-01

    The changes in Ca{sup 2+} homeostasis and phosphoinositide hydrolysis induced by EGF were studied in human epidermoid carcinoma A431 cells both when attached to a substratum and after detachment and suspension. The cytosolic Ca{sup 2+} concentration was measured by the conventional fluorimetric technique, using the specific probe, quin2, as well as by a new microscopic technique in which single cells are investigated after loading with another probe, fura-2. EGF applied in the complete, Ca{sup 2+}-containing medium caused a rapid rise in the cytosolic {sup 45}Ca{sup 2+} concentration, that remained elevated for several minutes. In Ca{sup 2+}-free, EGTA-containing medium, part of this response persisted, as revealed by quin2 results in suspended cells and microscopic results with fura-2. These results, as well as additional microscopic fura-2 results in Swiss 3T3 fibroblasts, demonstrate that the Ca{sup 2+} signal elicited by EGF is due to two components: redistribution from an intracellular store and stimulated influx across the plasmalemma. This latter process was not detected in 3T3 cells treated with either PDGF or bombesin. It is therefore suggested that the {sup 45}Ca{sup 2+} influx effect of EGF is under the control of a separate, as yet unidentified mechanism.

  4. Comparison of the metabolic activation of environmental carcinogens in mouse embryonic stem cells and mouse embryonic fibroblasts

    PubMed Central

    Krais, Annette M.; Mühlbauer, Karl-Rudolf; Kucab, Jill E.; Chinbuah, Helena; Cornelius, Michael G.; Wei, Quan-Xiang; Hollstein, Monica; Phillips, David H.; Arlt, Volker M.; Schmeiser, Heinz H.

    2015-01-01

    We compared mouse embryonic stem (ES) cells and fibroblasts (MEFs) for their ability to metabolically activate the environmental carcinogens benzo[a]pyrene (BaP), 3-nitrobenzanthrone (3-NBA) and aristolochic acid I (AAI), measuring DNA adduct formation by 32P-postlabelling and expression of xenobiotic-metabolism genes by quantitative real-time PCR. At 2 μM, BaP induced Cyp1a1 expression in MEFs to a much greater extent than in ES cells and formed 45 times more adducts. Nqo1 mRNA expression was increased by 3-NBA in both cell types but induction was higher in MEFs, as was adduct formation. For AAI, DNA binding was over 450 times higher in MEFs than in ES cells, although Nqo1 and Cyp1a1 transcriptional levels did not explain this difference. We found higher global methylation of DNA in ES cells than in MEFs, which suggests higher chromatin density and lower accessibility of the DNA to DNA damaging agents in ES cells. However, AAI treatment did not alter DNA methylation. Thus mouse ES cells and MEFs have the metabolic competence to activate a number of environmental carcinogens, but MEFs have lower global DNA methylation and higher metabolic capacity than mouse ES cells. PMID:25230394

  5. Dielectrophoretic differentiation of mouse ovarian surface epithelial cells, macrophages, and fibroblasts using contactless dielectrophoresis

    PubMed Central

    Salmanzadeh, Alireza; Kittur, Harsha; Sano, Michael B.; C. Roberts, Paul; Schmelz, Eva M.; Davalos, Rafael V.

    2012-01-01

    Ovarian cancer is the leading cause of death from gynecological malignancies in women. The primary challenge is the detection of the cancer at an early stage, since this drastically increases the survival rate. In this study we investigated the dielectrophoretic responses of progressive stages of mouse ovarian surface epithelial (MOSE) cells, as well as mouse fibroblast and macrophage cell lines, utilizing contactless dielectrophoresis (cDEP). cDEP is a relatively new cell manipulation technique that has addressed some of the challenges of conventional dielectrophoretic methods. To evaluate our microfluidic device performance, we computationally studied the effects of altering various geometrical parameters, such as the size and arrangement of insulating structures, on dielectrophoretic and drag forces. We found that the trapping voltage of MOSE cells increases as the cells progress from a non-tumorigenic, benign cell to a tumorigenic, malignant phenotype. Additionally, all MOSE cells display unique behavior compared to fibroblasts and macrophages, representing normal and inflammatory cells found in the peritoneal fluid. Based on these findings, we predict that cDEP can be utilized for isolation of ovarian cancer cells from peritoneal fluid as an early cancer detection tool. PMID:22536308

  6. Expression of the invertebrate sea urchin P16 protein into mammalian MC3T3 osteoblasts transforms and reprograms them into “osteocyte-like” cells

    PubMed Central

    Alvares, Keith; Ren, Yinshi; Feng, Jian Q.; Veis, Arthur

    2015-01-01

    P16 is an acidic phosphoprotein important in both sea urchin embryonic spicule development and transient mineralization during embryogenesis, and syncytium formation and mineralization in mature urchin tooth. Anti-P16 has been used to localize P16 to the syncytial membranes and the calcite mineral. Specific amino acid sequence motifs in P16 are similar to sequences in DSPP a protein common to all vertebrate teeth, and crucial for their mineralization. Here we examine the effect of P16 on vertebrate fibroblastic NIH3T3 cells and osteoblastic MC3T3 cells. Transfection of NIH3T3 cells with P16 cDNA resulted in profound changes in the morphology of the cells. In culture the transfected cells sent out long processes that contacted processes from neighboring cells forming networks or syncytia. There was a similar change in morphology in cultured osteoblastic MC3T3 cells. In addition, the MC3T3 developed numerous dendrites as found in osteocytes. Importantly, there was also a change in the expression of the osteoblast and osteocyte specific genes. MC3T3 cells transfected with P16 showed an 18 fold increase in expression of the osteocyte specific Dentin matrix protein (DMP1) gene, accompanied by decreased expression of osteoblast specific genes: Bone sialoprotein (BSP), osteocalcin (OCN) and β-catenin decreased by 70%, 64% and 68 %, respectively. Thus, invertebrate urchin P16 with no previously known analog in vertebrates was able to induce changes in both cell morphology and gene expression, converting vertebrate-derived osteoblast-like precursor cells to an “osteocyte-like” phenotype, an important process in bone biology. The mechanisms involved are presently under study. PMID:26581835

  7. MicroRNA-125b-5p inhibits proliferation and promotes adipogenic differentiation in 3T3-L1 preadipocytes.

    PubMed

    Ouyang, Dan; Ye, Yaqiong; Guo, Dongguang; Yu, Xiaofang; Chen, Jian; Qi, Junjie; Tan, Xiaotong; Zhang, Yuan; Ma, Yongjiang; Li, Yugu

    2015-05-01

    Previous evidence has indicated that the microRNA-125b (miR-125b) family plays important roles in the regulation of cancer cell growth, development, differentiation, and apoptosis. However, whether they contribute to the process of adipocyte differentiation remains unclear. In the present study, we revealed that the expression level of miR-125b-5p, a member of miR-125b family, was dramatically up-regulated during differentiation of 3T3-L1 preadipocyte into mature adipocyte. Supplement of miR-125b-5p into 3T3-L1 cells promoted adipogenic differentiation as evidenced by increased lipid droplets and mRNA levels of adipocyte-specific molecular markers, including peroxisome proliferators-activated receptor γ, CCAAT/enhancer-binding protein α, fatty acid-binding protein 4, and lipoprotein lipase, and by triglyceride accumulation. CCK-8 assay showed that miR-125b-5p supplementation significantly inhibited cell proliferation. Flow cytometry analysis showed that miR-125b-5p impaired G1/S phase transition as well as the mRNA and protein expression of G1/S-related genes, such as Cyclin D2, Cyclin D3, and CDK4. Nevertheless, it had no effect on apoptosis. Additionally, by target gene prediction, we demonstrated that smad4 may be a potential target of miR-125b-5p in mouse 3T3-L1 preadipocytes, accounting for some of miR-125b-5p's functions. Taken together, these data indicated that miR-125b-5p may serve as an important positive regulator in adipocyte differentiation, at least partially through down-regulating smad4.

  8. Neuronal and astrocyte dysfunction diverges from embryonic fibroblasts in the Ndufs4fky/fky mouse

    PubMed Central

    Bird, Matthew J.; Wijeyeratne, Xiaonan W.; Komen, Jasper C.; Laskowski, Adrienne; Ryan, Michael T.; Thorburn, David R.; Frazier, Ann E.

    2014-01-01

    Mitochondrial dysfunction causes a range of early-onset neurological diseases and contributes to neurodegenerative conditions. The mechanisms of neurological damage however are poorly understood, as accessing relevant tissue from patients is difficult, and appropriate models are limited. Hence, we assessed mitochondrial function in neurologically relevant primary cell lines from a CI (complex I) deficient Ndufs4 KO (knockout) mouse (Ndufs4fky/fky) modelling aspects of the mitochondrial disease LS (Leigh syndrome), as well as MEFs (mouse embryonic fibroblasts). Although CI structure and function were compromised in all Ndufs4fky/fky cell types, the mitochondrial membrane potential was selectively impaired in the MEFs, correlating with decreased CI-dependent ATP synthesis. In addition, increased ROS (reactive oxygen species) generation and altered sensitivity to cell death were only observed in Ndufs4fky/fky primary MEFs. In contrast, Ndufs4fky/fky primary isocortical neurons and primary isocortical astrocytes displayed only impaired ATP generation without mitochondrial membrane potential changes. Therefore the neurological dysfunction in the Ndufs4fky/fky mouse may partly originate from a more severe ATP depletion in neurons and astrocytes, even at the expense of maintaining the mitochondrial membrane potential. This may provide protection from cell death, but would ultimately compromise cell functionality in neurons and astrocytes. Furthermore, RET (reverse electron transfer) from complex II to CI appears more prominent in neurons than MEFs or astrocytes, and is attenuated in Ndufs4fky/fky cells. PMID:25312000

  9. Platelet-derived growth factor stimulation of (/sup 3/H)-glucosamine incorporation in density-arrested BALB/c-3T3 cells

    SciTech Connect

    Harrington, M.A.; Wharton, W.; Pledger, W.J.

    1987-01-01

    G/sub 0//G/sub 1/ traverse in density-arrested BALB/c-3T3 cells is controlled by multiple serum-derived growth factors. Platelet-derived growth factor (PDGF) initiates a proliferative response, whereas factors present in plasma facilitate progression through G/sub 0//G/sub 1/. In the absence of competence formation, progression factors are unable to stimulate cell cycle traverse. The authors have identified the stimulation of a biochemical process specific to competence formation in BALB/c-3T3 cells. PDGF treated BALB/c-3T3 cells incorporated 5-10 fold more (/sup 3/H)-glucosamine (GlcN) into acid-insoluble material as compared to platelet-poor plasma (PPP) treated cultures. Increased GlcN incorporation occurred in density-arrested BALB/c-3T3 cells in response to treatment with other competence factors, fibroblast growth factor, and Ca/sub 3/ (PO/sub 4/)/sub 2/ and was not due to cell-cycle traverse. Stimulation of (/sup 3/H)-GlcN incorporation by PDGF was time dependent, and increased incorporation of (/sup 3/H)-GlcN into protein required de novo protein synthesis. Several mechanisms through which PDGF could increase GlcN incorporation into cellular material were examined. Results of these studies suggest an increase in the cellular capacity to glycosylate proteins is a response to or a part of competence formation.

  10. Cytotoxicity of Titanate-Calcium Complexes to MC3T3 Osteoblast-Like Cells

    PubMed Central

    Drury, Jeanie L.; Moussi, Joelle; Taylor-Pashow, Kathryn M. L.

    2016-01-01

    Monosodium titanates (MST) are a relatively novel form of particulate titanium dioxide that have been proposed for biological use as metal sorbents or delivery agents, most recently calcium (II). In these roles, the toxicity of the titanate or its metal complex is crucial to its biological utility. The aim of this study was to determine the cytotoxicity of MST and MST-calcium complexes with MC3T3 osteoblast-like cells; MST-Ca(II) complexes could be useful to promote bone formation in various hard tissue applications. MC3T3 cells were exposed to native MST or MST-Ca(II) complexes for 24–72 h. A CellTiter-Blue® assay was employed to assess the metabolic activity of the cells. The results showed that MST and MST-Ca(II) suppressed MC3T3 metabolic activity significantly in a dose-, time-, and cell-density-dependent fashion. MST-Ca(II) suppressed MC3T3 metabolism in a statistically identical manner as native MST at all concentrations. We concluded that MST and MST-Ca(II) are significantly cytotoxic to MC3T3 cells through a mechanism yet unknown; this is a potential problem to the biological utility of these complexes. PMID:28044136

  11. Protein turnover and cellular autophagy in growing and growth-inhibited 3T3 cells

    SciTech Connect

    Papadopoulos, T.; Pfeifer, U. )

    1987-07-01

    The relationship between growth, protein degradation, and cellular autophagy was tested in growing and in growth-inhibited 3T3 cell monolayers. For the biochemical evaluation of DNA and protein metabolism, growth-inhibited 3T3 cell monolayers with high cell density and growing 3T3 cell monolayers with low cell density were labeled simultaneously with ({sup 14}C)thymidine and ({sup 3}H)leucine. The evaluation of the DNA turnover and additional ({sup 3}H)thymidine autoradiography showed that 24 to 5% of 3T3 cells continue to replicate even in the growth-inhibited state, where no accumulation of protein and DNA can be observed. Cell loss, therefore, has to be assumed to compensate for the ongoing cell proliferation. When the data of protein turnover were corrected for cell loss, it was found that the rate constant of protein synthesis in nongrowing monolayers was reduced to half the value found in growing monolayers. Simultaneously, the rate constant of protein degradation in nongrowing monolayers was increased to about 1.5-fold the value of growing monolayers. These data are in agreement with the assumption that cellular autophagy represents a major pathway of regulating protein degradation in 3T3 cells and that the regulation of autophagic protein degradation is of relevance for the transition from a growing to a nongrowing state.

  12. Spindlin1, a novel nuclear protein with a role in the transformation of NIH3T3 cells.

    PubMed

    Gao, Yanhong; Yue, Wen; Zhang, Peng; Li, Li; Xie, Xiaoyan; Yuan, Hongfeng; Chen, Lin; Liu, Daqing; Yan, Fang; Pei, Xuetao

    2005-09-23

    spindlin1, a novel human gene recently isolated by our laboratory, is highly homologous to mouse spindlin gene. In this study, we cloned cDNA full-length of this novel gene and send it to GenBank database as spindlin1 (Homo sapiens spindlin1) with Accession No. AF317228. In order to investigate the function of spindlin1, we studied further the subcellular localization of Spindlin1 protein and the effects of spindlin1 overexpression in NIH3T3 cells. The results showed that the fusion protein pEGFP-N1-spindlin1 was located in the nucleus and the C-terminal is correlated with nuclear localization of Spindlin1 protein. NIH3T3 cells which could stably express spindlin1 as a result of RT-PCR analysis compared with the control cells displayed a complete morphological change; made cell growth faster; and increased the percentage of cells in G2/M and S phase. Furthermore, overexpressed spindlin1 cells formed colonies in soft agar in vitro and formed tumors in nude mice. Our findings provide direct evidence that spindlin1 gene may contribute to tumorigenesis.

  13. Panax notoginseng stimulates alkaline phosphatase activity, collagen synthesis, and mineralization in osteoblastic MC3T3-E1 cells.

    PubMed

    Ji, Zhe; Cheng, Yizhao; Yuan, Puwei; Dang, Xiaoqian; Guo, Xiong; Wang, Weizhuo

    2015-10-01

    Total Panax notoginseng saponin (PNS) has been extensively used to treat a variety of diseases, such as bone fractures, soft tissue injuries, etc. In this study, mouse calvaria-original osteoblastic MC3T3-E1 cells were cultured in various concentrations of PNS (0.005-5 mg/mL) during the period (1, 5, 14, and 23 d). At the endpoint, the osteogenic capacity of MC3T3-E1 cells was investigated by measuring the alkaline phosphatase (ALP) activity, the deposited calcium, and the expression of osteogenic-related markers, including bone collagen type 1 (Col1) and osteocalcin (OCN). Compared with all groups in each period, the most pronounced effect was observed at the concentration range between 0.05 and 0.5 mg/mL (P < 0.05) and the cell proliferation with PNS treatment was found during the whole osteogenic period. Moreover, cellular ALP activity with PNS was increased during 7, 14, and 21 d and cell mineralization with PNS was enhanced in 14 and 21 d. Furthermore, the differentiation markers Col1 and OCN increased in the PNS-treated cells. Our work suggests that PNS may stimulate the osteogenesis process which contains osteoblastic proliferation, differentiation, and mineralization by increasing cellular ALP activity, extracellular matrix mineralization, and osteoblast-associated molecules in the osteoblasts.

  14. Chromium and vanadate combination increases insulin-induced glucose uptake by 3T3-L1 adipocytes.

    PubMed

    Brautigan, David L; Kruszewski, Allison; Wang, Hong

    2006-09-01

    Insulin activates signaling pathways in target tissues through the insulin receptor and Tyr phosphorylation of intracellular proteins. Vanadate mimics insulin and enhances its actions through inhibition of protein Tyr phosphatases. Chromium is a micronutrient that enhances insulin action to normalize blood glucose, but the mechanism is not understood. Here we show that either vanadate or chromium stimulates Tyr phosphorylation of insulin receptor in mouse 3T3-L1 adipocytes compared to insulin alone, but a combination of vanadate and chromium is not additive. Phosphorylation of MAPK or 4E-BP1 as markers for insulin signaling is stimulated by vanadate plus insulin, and chromium does not enhance the effects. Vanadate robustly activates glucose uptake by 3T3-L1 adipocytes even without added insulin and increases insulin-stimulated glucose uptake. Chromium pretreatment of adipocytes slightly enhances glucose uptake in response to insulin, but significantly increases glucose uptake above that induced by insulin plus vanadate. These data show that chromium enhances glucose uptake even when Tyr phosphorylation levels are elevated by vanadate plus insulin, suggesting separate mechanisms of action for vanadate and chromium.

  15. Influence of novel resin monomer on viability of L-929 mouse fibroblasts in vitro.

    PubMed

    Jinno, Satoshi; Kawai, Tatsushi; Ishikawa, Atsuko; Suzuki, Tomoo; Hattori, Nobuaki; Okeya, Hiroyuki; Hayashi, Tatsuhide; Maeda, Hatsuhiko; Ohno, Yuzo; Ito, Masamitsu; Noguchi, Toshihide

    2006-12-01

    We have previously synthesized a novel acrylic resin monomer, methacryloyloxyethyl methyl succinate (TA). The aim of this in vitro study, therefore, was to examine its influence on cell viability using L-929 mouse fibroblasts and then compare the results with MMA, EMA, and LMA. Medium containing each monomer was changed every 15 minutes as some monomers were volatile. After one hour of exposure, these mediums were replaced with a normal medium and cells were further incubated for 72 hours. IC50 value for each monomer was determined, and chronological cell viability and cytomorphologic observation were evaluated. Viability was impaired in a dose-dependent manner. All monomers, except TA, tended to correlate between molecular weight and cell viability. On the other hand, TA showed excellent viability and did not impair growth abruptly. These results thus demonstrated that cellular damage by TA was much lower than that by other monomers.

  16. [The growth behavior of mouse fibroblasts on intraocular lens surface of various silicone and PMMA materials].

    PubMed

    Kammann, J; Kreiner, C F; Kaden, P

    1994-08-01

    Experience with intraocular lenses (IOL) made of PMMA dates back ca. 40 years, while silicone IOLs have been in use for only about 10 years. The biocompatibility of PMMA and silicone caoutchouc was tested in a comparative study investigating the growth of mouse fibroblasts on different IOL materials. Spectrophotometric determination of protein synthesis and liquid scintillation counting of DNA synthesis were carried out. The spreading of cells was planimetrically determined, and the DNA synthesis of individual cells in direct contact with the test sample was tested. The results showed that the biocompatibility of silicone lenses made of purified caoutchouc is comparable with that of PMMA lenses; there is no statistically significant difference. However, impurities arising during material synthesis result in a statistically significant inhibition of cell growth on the IOL surfaces.

  17. Reversal of senescence in mouse fibroblasts through lentiviral suppression of p53.

    PubMed

    Dirac, Annette M G; Bernards, René

    2003-04-04

    Senescence is generally defined as an irreversible state of G(1) cell cycle arrest in which cells are refractory to growth factor stimulation. In mouse embryo fibroblasts (MEFs), induction of senescence requires the presence of p19(ARF) and p53, as genetic ablation of either of these genes allows escape from senescence and leads to immortalization. We have developed a lentiviral vector that directs the synthesis of a p53-specific short hairpin transcript, which mediates stable suppression of p53 expression through RNA interference. We show that suppression of p53 expression in senescent MEFs leads to rapid cell cycle re-entry, is associated with loss of expression of senescence-associated genes, and leads to immortalization. These data indicate that senescence in MEFs is reversible and demonstrate that both initiation and maintenance of senescence is p53-dependent.

  18. Cell cycle regulation of embryonic stem cells and mouse embryonic fibroblasts lacking functional Pax7

    PubMed Central

    Czerwinska, Areta M.; Nowacka, Joanna; Aszer, Magdalena; Fogtman, Anna; Iwanicka-Nowicka, Roksana; Jańczyk-Ilach, Katarzyna; Ciemerych, Maria A.; Grabowska, Iwona

    2016-01-01

    ABSTRACT The transcription factor Pax7 plays a key role during embryonic myogenesis and in adult organisms in that it sustains the proper function of satellite cells, which serve as adult skeletal muscle stem cells. Recently we have shown that lack of Pax7 does not prevent the myogenic differentiation of pluripotent stem cells. In the current work we show that the absence of functional Pax7 in differentiating embryonic stem cells modulates cell cycle facilitating their proliferation. Surprisingly, deregulation of Pax7 function also positively impacts at the proliferation of mouse embryonic fibroblasts. Such phenotypes seem to be executed by modulating the expression of positive cell cycle regulators, such as cyclin E. PMID:27610933

  19. A synthetic small molecule for rapid induction of multiple pluripotency genes in mouse embryonic fibroblasts

    NASA Astrophysics Data System (ADS)

    Pandian, Ganesh N.; Nakano, Yusuke; Sato, Shinsuke; Morinaga, Hironobu; Bando, Toshikazu; Nagase, Hiroki; Sugiyama, Hiroshi

    2012-07-01

    Cellular reprogramming involves profound alterations in genome-wide gene expression that is precisely controlled by a hypothetical epigenetic code. Small molecules have been shown to artificially induce epigenetic modifications in a sequence independent manner. Recently, we showed that specific DNA binding hairpin pyrrole-imidazole polyamides (PIPs) could be conjugated with chromatin modifying histone deacetylase inhibitors like SAHA to epigenetically activate certain pluripotent genes in mouse fibroblasts. In our steadfast progress to improve the efficiency of SAHA-PIPs, we identified a novel compound termed, δ that could dramatically induce the endogenous expression of Oct-3/4 and Nanog. Genome-wide gene analysis suggests that in just 24 h and at nM concentration, δ induced multiple pluripotency-associated genes including Rex1 and Cdh1 by more than ten-fold. δ treated MEFs also rapidly overcame the rate-limiting step of epithelial transition in cellular reprogramming by switching ``'' the complex transcriptional gene network.

  20. Genomic organization of the mouse fibroblast growth factor receptor 3 (Fgfr3) gene

    SciTech Connect

    Perez-Castro, A.V.; Wilson, J.; Altherr, M.R.

    1995-11-20

    The fibroblast growth factor receptor 3 (Fgfr3) protein is a tyrosine kinase receptor involved in the signal transduction of various fibroblast growth factors. Recent studies suggest its important role in normal development. In humans, mutation in Fgfr3 is responsible for growth disorders such as achondroplasia, hypoachondroplasia, and thanatophoric dysplasia. Here, we report the complete genomic organization of the mouse Fgfr3 gene. The murine gene spans approximately 15 kb and consists of 19 exons and 18 introns. One major and one minor transcription initiation site were identified. Position +1 is located 614 nucleotides upstream from the ATG initiation codon. The translation initiation and termination sites are located in exons 2 and 19, respectively. Five Sp1 sites, two AP2 sites, one Zeste site, and one Krox 24 site were observed in the 5{prime}-flanking region. The Fgfr3 promoter appears to be contained within a CpG island and, as is common in genes having multiple Sp1-binding sites, lacks a TATA box. 35 refs., 3 figs., 1 tab.

  1. Cranberries (Oxycoccus quadripetalus) inhibit adipogenesis and lipogenesis in 3T3-L1 cells.

    PubMed

    Kowalska, Katarzyna; Olejnik, Anna; Rychlik, Joanna; Grajek, Włodzimierz

    2014-04-01

    Cranberries (Oxycoccus quadripetalus) are a valuable source of bioactive substances with high antioxidant potential and well documented beneficial health properties. In the present study, the activity of cranberries, in terms of the inhibiting effects of adipogenesis, was investigated using the 3T3-L1 cell line. The obtained results showed that cranberries reduced proliferation and viability of 3T3-L1 preadipocytes in a dose-dependent manner. Treatment with cranberries decreased the number of adipocytes and reduced lipid accumulation in maturing 3T3-L1 preadipocytes, demonstrating an inhibitory effect on lipogenesis. Moreover, it was found that cranberries directly induced lipolysis in adipocytes and down-regulated the expression of major transcription factors of the adipogenesis pathway, such as PPARγ, C/EBPα and SREBP1. These findings indicate that cranberries are capable of suppressing adipogenesis and therefore they seem to be natural bioactive factors effective in adipose tissue mass modulation.

  2. Effect of Biodegradable Shape-Memory Polymers on Proliferation of 3T3 Cells

    NASA Astrophysics Data System (ADS)

    Xu, Shuo-Gui; Zhang, Peng; Zhu, Guang-Ming; Jiang, Ying-Ming

    2011-07-01

    This article evaluates the in vitro biocompatibility for biodegradable shape-memory polymers (BSMP) invented by the authors. 3T3 cells (3T3-Swiss albino GNM 9) of primary and passaged cultures were inoculated into two kinds of carriers: the BSMP carrier and the control group carrier. Viability, proliferation, and DNA synthesis (the major biocompatibility parameters), were measured and evaluated for both the BSMP and naked carrier via cell growth curve analysis, MTT colorimetry and addition of 3H-TdR to culture media. The results showed that there was no difference between the BSMP carrier and the control dish in terms of viability, proliferation, and metabolism of the 3T3 cells. Overall, the BSMP carrier provides good biocompatibility and low toxicity to cells in vitro, and could indicate future potential for this medium as a biological material for implants in vivo.

  3. Calcification of MC3T3-E1 cells on titanium and zirconium.

    PubMed

    Umezawa, Takayuki; Chen, Peng; Tsutsumi, Yusuke; Doi, Hisashi; Ashida, Maki; Suzuki, Shoichi; Moriyama, Keiji; Hanawa, Takao

    2015-01-01

    To confirm similarity of hard tissue compatibility between titanium and zirconium, calcification of MC3T3-E1 cells on titanium and zirconium was evaluated in this study. Mirror-polished titanium (Ti) and zirconium (Zr) disks and zirconium-sputter deposited titanium (Zr/Ti) were employed in this study. The surface of specimens were characterized using scanning electron microscopy and X-ray diffraction. Then, the cellular proliferation, differentiation and calcification of MC3T3-E1 cells on specimens were investigated. The surface of Zr/Ti was much smoother and cleaner than those of Ti and Zr. The proliferation of the cell was the same among three specimens, while the differentiation and calcification on Zr/Ti were faster than those on Ti and Zr. Therefore, Ti and Zr showed the identical hard tissue compatibility according to the evaluation with MC3T3-E1 cells. Sputter deposition may improve cytocompatibility.

  4. Uncoupling of 3T3-L1 gene expression from lipid accumulation during adipogenesis.

    PubMed

    Temple, Karla A; Basko, Xheni; Allison, Margaret B; Brady, Matthew J

    2007-02-06

    Adipocyte differentiation comprises altered gene expression and increased triglyceride storage. To investigate the interdependency of these two events, 3T3-L1 cells were differentiated in the presence of glucose or pyruvate. All adipocytic proteins examined were similarly increased between the two conditions. In contrast, 3T3-L1 adipocytes differentiated with glucose exhibited significant lipid accumulation, which was largely suppressed in the presence of pyruvate. Subsequent addition of glucose to the latter cells restored lipid accumulation and acute rates of insulin-stimulated lipogenesis. These data indicate that extracellular energy is required for induction of adipocytic proteins, while only glucose sustained the parallel increase in triglyceride storage.

  5. Chromosomal instability in mouse embryonic fibroblasts null for the transcriptional co-repressor Ski.

    PubMed

    Marcelain, Katherine; Armisen, Ricardo; Aguirre, Adam; Ueki, Nobuhide; Toro, Jessica; Colmenares, Clemencia; Hayman, Michael J

    2012-01-01

    Ski is a transcriptional regulator that has been considered an oncoprotein given its ability to induce oncogenic transformation in avian model systems. However, studies in mouse and in some human tumor cells have also indicated a tumor suppressor activity for this protein. We found that Ski-/- mouse embryo fibroblasts exhibit high levels of genome instability, namely aneuploidy, consistent with a tumor suppressor function for Ski. Time-lapse microscopy revealed lagging chromosomes and chromatin/chromosome bridges as the major cause of micronuclei (MN) formation and the subsequent aneuploidy. Although these cells arrested in mitosis after treatment with spindle disrupting drugs and exhibited a delayed metaphase/anaphase transition, spindle assembly checkpoint (SAC) was not sufficient to prevent chromosome missegregation, consistent with a weakened SAC. Our in vivo analysis also showed dynamic metaphase plate rearrangements with switches in polarity in cells arrested in metaphase. Importantly, after ectopic expression of Ski the cells that displayed this metaphase arrest died directly during metaphase or after aberrant cell division, relating SAC activation and mitotic cell death. This increased susceptibility to undergo mitosis-associated cell death reduced the number of MN-containing cells. The presented data support a new role for Ski in the mitotic process and in maintenance of genetic stability, providing insights into the mechanism of tumor suppression mediated by this protein.

  6. Chromosomal instability in mouse embryonic fibroblasts null for the transcriptional co-repressor Ski

    PubMed Central

    Marcelain, Katherine; Armisen, Ricardo; Aguirre, Adam; Ueki, Nobuhide; Toro, Jessica; Colmenares, Clemencia; Hayman, Michael J

    2011-01-01

    Ski is a transcriptional regulator that has been considered an oncoprotein, given its ability to induce oncogenic transformation in avian model systems. However, studies in mouse and in some human tumor cells have also indicated a tumor suppressor activity for this protein. We found that Ski−/− mouse embryo fibroblasts exhibit high levels of genome instability, namely aneuploidy, consistent with a tumor suppressor function for Ski. Time-lapse microscopy revealed lagging chromosomes and chromatin/chromosome bridges as the major cause of micronuclei formation and the subsequent aneuploidy. Although these cells arrested in mitosis after treatment with spindle disrupting drugs and exhibited a delayed metaphase/anaphase transition, Spindle Assembly Checkpoint (SAC) was not sufficient to prevent chromosome missegregation, consistent with a weakened SAC. Our in vivo analysis also showed dynamic metaphase plate rearrangements with switches in polarity in cells arrested in metaphase. Importantly, after ectopic expression of Ski the cells that displayed this metaphase arrest died directly during metaphase or after aberrant cell division, relating SAC activation and mitotic cell death. This increased susceptibility to undergo mitosis-associated cell death reduced the number of micronuclei-containing cells. The presented data support a new role for Ski in the mitotic process and in maintenance of genetic stability, providing insights into the mechanism of tumor suppression mediated by this protein. PMID:21412778

  7. Methylglyoxal induces oxidative stress and mitochondrial dysfunction in osteoblastic MC3T3-E1 cells.

    PubMed

    Suh, K S; Choi, E M; Rhee, S Y; Kim, Y S

    2014-02-01

    Methylglyoxal is a reactive dicarbonyl compound produced by glycolytic processing and identified as a precursor of advanced glycation end products. The elevated methylglyoxal levels in patients with diabetes are believed to contribute to diabetic complications, including bone defects. The objective of this study was to evaluate the effect of methylglyoxal on the function of osteoblastic MC3T3-E1 cells. The data indicated that methylglyoxal decreased osteoblast differentiation and induced osteoblast cytotoxicity. Pretreatment of MC3T3-E1 cells with aminoguanidine (a carbonyl scavenger), Trolox (an antioxidant), and cyclosporin A (a blocker of the mitochondrial permeability transition pore) prevented methylglyoxal-induced cytotoxicity in MC3T3-E1 cells. However, BAPTA/AM (an intracellular Ca(2+) chelator) and dantrolene (an inhibitor of endoplasmic reticulum Ca(2+) release) did not reverse the cytotoxic effect of methylglyoxal. Methylglyoxal increased the formation of intracellular reactive oxygen species, mitochondrial superoxide, and cardiolipin peroxidation in osteoblastic MC3T3-E1 cells. Methylglyoxal also decreased the mitochondrial membrane potential and intracellular ATP and nitric oxide levels, suggesting that carbonyl stress-induced loss of mitochondrial integrity contributes to the cytotoxicity of methylglyoxal. Furthermore, the results demonstrated that methylglyoxal induced protein adduct formation, inactivation of glyoxalase I, and activation of glyoxalase II. Aminoguanidine reversed all aforementioned effects of methylglyoxal. Taken together, these data support the notion that high methylglyoxal concentrations have detrimental effects on osteoblasts through a mechanism involving oxidative stress and mitochondrial dysfunction.

  8. Cirsium japonicum flavones enhance adipocyte differentiation and glucose uptake in 3T3-L1 cells.

    PubMed

    Liao, Zhiyong; Wu, Zhihua; Wu, Mingjiang

    2012-01-01

    Cirsium japonicum flavones have been demonstrated to possess anti-diabetic effects in diabetic rats, but the functional mechanism remains unknown. The nuclear receptor peroxisome proliferator-activated receptor γ (PPARγ) plays an important role in glucose and lipid homeostasis. In this study, we report the effects of Cirsium japonicum flavones (pectolinarin and 5,7-dihydroxy-6,4-dimethoxy flavone) on PPARγ activation, adipocyte differentiation, and glucose uptake in 3T3-L1 cells. Reporter gene assays and Oil Red O staining showed that Cirsium japonicum flavones induced PPARγ activation and enhanced adipocyte differentiation of 3T3-L1 cells in a dose-dependent manner. In addition, Cirsium japonicum flavones increased the expression of PPARγ target genes, such as adiponectin and glucose transporter 4 (GLUT4), and enhanced the translocation of intracellular GLUT4 to the plasma membrane. In mature 3T3-L1 adipocytes, Cirsium japonicum flavones significantly enhanced the basal and insulin-stimulated glucose uptake. The flavones-induced effects in 3T3-L1 cells were abolished by the PPARγ antagonist, GW9662, and by the phosphatidylinositol 3-kinase (PI3K) inhibitor, wortmannin. This study suggests that Cirsium japonicum flavones promote adipocyte differentiation and glucose uptake by inducing PPARγ activation and then modulating the insulin signaling pathway in some way, which could benefit diabetes patients.

  9. Fluorescence lifetime imaging of lipids during 3T3-L1 cell differentiation

    NASA Astrophysics Data System (ADS)

    Song, Young Sik; Won, Young Jae; Lee, Sang-Hak; Kim, Dug Young

    2014-03-01

    Obesity is becoming a big health problem in these days. Since increased body weight is due to increased number and size of the triglyceride-storing adipocytes, many researchers are working on differentiation conditions and processes of adipocytes. Adipocytes also work as regulators of whole-body energy homeostasis by secreting several proteins that regulate processes as diverse as haemostasis, blood pressure, immune function, angiogenesis and energy balance. 3T3-L1 cells are widely used cell line for studying adipogenesis because it can differentiate into an adipocyte-like phenotype under appropriate conditions. In this paper, we propose an effective fluorescence lifetime imaging technique which can easily distinguish lipids in membrane and those in lipid droplets. Nile red dyes are attached to lipids in 3T3-L1 cells. Fluorescence lifetime images were taken for 2 week during differentiation procedure of 3T3-L1 cells into adipocytes. We used 488 nm pulsed laser with 5MHz repetition rate and emission wavelength is 520 nm of Nile Red fluorescent dye. Results clearly show that the lifetime of Nile red in lipid droplets are smaller than those in cell membrane. Our results suggest that fluorescence lifetime imaging can be a very powerful tool to monitor lipid droplet formation in adipocytes from 3T3-L1 cells.

  10. Increased association of dynamin II with myosin II in ras transformed NIH3T3 cells.

    PubMed

    Jeong, Soon-Jeong; Kim, Su-Gwan; Yoo, Jiyun; Han, Mi-Young; Park, Joo-Cheol; Kim, Heung-Joong; Kang, Seong-Soo; Choi, Baik-Dong; Jeong, Moon-Jin

    2006-08-01

    Dynamin has been implicated in the formation of nascent vesicles through both endocytic and secretory pathways. However, dynamin has recently been implicated in altering the cell membrane shape during cell migration associated with cytoskeleton-related proteins. Myosin II has been implicated in maintaining cell morphology and in cellular movement. Therefore, reciprocal immunoprecipitation was carried out to identify the potential relationship between dynamin II and myosin II. The dynamin II expression level was higher when co-expressed with myosin II in Ras transformed NIH3T3 cells than in normal NIH3T3 cells. Confocal microscopy also confirmed the interaction between these two proteins. Interestingly, exposing the NIH3T3 cells to platelet-derived growth factor altered the interaction and localization of these two proteins. The platelet-derived growth factor treatment induced lamellipodia and cell migration, and dynamin II interacted with myosin II. Grb2, a 24 kDa adaptor protein and an essential element of the Ras signaling pathway, was found to be associated with dynamin II and myosin II gene expression in the Ras transformed NIH3T3 cells. These results suggest that dynamin II acts as an intermediate messenger in the Ras signal transduction pathway leading to membrane ruffling and cell migration.

  11. Antiadopogenic effects of rice hull smoke extract in 3T3-L1 cells

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The present study investigates the inhibitory effects of a rice hull smoke extract (RHSE) against adipogenesis in 3T3-L1 pre-adipocyte cells. At concentrations of 0.1% and 0.5% RHSE, MDI-induced cells were shown to reduce their cellular lipid content by about 72% and 88%, respectively, compared to ...

  12. Effect of Gambisan on the Inhibition of Adipogenesis in 3T3-L1 Adipocytes

    PubMed Central

    Kang, Jung Won; Nam, Dongwoo; Kim, Kun Hyung; Huh, Jeong-Eun; Lee, Jae-Dong

    2013-01-01

    This study was conducted to explore the antiadipogenic effect and possible mechanism of Gambisan on 3T3-L1 cells. For quality control, Gambisan was standardized by HPLC and the standard compounds ephedrine, epigallocatechin-3-gallate, and caffeine were screened. Cultured 3T3-L1 cells that had been induced to differentiate were treated with various concentrations of Gambisan or its major component extracts (Ephedra intermedia Schrenk, Atractylodes lancea DC., and Thea sinensis L.) for 72 hours for MTT assay to determine cell viability or 10 days for LDH assay, triglyceride assay, DNA content measurement, Oil red O staining, RT-PCR, and western blot. Gambisan significantly inhibited adipogenesis in 3T3-L1 cells by reducing triglyceride contents and lipid accumulation in a dose-dependent manner without obvious cytotoxicity. Viability and DNA content in 3T3-L1 cells treated with Gambisan were significantly higher than cells treated with the major component extracts at every concentration. The anti-adipogenic effects of Gambisan appeared to be mediated by a significant downregulation of the expression of lipoprotein lipase mRNA and PPARγ, C/EBPα, and SREBP-1 protein apart from the expression of hormone-sensitive lipase. Gambisan could act as a possible therapeutic agent for obesity. However, further studies including in vivo assays and clinical trials are needed to confirm the efficacy, safety and mechanisms of the antiobesity effects of Gambisan. PMID:24069055

  13. Occurrence of cGMP/nitric oxide-sensitive store-operated calcium entry in fibroblasts and its effect on matrix metalloproteinase secretion

    PubMed Central

    Huang, Yong; Lu, Min-Qiang; Li, Hua; Xu, Chi; Yi, Shu-Hong; Chen, Gui-Hua

    2006-01-01

    AIM: To examine the existence of Nitric oxide/cGMP sensitive store-operated Ca2+ entry in mouse fibroblast NIH/3T3 cells and its influence on matrix metalloproteinase (MMP) production and adhesion ability of fibroblasts. METHODS: NIH/3T3 cells were cultured. Confocal laser scanning microscopy was used to examine the existence of thapsigargin-induced store-operated Ca2+ entry in fibroblasts. Gelatin zymography and semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) were employed to detect the involvement of [Ca2+]i and NO/cGMP in MMP secretion. The involvement of NO/cGMP-sensitive Ca2+ entry in adhesion was determined using matrigel-coated culture plates. RESULTS: 8-bromo-cGMP inhibited the thapsigargin-induced Ca2+ entry in 3T3 cells. The cGMP-induced inhibition was abolished by an inhibitor of protein kinase G, KT5823 (1μmol/L). A similar effect on the Ca2+ entry was observed in 3T3 cells in response to a NO donor, (±)-S-nitroso-N-acetylpenicillamine (SNAP). The inhibitory effect of SNAP on the thapsigargin-induced Ca2+ entry was also observed, indicating NO/cGMP-regulated Ca2+ entry in 3T3 cells. Results of gelatin zymography assay showed that addition of extracellular Ca2+ concentration induced MMP release and activation in a dose-dependent manner. RT-PCR also showed that cGMP and SNAP reduced the production of MMP mRNA in 3T3 cells. Experiments investigating adhesion potentials demonstrated that cGMP and SNAP could upgrade 3T3 cell attachment rate to the matrigel-coated culture plates. CONCLUSION: NO/cGMP sensitive store-operated Ca2+ entry occurs in fibroblasts, and attenuates their adhesion potentials through its influence on MMP secretion. PMID:17006985

  14. Osteogenic gene expression of murine osteoblastic (MC3T3-E1) cells under cyclic tension

    NASA Astrophysics Data System (ADS)

    Kao, C. T.; Chen, C. C.; Cheong, U.-I.; Liu, S. L.; Huang, T. H.

    2014-08-01

    Low-level laser therapy (LLLT) can promote cell proliferation. The remodeling ability of the tension side of orthodontic teeth affects post-orthodontic stability. The purpose of the present study was to investigate the osteogenic effects of LLLT on osteoblast-like cells treated with a simulated tension system that provides a mechanical tension regimen. Murine osteoblastic (MC3T3-E1) cells were cultured in a Flexcell strain unit with programmed loads of 12% elongation at a frequency of 0.5 Hz for 24 and 48 h. The cultured cells were treated with a low-level diode laser using powers of 5 J and 10 J. The proliferation of MC3T3-E1 cells was determined using the Alamar Blue assay. The expression of osteogenic genes (type I collagen (Col-1), osteopontin (OPN), osteocalcin (OC), osteoprotegerin (OPG), receptor activator of nuclear factor kappa B ligand (RANKL), bone morphologic protein (BMP-2), and bone morphologic protein (BMP-4)) in MC3T3-E1 cells was analyzed using reverse transcription polymerase chain reaction (RT-PCR). The data were analyzed using one-way analysis of variance. The proliferation rate of tension-cultured MC3T3-E1 cells under 5 J and 10 J LLLT increased compared with that of the control group (p < 0.05). Prominent mineralization of the MC3T3-E1 cells was visible using a von Kossa stain in the 5 J LLLT group. Osteogenic genes (Col-1, OC, OPG and BMP-2) were significantly expressed in the MC3T3-E1 cells treated with 5 J and 10 J LLLT (p < 0.05). LLLT in tension-cultured MC3T3-E1 cells showed synergistic osteogenic effects, including increases in cell proliferation and Col-1, OPN, OC, OPG and BMP-2 gene expression. LLLT might be beneficial for bone remodeling on the tension side of orthodontics.

  15. Human c-fgr induces a monocyte-specific enzyme in NIH 3T3 cells

    SciTech Connect

    Inoue, Kazushi; Akiyama, Tetsu; Toyoshima, Kumao ); Wongsasant, Budsaba )

    1991-12-01

    The mutant c-fgr protein (p58{sup c-fgr/F523}) containing Phe-523 instead of Tyr-523 exhibited transforming activity in NIH 3T3 cells like other protein-tyrosine kinases of the src family, but normal p58{sup c-fgr} (p58{sup c-fgr/wt}) did not. The mutant protein showed tyrosine kinase activity threefold higher than that of the normal protein in vitro. Surprisingly, transfection of the normal c-fgr gene into NIH 3T3 cells resulted in induction of sodium fluoride (NaF)-sensitive {alpha}-naphthyl butyrate esterase ({alpha}-NBE), marker enzyme of cells of monocytic origin, which was not induced in v-src-, v-fgr-, or lyn-transfected NIH 3T3 cells. The NaF-sensitive {alpha}-NBE induced in c-fgr transfectants was shown by isoelectric focusing to have a pI of 5.2 to 5.4, a range which was the same as those for thioglycolate-induced murine peritoneal macrophages and 1{alpha}, 25-dihydroxyvitamin D{sub 3}-treated WEHI-3B cells. Immunoblotting studies with antophosphotyrosine antibodies revealed that 58-, 62-, 75-, 120-, 200-, and 230-kDa proteins were commonly phosphorylated at tyrosine residues in NIH 3T3 cells transfected with normal and mutated c-fgr, while 95-kDa protein was significantly phosphorylated at tyrosine residues in NIH 3T3 cells transfected with normal and mutated c-fgr, while 95-kDa protein was significantly phosphorylated at tyrosine residues in cells transfected with the mutated c-fgr. These findings suggest that tyrosine phosphorylation of specific cellular substrate proteins is important in induction of NaF-sensitive {alpha}-NBE and cell transformation by p58{sup c-fgr}.

  16. Berberine activates GLUT1-mediated glucose uptake in 3T3-L1 adipocytes.

    PubMed

    Kim, So Hui; Shin, Eun-Jung; Kim, Eun-Do; Bayaraa, Tsenguun; Frost, Susan Cooke; Hyun, Chang-Kee

    2007-11-01

    It has recently been known that berberine, an alkaloid of medicinal plants, has anti-hyperglycemic effects. To explore the mechanism underlying this effect, we used 3T3-L1 adipocytes for analyzing the signaling pathways that contribute to glucose transport. Treatment of berberine to 3T3-L1 adipocytes for 6 h enhanced basal glucose uptake both in normal and in insulin-resistant state, but the insulin-stimulated glucose uptake was not augmented significantly. Inhibition of phosphatidylinositol 3-kinase (PI 3-K) by wortmannin did not affect the berberine effect on basal glucose uptake. Berberine did not augment tyrosine phosphorylation of insulin receptor (IR) and insulin receptor substrate (IRS)-1. Further, berberine had no effect on the activity of the insulin-sensitive downstream kinase, atypical protein kinase C (PKCzeta/lambda). However, interestingly, extracellular signal-regulated kinases (ERKs), which have been known to be responsible for the expression of glucose transporter (GLUT)1, were significantly activated in berberine-treated 3T3-L1 cells. As expected, the level of GLUT1 protein was increased both in normal and insulin-resistant cells in response to berberine. But berberine affected the expression of GLUT4 neither in normal nor in insulin-resistant cells. In addition, berberine treatment increased AMP-activated protein kinase (AMPK) activity in 3T3-L1 cells, which has been reported to be associated with GLUT1-mediated glucose uptake. Together, we concluded that berberine increases glucose transport activity of 3T3-L1 adipocytes by enhancing GLUT1 expression and also stimulates the GLUT1-mediated glucose uptake by activating GLUT1, a result of AMPK stimulation.

  17. Isolation and characterization of NIH 3T3 cells expressing polyomavirus small T antigen

    SciTech Connect

    Noda, T.; Satake, M.; Robins, T.; Ito, Y.

    1986-10-01

    The polyomavirus small T-antigen gene, together with the polyomavirus promoter, was inserted into retrovirus vector pGV16 which contains the Moloney sarcoma virus long terminal repeat and neomycin resistance gene driven by the simian virus 40 promoter. This expression vector, pGVST, was packaged into retrovirus particles by transfection of PSI2 cells which harbor packaging-defective murine retrovirus genome. NIH 3T3 cells were infected by this replication-defective retrovirus containing pGVST. Of the 15 G418-resistant cell clones, 8 express small T antigen at various levels as revealed by immunoprecipitation. A cellular protein with an apparent molecular weight of about 32,000 coprecipitates with small T antigen. Immunofluorescent staining shows that small T antigen is mainly present in the nuclei. Morphologically, cells expressing small T antigen are indistinguishable from parental NIH 3T3 cells and have a microfilament pattern similar to that in parental NIH 3T3 cells. Cells expressing small T antigen form a flat monolayer but continue to grow beyond the saturation density observed for parental NIH 3T3 cells and eventually come off the culture plate as a result of overconfluency. There is some correlation between the level of expression of small T antigen and the growth rate of the cells. Small T-antigen-expressing cells form small colonies in soft agar. However, the proportion of cells which form these small colonies is rather small. A clone of these cells tested did not form tumors in nude mice within 3 months after inoculation of 10/sup 6/ cells per animal. Thus, present studies establish that the small T antigen of polyomavirus is a second nucleus-localized transforming gene product of the virus (the first one being large T antigen) and by itself has a function which is to stimulate the growth of NIH 3T3 cells beyond their saturation density in monolayer culture.

  18. Absence of AMPKα2 accelerates cellular senescence via p16 induction in mouse embryonic fibroblasts.

    PubMed

    Ding, Ye; Chen, Jie; Okon, Imoh Sunday; Zou, Ming-Hui; Song, Ping

    2016-02-01

    Emerging evidence suggests that activation of adenosine monophosphate-activated protein kinase (AMPK), an energy gauge and redox sensor, delays aging process. However, the molecular mechanisms by which AMPKα isoform regulates cellular senescence remain largely unknown. The aim of this study was to determine if AMPKα deletion contributes to the accelerated cell senescence by inducing p16(INK4A) (p16) expression thereby arresting cell cycle. The markers of cellular senescence, cell cycle proteins, and reactive oxygen species (ROS) were monitored in cultured mouse embryonic fibroblasts (MEFs) isolated from wild type (WT, C57BL/6J), AMPKα1, or AMPKα2 homozygous deficient (AMPKα1(-/-), AMPKα2(-/-)) mice by Western blot and cellular immunofluorescence staining, as well as immunohistochemistry (IHC) in skin tissue of young and aged mice. Deletion of AMPKα2, the minor isoform of AMPKα, but not AMPKα1 in high-passaged MEFs led to spontaneous cell senescence demonstrated by accumulation of senescence-associated-β-galactosidase (SA-β-gal) staining and foci formation of heterochromatin protein 1 homolog gamma (HP1γ). It was shown here that AMPKα2 deletion upregulates cyclin-dependent kinase (CDK) inhibitor, p16, which arrests cell cycle. Furthermore, AMPKα2 null cells exhibited elevated ROS production. Interestingly, knockdown of HMG box-containing protein 1 (HBP1) partially blocked the cellular senescence of AMPKα2-deleted MEFs via the reduction of p16. Finally, dermal cells senescence, including fibroblasts senescence evidenced by the staining of p16, HBP1, and Ki-67, in the skin of aged AMPKα2(-/-) mice was enhanced when compared with that in wild type mice. Taken together, our results suggest that AMPKα2 isoform plays a fundamental role in anti-oxidant stress and anti-senescence.

  19. Peptide-enhanced mRNA transfection in cultured mouse cardiac fibroblasts and direct reprogramming towards cardiomyocyte-like cells.

    PubMed

    Lee, Kunwoo; Yu, Pengzhi; Lingampalli, Nithya; Kim, Hyun Jin; Tang, Richard; Murthy, Niren

    2015-01-01

    The treatment of myocardial infarction is a major challenge in medicine due to the inability of heart tissue to regenerate. Direct reprogramming of endogenous cardiac fibroblasts into functional cardiomyocytes via the delivery of transcription factor mRNAs has the potential to regenerate cardiac tissue and to treat heart failure. Even though mRNA delivery to cardiac fibroblasts has the therapeutic potential, mRNA transfection in cardiac fibroblasts has been challenging. Herein, we develop an efficient mRNA transfection in cultured mouse cardiac fibroblasts via a polyarginine-fused heart-targeting peptide and lipofectamine complex, termed C-Lipo and demonstrate the partial direct reprogramming of cardiac fibroblasts towards cardiomyocyte cells. C-Lipo enabled the mRNA-induced direct cardiac reprogramming due to its efficient transfection with low toxicity, which allowed for multiple transfections of Gata4, Mef2c, and Tbx5 (GMT) mRNAs for a period of 2 weeks. The induced cardiomyocyte-like cells had α-MHC promoter-driven GFP expression and striated cardiac muscle structure from α-actinin immunohistochemistry. GMT mRNA transfection of cultured mouse cardiac fibroblasts via C-Lipo significantly increased expression of the cardiomyocyte marker genes, Actc1, Actn2, Gja1, Hand2, and Tnnt2, after 2 weeks of transfection. Moreover, this study provides the first direct evidence that the stoichiometry of the GMT reprogramming factors influence the expression of cardiomyocyte marker genes. Our results demonstrate that mRNA delivery is a potential approach for cardiomyocyte generation.

  20. Dehydrocostus lactone prevents mitochondrial dysfunction in osteoblastic MC3T3-E1 cells.

    PubMed

    Choi, Eun Mi

    2011-08-16

    The dried root of Saussurea lappa Clarke (Compositae) has been used as a traditional medicine. Dehydrocostus lactone is one of the main bioactive constituents of this medicinal plant. In the present study, the protective effect of dehydrocostus lactone against antimycin A (an inhibitor of mitochondrial complex III)-induced cytotoxicity was investigated in osteoblastic MC3T3-E1 cells. Pre-treatment with dehydrocostus lactone prior to antimycin A exposure significantly prevented mitochondrial membrane potential dissipation, complex IV inactivation, ATP loss, cytochrome c release, intracellular calcium elevation and potassium loss, and reactive oxygen species production induced by antimycin A. These results suggest that dehydrocostus lactone protects osteoblastic MC3T3-E1 cells from antimycin A-induced cell damage through the improved mitochondrial function.

  1. Expression of Nanog gene promotes NIH3T3 cell proliferation

    SciTech Connect

    Zhang Jingyu; Wang Xia; Chen Bing; Suo Guangli; Zhao Yanhong; Duan Ziyuan; Dai Jianwu . E-mail: jwdai@genetics.ac.cn

    2005-12-16

    Cells are the functional elements in tissue engineering and regenerative medicine. A large number of cells are usually needed for these purposes. However, there are numbers of limitations for in vitro cell proliferation. Nanog is an important self-renewal determinant in embryonic stem cells. However, it remains unknown whether Nanog will influence the cell cycle and cell proliferation of mature cells. In this study, we expressed Nanog in NIH3T3 cells and showed that expression of Nanog in NIH3T3 promoted cells to enter into S phase and enhanced cell proliferation. This suggests that Nanog gene might function in a similar fashion in mature cells as in ES cells. In addition, it may provide an approach for in vitro cell expansion.

  2. Human papillomavirus type 16 DNA-induced malignant transformation of NIH 3T3 cells

    SciTech Connect

    Yasumoto, S.; Burkhardt, A.L.; Doniger, J.; DiPaolo, J.A.

    1986-02-01

    A biological function for human papillomavirus 16 (HPV 16) DNA was demonstrated by transformation of NIH 3T3 cells. HPV 16 DNA has been found frequently in genital cancer and has been classified as a papillomavirus on the basis of DNA homology. A recombinant HPV 16 DNA (pSHPV16d), which contains a head-to-tail dimer of the full-length HPV 16 genome, induced morphologic transformation; the transformed cells were tumorigenic in nude mice. Expression of transforming activity was unique because of the long latency period (more than 4 weeks) required for induction of morphologic transformation and because the transfected DNA existed primarily in a multimeric form with some rearrangement. Furthermore, virus-specific RNAs were expressed in the transformants. The transformation of NIH 3T3 cells provides a model for analyzing the functions of HPV 16, which is associated with cervical carcinomas.

  3. Restoration of Hypoxanthine Phosphoribosyl Transferase Activity in Mouse 1R Cells After Fusion with Chick-Embryo Fibroblasts

    PubMed Central

    Bakay, Bohdan; Croce, Carlo M.; Koprowski, Hilary; Nyhan, William L.

    1973-01-01

    Fusion of the 1R mouse cell, which lacks activity of hypoxanthine phosphoribosyl transferase (EC 2.4.2.8), with chick-embryo fibroblasts yielded progeny cells that survived in hypoxanthine-aminopterin-thymidine selective medium. This property and the failure of the progeny to survive in 8-azaguanine indicated that hypoxanthine phosphoribosyl transferase activity was present. Electrophoretic analysis revealed that the enzyme was of mouse, not chick, origin. These observations are consistent with the operation of a regulator gene responsible for the absence of hypoxanthine phosphoribosyl-transferase activity in the 1R cell and its presence in the progeny. Images PMID:4516198

  4. Effects of 6-Hydroxyflavone on Osteoblast Differentiation in MC3T3-E1 Cells

    PubMed Central

    Wu, Yu-Wei; Yeh, Shauh-Der; Lin, Yu-Hsaing; Tsai, Yu-Hui

    2014-01-01

    Osteoblast differentiation plays an essential role in bone integrity. Isoflavones and some flavonoids are reported to have osteogenic activity and potentially possess the ability to treat osteoporosis. However, limited information concerning the osteogenic characteristics of hydroxyflavones is available. This study investigates the effects of various hydroxyflavones on osteoblast differentiation in MC3T3-E1 cells. The results showed that 6-hydroxyflavone (6-OH-F) and 7-hydroxyflavone (7-OH-F) stimulated ALP activity. However, baicalein and luteolin inhibited ALP activity and flavone showed no effect. Up to 50 μM of each compound was used for cytotoxic effects study; flavone, 6-OH-F, and 7-OH-F had no cytotoxicity on MC3T3-E1 cells. Moreover, 6-OH-F activated AKT and serine/threonine kinases (also known as protein kinase B or PKB), extracellular signal-regulated kinases (ERK 1/2), and the c-Jun N-terminal kinase (JNK) signaling pathways. On the other hand, 7-OH-F promoted osteoblast differentiation mainly by activating ERK 1/ 2 signaling pathways. Finally, after 5 weeks of 6-OH-F induction, MC3T3-E1 cells showed a significant increase in the calcein staining intensity relative to merely visible mineralization observed in cells cultured in the osteogenic medium only. These results suggested that 6-OH-F could activate AKT, ERK 1/2, and JNK signaling pathways to effectively promote osteoblastic differentiation. PMID:24795772

  5. Traditional Herbal Formula Oyaksungi-San Inhibits Adipogenesis in 3T3-L1 Adipocytes

    PubMed Central

    Seo, Chang-Seob; Shin, Hyeun-Kyoo

    2015-01-01

    Background. Oyaksungi-san (OYSGS) is a herbal formula that has been used for treating cardiovascular diseases in traditional Asian medicine. Here, we investigated the antiadipogenic effect of OYSGS extract in 3T3-L1 adipose cells. Methods. 3T3-L1 preadipocytes were differentiated into adipocytes with or without OYSGS. After differentiation, we measured Oil Red O staining, glycerol-3-phosphate dehydrogenase (GPDH) activity, leptin production, mRNA, and protein levels of adipogenesis-related factors. Results. OYSGS extract dramatically inhibited intracellular lipid accumulation in the differentiated adipocytes. It also significantly suppressed the (GPDH) activity, triglyceride (TG) content, and leptin production by reducing the expression of adipogenesis-related genes including lipoprotein lipase, fatty acid binding protein 4, CCAAT/enhancer-binding protein-alpha (C/EBP-α), and peroxisome proliferator-activated receptor gamma (PPAR-γ). Furthermore, OYSGS clearly enhanced phosphorylation of AMP-activated protein kinase (AMPK) as well as its substrate acetyl CoA (ACC) carboxylase. Conclusions. Our results demonstrate that OYSGS negatively controls TG accumulation in 3T3-L1 adipocytes. We suggest antiadipogenic activity of OYSGS and its potential benefit in preventing obesity. PMID:25802547

  6. Ginkgolide C Suppresses Adipogenesis in 3T3-L1 Adipocytes via the AMPK Signaling Pathway

    PubMed Central

    Liou, Chian-Jiun; Lai, Xuan-Yu; Chen, Ya-Ling; Wang, Chia-Ling; Wei, Ciao-Han; Huang, Wen-Chung

    2015-01-01

    Ginkgolide C, isolated from Ginkgo biloba leaves, is a flavone reported to have multiple biological functions, from decreased platelet aggregation to ameliorating Alzheimer disease. The study aim was to evaluate the antiadipogenic effect of ginkgolide C in 3T3-L1 adipocytes. Ginkgolide C was used to treat differentiated 3T3-L1 cells. Cell supernatant was collected to assay glycerol release, and cells were lysed to measure protein and gene expression related to adipogenesis and lipolysis by western blot and real-time PCR, respectively. Ginkgolide C significantly suppressed lipid accumulation in differentiated adipocytes. It also decreased adipogenesis-related transcription factor expression, including peroxisome proliferator-activated receptor and CCAAT/enhancer-binding protein. Furthermore, ginkgolide C enhanced adipose triglyceride lipase and hormone-sensitive lipase production for lipolysis and increased phosphorylation of AMP-activated protein kinase (AMPK), resulting in decreased activity of acetyl-CoA carboxylase for fatty acid synthesis. In coculture with an AMPK inhibitor (compound C), ginkgolide C also improved activation of sirtuin 1 and phosphorylation of AMPK in differentiated 3T3-L1 cells. The results suggest that ginkgolide C is an effective flavone for increasing lipolysis and inhibiting adipogenesis in adipocytes through the activated AMPK pathway. PMID:26413119

  7. Stevioside from Stevia rebaudiana Bertoni Increases Insulin Sensitivity in 3T3-L1 Adipocytes

    PubMed Central

    Mohd-Radzman, Nabilatul Hani; Ismail, Wan Iryani Wan; Jaapar, Siti Safura; Adam, Zainah; Adam, Aishah

    2013-01-01

    Stevioside from Stevia rebaudiana has been reported to exert antihyperglycemic effects in both rat and human subjects. There have been few studies on these effects in vitro. In this paper, radioactive glucose uptake assay was implemented in order to assess improvements in insulin sensitivity in 3T3-L1 cells by elevation of glucose uptake following treatment with stevioside. Oil Red-O staining and MTT assay were utilized to confirm adipocyte differentiation and cell viability, respectively. Findings from this research showed a significant increase in absorbance values in mature adipocytes following Oil Red-O staining, confirming the differentiation process. Stevioside was noncytotoxic to 3T3-L1 cells as cell viability was reduced by a maximum of 17%, making it impossible to determine its IC50. Stevioside increased glucose uptake activities by 2.1 times (p < 0.001) in normal conditions and up to 4.4 times (p < 0.001) in insulin-resistant states. At times, this increase was higher than that seen in positive control group treated with rosiglitazone maleate, an antidiabetic agent. Expressions of pY20 and p-IRS1 which were measured via Western blot were improved by stevioside treatment. In conclusion, stevioside has direct effects on 3T3-L1 insulin sensitivity via increase in glucose uptake and enhanced expression of proteins involved in insulin-signalling pathway. PMID:24391675

  8. Stevioside from Stevia rebaudiana Bertoni Increases Insulin Sensitivity in 3T3-L1 Adipocytes.

    PubMed

    Mohd-Radzman, Nabilatul Hani; Ismail, Wan Iryani Wan; Jaapar, Siti Safura; Adam, Zainah; Adam, Aishah

    2013-01-01

    Stevioside from Stevia rebaudiana has been reported to exert antihyperglycemic effects in both rat and human subjects. There have been few studies on these effects in vitro. In this paper, radioactive glucose uptake assay was implemented in order to assess improvements in insulin sensitivity in 3T3-L1 cells by elevation of glucose uptake following treatment with stevioside. Oil Red-O staining and MTT assay were utilized to confirm adipocyte differentiation and cell viability, respectively. Findings from this research showed a significant increase in absorbance values in mature adipocytes following Oil Red-O staining, confirming the differentiation process. Stevioside was noncytotoxic to 3T3-L1 cells as cell viability was reduced by a maximum of 17%, making it impossible to determine its IC50. Stevioside increased glucose uptake activities by 2.1 times (p < 0.001) in normal conditions and up to 4.4 times (p < 0.001) in insulin-resistant states. At times, this increase was higher than that seen in positive control group treated with rosiglitazone maleate, an antidiabetic agent. Expressions of pY20 and p-IRS1 which were measured via Western blot were improved by stevioside treatment. In conclusion, stevioside has direct effects on 3T3-L1 insulin sensitivity via increase in glucose uptake and enhanced expression of proteins involved in insulin-signalling pathway.

  9. Menaquinone-7 regulates gene expression in osteoblastic MC3T3E1 cells.

    PubMed

    Katsuyama, Hironobu; Saijoh, Kiyofumi; Otsuki, Takemi; Tomita, Masafumi; Fukunaga, Masao; Sunami, Shigeo

    2007-02-01

    Previous study has shown that the vitamin K2 analog menaquinone-7 (MK-7) induces expression of the osteoblast-specific genes osteocalcin, osteoprotegerin, receptor activator of NFkappaB, and its ligand. Since MK-7 may also regulate osteoblast cell function, we examined the expression of osteoblast genes regulated by MK-7 administration. Differences between gene expression in control and MK-7-administered MC3T3E1 cells were analyzed using the suppression subtractive hybridization method. After 24 h of MK-7 administration, genes upregulated by MK-7 included tenascin C and BMP2. Genes downregulated by MK-7 administration included biglycan and butyrophilin. Real-time PCR showed a marked increase in tenascin C. When the protein level was examined using Western blot analysis, tenascin C was higher in MK-7-administered cells than in control cells. These results indicated that MK-7 affected the cellular function of osteoblastic MC3T3E1 cells. Considering BMP2 mRNA expression was higher in MK-7-administered cells than in control cells, the effect of MK-7 administration on the signal transduction system was examined. Western blot analysis showed that cells administered MK-7 displayed a higher phosphorylated Smad1 level than control cells. Because MC3T3E1 cells have a nuclear binding receptor for MK-7, this result might indicate an indirect effect of MK-7 through BMP2 production.

  10. Retinoic acid inhibits inducible nitric oxide synthase expression in 3T3-L1 adipocytes.

    PubMed

    Yang, Jeong-Yeh; Koo, Bon-Sun; Kang, Mi-Kyung; Rho, Hye-Won; Sohn, Hee-Sook; Jhee, Eun-Chung; Park, Jin-Woo

    2002-11-30

    The present study was undertaken to explore whether retinoids, which are known to have immunomodulatory actions, could attenuate tumor necrosis factor-alpha (TNF)-stimulated inducible nitric oxide synthase (iNOS) expression in 3T3-L1 adipocytes. Adipocytes incubated with TNF induced dose- and time-dependent accumulation of nitrite in the culture medium through the iNOS induction as confirmed by Western blotting. Treatment of cells with TNF in the presence of all-trans-retinoic acid (RA) significantly decreased their ability to produce nitrite and iNOS induction. Both 13-cis- and all- trans-RA-induced suppression was dose-dependent, and all-trans-RA was somewhat potent than 13-cis-RA. The inhibitory effect of RA on TNF-induced iNOS induction was reversible, completely recovered after 2 days, and was exerted through the inhibition of NF-kappaB activation. TNF also suppressed the lipoprotein lipase (LPL) activity of 3T3-L1 adipocytes. RA could not reverse the TNF- induced LPL suppression at RA levels causing near complete inhibition of the TNF-induced NO production. These results indicate that RAs attenuate iNOS expression reversibly in TNF-stimulated 3T3-L1 adipocytes, and that the TNF-induced LPL suppression is not the result of NO overproduction.

  11. Extract of Chaga mushroom (Inonotus obliquus) stimulates 3T3-L1 adipocyte differentiation.

    PubMed

    Joo, Jeong In; Kim, Dong Hyun; Yun, Jong Won

    2010-11-01

    Chaga mushroom (Inonotus obliquus) has long been used as a folk medicine due to its numerous biological functions such as antibacterial, antiallergic, antiinflammatory and antioxidative activities. In the present study, it was found that the I. obliquus hot water extract (IOWE) activated adipogenesis of 3T3-L1 preadipocytes. Even in the absence of adipogenic stimuli by insulin, the IOWE strongly induced adipogenesis of 3T3-L1 preadipocytes. The major constituent of IOWE was glucose-rich polysaccharides with a molecular mass of 149  kDa. IOWE enhanced the differentiation of 3T3-L1 preadipocytes, increasing TG (triacylglycerol) accumulation that is critical for acquisition of the adipocyte phenotype, in a dose-dependent manner. IOWE stimulated gene expression of C/EBPα (CCAAT/enhancer-binding protein α) and PPARγ (peroxisome proliferator-activated receptors γ) during adipocyte differentiation, and induced the expression of PPARγ target genes such as aP2 (adipocyte protein 2), LPL (lipoprotein lipase) and CD36 (fatty acid translocase). Immunoblot analysis revealed that IOWE increased the expression of adipogenic makers such as PPARγ and GLUT4 (glucose transporter 4). The luciferase reporter assay demonstrated that IOWE did not exhibit PPARγ ligand activity. Although these results require further investigation, the ability of natural mushroom product to increase PPARγ transcriptional activities may be expected to be therapeutic targets for dyslipidemia and type 2 diabetes.

  12. Endoplasmic reticulum stress suppresses lipin-1 expression in 3T3-L1 adipocytes

    SciTech Connect

    Takahashi, Nobuhiko; Hiranaka, Natsumi; Suzuki, Takeshi; Yui, Tomoo; Akanuma, Masayoshi; Kanazawa, Kaoru; Yoshida, Mika; Naito, Sumiyoshi; Fujiya, Mikihiro; Kohgo, Yutaka

    2013-02-01

    Highlights: ► Lipin-1 involves lipid metabolism, adipocyte differentiation, and inflammation. ► Adipose lipin-1 expression is reduced in obesity. ► ER stress suppresses lipin-1 expression in 3T3-L1 adipocytes. ► Activation of PPAR-γ recovers ER stress-induced lipin-1 reduction. -- Abstract: Lipin-1 plays crucial roles in the regulation of lipid metabolism and cell differentiation in adipocytes. In obesity, adipose lipin-1 mRNA expression is decreased and positively correlated with systemic insulin sensitivity. Amelioration of the lipin-1 depletion might be improved dysmetabolism. Although some cytokines such as TNF-α and interleukin-1β reduces adipose lipin-1 expression, the mechanism of decreased adipose lipin-1 expression in obesity remains unclear. Recently, endoplasmic reticulum (ER) stress is implicated in the pathogenesis of obesity. Here we investigated the role of ER stress on the lipin-1 expression in 3T3-L1 adipocytes. We demonstrated that lipin-1 expression was suppressed by the treatment with ER stress inducers (tunicamycin and thapsigargin) at transcriptional level. We also showed that constitutive lipin-1 expression could be maintained by peroxisome proliferator-activated receptor-γ in 3T3-L1 adipocytes. Activation of peroxisome proliferator-activated receptor-γ recovered the ER stress-induced lipin-1 suppression. These results suggested that ER stress might be involved in the pathogenesis of obesity through lipin-1 depletion.

  13. PPARγ partial agonist GQ-16 strongly represses a subset of genes in 3T3-L1 adipocytes

    SciTech Connect

    Milton, Flora Aparecida; Cvoro, Aleksandra; Amato, Angelica A.; Sieglaff, Douglas H.; Filgueira, Carly S.; Arumanayagam, Anithachristy Sigamani; Caro Alves de Lima, Maria do; Rocha Pitta, Ivan; Assis Rocha Neves, Francisco de; Webb, Paul

    2015-08-28

    Thiazolidinediones (TZDs) are peroxisome proliferator-activated receptor gamma (PPARγ) agonists that improve insulin resistance but trigger side effects such as weight gain, edema, congestive heart failure and bone loss. GQ-16 is a PPARγ partial agonist that improves glucose tolerance and insulin sensitivity in mouse models of obesity and diabetes without inducing weight gain or edema. It is not clear whether GQ-16 acts as a partial agonist at all PPARγ target genes, or whether it displays gene-selective actions. To determine how GQ-16 influences PPARγ activity on a gene by gene basis, we compared effects of rosiglitazone (Rosi) and GQ-16 in mature 3T3-L1 adipocytes using microarray and qRT-PCR. Rosi changed expression of 1156 genes in 3T3-L1, but GQ-16 only changed 89 genes. GQ-16 generally showed weak effects upon Rosi induced genes, consistent with partial agonist actions, but a subset of modestly Rosi induced and strongly repressed genes displayed disproportionately strong GQ-16 responses. PPARγ partial agonists MLR24 and SR1664 also exhibit disproportionately strong effects on transcriptional repression. We conclude that GQ-16 displays a continuum of weak partial agonist effects but efficiently represses some negatively regulated PPARγ responsive genes. Strong repressive effects could contribute to physiologic actions of GQ-16. - Highlights: • GQ-16 is an insulin sensitizing PPARγ ligand with reduced harmful side effects. • GQ-16 displays a continuum of weak partial agonist activities at PPARγ-induced genes. • GQ-16 exerts strong repressive effects at a subset of genes. • These inhibitor actions should be evaluated in models of adipose tissue inflammation.

  14. Tunable swelling of polyelectrolyte multilayers in cell culture media for modulating NIH-3T3 cells adhesion.

    PubMed

    Qi, Wei; Cai, Peng; Yuan, Wenjing; Wang, Hua

    2014-11-01

    For polyelectrolyte multilayers (PEMs) assembled by the layer-by-layer (LbL) assembly technique, their nanostructure and properties can be governed by many parameters during the building process. Here, it was demonstrated that the swelling of the PEMs containing poly(diallyldimethylammonium chloride) (PDDA) and poly(sodium 4-styrenesulfonate) (PSS) in cell culture media could be tuned with changing supporting salt solutions during the assembly process. Importantly, the influence of the PEMs assembled in different salt solutions on NIH-3T3 cell adhesion was observable. Specifically, the cells could possess a higher affinity for the films assembled in low salt concentration (i.e. 0.15M NaCl) or no salt, the poorly swelling films in cell culture media, which was manifested by the large cell spreading area and focal adhesions. In contrast, those were assembled in higher salt concentration, highly swelling films in cell culture media, were less attractive for the fibroblasts. As a result, the cell adhesion behaviors may be manipulated by tailoring the physicochemical properties of the films, which could be performed by changing the assembly conditions such as supporting salt concentration. Such a finding might promise a great potential in designing desired biomaterials for tissue engineering and regenerative medicine.

  15. Electrical Stimulation of NIH-3T3 Cells with Platinum-PEDOT-Electrodes Integrated in a Bioreactor

    PubMed Central

    Blume, Grit; Müller-Wichards, Wiebke; Goepfert, Christiane; Pörtner, Ralf; Müller, Jörg

    2013-01-01

    The objective of this work involves the development and integration of electrodes for the electrical stimulation of cells within a bioreactor. Electrodes need to fit properties such as biocompatibility, large reversible charge transfer and high flexibility in view of their future application as implants on the tympanic membrane. Flexible thin-film platinum-poly(3,4-ethylene-dioxythiophene)-electrodes on a poly(ethylene terephthalate)-foil manufactured using microsystems technology were integrated into a bioreactor based on the design of a 24 well plate. The murine fibroblast cell line NIH-3T3 was cultured on the foil electrodes and the cells were stimulated with direct voltage and unipolar pulsed voltage. The amplitude, the pulse length and the ratio of pulse to pause were varied. The stimulated cells were stained in order to determine the angle between the cell cleavage plane of the dividing cells and the vector of the electric field. These angles were subsequently used to calculate the polarization index, which is a measure of the orientation of the metaphase plane of dividing cells that occurs for example during wound healing or embryonic morphogenesis. PMID:24358059

  16. Influence of zinc deficiency on cell-membrane fluidity in Jurkat, 3T3 and IMR-32 cells.

    PubMed Central

    Verstraeten, Sandra V; Zago, M Paola; MacKenzie, Gerardo G; Keen, Carl L; Oteiza, Patricia I

    2004-01-01

    We investigated whether zinc deficiency can affect plasma membrane rheology. Three cell lines, human leukaemia T-cells (Jurkat), rat fibroblasts (3T3) and human neuroblastoma cells (IMR-32), were cultured for 48 h in control medium, in zinc-deficient medium (1.5 microM zinc; 1.5 Zn), or in the zinc-deficient medium supplemented with 15 microM zinc (15 Zn). The number of viable cells was lower in the 1.5 Zn group than in the control and 15 Zn groups. The frequency of apoptosis was higher in the 1.5 Zn group than in the control and 15 Zn groups. Membrane fluidity was evaluated using the 6-(9-anthroyloxy)stearic acid and 16-(9-anthroyloxy)palmitic acid probes. Membrane fluidity was higher in 1.5 Zn cells than in the control cells; no differences were observed between control cells and 15 Zn cells. The effect of zinc deficiency on membrane fluidity at the water/lipid interface was associated with a higher phosphatidylserine externalization. The higher membrane fluidity in the hydrophobic region of the bilayer was correlated with a lower content of arachidonic acid. We suggest that the increased fluidity of the membrane secondary to zinc deficiency is in part due to a decrease in arachidonic acid content and the apoptosis-related changes in phosphatidylserine distribution. PMID:14629198

  17. Early tissue patterning recreated by mouse embryonic fibroblasts in a three-dimensional environment.

    PubMed

    Quintana, Lluís; Muiños, Teresa Fernández; Genove, Elsa; Del Mar Olmos, María; Borrós, Salvador; Semino, Carlos E

    2009-01-01

    Cellular self-organization studies have been mainly focused on models such as Volvox, the slime mold Dictyostelium discoideum, and animal (metazoan) embryos. Moreover, animal tissues undergoing regeneration also exhibit properties of embryonic systems such as the self-organization process that rebuilds tissue complexity and function. We speculated that the recreation in vitro of the biological, biophysical, and biomechanical conditions similar to those of a regenerative milieu could elicit the intrinsic capacity of differentiated cells to proceed to the development of a tissue-like structure. Here we show that, when primary mouse embryonic fibroblasts are cultured in a soft nanofiber scaffold, they establish a cellular network that causes an organized cell contraction,proliferation, and migration that ends in the formation of a symmetrically bilateral structure with a distinct central axis. A subset of mesodermal genes (brachyury, Sox9, Runx2) is upregulated during this morphogenetic process. The expression of brachyury was localized first at the central axis, extending then to both sides of the structure. The spontaneous formation of cartilage-like tissue mainly at the paraxial zone followed expression ofSox9 and Runx2. Because cellular self-organization is an intrinsic property of the tissues undergoing development,this model could lead to new ways to consider tissue engineering and regenerative medicine.

  18. Mitochondrial fission and fusion factors reciprocally orchestrate mitophagic culling in mouse hearts and cultured fibroblasts

    PubMed Central

    Song, Moshi; Mihara, Katsuyoshi; Chen, Yun; Scorrano, Luca; Dorn, Gerald W

    2014-01-01

    Summary How mitochondrial dynamism (fission and fusion) affects mitochondrial quality control is unclear. We uncovered distinct effects on mitophagy of inhibiting Drp1-mediated mitochondrial fission versus mitofusin-mediated mitochondrial fusion. Conditional cardiomyocyte-specific Drp1 ablation evoked mitochondrial enlargement, lethal dilated cardiomyopathy, and cardiomyocyte necrosis. Conditionally ablating cardiomyocyte mitofusins (Mfn) caused mitochondrial fragmentation with eccentric remodeling and no cardiomyocyte dropout. Parallel studies in cultured murine embryonic fibroblasts (MEFs) and in vivo mouse hearts revealed that Mfn1/Mfn2 deletion provoked accumulation of defective mitochondria exhibiting an unfolded protein response, without appropriately increasing mitophagy. Conversely, interrupting mitochondrial fission by Drp1 ablation increased mitophagy and caused a generalized loss of mitochondria. Mitochondrial permeability transition pore (MPTP) opening in Drp1 null mitochondria was associated with mitophagy in MEFs and contributed to cardiomyocyte necrosis and dilated cardiomyopathy in mice. Drp1, MPTP, and cardiomyocyte mitophagy are functionally integrated. Mitochondrial fission and fusion have opposing roles during in vivo cardiac mitochondrial quality control. PMID:25600785

  19. Enhanced adherence of mouse fibroblast and vascular cells to plasma modified polyethylene.

    PubMed

    Reznickova, Alena; Novotna, Zdenka; Kolska, Zdenka; Kasalkova, Nikola Slepickova; Rimpelova, Silvie; Svorcik, Vaclav

    2015-01-01

    Since the last decade, tissue engineering has shown a sensational promise in providing more viable alternatives to surgical procedures for harvested tissues, implants and prostheses. Biomedical polymers, such as low-density polyethylene (LDPE), high-density polyethylene (HDPE) and ultra-high molecular weight polyethylene (UHMWPE), were activated by Ar plasma discharge. Degradation of polymer chains was examined by determination of the thickness of ablated layer. The amount of an ablated polymer layer was measured by gravimetry. Contact angle, measured by goniometry, was studied as a function of plasma exposure and post-exposure aging times. Chemical structure of modified polymers was characterized by angle resolved X-ray photoelectron spectroscopy. Surface chemistry and polarity of the samples were investigated by electrokinetic analysis. Changes in surface morphology were followed using atomic force microscopy. Cytocompatibility of plasma activated polyethylene foils was studied using two distinct model cell lines; VSMCs (vascular smooth muscle cells) as a model for vascular graft testing and connective tissue cells L929 (mouse fibroblasts) approved for standardized material cytotoxicity testing. Specifically, the cell number, morphology, and metabolic activity of the adhered and proliferated cells on the polyethylene matrices were studied in vitro. It was found that the plasma treatment caused ablation of the polymers, resulting in dramatic changes in their surface morphology and roughness. ARXPS and electrokinetic measurements revealed oxidation of the polymer surface. It was found that plasma activation has a positive effect on the adhesion and proliferation of VSMCs and L929 cells.

  20. Cytotoxicity of latex and non-latex orthodontic elastomeric ligatures on L929 mouse fibroblasts.

    PubMed

    Santos, Rogério Lacerda dos; Pithon, Matheus Melo; Martins, Fernanda Otaviano; Romanos, Maria Teresa Villela; Ruellas, Antônio Carlos de Oliveira

    2010-01-01

    This study investigated the cytotoxicity exists between latex and non-latex Orthodontic elastomeric ligatures. Six elastomeric ligatures (1 latex, 2 latex-free and 3 polyurethane) from different manufacturers were divided into 6 groups of 15 elastics each: A (Latex-free, American Orthodontics), M (Polyurethane, Morelli), G (Polyurethane,GAC International), Te (Polyurethane, Tecnident), TP (Natural latex,TP Orthodontics) and U (Latex-free,3M Unitek). The cytotoxicity assay was performed using cell cultures (L929 mouse fibroblast cell line), which were subjected to the cell viability test with neutral red ("dye-uptake") at 1, 2, 3, 7 and 28 days. Data were analyzed statistically by ANOVA and Tukey's test (α=0.05). No statistically significant differences (p>0.05) were observed between Groups M and Te in all experimental periods, except at 2 days. No significant differences (p>0.05) in cell viability were found either among Groups A, G, TP and U or between Groups M and Te at 24 h or among Groups CC, A, G, TP and U at 2 and 28 days. It may be concluded that latex-free elastomeric ligatures from American Orthodontics and Unitek trademarks induced less cell lysis compared to latex and polyurethane ligatures.

  1. Comparing the mechanical influence of vinculin, focal adhesion kinase and p53 in mouse embryonic fibroblasts

    SciTech Connect

    Klemm, Anna H.; Diez, Gerold; Alonso, Jose-Luis

    2009-02-13

    Cytoskeletal reorganization is an ongoing process when cells adhere, move or invade extracellular substrates. The cellular force generation and transmission are determined by the intactness of the actomyosin-(focal adhesion complex)-integrin connection. We investigated the intracellular course of action in mouse embryonic fibroblasts deficient in the focal adhesion proteins vinculin and focal adhesion kinase (FAK) and the nuclear matrix protein p53 using magnetic tweezer and nanoparticle tracking techniques. Results show that the lack of these proteins decrease cellular stiffness and affect cell rheological behavior. The decrease in cellular binding strength was higher in FAK- to vinculin-deficient cells, whilst p53-deficient cells showed no effect compared to wildtype cells. The intracellular cytoskeletal activity was lowest in wildtype cells, but increased in the following order when cells lacked FAK+p53 > p53 > vinculin. In summary, cell mechanical processes are differently affected by the focal adhesion proteins vinculin and FAK than by the nuclear matrix protein, p53.

  2. Biocompatibility of microbially reduced graphene oxide in primary mouse embryonic fibroblast cells.

    PubMed

    Gurunathan, Sangiliyandi; Han, Jae Woong; Eppakayala, Vasuki; Kim, Jin-Hoi

    2013-05-01

    Graphene nanosheet is a one-atom thick planar sheet of sp(2)-bonded carbon atoms, which are densely packed in a honeycomb crystal lattice, attracting tremendous attention from both fundamental research and industrial applications. The synthesis of graphene from graphene oxide (GO) using a biological method is one of the important topics in the areas of nanotechnology, because graphene-based nanomaterials have potential applications. A green, simple and non-toxic method for preparing graphene using biomass of Pseudomonas aeruginosa as the reducing reagent is proposed. The resulting microbially reduced graphene oxide (M-rGO) was characterized using a range of analytical techniques. UV-visible spectroscopy confirms the transition of graphene oxide to graphene. Fourier transform infrared spectroscopy (FT-IR) was used to study the changes in surface functionalities. X-ray diffraction (XRD) and high resolution scanning electron microscopy (SEM) were used to investigate the crystalline nature and the morphologies of prepared graphene respectively. Furthermore, the biocompatibility of the M-rGO was investigated using primary mouse embryonic fibroblast (PMEF) cells. The present study suggests that the M-rGO has significant biocompatibility for PMEF cells, even at a high concentration of 100 μg ml(-1). Therefore, the proposed safe and green method confers the M-rGO with a great potential for various biomedical applications.

  3. Suppression of oxidative phosphorylation in mouse embryonic fibroblast cells deficient in apurinic/apyrimidinic endonuclease

    PubMed Central

    Suganya, Rangaswamy; Chakraborty, Anirban; Miriyala, Sumitra; Hazra, Tapas K.; Izumi, Tadahide

    2015-01-01

    The mammalian apurinic/apyrimidinic (AP) endonuclease 1 (APE1) is an essential DNA repair/gene regulatory protein. Decrease of APE1 in cells by inducible shRNA knockdown or by conditional gene knockout caused apoptosis. Here we succeeded in establishing a unique mouse embryonic fibroblast (MEF) line expressing APE1 at a level far lower than those achieved with shRNA knockdown. The cells, named MEFla (MEFlowAPE1), were hypersensitive to methyl methanesulfonate (MMS), and showed little activity for repairing AP-sites and MMS induced DNA damage. While these results were consistent with the essential role of APE1 in repair of AP sites, the MEFla cells grew normally and the basal activation of poly(ADP-ribose) polymerases in MEFla was lower than that in the wild-type MEF (MEFwt), indicating the low DNA damage stress in MEFla under the normal growth condition. Oxidative phosphorylation activity in MEFla was lower than in MEFwt, while the glycolysis rates in MEFla were higher than in MEFwt. In addition, we observed decreased intracellular oxidative stress in MEFla. These results suggest that cells with low APE1 reversibly suppress mitochondrial respiration and thereby reduce DNA damage stress and increases the cell viability. PMID:25645679

  4. Cerium oxide nanoparticles stimulate proliferation of primary mouse embryonic fibroblasts in vitro.

    PubMed

    Popov, Anton L; Popova, Nelly R; Selezneva, Irina I; Akkizov, Azamat Y; Ivanov, Vladimir K

    2016-11-01

    The increasing application of cell therapy technologies in the treatment of various diseases requires the development of new effective methods for culturing primary cells. The major limitation for the efficient use of autologous cell material is the low rate of cell proliferation. Successful cell therapy requires sufficient amounts of cell material over a short period of time with the preservation of their differentiation and proliferative potential. In this regard, the development of novel, highly efficient stimulators of proliferative activity in stem cells is a truly urgent task. In this paper we have demonstrated that citrate-stabilized cerium oxide nanoparticles (nanoceria) enhance the proliferative activity of primary mouse embryonic fibroblasts in vitro. Cerium oxide nanoparticles stimulate cell proliferation in a wide range of concentrations (10(-3)М-10(-9)M) through reduction of intracellular levels of reactive oxygen species (ROS) during the lag phase of cell growth and by modulating the expression level of the major antioxidant enzymes. We found the optimal concentration of nanoceria, which provides the greatest acceleration of cell proliferation in vitro, while maintaining the levels of intracellular ROS and mRNA of antioxidant enzymes in the physiological range. Our results confirm that nanocrystalline ceria can be considered as a basis for effective and inexpensive supplements in cell culturing.

  5. Metabolomic Analysis of Mouse Embryonic Fibroblast Cells in Response to Autophagy Induced by Acute Starvation

    PubMed Central

    Shen, Sensen; Weng, Rui; Li, Linnan; Xu, Xinyuan; Bai, Yu; Liu, Huwei

    2016-01-01

    Autophagy-related protein 7 (Atg7) is essential in the formation of the autophagophore and is indispensable for autophagy induction. Autophagy will exist in lower level or even be blocked in cells without Atg7. Even though the possible signaling pathways of Atg7 have been proposed, the metabolomic responses under acute starvation in cells with and without Atg7 have not been elucidated. This study therefore was designed and aimed to reveal the metabolomics of Atg7-dependent autophagy through metabolomic analysis of Atg7−/− mouse embryonic fibroblast cells (MEFs) and wild-type MEFs along with the starvation time. 30 significantly altered metabolites were identified in response to nutrient stress, which were mainly associated with amino acid, energy, carbohydrate, and lipid metabolism. For the wild-type MEFs, the induction of autophagy protected cell survival with some up-regulated lipids during the first two hours’ starvation, while the subsequent apoptosis resulted in the decrease of cell viability after four hours’ starvation. For the Atg7−/− MEFs, apoptosis perhaps led to the deactivation of tricarboxylic acid (TCA) cycle due to the lack of autophagy, which resulted in the immediate drop of cellular viability under starvation. These results contributed to the metabolomic study and provided new insights into the mechanism associated with Atg7-dependent autophagy. PMID:27703171

  6. Biocompatibility effects of biologically synthesized graphene in primary mouse embryonic fibroblast cells

    NASA Astrophysics Data System (ADS)

    Gurunathan, Sangiliyandi; Han, Jae Woong; Eppakayala, Vasuki; Dayem, Ahmed Abdal; Kwon, Deug-Nam; Kim, Jin-Hoi

    2013-09-01

    Due to unique properties and unlimited possible applications, graphene has attracted abundant interest in the areas of nanobiotechnology. Recently, much work has focused on the synthesis and properties of graphene. Here we show that a successful reduction of graphene oxide (GO) using spinach leaf extract (SLE) as a simultaneous reducing and stabilizing agent. The as-prepared SLE-reduced graphene oxide (S-rGO) was characterized by ultraviolet-visible spectroscopy and Fourier transform infrared spectroscopy. Dynamic light scattering technique was used to determine the average size of GO and S-rGO. Scanning electron microscopy and atomic force microscopy images provide clear surface morphological evidence for the formation of graphene. The resulting S-rGO has a mostly single-layer structure, is stable, and has significant water solubility. In addition, the biocompatibility of graphene was investigated using cell viability, leakage of lactate dehydrogenase and alkaline phosphatase activity in primary mouse embryonic fibroblast (PMEFs) cells. The results suggest that the biologically synthesized graphene has significant biocompatibility with PMEF cells, even at a higher concentration of 100 μg/mL. This method uses a `green', natural reductant and is free of additional stabilizing reagents; therefore, it is an environmentally friendly, simple, and cost-effective method for the fabrication of soluble graphene. This study could open up a promising view for substitution of hydrazine by a safe, biocompatible, and powerful reduction for the efficient deoxygenation of GO, especially in large-scale production and potential biomedical applications.

  7. Random mtDNA mutations modulate proliferation capacity in mouse embryonic fibroblasts

    SciTech Connect

    Kukat, Alexandra; Edgar, Daniel; Bratic, Ivana; Maiti, Priyanka; Trifunovic, Aleksandra

    2011-06-10

    Highlights: {yields} Increased mtDNA mutations in MEFs lead to high level of spontaneous immortalization. {yields} This process is independent of endogenous ROS production. {yields} Aerobic glycolysis significantly contributes to spontaneous immortalization of MEFs. -- Abstract: An increase in mtDNA mutation load leads to a loss of critical cells in different tissues thereby contributing to the physiological process of organismal ageing. Additionally, the accumulation of senescent cells that display changes in metabolic function might act in an active way to further disrupt the normal tissue function. We believe that this could be the important link missing in our understanding of the molecular mechanisms of premature ageing in the mtDNA mutator mice. We tested proliferation capacity of mtDNA mutator cells in vitro. When cultured in physiological levels of oxygen (3%) their proliferation capacity is somewhat lower than wild-type cells. Surprisingly, in conditions of increased oxidative stress (20% O{sub 2}) mtDNA mutator mouse embryonic fibroblasts exhibit continuous proliferation due to spontaneous immortalization, whereas the same conditions promote senescence in wild-type cells. We believe that an increase in aerobic glycolysis observed in mtDNA mutator mice is a major mechanism behind this process. We propose that glycolysis promotes proliferation and allows a fast turnover of metabolites, but also leads to energy crisis due to lower ATP production rate. This could lead to compromised replication and/or repair and therefore, in rare cases, might lead to mutations in tumor suppressor genes and spontaneous immortalization.

  8. Ontogeny of basic fibroblast growth factor binding sites in mouse ocular tissues

    SciTech Connect

    Fayein, N.A.; Courtois, Y.; Jeanny, J.C. )

    1990-05-01

    Basic fibroblast growth factor (bFGF) binding to ocular tissues has been studied by autoradiographical and biochemical approaches directly performed on sections during mouse embryonic and postnatal development. Frozen sections of embryos (9 to 18 days), newborns, and adults (1 day to 6 months) were incubated with iodinated bFGF. One specific FGF binding site (KD = 2.5 nM) is colocalized with heparan sulfate proteoglycans of the basement membranes and is heparitinase sensitive. It first appears at Day 9 around the neural tube, the optic vesicles, and below the head ectoderm and by Day 14 of embryonic development is found in all basement membranes of the eye. At Day 16, very intensely labeled patches appear, corresponding to mast cells which have been characterized by metachromatic staining of their heparin-rich granulations with toluidine blue. In addition to the latter binding, we have also observed a general diffuse distribution of silver grains on all tissues and preferentially in the ecto- and neuroectodermic tissues. From Days 17-18, there is heterogeneous labeling inside the retina, localized in the pigmented epithelium and in three different layers colocalized with the inner and outer plexiform layers and with the inner segments of the photoreceptors. This binding is heparitinase resistant but N-glycanase sensitive and may represent a second specific binding site corresponding to cellular FGF receptors (KD = 280 pM). Both types of binding patterns observed suggest a significant role for bFGF in eye development and physiology.

  9. Research Resource: Comprehensive Expression Atlas of the Fibroblast Growth Factor System in Adult Mouse

    PubMed Central

    Fon Tacer, Klementina; Bookout, Angie L.; Ding, Xunshan; Kurosu, Hiroshi; John, George B.; Wang, Lei; Goetz, Regina; Mohammadi, Moosa; Kuro-o, Makoto; Mangelsdorf, David J.; Kliewer, Steven A.

    2010-01-01

    Although members of the fibroblast growth factor (FGF) family and their receptors have well-established roles in embryogenesis, their contributions to adult physiology remain relatively unexplored. Here, we use real-time quantitative PCR to determine the mRNA expression patterns of all 22 FGFs, the seven principal FGF receptors (FGFRs), and the three members of the Klotho family of coreceptors in 39 different mouse tissues. Unsupervised hierarchical cluster analysis of the mRNA expression data reveals that most FGFs and FGFRs fall into two groups the expression of which is enriched in either the central nervous system or reproductive and gastrointestinal tissues. Interestingly, the FGFs that can act as endocrine hormones, including FGF15/19, FGF21, and FGF23, cluster in a third group that does not include any FGFRs, underscoring their roles in signaling between tissues. We further show that the most recently identified Klotho family member, Lactase-like, is highly and selectively expressed in brown adipose tissue and eye and can function as an additional coreceptor for FGF19. This FGF atlas provides an important resource for guiding future studies to elucidate the physiological functions of FGFs in adult animals. PMID:20667984

  10. Reprogramming of Mouse, Rat, Pig, and Human Fibroblasts into iPS Cells

    PubMed Central

    Wu, Sean M.

    2012-01-01

    The induction of pluripotency in somatic cells by transcription factor overexpression has been widely regarded as one of the major breakthroughs in stem cell biology within this decade. The generation of these induced pluripotent stem cells (iPSCs) has enabled investigators to develop in vitro disease models for biological discovery and drug screening, and in the future, patient-specific therapy for tissue or organ regeneration. While new technologies for reprogramming are continually being discovered, the availability of iPSCs from different species is also increasing rapidly. Comparison of iPSCs across species may provide new insights into key aspects of pluripotency and early embryonic development. iPSCs from large animals may enable the generation of genetically-modified large animal models or potentially transplantable donor tissues or organs. In this unit, we describe the procedure for the generation of iPSCs from mouse, rat, pig and human fibroblasts. We focus on lenti- and retroviral infection as the main platform for pluripotent transcription factor overexpression since these reagents are widely-available and remain the most efficient way to generate iPSC colonies. We hope to illustrate the basic process for iPSC generation in these four species in such a way that would enable the lowering of the entry barrier into iPSC biology by new investigators. PMID:22237859

  11. Early Tissue Patterning Recreated by Mouse Embryonic Fibroblasts in a Three-Dimensional Environment

    PubMed Central

    Quintana, Lluís; Muiños, Teresa Fernández; Genové, Elsa; Del Mar Olmos, María; Borrós, Salvador

    2009-01-01

    Cellular self-organization studies have been mainly focused on models such as Volvox, the slime mold Dictyostelium discoideum, and animal (metazoan) embryos. Moreover, animal tissues undergoing regeneration also exhibit properties of embryonic systems such as the self-organization process that rebuilds tissue complexity and function. We speculated that the recreation in vitro of the biological, biophysical, and biomechanical conditions similar to those of a regenerative milieu could elicit the intrinsic capacity of differentiated cells to proceed to the development of a tissue-like structure. Here we show that, when primary mouse embryonic fibroblasts are cultured in a soft nanofiber scaffold, they establish a cellular network that causes an organized cell contraction, proliferation, and migration that ends in the formation of a symmetrically bilateral structure with a distinct central axis. A subset of mesodermal genes (brachyury, Sox9, Runx2) is upregulated during this morphogenetic process. The expression of brachyury was localized first at the central axis, extending then to both sides of the structure. The spontaneous formation of cartilage-like tissue mainly at the paraxial zone followed expression of Sox9 and Runx2. Because cellular self-organization is an intrinsic property of the tissues undergoing development, this model could lead to new ways to consider tissue engineering and regenerative medicine. PMID:19025338

  12. Wound Healing Activity of Extracts and Formulations of Aloe vera, Henna, Adiantum capillus-veneris, and Myrrh on Mouse Dermal Fibroblast Cells

    PubMed Central

    Negahdari, Samira; Galehdari, Hamid; Kesmati, Mahnaz; Rezaie, Anahita; Shariati, Gholamreza

    2017-01-01

    Background: Among the most important factors in wound healing pathways are transforming growth factor beta1 and vascular endothelial growth factor. Fibroblasts are the main cell in all phases wound closure. In this study, the extracts of plant materials such as Adiantum capillus-veneris, Commiphora molmol, Aloe vera, and henna and one mixture of them were used to treatment of normal mouse skin fibroblasts. Methods: Cytotoxic effects of each extract and their mixture were assessed on mouse skin fibroblasts cells using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. We performed migration assays to assess migration properties of mouse skin fibroblasts cells in response to the extracts. Changes in the gene expression of the Tgfβ1 and Vegf-A genes were monitored by real-time polymerase chain reaction. Results: A. capillus-veneris, C. molmol and henna extract improved the expression of Tgfβ1 gene. All used extracts upregulated the expression of Vegf-A gene and promoted the migration of mouse fibroblast cells in vitro. Conclusions: The present study demonstrated that the mentioned herbal extracts might be effective in wound healing, through the improvement in the migration of fibroblast cells and regulating the gene expression of Tgfβ1 and Vegf-A genes in fibroblast cells treated with extracts. PMID:28382194

  13. Tea catechins modulate the glucose transport system in 3T3-L1 adipocytes.

    PubMed

    Ueda, Manabu; Furuyashiki, Takashi; Yamada, Kayo; Aoki, Yukiko; Sakane, Iwao; Fukuda, Itsuko; Yoshida, Ken-Ichi; Ashida, Hitoshi

    2010-11-01

    In this study, we investigated the effects of tea catechins on the translocation of glucose transporter (GLUT) 4 in 3T3-L1 adipocytes. We found that the ethyl acetate fraction of green tea extract, containing abundant catechins, most decreased insulin-induced glucose uptake activity in 3T3-L1 cells. When the cells were treated with 50 μM catechins in the absence or presence of insulin for 30 min, nongallate-type catechins increased glucose uptake activity without insulin, whereas gallate-type catechins decreased insulin-induced glucose uptake activity. (-)-Epicatechin (EC) and (-)-epigallocatechin (EGC), nongallate-type catechins, increased glucose uptake activity in the dose- and time-dependent manner, whereas (-)-catechin 3-gallate (Cg) and (-)-epigallocatechin 3-gallate (EGCg), gallate-type catechins, decreased insulin-induced glucose uptake activity in the dose- and time-dependent manner. When the cells were treated with 50 μM catechins for 30 min, EC and EGC promoted GLUT4 translocation, whereas Cg and EGCg decreased the insulin-induced translocation in the cells. EC and EGC increased phosphorylation of PKCλ/ζ without phosphorylation of insulin receptor (IR) and Akt. Wortmannin and LY294002, inhibitors for phosphatidylinositol 3'-kinase (PI3K), decreased EC- and EGC-induced glucose uptake activity in the cells. Cg and EGCg decreased phosphorylation of PKCλ/ζ in the presence of insulin without affecting insulin-induced phosphorylation of IR, and Akt. Therefore, EC and EGC promote the translocation of GLUT4 through activation of PI3K, and Cg and EGCg inhibit insulin-induced translocation of GLUT4 by the insulin signaling pathway in 3T3-L1 cells.

  14. The aporphine alkaloid boldine induces adiponectin expression and regulation in 3T3-L1 cells.

    PubMed

    Yu, Bangning; Cook, Carla; Santanam, Nalini

    2009-10-01

    Adiponectin is an adipokine secreted by differentiated adipocytes. Clinical studies suggest a negative correlation between oxidative stress and adiponectin levels in patients with metabolic syndrome or cardiovascular disease. Natural compounds that can prevent oxidative stress mediated inhibition of adiponectin may be potentially therapeutic. Boldine, an aporphine alkaloid abundant in the medicinal plant Peumus boldus, is a powerful antioxidant. The current study demonstrates the effects of boldine on the expression of adiponectin and its regulators, CCAAT/enhancer binding protein-alpha (C/EBPalpha) and peroxisome proliferator-activated receptor (PPAR)-gamma, in 3T3-L1 cells. Differentiated 3T3-L1 adipocytes were exposed to either hydrogen peroxide (H(2)O(2)) (100 microM) or tumor necrosis factor-alpha (TNFalpha) (1 ng/mL) for 24 hours in the presence or absence of increasing concentrations of boldine (5-100 microM). Quantitative polymerase chain reaction showed that both the oxidants decreased the mRNA levels of adiponectin, PPARgamma, and C/EBPalpha to half of the control levels. Boldine, at all concentrations, counteracted the inhibitory effect of H(2)O(2) or TNFalpha and increased the expression of adiponectin and its regulators. The effect of boldine on adiponectin expression was biphasic, with the lower concentrations (5-25 microM) having a larger inductive effect compared to higher concentrations (50-100 microM). Boldine treatment alone in the absence of H(2)O(2) or TNFalpha was also able to induce adiponectin at the inductive phase of adipogenesis. Peroxisome proliferator response element-luciferase promoter transactivity analysis showed that boldine interacts with the PPAR response element and could potentially modulate PPAR responsive genes. Our results indicate that boldine is able to modulate the expression of adiponectin and its regulators in 3T3-L1 cells and has the potential to be beneficial in obesity-related cardiovascular disease.

  15. The Aporphine Alkaloid Boldine Induces Adiponectin Expression and Regulation in 3T3-L1 Cells

    PubMed Central

    Yu, Bangning; Cook, Carla

    2009-01-01

    Abstract Adiponectin is an adipokine secreted by differentiated adipocytes. Clinical studies suggest a negative correlation between oxidative stress and adiponectin levels in patients with metabolic syndrome or cardiovascular disease. Natural compounds that can prevent oxidative stress mediated inhibition of adiponectin may be potentially therapeutic. Boldine, an aporphine alkaloid abundant in the medicinal plant Peumus boldus, is a powerful antioxidant. The current study demonstrates the effects of boldine on the expression of adiponectin and its regulators, CCAAT/enhancer binding protein-α (C/EBPα) and peroxisome proliferator-activated receptor (PPAR)-γ, in 3T3-L1 cells. Differentiated 3T3-L1 adipocytes were exposed to either hydrogen peroxide (H2O2) (100 μM) or tumor necrosis factor-α (TNFα) (1 ng/mL) for 24 hours in the presence or absence of increasing concentrations of boldine (5–100 μM). Quantitative polymerase chain reaction showed that both the oxidants decreased the mRNA levels of adiponectin, PPARγ, and C/EBPα to half of the control levels. Boldine, at all concentrations, counteracted the inhibitory effect of H2O2 or TNFα and increased the expression of adiponectin and its regulators. The effect of boldine on adiponectin expression was biphasic, with the lower concentrations (5–25 μM) having a larger inductive effect compared to higher concentrations (50–100 μM). Boldine treatment alone in the absence of H2O2 or TNFα was also able to induce adiponectin at the inductive phase of adipogenesis. Peroxisome proliferator response element-luciferase promoter transactivity analysis showed that boldine interacts with the PPAR response element and could potentially modulate PPAR responsive genes. Our results indicate that boldine is able to modulate the expression of adiponectin and its regulators in 3T3-L1 cells and has the potential to be beneficial in obesity-related cardiovascular disease. PMID:19857072

  16. Lysophosphatidic acid induces chemotaxis in MC3T3-E1 osteoblastic cells

    SciTech Connect

    Masiello, Lisa M.; Fotos, Joseph S.; Galileo, Deni S.; Karin, Norm J.

    2006-07-01

    Lysophosphatidic acid (LPA) is a bioactive lipid that has pleiotropic effects on a variety of cell types and enhances the migration of endothelial and cancer cells, but it is not known if this lipid can alter osteoblast motility. We performed transwell migration assays using MC3T3-E1 osteoblastic cells and found LPA to be a potent chemotactic agent. Quantitative time-lapse video analysis of osteoblast migration after wounds were introduced into cell monolayers indicated that LPA stimulated both migration velocity and the average migration distance per cell. LPA also elicited substantial changes in cell shape and actin cytoskeletal structure; lipid-treated cells contained fewer stress fibers and displayed long membrane processes that were enriched in F-actin. Quantitative RT-PCR analysis showed that MC3T3-E1 cells express all four known LPA-specific G protein-coupled receptors (LPA1-LPA4) with a relative mRNA abundance of LPA1 > LPA4 > LPA2 >> LPA3. LPA-induced changes in osteoblast motility and morphology were antagonized by both pertussis toxin and Ki16425, a subtype-specific blocker of LPA1 and LPA3 receptor function. Cell migration in many cell types is linked to changes in intracellular Ca2+. Ki16425 also inhibited LPA-induced Ca2+ signaling in a dose-dependent manner, suggesting a link between LPA-induced Ca2+ transients and osteoblast chemotaxis. Our data show that LPA stimulates MC3T3-E1 osteoblast motility via a mechanism that is linked primarily to the G protein-coupled receptor LPA1.

  17. Nebivolol stimulates mitochondrial biogenesis in 3T3-L1 adipocytes

    SciTech Connect

    Huang, Chenglin; Chen, Dongrui; Xie, Qihai; Yang, Ying; Shen, Weili

    2013-08-16

    Highlights: •Nebivolol may act as a partial agonist of β3-adrenergic receptor (AR). •Nebivolol stimulates mitochondrial DNA replication and protein expression. •Nebivolol promotes mitochondrial synthesis via activation of eNOS by β3-AR. -- Abstract: Nebivolol is a third-generation β-adrenergic receptor (β-AR) blocker with additional beneficial effects, including the improvement of lipid and glucose metabolism in obese individuals. However, the underlying mechanism of nebivolol’s role in regulating the lipid profile remains largely unknown. In this study, we investigated the role of nebivolol in mitochondrial biogenesis in 3T3-L1 adipocytes. Exposure of 3T3-L1 cells to nebivolol for 24 h increased mitochondrial DNA copy number, mitochondrial protein levels and the expression of transcription factors involved in mitochondrial biogenesis, including PPAR-γ coactivator-1α (PGC-1α), Sirtuin 3 (Sirt3), mitochondrial transcription factor A (Tfam) and nuclear related factor 1 (Nrf1). These changes were accompanied by an increase in oxygen consumption and in the expression of genes involved in fatty acid oxidation and antioxidant enzymes in 3T3-L1 adipocytes, including nebivolol-induced endothelial nitric oxide synthase (eNOS), as well as an increase in the formation of cyclic guanosine monophosphate (cGMP). Pretreatment with NG-nitro-L-arginine methyl ester (l-NAME) attenuated nebivolol-induced mitochondrial biogenesis, as did the soluble guanylate cyclase inhibitor, ODQ. Treatment with nebivolol and β3-AR blocker SR59230A markedly attenuated PGC-1α, Sirt3 and manganese superoxide dismutase (MnSOD) protein levels in comparison to treatment with nebivolol alone. These data indicate that the mitochondrial synthesis and metabolism in adipocytes that is promoted by nebivolol is primarily mediated through the eNOS/cGMP-dependent pathway and is initiated by the activation of β3-AR receptors.

  18. Gsalpha signalling suppresses PPARgamma2 generation and inhibits 3T3L1 adipogenesis.

    PubMed

    Zhang, Lei; Paddon, Carol; Lewis, Mark D; Grennan-Jones, Fiona; Ludgate, Marian

    2009-08-01

    Since TSH receptor (TSHR) expression increases during adipogenesis and signals via cAMP/phospho-cAMP-response element binding protein (CREB), reported to be necessary and sufficient for adipogenesis, we hypothesised that TSHR activation would induce preadipocyte differentiation. Retroviral vectors introduced constitutively active TSHR (TSHR*) into 3T3L1 preadipocytes; despite increased cAMP (RIA) and phospho-CREB (western blot) there was no spontaneous adipogenesis (assessed morphologically, using oil red O and QPCR measurement of adipogenesis markers). We speculated that Gbetagamma signalling may be inhibitory but failed to induce adipogenesis using activated Gsalpha (gsp*). Inhibition of phosphodiesterases did not promote adipogenesis in TSHR* or gsp* populations. Furthermore, differentiation induced by adipogenic medium with pioglitazone was reduced in TSHR* and abolished in gsp* expressing 3T3L1 cells. TSHR* and gsp* did not inactivate PPARgamma (PPARG as listed in the HUGO database) by phosphorylation but expression of PPARgamma1 was reduced and PPARgamma2 undetectable in gsp*. FOXO1 phosphorylation (required to inactivate this repressor of adipogenesis) was lowest in gsp* despite the activation of AKT by phosphorylation. PROF is a mediator that facilitates FOXO1 phosphorylation by phospho-Akt. Its transcript levels remained constantly low in the gsp* population. In most measurements, the TSHR* cells were between the gsp* and control 3T3L1 preadipocytes. The enhanced down-regulation of PREF1 (adipogenesis inhibitor) permits retention of some adipogenic potential in the TSHR* population. We conclude that Gsalpha signalling impedes FOXO1 phosphorylation and thus inhibits PPARgamma transcription and the alternative promoter usage required to generate PPARgamma2, the fat-specific transcription factor necessary for adipogenesis.

  19. Cytotoxicity, oxidative stress, apoptosis and the autophagic effects of silver nanoparticles in mouse embryonic fibroblasts.

    PubMed

    Lee, Yu-Hsuan; Cheng, Fong-Yu; Chiu, Hui-Wen; Tsai, Jui-Chen; Fang, Chun-Yong; Chen, Chun-Wan; Wang, Ying-Jan

    2014-05-01

    With the advancement of nanotechnology, nanomaterials have been comprehensively applied in our modern society. However, the hazardous impacts of nanoscale particles on organisms have not yet been thoroughly clarified. Currently, there exist numerous approaches to perform toxicity tests, but common and reasonable bio-indicators for toxicity evaluations are lacking. In this study, we investigated the effects of silver nanoparticles (AgNPs) on NIH 3T3 cells to explore the potential application of these nanoparticles in consumer products. Our results demonstrated that AgNPs were taken up by NIH 3T3 cells and localized within the intracellular endosomal compartments. Exposure to AgNPs is a potential source of oxidative stress, which leads to the induction of reactive oxygen species (ROS), the up-regulation of Heme oxygenase 1 (HO-1) expression, apoptosis and autophagy. Interestingly, AgNPs induced morphological and biochemical markers of autophagy in NIH 3T3 cells and induced autophagosome formation, as evidenced by transmission electron microscopic analysis, the formation of microtubule-associated protein-1 light chain-3 (LC3) puncta and the expression of LC3-II protein. Thus, autophagy activation may be a key player in the cellular response against nano-toxicity.

  20. Tocotrienol suppresses adipocyte differentiation and Akt phosphorylation in 3T3-L1 preadipocytes.

    PubMed

    Uto-Kondo, Harumi; Ohmori, Reiko; Kiyose, Chikako; Kishimoto, Yoshimi; Saito, Hisako; Igarashi, Osamu; Kondo, Kazuo

    2009-01-01

    In vivo studies show that alpha-tocotrienol and gamma-tocotrienol accumulate in adipose tissue. Furthermore, a recent study reports that the oral administration of gamma-tocotrienol from a tocotrienol-rich fraction from palm oil (TRF) decreases body fat levels in rats. The objective of this study was to evaluate the effect of TRF and its components on adipocyte differentiation in 3T3-L1 preadipocytes, which differentiated into adipocytes in the presence of 1.8 micromol/L insulin. TRF suppressed the insulin-induced mRNA expression of adipocyte-specific genes such as PPARgamma, adipocyte fatty acid-binding protein (aP2), and CCAAT/enhancer-binding protein-alpha (C/EBPalpha) compared with the differentiation of 3T3-L1 preadipocytes into adipocytes only in the presence of insulin. To confirm the suppressive effect of TRF, the major components of TRF, such as alpha-tocotrienol, gamma-tocotrienol, and alpha-tocopherol, were investigated. Alpha-tocotrienol and gamma-tocotrienol decreased the insulin-induced PPARgamma mRNA expression by 55 and 90%, respectively, compared with insulin, whereas alpha-tocopherol increased the mRNA expression. In addition, gamma-tocotrienol suppressed the insulin-induced aP2 and C/EBPalpha mRNA expression, triglyceride accumulation, and PPARgamma protein levels compared with insulin. The current results also revealed that gamma-tocotrienol inhibited the insulin-stimulated phosphorylation of Akt but not extracellular signal-regulated kinase (ERK)1/2 in the insulin signaling pathway of 3T3-L1 preadipocytes. Thus, the antiadipogenic effect of TRF depends on alpha-tocotrienol and gamma-tocotrienol, and gamma-tocotrienol may be a more potent inhibitor of adipogenesis than alpha-tocotrienol. Therefore, the results of this study suggest that tocotrienol suppresses insulin-induced differentiation and Akt phosphorylation in 3T3-L1 preadipocytes. Furthermore, tocotrienol could act as an antiadipogenic vitamin in the nutrient-mediated regulation of body

  1. Biocompatibility effects of biologically synthesized graphene in primary mouse embryonic fibroblast cells

    PubMed Central

    2013-01-01

    Due to unique properties and unlimited possible applications, graphene has attracted abundant interest in the areas of nanobiotechnology. Recently, much work has focused on the synthesis and properties of graphene. Here we show that a successful reduction of graphene oxide (GO) using spinach leaf extract (SLE) as a simultaneous reducing and stabilizing agent. The as-prepared SLE-reduced graphene oxide (S-rGO) was characterized by ultraviolet–visible spectroscopy and Fourier transform infrared spectroscopy. Dynamic light scattering technique was used to determine the average size of GO and S-rGO. Scanning electron microscopy and atomic force microscopy images provide clear surface morphological evidence for the formation of graphene. The resulting S-rGO has a mostly single-layer structure, is stable, and has significant water solubility. In addition, the biocompatibility of graphene was investigated using cell viability, leakage of lactate dehydrogenase and alkaline phosphatase activity in primary mouse embryonic fibroblast (PMEFs) cells. The results suggest that the biologically synthesized graphene has significant biocompatibility with PMEF cells, even at a higher concentration of 100 μg/mL. This method uses a ‘green’, natural reductant and is free of additional stabilizing reagents; therefore, it is an environmentally friendly, simple, and cost-effective method for the fabrication of soluble graphene. This study could open up a promising view for substitution of hydrazine by a safe, biocompatible, and powerful reduction for the efficient deoxygenation of GO, especially in large-scale production and potential biomedical applications. PMID:24059222

  2. Connective tissue fibroblast properties are position-dependent during mouse digit tip regeneration.

    PubMed

    Wu, Yuanyuan; Wang, Karen; Karapetyan, Adrine; Fernando, Warnakulusuriya Akash; Simkin, Jennifer; Han, Manjong; Rugg, Elizabeth L; Muneoka, Ken

    2013-01-01

    A key factor that contributes to the regenerative ability of regeneration-competent animals such as the salamander is their use of innate positional cues that guide the regeneration process. The limbs of mammals has severe regenerative limitations, however the distal most portion of the terminal phalange is regeneration competent. This regenerative ability of the adult mouse digit is level dependent: amputation through the distal half of the terminal phalanx (P3) leads to successful regeneration, whereas amputation through a more proximal location, e.g. the subterminal phalangeal element (P2), fails to regenerate. Do the connective tissue cells of the mammalian digit play a role similar to that of the salamander limb in controlling the regenerative response? To begin to address this question, we isolated and cultured cells of the connective tissue surrounding the phalangeal bones of regeneration competent (P3) and incompetent (P2) levels. Despite their close proximity and localization, these cells show very distinctive profiles when characterized in vitro and in vivo. In vitro studies comparing their proliferation and position-specific interactions reveal that cells isolated from the P3 and P2 are both capable of organizing and differentiating epithelial progenitors, but with different outcomes. The difference in interactions are further characterized with three-dimension cultures, in which P3 regenerative cells are shown to lack a contractile response that is seen in other fibroblast cultures, including the P2 cultures. In in vivo engraftment studies, the difference between these two cell lines is made more apparent. While both P2 and P3 cells participated in the regeneration of the terminal phalanx, their survival and proliferative indices were distinct, thus suggesting a key difference in their ability to interact within a regeneration permissive environment. These studies are the first to demonstrate distinct positional characteristics of connective tissue

  3. JMV641: a potent bombesin receptor antagonist that inhibits Swiss 3T3 cell proliferation.

    PubMed

    Azay, J; Gagne, D; Devin, C; Llinares, M; Fehrentz, J A; Martinez, J

    1996-08-27

    The peptides of the bombesin family are involved in stimulation of mitogenesis in various cell lines, including cancerous cell lines. Bombesin receptor antagonists are of great interest to inhibit this proliferation. We have synthesized a potent bombesin receptor antagonist, e.g., compound JMV641 [H-DPhe-Gln-Trp-Ala-Val-Gly-His-NH-*CH[CH2-CH(CH3)2]-**CHOH- (CH2)3-CH3 [*(S); **92% of (S) isomer], in which a pseudopeptide bond mimicking the transition state analogue replaced the peptide bond between the two C-terminal residues. This compound was highly potent to dose-dependently inhibit binding of 125I-GRP to Swiss 3T3 cells (IC50 = 0.85 +/- 0.15 nM) and bombesin-stimulated Swiss 3T3 proliferation (pA2 = 8.78). However, compound JMV641 can inhibit bombesin-induced AP-1 regulated genes that are nuclear messengers mediating the actions of signal transduction pathways stimulated by growth factors.

  4. Combination therapy with catechins and caffeine inhibits fat accumulation in 3T3-L1 cells

    PubMed Central

    Zhu, Xiaojuan; Yang, Licong; Xu, Feng; Lin, Lezhen; Zheng, Guodong

    2017-01-01

    Catechins and caffeine, which are green tea components, have a slimming effect; however, the combinational effect of fat metabolism in 3T3-L1 cells remains unclear. In the present study, 3T3-L1 cells were treated with catechins and caffeine in combination, and it was found that combination therapy with catechins and caffeine markedly reduced intracellular fat accumulation, mRNA expression levels of peroxisome proliferator-activated receptor-γ and CCAAT/enhancer-binding protein α in the early stage of cell differentiation were significantly reduced, and mRNA expression of fatty acid synthetase(FAS) andglycerol-3-phosphate dehydrogenase protein expression levels of FAS were downregulated. Noradrenaline-induced lipolysis was enhanced by caffeine, which markedly increased the protein expression of adipose triglyceride lipase and hormone sensitive lipase. These results indicated that combination therapy with catechins and caffeine synergistically inhibited lipid accumulation by regulating the gene and protein expression levels of lipid metabolism-related enzymes. Therefore, catechins and caffeine combination therapy has potential as a functional food that may be used to prevent obesity and lifestyle-associated diseases. PMID:28352352

  5. Berberine inhibits 3T3-L1 adipocyte differentiation through the PPARgamma pathway.

    PubMed

    Huang, Cheng; Zhang, Yuebo; Gong, Zhenwei; Sheng, Xiaoyan; Li, Zongmeng; Zhang, Wei; Qin, Ying

    2006-09-22

    Berberine (BBR), a compound purified from Cortidis rhizoma, reduces serum cholesterol, triglycerides, and LDL-cholesterol of hypercholesterolemic patients and high fat diet fed animals, and increases hepatic LDLR mRNA and protein levels through a post-transcriptional mechanism. BBR also enhances the hypoglycemic action of insulin in diabetic animal models. Here, we show that BBR inhibits the differentiation of 3T3-L1 preadipocytes induced by DM and suppresses the mitotic clonal expansion of 3T3-L1 preadipocytes in a time- and dose-dependent manner. Gene expression analysis and Western blot analysis reveal that the BBR inhibits the mRNA and protein levels of adipogenesis related transcription factors PPARgamma and C/EBPalpha and their upstream regulator, C/EBPbeta. Reporter gene assays demonstrate that the full-length PPARgamma and alpha transcription activities are inhibited by BBR. Using real-time PCR, we have also found that the PPAR target genes that are involved in adipocyte differentiation, such as aP2, CD36, ACO, LPL, and other adipocyte markers, are suppressed by BBR. These studies suggest that BBR works on multiple molecular targets as an inhibitor of PPARgamma and alpha, and is a potential weight reducing, hypolipidemic, and hypoglycemic drug.

  6. Effect of pycnogenol on glucose transport in mature 3T3-L1 adipocytes.

    PubMed

    Lee, Hee-Hyun; Kim, Kui-Jin; Lee, Ok-Hwan; Lee, Boo-Yong

    2010-08-01

    Pycnogenol, a procyanidins-enriched extract of Pinus maritima bark, possesses antidiabetic properties, which improves the altered parameters of glucose metabolism that are associated with type 2 diabetes mellitus (T2DM). Since the insulin-stimulated antidiabetic activities of natural bioactive compounds are mediated by GLUT4 via the phosphatidylinositol-3-kinase (PI3K) and/or p38 mitogen activated protein kinase (p38-MAPK) pathway, the effects of pycnogenol were examined on the molecular mechanism of glucose uptake by the glucose transport system. 3T3-L1 adipocytes were treated with various concentrations of pycnogenol, and glucose uptake was examined using a non-radioisotope enzymatic assay and by molecular events associated with the glucose transport system using semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR). The results show that pycnogenol increased glucose uptake in fully differentiated 3T3-L1 adipocytes and increased the relative abundance of both GLUT4 and Akt mRNAs through the PI3K pathway in a dose dependent manner. Furthermore, pycnogenol restored the PI3K antagonist-induced inhibition of glucose uptake in the presence of wartmannin, an inhibitor of the PI3K. Overall, these results indicate that pycnogenol may stimulate glucose uptake via the PI3K dependent tyrosine kinase pathways involving Akt. Further the results suggest that pycnogenol might be useful in maintaining blood glucose control.

  7. Paprika Pigments Attenuate Obesity-Induced Inflammation in 3T3-L1 Adipocytes

    PubMed Central

    Maeda, Hayato; Saito, Shuuichi; Nakamura, Nozomi; Maoka, Takashi

    2013-01-01

    Obesity is related to various diseases, such as diabetes, hyperlipidemia, and hypertension. Adipocytokine, which is released from adipocyte cells, affects insulin resistance and blood lipid level disorders. Further, adipocytokine is related to chronic inflammation in obesity condition adipocyte cells. Paprika pigments (PPs) contain large amounts of capsanthin and capsorubin. These carotenoids affect the liver and improve lipid disorders of the blood. However, how these carotenoids affect adipocyte cells remains unknown. Present study examined the effects of PP on adipocytokine secretion, which is related to improvement of metabolic syndrome. In addition, suppressive effects of PP on chronic inflammation in adipocyte cells were analyzed using 3T3-L1 adipocyte cells and macrophage cell coculture experiments. PP promoted 3T3-L1 adipocyte cells differentiation upregulated adiponectin mRNA expression and secretion. Further, coculture of adipocyte and macrophage cells treated with PP showed suppressed interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), monocyte chemotactic protein-1 (MCP-1), and resistin mRNA expression, similarly to treatment with troglitazone, which is a PPARγ ligand medicine. Conclusion. These results suggest that PP ameliorates chronic inflammation in adipocytes caused by obesity. PP adjusts adipocytokine secretion and might, therefore, affect antimetabolic syndrome diseases. PMID:24049664

  8. Simvastatin enhances induction of inducible nitric oxide synthase in 3T3-L1 adipocytes.

    PubMed

    Araki, Shunsuke; Dobashi, Kazushige; Asayama, Kohtaro; Shirahata, Akira

    2007-09-01

    The present study was designed to determine whether hydroxymethylglutaryl-CoA reductase inhibitors (statins) modulate the NO production via iNOS in adipocytes stimulated by lipopolysaccharide (L) and tumour necrosis factor-alpha (T). Well-differentiated 3T3-L1 adipocytes significantly produced NO by LT-treatment. Pre-incubation with simvastatin, a lipophilic statin, pravastatin, a hydrophilic one, or Y27632, an inhibitor of Rho kinase, further enhanced the production of NO. The effect of simvastatin was offset by mevalonate and geranylgeranyl pyrophosphate (GGPP) but not by squalene. The mRNA level for iNOS parallelled the NO production. The NF-kappaB was activated by the LT-treatment and was further enhanced by simvastatin, pravastatin or Y27632 addition. Mevalonate and GGPP completely offset the effect of simvastatin. Statins and Y27632 also further increased the interleukin-6 secretion in the LT-treated 3T3-L1 adipocytes. These results suggest that statins, especially lipophilic type, enhance induction of iNOS by inhibiting the small GTP-binding protein signal in adipocytes.

  9. Cellular uptake and fate of fibroin microspheres loaded with randomly fragmented DNA in 3T3 cells.

    PubMed

    Lee, Jin Sil; Hur, Won

    2016-01-01

    Purified fibroin protein can be obtained in large quantities from silk fibers and processed to form microscopic particles as delivery vehicles for therapeutic agents. In this study, we demonstrated that fibroin microspheres were taken up by 3T3 cells, localized in the nonlysosomal compartment, and secreted from the cytoplasm after medium replenishment. DNA-loaded microspheres were taken up by >95% of 3T3 cells. DNA cargo had no influence on the intracellular trafficking of microspheres, while fluorescently labeled cargo DNA was observed in the lysosomal compartment and in the microspheres. These results indicate that fibroin microspheres can travel through 3T3 cells without making any contact with the lysosomal compartments. The amount of DNA loaded in the microspheres taken up by 3T3 cells was estimated up to 831.0 pg/cell. Thus, fibroin microspheres can deliver a large amount of randomly fragmented DNA (<10 kb) into the cytoplasmic compartment of 3T3 cells.

  10. An electron microscopic radioautographic study of collagen secretion in periodontal ligament fibroblasts of the mouse: II. Colchicine-treated fibroblasts

    SciTech Connect

    Cho, M.I.; Garant, P.R.

    1981-12-01

    Colchicine administered intravenously depolymerized microtubules and disrupted the normal organization of the Golgi apparatus in periodontal ligament fibroblasts. Radioautography with /sup 3/H-proline indicated that collagen secretion was completely inhibited during a period of approximately 4 hours following the onset of the colchicine effect. During this period of secretory inhibition, labeled collagen precursors were present within a variety of dense bodies, primarily located in a juxtanuclear location replacing the normal Golgi complex. The time course of /sup 3/H-proline labeling from 2 to 8 hours suggested that small, newly formed dense bodies fused to form larger dense bodies and pleomorphic structures (zebra bodies), within which collagen precursors appeared to undergo partial polymerization. Autophagosomes, many labeled with /sup 3/H-proline, also increased in number after colchicine administration. A gradual decline in /sup 3/H-proline label occurred from 4 to 24 hours, presumably due to exocytosis of dense bodies or by the digestion of labeled collagen precursors within autophagosomes. These results support the concept that an intact microtubular network is essential for the organized transport of collagen precursors, from the rough endoplasmic reticulum to the Golgi apparatus, and the eventual transport and exocytosis of collagen secretory granules.

  11. In vitro MC3T3 osteoblast adhesion with respect to surface roughness of Ti6Al4V substrates.

    PubMed

    Linez-Bataillon, P; Monchau, F; Bigerelle, M; Hildebrand, H F

    2002-08-01

    This work investigates the role of the surface roughness of Ti6Al4V on the cell morphology, proliferation and adhesion, and in particular on the variation of the expression of cell adhesion proteins. Standardised test samples with five different surface preparations are used: sandblasted, 80, 1200, and 4000 grade polished, mirror polished. Surface roughness is analysed by Scanning Electron Microscopy and LASER Confocal Microscopy. Cell culture experiments are performed with MC3T3-E1 mouse osteoblasts after 3 days culture: proliferation rate, morphology and adhesion are assessed. The variations of expression of cell adhesion proteins are evidenced by indirect immune fluorescence method: actin from the cytoskeleton, vinculin from the focal adhesion complex, fibronectin and collagen I from the extracellular matrix. The results reveal a clear influence of surface roughness of Ti6Al4V on cell proliferation, morphology and adhesion. A significant correlation is established between surface roughness and cell growth. More the surface is smooth more the osteoblasts proliferate and appear spread out on the test samples. In addition, the expression of adhesion proteins varies with respect to the surface roughness. These results indicate a direct relationship between the decrease of cell adhesion and the increase of cell proliferation on mirror polished materials.

  12. Traf2 interacts with Smad4 and regulates BMP signaling pathway in MC3T3-E1 osteoblasts

    SciTech Connect

    Shimada, Koichi; Ikeda, Kyoko; Ito, Koichi

    2009-12-18

    Bone morphogenetic proteins (BMPs) play important roles in osteoblast differentiation and maturation. In mammals, the BMP-induced receptor-regulated Smads form complexes with Smad4. These complexes translocate and accumulate in the nucleus, where they regulate the transcription of various target genes. However, the function of Smad4 remains unclear. We performed a yeast two-hybrid screen using Smad4 as bait and a cDNA library derived from bone marrow, to indentify the proteins interacting with Smad4. cDNA clones for Tumor necrosis factor (TNF) receptor-associated factor 2 (Traf2) were identified, and the interaction between the endogenous proteins was confirmed in the mouse osteoblast cell line MC3T3-E1. To investigate the function of Traf2, we silenced it with siRNA. The level of BMP-2 protein in the medium, the expression levels of the Bmp2 gene and BMP-induced transcription factor genes, including Runx2, Dlx5, Msx2, and Sp7, and the phosphorylated-Smad1 protein level were increased in cells transfected with Traf2 siRNA. The nuclear accumulation of Smad1 increased with TNF-{alpha} stimulation for 30 min at Traf2 silencing. These results suggest that the TNF-{alpha}-stimulated nuclear accumulation of Smad1 may be dependent on Traf2. Thus, the interaction between Traf2 and Smad4 may play a role in the cross-talk between TNF-{alpha} and BMP signaling pathways.

  13. The Effects of Fibroblast Co-Culture and Activin A on in vitro Growth of Mouse Preantral Follicles

    PubMed Central

    Karimpour Malekshah, Abbasali; Heidari, Mahmoud; Parivar, Kazem; Azami, Nasrin Sadat

    2014-01-01

    Background: This study was conducted to evaluate fibroblast co-culture and Activin A on in vitro maturation and fertilization of mouse preantral follicles. Methods: The ovaries from 12-14-day-old mice were dissected, and 120-150 μm preantral follicles were cultured individually in α-MEM as based medium for 12 days. A total number of 456 follicles were cultured in four conditions: (i) base medium as control group (n = 113), (ii) base medium supplemented with 30 ng/ml Activin A (n = 115), (iii) base medium co-cultured with mouse embryonic fibroblast (n = 113), and (iv) base medium supplemented with 30 ng/ml Activin A and co-cultured with fibroblast (n = 115). Rate of growth, survivability, antrum formation, ovulation, embryonic development and steroid production were evaluated. Analysis of Variance and Duncan test were applied for analyzing. Results: Both co-culture and co-culture + Activin A groups showed significant difference (P<0.05) in growth (on days 4, 6, and 8 of culture period) and survival rates. However, there was no significant difference in antrum formation, ovulation rate, and embryonic development of ovulated oocytes. There were significant differences (P<0.05) in the estradiol production on days 8, 10, and 12 between co-culture + Activin A and the control group. Progesterone production also was significant (P<0.05) in co-culture + Activin A group on days 6, 8, 10, and 12 compared to control group. Conclusion: Fibroblast co-culture and Activin A promoted growth and survivability of preantral follicles. However, simultaneous use of them was more efficient. PMID:24375163

  14. A growth factor-responsive gene of murine BALB/c 3T3 cells encodes a protein homologous to human tissue factor

    SciTech Connect

    Hartzell, S.; Ryder, K.; Lanahan, A.; Nathans, D.; Lau, L.F.

    1989-06-01

    Polypeptide growth factors rapidly induce the transcription of a set of genes that appear to mediate cell growth. The authors report that one of the genes induced in BALB/c mouse 3T3 cells encodes a transmembrane protein (mTF) homologous to human tissue factor, which is involved in the proteolytic activation of blood clotting. mTF mRNA is present in many murine tissues and cell lines. The authors' results raise the possibility that mTF may also play a role in cell growth.

  15. Cell volume increase in murine MC3T3-E1 pre-osteoblasts attaching onto biocompatible tantalum observed by magnetic AC mode atomic force microscopy.

    PubMed

    Andersen, L Klembt; Contera, S Antoranz; Justesen, J; Duch, M; Hansen, O; Chevallier, J; Foss, M; Pedersen, F S; Besenbacher, F

    2005-12-02

    Magnetic AC mode (MACmode) atomic force microscopy (AFM) was used to study murine (mouse) MC3T3-E1 preosteoblastic cells attached to biocompatible tantalum substrates. Cell volumes of attached cells derived from AFM images were compared to volumes of detached cells in suspension measured by the Coulter sizing technique. An increase of approximately 50% in cell volume was observed when the cells attached to planar tantalum substrates and developed a flattened structure including lamellipodia. We address thoroughly the issues general to the AFM determination of absolute cell volumes, and compare our magnetic AC mode AFM measurements to hitherto reported cell volume determinations by contact mode AFM.

  16. 3T3 cell motility and morphology before, during, and after exposure to extremely-low-frequency magnetic fields

    SciTech Connect

    Spadinger, I.; Palcic, B.; Agnew, D.

    1995-08-01

    Automated image cytometry techniques were used to measure motility and morphology in 3T3 fibro-blasts exposed to extremely-low-frequency (ELF) magnetic fields. Cell motility and morphology were measured as a function of time before, during, and after 3--4 hour exposures to vertically oriented, 100 {mu}T{sub RMS} sinusoidal magnetic fields at various frequencies in the 10--63 Hz range. Sham exposures were also carried out. No static DC fields were applied, but the geomagnetic field was almost vertical and, therefore, had a large component (28.3 {mu}T) parallel to the applied AC field. The morphology and motile behavior of the cells were characterized by mathematically defined descriptors, which were calculated and averaged for the exposure period as well as for control periods that preceded and followed the exposure period. Each experiment involved the tracking of 100 cells that were subjected to one of the test frequencies (unless a sham exposure was being conducted). Statistical analysis of the results showed that even small changes of 10--20% could be significant at the P < .05 level. Changes on this order were measured in a significant proportion of the experiments. However, because such results were seen for both the sham-exposed and the ELF-exposed cells, and because the range of values that was obtained for the sham exposures was the same as that obtained for the ELF exposures, the authors concluded that there was no evidence to show that any of the measured changes were attributable to the applied ELF magnetic field.

  17. Capsaicin Induces “Brite” Phenotype in Differentiating 3T3-L1 Preadipocytes

    PubMed Central

    Baboota, Ritesh K.; Singh, Dhirendra P.; Sarma, Siddhartha M.; Kaur, Jaspreet; Sandhir, Rajat; Boparai, Ravneet K.; Kondepudi, Kanthi K.; Bishnoi, Mahendra

    2014-01-01

    Objective Targeting the energy storing white adipose tissue (WAT) by pharmacological and dietary means in order to promote its conversion to energy expending “brite” cell type holds promise as an anti-obesity approach. Present study was designed to investigate/revisit the effect of capsaicin on adipogenic differentiation with special reference to induction of “brite” phenotype during differentiation of 3T3-L1 preadipocytes. Methods Multiple techniques such as Ca2+ influx assay, Oil Red-O staining, nutrigenomic analysis in preadipocytes and matured adipocytes have been employed to understand the effect of capsaicin at different doses. In addition to in-vitro experiments, in-vivo studies were carried out in high-fat diet (HFD) fed rats treated with resiniferatoxin (RTX) (a TRPV1 agonist) and in mice administered capsaicin. Results TRPV1 channels are expressed in preadipocytes but not in adipocytes. In preadipocytes, both capsaicin and RTX stimulate Ca2+ influx in dose-dependent manner. This stimulation may be prevented by capsazepine, a TRPV1 antagonist. At lower doses, capsaicin inhibits lipid accumulation and stimulates TRPV1 gene expression, while at higher doses it enhances accumulation of lipids and suppresses expression of its receptor. In doses of 0.1–100 µM, capsaicin promotes expression of major pro-adipogenic factor PPARγ and some of its downstream targets. In concentrations of 1 µM, capsaicin up-regulates anti-adipogenic genes. Low-dose capsaicin treatment of 3T3-L1 preadipocytes differentiating into adipocytes results in increased expression of brown fat cell marker genes. In white adipose of mice, capsaicin administration leads to increase in browning-specific genes. Global TRPV1 ablation (i.p. by RTX administration) leads to increase in locomotor activity with no change in body weight. Conclusion Our findings suggest the dual modulatory role of capsaicin in adipogenesis. Capsaicin inhibits adipogenesis in 3T3-L1 via TRPV1 activation and

  18. Ultrasound stimulation increases proliferation of MC3T3-E1 preosteoblast-like cells

    PubMed Central

    2014-01-01

    Background Mechanical stimulation of bone increases bone mass and fracture healing, at least in part, through increases in proliferation of osteoblasts and osteoprogenitor cells. Researchers have previously performed in vitro studies of ultrasound-induced osteoblast proliferation but mostly used fixed ultrasound settings and have reported widely varying and inconclusive results. Here we critically investigated the effects of the excitation parameters of low-intensity pulsed ultrasound (LIPUS) stimulation on proliferation of MC3T3-E1 preosteoblastic cells in monolayer cultures. Methods We used a custom-designed ultrasound exposure system to vary the key ultrasound parameters—intensity, frequency and excitation duration. MC3T3-E1 cells were seeded in 12-well cell culture plates. Unless otherwise specified, treated cells, in groups of three, were excited twice for 10 min with an interval of 24 h in between after cell seeding. Proliferation rates of these cells were determined using BrdU and MTS assays 24 h after the last LIPUS excitation. All data are presented as the mean ± standard error. The statistical significance was determined using Student's two-sample two-tailed t tests. Results Using discrete LIPUS intensities ranging from 1 to 500 mW/cm2 (SATA, spatial average-temporal average), we found that approximately 75 mW/cm2 produced the greatest increase in osteoblast proliferation. Ultrasound exposures at higher intensity (approximately 465 mW/cm2) significantly reduced proliferation in MC3T3-E1 cells, suggesting that high-intensity pulsed ultrasound may increase apoptosis or loss of adhesion in these cells. Variation in LIPUS frequency from 0.5 MHz to 5 MHz indicated that osteoblast proliferation rate was not frequency dependent. We found no difference in the increase in proliferation rate if LIPUS was applied for 30 min/day or 10 min/day, indicating a habituation response. Conclusion This study concludes that a short-term stimulation with optimum intensity

  19. Comparison of the Transcriptomes of Mouse Skin Derived Precursors (SKPs) and SKP-Derived Fibroblasts (SFBs) by RNA-Seq

    PubMed Central

    Mao, Yujie; Xiong, Lidan; Wang, Siyu; Zhong, Jianqiao; Zhou, Rongying; Li, Li

    2015-01-01

    Skin-derived precursors (SKPs) from dermis possess the capacities of self-renewal and multipotency. In vitro and in vivo studies demonstrated that they can differentiate into fibroblasts. However, little is known about the molecular mechanism of the differentiation of SKPs into fibroblasts. Here we compare the transcriptomes of mouse SKPs and SKP-derived fibroblasts (SFBs) by RNA-Seq analysis, trying to find differences in gene expression between the two kinds of cells and then elucidate the candidate genes that may play important roles in the differentiation of SKPs into fibroblasts. A total of 1971 differentially expressed genes (DEGs) were identified by RNA-Seq, which provided abundant data for further analysis. Gene Ontology enrichment analysis revealed that genes related to cell differentiation, cell proliferation, protein binding, transporter activity and membrane were significantly enriched. The most significantly up-regulated genes Wnt4, Wisp2 and Tsp-1 and down-regulated genes Slitrk1, Klk6, Agtr2, Ivl, Msx1, IL15, Atp6v0d2, Kcne1l and Thbs4 may play important roles in the differentiation of SKPs into fibroblasts. KEGG analysis showed that DEGs were significantly enriched in the TGF-β signaling pathway, Wnt signaling pathway and Notch signaling pathway, which have been previously proven to regulate the differentiation and self-renewal of various stem cells. These identified DEGs and pathways could facilitate further investigations of the detailed molecular mechanisms, making it possible to take advantage of the potential therapeutic applications of SKPs in skin regeneration in the future. PMID:25719759

  20. Averrhoa carambola L. peel extract suppresses adipocyte differentiation in 3T3-L1 cells.

    PubMed

    Rashid, Asyifah Mohamed; Lu, Kaihui; Yip, Yew Mun; Zhang, Dawei

    2016-02-01

    Obesity is associated with an increased risk of many chronic diseases. Recently, a growing body of evidence has shown that phytochemicals may inhibit adipogenesis and obesity. In this study, we report for the first time, the ability of Averrhoa carambola L. peel extract commonly known as star fruit (SFP) to effectively suppress adipocyte differentiation in 3T3-L1 preadipocytes and therefore, address it as a potential candidate to treat obesity and its related diseases. (-)-Epicatechin was identified as a bioactive compound likely responsible for this suppression. As the genetic expression studies revealed that the adipogenic activity of SFP extract was due to the simultaneous downregulation of the C/EBPα and PPARγ as well as the upregulation of PPARα receptor genes, a detailed computational docking study was also elucidated to reveal the likely binding mode of (-)-epicatechin to the receptor of interest, accounting for the likely mechanism that results in the overall suppression of adipocyte differentiation.

  1. Prednisolone induces the Wnt signalling pathway in 3T3-L1 adipocytes.

    PubMed

    Fleuren, Wilco W M; Linssen, Margot M L; Toonen, Erik J M; van der Zon, Gerard C M; Guigas, Bruno; de Vlieg, Jacob; Dokter, Wim H A; Ouwens, D Margriet; Alkema, Wynand

    2013-05-01

    Synthetic glucocorticoids are potent anti-inflammatory drugs but show dose-dependent metabolic side effects such as the development of insulin resistance and obesity. The precise mechanisms involved in these glucocorticoid-induced side effects, and especially the participation of adipose tissue in this are not completely understood. We used a combination of transcriptomics, antibody arrays and bioinformatics approaches to characterize prednisolone-induced alterations in gene expression and adipokine secretion, which could underlie metabolic dysfunction in 3T3-L1 adipocytes. Several pathways, including cytokine signalling, Akt signalling, and Wnt signalling were found to be regulated at multiple levels, showing that these processes are targeted by prednisolone. These results suggest that mechanisms by which prednisolone induce insulin resistance include dysregulation of wnt signalling and immune response processes. These pathways may provide interesting targets for the development of improved glucocorticoids.

  2. Prednisolone induces the Wnt signalling pathway in 3T3-L1 adipocytes

    PubMed Central

    Fleuren, Wilco W. M.; Linssen, Margot M. L.; Toonen, Erik J. M.; van der Zon, Gerard C. M.; Guigas, Bruno; de Vlieg, Jacob; Dokter, Wim H. A.; Ouwens, D. Margriet

    2013-01-01

    Synthetic glucocorticoids are potent anti-inflammatory drugs but show dose-dependent metabolic side effects such as the development of insulin resistance and obesity. The precise mechanisms involved in these glucocorticoid-induced side effects, and especially the participation of adipose tissue in this are not completely understood. We used a combination of transcriptomics, antibody arrays and bioinformatics approaches to characterize prednisolone-induced alterations in gene expression and adipokine secretion, which could underlie metabolic dysfunction in 3T3-L1 adipocytes. Several pathways, including cytokine signalling, Akt signalling, and Wnt signalling were found to be regulated at multiple levels, showing that these processes are targeted by prednisolone. These results suggest that mechanisms by which prednisolone induce insulin resistance include dysregulation of wnt signalling and immune response processes. These pathways may provide interesting targets for the development of improved glucocorticoids. PMID:23506355

  3. Single Synonymous Mutations in KRAS Cause Transformed Phenotypes in NIH3T3 Cells

    PubMed Central

    Waters, Andrew M.; Bagni, Rachel; Portugal, Franklin; Hartley, James L.

    2016-01-01

    Synonymous mutations in the KRAS gene are clustered at G12, G13, and G60 in human cancers. We constructed 9 stable NIH3T3 cell lines expressing KRAS, each with one of these synonymous mutations. Compared to the negative control cell line expressing the wild type human KRAS gene, all the synonymous mutant lines expressed more KRAS protein, grew more rapidly and to higher densities, and were more invasive in multiple assays. Three of the cell lines showed dramatic loss of contact inhibition, were more refractile under phase contrast, and their refractility was greatly reduced by treatment with trametinib. Codon usage at these glycines is highly conserved in KRAS compared to HRAS, indicating selective pressure. These transformed phenotypes suggest that synonymous mutations found in driver genes such as KRAS may play a role in human cancers. PMID:27684555

  4. Surface extensions of 3T3 cells towards distant infrared light sources

    PubMed Central

    1991-01-01

    Using a specially designed phase-contrast light microscope with an infrared spot illuminator we found that approximately 25% of 3T3 cells were able to extend pseudopodia towards single microscopic infrared light sources nearby. If the cells were offered a pair of such light sources next to each other, 47% of the cells extended towards them. In the latter case 30% of the responding cells extended separate pseudopodia towards each individual light source of a pair. The strongest responses were observed if the infrared light sources emitted light of wavelengths in the range of 800-900 nm intermittently at rates of 30-60 pulses per min. The temperature increases of the irradiated spots can be shown to be negligible. The results suggest that the cells are able to sense specific infrared wavelengths and to determine the direction of individual sources. PMID:1860881

  5. Melatonin Suppresses Autophagy Induced by Clinostat in Preosteoblast MC3T3-E1 Cells.

    PubMed

    Yoo, Yeong-Min; Han, Tae-Young; Kim, Han Sung

    2016-04-08

    Microgravity exposure can cause cardiovascular and immune disorders, muscle atrophy, osteoporosis, and loss of blood and plasma volume. A clinostat device is an effective ground-based tool for simulating microgravity. This study investigated how melatonin suppresses autophagy caused by simulated microgravity in preosteoblast MC3T3-E1 cells. In preosteoblast MC3T3-E1 cells, clinostat rotation induced a significant time-dependent increase in the levels of the autophagosomal marker microtubule-associated protein light chain (LC3), suggesting that autophagy is induced by clinostat rotation in these cells. Melatonin treatment (100, 200 nM) significantly attenuated the clinostat-induced increases in LC3 II protein, and immunofluorescence staining revealed decreased levels of both LC3 and lysosomal-associated membrane protein 2 (Lamp2), indicating a decrease in autophagosomes. The levels of phosphorylation of mammalian target of rapamycin (p-mTOR) (Ser2448), phosphorylation of extracellular signal-regulated kinase (p-ERK), and phosphorylation of serine-threonine protein kinase (p-Akt) (Ser473) were significantly reduced by clinostat rotation. However, their expression levels were significantly recovered by melatonin treatment. Also, expression of the Bcl-2, truncated Bid, Cu/Zn- superoxide dismutase (SOD), and Mn-SOD proteins were significantly increased by melatonin treatment, whereas levels of Bax and catalase were decreased. The endoplasmic reticulum (ER) stress marker GRP78/BiP, IRE1α, and p-PERK proteins were significantly reduced by melatonin treatment. Treatment with the competitive melatonin receptor antagonist luzindole blocked melatonin-induced decreases in LC3 II levels. These results demonstrate that melatonin suppresses clinostat-induced autophagy through increasing the phosphorylation of the ERK/Akt/mTOR proteins. Consequently, melatonin appears to be a potential therapeutic agent for regulating microgravity-related bone loss or osteoporosis.

  6. Melatonin Suppresses Autophagy Induced by Clinostat in Preosteoblast MC3T3-E1 Cells

    PubMed Central

    Yoo, Yeong-Min; Han, Tae-Young; Kim, Han Sung

    2016-01-01

    Microgravity exposure can cause cardiovascular and immune disorders, muscle atrophy, osteoporosis, and loss of blood and plasma volume. A clinostat device is an effective ground-based tool for simulating microgravity. This study investigated how melatonin suppresses autophagy caused by simulated microgravity in preosteoblast MC3T3-E1 cells. In preosteoblast MC3T3-E1 cells, clinostat rotation induced a significant time-dependent increase in the levels of the autophagosomal marker microtubule-associated protein light chain (LC3), suggesting that autophagy is induced by clinostat rotation in these cells. Melatonin treatment (100, 200 nM) significantly attenuated the clinostat-induced increases in LC3 II protein, and immunofluorescence staining revealed decreased levels of both LC3 and lysosomal-associated membrane protein 2 (Lamp2), indicating a decrease in autophagosomes. The levels of phosphorylation of mammalian target of rapamycin (p-mTOR) (Ser2448), phosphorylation of extracellular signal-regulated kinase (p-ERK), and phosphorylation of serine-threonine protein kinase (p-Akt) (Ser473) were significantly reduced by clinostat rotation. However, their expression levels were significantly recovered by melatonin treatment. Also, expression of the Bcl-2, truncated Bid, Cu/Zn- superoxide dismutase (SOD), and Mn-SOD proteins were significantly increased by melatonin treatment, whereas levels of Bax and catalase were decreased. The endoplasmic reticulum (ER) stress marker GRP78/BiP, IRE1α, and p-PERK proteins were significantly reduced by melatonin treatment. Treatment with the competitive melatonin receptor antagonist luzindole blocked melatonin-induced decreases in LC3 II levels. These results demonstrate that melatonin suppresses clinostat-induced autophagy through increasing the phosphorylation of the ERK/Akt/mTOR proteins. Consequently, melatonin appears to be a potential therapeutic agent for regulating microgravity-related bone loss or osteoporosis. PMID:27070587

  7. Honokiol enhances adipocyte differentiation by potentiating insulin signaling in 3T3-L1 preadipocytes.

    PubMed

    Choi, Sun-Sil; Cha, Byung-Yoon; Iida, Kagami; Sato, Masako; Lee, Young-Sil; Teruya, Toshiaki; Yonezawa, Takayuki; Nagai, Kazuo; Woo, Je-Tae

    2011-07-01

    Adipose tissue plays an essential role in energy homeostasis as a metabolic and endocrine organ. Accordingly, adipocytes are emerging as a major drug target for obesity and obesity-mediated metabolic syndrome. Dysfunction of enlarged adipocytes in obesity is involved in obesity-mediated metabolic syndrome. Adipocytokines, such as adiponectin released from small adipocytes, are able to prevent these disorders. In this study, we found that honokiol, an ingredient of Magnolia officinalis used in traditional Chinese and Japanese medicines, enhanced adipocyte differentiation in 3T3-L1 preadipocytes. Oil Red O staining showed that treatment with honokiol in the presence of insulin dose-dependently increased lipid accumulation in 3T3-L1 preadipoyctes although its activity was weak compared with rosiglitazone. During adipocyte differentiation, the expression of peroxisome proliferator-activated receptor γ2 (PPARγ2) mRNA and PPARγ target genes such as adipocyte protein 2 (aP2), adiponectin, and GLUT4 was induced by treatment with 10 μM honokiol. However, honokiol failed to show direct binding to the PPARγ ligand-binding domain in vitro. In preadipocytes, treatment with honokiol in the presence of insulin increased the phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 protein and Akt protein, early insulin signaling pathways related to adipocyte differentiation, compared with insulin-only treatment. Taken together, our results suggest that honokiol promotes adipocyte differentiation through increased expression of PPARγ2 mRNA and potentiation of insulin signaling pathways such as the Ras/ERK1/2 and phosphoinositide-3-kinase (PI3K)/Akt signaling pathways.

  8. Dihydrocytochalasin B. Biological effects and binding to 3T3 cells

    PubMed Central

    Atlas, S. J.; Lin, S.

    1978-01-01

    Dihydrocytochalasin B (H2CB) does not inhibit sugar uptake in BALB/c 3T3 cells. Excess H2CB does not affect inhibition of sugar uptake by cytochalasin B (CB), indicating that it does not compete with CB for binding to high-affinity sites. As in the case of CB, H2CB inhibits cytokinesis and changes the morphology of the cells. These results demonstrate that the effects of CB on sugar transport and on cell motility and morphology involve separate and independent sites. Comparison of the effects of H2CB, CB, and cytochalasin D (CD) indicates that treatment of cells with any one of the compounds results in the same series of morphological changes; the cells undergo zeiosis and elongation at 2-4 microM CB and become arborized and rounded up at 10-50 microM CB. H2CB is slightly less potent than CB, whereas CD is five to eight times more potent than CB in causing a given state of morphological change. These results indicate that the cytochalasin-induced changes in cell morphology are mediated by a specific site(s) which can distinguish the subtle differences in the structures of the three compounds. Competitive binding studies indicate that excess H2CB displaces essentially all of the high-affinity bound [3H]CB, but, at less than 5 x 10(-5) M H2CB is not so efficient as unlabeled CB in the displacement reaction. In contrast, excess CD displaces up to 40% of the bound [3H]CB. These results suggest that three different classes of high-affinity CB binding sites exist in 3T3 cells: sites related to sugar transport, sites related to cell motility and morphology, and sites with undetermined function. PMID:10605443

  9. Cellular Transformation of Mouse Embryo Fibroblasts in the Absence of Activator E2Fs

    PubMed Central

    Gupta, Tushar; Sáenz Robles, Maria Teresa

    2015-01-01

    ABSTRACT The E2F family of transcription factors, broadly divided into activator and repressor E2Fs, regulates cell cycle genes. Current models indicate that activator E2Fs are necessary for cell cycle progression and tumorigenesis and are also required to mediate transformation induced by DNA tumor viruses. E2Fs are negatively regulated by the retinoblastoma (RB) family of tumor suppressor proteins, and virus-encoded oncogenes disrupt the RB-E2F repressor complexes. This results in the release of activator E2Fs and induction of E2F-dependent genes. In agreement, expression of large tumor T antigens (TAg) encoded by polyomaviruses in mammalian cells results in increased transcriptional levels of E2F target genes. In addition, tumorigenesis induced by transgenic expression of simian virus 40 (SV40) TAg in choroid plexus or intestinal villi requires at least one activator E2F. In contrast, we show that SV40 TAg-induced transformation in mouse embryonic fibroblasts is independent of activator E2Fs. This work, coupled with recent studies showing that proliferation in stem and progenitor cells is independent of activator E2Fs, suggests the presence of parallel pathways governing cell proliferation and tumorigenesis. IMPORTANCE The RB-E2F pathway is altered in many cancers and is also targeted by DNA tumor viruses. Viral oncoprotein action on RBs results in the release of activator E2Fs and upregulation of E2F target genes; thus, activator E2Fs are considered essential for normal and tumorigenic cell proliferation. However, we have observed that SV40 large T antigen can induce cell proliferation and transformation in the absence of activator E2Fs. Our results also suggest that TAg action on pRBs regulates both E2F-dependent and -independent pathways that govern proliferation. Thus, specific cell proliferation pathways affected by RB alterations in cancer may be a factor in tumor behavior and response to therapy. PMID:25717106

  10. Short exposure to collagenase and coculture with mouse embryonic pancreas improve human dermal fibroblast culture.

    PubMed

    Pandamooz, Sareh; Hadipour, Abbas; Akhavan-Niaki, Haleh; Pourghasem, Mohsen; Abedian, Zeinab; Ardekani, Ali Motevallizadeh; Golpour, Monireh; Hassan, Zuhair Mohammad; Mostafazadeh, Amrollah

    2012-01-01

    The dermal fibroblast as a major component of connective tissue has attracted much attention in the past few years, and application of these very fast growing cells in several fields has been intensively studied. Isolating dermal fibroblasts is an appropriate way to expand these fast growing cells in vitro. Although using a dissociated fibroblast culture method is more convenient than skin explant culture, its enzymatic digestion is critical because a large number of cells can be lost over prolonged exposure to collagenase. This study was performed to increase the number of viable cells after digestion of fresh human foreskin of donors aged from 1 to 3 months with collagenase and also by to design a coculture system for resuscitation of the injured fibroblast. Our results demonstrate that we can maximize cell yield while maintaining cell viability by cutting the specimens into very small pieces (1-2 mm³) after removing the epidermal layer with dispase II and also by collecting released cells every 20 Min subsequent to digesting the dermal layer with collagenase. Moreover, our data strongly indicate that coculturing of isolated fibroblasts with embryonic pancreas explants can enhance the rate of proliferation in cultured fibroblasts.

  11. mTOR ensures increased release and reduced uptake of the organic osmolyte taurine under hypoosmotic conditions in mouse fibroblasts.

    PubMed

    Lambert, Ian Henry; Jensen, Jane Vendelbo; Pedersen, Per Amstrup

    2014-06-01

    Mammalian target of rapamycin (mTOR) is a serine/threonine kinase that modulates translation in response to growth factors and alterations in nutrient availability following hypoxia and DNA damage. Here we demonstrate that mTOR activity in Ehrlich Lettré ascites (ELA) cells is transiently increased within minutes following osmotic cell swelling and that inhibition of phosphatidylinositol-3-phosphatase (PTEN) counteracts the upstream phosphatidylinositol kinase and potentiates mTOR activity. PTEN inhibition concomitantly potentiates swelling-induced taurine release via the volume-sensitive transporter for organic osmolytes and anion channels (VSOAC) and enhances swelling-induced inhibition of taurine uptake via the taurine-specific transporter (TauT). Chronic osmotic stress, i.e., exposure to hypotonic or hypertonic media for 24 h, reduces and increases mTOR activity in ELA cells, respectively. Using rapamycin, we demonstrate that mTOR inhibition is accompanied by reduction in TauT activity and increase in VSOAC activity in cells expressing high (NIH3T3 fibroblasts) or low (ELA) amounts of mTOR protein. The effect of mTOR inhibition on TauT activity reflects reduced TauT mRNA, TauT protein abundance, and an overall reduction in protein synthesis, whereas the effect on VSOAC is mimicked by catalase inhibition and correlates with reduced catalase mRNA abundance. Hence, mTOR activity favors loss of taurine following hypoosmotic cell swelling, i.e., release via VSOAC and uptake via TauT during acute hypotonic exposure is potentiated and reduced, respectively, by phosphorylation involving mTOR and/or the kinases upstream to mTOR. Decrease in TauT activity during chronic hypotonic exposure, on the other hand, involves reduction in expression/activity of TauT and enzymes in antioxidative defense.

  12. Sheets of Vertically Aligned BaTiO3 Nanotubes Reduce Cell Proliferation but Not Viability of NIH-3T3 Cells

    PubMed Central

    Giannini, Marianna; Giannaccini, Martina; Sibillano, Teresa; Giannini, Cinzia; Liu, Dun; Wang, Zhigang; Baù, Andrea; Dente, Luciana; Cuschieri, Alfred; Raffa, Vittoria

    2014-01-01

    All biomaterials initiate a tissue response when implanted in living tissues. Ultimately this reaction causes fibrous encapsulation and hence isolation of the material, leading to failure of the intended therapeutic effect of the implant. There has been extensive bioengineering research aimed at overcoming or delaying the onset of encapsulation. Nanotechnology has the potential to address this problem by virtue of the ability of some nanomaterials to modulate interactions with cells, thereby inducing specific biological responses to implanted foreign materials. To this effect in the present study, we have characterised the growth of fibroblasts on nano-structured sheets constituted by BaTiO3, a material extensively used in biomedical applications. We found that sheets of vertically aligned BaTiO3 nanotubes inhibit cell cycle progression - without impairing cell viability - of NIH-3T3 fibroblast cells. We postulate that the 3D organization of the material surface acts by increasing the availability of adhesion sites, promoting cell attachment and inhibition of cell proliferation. This finding could be of relevance for biomedical applications designed to prevent or minimize fibrous encasement by uncontrolled proliferation of fibroblastic cells with loss of material-tissue interface underpinning long-term function of implants. PMID:25506693

  13. PAPSS2 Promotes Alkaline Phosphates Activity and Mineralization of Osteoblastic MC3T3-E1 Cells by Crosstalk and Smads Signal Pathways

    PubMed Central

    Wang, Weizhuo; Li, Fang; Wang, Kunzheng; Cheng, Bin; Guo, Xiong

    2012-01-01

    Several studies have indicated that PAPSS2 (3′-phosphoadenosine-5′-phosphosulfate synthetase 2) activity is important to normal skeletal development. Mouse PAPSS2 is predominantly expressed during the formation of the skeleton and cartilaginous elements of the mouse embryo and in newborn mice. However, the role and mechanism of PAPSS2 in bone formation remains largely unidentified. By analyzing the expression pattern of the PAPSS2 gene, we have found that PAPSS2 is expressed in bone tissue and bone formation. PAPSS2 transcripts increase during osteoblast differentiation and are in less level in RANKL-induced osteoclast like cells. By using lentivirus-mediated RNA interference (RNAi) technology, we knocked down PAPSS2 expression in MC3T3-E1 osteoblast. Silencing of PAPSS2 expression significantly decreases ALP activity and cell mineralization, inhibits expression of osteoblast marker osteopontin (OPN) and collagen I. Conversely, overexpression of PAPSS2 promotes the MC3T3-E1 to differentiate into osteoblast and mineralization. Moreover, compared to that in the control cells, the mRNA level and protein expression of phosphorylated Smad 2/3, which is a key transcriptional factor in the Smad osteoblast differentiation pathway, showed significant decreases in PAPSS2-silenced cells and increases in PAPSS2-overexpression cells. These results suggest that PAPSS2 might regulate osteoblast ALP activity and cell mineralization, probably through Smads signal pathways. PMID:22916269

  14. Connective tissue growth factor/CCN2-null mouse embryonic fibroblasts retain intact transforming growth factor-{beta} responsiveness

    SciTech Connect

    Mori, Yasuji; Hinchcliff, Monique; Wu, Minghua; Warner-Blankenship, Matthew; Lyons, Karen M.

    2008-03-10

    Background: The matricellular protein connective tissue growth factor (CCN2) has been implicated in pathological fibrosis, but its physiologic role remains elusive. In vitro, transforming growth factor-{beta} (TGF-{beta}) induces CCN2 expression in mesenchymal cells. Because CCN2 can enhance profibrotic responses elicited by TGF-{beta}, it has been proposed that CCN2 functions as an essential downstream signaling mediator for TGF-{beta}. To explore this notion, we characterized TGF-{beta}-induced activation of fibroblasts from CCN2-null (CCN2{sup -/-}) mouse embryos. Methods: The regulation of CCN2 expression was examined in vivo in a model of fibrosis induced by bleomycin. Cellular TGF-{beta} signal transduction and regulation of collagen gene expression were examined in CCN2{sup -/-} MEFs by immunohistochemistry, Northern, Western and RT-PCR analysis, immunocytochemistry and transient transfection assays. Results: Bleomycin-induced skin fibrosis in the mouse was associated with substantial CCN2 up-regulation in lesional fibroblasts. Whereas in vitro proliferation rate of CCN2{sup -/-} MEFs was markedly reduced compared to wild type MEFs, TGF-{beta}-induced activation of the Smad pathways, including Smad2 phosphorylation, Smad2/3 and Smad4 nuclear accumulation and Smad-dependent transcriptional responses, were unaffected by loss of CCN2. The stimulation of COL1A2 and fibronectin mRNA expression and promoter activity, and of corresponding protein levels, showed comparable time and dose-response in wild type and CCN2{sup -/-} MEFs, whereas stimulation of alpha smooth muscle actin and myofibroblast transdifferentiation showed subtle impairment in MEFs lacking CCN2. Conclusion: Whereas endogenous CCN2 plays a role in regulation of proliferation and TGF-{beta}-induced myofibroblast transdifferentiation, it appears to be dispensable for Smad-dependent stimulation of collagen and extracellular matrix synthesis in murine embryonic fibroblasts.

  15. In vitro BALB/3T3 cell transformation assay of nonoxynol-9 and 1,4-dioxane.

    PubMed

    Sheu, C W; Moreland, F M; Lee, J K; Dunkel, V C

    1988-01-01

    The spermicidal surfactant nonoxynol-9 (Igepal CO-630, GAF Corp.) and a potential impurity, 1,4-dioxane, were tested in the in vitro cell transformation assay using BALB/3T3 cells. Two treatment periods, 48 hr and 13 days, were used. Nonoxynol-9, tested at levels up to 10 micrograms/ml, did not induce transformation, whereas dioxane was very active in the induction of type III foci in the cultured BALB/3T3 cells.

  16. Berberine reduces the expression of adipogenic enzymes and inflammatory molecules of 3T3-L1 adipocyte.

    PubMed

    Choi, Bong-Hyuk; Ahn, In-Sook; Kim, Yu-Hee; Park, Ji-Won; Lee, So-Young; Hyun, Chang-Kee; Do, Myoung-Sool

    2006-12-31

    Berberine (BBR), an isoquinoline alkaloid, has a wide range of pharmacological effects, yet its exact mechanism is unknown. In order to understand the anti-adipogenic effect of BBR, we studied the change of expression of several adipogenic enzymes of 3T3-L1 cells by BBR treatment. First, we measured the change of leptin and glycerol in the medium of 3T3-L1 cells treated with 1 micrometer, 5 micrometer and 10 micrometer concentrations of BBR. We also measured the changes of adipogenic and lipolytic factors of 3T3-L1. In 3T3-L1 cells, both leptin and adipogenic factors (SREBP-1c, C/EBP-alpha, PPAR-gamma, fatty acid synthase, acetyl-CoA carboxylase, acyl-CoA synthase and lipoprotein lipase) were reduced by BBR treatment. Glycerol secretion was increased, whereas expression of lipolytic enzymes (hormone-sensitive lipase and perilipin) mRNA was slightly decreased. Next, we measured the change of inflammation markers of 3T3-L1 cells by BBR treatment. This resulted in the down-regulation of mRNA level of inflammation markers such as TNF-alpha, IL-6, C- reactive protein and haptoglobin. Taken together, our data shows that BBR has both anti-adipogenic and anti-inflammatory effects on 3T3-L1 adipocytes, and the anti-adipogenic effect seems to be due to the down-regulation of adipogenic enzymes and transcription factors.

  17. Resistance to oncogenic transformation in revertant R1 of human ras-transformed NIH 3T3 cells

    SciTech Connect

    Kuzumaki, N.; Ogiso, Y.; Oda, A.; Fujita, H.; Suzuki, H.; Sato, C.; Mullauer, L.

    1989-05-01

    A flat revertant, R1, was isolated from human activated c-Ha-ras-1 (hu-ac-Ha-ras) gene-transformed NIH 3T3 cells (EJ-NIH 3T3) treated with mutagens. R1 contained unchanged transfected hu-ac-Ha-ras DNA and expressed high levels of hu-ac-Ha-ras-specific mRNA and p21 protein. Transfection experiments revealed that NIH 3T3 cells could be transformed by DNA from R1 cells but R1 cells could not be retransformed by Kirsten sarcoma virus, DNA from EJ-NIH 3T3 cells, hu-ac-Ha-ras, v-src, v-mos, simian virus 40 large T antigen, or polyomavirus middle T antigen. Somatic cell hybridization studies showed that R1 was not retransformed by fusion with NIH 3T3 cells and suppressed anchorage independence of EJ-NIH 3T3 and hu-ac-Ha-ras gene-transformed rat W31 cells in soft agar. These results suggest that the reversion and resistance to several oncogenes in R1 is due n not to cellular defects in the production of the transformed phenotype but rather to enhancement of cellular mechanisms that suppress oncogenic transformation.

  18. Induction of fibrillin-2 and periostin expression in Osterix-knockdown MC3T3-E1 cells.

    PubMed

    Lee, So-Jeong; Lee, Eun-Hye; Park, Seung-Yoon; Kim, Jung-Eun

    2017-01-05

    Osteoporosis is the most common age-related bone disease that is characterized by an imbalance between osteoblasts for bone formation and osteoclasts for bone resorption. Anti-catabolic drugs have been developed to inhibit osteoclast activity and to prevent bone loss in osteoporosis. However, because it is difficult to increase bone mass in osteoporotic bone, it would be beneficial to simultaneously enhance osteoblast function and thus form bone. Osterix (Osx) is an essential transcription factor for osteoblast differentiation. To date, many studies have focused on discovering Osx target genes and on increasing osteoblast differentiation. However, Osx targets and the mechanisms controlling osteoblast differentiation, are not well known. Here, we generated stable Osx-knockdown cell lines by employing shRNA in MC3T3-E1 osteoblastic cells. Stable Osx-knockdown osteoblasts exhibited a significant reduction in cell differentiation and nodule formation, which was similar to the reduced osteoblast activity observed in an Osx-deficient mouse model. Using an Affymetrix GeneChip microarray, we determined the differential gene expression profile in response to Osx knockdown, which provided insight into molecular mechanisms underlying osteoblast differentiation. Of 2743 genes with roles in cell differentiation, 15 were upregulated and 2 were downregulated in Osx-knockdown osteoblasts. In particular, the expression of fibrillin-2 and periostin was significantly increased in Osx-knockdown osteoblasts compared to that in control cells, as validated by RT-PCR and quantitative real-time PCR. Finally, this study showed differential gene expression profiles for Osx-mediated osteoblast differentiation, suggesting that fibrillin-2 and periostin will be target candidates of Osx in osteoblast differentiation.

  19. Sphingosine-1-phosphate inhibits the adipogenic differentiation of 3T3-L1 preadipocytes.

    PubMed

    Moon, Myung-Hee; Jeong, Jae-Kyo; Lee, You-Jin; Seol, Jae-Won; Park, Sang-Youel

    2014-10-01

    Sphingosine-1-phosphate (S1P) is a pluripotent lipid mediator that transmits signals through G-protein-coupled receptors to control diverse biological processes. The novel biological activity of S1P in the adipogenesis of 3T3-L1 preadipocytes was identified in the present study. S1P significantly decreased lipid accumulation in maturing preadipocytes in a dose‑dependent manner. In order to understand the anti‑adipogenic effects of S1P, preadipocytes were treated with S1P, and the change in the expression of several adipogenic transcription factors and enzymes was investigated using quantitative RT-PCR. S1P downregulated the transcriptional levels of the peroxisome proliferator-activated receptor γ, CCAAT/enhancer binding proteins and adiponectin, which are markers of adipogenic differentiation. The effects of S1P on the levels of mitogen‑activated protein kinase (MAPK) signals in preadipocytes were also investigated. The activation of JNK and p38 were downregulated by S1P treatment in human preadipocytes. In conclusion, the results of this study suggest that S1P alters fat mass by directly affecting adipogenesis. This is mediated by the downregulation of adipogenic transcription factors and by inactivation of the JNK and p38 MAPK pathways. Thus, selective targeting of the S1P receptors and sphingosine kinases may have clinical applications for the treatment of obesity.

  20. Protein turnover in 3T3 cells transformed with the oncogene c-H-ras1.

    PubMed Central

    Gunn, J M; James, G

    1992-01-01

    We have examined protein turnover, growth, DNA synthesis and proliferation in three independent clones of 3T3-NR6 cells transformed with the oncogene c-H-ras1. We find that, firstly, the half-maximum concentration of serum and insulin regulating protein turnover in ras-transformed cells is significantly reduced from 0.5 to 0.3% for serum and from 4 nM to 0.5 nM for insulin, and, secondly, ras-transformed cells consistently have lower rates of protein degradation. The catabolic effect of conditioned medium or serum withdrawal is attenuated in transformed lines by maintaining lower basal rates of protein breakdown and higher basal rates of DNA and protein synthesis. Serum stimulation of growth in transformed cells is achieved in the short term by lower rates of protein breakdown rather than higher rates of protein synthesis: rates of protein synthesis become significantly higher 24 h after serum stimulation. Therefore transformed cells have higher rates of proliferation and grow to higher densities, but display characteristics common to normal cells because rates of protein synthesis decrease and protein degradation increase as a function of cell density. We conclude that higher basal rates of protein synthesis and growth with retention of the normal proliferative response to serum result from the pleiotropic nature of ras transformation, whereas lower rates of protein degradation and increased sensitivity to serum and insulin imply a direct regulatory role for ras. PMID:1575687

  1. Fisetin induces Sirt1 expression while inhibiting early adipogenesis in 3T3-L1 cells.

    PubMed

    Kim, Sang Chon; Kim, Yoo Hoon; Son, Sung Wook; Moon, Eun-Yi; Pyo, Suhkneung; Um, Sung Hee

    2015-11-27

    Fisetin (3,7,3',4'-tetrahydroxyflavone) is a naturally found flavonol in many fruits and vegetables and is known to have anti-aging, anti-cancer and anti-viral effects. However, the effects of fisetin on early adipocyte differentiation and the epigenetic regulator controlling adipogenic transcription factors remain unclear. Here, we show that fisetin inhibits lipid accumulation and suppresses the expression of PPARγ in 3T3-L1 cells. Fisetin suppressed early stages of preadipocyte differentiation, and induced expression of Sirt1. Depletion of Sirt1 abolished the inhibitory effects of fisetin on intracellular lipid accumulation and on PPARγ expression. Mechanistically, fisetin facilitated Sirt1-mediated deacetylation of PPARγ and FoxO1, and enhanced the association of Sirt1 with the PPARγ promoter, leading to suppression of PPARγ transcriptional activity, thereby repressing adipogenesis. Lowering Sirt1 levels reversed the effects of fisetin on deacetylation of PPARγ and increased PPARγ transactivation. Collectively, our results suggest the effects of fisetin in increasing Sirt1 expression and in epigenetic control of early adipogenesis.

  2. Serum-induced G0/G1 transition in chemically transformed 3T3 cells

    SciTech Connect

    Gray, H.E.; Buchou, T.; Mester, J.

    1987-03-01

    Quiescent, chemically transformed (benzo-a-pyrene) BALB/c 3T3 cells (BP A31) enter the cell division cycle when exposed to complete medium containing 10% fetal calf serum (FCS); the number of cells recruited is a function of the duration of serum exposure. The recruitment of cells by short (<4 h) serum pulses is not inhibited by simultaneous exposure to cycloheximide (CH), and therefore the initial commitment does not require protein synthesis. The cells enter S phase with a constant delay following the removal of CH, even if CH exposure has been continued for as long as 20 h after the end of the serum pulse. The cell recruitment by serum pulses was inhibited by 5,6-dichloro-1-..beta..-D-ribofuranosyl-benzimidazole (DRB), an inhibitor of cytoplasmic mRNA accumulation. These data suggest that serum exposure produces a stable memory that is necessary and sufficient for the eventual progression through G1 to S phase that occurs when protein synthesis is resumed after the removal of CH; this memory probably consists of mRNA species that are induced by serum and that are stable in the absence of protein synthesis. Unexpectedly, pretreatment of quiescent BP A31 cells with CH (8-24 h) dramatically increased the fraction of the total cell population that is recruited by a serum pulse of fixed duration.

  3. Inhibitory effects of tannic acid on fatty acid synthase and 3T3-L1 preadipocyte.

    PubMed

    Fan, Huijin; Wu, Dan; Tian, Weixi; Ma, Xiaofeng

    2013-07-01

    Tannic acid is a hydrolyzable tannin that exists in many widespread edible plants with a variety of biological activities. In this study, we found that tannic acid potently inhibited the activity of fatty acid synthase (FAS) in a concentration-dependent manner with a half-inhibitory concentration value (IC50) of 0.14 microM. The inhibition kinetic results showed that the inhibition of FAS by tannic acid was mixed competitive and noncompetitive manner with respect to acetyl-CoA and malonyl-CoA, but uncompetitive to NADPH. Tannic acid prevented the differentiation of 3T3-L1 pre-adipocytes, and thus repressed intracellular lipid accumulation. In the meantime, tannic acid decreased the expression of FAS and down-regulated the mRNA level of FAS and PPARgamma during adipocyte differentiation. Further studies showed that the inhibitory effect of tannic acid did not relate to FAS non-specific sedimentation. Since FAS was believed to be a therapeutic target of obesity, these findings suggested that tannic acid was considered having potential in the prevention of obesity.

  4. Nobiletin enhances differentiation and lipolysis of 3T3-L1 adipocytes

    SciTech Connect

    Saito, Takeshi; Abe, Daigo; Sekiya, Keizo . E-mail: ksekiya@affrc.go.jp

    2007-06-01

    Nobiletin is a polymethoxylated flavone found in certain citrus fruits. Here we demonstrate that nobiletin enhance differentiation of 3T3-L1 preadipocytes. Nobiletin dose-dependently increased accumulation of lipid droplets in adipocytes. Quantitative RT-PCR analyses indicated that nobiletin increased the expression of genes critical for acquisition of the adipocyte phenotype. Some of them were known peroxisome proliferator activated receptor {gamma} (PPAR{gamma}) targets and PPAR{gamma} itself, however, nobiletin did not exhibit PPAR{gamma} ligand activity. We observed the expression of CCAAT/enhancer binding protein {beta} (C/EBP{beta}), a transcription factor for PPAR{gamma}, was increased by nobiletin. The activation of cAMP-responsive element binding protein (CREB) and extracellular signal-regulated kinase (ERK), which play important roles in C/EBP{beta} expression were also potentiated by nobiletin. Furthermore, nobiletin stimulated lipolysis in differentiated adipocytes, which is known to be stimulated by cAMP pathway. These results suggested that nobiletin enhanced both differentiation and lipolysis of adipocyte through activation of signaling cascades mediated by cAMP/CREB.

  5. Platelet-derived growth factor (PDGF) stimulates glycogen synthase activity in 3T3 cells

    SciTech Connect

    Chan, C.P.; Bowen-Pope, D.F.; Ross, R.; Krebs, E.G.

    1986-05-01

    Hormonal regulation of glycogen synthase, an enzyme that can be phosphorylated on multiple sites, is often associated with changes in its phosphorylation state. Enzyme activation is conventionally monitored by determining the synthase activity ratio ((activity in the absence of glucose 6-P)/(activity in the presence of glucose 6-P)). Insulin causes an activation of glycogen synthase with a concomitant decrease in its phosphate content. In a previous report, the authors showed that epidermal growth factor (EGF) increases the glycogen synthase activity ratio in Swiss 3T3 cells. The time and dose-dependency of this response was similar to that of insulin. Their recent results indicate that PDGF also stimulates glycogen synthase activity. Enzyme activation was maximal after 30 min. of incubation with PDGF; the time course observed was very similar to that with insulin and EGF. At 1 ng/ml (0.03nM), PDGF caused a maximal stimulation of 4-fold in synthase activity ratio. Half-maximal stimulation was observed at 0.2 ng/ml (6 pM). The time course of changes in enzyme activity ratio closely followed that of /sup 125/I-PDGF binding. The authors data suggest that PDGF, as well as EFG and insulin, may be important in regulating glycogen synthesis through phosphorylation/dephosphorylation mechanisms.

  6. Modulation of Osteogenesis in MC3T3-E1 Cells by Different Frequency Electrical Stimulation

    PubMed Central

    Wang, Yu; Cui, Haitao; Wu, Zhenxu; Wu, Naipeng; Wang, Zongliang; Chen, Xuesi; Wei, Yen; Zhang, Peibiao

    2016-01-01

    Electrical stimulation (ES) is therapeutic to many bone diseases, from promoting fracture regeneration to orthopedic intervention. The application of ES offers substantial therapeutic potential, while optimal ES parameters and the underlying mechanisms responsible for the positive clinical impact are poorly understood. In this study, we assembled an ES cell culture and monitoring device. Mc-3T3-E1 cells were subjected to different frequency to investigate the effect of osteogenesis. Cell proliferation, DNA synthesis, the mRNA levels of osteosis-related genes, the activity of alkaline phosphatase (ALP), and intracellular concentration of Ca2+ were thoroughly evaluated. We found that 100 Hz could up-regulate the mRNA levels of collagen I, collagen II and Runx2. On the contrary, ES could down-regulate the mRNA levels of osteopontin (OPN). ALP activity assay and Fast Blue RR salt stain showed that 100 Hz could accelerate cells differentiation. Compared to the control group, 100 Hz could promote cell proliferation. Furthermore, 1 Hz to 10 Hz could improve calcium deposition in the intracellular matrix. Overall, these results indicate that 100Hz ES exhibits superior potentialities in osteogenesis, which should be beneficial for the clinical applications of ES for the treatment of bone diseases. PMID:27149625

  7. Transformation of NIH 3T3 cells with cloned fragments of human cytomegalovirus strain AD169.

    PubMed Central

    Nelson, J A; Fleckenstein, B; Galloway, D A; McDougall, J K

    1982-01-01

    NIH 3T3 cells were transfected with restriction endonuclease and cloned human cytomegalovirus DNA fragments to identify the transforming region(s). Cleavage of human cytomegalovirus strain AD169 DNA with XbaI and HindIII left a transforming region intact whereas EcoRI inactivated this function. Transfection of cells with cosmids containing human cytomegalovirus DNA spanning the entire genome resulted in transformation by one cosmid, pCM1058, with the AD169 HindIII DNA fragments E, R, T, and a'. Cells were selected for their growth in 1.2% methylcellulose. The clones isolated had a significant replating efficiency and were oncogenic in BALB/c nu/nu mice. Transfection of cosmids and plasmids containing subsets of the viral sequences in pCM1058 identified a common region possessed by all of the transforming recombinant molecules. This region was in the HindIII E fragment with the left boundary defined by the EcoRI d-R junction and the right boundary defined by the HindIII E-T junction. Further mapping and transfection experiments determined that the transforming region was contained without a 2.9-kilobase fragment between map units 0.123 and 0.14 on the prototype molecule of the AD169 strain. Images PMID:6287019

  8. Metabolic Flux Analysis of Mitochondrial Uncoupling in 3T3-L1 Adipocytes

    PubMed Central

    Si, Yaguang; Shi, Hai; Lee, Kyongbum

    2009-01-01

    Background Increasing energy expenditure at the cellular level offers an attractive option to limit adiposity and improve whole body energy balance. In vivo and in vitro observations have correlated mitochondrial uncoupling protein-1 (UCP1) expression with reduced white adipose tissue triglyceride (TG) content. The metabolic basis for this correlation remains unclear. Methodology/Principal Findings This study tested the hypothesis that mitochondrial uncoupling requires the cell to compensate for the decreased oxidation phosphorylation efficiency by up-regulating lactate production, thus redirecting carbon flux away from TG synthesis. Metabolic flux analysis was used to characterize the effects of non-lethal, long-term mitochondrial uncoupling (up to 18 days) on the pathways of intermediary metabolism in differentiating 3T3-L1 adipocytes. Uncoupling was induced by forced expression of UCP1 and chemical (FCCP) treatment. Chemical uncoupling significantly decreased TG content by ca. 35%. A reduction in the ATP level suggested diminished oxidative phosphorylation efficiency in the uncoupled adipocytes. Flux analysis estimated significant up-regulation of glycolysis and down-regulation of fatty acid synthesis, with chemical uncoupling exerting quantitatively larger effects. Conclusions/Significance The results of this study support our hypothesis regarding uncoupling-induced redirection of carbon flux into glycolysis and lactate production, and suggest mitochondrial proton translocation as a potential target for controlling adipocyte lipid metabolism. PMID:19746157

  9. Cytoplasmic pH influences cytoplasmic calcium in MC3T3-E1 osteoblast cells

    NASA Technical Reports Server (NTRS)

    Lin, H. S.; Hughes-Fulford, M.; Kumegawa, M.; Pitts, A. C.; Snowdowne, K. W.

    1993-01-01

    We found that the cytoplasmic concentration of calcium (Cai) of MC3T3-E1 osteoblasts was influenced by the type of pH buffer we used in the perfusing medium, suggesting that intracellular pH (pHi) might influence Cai. To study this effect, the Cai and pHi were monitored as we applied various experimental conditions known to change pHi. Exposure to NH4Cl caused a transient increase in both pHi and Cai without a change in extracellular pH (pHo). Decreasing pHo and pHi by lowering the bicarbonate concentration of the medium decreased Cai, and increasing pHi by the removal of 5% CO2 increased Cai. Clamping pHi to known values with 10 microM nigericin, a potassium proton ionophore, also influenced Cai: acid pHi lowered Cai, whereas alkaline pHi increased it. The rise in Cai appears to be very sensitive to the extracellular concentration of calcium, suggesting the existence of a pH-sensitive calcium influx mechanism. We conclude that physiologic changes in pH could modulate Cai by controlling the influx of calcium ions and could change the time course of the Cai transient associated with hormonal activation.

  10. Interactions of silver nanoparticles with primary mouse fibroblasts and liver cells

    SciTech Connect

    Arora, S.; Jain, J.; Rajwade, J.M.; Paknikar, K.M.

    2009-05-01

    Primary cells are ideal for in vitro toxicity studies since they closely resemble tissue environment. Here, we report a detailed study on the in vitro interactions of 7-20 nm spherical silver nanoparticles (SNP) with primary fibroblasts and primary liver cells isolated from Swiss albino mice. The intended use of silver nanoparticles is in the form of a topical antimicrobial gel formulation for the treatment of burns and wounds. Upon exposure to SNP for 24 h, morphology of primary fibroblasts and primary liver cells remained unaltered up to 25 {mu}g/mL and 100 {mu}g/mL SNP, respectively, although with minor decrease in confluence. IC{sub 50} values for primary fibroblasts and primary liver cells as revealed by XTT assay were 61 {mu}g/mL and 449 {mu}g/mL, respectively. Ultra-thin sections of primary cells exposed to 1/2 IC{sub 50} SNP for 24 h, visualized under Transmission electron microscope showed the presence of dark, electron dense, spherical aggregates inside the mitochondria, and cytoplasm, probably representing the intracellular SNP. When the cells were challenged with {approx} 1/2 IC{sub 50} concentration of SNP (i.e. 30 {mu}g/mL and 225 {mu}g/mL for primary fibroblasts and primary liver cells, respectively), enhancement of GSH ({approx} 1.2 fold) and depletion of lipid peroxidation ({approx} 1.4 fold) were seen in primary fibroblasts which probably protect the cells from functional damage. In case of primary liver cells; increased levels of SOD ({approx} 1.4 fold) and GSH ({approx} 1.1 fold) as compared to unexposed cells were observed. Caspase-3 activity assay indicated that the SNP concentrations required for the onset of apoptosis were found to be much lower (3.12 {mu}g/mL in primary fibroblasts, 12.5 {mu}g/mL in primary liver cells) than the necrotic concentration (100 {mu}g/mL in primary fibroblasts, 500 {mu}g/mL in primary liver cells). These observations were confirmed by CLSM studies by exposure of cells to 1/2 IC{sub 50} SNP (resulting in apoptosis

  11. Nitric Oxide-Induced Autophagy in MC3T3-E1 Cells is Associated with Cytoprotection via AMPK Activation

    PubMed Central

    Yang, Jung Yoon; Park, Min Young; Park, Sam Young; Yoo, Hong Il; Kim, Min Seok; Kim, Jae Hyung

    2015-01-01

    Nitric oxide (NO) is important in the regulation of bone remodeling, whereas high concentration of NO promotes cell death of osteoblast. However, it is not clear yet whether NO-induced autophagy is implicated in cell death or survival of osteoblast. The present study is aimed to examine the role of NO-induced autophagy in the MC3T3-E1 cells and their underlying molecular mechanism. The effect of sodium nitroprusside (SNP), an NO donor, on the cytotoxicity of the MC3T3-E1 cells was determined by MTT assay and expression of apoptosis or autophagy associated molecules was evaluated by western blot analysis. The morphological observation of autophagy and apoptosis by acridine orange stain and TUNEL assay were performed, respectively. Treatment of SNP decreased the cell viability of the MC3T3-E1 cells in dose- and time-dependent manner. SNP increased expression levels of p62, ATG7, Beclin-1 and LC3-II, as typical autophagic markers and augmented acidic autophagolysosomal vacuoles, detected by acridine orange staining. However, pretreatment with 3-methyladenine (3MA), the specific inhibitor for autophagy, decreased cell viability, whereas increased the cleavage of PARP and caspase-3 in the SNP-treated MC3T3-E1 cells. AMP-activated protein kinase (AMPK), a major autophagy regulatory kinase, was activated in SNP-treated MC3T3-E1 cells. In addition, pretreatment with compound C, an inhibitor of AMPK, decreased cell viability, whereas increased the number of apoptotic cells, cleaved PARP and caspase-3 levels compared to those of SNP-treated MC3T3-E1 cells. Taken together, it is speculated that NO-induced autophagy functions as a survival mechanism via AMPK activation against apoptosis in the MC3T3-E1 cells. PMID:26557017

  12. Triiodothyronine modulates the expression of leptin and adiponectin in 3T3-L1 adipocytes

    PubMed Central

    de Oliveira, Miriane; Síbio, Maria Teresa De; Olimpio, Regiane Marques Castro; Moretto, Fernanda Cristina Fontes; Luvizotto, Renata de Azevedo Melo; Nogueira, Celia Regina

    2015-01-01

    Objective To study the effect of different doses of triiodothyronine on gene expression of the adipokines leptin and adiponectin, at different times, and to evaluate the difference in expression between the two adipokines in each group. Methods 3T3-L1 adipocytes were incubated with triiodothyronine at physiological dose (10nM) and supraphysiological doses (100nM or 1,000nM), or without triiodothyronine (control, C) for 0.5, 6, or 24 hours. Leptin and adiponectin mRNA was detected using real-time polymerase chain reaction (RT-PCR). One-way analyses of variance, Tukey’s test or Student’s t test, were used to analyze data, and significance level was set at 5%. Results Leptin levels decreased in the 1,000nM-dose group after 0.5 hour. Adiponectin levels dropped in the 10nM-dose group, but increased at the 100nM dose. After 6 hours, both genes were suppressed in all hormone concentrations. After 24 hours, leptin levels increased at 10, 100 and 1,000nM groups as compared to the control group; and adiponectin levels increased only in the 100nM group as compared to the control group. Conclusion These results demonstrated fast actions of triiodothyronine on the leptin and adiponectin expression, starting at 0.5 hour, at a dose of 1,000nM for leptin and 100nM for adiponectin. Triiodothyronine stimulated or inhibited the expression of adipokines in adipocytes at different times and doses which may be useful to assist in the treatment of obesity, assuming that leptin is increased and adiponectin is decreased, in obesity cases. PMID:25993072

  13. Substrate selectivity of diacylglycerol kinase in PDGF-stimulated 3T3 cells

    SciTech Connect

    MacDonald, M.L.; Mack, K.F.; Glomset, J.A.

    1987-05-01

    The authors investigated the properties of Diacylglycerol (DAG) Kinase in 3T3 cells. PDGF treatment caused an increase in DAG mass, an increase in incorporation of /sup 32/P into phosphatidic acid (PA) and phosphatidylinositol (PI), and an increase in the rate of phosphorylation of membrane DAG in vitro. The mechanism of enhanced phosphorylation of DAG was studied with dicaprylin (diC/sub 10/) as a probe. Cells were prelabeled with /sup 32/P and treated with PDGF or carrier. DiC/sub 10/ was added to the cell medium before harvesting. With PDGF treatment, the radioactivity in endogenous PA increased fourfold, whereas the radioactivity in PA/sub 10/ and PI/sub 10/ was consistently decreased. To verify that the PDGF effect on PA/sub 10/ formation in intact cells was due to reduced phosphorylation of diC/sub 10/ by DAG kinase, cells were treated with PDGF and/or diC/sub 10/, freeze-thawed, and then incubated with Mg(/sup 32/P)ATP. The rate of phosphorylation of cell-associated diC/sub 10/ was decreased 50% by PDGF treatment. This effect could not be explained by decreased intracellular levels of diC/sub 10/, or by saturation of DAG kinase with endogenous DAGs. Therefore, it seemed that endogenous DAGs, derived from PI, might be better substrates for DAG kinase than is diC/sub 10/. In studies of the properties of DAG kinase with pure DAGs in mixed detergent micelles, they found that the enzyme phosphorylated arachidonoyl-DAG more readily than diC/sub 10/. The selectivity of DAG kinase may play a key role in the formation of arachidonoyl species of PI.

  14. Mango fruit peel and flesh extracts affect adipogenesis in 3T3-L1 cells.

    PubMed

    Taing, Meng-Wong; Pierson, Jean-Thomas; Hoang, Van L T; Shaw, Paul N; Dietzgen, Ralf G; Gidley, Michael J; Roberts-Thomson, Sarah J; Monteith, Gregory R

    2012-08-01

    Obesity is associated with many chronic disease states, such as diabetes mellitus, coronary disease and certain cancers, including those of the breast and colon. There is a growing body of evidence that links phytochemicals with the inhibition of adipogenesis and protection against obesity. Mangoes (Mangifera indica L.) are tropical fruits that are rich in a diverse array of bioactive phytochemicals. In this study, methanol extracts of peel and flesh from three archetypal mango cultivars; Irwin, Nam Doc Mai and Kensington Pride, were assessed for their effects on a 3T3-L1 pre-adipocyte cell line model of adipogenesis. High content imaging was used to assess: lipid droplets per cell, lipid droplet area per cell, lipid droplet integrated intensity, nuclei count and nuclear area per cell. Mango flesh extracts from the three cultivars did not inhibit adipogenesis; peel extracts from both Irwin and Nam Doc Mai, however, did so with the Nam Doc Mai extract most potent at inhibiting adipogenesis. Peel extract from Kensington Pride promoted adipogenesis. The inhibition of adipogenesis by Irwin (100 μg mL(-1)) and Nam Doc Mai peel extracts (50 and 100 μg mL(-1)) was associated with an increase in the average nuclear area per cell; similar effects were seen with resveratrol, suggesting that these extracts may act through pathways similar to resveratrol. These results suggest that differences in the phytochemical composition between mango cultivars may influence their effectiveness in inhibiting adipogenesis, and points to mango fruit peel as a potential source of nutraceuticals.

  15. Differential expression of fatty acid uptake in 3T3-L1 cells

    SciTech Connect

    Waggoner, D.; Bernlohr, D.A.

    1987-05-01

    Cultured 3T3-L1 cells have been used as a model system to investigate the mechanism of fatty acid uptake by adipose tissue. Using a 1:1 molar ratio of /sup 14/C-oleate and defatted bovine serum albumin (BSA), fatty acid (FA) uptake was quantitated at 4/sup 0/ and 37/sup 0/ as cell associated radioactivity. The profile of FA uptake in preadipocytes and adipocytes was biphasic; an initial rapid phase (1-20s) followed by a second slower phase (60-480s). At 37/sup 0/ the initial rate of FA accumulation in preadipocytes was identical to that in adipocytes, whereas the rate of accumulation during the second phase increased 7-fold (100 ..mu..M total FA) as a consequence of adipose conversion. When uptake measurements were made at 4/sup 0/ in adipocytes, the initial rate was identical to that at 37/sup 0/, however the rate of second phase decreased 5-fold. Incubation of /sup 14/C-BSA and nonradiolabeled FA with adipocyte monolayers (100 ..mu..M total FA) resulted in the rapid association (t/sub 1/2/ = 20s) of the BSA-FA complex with the cell surface. Incubation of 100, 10, and 1 ..mu..M total FA with adipocytes resulted in a 50-fold change in FA accumulation during the second phase. These results suggest that (1) FA uptake is significantly increased after differentiation, suggesting the participation of specialized proteins, (2) the temperature-insensitive initial FA accumulation can be attributed to rapid association of the BSA-FA complex to the cell surface, (3) the second phase of FA accumulation represents uptake.

  16. Hormone and pharmaceutical regulation of ASP production in 3T3-L1 adipocytes.

    PubMed

    Gao, Ying; Gauvreau, Danny; Cianflone, Katherine

    2010-04-01

    Several studies have demonstrated increases in acylation stimulating protein (ASP), and precursor protein C3 in obesity, diabetes and dyslipidemia, however the nature of the regulation is unknown. To evaluate chronic hormonal and pharmaceutical mediated changes in ASP and potential mechanisms, 3T3-L1 adipocytes were treated with physiological concentrations of relevant hormones and drugs currently used in treatment of metabolic diseases for 48 h. Medium ASP production and C3 secretion were evaluated in relation to changes in adipocyte lipid metabolism (cellular triglyceride (TG) mass, non-esterified fatty acid (NEFA) release and real-time FA uptake). Chylomicrons increased ASP production (up to 411 +/- 133% P < 0.05), while leptin, triiodothyronine, and beta-blockers atenolol and propranolol had no effect. Dexamethasone, lovastatin, rosiglitazone and rimonabant decreased ASP production (-53 to -85%, P < 0.05), associated with a decrease in the precursor protein C3 (-37% to -65%, P < 0.01). By contrast, epinephrine, progesterone, testosterone, angiotensin II and metformin also decreased ASP (-54% to -100%, P < 0.05), but without change in precursor protein C3, suggesting a direct effect on convertase activity, possibly mediated by interference (except metformin) due to marked increases in NEFA (5.6-31-fold, increased P < 0.05). Both lovastatin and metformin induced decreases in ASP were also associated with decreased TG mass (maximal -60%, P < 0.05) and real-time FA uptake (maximum -75%, P < 0.05), suggesting a change in adipocyte differentiation status. These in vitro results are consistent with in vivo ASP profiles in subjects, and suggest that ASP may be regulated through precursor C3 availability, convertase activity and differentiation status.

  17. Advanced oxidation protein products inhibit differentiation and activate inflammation in 3T3-L1 preadipocytes.

    PubMed

    Zhou, Qiu Gen; Peng, Xin; Hu, Li Li; Xie, Di; Zhou, Min; Hou, Fan Fan

    2010-10-01

    Accumulation of advanced oxidation protein products (AOPPs) is prevalent in metabolic syndromes, a condition with impaired preadipocytes differentiation. In the present study, we tested the hypothesis that AOPPs disturb preadipocyte differentiation. Exposure of 3T3-L1 preadipocytes to increased levels of AOPPs inhibited accumulation of intracellular triglyceride and decreased the expression of the essential markers of matured adipocytes, such as adipocyte fatty-acid-binding protein (aP2), CAAT/enhancer-binding protein (C/EBP)-alpha, and peroxisome proliferator-activated receptor (PPAR)-gamma, in response to standard adipogenic induction. Inhibitory effects of AOPPs on preadipocytes differentiation was time sensitive, which occurred at the early stage of differentiation. In the presence of AOPPs, induction of preadipocytes differentiation resulted in upregulated expression of C/EBP homologous protein (CHOP) and CUG-Triplet repeat-binding protein (CUGBP), two important inhibitors of preadipocytes differentiation. In addition, treatment with AOPPs increased abundance of C/EBP-beta-liver enriched inhibitory protein (C/EBP-beta-LIP), a truncated C/EBP-beta isoform without adipogenic activity. Moreover, AOPPs-treated preadipocytes expressed a macrophage marker F4/80 and overexpressed tumor necrosis factor-alpha and interleukin-6 via nuclear factor-kappaB (NF-kappaB)-dependent pathway. However, blocking inflammation with NF-kappaB inhibitor failed to improve AOPPs-induced inhibition of preadipocytes differentiation. These data suggest that accumulation of AOPPs may inhibit differentiation of preadipocytes and activate inflammation in these cells. This information might have implication for understanding the impairment of preadipocytes differentiation and fat inflammation seen in metabolic syndrome.

  18. Actions of β-apo-carotenoids in differentiating cells: differential effects in P19 cells and 3T3-L1 adipocytes.

    PubMed

    Wang, Cynthia X; Jiang, Hongfeng; Yuen, Jason J; Lee, Seung-Ah; Narayanasamy, Sureshbabu; Curley, Robert W; Harrison, Earl H; Blaner, William S

    2015-04-15

    β-Apo-carotenoids, including β-apo-13-carotenone and β-apo-14'-carotenal, are potent retinoic acid receptor (RAR) antagonists in transactivation assays. We asked how these influence RAR-dependent processes in living cells. Initially, we explored the effects of β-apo-13-carotenone and β-apo-14'-carotenal on P19 cells, a mouse embryonal carcinoma cell line that differentiates into neurons when treated with all-trans-retinoic acid. Treatment of P19 cells with either compound failed to block all-trans-retinoic acid induced differentiation. Liquid chromatography tandem mass spectrometry studies, however, established that neither of these β-apo-carotenoids accumulates in P19 cells. All-trans-retinoic acid accumulated to high levels in P19 cells. This suggests that the uptake and metabolism of β-apo-carotenoids by some cells does not involve the same processes used for retinoids and that these may be cell type specific. We also investigated the effects of two β-apo-carotenoids on 3T3-L1 adipocyte marker gene expression during adipocyte differentiation. Treatment of 3T3-L1 adipocytes with either β-apo-13-carotenone or β-apo-10'-carotenoic acid, which lacks RAR antagonist activity, stimulated adipocyte marker gene expression. Neither blocked the inhibitory effects of a relatively large dose of exogenous all-trans-retinoic acid on adipocyte differentiation. Our data suggest that in addition to acting as transcriptional antagonists, some β-apo-carotenoids act through other mechanisms to influence 3T3-L1 adipocyte differentiation.

  19. Soshiho-Tang Aqueous Extract Exerts Antiobesity Effects in High Fat Diet-Fed Mice and Inhibits Adipogenesis in 3T3-L1 Adipocytes

    PubMed Central

    Lee, Mee-young; Kang, Byoung-Kab

    2016-01-01

    Soshiho-tang (SST; sho-saiko-to in Japanese; xiaochaihu-tang in Chinese) has generally been used to improve liver fibrosis- and cirrhosis-related symptoms in traditional Korean medicine. Although many studies have investigated the pharmacological properties of SST, its antiobesity effect has not been elucidated. Thus, our present study was carried out to evaluate the antiobesity effect of SST using a high fat diet- (HFD) induced obese mouse model and 3T3-L1 adipose cells. C57BL/6J mice were randomly divided into four groups (n = 6/group), normal diet (ND), HFD-fed group, and HFD- and SST-fed groups (S200: 200 mg/kg of SST; S600: 600 mg/kg of SST) and given HFD with or without SST extract for 8 weeks. 3T3-L1 preadipocytes were differentiated into adipocytes for 8 days with or without SST. In the HFD-fed obese mice, body weight and fat accumulation in adipose tissue were significantly reduced by SST administration. Compared with control-differentiated adipocytes, SST significantly inhibited lipid accumulation by decreasing the triglyceride (TG) content and leptin concentration in 3T3-L1 adipocytes. SST also decreased the expression of adipogenesis-related genes including lipoprotein lipase (LPL), fatty acid binding protein 4 (FABP4), CCAAT/enhancer-binding protein-alpha (C/EBP-α), and peroxisome proliferator-activated receptor-gamma (PPAR-γ). Our findings suggest that SST has potential as a nontoxic antiobesity medication. PMID:27777595

  20. EVALUATION OF BENZO[C]CHRYSENE DIHYDRODIOLS IN THE MORPHOLOGICAL CELL TRANSFORMATION OF MOUSE EMBRYO FIBROBLAST C3H10T1/2CL8 CELLS

    EPA Science Inventory

    EVALUATION OF BENZO[c]CHRYSENE DIHYDRODIOLS IN THE MORPHOLOGICAL CELL TRANSFORMATION OF MOUSE EMBRYO FIBROBLAST C3H10T?CL8 CELLS

    Abstract The morphological cell transforming activities of three dihydrodiols of benzo[c]chrysene (B[c]C), trans-B[c]C-7,8-diol, trans-B[c]C-9...

  1. Plasma treated polyethylene grafted with adhesive molecules for enhanced adhesion and growth of fibroblasts.

    PubMed

    Rimpelová, Silvie; Kasálková, Nikola Slepičková; Slepička, Petr; Lemerová, Helena; Švorčík, Václav; Ruml, Tomáš

    2013-04-01

    The cell-material interface plays a crucial role in the interaction of cells with synthetic materials for biomedical use. The application of plasma for tailoring polymer surfaces is of abiding interest and holds a great promise in biomedicine. In this paper, we describe polyethylene (PE) surface tuning by Ar plasma irradiating and subsequent grafting of the chemically active PE surface with adhesive proteins or motives to support cell attachment. These simple modifications resulted in changed polymer surface hydrophilicity, roughness and morphology, which we thoroughly characterized. The effect of our modifications on adhesion and growth was tested in vitro using mouse embryonic fibroblasts (NIH 3T3 cell line). We demonstrate that the plasma treatment of PE had a positive effect on the adhesion, spreading, homogeneity of distribution and moderately on proliferation activity of NIH 3T3 cells. This effect was even more pronounced on PE coated with biomolecules.

  2. Mechanisms of Multi-walled Carbon Nanotubes-Induced Oxidative Stress and Genotoxicity in Mouse Fibroblast Cells.

    PubMed

    Alarifi, Saud; Ali, Daoud

    2015-01-01

    The extensive production and wide application of carbon nanotubes have made investigations of its toxic potentials necessary. In the present study, we explored the underlying mechanism through which multi-walled carbon nanotubes (MWCNTs) induce toxicity in mouse fibroblast cells (L929). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and neutral red uptake viability assays were used to examine mechanisms of cytotoxicity. Dose and time-dependent cytotoxicity was observed in L929 cells. The MWCNTs significantly increased the generation of reactive oxygen species, lipid peroxidation, superoxide dismutase, and decreased glutathione. It was observed that the MWCNTs induced caspase 3 activity. The highest DNA strand breakage was detected by comet assay at 300 µg/mL of MWCNTs. Thus, the data indicate that MWCNTs induced cytotoxicity and apoptosis in L929 cells via oxidative stress.

  3. Hmga1 null mouse embryonic fibroblasts display downregulation of spindle assembly checkpoint gene expression associated to nuclear and karyotypic abnormalities

    PubMed Central

    Pierantoni, Giovanna Maria; Conte, Andrea; Rinaldo, Cinzia; Tornincasa, Mara; Gerlini, Raffaele; Valente, Davide; Izzo, Antonella; Fusco, Alfredo

    2016-01-01

    ABSTRACT The High Mobility Group A1 proteins (HMGA1) are nonhistone chromatinic proteins with a critical role in development and cancer. We have recently reported that HMGA1 proteins are able to increase the expression of spindle assembly checkpoint (SAC) genes, thus impairing SAC function and causing chromosomal instability in cancer cells. Moreover, we found a significant correlation between HMGA1 and SAC genes expression in human colon carcinomas. Here, we report that mouse embryonic fibroblasts null for the Hmga1 gene show downregulation of Bub1, Bub1b, Mad2l1 and Ttk SAC genes, and present several features of chromosomal instability, such as nuclear abnormalities, binucleation, micronuclei and karyotypic alterations. Interestingky, also MEFs carrying only one impaired Hmga1 allele present karyotypic alterations. These results indicate that HMGA1 proteins regulate SAC genes expression and, thereby, genomic stability also in embryonic cells. PMID:26889953

  4. Activation of PPAR-γ inhibits PDGF-induced proliferation of mouse renal fibroblasts.

    PubMed

    Lu, Jiamei; Shi, Jianhua; Gui, Baosong; Yao, Ganglian; Wang, Li; Ou, Yan; Zhu, Dan; Ma, Liqun; Ge, Heng; Fu, Rongguo

    2016-10-15

    Recent studies have shown that activation of peroxisome proliferators activated receptor-γ (PPAR-γ) ameliorates renal interstitial fibrosis (RIF) in animal model. Yet, the underlying molecular mechanisms remain still largely unknown. Here, we investigated the hypothesis that activation of PPAR-γ regulates renal remodeling by modulating proliferation of primary cultured renal fibroblasts. In our present study, platelet-derived growth factor-AA (PDGF-AA), a key isoform of PDGF superfamily as mitogen in RIF, was applied to stimulate renal fibroblasts, the selective inhibitor or sequence specific siRNA of PI3K, skp2 or PPAR-γ was used to investigate the involvement of above molecular mediators in PDGF-AA-induced cell proliferation. Our results demonstrate that PDGF-AA induced proliferation of renal fibroblasts by activating PI3K/AKT signaling and resultant skp2 production. Pre-stimulation of cells with rosiglitazone or adenovirus carrying PPAR-γ cDNA (AdPPAR-γ) blocked PDGF-AA-stimulated cell proliferation, this effect was particularly coupled to PPAR-γ inhibition of AKT phosphorylation and skp2 expression. Inhibition of PPAR-γ by GW9662 restored the suppression of activated PPAR-γ on phosphorylation of AKT and subsequent skp2 production. Our results indicate that activation of PI3K/AKT signaling and resultant skp2 generation mediated PDGF-induced proliferation of renal fibroblasts. Activation of PPAR-γ inhibited cell proliferation by inhibition of AKT phosphorylation and its down-streams.

  5. Lentivirus-meditated frataxin gene delivery reverses genome instability in Friedreich ataxia patient and mouse model fibroblasts

    PubMed Central

    Khonsari, H; Schneider, M; Al-Mahdawi, S; Chianea, Y G; Themis, M; Parris, C; Pook, M A; Themis, M

    2016-01-01

    Friedreich ataxia (FRDA) is a progressive neurodegenerative disease caused by deficiency of frataxin protein, with the primary sites of pathology being the large sensory neurons of the dorsal root ganglia and the cerebellum. FRDA is also often accompanied by severe cardiomyopathy and diabetes mellitus. Frataxin is important in mitochondrial iron–sulfur cluster (ISC) biogenesis and low-frataxin expression is due to a GAA repeat expansion in intron 1 of the FXN gene. FRDA cells are genomically unstable, with increased levels of reactive oxygen species and sensitivity to oxidative stress. Here we report the identification of elevated levels of DNA double strand breaks (DSBs) in FRDA patient and YG8sR FRDA mouse model fibroblasts compared to normal fibroblasts. Using lentivirus FXN gene delivery to FRDA patient and YG8sR cells, we obtained long-term overexpression of FXN mRNA and frataxin protein levels with reduced DSB levels towards normal. Furthermore, γ-irradiation of FRDA patient and YG8sR cells revealed impaired DSB repair that was recovered on FXN gene transfer. This suggests that frataxin may be involved in DSB repair, either directly by an unknown mechanism, or indirectly via ISC biogenesis for DNA repair enzymes, which may be essential for the prevention of neurodegeneration. PMID:27518705

  6. Selective de-repression of germ cell-specific genes in mouse embryonic fibroblasts in a permissive epigenetic environment

    PubMed Central

    Sekinaka, Tamotsu; Hayashi, Yohei; Noce, Toshiaki; Niwa, Hitoshi; Matsui, Yasuhisa

    2016-01-01

    Epigenetic modifications play crucial roles on establishment of tissue-specific transcription profiles and cellular characteristics. Direct conversions of fibroblasts into differentiated tissue cells by over-expression of critical transcription factors have been reported, but the epigenetic mechanisms underlying these conversions are still not fully understood. In addition, conversion of somatic cells into germ cells has not yet been achieved. To understand epigenetic mechanisms that underlie germ cell characteristics, we attempted to use defined epigenetic factors to directly convert mouse embryonic fibroblasts (MEFs) into germ cells. Here, we successfully induced germ cell-specific genes by inhibiting repressive epigenetic modifications via RNAi or small-molecule compounds. Under these conditions, some tissue-specific genes and stimulus-inducible genes were also induced. Meanwhile, the treatments did not result in genome-wide transcriptional activation. These results suggested that a permissive epigenetic environment resulted in selective de-repression of stimulus- and differentiation-inducible genes including germ cell-specific genes in MEFs. PMID:27608931

  7. Lipid droplets fusion in adipocyte differentiated 3T3-L1 cells: A Monte Carlo simulation

    SciTech Connect

    Boschi, Federico; Rizzatti, Vanni; Zamboni, Mauro; Sbarbati, Andrea

    2014-02-15

    Several human worldwide diseases like obesity, type 2 diabetes, hepatic steatosis, atherosclerosis and other metabolic pathologies are related to the excessive accumulation of lipids in cells. Lipids accumulate in spherical cellular inclusions called lipid droplets (LDs) whose sizes range from fraction to one hundred of micrometers in adipocytes. It has been suggested that LDs can grow in size due to a fusion process by which a larger LD is obtained with spherical shape and volume equal to the sum of the progenitors’ ones. In this study, the size distribution of two populations of LDs was analyzed in immature and mature (5-days differentiated) 3T3-L1 adipocytes (first and second populations, respectively) after Oil Red O staining. A Monte Carlo simulation of interaction between LDs has been developed in order to quantify the size distribution and the number of fusion events needed to obtain the distribution of the second population size starting from the first one. Four models are presented here based on different kinds of interaction: a surface weighted interaction (R2 Model), a volume weighted interaction (R3 Model), a random interaction (Random model) and an interaction related to the place where the LDs are born (Nearest Model). The last two models mimic quite well the behavior found in the experimental data. This work represents a first step in developing numerical simulations of the LDs growth process. Due to the complex phenomena involving LDs (absorption, growth through additional neutral lipid deposition in existing droplets, de novo formation and catabolism) the study focuses on the fusion process. The results suggest that, to obtain the observed size distribution, a number of fusion events comparable with the number of LDs themselves is needed. Moreover the MC approach results a powerful tool for investigating the LDs growth process. Highlights: • We evaluated the role of the fusion process in the synthesis of the lipid droplets. • We compared the

  8. Five-parameter fluorescence imaging: wound healing of living Swiss 3T3 cells

    PubMed Central

    1987-01-01

    Cellular functions involve the temporal and spatial interplay of ions, metabolites, macromolecules, and organelles. To define the mechanisms responsible for completing cellular functions, we used methods that can yield both temporal and spatial information on multiple physiological parameters and chemical components in the same cell. We demonstrated that the combined use of selected fluorescent probes, fluorescence microscopy, and imaging methods can yield information on at least five separate cellular parameters and components in the same living cell. Furthermore, the temporal and spatial dynamics of each of the parameters and/or components can be correlated with one or more of the others. Five parameters were investigated by spectrally isolating defined regions of the ultraviolet, visible, and near-infrared spectrum based on five distinct fluorescent probes. The parameters included nuclei (Hoechst 33342), mitochondria (diIC1-[5] ), endosomes (lissamine rhodamine B-dextran), actin (fluorescein), and the cell volume Cy7- dextran). Nonmotile, confluent Swiss 3T3 cells did not show any detectable polarity of cell shape, or distribution of nuclei, endosomes, or mitochondria. These cells also organized a large percentage of the actin into stress fibers. In contrast, cells migrating into an in vitro wound exhibited at least two stages of reorganization of organelles and cytoplasm. During the first 3 h after wounding, the cells along the edge of the wound assumed a polarized shape, carried the nuclei in the rear of the cells, excluded endosomes and mitochondria from the lamellipodia, and lost most of the highly organized stress fibers. The cell showed a dramatic change between 3 and 7 h after producing the wound. The cells became highly elongated and motile; both the endosomes and the mitochondria penetrated into the lamellipodia, while the nuclei remained in the rear and the actin remained in less organized structures. Defining the temporal and spatial dynamics and

  9. Expression profiling and pathway analysis of Krüppel-like factor 4 in mouse embryonic fibroblasts

    PubMed Central

    Hagos, Engda G; Ghaleb, Amr M; Kumar, Amrita; Neish, Andrew S; Yang, Vincent W

    2011-01-01

    Background: Krüppel-like factor 4 (KLF4) is a zinc-finger transcription factor with diverse regulatory functions in proliferation, differentiation, and development. KLF4 also plays a role in inflammation, tumorigenesis, and reprogramming of somatic cells to induced pluripotent stem (iPS) cells. To gain insight into the mechanisms by which KLF4 regulates these processes, we conducted DNA microarray analyses to identify differentially expressed genes in mouse embryonic fibroblasts (MEFs) wild type and null for Klf4. Methods: Expression profiles of fibroblasts isolated from mouse embryos wild type or null for the Klf4 alleles were examined by DNA microarrays. Differentially expressed genes were subjected to the Database for Annotation, Visualization and Integrated Discovery (DAVID). The microarray data were also interrogated with the Ingenuity Pathway Analysis (IPA) and Gene Set Enrichment Analysis (GSEA) for pathway identification. Results obtained from the microarray analysis were confirmed by Western blotting for select genes with biological relevance to determine the correlation between mRNA and protein levels. Results: One hundred and sixty three up-regulated and 88 down-regulated genes were identified that demonstrated a fold-change of at least 1.5 and a P-value < 0.05 in Klf4-null MEFs compared to wild type MEFs. Many of the up-regulated genes in Klf4-null MEFs encode proto-oncogenes, growth factors, extracellular matrix, and cell cycle activators. In contrast, genes encoding tumor suppressors and those involved in JAK-STAT signaling pathways are down-regulated in Klf4-null MEFs. IPA and GSEA also identified various pathways that are regulated by KLF4. Lastly, Western blotting of select target genes confirmed the changes revealed by microarray data. Conclusions: These data are not only consistent with previous functional studies of KLF4's role in tumor suppression and somatic cell reprogramming, but also revealed novel target genes that mediate KLF4's

  10. The role of hypoxia inducible factor 1alpha in cobalt chloride induced cell death in mouse embryonic fibroblasts.

    PubMed

    Vengellur, A; LaPres, J J

    2004-12-01

    Cobalt has been widely used in the treatment of anemia and as a hypoxia mimic in cell culture and it is known to activate hypoxic signaling by stabilizing the hypoxia inducible transcription factor 1alpha (HIF1alpha). However, cobalt exposure can lead to tissue and cellular toxicity. These studies were conducted to determine the role of HIF1alpha in mediating cobalt-induced toxicity. Mouse embryonic fibroblasts (MEFs) that were null for the HIF1alpha protein were used to show that HIF1alpha protein plays a major role in mediating cobalt-induced cytotoxicity. Previous work from our lab and others has shown that two BH3 domain containing cell death genes, BNip3 and NIX, are targets of hypoxia signaling. These experiments document that BNip3 and NIX expression is HIF1alpha-dependent, and cobalt induces their expression in a time and dose dependent manner. In addition, their expression is correlated with an increase in BNIP3 and NIX protein. Characteristically, the elevated level of BNIP3 was correlated with an increased presence of chromatin condensation, one marker for cell injury. Interestingly, this increased chromosomal condensation was not coupled to caspase-3 activation as usually seen in a typical apoptotic response. These results show that HIF1alpha is playing a major role in mediating cobalt-induced toxicity in mouse embryonic fibroblasts and may offer a possible mechanism for the underlying pathology of injuries seen in workers exposed to environmental contaminants that can influence the hypoxia signaling system, such as cobalt.

  11. Molybdenum(VI) salts convert the xanthine oxidoreductase apoprotein into the active enzyme in mouse L929 fibroblastic cells.

    PubMed Central

    Falciani, F; Terao, M; Goldwurm, S; Ronchi, A; Gatti, A; Minoia, C; Li Calzi, M; Salmona, M; Cazzaniga, G; Garattini, E

    1994-01-01

    The mouse L929 fibroblastic cell line presents low, but detectable, levels of the mRNA encoding xanthine oxidoreductase under basal conditions, and it responds to type I and type II interferons by inducing the expression of the transcript [Falciani, Ghezzi, Terao, Cazzaniga, and Garattini (1992) Biochem. J. 285, 1001-1008]. This cell line, however, does not show any detectable amount of xanthine oxidoreductase enzymic activity, either before or after treatment with the cytokines. Molybdenum(VI) salts, in the millimolar range, are capable of activating xanthine oxidoreductase in L929 cells both under basal conditions and after treatment with interferon-alpha. The increase is observed in mouse L929 as well as in clones derived from it, but not in many other human and mouse cell lines. The induction observed in L929 cells is post-translational in nature and it is insensitive to cycloheximide, indicating that the molybdenum ion converts a pool of inactive xanthine oxidoreductase apoenzyme into its holoenzymic form. When grown in the absence of sodium molybdate, the L929 cell line has undetectable intracellular levels of the molybdenum cofactor, since the cell extracts are unable to complement the nitrate reductase defect of the nit-1 mutant of Neurospora crassa. L929 cells grown in the presence of millimolar concentrations of sodium molybdate, however, become competent to complement the nit-1 defect. L929 cells accumulate molybdenum ion inside the intracellular compartment as efficiently as TEnd cells, a mouse endothelial cell line that expresses xanthine oxidoreductase activity both under basal conditions and after treatment with interferon-gamma, suggesting that L929 cells have a defect in one or more of the metabolic steps leading to the synthesis of the molybdenum cofactor. Images Figure 1 Figure 2 Figure 4 PMID:8129733

  12. Fibroblast growth factor 10 alters the balance between goblet and Paneth cells in the adult mouse small intestine.

    PubMed

    Al Alam, Denise; Danopoulos, Soula; Schall, Kathy; Sala, Frederic G; Almohazey, Dana; Fernandez, G Esteban; Georgia, Senta; Frey, Mark R; Ford, Henri R; Grikscheit, Tracy; Bellusci, Saverio

    2015-04-15

    Intestinal epithelial cell renewal relies on the right balance of epithelial cell migration, proliferation, differentiation, and apoptosis. Intestinal epithelial cells consist of absorptive and secretory lineage. The latter is comprised of goblet, Paneth, and enteroendocrine cells. Fibroblast growth factor 10 (FGF10) plays a central role in epithelial cell proliferation, survival, and differentiation in several organs. The expression pattern of FGF10 and its receptors in both human and mouse intestine and their role in small intestine have yet to be investigated. First, we analyzed the expression of FGF10, FGFR1, and FGFR2, in the human ileum and throughout the adult mouse small intestine. We found that FGF10, FGFR1b, and FGFR2b are expressed in the human ileum as well as in the mouse small intestine. We then used transgenic mouse models to overexpress Fgf10 and a soluble form of Fgfr2b, to study the impact of gain or loss of Fgf signaling in the adult small intestine. We demonstrated that overexpression of Fgf10 in vivo and in vitro induces goblet cell differentiation while decreasing Paneth cells. Moreover, FGF10 decreases stem cell markers such as Lgr5, Lrig1, Hopx, Ascl2, and Sox9. FGF10 inhibited Hes1 expression in vitro, suggesting that FGF10 induces goblet cell differentiation likely through the inhibition of Notch signaling. Interestingly, Fgf10 overexpression for 3 days in vivo and in vitro increased the number of Mmp7/Muc2 double-positive cells, suggesting that goblet cells replace Paneth cells. Further studies are needed to determine the mechanism by which Fgf10 alters cell differentiation in the small intestine.

  13. Fibroblasts maintain the phenotype and viability of the rat heparin-containing mast cell in vitro

    SciTech Connect

    Levi-Schaffer, F.; Austen, K.F.; Caulfield, J.P.; Hein, A.; Bloes, W.F.; Stevens, R.L.

    1985-11-01

    Rat serosal heparin-containing mast cells (HP-MC) were maintained in vitro for as long as 30 days when co-cultured with mouse skin-derived 3T3 fibroblasts. In contrast, when the mast cells were cultured alone, on fibronectin-, gelatin-, or dermal-collagen-coated dishes, on acid and heat-killed fibroblasts in the presence or absence of 24 hr fibroblast-conditioned medium, or on a monolayer of mouse serosal macrophages, they failed to adhere to the dishes, released significant amounts of their histamine and lactate dehydrogenase, and stained with trypan blue, indicating a loss of viability. The rat serosal HP-MC cultured with the 3T3 fibroblasts became so adherent to the fibroblasts that the two cell types could be separated from one another only by trypsinization. The cultured HP-MC stained with both alcian blue and safranin and continued to synthesize proteoglycan at a rate comparable to that of freshly isolated cells. The /sup 35/S-labeled proteoglycan synthesized by these cultured cells, like that produced by freshly isolated rat serosal HP-MC, was a 750,000 to 1,000,000 m.w. proteoglycan containing only heparin glycosaminoglycans of 50,000 to 100,000 m.w. As assessed by electron microscopy, many of the cultured HP-MC resembled freshly isolated cells except that some secretory granules had fused with one another in some cells. These results demonstrate that the in vivo differentiated rat HP-MC maintain their histology, morphology, immunologic responsiveness, histamine content, and ability to synthesize heparin proteoglycan when co-cultured with living fibroblasts.

  14. Mouse Lung Fibroblast Resistance to Fas-Mediated Apoptosis Is Dependent on the Baculoviral Inhibitor of Apoptosis Protein 4 and the Cellular FLICE-Inhibitory Protein

    PubMed Central

    Predescu, Sanda A.; Zhang, Jian; Bardita, Cristina; Patel, Monal; Godbole, Varun; Predescu, Dan N.

    2017-01-01

    A characteristic feature of idiopathic pulmonary fibrosis (IPF) is accumulation of apoptotic resistant fibroblasts/myofibroblasts in the fibroblastic foci. As caveolin (Cav)-null mice develop pulmonary fibrosis (PF), we hypothesized that the participating fibroblasts display an apoptosis-resistant phenotype. To test this hypothesis and identify the molecular mechanisms involved we isolated lung fibroblasts from Cav-null mice and examined the expression of several inhibitors of apoptosis (IAPs), of c-FLIP, of Bcl-2 proteins and of the death receptor CD95/Fas. We found significant increase in XIAP and c-FLIP constitutive protein expression with no alteration of Bcl-2 and lower levels of CD95/Fas. The isolated fibroblasts were then treated with the CD95/Fas ligand (FasL) to induce apoptosis. While the morphological and biochemical alterations induced by FasL were similar in wild-type (wt) and Cav-null mouse lung fibroblasts, the time course and the extent of the alterations were greater in the Cav-null fibroblasts. Several salient features of Cav-null fibroblasts response such as loss of membrane potential, fragmentation of the mitochondrial continuum concurrent with caspase-8 activation, and subsequent Bid cleavage, prior to caspase-3 activation were detected. Furthermore, M30 antigen formation, phosphatidylserine expression and DNA fragmentation were caspase-3 dependent. SiRNA-mediated silencing of XIAP and c-FLIP, individually or combined, enhanced the sensitivity of lung fibroblasts to FasL-induced apoptosis. Pharmacological inhibition of Bcl-2 had no effect. Together our findings support a mechanism in which CD95/Fas engagement activates caspase-8, inducing mitochondrial apoptosis through Bid cleavage. XIAP and c-FLIP fine tune this process in a cell-type specific manner. PMID:28352235

  15. A high G418-resistant neo(R) transgenic mouse and mouse embryonic fibroblast (MEF) feeder layers for cytotoxicity and gene targeting in vivo and in vitro.

    PubMed

    Aubrecht, Jiri; Goad, Mary E P; Czopik, Agnieszka K; Lerner, Charles P; Johnson, Kevin A; Simpson, Elizabeth M; Schiestl, Robert H

    2011-10-01

    Aminoglycoside antibiotics have been in use since 1944 with the discovery of streptomycin. The aim of this study was to derive a new, highly resistant multicopy neo(R) transgenic mouse strain, named TgN3Ems, by random insertion of the plasmid, pPGKneobpA, and compare the level of drug resistance of wild-type and transgenic mice in vivo and corresponding primary mouse embryonic fibroblasts (MEFs) in vitro to a model neomycin analog, G418. The expression neoR in transgenic animals caused a 5-fold increase in the approximate lethal dose of G418, compared to wild type. No adverse pathological changes were found for the transgenic mice treated with G418, as they all died within minutes after injection. In contrast, the G418 treatment of wild-type mice resulted in a marked liver and kidney toxicity detected microscopically and via increases of serum biomarkers for liver and kidney damage. In addition, there was a mild bone marrow and lymphoid depletion. In in vitro studies, the transgenic MEFs survived 20-fold higher G418 levels, compared to the wild-type MEF cells. Therefore, TgN3Ems transgenic mice could be used as a source of G418-resistant feeder cells for gene targeting. Since the expression of drug-resistance genes in transgenic animals confers resistance to toxicity, the TgN3Ems mice might serve as a tool applicable in drug design.

  16. Expression of the small T antigen of Lymphotropic Papovavirus is sufficient to transform primary mouse embryo fibroblasts

    SciTech Connect

    Gupta, Tushar; Robles, Maria Teresa Sáenz; Schowalter, Rachel M.; Buck, Christopher B.; Pipas, James M.

    2016-01-15

    Polyomaviruses induce cell proliferation and transformation through different oncoproteins encoded within the early region (ER): large T antigen (LT), small T antigen (sT) and, in some cases, additional components. Each virus utilizes different mechanisms to achieve transformation. For instance, the LTs of Simian virus 40 (SV40), BK and/or JC virus can induce transformation; but Merkel Cell Polyomavirus (MCPyV) requires expression of sT. Lymphotropic Papovavirus (LPV) is closely related to Human Polyomavirus 9 (HuPyV9) and, under similar conditions, mice expressing LPV.ER exhibit higher rates of tumor formation than mice expressing SV40.ER. We have investigated the contributions of individual LPV.ER components to cell transformation. In contrast to SV40, LPV.ER transforms mouse embryonic fibroblasts (MEFs), but expression of LPV LT is insufficient to transform MEFs. Furthermore, LPV sT induces immortalization and transformation of MEFs. Thus, in the case of LPV, sT is the main mediator of oncogenesis. - Highlights: • Characterization of early region products from the Lymphotropic Polyomavirus (LPV). • On its own, sT immortalizes and transforms mouse primary cells, and is able to block p53 activation. • Combined LT and sT expression induces a greater rate of proliferation than either LT or sT alone.

  17. Case Report of S1Q3T3 Electrocardiographic Abnormality in a Pregnant Asthmatic Patient During Acute Bronchospasm

    PubMed Central

    Arshad, Hafiza; Khan, Rana Rahel; Khaja, Misbahuddin

    2017-01-01

    Patient: Female, 33 Final Diagnosis: S1Q3T3 electrocardiographic abnormality in a pregnant asthmatic during acute bronchospasm Symptoms: Cough • shortness of breath Medication: — Clinical Procedure: EKG Specialty: Pulmonology Objective: Rare co-existance of disease or pathology Background: Asthma is the most common chronic pulmonary disease during pregnancy. Several previous reports have documented reversible electrocardiographic changes during severe acute asthma attacks, including tachycardia, P pulmonale, right bundle branch block, right axis deviation, and ST segment and T wave abnormalities. Case Report: We present the case of a pregnant patient with asthma exacerbation in which acute bronchospasm caused S1Q3T3 abnormality on an electrocardiogram (ECG). The complete workup of ECG findings of S1Q3T3 was negative and correlated with bronchospasm. The S1Q3T3 electrocardiographic abnormality can be seen in acute bronchospasm in pregnant women. The other causes like pulmonary embolism, pneumothorax, acute lung disease, cor pulmonale, and left posterior fascicular block were excluded. Conclusions: Asthma exacerbations are of considerable concern during pregnancy due to their adverse effect on the fetus, and optimization of asthma treatment during pregnancy is vital for achieving good outcomes. Prompt recognition of electrocardiographic abnormality and early treatment can prevent adverse perinatal outcomes. PMID:28144025

  18. Kaempferol and quercetin isolated from Euonymus alatus improve glucose uptake of 3T3-L1 cells without adipogenesis activity.

    PubMed

    Fang, Xian-Kang; Gao, Jie; Zhu, Dan-Ni

    2008-03-12

    Euonymus alatus as a folk medicine in China has been clinically used to treat type 2 diabetes for many years, and also exerts beneficial effects on hyperglycemia of diabetic animals. Our previous studies have isolated kaempferol and quercetin from the extract of E. alatus. In the present study, we investigated the possible mechanism of antidiabetic activity of these compounds. Kaempferol and quercetin could significantly improve insulin-stimulated glucose uptake in mature 3T3-L1 adipocytes. In addition, further experiments showed that kaempferol and quercetin served as weak partial agonists in the peroxisome proliferator-agonist receptor gamma (PPARgamma) reporter gene assay. Kaempferol and quercetin could not induce differentiation of 3T3-L1 preadipocytes as traditional PPARgamma agonist. When added together with the PPARgamma agonist rosiglitazone to 3T3-L1 preadipocytes, they could inhibit 3T3-L1 differentiation in a dose-dependent manner. Competitive ligand-binding assay confirmed that kaempferol and quercetin could compete with rosiglitazone at the same binding pocket site as PPARgamma. Kaempferol and quercetin showed significant inhibitory effects on NO production in response to lipopolysaccharide treatment in macrophage cells in which the PPARgamma was overexpressed; rosiglitazone was less potent than kaempferol and quercetin. These observations suggest that kaempferol and quercetin potentially act at multiple targets to ameliorate hyperglycemia, including by acting as partial agonists of PPARgamma.

  19. Knockdown of LYRM1 rescues insulin resistance and mitochondrial dysfunction induced by FCCP in 3T3-L1 adipocytes.

    PubMed

    Zhang, Min; Qin, Zhen-Ying; Dai, Yong-mei; Wang, Yu-Mei; Zhu, Guan-zhong; Zhao, Ya-Ping; Ji, Chen-Bo; Zhu, Jin-Gai; Shi, Chun-Mei; Qiu, Jie; Cao, Xin-Guo; Guo, Xi-Rong

    2014-09-01

    LYR motif-containing 1 (LYRM1) was recently discovered to be involved in adipose tissue homeostasis and obesity-associated insulin resistance. We previously demonstrated that LYRM1 overexpression might contribute to insulin resistance and mitochondrial dysfunction. Additionally, knockdown of LYRM1 enhanced insulin sensitivity and mitochondrial function in 3T3-L1 adipocytes. We investigated whether knockdown of LYRM1 in 3T3-L1 adipocytes could rescue insulin resistance and mitochondrial dysfunction induced by the cyanide p-trifluoromethoxyphenyl-hydrazone (FCCP), a mitochondrion uncoupler, to further ascertain the mechanism by which LYRM1 is involved in obesity-associated insulin resistance. Incubation of 3T3-L1 adipocytes with 1 µM FCCP for 12 h decreased insulin-stimulated glucose uptake, reduced intracellular ATP synthesis, increased intracellular reactive oxygen species (ROS) production, impaired insulin-stimulated Glucose transporter type 4 (GLUT4) translocation, and diminished insulin-stimulated tyrosine phosphorylation of Insulin receptor substrate-1 (IRS-1) and serine phosphorylation of Protein Kinase B (Akt). Knockdown of LYRM1 restored insulin-stimulated glucose uptake, rescued intracellular ATP synthesis, reduced intracellular ROS production, restored insulin-stimulated GLUT4 translocation, and rescued insulin-stimulated tyrosine phosphorylation of IRS-1 and serine phosphorylation of Akt in FCCP-treated 3T3-L1 adipocytes. This study indicates that FCCP-induced mitochondrial dysfunction and insulin resistance are ameliorated by knockdown of LYRM1.

  20. Piromelatine decreases triglyceride accumulation in insulin resistant 3T3-L1 adipocytes: role of ATGL and HSL.

    PubMed

    Wang, Ping-Ping; She, Mei-Hua; He, Ping-Ping; Chen, Wu-Jun; Laudon, Moshe; Xu, Xuan-Xuan; Yin, Wei-Dong

    2013-08-01

    Piromelatine, a novel investigational multimodal sleep medicine, is developed for the treatment of patients with primary and co-morbid insomnia. Piromelatine has been shown to inhibit weight gain and improve insulin sensitivity in high-fat/high-sucrose-fed (HFHS) rats. Considering that piromelatine has also been implicated in lowering of triglyceride levels in HFHS rats, this work elucidated whether this effect involves in the regulation of adipose triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL) in triglyceride (TG) metabolism. In this study, we investigated the effects of piromelatine and MT2 receptors inhibition on TG content, insulin-stimulated glucose uptake, and the expressions of ATGL and HSL in 3T3-L1 adipocytes preincubated in high glucose and high insulin (HGI) conditions. Our results showed that culturing 3T3-L1 adipocytes under HGI conditions increased triglyceride accumulation with concomitant decrease of ATGL and HSL expression, inducing insulin resistance in 3T3-L1 adipocytes. We also found that triglyceride accumulation was significantly inhibited and the levels of ATGL/HSL increased after melatonin or piromelatine treatment. The effects of melatonin/piromelatine (10 nM) were counteracted by pretreatment with the relatively selective MT2 receptor antagonist luzindole (100 nM). In this study, our data demonstrate that piromelatine reverses high glucose and high insulin-induced triglyceride accumulation in 3T3-L1 adipocytes, possibly through up-regulating of ATGL and HSL expression via a melatonin-dependent manner.

  1. The role and possible mechanism of lncRNA U90926 in modulating 3T3-L1 preadipocyte differentiation

    PubMed Central

    Chen, J; Liu, Y; Lu, S; Yin, L; Zong, C; Cui, S; Qin, D; Yang, Y; Guan, Q; Li, X; Wang, X

    2017-01-01

    Background: Obesity is a risk factor for metabolic diseases, while preadipocyte differentiation or adipogenesis is closely related to obesity occurrence. Long noncoding RNAs (lncRNAs) are a unique class of transcripts in regulation of a variety of biological processes. Using cDNA microarray, we found lncRNA U90926 is negatively correlated with 3T3-L1 preadipocyte differentiation. Objective: The aim of this study was to explore the role of lncRNA U90926 (lnc-U90926) in adipogenesis and the underlying mechanisms. Methods: Quantitative real-time PCR (qPCR) was performed to determine lnc-U90926 expression in 3T3-L1 preadipocytes, differentiated adipocytes, and in adipose tissues form mice. RNA fluorescent in situ hybridization (FISH) was performed to determine the localization of lnc-U90926 in 3T3-L1 preadipocytes. The effects of lnc-U90926 on 3T3-L1 adipogenesis were analyzed with lentivirus-mediated gain- and loss-of-function experiments. Lipid accumulation was evaluated by oil red O staining; several adipogenesis makers were analyzed by qPCR and western blotting. Dual luciferase assay was applied to explore the transactivation of target genes modulated by lnc-U90926. All measurements were performed at least for three times. Results: Lnc-U90926 expression decreased along the differentiation of 3T3-L1 preadipocytes. In mice, lnc-U90926 is predominantly expressed in adipose tissue. Obese mice have lower lnc-U90926 expression in subcutaneous and visceral adipose tissue than non-obese mice. FISH results showed that lnc-U90926 was mainly located in the cytoplasm. Overexpression lnc-U90926 attenuated 3T3-L1 adipocyte differentiation as evidenced by its ability to inhibit lipid accumulation, to decrease the mRNA levels of peroxisome proliferator-activated receptor gamma 2 (PPARγ2), fatty acid binding protein 4 (FABP4) and adiponectin (AdipoQ) as well as to reduce the protein levels of PPARγ and FABP4 (P<0.05). Knockdown of lnc-U90926 showed opposite effects, which

  2. Ghrelin inhibits the apoptosis of MC3T3-E1 cells through ERK and AKT signaling pathway

    SciTech Connect

    Liang, Qiu-Hua; Liu, Yuan; Wu, Shan-Shan; Cui, Rong-Rong; Yuan, Ling-Qing Liao, Er-Yuan

    2013-11-01

    Ghrelin is a 28-amino-acid peptide that acts as a natural endogenous ligand of the growth hormone secretagogue receptor (GHSR) and strongly stimulates the release of growth hormone from the hypothalamus–pituitary axis. Previous studies have identified the important physiological effects of ghrelin on bone metabolism, such as regulating proliferation and differentiation of osteoblasts, independent of GH/IGF-1 axis. However, research on effects and mechanisms of ghrelin on osteoblast apoptosis is still rare. In this study, we identified expression of GHSR in MC3T3-E1 cells and determined the effects of ghrelin on the apoptosis of osteoblastic MC3T3-E1 cells and the mechanism involved. Our data demonstrated that ghrelin inhibited the apoptosis of osteoblastic MC3T3-E1 cells induced by serum deprivation, as determined by terminal deoxynucleotidyl transferase-mediated deoxyribonucleotide triphosphate nick end-labeling (TUNEL) and ELISA assays. Moreover, ghrelin upregulated Bcl-2 expression and downregulated Bax expression in a dose-dependent manner. Our study also showed decreased activated caspase-3 activity under the treatment of ghrelin. Further study suggested that ghrelin stimulated the phosphorylation of ERK and AKT. Pretreatment of cells with the ERK inhibitor PD98059, PI3K inhibitor LY294002, and GHSR-siRNA blocked the ghrelin-induced activation of ERK and AKT, respectively; however, ghrelin did not stimulate the phosphorylation of p38 or JNK. PD90859, LY294002 and GHSR-siRNA attenuated the anti-apoptosis effect of ghrelin in MC3T3-E1 cells. In conclusion, ghrelin inhibits the apoptosis of osteoblastic MC3T3-E1 cells induced by serum deprivation, which may be mediated by activating the GHSR/ERK and GHSR/PI3K/AKT signaling pathways. - Highlights: • We explored the effects of ghrelin on serum deprivation-induced MC3T3-E1 cells apoptosis. • Both ELISA and TUNEL were used to detect the apoptosis. • The receptor of ghrelin, GHSR, was expressed in MC3T3-E1

  3. Anti-adipogenic activity of an olive seed extract in mouse fibroblasts.

    PubMed

    Veciana-Galindo, Carmen; Cortés-Castell, Ernesto; Torró-Montell, Luis; Palazón-Bru, Antonio; Sirvent-Segura, Elia; Rizo-Baeza, María M; Gil-Guillén, Vicente F

    2015-06-01

    La administración de diferentes polifenoles protege contra el incremento de peso y la acumulación de grasa. Objetivo: comprobar la actividad anti-adipogénica de un extracto polifenólico de huesos de aceituna, utilizando la diferenciación a adipocitos de la línea celular 3T3-L1 de fibroblastos de ratón. Material y métodos: se cultivan y diferencian las células (6.000 células/pocillo) en presencia del extracto de huesos de aceitunas a 10 y 50 mg/l de polifenoles, concentraciones bioseguras, y sin extracto como control. A los 5-7 días se forman los adipocitos maduros. Se cuantifican los cúmulos de grasa formados mediante tinción con Oil- Red y medida de la absorbancia a 490 nm y la expresión de los genes de leptina y PPARg, relacionándolos con los valores en los cultivos antes y después de diferenciarse a adipocitos. Resultados: las muestras control, sin extracto, se consideran el 100% de acumulación de grasas. En contraste, la adición de 50 mg/l de extracto de polifenoles de huesos de aceituna muestra un cúmulo de grasa de alrededor del 50%, semejante a las células no diferenciadas. Con 10 mg/l de extracto no se muestra efecto. Se confirma la actividad antiadipogénica, observándose disminución en la expresión de los genes PPARg y de leptina en la diferenciación a adipocitos en presencia del extracto a 50 mg/l. En conclusión, la formación de los cuerpos grasos característicos de la adipogénesis queda inhibida previa adición de 50 mg/l de polifenoles de extracto de huesos de aceituna, así como la expresión de los genes adipogénicos PPARg y de leptina.

  4. Mechanisms of stress resistance in Snell dwarf mouse fibroblasts: enhanced antioxidant and DNA base excision repair capacity, but no differences in mitochondrial metabolism.

    PubMed

    Page, Melissa M; Salmon, Adam B; Leiser, Scott F; Robb, Ellen L; Brown, Melanie F; Miller, Richard A; Stuart, Jeffrey A

    2009-04-15

    Dermal fibroblasts from long-lived Snell dwarf mice can withstand a variety of oxidative and non-oxidative stressors compared to normal littermate controls. Here, we report differences in the levels and activities of intracellular antioxidant and DNA repair enzymes between normal and Snell dwarf mice fibroblasts cultured under a variety of conditions, including: 3% and 20% ambient O(2); the presence and absence of serum; and the addition of an exogenous oxidative stress. The only significant difference between normal and dwarf cells cultured in complete medium, at 20% O(2), was an approximately 40% elevation of glutathione peroxidase (GPx) activity in the mutant cells. Serum deprivation elicited increases in GPx in both genotypes, but these activities remained higher in dwarf mouse cells. Dwarf mouse cells deprived of serum and challenged with exposure to paraquat or hydrogen peroxide showed a generally greater upregulation of catalase and DNA base excision repair enzymes. As these toxins can interact with mitochondria to increase mitochondrial ROS production, we explored whether there were differences in mitochondrial metabolism between normal and dwarf mouse cells. However, neither mitochondrial content nor the apparent mitochondrial membrane potential differed between genotypes. Overall, the results suggest that superior hydrogen peroxide metabolism and a marginally greater DNA base excision repair capacity contribute to the stress resistance phenotype of Snell dwarf mouse fibroblasts.

  5. Identification of the early and late responder genes during the generation of induced pluripotent stem cells from mouse fibroblasts

    PubMed Central

    Ham, Seokjin; Hong, Chang-Pyo; Seo, Seonghye; Choe, Moon Kyung; Shin, So-I; Lee, Choon-Soo; Kim, Hyo-Soo

    2017-01-01

    Background The generation of induced pluripotent stem cell (iPSC), a substitute for embryonic stem cell (ESC), requires the proper orchestration of a transcription program at the chromatin level. Our recent approach for the induction of pluripotent stem cells from fibroblasts using protein extracts from mouse ESCs could overcome the potential tumorigenicity risks associated with random retroviral integration. Here, we examine the epigenetic modifications and the transcriptome of two types of iPSC and of partially reprogrammed iPSCs (iPSCp) generated independently from adult cardiac and skin fibroblasts to assess any perturbations of the transcription program during reprogramming. Results The comparative dissection of the transcription profiles and histone modification patterns at lysines 4 and 27 of histone H3 of the iPSC, iPSCp, ESC, and somatic cells revealed that the iPSC was almost completely comparable to the ESC, regardless of their origins, whereas the genes of the iPSCp were dysregulated to a larger extent. Regardless of the origins of the somatic cells, the fibroblasts induced using the ESC protein extracts appear to be completely reprogrammed into pluripotent cells, although they show unshared marginal differences in their gene expression programs, which may not affect the maintenance of stemness. A comparative investigation of the iPSCp generated by unwanted reprogramming showed that the two groups of genes on the pathway from somatic cells to iPSC might function as sequential reprogramming-competent early and late responders to the induction stimulus. Moreover, some of the divergent genes expressed only in the iPSCp were associated with many tumor-related pathways. Conclusions Faithful transcriptional reprogramming should follow epigenetic alterations to generate induced pluripotent stem cells from somatic cells. This genome-wide comparison enabled us to define the early and late responder genes during the cell reprogramming process to iPSC. Our results

  6. Endothelial dysfunction exacerbates renal interstitial fibrosis through enhancing fibroblast Smad3 linker phosphorylation in the mouse obstructed kidney.

    PubMed

    Sun, Yu Bo Yang; Qu, Xinli; Li, Xueling; Nikolic-Paterson, David J; Li, Jinhua

    2013-01-01

    Endothelial dysfunction and enhanced transforming growth factor-β (TGF-β)/Smad3 signalling are common features of progressive renal fibrosis. This study investigated a potential link between these mechanisms. In unilateral ureteric obstruction (UUO) we observed an acute (6 hr) down-regulation of nitric oxide synthase 3 (NOS3/eNOS) levels and increased phosphorylation of the linker region of Smad3 at T179 and S208 in Smad3/JNK complexes. These events preceded Smad3 C-terminal domain phosphorylation and the induction of myofibroblast proliferation at 48 hrs. Mice deficient in NOS3 showed enhanced myofibroblast proliferation and collagen accumulation compared to wild type mice in a 7 day UUO model. This was associated with enhanced phosphorylation of Smad3 T179 and S208 by 92% and 88%, respectively, whereas Smad3-C-terminal phosphorylation was not affected. Resolvin D1 (RvD1) can suppress renal fibrosis in the UUO model, and further analysis herein showed that RvD1 protected against endothelial dysfunction and suppressed Smad3/JNK complex formation with a consequent reduction in phosphorylation of Smad3 T179 and S208 by 78% and 65%, respectively, while Smad3 C-terminal phosphorylation was unaltered. In vitro, conditioned media from mouse microvascular endothelial cells (MMEC) treated with a general inhibitor of nitric oxide synthase (L-NAME) augmented the proliferation and collagen production of renal fibroblasts (NRK49F cells) compared to control MMEC media and this was associated with increased phosphorylation of JNK and Smad3 T179 and S208, whereas Smad3-C-terminal domain phosphorylation was unaffected. The addition of RvD1 to L-NAME treated MMEC abrogated these effects of the conditioned media on renal fibroblasts. Finally, Smad3 T179/V and S208/A mutations significantly inhibit TGF-β1 induced up-regulation collagen I promoter. In conclusion, these data suggest that endothelial dysfunction can exacerbate renal interstitial fibrosis through increased fibroblast

  7. Mitochondrial bioenergetics and drug-induced toxicity in a panel of mouse embryonic fibroblasts with mitochondrial DNA single nucleotide polymorphisms

    SciTech Connect

    Pereira, Claudia V.; Oliveira, Paulo J.; Will, Yvonne; Nadanaciva, Sashi

    2012-10-15

    Mitochondrial DNA (mtDNA) variations including single nucleotide polymorphisms (SNPs) have been proposed to be involved in idiosyncratic drug reactions. However, current in vitro and in vivo models lack the genetic diversity seen in the human population. Our hypothesis is that different cell strains with distinct mtDNA SNPs may have different mitochondrial bioenergetic profiles and may therefore vary in their response to drug-induced toxicity. Therefore, we used an in vitro system composed of four strains of mouse embryonic fibroblasts (MEFs) with mtDNA polymorphisms. We sequenced mtDNA from embryonic fibroblasts isolated from four mouse strains, C57BL/6J, MOLF/EiJ, CZECHII/EiJ and PERA/EiJ, with the latter two being sequenced for the first time. The bioenergetic profile of the four strains of MEFs was investigated at both passages 3 and 10. Our results showed that there were clear differences among the four strains of MEFs at both passages, with CZECHII/EiJ having a lower mitochondrial robustness when compared to C57BL/6J, followed by MOLF/EiJ and PERA/EiJ. Seven drugs known to impair mitochondrial function were tested for their effect on the ATP content of the four strains of MEFs in both glucose- and galactose-containing media. Our results showed that there were strain-dependent differences in the response to some of the drugs. We propose that this model is a useful starting point to study compounds that may cause mitochondrial off-target toxicity in early stages of drug development, thus decreasing the number of experimental animals used. -- Highlights: ► mtDNA SNPs may be linked to individual predisposition to drug-induced toxicity. ► CZECHII/EiJ and PERA/EiJ mtDNA was sequenced for the first time in this study. ► Strain-dependent mitochondrial capacity differences were measured. ► Strain-dependent differences in response to mitochondrial toxicants were observed.

  8. Tyrosine kinase inhibitor BIBF1120 ameliorates inflammation, angiogenesis and fibrosis in CCl4-induced liver fibrogenesis mouse model

    PubMed Central

    Öztürk Akcora, Büsra; Storm, Gert; Prakash, Jai; Bansal, Ruchi

    2017-01-01

    Hepatic fibrosis, a progressive chronic disease mainly caused by hepatitis viral infections, alcohol abuse or metabolic syndrome leading to liver dysfunction and is the growing cause of mortality worldwide. Tyrosine kinase inhibitor BIBF1120 (Nintedanib) has been evaluated in clinical trials for idiopathic pulmonary fibrosis and advanced Hepatocellular carcinoma, but has not been explored for liver fibrosis yet. In this study, we aimed to investigate the therapeutic effects and mechanism of BIBF1120 in liver fibrogenesis. The effects of BIBF1120 were evaluated in TGFβ-activated mouse 3T3 fibroblasts, LX2 cells, primary human hepatic stellate cells (HSCs) and CCl4-induced liver fibrogenesis mouse model. Fibroblasts-conditioned medium studies were performed to assess the paracrine effects on macrophages and endothelial cells. In-vitro in TGFβ-activated fibroblasts, BIBF1120 significantly inhibited expression of major fibrotic parameters, wound-healing and contractility. In vivo in CCl4-induced acute liver injury model, post-disease BIBF1120 administration significantly attenuated collagen accumulation and HSC activation. Interestingly, BIBF1120 drastically inhibited intrahepatic inflammation and angiogenesis. To further elucidate the mechanism of action, 3T3-conditioned medium studies demonstrated increased 3T3-mediated macrophage chemotaxis and endothelial cells tube formation and activation, which was significantly decreased by BIBF1120. These results suggests that BIBF1120 can be a potential therapeutic approach for the treatment of liver fibrosis. PMID:28291245

  9. Serotonin derivative, N-(p-Coumaroyl)serotonin, isolated from safflower (Carthamus tinctorius L.) oil cake augments the proliferation of normal human and mouse fibroblasts in synergy with basic fibroblast growth factor (bFGF) or epidermal growth factor (EGF).

    PubMed

    Takii, T; Hayashi, M; Hiroma, H; Chiba, T; Kawashima, S; Zhang, H L; Nagatsu, A; Sakakibara, J; Onozaki, K

    1999-05-01

    N-(p-Coumaroyl)serotonin (CS) with antioxidative activity is present in safflower oil. We have reported that CS inhibits proinflammatory cytokine generation from human monocytes in vitro. As reactive oxygen species (ROS) affect cell proliferation, in this study the effect of CS on the proliferation of various cell types was examined. CS augments the proliferation of normal human and mouse fibroblast cells. The cells continue to proliferate in the presence of CS and form a transformed cell-like focus without transformation. CS, however, does not augment the proliferation of other cell types, either normal or tumor cells. CS augments the proliferation of fibroblasts in synergy with basic fibroblast growth factor (bFGF) or epidermal growth factor (EGF), but not with acidic FGF(aFGF) or platelet-derived growth factor (PDGF). This study using synthesized derivatives of CS reveals that the growth-promoting activity is not due to antioxidative activity. These findings indicate that CS is a natural compound with unique growth-promoting activity for fibroblasts.

  10. Mouse embryonic fibroblasts accumulate differentially on titanium surfaces treated with nanosecond laser pulses.

    PubMed

    Radmanesh, Mitra; Ektesabi, Amin M; Wyatt, Rachael A; Crawford, Bryan D; Kiani, Amirkianoosh

    2016-10-01

    Biomaterial engineering, specifically in bone implant and osseointegration, is currently facing a critical challenge regarding the response of cells to foreign objects and general biocompatibility of the materials used in the production of these implants. Using the developing technology of the laser surface treatment, this study investigates the effects of the laser repetition rate (frequency) on cell distribution across the surface of the titanium substrates. The main objective of this research is building a fundamental understanding of how cells interact with treated titanium and how different treatments affect cell accumulation. Cells respond differently to surfaces treated with different frequency lasers. The results of this research identify the influence of frequency on surface topography properties and oxidation of titanium, and their subsequent effects on the pattern of cell accumulation on its surface. Despite increased oxidation in laser-treated regions, the authors observe that fibroblast cells prefer untreated titanium to laser-treated regions, except the regions treated with 25 kHz pulses, which become preferentially colonized after 72 h.

  11. Role of MSK1 in the Malignant Phenotype of Ras-transformed Mouse Fibroblasts*

    PubMed Central

    Pérez-Cadahía, Beatriz; Drobic, Bojan; Espino, Paula S.; He, Shihua; Mandal, Soma; Healy, Shannon; Davie, James R.

    2011-01-01

    Activated by the RAS-MAPK signaling pathway, MSK1 is recruited to immediate-early gene (IEG) regulatory regions, where it phosphorylates histone H3 at Ser-10 or Ser-28. Chromatin remodelers and modifiers are then recruited by 14-3-3 proteins, readers of phosphoserine marks, leading to the occupancy of IEG promoters by the initiation-engaged form of RNA polymerase II and the onset of transcription. In this study, we show that this mechanism of IEG induction, initially elucidated in parental 10T1/2 murine fibroblast cells, applies to metastatic Hras1-transformed Ciras-3 cells. As the RAS-MAPK pathway is constitutively activated in Ciras-3 cells, MSK1 activity and phosphorylated H3 steady-state levels are elevated. We found that steady-state levels of the IEG products AP-1 and COX-2 were also elevated in Ciras-3 cells. When MSK1 activity was inhibited or MSK1 expression was knocked down in Ciras-3 cells, the induction of IEG expression and the steady-state levels of COX-2, FRA-1, and JUN were greatly reduced. Furthermore, MSK1 knockdown Ciras-3 cells lost their malignant phenotype, as reflected by the absence of anchorage-independent growth. PMID:21071437

  12. Trace formation during locomotion of L929 mouse fibroblasts continuously recorded by interference reflection microscopy (IRM).

    PubMed

    Richter, E; Hitzler, H; Zimmermann, H; Hagedorn, R; Fuhr, G

    2000-09-01

    The recently reported formation of highly ordered traces by migrating cells has been studied on L929 fibroblasts in time lapse experiments by means of interference reflection microscopy (IRM) as well as by conventional microscopy. Formation of pronounced traces on glass substrates correlates to migration after cell division, and the trace arrangement on the substrate depends on migration velocity: slow migration results in a highly branched, broad, and relatively short trace, while fast migration yields a slim and long trace with few branches. IRM-irradiation caused cessation of locomotion and trace formation and accelerated degradation of existing traces. Traces consist of cord-like cytoplasmic strands, which contain F-actin filaments and they seem to be enveloped by a membrane. It is supposed that cell traces are homologous to filopodia. Traces arise mainly from non-retracted filopodia at the rear margin of the migrating cell. The branches within the traces are the result of the repeated stretching out of a backwardly directed lamellipodium. They arise from the formation of new filopodia that emerge at the actin ribs of the lamellipodium.

  13. FADD null mouse embryonic fibroblasts undergo apoptosis after photosensitization with the silicon phthalocyanine Pc 4.

    PubMed

    Nagy, B; Yeh, W C; Mak, T W; Chiu, S M; Separovic, D

    2001-01-01

    Oxidative stress, such as photodynamic therapy with the silicon phthalocyanine Pc 4 (Pc 4-PDT), can induce apoptosis and tumor necrosis factor alpha (TNF) production. TNF receptors, as well as other death receptors, have been implicated in stress-induced apoptosis. To assess directly the role of FADD, a death receptor-associated protein, in induction of apoptosis post-Pc 4-PDT, embryonic fibroblasts from FADD knock out (k/o) and wild-type (wt) mice were used. Pc 4-PDT induced casp-3 activation and apoptosis in both cell types. In the presence of zVAD, a pancaspase inhibitor, Pc 4-PDT-induced apoptosis was abrogated in both cell lines. Fumonisin B1 (FB), an inhibitor of ceramide synthase, had no effect on apoptosis after Pc 4-PDT in either cell line. Similar to Pc 4-PDT, exogenous C6-ceramide bypassed FADD deficiency and induced zVAD-sensitive apoptosis. In contrast to Pc 4 photosensitization, TNF did not induce either apoptosis or ceramide accumulation in FADD k/o cells. In the absence of FADD deficiency, TNF-induced apoptosis was zVAD-sensitive and FB-insensitive. Induced ceramide levels remained elevated after cotreatment with TNF and zVAD in FADD wt cells. Taken together, these data provide genetic evidence for a lack of FADD requirement in Pc 4-PDT- or C6-ceramide-induced apoptosis. FB-sensitive ceramide production accompanies, but does not suffice, for apoptosis after Pc 4 photosensitization or TNF.

  14. Mouse embryonic fibroblasts (MEF) exhibit a similar but not identical phenotype to bone marrow stromal stem cells (BMSC).

    PubMed

    Saeed, Hamid; Taipaleenmäki, Hanna; Aldahmash, Abdullah M; Abdallah, Basem M; Kassem, Moustapha

    2012-06-01

    Mouse embryonic fibroblasts have been utilized as a surrogate stem cell model for the postnatal bone marrow-derived stromal stem cells (BMSC) to study mesoderm-type cell differentiation e.g. osteoblasts, adipocytes and chondrocytes. However, no formal characterization of MEF phenotype has been reported. Utilizing standard in vitro and in vivo assays we performed a side-by-side comparison of MEF and BMSC to determine their ability to differentiate into mesoderm-type cells. BMSC were isolated from 8-10 weeks old mouse bone marrow by plastic adherence. MEF were established by trypsin/EDTA digestion from E13.5 embryos after removing heads and viscera, followed by plastic adherence. Compared to BMSC, MEF exhibited telomerase activity and improved cell proliferation as assessed by q-PCR based TRAP assay and cell number quantification, respectively. FACS analysis revealed that MEF exhibited surface markers characteristic of the BMSC: Sca-1(+), CD73(+), CD105(+), CD29(+), CD44(+), CD106(+), CD11b(-), and CD45(-). In contrast to BMSC, ex vivo osteoblast (OB) differentiation of MEF exhibited a less mature osteoblastic phenotype (less alkaline phosphatase, collagen type I and osteocalcin) as assessed by real-time PCR analysis. Compared to BMSC, MEF exhibited a more enhanced differentiation into adipocyte and chondrocyte lineages. Interestingly, both MEF and BMSC formed the same amount of heterotopic bone and bone marrow elements upon in vivo subcutaneous implantation with hydroxyapatite/tricalcium phosphate, in immune deficient mice. In conclusion, MEF contain a population of stem cells that behave in ex vivo and in vivo assays, similar but not identical, to BMSC. Due to their enhanced cell growth, they may represent a good alternative for BMSC in studying molecular mechanisms of stem cell commitment and differentiation to osteoblasts, adipocytes and chondrocytes.

  15. Magnetic Levitation of MC3T3 Osteoblast Cells as a Ground-Based Simulation of Microgravity.

    PubMed

    Hammer, Bruce E; Kidder, Louis S; Williams, Philip C; Xu, Wayne Wenzhong

    2009-11-01

    Diamagnetic samples placed in a strong magnetic field and a magnetic field gradient experience a magnetic force. Stable magnetic levitation occurs when the magnetic force exactly counter balances the gravitational force. Under this condition, a diamagnetic sample is in a simulated microgravity environment. The purpose of this study is to explore if MC3T3-E1 osteoblastic cells can be grown in magnetically simulated hypo-g and hyper-g environments and determine if gene expression is differentially expressed under these conditions. The murine calvarial osteoblastic cell line, MC3T3-E1, grown on Cytodex-3 beads, were subjected to a net gravitational force of 0, 1 and 2 g in a 17 T superconducting magnet for 2 days. Microarray analysis of these cells indicated that gravitational stress leads to up and down regulation of hundreds of genes. The methodology of sustaining long-term magnetic levitation of biological systems are discussed.

  16. FGF-2 signaling induces downregulation of TAZ protein in osteoblastic MC3T3-E1 cells

    SciTech Connect

    Eda, Homare; Aoki, Katsuhiko; Marumo, Keishi; Fujii, Katsuyuki; Ohkawa, Kiyoshi

    2008-02-08

    Transcriptional coactivator with PDZ-binding motif (TAZ) protein is a coactivator of Runx2 and corepressor of PPAR{gamma}. It also induces differentiation of mesenchymal cells into osteoblasts. In this study, we found that FGF-2, which inhibits bone mineralization and stimulates cell proliferation, reduced the TAZ protein expression level in osteoblast-like cells, MC3T3-E1. This reduction was recovered by removing FGF-2 from the culture medium, which also restored the osteoblastic features of MC3T3-E1 cells. Furthermore, FGF-2-induced reduction of TAZ is blocked by a SAPK/JNK-specific inhibitor. These findings suggest that the expression of TAZ protein is involved in osteoblast proliferation and differentiation. This may help elucidate the discrepancies in the effect of FGF-2 and contribute to the understanding of FGF/FGFR-associated craniosynostosis syndrome etiology and treatment.

  17. Dimethylfumarate suppresses adipogenic differentiation in 3T3-L1 preadipocytes through inhibition of STAT3 activity.

    PubMed

    Kang, Hyeon-Ji; Seo, Hyun-Ae; Go, Younghoon; Oh, Chang Joo; Jeoung, Nam Ho; Park, Keun-Gyu; Lee, In-Kyu

    2013-01-01

    The excessive accumulation of adipocytes contributes to the development of obesity and obesity-related diseases. The interactions of several transcription factors, such as C/EBPβ, PPARγ, C/EBPα, Nrf2, and STAT3, are required for adipogenic differentiation. Dimethylfumarate (DMF), an immune modulator and antioxidant, may function as an inhibitor of STAT3 and an activator of Nrf2. This study examined whether DMF inhibits adipogenic differentiation of 3T3-L1 preadipocytes by inhibiting STAT3 or activating Nrf2. DMF suppressed 3T3-L1 preadipocyte differentiation to mature adipocytes in a dose-dependent manner as determined by Oil Red O staining. The mRNA and protein levels of adipogenic genes, including C/EBPβ, C/EBPα, PPARγ, SREBP-1c, FAS, and aP2, were significantly lower in DMF-treated 3T3-L1 preadipocytes. Suppression of adipogenic differentiation by DMF treatment resulted primarily from inhibition of the early stages of differentiation. DMF inhibits clonal expansion during adipogenic differentiation through induction of a G1 cell cycle arrest. Additionally, DMF regulates cell cycle-related proteins, such as p21, pRb, and cyclin D. DMF treatment markedly inhibited differentiation medium-induced STAT3 phosphorylation and inhibited STAT3 transcriptional activation of a reporter construct composed of four synthetic STAT3-response elements. Moreover, inhibition of endogenous Nrf2 activity using a dominant negative Nrf2 did not abolish the DMF-induced inhibition of adipogenic differentiation of 3T3-L1 preadipocytes. In summary, DMF is a negative regulator of adipogenic differentiation based on its regulation of adipogenic transcription factors and cell cycle proteins. This negative regulation by DMF is mediated by STAT3 inhibition, but is unlikely to involve Nrf2 activation.

  18. Inhibition of adipogenesis and leptin production in 3T3-L1 adipocytes by a derivative of meridianin C

    SciTech Connect

    Park, Yu-Kyoung; Lee, Tae-Yoon; Choi, Jong-Soon; Hong, Victor Sukbong; Lee, Jinho; Park, Jong-Wook; Jang, Byeong-Churl

    2014-10-03

    Highlights: • Compound 7b, a meridianin C derivative, inhibits adipogenesis. • Compound 7b inhibits C/EBP-α, PPAR-γ, FAS, STAT-3, and STAT-5 in 3T3-L1 adipocytes. • Compound 7b inhibits leptin, but not adiponectin, expression in 3T3-L1 adipocytes. • Compound 7b thus may have therapeutic potential against obesity. - Abstract: Meridianin C, a marine alkaloid, is a potent protein kinase inhibitor and has anti-cancer activity. We have recently developed a series of meridianin C derivatives (compound 7a–7j) and reported their proviral integration Moloney Murine Leukemia Virus (pim) kinases’ inhibitory and anti-proliferative effects on human leukemia cells. Here we investigated the effect of these meridianin C derivatives on adipogenesis. Strikingly, among the derivatives tested, compound 7b most strongly inhibited lipid accumulation during the differentiation of 3T3-L1 preadipocytes into adipocytes. However, meridianin C treatment was largely cytotoxic to 3T3-L1 adipocytes. On mechanistic levels, compound 7b reduced not only the expressions of CCAAT/enhancer-binding protein-α (C/EBP-α), peroxisome proliferator-activated receptor-γ (PPAR-γ), and fatty acid synthase (FAS) but also the phosphorylation levels of signal transducer and activator of transcription-3 (STAT-3) and STAT-5 during adipocyte differentiation. Moreover, compound 7b repressed leptin, but not adiponectin, expression during adipocyte differentiation. Collectively, these findings demonstrate that a meridianin C derivative inhibits adipogenesis by down-regulating expressions and/or phosphorylations of C/EBP-α, PPAR-γ, FAS, STAT-3 and STAT-5.

  19. Effects of C-reactive protein on adipokines genes expression in 3T3-L1 adipocytes

    SciTech Connect

    Yuan, Guoyue; Jia, Jue; Di, Liangliang; Zhou, Libin; Dong, Sijing; Ye, Jingjing; Wang, Dong; Yang, Ling; Wang, Jifang; Li, Lianxi; Yang, Ying; Mao, Chaoming; Chen, Mingdao

    2012-08-03

    Highlights: Black-Right-Pointing-Pointer CRP increases TNF-{alpha} and IL-6 genes expression in matured 3T3-L1 adipocytes. Black-Right-Pointing-Pointer CRP suppresses adiponectin, leptin and PPAR-{gamma} mRNA levels in matured 3T3-L1 cells. Black-Right-Pointing-Pointer Wortmannin reverses effects of CRP on adiponectin, TNF-{alpha} and leptin mRNA levels. Black-Right-Pointing-Pointer CRP may regulate IR, obesity and metabolic syndrome by this mechanism. -- Abstract: Adipose tissue is now recognized to be an important endocrine organ, secreting a variety of adipokines that are involved in the regulation of energy metabolism, insulin resistance and metabolic syndrome. C-reactive protein (CRP) is considered as one of the most sensitive markers of inflammation. A number of studies have shown that elevation of CRP concentrations is an independent predictive parameter of type 2 diabetes mellitus, which is also strongly associated with various components of the metabolic syndrome. The aim of the present study is to investigate the effects of CRP on adipokines genes expression in 3T3-L1 adipocytes. Quantitative real-time PCR analysis revealed that CRP inhibited adiponectin, leptin and peroxisome proliferator-activated receptor-gamma (PPAR-{gamma}) genes expression and raised tumor necrosis factor-{alpha} (TNF-{alpha}) and interleukin-6 (IL-6) mRNA levels in matured 3T3-L1 adipocytes in a dose and time-dependent manner. Pharmacological inhibition of phosphatidylinositol (PI)-3 kinase by wortmannin partially reversed the effects of CRP on adiponectin, TNF-{alpha} and leptin genes expression. These results collectively suggest that CRP regulates adiponectin, TNF-{alpha}, leptin, IL-6 and PPAR-{gamma} genes expression, and that might represent a mechanism by which CRP regulates insulin resistance, obesity and metabolic syndrome.

  20. Inhibition of NIH 3T3 cell proliferation by a mutant ras protein with preferential affinity for GDP.

    PubMed Central

    Feig, L A; Cooper, G M

    1988-01-01

    Substitution of asparagine for serine at position 17 decreased the affinity of rasH p21 for GTP 20- to 40-fold without significantly affecting its affinity for GDP. Transfection of NIH 3T3 cells with a mammalian expression vector containing the Asn-17 rasH gene and a Neor gene under the control of the same promoter yielded only a small fraction of the expected number of G418-resistant colonies, indicating that expression of Asn-17 p21 inhibited cell proliferation. The inhibitory effect of Asn-17 p21 required its localization to the plasma membrane and was reversed by coexpression of an activated ras gene, indicating that the mutant p21 blocked the endogenous ras function required for NIH 3T3 cell proliferation. NIH 3T3 cells transformed by v-mos and v-raf, but not v-src, were resistant to inhibition by Asn-17 p21, indicating that the requirement for normal ras function can be bypassed by these cytoplasmic oncogenes. The Asn-17 mutant represents a novel reagent for the study of ras function by virtue of its ability to inhibit cellular ras activity in vivo. Since this phenotype is likely associated with the preferential affinity of the mutant protein for GDP, analogous mutations might also yield inhibitors of other proteins whose activities are regulated by guanine nucleotide binding. Images PMID:3145408

  1. Inhibitory effect of apocynin on methylglyoxal-mediated glycation in osteoblastic MC3T3-E1 cells.

    PubMed

    Suh, Kwang Sik; Rhee, Sang Youl; Kim, Young Seol; Choi, Eun Mi

    2015-04-01

    Methylglyoxal (MG), a highly reactive metabolite of hyperglycemia, can enhance protein glycation, oxidative stress or inflammation. The present study investigated the effects of apocynin on the mechanisms associated with MG toxicity in osteoblastic MC3T3-E1 cells. Pretreatment of MC3T3-E1 cells with apocynin prevented the MG-induced protein glycation and formation of intracellular reactive oxygen species and mitochondrial superoxide in MC3T3-E1 cells. In addition, apocynin increased glutathione levels and restored the activity of glyoxalase I inhibited by MG. These findings suggest that apocynin provide a protective action against MG-induced cell damage by reducing oxidative stress and by increasing the MG detoxification system. Apocynin treatment decreased the levels of proinflammatory cytokines such as tumor necrosis factor-α and interleukin-6 induced by MG. Additionally, the nitric oxide level reduced by MG was significantly increased by apocynin. These findings indicate that apocynin might exert its therapeutic effects via upregulation of glyoxalase system and antioxidant activity. Taken together, apocynin may prove to be an effective treatment for diabetic osteopathy.

  2. Rapamycin/sodium hyaluronate binding on nano-hydroxyapatite coated titanium surface improves MC3T3-E1 osteogenesis

    PubMed Central

    Liu, Chao; Dong, Jian Yong; Yue, Lin Lin; Liu, Shao Hua; Wan, Yi; Liu, Hong; Tan, Wan Ye; Guo, Qian Qian; Zhang, Dong

    2017-01-01

    Endosseous titanium (Ti) implant failure due to poor biocompatibility of implant surface remains a major problem for osseointegration. Improving the topography of Ti surface may enhance osseointegration, however, the mechanism remains unknown. To investigate the effect of modified Ti surface on osteogenesis, we loaded rapamycin (RA) onto nano-hydroxyapatite (HAp) coated Ti surface which was acid-etched, alkali-heated and HAp coated sequentially. Sodium hyaluronate (SH) was employed as an intermediate layer for the load of RA, and a steady release rate of RA was maintained. Cell vitality of MC3T3-E1 was assessed by MTT. Osteogenesis of MC3T3-E1 on this modified Ti surface was evaluated by alkaline phosphatase (ALP) activity, mineralization and related osteogenesis genes osteocalcin (OCN), osteopontin (OPN), Collagen-I and Runx2. The result revealed that RA/SH-loaded nano-HAp Ti surface was innocent for cell vitality and even more beneficial for cell osteogenesis in vitro. Furthermore, osteogenesis of MC3T3-E1 showed significant association with the mammalian target of rapamycin (mTOR) phosphorylation by RA, which required further study about the mechanism. The approach to this modified Ti surface presented in this paper has high research value for the development of Ti-based implant. PMID:28182765

  3. Enhanced osteogenic differentiation of MC3T3-E1 on rhBMP-2-immobilized titanium via click reaction.

    PubMed

    Kim, Eun-Cheol; Kim, Tae-Hee; Jung, Jae-Hoon; Hong, Sung Ok; Lee, Deok-Won

    2014-03-15

    In the present study, we report about the efficacy of titanium surface-immobilized with bone morphogenetic protein-2 (BMP-2) via click reaction on enhanced osteogenic differentiation of MC3T3-E1 cells. The surface was characterized by static contact angles and XPS measurements, which indicated that pristine titanium (Ti-1) was successfully surface-modified via click chemistry (aminated titanium, Ti-4). By quantitative analysis of heparin immobilized on aminated titanium (Ti-4), we found that the Ti-4 can be used as a good candidate to immobilize biomolecules such as heparin. BMP-2 from titanium immobilized with BMP-2 (Ti-6) was released for a period of 28 days in a sustained manner. The highest proliferation rate of MC3T3-E1 cells was observed on Ti-6. Through in vitro tests including alkaline phosphatase (ALP) activity, calcium deposition and real-time polymerase chain reaction (real-time PCR), we found that Ti-6 can be used as a good implant to enhance the osteogenic differentiation of MC3T3-E1 cells.

  4. Nickel-Refining Fumes Induced DNA Damage and Apoptosis of NIH/3T3 Cells via Oxidative Stress

    PubMed Central

    Wang, Yue; Wang, Sheng-Yuan; Jia, Li; Zhang, Lin; Ba, Jing-Chong; Han, Dan; Yu, Cui-Ping; Wu, Yong-Hui

    2016-01-01

    Although there have been numerous studies examining the toxicity and carcinogenicity of nickel compounds in humans and animals, its molecular mechanisms of action are not fully elucidated. In our research, NIH/3T3 cells were exposed to nickel-refining fumes at the concentrations of 0, 6.25, 12.50, 25, 50 and 100 μg/mL for 24 h. Cell viability, cell apoptosis, reactive oxygen species (ROS) level, lactate dehydrogenase (LDH) assay, the level of glutathione (GSH), activities of superoxide dismutase (SOD), catalase (CAT), and malondialdehyde (MDA) level were detected. The exposure of NIH/3T3 cells to nickel-refining fumes significantly reduced cell viability and induced cell apoptotic death in a dose-dependent manner. Nickel-refining fumes significantly increased ROS levels and induced DNA damage. Nickel-refining fumes may induce the changes in the state of ROS, which may eventually initiate oxidative stress, DNA damage and apoptosis of NIH/3T3 cells. PMID:27347984

  5. Monascus ruber-Fermented Buckwheat (Red Yeast Buckwheat) Suppresses Adipogenesis in 3T3-L1 Cells.

    PubMed

    Hong, Heeok; Park, Jiyoung; Lumbera, Wenchie L; Hwang, Seong Gu

    2017-03-23

    Although various treatments have been used for weight loss to date, obese people rarely have safe and effective treatment options. Therefore, the antiobesity effects of several natural compounds are being actively investigated. This study was conducted to investigate the antiadipogenic effects of Monascus ruber-fermented Fagopyrum esculentum (red yeast buckwheat, RYB) in 3T3-L1 cells. We assessed the intracellular lipid content and adipocyte differentiation by oil red O staining and the expression of genes and proteins associated with adipocyte differentiation by reverse transcription-polymerase chain reaction and western blotting in 3T3-L1 cells. RYB dose dependently inhibited 3T3-L1 cell differentiation at concentrations of 50-800 μg/mL, without cytotoxic effects. It also suppressed the expression of adipogenic transcription factors, including peroxisome proliferator-activated receptor γ and CCAAT/enhancer-binding protein α, and adipocyte-specific genes, such as adipocyte fatty acid-binding protein (aP2), fatty acid synthase, and leptin, during preadipocyte differentiation into adipocytes. Furthermore, RYB reduced cyclin-dependent kinase 2 and cyclin expression and increased p21 and p27 expression, thus causing cell cycle arrest at the G1/S phase. Collectively, these results suggest that RYB may be an effective nutraceutical for weight loss as indicated by its ability to suppress adipogenesis-specific gene expression and cause cell cycle arrest at the G1/S interphase.

  6. Collagen-derived dipeptide prolyl-hydroxyproline promotes differentiation of MC3T3-E1 osteoblastic cells

    SciTech Connect

    Kimira, Yoshifumi; Ogura, Kana; Taniuchi, Yuri; Kataoka, Aya; Inoue, Naoki; Sugihara, Fumihito; Nakatani, Sachie; Shimizu, Jun; Wada, Masahiro; Mano, Hiroshi

    2014-10-24

    Highlights: • Pro-Hyp did not affect MC3T3-E1 cell proliferation and matrix mineralization. • Pro-Hyp significantly increased alkaline phosphatase activity. • Pro-Hyp significantly upregulated gene expression of Runx2, Osterix, and Col1α1. - Abstract: Prolyl-hydroxyproline (Pro-Hyp) is one of the major constituents of collagen-derived dipeptides. The objective of this study was to investigate the effects of Pro-Hyp on the proliferation and differentiation of MC3T3-E1 osteoblastic cells. Addition of Pro-Hyp did not affect MC3T3-E1 cell proliferation and matrix mineralization but alkaline phosphatase activity was significantly increased. Furthermore, cells treated with Pro-Hyp significantly upregulated gene expression of Runx2, Osterix, and Col1α1. These results indicate that Pro-Hyp promotes osteoblast differentiation. This study demonstrates for the first time that Pro-Hyp has a positive effect on osteoblast differentiation with upregulation of Runx2, Osterix, and Collα1 gene expression.

  7. Aculeatin, a coumarin derived from Toddalia asiatica (L.) Lam., enhances differentiation and lipolysis of 3T3-L1 adipocytes

    SciTech Connect

    Watanabe, Akio; Kato, Tsuyoshi; Ito, Yusuke; Yoshida, Izumi; Harada, Teppei; Mishima, Takashi; Fujita, Kazuhiro; Watai, Masatoshi; Nakagawa, Kiyotaka; Miyazawa, Teruo

    2014-10-31

    Highlights: • Aculeatin promoted adipocyte differentiation. • Aculeatin improved glucose uptake. • Aculeatin enhanced adipocyte lipolysis. - Abstract: Toddalia asiatica (L.) Lam. (T. asiatica) has been utilized traditionally for medicinal purposes such as the treatment of diabetes. Currently, the extract is considered to be a good source of anti-diabetic agents, but the active compounds have yet to be identified. In this study, we investigated the effects of fractionated T. asiatica extracts on the differentiation of 3T3-L1 preadipocytes and identified aculeatin as a potential active agent. When 3T3-L1 preadipocytes were treated with aculeatin isolated from T. asiatica in the presence of insulin, aculeatin increased cellular triglyceride levels and glycerol-3-phosphate dehydrogenase activity. This indicated that aculeatin could enhance the differentiation of preadipocytes into adipocytes. Further analyses using a DNA microarray and real-time quantitative reverse-transcription PCR showed an increase in the expression of peroxisome proliferator-activated receptor-γ target genes (Pparg, Ap2, Cd36, Glut4 and Adipoq) by aculeatin, suggesting that aculeatin enhances the differentiation of 3T3-L1 cells by modulating the expression of genes critical for adipogenesis. Interestingly, after treatment of differentiated adipocytes with aculeatin, glucose uptake and lipolysis were enhanced. Overall, our results suggested that aculeatin is an active compound in T. asiatica for enhancing both differentiation and lipolysis of adipocytes, which are useful for the treatment of lipid abnormalities as well as diabetes.

  8. Suppressive Effects of Barley β-Glucans with Different Molecular Weight on 3T3-L1 Adipocyte Differentiation.

    PubMed

    Zhu, Yingying; Yao, Yang; Gao, Yue; Hu, Yibo; Shi, Zhenxing; Ren, Guixing

    2016-03-01

    In this study, 2 β-glucans with different molecular weight were prepared and purified from hull-less barley bran. The aim was to evaluate their effects on the differentiation of 3T3-L1 pre-adipocytes. Results showed that barley β-glucans inhibited the differentiation of 3T3-L1 pre-adipocytes induced by differentiation medium in a dose-dependent manner, the suppressive effect of high-molecular-weight barley β-glucans (552 kDa, BGH) was stronger (P < 0.05) than that of low-molecular-weight barley β-glucan (32 kDa, BGL), evidenced by the significantly decrease (P < 0.05) of Oil-red O staining and intracellular triglyceride content in the mature adipocytes. Besides, gene expression analysis and Western Blot analysis revealed that both BGH and BGL inhibited the mRNA and protein levels of adipogenesis related transcription factors peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT-enhancer-binding protein α (C/EBPα) which are principal regulators of adipogenesis. Furthermore, the mRNA and protein expression levels of PPARγ target genes in adipose tissue including adipocyte fatty acid binding protein (ap2), lipoprotein lipase (LPL), uncoupling protein-2 (UCP-2), and glucose-transporter 4 (Glut4) in 3T3-L1 cells was also markedly downregulated (P < 0.05). These findings were anticipated to help develop barley β-glucans based functional food for the management of obesity.

  9. Interferon-stimulated gene ISG12b1 inhibits adipogenic differentiation and mitochondrial biogenesis in 3T3-L1 cells.

    PubMed

    Li, Bing; Shin, Jonghyun; Lee, Kichoon

    2009-03-01

    Microarray analysis was performed to find a new group of genes or pathways that might be important in adipocyte development and metabolism. Among them, a mouse interferon-stimulated gene 12b1 (ISG12b1) is expressed at a 400-fold higher level in adipocytes compared with stromal-vascular cells. It is predominantly expressed in adipose tissue among other tissues we tested. Developmentally, ISG12b1 mRNA expression was initially inhibited followed by a dramatic induction during both in vivo and in vitro adipogenic differentiation. Adenovirus-mediated overexpression of ISG12b1 inhibited adipogenic differentiation in 3T3-L1 cells as shown by decreased lipid staining with Oil-Red-O and reduction in adipogenic marker proteins including peroxisome proliferator-activated receptor-gamma (PPARgamma), and CCAAT/enhancer-binding protein-alpha (C/EBPalpha). Our bioinformatics analysis for the predicted localization of ISG12b1 protein suggested the mitochondrial localization, which was confirmed by the colocalization of hemagglutinin-tagged ISG12b1 protein with mitochondrial marker MitoTracker. In addition, ISG12b1 protein was exclusively detected in protein extract from the fractionated mitochondria by Western blot analysis. Furthermore, overexpression of ISG12b1 in adipocytes reduced mitochondrial DNA content and gene expression of mitochondrial transcription factor A (mtTFA), nuclear respiratory factor 1 (NRF1), and cytochrome oxidase II, suggesting an inhibitory role of ISG12b1 in mitochondrial biogenesis and function. Activation of mitochondrial biogenesis and function by treatment with PPARgamma and PPARalpha agonists in 3T3-L1 cells and cold exposure in mice induced mitochondrial transcription factors and reduced ISG12 expression. These data demonstrated that mitochondrial-localized ISG12b1 protein inhibits adipocyte differentiation and mitochondrial biogenesis and function, implying the important role of mitochondrial function in adipocyte development and associated

  10. Loss of platelet-derived growth factor-stimulated phospholipase activity in NIH-3T3 cells expressing the EJ-ras oncogene

    SciTech Connect

    Benjamin, C.W.; Tarpley, W.G.; Gorman, R.R.

    1987-01-01

    Data indicating that the 21-kDa protein (p21) Harvey-ras gene product shares sequence homology with guanine nucleotide-binding proteins (G proteins) has stimulated research on the influence(s) of p21 on G-protein-regulated systems in vertebrate cells. Previous work demonstrated that NIH-3T3 mouse cells expressing high levels of the cellular ras oncogene isolated from the EJ human bladder carcinoma (EJ-ras) exhibited reduced hormone-stimulated adenylate cyclase activity. The authors now report that in these cells another enzyme system thought to be regulated by G proteins is inhibited, namely phospholipases A/sub 2/ and C. NIH-3T3 cells incubated in plasma-derived serum release significant levels of prostaglandin E/sub 2/ (PGE/sub 2/) as determined by radioimmunoassay when exposed to platelet-derived growth factor (PDGF) at 2 units/ml. The lack of PDGF-stimulated PGE/sub 2/ release from EJ-ras-transfected cells is not due to a defect in the prostaglandin cyclooxygenase enzyme, since incubation of control cells and EJ-ras-transfected cells in 0.33, 3.3, or 33 ..mu..M arachidonate resulted in identical levels of PGE/sub 2/ release. The lack of PDGF-stimulated PGE/sub 2/ release from EJ-ras-transfected cells also does not result from the loss of functional PDGF receptors. EJ-ras-transformed cells bind 70% as much /sup 125/I-labeled PDGF as control cells and are stimulated to incorporate (/sup 3/H)thymidine and to proliferate after exposure to PDGF. Determination of total water-soluble inositolphospholipids and changes in the specific activities of phosphatidylcholine in control and EJ-ras-transfected cells demonstrated that PDGF-stimulated phospholipase C and A/sub 2/ activities are inhibited in the EJ-ras-transfected cells.

  11. Phytosphingosine-1-phosphate stimulates chemotactic migration of L2071 mouse fibroblasts via pertussis toxin-sensitive G-proteins.

    PubMed

    Kim, Mi-Kyoung; Park, Kyoung Sun; Lee, Hyuck; Kim, Young Dae; Yun, Jeanho; Bae, Yoe-Sik

    2007-04-30

    Phytosphingosine-1-phosphate (PhS1P) was found to stimulate an intracellular calcium increase via phospholipase C but not pertussis toxin (PTX)-sensitive G-proteins in L2071 mouse fibroblasts. PhS1P also activated ERK and p38 kinase, and these activations by PhS1P were inhibited by PTX. Moreover, PhS1P stimulated the chemotactic migration of L2071 cells via PTX-sensitive Gi protein(s). In addition, the PhS1P-induced chemotactic migration of L2071 cells was also dramatically inhibited by LY294002 and SB203580 (inhibitors of phosphoinositide 3-kinase and p38 kinase, respectively). L2071 cells are known to express four S1P receptors, i.e., S1P1, S1P2, S1P3, and S1P4, and pretreatment with an S1P1 and S1P3 antagonist (VPC 23019) did not affect on PhS1P-induced chemotaxis. This study demonstrates that PhS1P stimulates at least two different signaling cascades, one is a PTX-insensitive but phospholipase C dependent intracellular calcium increase, and the other is a PTX-sensitive chemotactic migration mediated by phosphoinositide 3-kinase and p38 kinase.

  12. Skp2 promotes adipocyte differentiation via a p27{sup Kip1}-independent mechanism in primary mouse embryonic fibroblasts

    SciTech Connect

    Okada, Mitsuru; Sakai, Tamon; Nakamura, Takehiro; Tamamori-Adachi, Mimi; Kitajima, Shigetaka; Matsuki, Yasushi; Watanabe, Eijiro; Hiramatsu, Ryuji; Sakaue, Hiroshi Kasuga, Masato

    2009-02-06

    Skp2, the substrate-binding subunit of an SCF ubiquitin ligase complex, is a key regulator of cell cycle progression that targets substrates for degradation by the 26S proteasome. We have now shown that ablation of Skp2 in primary mouse embryonic fibroblasts (MEFs) results both in impairment of adipocyte differentiation and in the accumulation of the cyclin-dependent kinase inhibitor p27{sup Kip1}, a principal target of the SCF{sup Skp2} complex. Genetic ablation of p27{sup Kip1} in MEFs promoted both lipid accumulation and adipocyte-specific gene expression. However, depletion of p27{sup Kip1} by adenovirus-mediated RNA interference failed to correct the impairment of adipocyte differentiation in Skp2{sup -/-} MEFs. In contrast, troglitazone, a high-affinity ligand for peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}), largely restored lipid accumulation and PPAR{gamma} gene expression in Skp2{sup -/-} MEFs. Our data suggest that Skp2 plays an essential role in adipogenesis in MEFs in a manner that is at least in part independent of regulation of p27{sup Kip1} expression.

  13. Quantitative Proteomic Analysis of Mouse Embryonic Fibroblasts and Induced Pluripotent Stem Cells Using 16O /18O labeling

    SciTech Connect

    Huang, Xin; Tian, Changhai; Liu, Miao; Wang, Yongxiang; Tolmachev, Aleksey V.; Sharma, Seema; Yu, Fang; Fu, Kai; Zheng, Jialin; Ding, Shi-Jian

    2012-04-06

    Induced pluripotent stem cells (iPSC) hold great promise for regenerative medicine as well as for investigations into the pathogenesis and treatment of various diseases. Understanding of key intracellular signaling pathways and protein targets that control development of iPSC from somatic cells is essential for designing new approaches to improve reprogramming efficiency. Here we report the development and application of an integrated quantitative proteomics platform for investigating differences in protein expressions between mouse embryonic fibroblasts (MEF) and MEF-derived iPSC. This platform consists of 16O/18O labeling, multidimensional peptide separation coupled with tandem mass spectrometry, and data analysis with UNiquant software. Using this platform a total of 2,481 proteins were identified and quantified from the 16O/18O-labeled MEF-iPSC proteome mixtures with a false discovery rate of 0.01. Among them, 218 proteins were significantly upregulated, while 247 proteins were significantly downregulated in iPSC compared to MEF. Many nuclear proteins, including Hdac1, Dnmt1, Pcna, Ccnd1, Smarcc1, and subunits in DNA replication and RNA polymerase II complex were found to be enhanced in iPSC. Protein network analysis revealed that Pcna functions as a hub orchestrating complicated mechanisms including DNA replication, epigenetic inheritance (Dnmt1) and chromatin remodeling (Smarcc1) to reprogram MEF and maintain stemness of iPSC.

  14. Export-deficient monoubiquitinated PEX5 triggers peroxisome removal in SV40 large T antigen-transformed mouse embryonic fibroblasts

    PubMed Central

    Nordgren, Marcus; Francisco, Tânia; Lismont, Celien; Hennebel, Lore; Brees, Chantal; Wang, Bo; Van Veldhoven, Paul P; Azevedo, Jorge E; Fransen, Marc

    2015-01-01

    Peroxisomes are ubiquitous cell organelles essential for human health. To maintain a healthy cellular environment, dysfunctional and superfluous peroxisomes need to be selectively removed. Although emerging evidence suggests that peroxisomes are mainly degraded by pexophagy, little is known about the triggers and molecular mechanisms underlying this process in mammalian cells. In this study, we show that PEX5 proteins fused to a bulky C-terminal tag trigger peroxisome degradation in SV40 large T antigen-transformed mouse embryonic fibroblasts. In addition, we provide evidence that this process is autophagy-dependent and requires monoubiquitination of the N-terminal cysteine residue that marks PEX5 for recycling. As our findings also demonstrate that the addition of a bulky tag to the C terminus of PEX5 does not interfere with PEX5 monoubiquitination but strongly inhibits its export from the peroxisomal membrane, we hypothesize that such a tag mimics a cargo protein that cannot be released from PEX5, thus keeping monoubiquitinated PEX5 at the membrane for a sufficiently long time to be recognized by the autophagic machinery. This in turn suggests that monoubiquitination of the N-terminal cysteine of peroxisome-associated PEX5 not only functions to recycle the peroxin back to the cytosol, but also serves as a quality control mechanism to eliminate peroxisomes with a defective protein import machinery. PMID:26086376

  15. In vivo injection of fibroblast growth factor-2 into the cisterna magna induces glypican-6 expression in mouse brain tissue.

    PubMed

    Salehi, Zivar

    2009-05-01

    The proteoglycans (PGs) are multifunctional macromolecules composed of a core polypeptide and a variable number of glycosaminoglycan chains. In the nervous system, PGs regulate the structural organization of the extracellular matrix (ECM) and modulate growth factor activities and cell proliferation and migration. Most cortical neurons are generated from neural precursor cells that reside in the ventricular zone of the embryonic brain. The proliferation and differentiation of neural precursor cells are regulated by various growth and neurotrophic factors. Fibroblast growth factor-2 (FGF-2) is an important mitogen for cortical neural precursor cells, and glypicans regulate the action of FGF-2 on neural precursor cells. Glypican-6 is one of the most abundant ECM molecules in the brain. In this study the effects of FGF-2 on glypican-6 expression in brain tissue have been investigated. FGF-2 was injected into the cerebrospinal fluid (CSF) through the cisterna magna of mouse pups. Using Western blotting, it was shown that the expression of glypican-6 is increased in response to infusion of FGF-2 into the CSF. The injection of anti-FGF-2 antibody into the cisterna magna decreased glypican-6 expression in brain tissue. The results from this study suggest that glypican-6 is important in regulating FGF-2 activity during cerebral cortical development.

  16. Expression of the Small T antigen of Lymphotropic Papovavirus is Sufficient to Transform Primary Mouse Embryo Fibroblasts

    PubMed Central

    Gupta, Tushar; Robles, Maria Teresa Sáenz; Schowalter, Rachel M.; Buck, Christopher B.; Pipas, James M.

    2015-01-01

    Polyomaviruses induce cell proliferation and transformation through different oncoproteins encoded within the early region (ER): large T antigen (LT), small T antigen (sT) and, in some cases, additional components. Each virus utilizes different mechanisms to achieve transformation. For instance, the LTs of Simian virus 40 (SV40), BK and/or JC virus can induce transformation; but Merkel Cell Polyomavirus (MCPyV) requires expression of sT. Lymphotropic Papovavirus (LPV) is closely related to Human Polyomavirus 9 (HuPyV9) and, under similar conditions, mice expressing LPV.ER exhibit higher rates of tumor formation than mice expressing SV40.ER. We have investigated the contributions of individual LPV.ER components to cell transformation. In contrast to SV40, LPV.ER transforms mouse embryonic fibroblasts (MEFs), but expression of LPV LT is insufficient to transform MEFs. Furthermore, LPV sT induces immortalization and transformation of MEFs. Thus, in the case of LPV, sT is the main mediator of oncogenesis. PMID:26517398

  17. Histone deacetylase inhibitor valproic acid promotes the induction of pluripotency in mouse fibroblasts by suppressing reprogramming-induced senescence stress

    SciTech Connect

    Zhai, Yingying; Chen, Xi; Yu, Dehai; Li, Tao; Cui, Jiuwei; Wang, Guanjun; Hu, Ji-Fan; Li, Wei

    2015-09-10

    Histone deacetylase inhibitor valproic acid (VPA) has been used to increase the reprogramming efficiency of induced pluripotent stem cell (iPSC) from somatic cells, yet the specific molecular mechanisms underlying this effect is unknown. Here, we demonstrate that reprogramming with lentiviruses carrying the iPSC-inducing factors (Oct4-Sox2-Klf4-cMyc, OSKM) caused senescence in mouse fibroblasts, establishing a stress barrier for cell reprogramming. Administration of VPA protected cells from reprogramming-induced senescent stress. Using an in vitro pre-mature senescence model, we found that VPA treatment increased cell proliferation and inhibited apoptosis through the suppression of the p16/p21 pathway. In addition, VPA also inhibited the G2/M phase blockage derived from the senescence stress. These findings highlight the role of VPA in breaking the cell senescence barrier required for the induction of pluripotency. - Highlights: • Histone deacetylase inhibitor valproic acid enhances iPSC induction. • Valproic acid suppresses reprogramming-induced senescence stress. • Valproic acid downregulates the p16/p21 pathway in reprogramming. • This study demonstrates a new mechanistic role of valproic acid in enhancing reprogramming.

  18. Inhibitory effect of peroxiredoxin II (Prx II) on Ras-ERK-NFkappaB pathway in mouse embryonic fibroblast (MEF) senescence.

    PubMed

    Han, Ying-Hao; Kwon, Jeong-Hoon; Yu, Dae-Yeul; Moon, Eun-Yi

    2006-11-01

    Intracellular reactive oxygen species (ROS) were attenuated by the expression of peroxiredoxin II (Prx II). Cellular senescence as judged by senescence-associated (SA)-beta-galactosidase (Gal) positive cell formation was increased in Prx II-deficient mouse embryonic fibroblast (MEF). Ras expression was increased following passages. The level of Ras expression was higher in Prx II-/- MEF than wild type MEF. ERK activity was also augmented by the deletion of Prx II. SA-beta-Gal-positive cell formation was reduced by PD98059, ERK inhibitor. Activated nuclear transcription factor, nuclear factor-kappaB (NFkappaB) by the deletion of Prx II was inhibited by the treatment with PD98059. In contrast, no changes in SA-beta-Gal-positive cell formation were detected by NFkappaB inhibitor, N-alpha-tosyl-L-phenylalanyl chloromethyl ketone (TPCK). Collectively, results suggest that Prx II deletion activate Ras-ERK-NFkappaB pathways and cellular senescence in Prx II-/- MEF cells was mediated by ERK activation but not by NFkappaB activation.

  19. The docking protein FRS2alpha is an essential component of multiple fibroblast growth factor responses during early mouse development.

    PubMed

    Gotoh, N; Manova, K; Tanaka, S; Murohashi, M; Hadari, Y; Lee, A; Hamada, Y; Hiroe, T; Ito, M; Kurihara, T; Nakazato, H; Shibuya, M; Lax, I; Lacy, E; Schlessinger, J

    2005-05-01

    The docking protein FRS2alpha is a major mediator of fibroblast growth factor (FGF) signaling. However, the physiological role of FRS2alpha in vivo remains unknown. In this report, we show that Frs2alpha-null mouse embryos have a defect in anterior-posterior (A-P) axis formation and are developmentally retarded, resulting in embryonic lethality by embryonic day 8. We demonstrate that FRS2alpha is essential for the maintenance of self-renewing trophoblast stem (TS) cells in response to FGF4 in the extraembryonic ectoderm (ExE) that gives rise to tissues of the placenta. By analyzing chimeric embryos, we found that FRS2alpha also plays a role in cell movement through the primitive streak during gastrulation. In addition, experiments are presented demonstrating that Bmp4 expression in TS cells is controlled by mitogen-activated protein kinase-dependent FGF4 stimulation. Moreover, both the expression of Bmp4 in ExE and activation of Smad1/5 in epiblasts are reduced in Frs2alpha-null embryos. These experiments underscore the critical role of FRS2alpha in mediating multiple processes during embryonic development and reveal a potential new link between FGF and Bmp4 signaling pathways in early embryogenesis.

  20. UV light induces premature senescence in Akt1-null mouse embryonic fibroblasts by increasing intracellular levels of ROS

    SciTech Connect

    Jee, Hye Jin; Kim, Hyun-Ju; Kim, Ae Jeong; Bae, Yoe-Sik; Bae, Sun Sik; Yun, Jeanho

    2009-06-05

    Akt/PKB plays a pivotal role in cell survival and proliferation. Previously, we reported that UV-irradiation induces extensive cell death in Akt2{sup -/-} mouse embryonic fibroblasts (MEFs) while Akt1{sup -/-} MEFs show cell cycle arrest. Here, we find that Akt1{sup -/-} MEFs exhibit phenotypic changes characteristics of senescence upon UV-irradiation. An enlarged and flattened morphology, a reduced cell proliferation and an increased senescence-associated {beta}-galactosidase (SA {beta}-gal) staining indicate that Akt1{sup -/-} MEFs undergo premature senescence after UV-irradiation. Restoring Akt1 expression in Akt1{sup -/-} MEFs suppressed SA {beta}-gal activity, indicating that UV-induced senescence is due to the absence of Akt1 function. Notably, levels of ROS were rapidly increased upon UV-irradiation and the ROS scavenger NAC inhibits UV-induced senescence of Akt1{sup -/-} MEFs, suggesting that UV light induces premature senescence in Akt1{sup -/-} MEFs by modulating intracellular levels of ROS. In conjunction with our previous work, this indicates that different isoforms of Akt have distinct function in response to UV-irradiation.

  1. Mobile phone signal exposure triggers a hormesis-like effect in Atm(+/+) and Atm(-/-) mouse embryonic fibroblasts.

    PubMed

    Sun, Chuan; Wei, Xiaoxia; Fei, Yue; Su, Liling; Zhao, Xinyuan; Chen, Guangdi; Xu, Zhengping

    2016-11-18

    Radiofrequency electromagnetic fields (RF-EMFs) have been classified by the International Agency for Research on Cancer as possible carcinogens to humans; however, this conclusion is based on limited epidemiological findings and lacks solid support from experimental studies. In particular, there are no consistent data regarding the genotoxicity of RF-EMFs. Ataxia telangiectasia mutated (ATM) is recognised as a chief guardian of genomic stability. To address the debate on whether RF-EMFs are genotoxic, we compared the effects of 1,800 MHz RF-EMF exposure on genomic DNA in mouse embryonic fibroblasts (MEFs) with proficient (Atm(+/+)) or deficient (Atm(-/-)) ATM. In Atm(+/+) MEFs, RF-EMF exposure for 1 h at an average special absorption rate of 4.0 W/kg induced significant DNA single-strand breaks (SSBs) and activated the SSB repair mechanism. This effect reduced the DNA damage to less than that of the background level after 36 hours of exposure. In the Atm(-/-) MEFs, the same RF-EMF exposure for 12 h induced both SSBs and double-strand breaks and activated the two repair processes, which also reduced the DNA damage to less than the control level after prolonged exposure. The observed phenomenon is similar to the hormesis of a toxic substance at a low dose. To the best of our knowledge, this study is the first to report a hormesis-like effect of an RF-EMF.

  2. Specific fixation of bovine brain and retinal acidic and basic fibroblast growth factors to mouse embryonic eye basement membranes

    SciTech Connect

    Jeanny, J.C.; Fayein, N.; Courtois, Y. ); Moenner, M.; Chevallier, B.; Barritault, D. )

    1987-07-01

    The labeling pattern of mouse embryonic eye frozen sections incubated with radioiodinated brain acidic and basic fibroblasts growth factors (aFGF and bFGF) was investigated by autoradiography. Both growth factors bind to basement membranes in a dose-dependent way, with a higher affinity for bFGF. Similar data were obtained with eye-derived growth factors (EDGF), the retinal forms of FGF. There was a heterogeneity in the affinity of the various basement membranes toward these growth factors. The specificity of the growth factor-basement membrane interaction was demonstrated by the following experiments: (i) an excess of unlabeled growth factor displaced the labeling; (ii) unrelated proteins with different isoelectric points did not modify the labeling; and (iii) iodinated EGF or PDGF did not label basement membrane. In order to get a better understanding of the nature of this binding, the authors performed the incubation of the frozen sections with iodinated FGFs preincubated with various compounds. These results demonstrate that FGFs bind specifically to basement membranes, probably on the polysaccharidic part of the proteoheparan sulfate, and suggest that this type of interaction may be a general feature of the mechanism of action of these growth factors.

  3. Alkaline Phosphatase-Positive Immortal Mouse Embryo Fibroblasts Are Cells in a Transitional Reprogramming State Induced to Face Environmental Stresses

    PubMed Central

    Evangelista, Monica; Baroudi, Mariama El; Rizzo, Milena; Tuccoli, Andrea; Poliseno, Laura; Pellegrini, Marco; Rainaldi, Giuseppe

    2015-01-01

    In this study, we report that immortal mouse embryonic fibroblasts (I-MEFs) have a baseline level of cells positive for alkaline phosphatase (AP+) staining. Environmental stresses, including long-lasting growth in the absence of expansion and treatment with drugs, enhance the frequency of AP+ I-MEFs. By adapting fast red AP staining to the sorting procedure, we separated AP+ and AP− I-MEFs and demonstrated that the differentially expressed genes are consistent with a reprogrammed phenotype. In particular, we found that sestrin 1 is upregulated in AP+ I-MEFs. We focused on this gene and demonstrated that increased sestrin 1 expression is accompanied by the growth of I-MEFs in the absence of expansion and occurs before the formation of AP+ I-MEFs. Together with sestrin 1 upregulation, we found that AP+ I-MEFs accumulated in the G1 phase of the cell cycle, suggesting that the two events are causally related. Accordingly, we found that silencing sestrin 1 expression reduced the frequency and G1 accumulation of AP+ I-MEFs. Taken together, our data suggested that I-MEFs stressed by environmental changes acquire the AP+ phenotype and achieve a quiescent state characterized by a new transcriptional network. PMID:26740745

  4. Type I pro-collagen promoting and anti-collagenase activities of Phyllanthus emblica extract in mouse fibroblasts.

    PubMed

    Chanvorachote, Pithi; Pongrakhananon, Varisa; Luanpitpong, Sudjit; Chanvorachote, Boontarika; Wannachaiyasit, Sumalee; Nimmannit, Ubonthip

    2009-01-01

    As part of an ongoing search for the novel pharmacological activities of Phyllanthus emblica, the present study has shown its type I collagen promoting and anti-collagenase effects on primary mouse fibroblast cells. At a concentration of 0.1 mg/ml, emblica extract significantly increased the type I pro-collagen level up to 1.65-fold, and 6.78-fold greater than that of an untreated control, determined by immunocytochemistry and Western blot analysis, respectively. Emblica extract caused an approximately 7.75-fold greater type I pro-collagen induction compared to the known herbal collagen enhancer asiaticoside at the same treatment concentration (0.1 mg/ml). Moreover, emblica extract inhibited collagenase activity in a dose-dependent manner. Maximal inhibition was observed (78.67 +/- 3.51%) at a concentration of 1 mg/ml. In summary, emblica extract has a promising pharmacological effect that benefits collagen synthesis and protects against its degradation and could be used as a natural anti-aging ingredient.

  5. Mobile phone signal exposure triggers a hormesis-like effect in Atm+/+ and Atm−/− mouse embryonic fibroblasts

    PubMed Central

    Sun, Chuan; Wei, Xiaoxia; Fei, Yue; Su, Liling; Zhao, Xinyuan; Chen, Guangdi; Xu, Zhengping

    2016-01-01

    Radiofrequency electromagnetic fields (RF-EMFs) have been classified by the International Agency for Research on Cancer as possible carcinogens to humans; however, this conclusion is based on limited epidemiological findings and lacks solid support from experimental studies. In particular, there are no consistent data regarding the genotoxicity of RF-EMFs. Ataxia telangiectasia mutated (ATM) is recognised as a chief guardian of genomic stability. To address the debate on whether RF-EMFs are genotoxic, we compared the effects of 1,800 MHz RF-EMF exposure on genomic DNA in mouse embryonic fibroblasts (MEFs) with proficient (Atm+/+) or deficient (Atm−/−) ATM. In Atm+/+ MEFs, RF-EMF exposure for 1 h at an average special absorption rate of 4.0 W/kg induced significant DNA single-strand breaks (SSBs) and activated the SSB repair mechanism. This effect reduced the DNA damage to less than that of the background level after 36 hours of exposure. In the Atm−/− MEFs, the same RF-EMF exposure for 12 h induced both SSBs and double-strand breaks and activated the two repair processes, which also reduced the DNA damage to less than the control level after prolonged exposure. The observed phenomenon is similar to the hormesis of a toxic substance at a low dose. To the best of our knowledge, this study is the first to report a hormesis-like effect of an RF-EMF. PMID:27857169

  6. The apoptotic effect of nanosilver is mediated by a ROS- and JNK-dependent mechanism involving the mitochondrial pathway in NIH3T3 cells.

    PubMed

    Hsin, Yi-Hong; Chen, Chun-Feng; Huang, Shing; Shih, Tung-Sheng; Lai, Ping-Shan; Chueh, Pin Ju

    2008-07-10

    Nanomaterials and nanoparticles have received considerable attention recently because of their unique properties and diverse biotechnology and life sciences applications. Nanosilver products, which have well-known antimicrobial properties, have been used extensively in a range of medical settings. Despite the widespread use of nanosilver products, relatively few studies have been undertaken to determine the biological effects of nanosilver exposure. The purpose of this study was to evaluate the toxicity of nanosilver and to elucidate possible molecular mechanisms underlying the biological effects of nanosilver. Here, we show that nanosilver is cytotoxic, inducing apoptosis in NIH3T3 fibroblast cells. Treatment with nanosilver induced the release of cytochrome c into the cytosol and translocation of Bax to mitochondria, indicating that nanosilver-mediated apoptosis is mitochondria-dependent. Nanosilver-induced apoptosis was associated with the generation of reactive oxygen species (ROS) and JNK activation, and inhibition of either ROS or JNK attenuated nanosilver-induced apoptosis. In nanosilver-resistant HCT116 cells, up-regulation of the anti-apoptotic proteins, Bcl-2 appeared to be associated with a diminished apoptotic response. Taken together, our results provide the first evidence for a molecular mechanism of nanosilver cytotoxicity, showing that nanosilver acts through ROS and JNK to induce apoptosis via the mitochondrial pathway.

  7. Modest hypoxia significantly reduces triglyceride content and lipid droplet size in 3T3-L1 adipocytes

    SciTech Connect

    Hashimoto, Takeshi; Yokokawa, Takumi; Endo, Yuriko; Iwanaka, Nobumasa; Higashida, Kazuhiko; Taguchi, Sadayoshi

    2013-10-11

    Highlights: •Long-term hypoxia decreased the size of LDs and lipid storage in 3T3-L1 adipocytes. •Long-term hypoxia increased basal lipolysis in 3T3-L1 adipocytes. •Hypoxia decreased lipid-associated proteins in 3T3-L1 adipocytes. •Hypoxia decreased basal glucose uptake and lipogenic proteins in 3T3-L1 adipocytes. •Hypoxia-mediated lipogenesis may be an attractive therapeutic target against obesity. -- Abstract: Background: A previous study has demonstrated that endurance training under hypoxia results in a greater reduction in body fat mass compared to exercise under normoxia. However, the cellular and molecular mechanisms that underlie this hypoxia-mediated reduction in fat mass remain uncertain. Here, we examine the effects of modest hypoxia on adipocyte function. Methods: Differentiated 3T3-L1 adipocytes were incubated at 5% O{sub 2} for 1 week (long-term hypoxia, HL) or one day (short-term hypoxia, HS) and compared with a normoxia control (NC). Results: HL, but not HS, resulted in a significant reduction in lipid droplet size and triglyceride content (by 50%) compared to NC (p < 0.01). As estimated by glycerol release, isoproterenol-induced lipolysis was significantly lowered by hypoxia, whereas the release of free fatty acids under the basal condition was prominently enhanced with HL compared to NC or HS (p < 0.01). Lipolysis-associated proteins, such as perilipin 1 and hormone-sensitive lipase, were unchanged, whereas adipose triglyceride lipase and its activator protein CGI-58 were decreased with HL in comparison to NC. Interestingly, such lipogenic proteins as fatty acid synthase, lipin-1, and peroxisome proliferator-activated receptor gamma were decreased. Furthermore, the uptake of glucose, the major precursor of 3-glycerol phosphate for triglyceride synthesis, was significantly reduced in HL compared to NC or HS (p < 0.01). Conclusion: We conclude that hypoxia has a direct impact on reducing the triglyceride content and lipid droplet size via

  8. Molecular mechanism of extinction of liver-specific functions in mouse hepatoma x rat fibroblast hybrids: extinction of the albumin gene

    SciTech Connect

    Papaconstantinou, J.; Wong, E.; Ratrie, H.; Szpirer, C.; Szpirer, J.

    1982-01-01

    Hybrids formed by the fusion of mouse hepatoma (BWTG3) and rat fibroblast (JF1) cells exhibit the extinction of mouse albumin and ..cap alpha..-fetoprotein synthesis. Karyotype analyses suggest that all parental chromosomes are present in the hybrids. The extinction, therefore, of mouse hepatocyte genes is attributed to the inhibitory action of the rat genome. In these studies, we show that these hybrids possess and express the mouse ..beta..-glucyronidase gene (which is encoded on the same chromosome as the mouse albumin and ..cap alpha..-fetoprotein gene), and we present data of Southern blot analysis which demonstrate that such hybrids have indeed retained both mouse and rat albumin DNA sequences. In addition, using mouse albumin cDNA, we have shown by cDNA-RNA reassociation kinetics that albumin mRNA is virtually absent in these hybrids. We conclude from these studies that the extinction of albumin synthesis involves a mechanism which results in the loss of cytoplasmic albumin mRNA.

  9. Model-based investigation of the circadian clock and cell cycle coupling in mouse embryonic fibroblasts: Prediction of RevErb-α up-regulation during mitosis.

    PubMed

    Traynard, Pauline; Feillet, Céline; Soliman, Sylvain; Delaunay, Franck; Fages, François

    2016-11-01

    Experimental observations have put in evidence autonomous self-sustained circadian oscillators in most mammalian cells, and proved the existence of molecular links between the circadian clock and the cell cycle. Some mathematical models have also been built to assess conditions of control of the cell cycle by the circadian clock. However, recent studies in individual NIH3T3 fibroblasts have shown an unexpected acceleration of the circadian clock together with the cell cycle when the culture medium is enriched with growth factors, and the absence of such acceleration in confluent cells. In order to explain these observations, we study a possible entrainment of the circadian clock by the cell cycle through a regulation of clock genes around the mitosis phase. We develop a computational model and a formal specification of the observed behavior to investigate the conditions of entrainment in period and phase. We show that either the selective activation of RevErb-α or the selective inhibition of Bmal1 transcription during the mitosis phase, allow us to fit the experimental data on both period and phase, while a uniform inhibition of transcription during mitosis seems incompatible with the phase data. We conclude on the arguments favoring the RevErb-α up-regulation hypothesis and on some further predictions of the model.

  10. Liraglutide attenuates the osteoblastic differentiation of MC3T3-E1 cells by modulating AMPK/mTOR signaling

    PubMed Central

    Hu, Xiong-Ke; Yin, Xin-Hua; Zhang, Hong-Qi; Guo, Chao-Feng; Tang, Ming-Xing

    2016-01-01

    Liraglutide, a synthetic analogue of glucagon-like peptide-1, is utilized in the treatment of type 2 diabetes and obesity. Liraglutide has been previously demonstrated to prevent osteoblastic differentiation of human vascular smooth muscle cells, resulting in the slowing of arterial calcification, however, its effect on bone formation remains unclear. The present study investigated the effect of liraglutide on osteoblastic differentiation using Alizarin Red S staining, and examined the molecular mechanisms underlying the regulatory effect by western blot analysis. The present study demonstrated that protein expression levels of phosphorylated adenosine monophosphate-activated protein kinase (p-AMPK) were downregulated in MC3T3-E1 cells during osteoblastic differentiation in commercial osteogenic differentiation medium, whereas protein expression levels of transforming growth factor-β (TGF-β) and phosphorylated mammalian target of rapamycin (p-mTOR) increased. Liraglutide was subsequently demonstrated to dose-dependently attenuate the osteoblastic differentiation of MC3T3-E1 cells, to upregulate p-AMPK, and downregulate p-mTOR and TGF-β protein expression levels. Treatment with an AMPK-specific inhibitor, Compound C, eradicated the effect of liraglutide on osteoblastic differentiation, and p-mTOR and TGF-β downregulation. An mTOR activator, MHY1485, also abolished the inhibitory effect of liraglutide on osteoblastic differentiation, and resulted in p-mTOR and TGF-β downregulation, but did not attenuate the liraglutide-induced increase in p-AMPK protein expression levels. The results of the present study demonstrate that liraglutide attenuates osteoblastic differentiation of MC3T3-E1 cells via modulation of AMPK/mTOR signaling. The present study revealed a novel function of liraglutide, which contributes to the understanding of its pharmacological and physiological effects in clinical settings. PMID:27600753

  11. Nano-hydroxyapatite particles induce apoptosis on MC3T3-E1 cells and tissue cells in SD rats

    NASA Astrophysics Data System (ADS)

    Wang, Liting; Zhou, Gang; Liu, Haifeng; Niu, Xufeng; Han, Jingyun; Zheng, Lisha; Fan, Yubo

    2012-04-01

    While the advantages of nanomaterials are being increasingly recognized, their potential toxicity is drawing more and more attention and concern. In this study, we explore the toxicity mechanism of 20-30 nm rod-shaped hydroxyapatite (HA) nanoparticles in vitro and in vivo. The nanoparticles were prepared by precipitation and characterized by IR, XRD and TEM. Concentrations of 0 μg mL-1, 10 μg mL-1, 100 μg mL-1, 1 mg mL-1, and 10 mg mL-1 were applied to the MC3T3-E1 cells for viability (MTT-test). Based on the characteristic differences of the two methods of cell death, the morphological features of the MC3T3-E1 cell line co-cultured with nano-hydroxyapatite (n-HA) (10 mg mL-1) for 24 h were also observed by TEM. Furthermore, important serum biochemical markers and histopathological examinations were used to evaluate the potential toxicological effect of n-HA on the major organs of SD rats injected intraperitoneally with n-HA (33.3 mg kg-1 body weight). In the results, we found cell growth inhibition and apoptosis in MC3T3-E1 cells co-cultured with n-HA. Moreover, apoptosis but not necrosis was illustrated in liver and renal tissue by using histopathology slices and serum biochemical markers. It suggests that apoptosis may be the possible mechanism of n-HA toxicity and provides a better understanding of the biocompatibility of nanomaterials applied in human bone repair.

  12. Radicicol, a heat shock protein 90 inhibitor, inhibits differentiation and adipogenesis in 3T3-L1 preadipocytes

    SciTech Connect

    He, Yonghan; Li, Ying; Zhang, Shuocheng; Perry, Ben; Zhao, Tiantian; Wang, Yanwen; Sun, Changhao

    2013-06-28

    Highlights: •Radicicol suppressed intracellular fat accumulation in 3T3-L1 adipocytes. •Radicicol inhibited the expression of FAS and FABP4. •Radicicol blocked cell cycle at the G1-S phase during cell differentiation. •Radicicol inhibited the PDK1/Akt pathway in adipocyte differentiation. -- Abstract: Heat shock protein 90 (Hsp90) is involved in various cellular processes, such as cell proliferation, differentiation and apoptosis. As adipocyte differentiation plays a critical role in obesity development, the present study investigated the effect of an Hsp90 inhibitor radicicol on the differentiation of 3T3-L1 preadipocytes and potential mechanisms. The cells were treated with different concentrations of radicicol during the first 8 days of cell differentiation. Adipogenesis, the expression of adipogenic transcriptional factors, differentiation makers and cell cycle were determined. It was found that radicicol dose-dependently decreased intracellular fat accumulation through down-regulating the expression of peroxisome proliferator-activated receptor γ (PPAR{sub γ}) and CCAAT element binding protein α (C/EBP{sub α}), fatty acid synthase (FAS) and fatty acid-binding protein 4 (FABP4). Flow cytometry analysis revealed that radicicol blocked cell cycle at G1-S phase. Radicicol redcued the phosphorylation of Akt while showing no effect on β-catenin expression. Radicicol decreased the phosphorylation of phosphoinositide-dependent kinase 1 (PDK1). The results suggest that radicicol inhibited 3T3-L1 preadipocyte differentiation through affecting the PDK1/Akt pathway and subsequent inhibition of mitotic clonal expansion and the expression/activity of adipogenic transcriptional factors and their downstream adipogenic proteins.

  13. Pasteurella multocida toxin (PMT) upregulates CTGF which leads to mTORC1 activation in Swiss 3T3 cells.

    PubMed

    Oubrahim, Hammou; Wong, Allison; Wilson, Brenda A; Chock, P Boon

    2013-05-01

    Pasteurella multocida toxin (PMT) is a mitogenic protein that hijacks cellular signal transduction pathways via deamidation of heterotrimeric G proteins. We previously showed that rPMT activates mTOR signaling via a Gαq/11/PLCβ/PKC mediated pathway, leading in part to cell proliferation and migration. Herein, we show that mTOR and MAPK, but not membrane-associated tyrosine kinases, are activated in serum-starved 3T3 cells by an autocrine/paracrine substance(s) secreted into the conditioned medium following rPMT treatment. Surprisingly, this diffusible factor(s) is capable of activating mTOR and MAPK pathways even in MEF Gαq/11 double knockout cells. Microarray analysis identified connective tissue growth factor (CTGF) mRNA as the most upregulated gene in rPMT-treated serum-starved 3T3 cells relative to untreated cells. These results were further confirmed using RT-PCR and Western blot analyses. In accord with rPMT-induced mTOR activation, upregulation of CTGF protein was observed in WT MEF, but not in Gαq/11 double knockout MEF cells. Although CTGF expression is regulated by TGFβ, rPMT did not activate TGFβ pathway. In addition, MEK inhibitors U0126 or PD98059, but not mTOR specific inhibitors, rapamycin and Torin 1, inhibited rPMT-induced upregulation of CTGF. Importantly, CTGF overexpression in serum-starved 3T3 cells using adenovirus led to phosphorylation of ribosomal protein S6, a downstream target of mTOR. However, despite the ability of CTGF to activate the mTOR pathway, upregulation of CTGF alone could not induce morphological changes as those observed in rPMT-treated cells. Our findings reveal that CTGF plays an important role, but there are additional factors involved in the mitogenic action of PMT.

  14. ClC-3 Promotes Osteogenic Differentiation in MC3T3-E1 Cell After Dynamic Compression.

    PubMed

    Wang, Dawei; Wang, Hao; Gao, Feng; Wang, Kun; Dong, Fusheng

    2017-06-01

    ClC-3 chloride channel has been proved to have a relationship with the expression of osteogenic markers during osteogenesis, persistent static compression can upregulate the expression of ClC-3 and regulate osteodifferentiation in osteoblasts. However, there was no study about the relationship between the expression of ClC-3 and osteodifferentiation after dynamic compression. In this study, we applied dynamic compression on MC3T3-E1 cells to detect the expression of ClC-3, runt-related transcription factor 2 (Runx2), bone morphogenic protein-2 (BMP-2), osteopontin (OPN), nuclear-associated antigen Ki67 (Ki67), and proliferating cell nuclear antigen (PCNA) in biopress system, then we investigated the expression of these genes after dynamic compression with Chlorotoxin (specific ClC-3 chloride channel inhibitor) added. Under transmission electron microscopy, there were more cell surface protrusions, rough surfaced endoplasmic reticulum, mitochondria, Golgi apparatus, abundant glycogen, and lysosomes scattered in the cytoplasm in MC3T3-E1 cells after dynamic compression. The nucleolus was more obvious. We found that ClC-3 was significantly up-regulated after dynamic compression. The compressive force also up-regulated Runx2, BMP-2, and OPN after dynamic compression for 2, 4 and 8 h. The proliferation gene Ki67 and PCNA did not show significantly change after dynamic compression for 8 h. Chlorotoxin did not change the expression of ClC-3 but reduced the expression of Runx2, BMP-2, and OPN after dynamic compression compared with the group without Cltx added. The data from the current study suggested that ClC-3 may promotes osteogenic differentiation in MC3T3-E1 cell after dynamic compression. J. Cell. Biochem. 118: 1606-1613, 2017. © 2016 Wiley Periodicals, Inc.

  15. Endothelin-1 inhibits TNF alpha-induced iNOS expression in 3T3-F442A adipocytes.

    PubMed

    Mérial-Kieny, Christelle; Lonchampt, Michel; Cogé, Francis; Verwaerde, Patrick; Galizzi, Jean-Pierre; Boutin, Jean A; Lafontan, Max; Levens, Nigel; Galitzky, Jean; Félétou, Michel

    2003-07-01

    1. Endothelin-1 (ET-1) and tumor necrosis factor alpha (TNFalpha) by their action on adipocytes have been independently linked to the pathogenesis of insulino-resistance. In isolated adipocytes, TNFalpha induces the expression of the inducible nitric oxide synthase (iNOS). The purpose of the present work was, in the 3T3-F442A adipocyte cell line, to characterise TNFalpha-induced iNOS expression and to determine whether or not ET-1 could influence TNFalpha-induced iNOS expression and NO production. 2. In differentiated 3T3-F442A, treatment with TNFalpha (20 ng ml(-1)) induced the expression of a functional iNOS as demonstrated by nitrite assay, Western blot, reverse transcription-polymerase chain reaction and Northern blot analysis. TNFalpha-induced iNOS expression requires nuclear factor kappaB activation, but does not necessitate the activation of the PI-3 kinase/Akt and P38-MAP kinase pathways. 3. ET-1, but not ET-3, inhibited the TNFalpha-induced expression of iNOS protein and mRNA as well as nitrite production. The effects of ET-1 were blocked by a specific ETA (BQ123, pA(2) 7.4) but not by a specific ETB receptor antagonist (BQ788). 3T3-F442A adipocytes express the mRNAs for prepro-ET-1 and the ET-A receptor subtype, but not for the ET-B subtype. 4. The inhibitory effect of ET-1 was not affected by bisindolylmaleimide, SB 203580 or indomethacin, inhibitors of protein kinase C, p38-MAP kinase and cyclooxygenase, respectively, and was not associated with cAMP production. However, the effect of ET-1 was partially reversed by wortmannin, suggesting the involvement of PI3 kinase in the transduction signal of ET-1. 5. Differentiated 3T3-F442A adipocytes did not release ET-1 with or without exposure to TNFalpha, although the mRNA for preproET-1 was detected in both pre- and differentiated adipocytes. 6. Thus, these results confirm that adipocytes are a target for circulating ET-1 and demonstrate that the activation of the ETA receptor subtype can prevent TNFalpha

  16. All-trans retinoic acid shifts rosiglitazone-induced adipogenic differentiation to osteogenic differentiation in mouse embryonic fibroblasts.

    PubMed

    Shao, Ying; Chen, Qian-Zhao; Zeng, Yu-Hua; Li, Yang; Ren, Wen-Yan; Zhou, Lin-Yun; Liu, Rong-Xin; Wu, Ke; Yang, Jun-Qing; Deng, Zhong-Liang; Yu, Yu; Sun, Wen-Juan; He, Bai-Cheng

    2016-12-01

    Rosiglitazone (RSG) is a potent drug used in the treatment of insulin resistance; however, it is associated with marked skeletal toxicity. RSG-induced osteoporosis may contribute to the promotion of adipogenic differentiation at the expense of osteogenic differentiation in bone marrow stromal cells. The aim of this study was to investigate whether RSG-induced bone toxicity can be reversed by combined treatment with all-trans retinoic acid (ATRA). We examined different osteogenic markers in mouse embryonic fibroblasts (MEFs) following treatment with RSG, ATRA, or RSG and ATRA in combination. We examined the effects of RSG and/or ATRA on ectopic bone formation, and dissected the possible molecular mechanisms underlying this process. We found that ATRA or RSG both induced alkaline phosphatase (ALP) activity in the MEFs, and that the ATRA-induced ALP activity was enhanced by RSG and vice versa. However, only the combination of RSG and ATRA increased the expression of osteopontin and osteocalcin, promoted matrix mineralization, and induced ectopic ossification in MEFs. Mechanistically, we found that the osteogenic differentiation induced by the combination of RSG and ATRA may be mediated partly by suppressing RSG-induced adipogenic differentiation and activating bone morphogenetic protein (BMP)/Smad signaling. On the whole, our findings demonstrate that RSG in combination with ATRA promotes the commitment of MEFs to the osteoblast lineage. Thus, the combination of these two agents may prove to be a promising and novel therapeutic regimen for insulin resistance without skeletal toxicity. It may also be a better strategy with which to prevent RSG-induced osteoporosis.

  17. Undifferentiated State Induced by Rb-p53 Double Inactivation in Mouse Thyroid Neuroendocrine Cells and Embryonic Fibroblasts.

    PubMed

    Kitajima, Shunsuke; Kohno, Susumu; Kondoh, Atsushi; Sasaki, Nobunari; Nishimoto, Yuuki; Li, Fengkai; Abdallah Mohammed, Mohammed Salah; Muranaka, Hayato; Nagatani, Naoko; Suzuki, Misa; Kido, Yukiharu; Takahashi, Chiaki

    2015-05-01

    Retinoblastoma tumor suppressor protein (RB) is inactivated more frequently during tumor progression than during tumor initiation. However, its exact role in controlling the malignant features associated with tumor progression is poorly understood. We established in vivo and in vitro models to investigate the undifferentiated state induced by Rb inactivation. Rb heterozygous mice develop well-differentiated thyroid medullary carcinoma. We found that additional deletion of Trp53, without change in lineage, converted these Rb-deficient tumors to a poorly differentiated type associated with higher self-renewal activity. Freshly prepared mouse embryonic fibroblasts (MEFs) of Rb(-/-) ; Trp53(-/-) background formed stem cell-like spheres that expressed significant levels of embryonic genes despite of lacking the ability to form colonies on soft agar or tumors in immune-deficient mice. This suggested that Rb-p53 double inactivation resulted in an undifferentiated status but without carcinogenic conversion. We next established Rb(-/-) ; N-ras(-/-) MEFs that harbored a spontaneous carcinogenic mutation in Trp53. These cells (RN6), in an Rb-dependent manner, efficiently generated spheres that expressed very high levels of embryonic genes, and appeared to be carcinogenic. We then screened an FDA-approved drug library to search for agents that suppressed the spherogenic activity of RN6 cells. Data revealed that RN6 cells were sensitive to specific agents including ones those are effective against cancer stem cells. Taken together, all these findings suggest that the genetic interaction between Rb and p53 is a critical determinant of the undifferentiated state in normal and tumor cells.

  18. Biological response to ionizing radiation in mouse embryo fibroblasts with a targeted disruption of the DNA polymerase beta gene.

    PubMed

    Miura, M; Watanabe, H; Okochi, K; Sasaki, T; Shibuya, H

    2000-06-01

    Base excision repair (BER) is carried out by two distinct pathways in mammalian cells, one dependent on DNA polymerase beta (Polb) and the other on proliferating cell nuclear antigen (Pcna). We studied whether the Polb-dependent pathway plays an important role in BER in vivo after exposure to ionizing radiation. For this purpose, we used mouse embryo fibroblasts derived from wild-type and Polb gene knockout littermates. Both cell lines had essentially the same clonogenic cell survival and low levels of apoptosis as determined by a colony formation assay and by a change in mitochondrial membrane potential, respectively. No significant cleavage of protein kinase C delta (Pkcd) in vivo, which is a substrate for caspase 3, was detected, and intact Pkcd was retained in both cell lines for at least 72 h after irradiation. Similar significant increases in caspase 3-like activities as measured by Asp-Glu-Val-Asp (DEVD) cleaving activity in vitro were observed in both cell lines after irradiation. Radiation induced cell cycle arrest in the form of a G(2)-phase block, and G(2)/M-phase fractions reached a peak approximately 10 h after irradiation and decreased thereafter with a similar time course in both cell lines. Similar levels of chromatin-bound Pcna were observed immediately after irradiation in non-S-phase cells of both cell lines and disappeared by 4 h after irradiation. We conclude that the deficiency in Polb does not have a significant influence on the radiation responses of these cells. Together with evidence accumulated in vitro, these results strongly support the idea that the Pcna-dependent pathway predominantly acts in BER of radiation-induced DNA damage in vivo.

  19. All-trans retinoic acid shifts rosiglitazone-induced adipogenic differentiation to osteogenic differentiation in mouse embryonic fibroblasts

    PubMed Central

    Shao, Ying; Chen, Qian-Zhao; Zeng, Yu-Hua; Li, Yang; Ren, Wen-Yan; Zhou, Lin-Yun; Liu, Rong-Xin; Wu, Ke; Yang, Jun-Qing; Deng, Zhong-Liang; Yu, Yu; Sun, Wen-Juan; He, Bai-Cheng

    2016-01-01

    Rosiglitazone (RSG) is a potent drug used in the treatment of insulin resistance; however, it is associated with marked skeletal toxicity. RSG-induced osteoporosis may contribute to the promotion of adipogenic differentiation at the expense of osteogenic differentiation in bone marrow stromal cells. The aim of this study was to investigate whether RSG-induced bone toxicity can be reversed by combined treatment with all-trans retinoic acid (ATRA). We examined different osteogenic markers in mouse embryonic fibroblasts (MEFs) following treatment with RSG, ATRA, or RSG and ATRA in combination. We examined the effects of RSG and/or ATRA on ectopic bone formation, and dissected the possible molecular mechanisms underlying this process. We found that ATRA or RSG both induced alkaline phosphatase (ALP) activity in the MEFs, and that the ATRA-induced ALP activity was enhanced by RSG and vice versa. However, only the combination of RSG and ATRA increased the expression of osteopontin and osteocalcin, promoted matrix mineralization, and induced ectopic ossification in MEFs. Mechanistically, we found that the osteogenic differentiation induced by the combination of RSG and ATRA may be mediated partly by suppressing RSG-induced adipogenic differentiation and activating bone morphogenetic protein (BMP)/Smad signaling. On the whole, our findings demonstrate that RSG in combination with ATRA promotes the commitment of MEFs to the osteoblast lineage. Thus, the combination of these two agents may prove to be a promising and novel therapeutic regimen for insulin resistance without skeletal toxicity. It may also be a better strategy with which to prevent RSG-induced osteoporosis. PMID:27779644

  20. Quantitation of fibroblast activation protein (FAP)-specific protease activity in mouse, baboon and human fluids and organs.

    PubMed

    Keane, Fiona M; Yao, Tsun-Wen; Seelk, Stefanie; Gall, Margaret G; Chowdhury, Sumaiya; Poplawski, Sarah E; Lai, Jack H; Li, Youhua; Wu, Wengen; Farrell, Penny; Vieira de Ribeiro, Ana Julia; Osborne, Brenna; Yu, Denise M T; Seth, Devanshi; Rahman, Khairunnessa; Haber, Paul; Topaloglu, A Kemal; Wang, Chuanmin; Thomson, Sally; Hennessy, Annemarie; Prins, John; Twigg, Stephen M; McLennan, Susan V; McCaughan, Geoffrey W; Bachovchin, William W; Gorrell, Mark D

    2013-01-01

    The protease fibroblast activation protein (FAP) is a specific marker of activated mesenchymal cells in tumour stroma and fibrotic liver. A specific, reliable FAP enzyme assay has been lacking. FAP's unique and restricted cleavage of the post proline bond was exploited to generate a new specific substrate to quantify FAP enzyme activity. This sensitive assay detected no FAP activity in any tissue or fluid of FAP gene knockout mice, thus confirming assay specificity. Circulating FAP activity was ∼20- and 1.3-fold less in baboon than in mouse and human plasma, respectively. Serum and plasma contained comparable FAP activity. In mice, the highest levels of FAP activity were in uterus, pancreas, submaxillary gland and skin, whereas the lowest levels were in brain, prostate, leukocytes and testis. Baboon organs high in FAP activity included skin, epididymis, bladder, colon, adipose tissue, nerve and tongue. FAP activity was greatly elevated in tumours and associated lymph nodes and in fungal-infected skin of unhealthy baboons. FAP activity was 14- to 18-fold greater in cirrhotic than in non-diseased human liver, and circulating FAP activity was almost doubled in alcoholic cirrhosis. Parallel DPP4 measurements concorded with the literature, except for the novel finding of high DPP4 activity in bile. The new FAP enzyme assay is the first to be thoroughly characterised and shows that FAP activity is measurable in most organs and at high levels in some. This new assay is a robust tool for specific quantitation of FAP enzyme activity in both preclinical and clinical samples, particularly liver fibrosis.

  1. Cell motility and local viscoelasticity of fibroblasts.

    PubMed

    Park, S; Koch, D; Cardenas, R; Käs, J; Shih, C K

    2005-12-01

    Viscoelastic changes of the lamellipodial actin cytoskeleton are a fundamental element of cell motility. Thus, the correlation between the local viscoelastic properties of the lamellipodium (including the transitional region to the cell body) and the speed of lamellipodial extension is studied for normal and malignantly transformed fibroblasts. Using our atomic force microscopy-based microrheology technique, we found different mechanical properties between the lamellipodia of malignantly transformed fibroblasts (H-ras transformed and SV-T2 fibroblasts) and normal fibroblasts (BALB 3T3 fibroblasts). The average elastic constants, K, in the leading edge of SV-T2 fibroblasts (0.48 +/- 0.51 kPa) and of H-ras transformed fibroblasts (0.42 +/- 0.35 kPa) are significantly lower than that of BALB 3T3 fibroblasts (1.01 +/- 0.40 kPa). The analysis of time-lapse phase contrast images shows that the decrease in the elastic constant, K, for malignantly transformed fibroblasts is correlated with the enhanced motility of the lamellipodium. The measured mean speeds are 6.1 +/- 4.5 microm/h for BALB 3T3 fibroblasts, 13.1 +/- 5.2 microm/h for SV-T2 fibroblasts, and 26.2 +/- 11.5 microm/h for H-ras fibroblasts. Furthermore, the elastic constant, K, increases toward the cell body in many instances which coincide with an increase in actin filament density toward the cell body. The correlation between the enhanced motility and the decrease in viscoelastic moduli supports the Elastic Brownian Ratchet model for driving lamellipodia extension.

  2. Macrophage-conditioned medium inhibits differentiation-induced Rb phosphorylation in 3T3-L1 preadipocytes

    SciTech Connect

    Yarmo, Michelle N.; Landry, Anne; Molgat, Andre S.D.; Gagnon, AnneMarie; Sorisky, Alexander

    2009-02-01

    This study examines the mechanisms underlying the anti-adipogenic effect of macrophage-secreted products. 3T3-L1 preadipocytes were induced to differentiate over 8 days in medium conditioned by murine J774 macrophages (MacCM). The inhibitory effect on lipid accumulation and expression of adipogenic markers was diminished when addition of MacCM was delayed to day 2 of differentiation. Clonal expansion, an early event required for 3T3-L1 adipogenesis, was reduced in the presence of MacCM (89%; n = 3; p < 0.001), and BrdU incorporation was impaired by 55% (n = 3; p < 0.01). Activation of ERK1/2 was not affected by MacCM, and neither was the expression of p27{sup kip1}, a cyclin-dependent kinase inhibitor. However, phosphorylation of the retinoblastoma protein (Rb), required for cell cycle progression, was impaired by MacCM (94% inhibition; n = 3; p < 0.01). Differentiation-dependent expression, nuclear localization, and DNA binding ability of C/EBP{beta} were not inhibited by MacCM. Alterations in cell cycle-associated proteins may be important with respect to the anti-adipogenic action of MacCM.

  3. Phosphoprotein phosphatase 1CB (PPP1CB), a novel adipogenic activator, promotes 3T3-L1 adipogenesis.

    PubMed

    Cho, Young-Lai; Min, Jeong-Ki; Roh, Kyung Min; Kim, Won Kon; Han, Baek Soo; Bae, Kwang-Hee; Lee, Sang Chul; Chung, Sang J; Kang, Hyo Jin

    2015-11-13

    Understanding the molecular networks that regulate adipogenesis is crucial for gaining insight into obesity and identifying medicinal targets thereof is necessary for pharmacological interventions. However, the identity and molecular actions of activators that promote the early development of adipocytes are still largely unknown. Here, we demonstrate a novel role for phosphoprotein phosphatase 1CB (PPP1CB) as a potent adipogenic activator that promotes adipocyte differentiation. PPP1CB expression increased in vitro during the early phase of 3T3-L1 adipogenesis and in the murine model of high-fat diet-induced obesity. Depletion of PPP1CB dramatically suppressed the differentiation of 3T3-L1 cells into mature adipocytes, with a concomitant change in adipocyte marker genes and significantly inhibited clonal expansion. We also showed that knockdown of PPP1CB caused a significant decrease in C/EBPδ expression, which in turn resulted in attenuation of PPARγ, C/EBPα, adiponectin, and aP2. In addition, we elucidated the functional significance of PPP1CB by linking p38 activation to C/EBPδ expression in early adipogenesis. Overall, our findings demonstrate a novel function of PPP1CB in promoting adipogenesis and suggest that PPP1CB may be a promising therapeutic target for treatment of obesity and obesity-related diseases.

  4. Testosterone stimulates glucose uptake and GLUT4 translocation through LKB1/AMPK signaling in 3T3-L1 adipocytes.

    PubMed

    Mitsuhashi, Kazuteru; Senmaru, Takafumi; Fukuda, Takuya; Yamazaki, Masahiro; Shinomiya, Katsuhiko; Ueno, Morio; Kinoshita, Shigeru; Kitawaki, Jo; Katsuyama, Masato; Tsujikawa, Muneo; Obayashi, Hiroshi; Nakamura, Naoto; Fukui, Michiaki

    2016-01-01

    Decreases in serum testosterone concentrations in aging men are associated with metabolic disorders. Testosterone has been reported to increase GLUT4-dependent glucose uptake in skeletal muscle cells and cardiomyocytes. However, studies on glucose uptake occurring in response to testosterone stimulation in adipocytes are currently not available. This study was designed to determine the effects of testosterone on glucose uptake in adipocytes. Glucose uptake was assessed with 2-[(3)H] deoxyglucose in 3T3-L1 adipocytes. GLUT4 translocation was evaluated in plasma membrane (PM) sheets and PM fractions by immunofluorescence and immunoblotting, respectively. Activation of GLUT4 translocation-related protein kinases, including Akt, AMPK, LKB1, CaMKI, CaMKII, and Cbl was followed by immunoblotting. Expression levels of androgen receptor (AR) mRNA and AR translocation to the PM were assessed by real-time RT-PCR and immunoblotting, respectively. The results showed that both high-dose (100 nM) testosterone and testosterone-BSA increased glucose uptake and GLUT4 translocation to the PM, independently of the intracellular AR. Testosterone and testosterone-BSA stimulated the phosphorylation of AMPK, LKB1, and CaMKII. The knockdown of LKB1 by siRNA attenuated testosterone- and testosterone-BSA-stimulated AMPK phosphorylation and glucose uptake. These results indicate that high-dose testosterone and testosterone-BSA increase GLUT4-dependent glucose uptake in 3T3-L1 adipocytes by inducing the LKB1/AMPK signaling pathway.

  5. Effects of Various 5,7-Dihydroxyflavone Analogs on Adipogenesis in 3T3-L1 Cells.

    PubMed

    Nishina, Atsuyoshi; Ukiya, Motohiko; Fukatsu, Makoto; Koketsu, Mamoru; Ninomiya, Masayuki; Sato, Daisuke; Yamamoto, Junpei; Kobayashi-Hattori, Kazuo; Okubo, Takeshi; Tokuoka, Hideyo; Kimura, Hirokazu

    2015-01-01

    We studied the effects of twelve 5,7-dihydroxyflavone analogs on adipogenesis in 3T3-L1 cells. Among the compounds, luteolin, diosmetin, and chrysoeriol partly inhibited adipogenesis by blocking the accumulation of triacylglycerol in the cells. Conversely, tricetin facilitated triacylglycerol accumulation in the cells. The induction of lipogenesis or lipolysis may depend on the number and bonding position of hydroxyl or methoxy groups on the B ring of 5,7-dihydroxyflavone. The mRNA expression levels of adipogenic and lipogenic genes were suppressed by luteolin treatment in the cells, while the mRNA levels of lipolytic genes were not affected. However, the expression levels of the adipogenic, lipogenic, and lipolytic genes, except for adipocyte protein 2 (aP2), were not affected by the addition of tricetin. Moreover, luteolin suppressed glucose transporter type 4 (GLUT4) gene and protein levels. These results indicate that luteolin decreased triacylglycerol levels in 3T3-L1 cells during adipogenesis through the suppression of adipogenic/lipogenic and GLUT4 genes and GLUT4 protein.

  6. Alliin, a garlic (Allium sativum) compound, prevents LPS-induced inflammation in 3T3-L1 adipocytes.

    PubMed

    Quintero-Fabián, Saray; Ortuño-Sahagún, Daniel; Vázquez-Carrera, Manuel; López-Roa, Rocío Ivette

    2013-01-01

    Garlic (Allium sativum L.) has been used to alleviate a variety of health problems due to its high content of organosulfur compounds and antioxidant activity. The main active component is alliin (S-allyl cysteine sulfoxide), a potent antioxidant with cardioprotective and neuroprotective actions. In addition, it helps to decrease serum levels of glucose, insulin, triglycerides, and uric acid, as well as insulin resistance, and reduces cytokine levels. However its potential anti-inflammatory effect is unknown. We examined the effects of alliin in lipopolysaccharide- (LPS-) stimulated 3T3-L1 adipocytes by RT-PCR, Western blot, and microarrays analysis of 22,000 genes. Incubation of cells for 24 h with 100 μmol/L alliin prevented the increase in the expression of proinflammatory genes, IL-6, MCP-1, and Egr-1 in 3T3-L1 adipocytes exposed to 100 ng/mL LPS for 1 h. Interestingly, the phosphorylation of ERK1/2, which is involved in LPS-induced inflammation in adipocytes, was decreased following alliin treatment. Furthermore, the gene expression profile by microarrays evidentiate an upregulation of genes involved in immune response and downregulation of genes related with cancer. The present results have shown that alliin is able to suppress the LPS inflammatory signals by generating an anti-inflammatory gene expression profile and by modifying adipocyte metabolic profile.

  7. Sodium acetate decreases phosphorylation of hormone sensitive lipase in isoproterenol-stimulated 3T3-L1 mature adipocytes.

    PubMed

    Aberdein, Nicola; Schweizer, Michael; Ball, Derek

    2014-04-01

    Lipolysis, the process of hydrolysis of stored triacylglycerol into glycerol and non-esterified fatty acids (NEFA), is reported to be reduced by short chain fatty acids (SCFA) but the mechanism of this inhibition is poorly understood. The aim of this study was to measure the phosphorylation at serine residue 563 of hormone sensitive lipase with and without exposure to sodium acetate. Using the 3T3-L1 cell line, we identified that stimulating the cells with isoproterenol increased phosphorylated hormone sensitive lipase (pHSL) expression by 60% compared with the basal state. In the presence of the SCFA acetate in stimulated cells, pHSL decreased by 15% compared with stimulated cells alone. These results were mirrored by the NEFA release from stimulated cells that had significantly decreased in the presence of sodium acetate after 60 min (from 0.53 µmol mg(-1) protein to 0.41 µmol mg(-1) protein, respectively, P = 0.004); and 180 min (1.73 µmol mg(-1) protein to 1.13 µmol mg(-1) protein, P = 0.020); however, treatment had no effect on glycerol release (P = 0.109). In conclusion, exposure to 4 mM acetate reduced the level of phosphorylation of HSL(SER563) in mature 3T3-L1 adipocytes and led to a significant reduction in NEFA release, although glycerol release was not affected.

  8. Catechin and quercetin attenuate adipose inflammation in fructose-fed rats and in 3T3-L1 adipocytes

    PubMed Central

    Vazquez Prieto, Marcela A.; Bettaieb, Ahmed; Rodriguez Lanzi, Cecilia; Soto, Verónica C.; Perdicaro, Diahann J.; Galmarini, Claudio R.; Haj, Fawaz G.; Miatello, Roberto M.; Oteiza, Patricia I.

    2015-01-01

    Scope This study evaluated the capacity of dietary catechin (C), quercetin (Q) and the combination of both (CQ), to attenuate adipose inflammation triggered by high fructose (HFr) consumption in rats and by tumor necrosis factor alpha (TNFα) in 3T3-L1 adipocytes. Methods and results In rats, HFr consumption for 6 wk caused dyslipidemia, insulin resistance, reduced plasma adiponectin, adiposity, and adipose tissue inflammation. Dietary supplementation with 20 mg/kg/d of C, Q and CQ improved all these parameters. In 3T3-L1 adipocytes, C and Q attenuated TNFα-induced elevated protein carbonyls, increased pro-inflammatory cytokine expression (MCP-1, resistin), and decreased adiponectin. The protective effects of C and Q on adipose inflammation are in part associated with their capacity to: i) decrease the activation of the mitogen activated kinases (MAPKs) JNK and p38; and ii) prevent the downregulation of PPARγ. In summary, C and Q, and to a larger extent the combination of both, attenuated adipose pro-inflammatory signaling cascades and regulated the balance of molecules that improve (adiponectin) or impair (TNFα, MCP-1, resistin) insulin sensitivity. Conclusion Together, these findings suggest that dietary Q and C may have potential benefits in mitigating MetS associated adipose inflammation, oxidative stress, and insulin resistance. PMID:25620282

  9. Resveratrol Metabolites Modify Adipokine Expression and Secretion in 3T3-L1 Pre-Adipocytes and Mature Adipocytes

    PubMed Central

    Eseberri, Itziar; Lasa, Arrate; Churruca, Itziar; Portillo, María P.

    2013-01-01

    Objective Due to the low bioavailability of resveratrol, determining whether its metabolites exert any beneficial effect is an interesting issue. Methods 3T3-L1 maturing pre-adipocytes were treated during differentiation with 25 µM of resveratrol or with its metabolites and 3T3-L1 mature adipocytes were treated for 24 hours with 10 µM resveratrol or its metabolites. The gene expression of adiponectin, leptin, visfatin and apelin was assessed by Real Time RT-PCR and their concentration in the incubation medium was quantified by ELISA. Results Resveratrol reduced mRNA levels of leptin and increased those of adiponectin. It induced the same changes in leptin secretion. Trans-resveratrol-3-O-glucuronide and trans-resveratrol-4′-O-glucuronide increased apelin and visfatin mRNA levels. Trans-resveratrol-3-O-sulfate reduced leptin mRNA levels and increased those of apelin and visfatin. Conclusions The present study shows for the first time that resveratrol metabolites have a regulatory effect on adipokine expression and secretion. Since resveratrol has been reported to reduce body-fat accumulation and to improve insulin sensitivity, and considering that these effects are mediated in part by changes in the analyzed adipokines, it may be proposed that resveratrol metabolites play a part in these beneficial effects of resveratrol. PMID:23717508

  10. Alliin, a Garlic (Allium sativum) Compound, Prevents LPS-Induced Inflammation in 3T3-L1 Adipocytes

    PubMed Central

    Quintero-Fabián, Saray; Ortuño-Sahagún, Daniel; Vázquez-Carrera, Manuel; López-Roa, Rocío Ivette

    2013-01-01

    Garlic (Allium sativum L.) has been used to alleviate a variety of health problems due to its high content of organosulfur compounds and antioxidant activity. The main active component is alliin (S-allyl cysteine sulfoxide), a potent antioxidant with cardioprotective and neuroprotective actions. In addition, it helps to decrease serum levels of glucose, insulin, triglycerides, and uric acid, as well as insulin resistance, and reduces cytokine levels. However its potential anti-inflammatory effect is unknown. We examined the effects of alliin in lipopolysaccharide- (LPS-) stimulated 3T3-L1 adipocytes by RT-PCR, Western blot, and microarrays analysis of 22,000 genes. Incubation of cells for 24 h with 100 μmol/L alliin prevented the increase in the expression of proinflammatory genes, IL-6, MCP-1, and Egr-1 in 3T3-L1 adipocytes exposed to 100 ng/mL LPS for 1 h. Interestingly, the phosphorylation of ERK1/2, which is involved in LPS-induced inflammation in adipocytes, was decreased following alliin treatment. Furthermore, the gene expression profile by microarrays evidentiate an upregulation of genes involved in immune response and downregulation of genes related with cancer. The present results have shown that alliin is able to suppress the LPS inflammatory signals by generating an anti-inflammatory gene expression profile and by modifying adipocyte metabolic profile. PMID:24453416

  11. Benzyl butyl phthalate promotes adipogenesis in 3T3-L1 preadipocytes: A High Content Cellomics and metabolomic analysis.

    PubMed

    Yin, Lei; Yu, Kevin Shengyang; Lu, Kun; Yu, Xiaozhong

    2016-04-01

    Benzyl butyl phthalate (BBP) has been known to induce developmental and reproductive toxicity. However, its association with dysregulation of adipogenesis has been poorly investigated. The present study aimed to examine the effect of BBP on the adipogenesis, and to elucidate the underlying mechanisms using the 3T3-L1 cells. The capacity of BBP to promote adipogenesis was evaluated by multiple staining approaches combined with a High Content Cellomics analysis. The dynamic changes of adipogenic regulatory genes and proteins were examined, and the metabolite profile was identified using GC/MC based metabolomic analysis. The High Content analysis showed BBP in contrast with Bisphenol A (BPA), a known environmental obesogen, increased lipid droplet accumulation in a similar dose-dependent manner. However, the size of the lipid droplets in BBP-treated cells was significantly larger than those in cells treated with BPA. BBP significantly induced mRNA expression of transcriptional factors C/EBPα and PPARγ, their downstream genes, and numerous adipogenic proteins in a dose and time-dependent manner. Furthermore, GC/MC metabolomic analysis revealed that BBP exposure perturbed the metabolic profiles that are associated with glyceroneogenesis and fatty acid synthesis. Altogether, our current study clearly demonstrates that BBP promoted the differentiation of 3T3-L1 through the activation of the adipogenic pathway and metabolic disturbance.

  12. Menaquinone-7 regulates the expressions of osteocalcin, OPG, RANKL and RANK in osteoblastic MC3T3E1 cells.

    PubMed

    Katsuyama, Hironobu; Otsuki, Takemi; Tomita, Masafumi; Fukunaga, Masao; Fukunaga, Tatsushige; Suzuki, Nobuo; Saijoh, Kiyofumi; Fushimi, Shigeko; Sunami, Shigeo

    2005-02-01

    Epidemiological studies show that dietary intake of natto, which contains significant amount of vitamin K(2), reduces the risk of bone formation loss. However, many confounding factors, such as calcium and isoflavone, are found in natto, because it is made from soybeans. In this study, the direct effects of MK-7, a vitamin K(2) analogue, were assessed in osteoblasts. Osteoblastic MC3T3E1 cells were cultured with or without MK-7 for 10 days and the number of cells was calculated. The cell count was not different between MK-7 treated cells and control cells for 1, 2, and 4 days. However, it was significantly suppressed in MK-7 treated cells at 10 days, suggesting that MK-7 suppressed cell proliferation. Real-time PCR analysis showed that mRNAs of osteocalcin (OC), osteoprotegerin (OPG), and the receptor activator of the NFkappaB ligand (RANKL) were induced after MK-7 administration to the culture medium. RANK mRNA expression was also enhanced by MK-7 administration. Immunocytochemical analysis showed that MK-7 increased the protein levels of OC and RANKL. RANK protein was also enhanced, but this induction was suppressed by anti-RANK antibody administration. This suppression was recovered when anti-RANK antibody and MK-7 were administered. These observations suggest that MK-7 may directly affect MC3T3E1 cells and stimulate osteoblastic differentiation, not proliferation.

  13. Modification of heterotrimeric G-proteins in Swiss 3T3 cells stimulated with Pasteurella multocida toxin.

    PubMed

    Babb, Rebecca C; Homer, Karen A; Robbins, Jon; Lax, Alistair J

    2012-01-01

    Many bacterial toxins covalently modify components of eukaryotic signalling pathways in a highly specific manner, and can be used as powerful tools to decipher the function of their molecular target(s). The Pasteurella multocida toxin (PMT) mediates its cellular effects through the activation of members of three of the four heterotrimeric G-protein families, G(q), G(12) and G(i). PMT has been shown by others to lead to the deamidation of recombinant Gα(i) at Gln-205 to inhibit its intrinsic GTPase activity. We have investigated modification of native Gα subunits mediated by PMT in Swiss 3T3 cells using 2-D gel electrophoresis and antibody detection. An acidic change in the isoelectric point was observed for the Gα subunit of the G(q) and G(i) families following PMT treatment of Swiss 3T3 cells, which is consistent with the deamidation of these Gα subunits. Surprisingly, PMT also induced a similar modification of Gα(11), a member of the G(q) family of G-proteins that is not activated by PMT. Furthermore, an alkaline change in the isoelectric point of Gα(13) was observed following PMT treatment of cells, suggesting differential modification of this Gα subunit by PMT. G(s) was not affected by PMT treatment. Prolonged treatment with PMT led to a reduction in membrane-associated Gα(i), but not Gα(q). We also show that PMT inhibits the GTPase activity of G(q).

  14. Suppression of lipin-1 expression increases monocyte chemoattractant protein-1 expression in 3T3-L1 adipocytes

    SciTech Connect

    Takahashi, Nobuhiko; Hiranaka, Natsumi; Suzuki, Takeshi; Yui, Tomoo; Akanuma, Masayasu; Oka, Kazuya; Kanazawa, Kaoru; Yoshida, Mika; Naito, Sumiyoshi; Fujiya, Mikihiro; Kohgo, Yutaka

    2011-11-11

    Highlights: Black-Right-Pointing-Pointer Lipin-1 affects lipid metabolism, adipocyte differentiation, and transcription. Black-Right-Pointing-Pointer Adipose lipin-1 expression is reduced in obesity. Black-Right-Pointing-Pointer Lipin-1 depletion using siRNA in 3T3-L1 adipocytes increased MCP-1 expression. Black-Right-Pointing-Pointer Lipin-1 is involved in adipose inflammation. -- Abstract: Lipin-1 plays a crucial role in the regulation of lipid metabolism and cell differentiation in adipocytes. Expression of adipose lipin-1 is reduced in obesity, and metabolic syndrome. However, the significance of this reduction remains unclear. This study investigated if and how reduced lipin-1 expression affected metabolism. We assessed mRNA expression levels of various genes related to adipocyte metabolism in lipin-1-depleted 3T3-L1 adipocytes by introducing its specific small interfering RNA. In lipin-1-depleted adipocytes, mRNA and protein expression levels of monocyte chemoattractant protein-1 (MCP-1) were significantly increased, although the other genes tested were not altered. The conditioned media from the cells promoted monocyte chemotaxis. The increase in MCP-1 expression was prevented by treatment with quinazoline or salicylate, inhibitors of nuclear factor-{kappa}B activation. Because MCP-1 is related to adipose inflammation and systemic insulin resistance, these results suggest that a reduction in adipose lipin-1 in obesity may exacerbate adipose inflammation and metabolism.

  15. SV40 transformation of Swiss 3T3 cells can cause a stable reduction in the calcium requirement for growth

    PubMed Central

    1984-01-01

    A well-characterized SV40-transformed Swiss 3T3 line, SV101, and its revertants were tested for the ability to grow in reduced Ca++ (0.01 mM). Transformants and revertants did not differ from the parent 3T3 line in their Ca++ requirements. All three classes of cells grew less well in low Ca++ than in regular Ca++ (2.0 mM). SV40 transformants were then selected for the ability to grow in reduced Ca++. This new class of transformants was found to grow in 1% serum, grow in soft agarose, have a reorganized actin cytoskeleton, and express viral T antigens, as well as grow well in low Ca++. One of the selected clones was found to be T antigen-negative, yet was transformed in the serum, anchorage, actin, and Ca++ assays. It is possible that this clone was a spontaneous transformant. However, Southern blot analysis revealed the presence of integrated SV40 DNA. In addition, this analysis revealed the absence of an intact early region fragment, which codes for the viral T antigens. One explanation of this result may be that the mechanism of viral transformation for growth in low Ca++ involves viral- host DNA interactions that may not require a fully functional T antigen. In this case SV40 integration may be acting as a nonspecific cellular mutagen. PMID:6094595

  16. Regulatory role of NADPH oxidase in glycated LDL-induced upregulation of plasminogen activator inhibitor-1 and heat shock factor-1 in mouse embryo fibroblasts and diabetic mice.

    PubMed

    Zhao, Ruozhi; Le, Khuong; Moghadasian, Mohammed H; Shen, Garry X

    2013-08-01

    Cardiovascular disease is the predominant cause of death in diabetic patients. Fibroblasts are one of the major types of cells in the heart or vascular wall. Increased levels of glycated low-density lipoprotein (glyLDL) were detected in diabetic patients. Previous studies in our group demonstrated that oxidized LDL increased the amounts of NADPH oxidase (NOX), plasminogen activator inhibitor-1 (PAI-1), and heat shock factor-1 (HSF1) in fibroblasts. This study examined the expression of NOX, PAI-1, and HSF1 in glyLDL-treated wild-type or HSF1-deficient mouse embryo fibroblasts (MEFs) and in leptin receptor-knockout (db/db) diabetic mice. Treatment with physiologically relevant levels of glyLDL increased superoxide and H2O2 release and the levels of NOX4 and p22phox (an essential component of multiple NOX complexes) in wild-type or HSF1-deficient MEFs. The levels of HSF1 and PAI-1 were increased by glyLDL in wild-type MEFs, but not in HSF1-deficient MEFs. Diphenyleneiodonium (a nonspecific NOX inhibitor) or small interfering RNA for p22phox prevented glyLDL-induced increases in the levels of NOX4, HSF1, or PAI-1 in MEFs. The amounts of NOX4, HSF1, and PAI-1 were elevated in hearts of db/db diabetic mice compared to wild-type mice. The results suggest that glyLDL increased the abundance of NOX4 or p22phox via an HSF1-independent pathway, but that of PAI-1 via an HSF1-dependent manner. NOX4 plays a crucial role in glyLDL-induced expression of HSF1 and PAI-1 in mouse fibroblasts. Increased expression of NOX4, HSF1, and PAI-1 was detected in cardiovascular tissue of diabetic mice.

  17. TNF-alpha inhibits 3T3-L1 adipocyte differentiation without downregulating the expression of C/EBPbeta and delta.

    PubMed

    Kurebayashi, S; Sumitani, S; Kasayama, S; Jetten, A M; Hirose, T

    2001-04-01

    Tumor necrosis factor-alpha (TNF-alpha) has been reported to inhibit adipocyte differentiation in which multiple transcription factors including CCAAT enhancer binding proteins (C/EBPs) and peroxisome proliferator-activated receptor (PPAR) gamma play an important role. Induction of C/EBPalpha and PPARgamma, which regulate the expression of many adipocyte-related genes, is dependent on the expression of C/EBPbeta and C/EBPdelta at the early phase of adipocyte differentiation. To elucidate the mechanism by which TNF-alpha inhibits adipocyte differentiation, we examined the effect of TNF-alpha on the expression of these transcription factors in mouse 3T3-L1 preadipocytes. TNF-alpha did not abrogate the induction of C/EBPbeta and C/EBPdelta in response to differentiation stimuli. In fully differentiated adipocytes, TNF-alpha rapidly induced C/EBPbeta and C/EBPdelta, whereas it downregulated the expression of C/EBPalpha and PPARgamma. Our results suggest that TNF-alpha inhibits adipocyte differentiation independently of the downregulation of C/EBPbeta and C/EBPdelta.

  18. Influence of MC3T3-E1 preosteoblast culture on the corrosion of a T6-treated AZ91 alloy

    PubMed Central

    Brooks, Emily K.; Tobias, Menachem E.; Yang, Shuying; Bone, Lawrence B.; Ehrensberger, Mark T.

    2015-01-01

    This study investigated the corrosion of artificially aged T6 heat-treated Mg-9%Al-1%Zn (AZ91) for biomedical applications. Corrosion tests and surface analysis were completed both with and without a monolayer of mouse preosteoblast MC3T3-E1 cells cultured on the sample. Electrochemical impedance spectroscopy (EIS) and inductively coupled plasma mass spectroscopy (ICPMS) were used to explore the corrosion processes after either 3 or 21 days of AZ91 incubation in cell culture medium (CCM). The EIS showed both the inner layer resistance (Rin) and outer layer resistance (Rout) were lower for samples without cells cultured on the surface at 3 days (Rin = 2.64 e4 Ω/cm2, Rout = 140 Ω/cm2) compared to 21 days (Rin = 3.60 e4 Ω/cm2, Rout = 287 Ω/cm2) due to precipitation of magnesium and calcium phosphates over time. Samples with preosteoblasts cultured on the surface had a slower initial corrosion (3 day, Rin = 1.88 e5 Ω/cm2, Rout = 1060 Ω/cm2) which was observed to increase over time (21 day, Rin = 2.99 e4 Ω/cm2, Rout = 287 Ω/cm2). Changes in the corrosion processes were thought to be related to changes in the coverage provided by the cell layer. Our results reveal that the presence of cells and biological processes are able to significantly influence the corrosion rate of AZ91. PMID:25715925

  19. Recombinant C3adesArg/acylation stimulating protein (ASP) is highly bioactive: a critical evaluation of C5L2 binding and 3T3-L1 adipocyte activation.

    PubMed

    Cui, Wei; Lapointe, Marc; Gauvreau, Danny; Kalant, David; Cianflone, Katherine

    2009-10-01

    C5L2 is a recently identified receptor for C5a/C5adesArg, C3a and C3adesArg (ASP). C5a/C5adesArg bind with high affinity, with no identified activation. By contrast, some studies demonstrate C3a/ASP binding/activation to C5L2; others do not. Our aim is to critically evaluate ASP/C3adesArg-C5L2 binding and bioactivity. Cell-associated fluorescent-ASP (Fl-ASP) binding to C5L2 increased from transiently transfectedmouse C5L2. Transfected C5L2-CHO cells had similar results. Endogenous C5L2 expression increased from 3T3-L1 preadipocytes<3T3-L1 adipocytesmouse adipocytes. Non-transfected cells+/-Fl-ASP demonstrated background fluorescence only. In adherent C5L2-HEK (Fl-ASP sorted) and 3T3-L1 cells, blocking with 10% fetal calf serum, protamine sulfate or ovalbumin prevented (125)I-ASP non-specific binding (NSB, no cells), while albumin increased NSB. Binding to non-transfected HEK was comparable to NSB. Optimal specific binding was obtained at 20 degrees C (vs. 4 degrees C) in PBS or serum-free medium with K(d) 83.7+/-23.7 nM (C5L2-HEK), 66+/-15 nM (C5L2-CHO) and 76+/-14.3 nM (3T3-L1 preadipocytes); (125)I-C5a binding had greater affinity. Fl-ASP-C5L2 binding was comparable and concentration dependent (K(d) 31 nM (direct binding) and IC(50) 35 nM (competition binding) regardless of conditions). Recombinant ASP (rASP) produced in modified Escherichia coli Origami (DE3) (allowing folding and disulphide bridge formation), purified under non-denaturing conditions demonstrated 10x greater bioactivity vs. proteolytically derived plasma ASP for triglyceride synthesis and fatty acid uptake in 3T3-L1 adipocytes and preadipocytes while adipose tissue from C5L2 KO mice was non-responsive. rASP stimulation of adipocyte BODIPY-fatty acid uptake demonstrated EC(50) 115+/-93 nM and maximal stimulation of 413+/-33%, p<0.001. ASP binding has distinct characteristics that lead to C5L2 activation and increased

  20. Differentiation of 3T3-L1 preadipocytes with 3-isobutyl-1-methylxanthine and dexamethasone stimulates cell-associated and soluble chondroitin 4-sulfate proteoglycans

    SciTech Connect

    Calvo, J.C.; Rodbard, D.; Katki, A.; Chernick, S.; Yanagishita, M. )

    1991-06-15

    The proteoglycans (cell-associated and culture media) in 3T3-L1 preadipocytes in culture were analyzed before and during differentiation into adipocytes. Cells were metabolically labeled with (35S)sulfate and (3H) glucosamine for 24 h and then extracted and analyzed. There was a 1.68 {plus minus} 0.07-fold increase in the 35S in medium proteoglycan during differentiation, whereas cell-associated proteoglycan radioactivity showed no increase. Analyses of radiolabeled molecules using ion-exchange chromatography, gel filtration, and high performance liquid chromatography after enzymatic or alkaline digestion indicated that all of the 35S label was recovered as two major species of chondroitin 4-sulfate proteoglycans (CSPG-I and CSPG-II) and 7% as heparan sulfate proteoglycan. CSPG-I has a mass of {approximately} 970 kDa with multiple chondroitin sulfate chains (average of 50 kDa each) and a core protein of {approximately} 370 kDa including oligosaccharides. CSPG-II has a mass of 140 kDa with one or two chondroitin sulfate chains (average of 68 kDa each) and a core protein of 41 kDa including oligosaccharides. CSPG-I appears to be similar to versican, whereas CSPG-II is similar to decorin and/or biglycan, found in other fibroblastic cells. Cell differentiation was associated with a specific increase in CSPG-I (4.0 {plus minus} 0.2-fold in media and 3.2 {plus minus} 0.5-fold in the cell-associated form). This system should facilitate study of the functional roles of proteoglycans during growth and differentiation.

  1. Early phosphorylation events following the treatment of Swiss 3T3 cells with bombesin and the mammalian bombesin-related peptide, gastrin-releasing peptide.

    PubMed Central

    Isacke, C M; Meisenhelder, J; Brown, K D; Gould, K L; Gould, S J; Hunter, T

    1986-01-01

    Bombesin and the related mammalian peptides, such as gastrin-releasing peptide (GRP), are potent mitogens for some fibroblast cell lines. Here we have examined the bombesin- and GRP-mediated changes in the phosphorylation of proteins in Swiss 3T3 cells and compared these to the events observed after platelet-derived growth factor (PDGF), epidermal growth factor (EGF) and tumor promoter treatment. In agreement with previous reports, bombesin, GRP and PDGF, but not EGF, increased the activity of protein kinase C. This was assayed by an inhibition of [125I]EGF binding, stimulation in phosphorylation of pp60c-src on serine 12 and stimulation in phosphorylation of a group of 80 kd proteins. The different phosphorylated forms of the 80 kd proteins were examined by tryptic peptide mapping and shown to contain multiple phosphorylation sites. An investigation of the tyrosine phosphorylation events following mitogen treatment revealed a significant difference between PDGF and the bombesin peptides. PDGF treatment caused a marked increase in total cellular phosphotyrosine levels, and tyrosine phosphorylation both of known substrates and its own receptor. In contrast, bombesin and GRP treatments resulted in only a weak or undetectable increase in tyrosine phosphorylation of total cellular protein or known substrates. In this respect bombesin and GRP were more similar to EGF. The fact that the bombesin peptides do not induce a phosphorylation response identical with either PDGF or EGF suggests that there is not a single common signal pathway which is activated by all these mitogens. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 5. Fig. 6. PMID:2431903

  2. Astragaloside IV suppresses transforming growth factor-β1 induced fibrosis of cultured mouse renal fibroblasts via inhibition of the MAPK and NF-κB signaling pathways

    SciTech Connect

    Che, Xiajing; Wang, Qin; Xie, Yuanyuan; Xu, Weijia; Shao, Xinghua; Mou, Shan Ni, Zhaohui

    2015-09-04

    Renal fibrosis, a progressive process characterized by the accumulation of extracellular matrix (ECM) leading to organ dysfunction, is a characteristic of chronic kidney diseases. Among fibrogenic factors known to regulate the renal fibrotic process, transforming growth factor-β (TGF-β) plays a central role. In the present study, we examined the effect of Astragaloside IV (AS-IV), a component of the traditional Chinese medicinal plant Astragalus membranaceus, on the processes associated with renal fibrosis in cultured mouse renal fibroblasts treated with TGF-β1. RT-PCR, western blotting, immunofluorescence staining and collagen assays showed that AS-IV suppressed TGF-β1 induced fibroblast proliferation, transdifferentiation, and ECM production in a dose-dependent manner. Examination of the underlying mechanisms showed that the effect of AS-IV on the inhibition of fibroblast differentiation and ECM formation were mediated by its modulation of the activity of the MAPK and NF-κB signaling pathways. Taken together, our results indicate that AS-IV alleviates renal interstitial fibrosis via a mechanism involving the MAPK and NF-κB signaling pathways and demonstrate the therapeutic potential of AS-IV for the treatment of chronic kidney diseases. - Highlights: • AS-IV suppressed TGF-β1 induced renal fibroblast proliferation. • AS-IV suppressed TGF-β1 induced renal fibroblast transdifferentiation. • AS-IV suppressed TGF-β1 induced ECM production. • AS-IV alleviates renal fibrosis via the MAPK and NF-κB signaling pathways.

  3. Matrix metalloproteinase-8 regulates transforming growth factor-β1 levels in mouse tongue wounds and fibroblasts in vitro.

    PubMed

    Aström, Pirjo; Pirilä, Emma; Lithovius, Riitta; Heikkola, Heidi; Korpi, Jarkko T; Hernández, Marcela; Sorsa, Timo; Salo, Tuula

    2014-10-15

    Matrix metalloproteinase-8 (MMP-8)-deficient mice (Mmp8-/-) exhibit delayed dermal wound healing, but also partly contradicting results have been reported. Using the Mmp8-/- mice we investigated the role of MMP-8 in acute wound healing of the mobile tongue, and analyzed the function of tongue fibroblasts in vitro. Interestingly, in the early phase the tongue wounds of Mmp8-/- mice healed faster than those of wild type (wt) mice resulting in significant difference in wound widths (P=0.001, 6-24h). The Mmp8-/- wounds showed no change in myeloperoxidase positive myeloid cell count, but the level of transforming growth factor (TGF)-β1 was significantly increased (P=0.007) compared to the wt tongues. Fibroblasts cultured from wt tongues expressed MMP-8 and TGF-β1. However, higher TGF-β1 levels were detected in Mmp8-/- fibroblasts, and MMP-8 treatment decreased phosphorylated Smad-2 levels and α-smooth muscle actin expression in these fibroblasts suggesting reduced TGF-β1 signaling. Consistently, a degradation of recombinant TGF-β1 by MMP-8 decreased its ability to activate the signaling cascade in fibroblasts. Moreover, collagen gels with Mmp8-/- fibroblasts reduced more in size. We conclude that MMP-8 regulates tongue wound contraction rate and TGF-β1 levels. In vitro analyses suggest that MMP-8 may also play a role in regulating TGF-β1 signaling of stromal fibroblasts.

  4. Expression of a connexin 43/beta-galactosidase fusion protein inhibits gap junctional communication in NIH3T3 cells

    PubMed Central

    1995-01-01

    Gap junctions contain membrane channels that mediate the cell-to-cell movement of ions, metabolites and cell signaling molecules. As gap junctions are comprised of a hexameric array of connexin polypeptides, the expression of a mutant connexin polypeptide may exert a dominant negative effect on gap junctional communication. To examine this possibility, we constructed a connexin 43 (Cx43)/beta-galactosidase (beta-gal) expression vector in which the bacterial beta-gal protein is fused in frame to the carboxy terminus of Cx43. This vector was transfected into NIH3T3 cells, a cell line which is well coupled via gap junctions and expresses high levels of Cx43. Transfectant clones were shown to express the fusion protein by northern and western analysis. X-Gal staining further revealed that all of the fusion protein containing cells also expressed beta-gal enzymatic activity. Double immunostaining with a beta-gal and Cx43 antibody demonstrated that the fusion protein is immunolocalized to the perinuclear region of the cytoplasm and also as punctate spots at regions of cell-cell contact. This pattern is similar to that of Cx43 in the parental 3T3 cells, except that in the fusion protein expressing cells, Cx43 expression was reduced at regions of cell-cell contact. Examination of gap junctional communication (GJC) with dye injection studies further showed that dye coupling was inhibited in the fusion protein expressing cells, with the largest reduction in coupling found in a clone exhibiting little Cx43 localization at regions of cell-cell contact. When the fusion protein expression vector was transfected into the communication poor C6 cell line, abundant fusion protein expression was observed, but unlike the transfected NIH3T3 cells, no fusion protein was detected at the cell surface. Nevertheless, dye coupling was inhibited in these C6 cells. Based on these observations, we propose that the fusion protein may inhibit GJC by sequestering the Cx43 protein intracellularly

  5. A Novel Regulatory Function of Sweet Taste-Sensing Receptor in Adipogenic Differentiation of 3T3-L1 Cells

    PubMed Central

    Masubuchi, Yosuke; Nakagawa, Yuko; Ma, Jinhui; Sasaki, Tsutomu; Kitamura, Tadahiro; Yamamoto, Yoritsuna; Kurose, Hitoshi; Kojima, Itaru; Shibata, Hiroshi

    2013-01-01

    Background Sweet taste receptor is expressed not only in taste buds but also in nongustatory organs such as enteroendocrine cells and pancreatic beta-cells, and may play more extensive physiological roles in energy metabolism. Here we examined the expression and function of the sweet taste receptor in 3T3-L1 cells. Methodology/Principal Findings In undifferentiated preadipocytes, both T1R2 and T1R3 were expressed very weakly, whereas the expression of T1R3 but not T1R2 was markedly up-regulated upon induction of differentiation (by 83.0 and 3.8-fold, respectively at Day 6). The α subunits of Gs (Gαs) and G14 (Gα14) but not gustducin were expressed throughout the differentiation process. The addition of sucralose or saccharin during the first 48 hours of differentiation considerably reduced the expression of peroxisome proliferator activated receptor γ (PPARγ and CCAAT/enhancer-binding protein α (C/EBPα at Day 2, the expression of aP2 at Day 4 and triglyceride accumulation at Day 6. These anti-adipogenic effects were attenuated by short hairpin RNA-mediated gene-silencing of T1R3. In addition, overexpression of the dominant-negative mutant of Gαs but not YM-254890, an inhibitor of Gα14, impeded the effects of sweeteners, suggesting a possible coupling of Gs with the putative sweet taste-sensing receptor. In agreement, sucralose and saccharin increased the cyclic AMP concentration in differentiating 3T3-L1 cells and also in HEK293 cells heterologously expressing T1R3. Furthermore, the anti-adipogenic effects of sweeteners were mimicked by Gs activation with cholera toxin but not by adenylate cyclase activation with forskolin, whereas small interfering RNA-mediated knockdown of Gαs had the opposite effects. Conclusions 3T3-L1 cells express a functional sweet taste-sensing receptor presumably as a T1R3 homomer, which mediates the anti-adipogenic signal by a Gs-dependent but cAMP-independent mechanism. PMID:23336004

  6. PDGFRalphaalpha signaling is regulated through the primary cilium in fibroblasts.

    PubMed

    Schneider, Linda; Clement, Christian A; Teilmann, Stefan C; Pazour, Gregory J; Hoffmann, Else K; Satir, Peter; Christensen, Søren T

    2005-10-25

    Recent findings show that cilia are sensory organelles that display specific receptors and ion channels, which transmit signals from the extracellular environment via the cilium to the cell to control tissue homeostasis and function. Agenesis of primary cilia or mislocation of ciliary signal components affects human pathologies, such as polycystic kidney disease and disorders associated with Bardet-Biedl syndrome. Primary cilia are essential for hedgehog ligand-induced signaling cascade regulating growth and patterning. Here, we show that the primary cilium in fibroblasts plays a critical role in growth control via platelet-derived growth factor receptor alpha (PDGFRalpha), which localizes to the primary cilium during growth arrest in NIH3T3 cells and primary cultures of mouse embryonic fibroblasts. Ligand-dependent activation of PDGFRalphaalpha is followed by activation of Akt and the Mek1/2-Erk1/2 pathways, with Mek1/2 being phosphorylated within the cilium and at the basal body. Fibroblasts derived from Tg737(orpk) mutants fail to form normal cilia and to upregulate the level of PDGFRalpha; PDGF-AA fails to activate PDGFRalphaalpha and the Mek1/2-Erk1/2 pathway. Signaling through PDGFRbeta, which localizes to the plasma membrane, is maintained at comparable levels in wild-type and mutant cells. We propose that ciliary PDGFRalphaalpha signaling is linked to tissue homeostasis and to mitogenic signaling pathways.

  7. Reactive oxygen species regulatory mechanisms associated with rapid response of MC3T3-E1 cells for vibration stress.

    PubMed

    Zhang, Ling; Gan, Xueqi; Zhu, Zhuoli; Yang, Yang; He, Yuting; Yu, Haiyang

    2016-02-12

    Although many previous studies have shown that refractory period-dependent memory effect of vibration stress is anabolic for skeletal homeostasis, little is known about the rapid response of osteoblasts simply derived from vibration itself. In view of the potential role of reactive oxygen species (ROS) in mediating differentiated activity of osteoblasts, whether and how ROS regulates the rapid effect of vibration deserve to be demonstrated. Our findings indicated that MC3T3-E1 cells underwent decreased gene expression of Runx2, Col-I and ALP and impaired ALP activity accompanied by increased mitochondrial fission immediately after vibration loading. Moreover, we also revealed the involvement of ERK-Drp1 signal transduction in ROS regulatory mechanisms responsible for the rapid effect of vibration stress.

  8. Peroxynitrite activates glucose uptake in 3T3-L1 adipocytes through a PI3-K-dependent mechanism.

    PubMed

    Guzman-Grenfell, Alberto M; Garcia-Macedo, Rebeca; Gonzalez-Martinez, Marco T; Hicks, Juan Jose; Medina-Navarro, Rafael

    2005-01-01

    Peroxynitrite, the product of the reaction between *NO and O2*-, is a strong oxidant and nitrating molecule, and it has been recently consideredas a component of some important signaling pathways. Herein, we report the effect of peroxynitrite on glucose uptake in 3T3-L1 adipocytes. Peroxynitrite stimulated glucose uptake and this effect was inhibited by citochalasin B, indicating the participation of facilitated GLUT transporters. Peroxynitrite-induced glucose uptake was not related to intracellular ATP, nor to external or internal calcium, but it was inhibited by the phosphatidylinositol 3-kinase (PI3-K) inhibitor, wortmannin. Additionally, we also found that peroxynitrite did not activate the insulin receptor nor the PI3-K downstream signaling protein kinase B (PKB/Akt). The dose-dependent inhibitory action of wortmannin suggests that peroxynitrite activates glucose transport without affecting GLUT transporters translocation.

  9. New anthracene glycosides from Rhodomyrtus tomentosa stimulate osteoblastic differentiation of MC3T3-E1 cells.

    PubMed

    Tung, Nguyen Huu; Ding, Yan; Choi, Eun Mi; Van Kiem, Phan; Van Minh, Chau; Kim, Young Ho

    2009-04-01

    Two new anthracene glycosides (1, 2) were isolated from aerial parts of Rhodomyrtus tomentosa, along with three known compounds (3-5). The structures of two new compounds were established to be 4,8,9,10-tetrahydroxy-2,3,7-trimethoxyanthracene-6-O-beta-D-glucopyranoside (1) and 2,4,7,8,9,10-hexahydroxy-3-methoxyanthracene-6-O-alpha-L-rhamnopyranoside (2) based on spectroscopic and chemical methods. Among them, compound 1, 2, and 5 significantly (P<0.05) increased the alkaline phosphatase activity, collagen synthesis, and mineralization of the nodules of MC3T3-E1 osteoblastic cells compared to those of the control, respectively.

  10. Ubiquitin Ligase NEDD4 Regulates PPARγ Stability and Adipocyte Differentiation in 3T3-L1 Cells

    PubMed Central

    Li, Jing Jing; Wang, Ruishan; Lama, Rati; Wang, Xinjiang; Floyd, Z. Elizabeth; Park, Edwards A.; Liao, Francesca-Fang

    2016-01-01

    Peroxisome proliferator–activated receptor-γ (PPARγ) is a ligand-activated nuclear receptor which controls lipid and glucose metabolism. It is also the master regulator of adipogenesis. In adipocytes, ligand-dependent PPARγ activation is associated with proteasomal degradation; therefore, regulation of PPARγ degradation may modulate its transcriptional activity. Here, we show that neural precursor cell expressed developmentally down-regulated protein 4 (NEDD4), an E3 ubiquitin ligase, interacts with the hinge and ligand binding domains of PPARγ and is a bona fide E3 ligase for PPARγ. NEDD4 increases PPARγ stability through the inhibition of its proteasomal degradation. Knockdown of NEDD4 in 3T3-L1 adipocytes reduces PPARγ protein levels and suppresses adipocyte conversion. PPARγ correlates positively with NEDD4 in obese adipose tissue. Together, these findings support NEDD4 as a novel regulator of adipogenesis by modulating the stability of PPARγ. PMID:27917940

  11. Ordered phosphorylation of 40S ribosomal protein S6 after serum stimulation of quiescent 3T3 cells.

    PubMed Central

    Martin-Pérez, J; Thomas, G

    1983-01-01

    The amino acids and tryptic peptides that become phosphorylated in 40S ribosomal protein S6 after serum stimulation of quiescent 3T3 cells were examined by two-dimensional thin-layer electrophoresis. In the maximally phosphorylated form of the protein, most of the phosphate was incorporated into serine and a small amount, into threonine. Digestion of this form of the protein with trypsin revealed 10 major phosphopeptides. All 10 contained phosphoserine and 2 of the 10 also contained phosphothreonine. Next, the five forms of increasingly phosphorylated S6 were individually separated on two-dimensional polyacrylamide gels or total S6 was isolated from cells that were stimulated for only a short time and their phosphotryptic maps were analyzed. The results showed that, as larger amounts of phosphate were added to S6, the phosphopeptides appeared in a specific order. Images PMID:6573662

  12. Stimulatory Effects of Cinnamon Extract (Cinnamomum cassia) during the Initiation Stage of 3T3-L1 Adipocyte Differentiation

    PubMed Central

    Lee, Sang Gil; Siaw, Joanna A.; Kang, Hye Won

    2016-01-01

    Cinnamon (Cinnamomum cassia) has an anti-diabetic effect by possibly increasing the lipid storage capacity of white adipocytes; however, this effect remains controversial. The aim of this study was to examine which stage of adipogenesis is critical for the stimulatory effect of cinnamon in adipogenesis using 3T3-L1 cells. Cells were treated with cinnamon extract during three different stages of adipogenesis. We found that genes related to adipogenesis and lipogenesis were enhanced when cinnamon extract was administered during the initiation stage of differentiation but not when administered during the preadipocyte and post stages of differentiation. At the same time, genes that were involved in the regulation of fatty acid oxidation were unexpectedly upregulated. Taken together, cinnamon may boost lipid storage in white adipocytes and increase the fatty acid oxidation capacity throughout the initiation stage of differentiation, which may be beneficial for the prevention of obesity-induced type II diabetes. PMID:28231178

  13. Characterization of the respiration of 3T3 cells by laser-induced fluorescence during a cyclic heating process

    NASA Astrophysics Data System (ADS)

    Beuthan, J.; Dressler, C.; Zabarylo, U.; Minet, O.

    2010-04-01

    The use of lasers in the near infrared spectral range for laser-induced tumor therapy (LITT) demands a new understanding of the thermal responses to repetitive heat stress. The analysis of laser-induced fluorescence during vital monitoring offers an excellent opportunity to solve many of the related issues in this field. The laser-induced fluorescence of the cellular coenzyme NADH was investigated for its time and intensity behavior under heat stress conditions. Heat was applied to vital 3T3 cells (from 22°C to 50°C) according to a typical therapeutical time regime. A sharp increase in temperature resulted in non-linear time behavior when the concentration of this vital coenzyme changed. There are indications that biological systems have a delayed reaction on a cellular level. These results are therefore important for further dosimetric investigations.

  14. Pseudoginsenoside F11, a Novel Partial PPARγ Agonist, Promotes Adiponectin Oligomerization and Secretion in 3T3-L1 Adipocytes

    PubMed Central

    Wu, Guoyu; Yi, Junyang; Liu, Ling; Wang, Pengcheng; Zhang, Zhijie

    2013-01-01

    PPARγ is a nuclear hormone receptor that functions as a master regulator of adipocyte differentiation and development. Full PPARγ agonists, such as the thiazolidinediones (TZDs), have been widely used to treat type 2 diabetes. However, they are characterized by undesirable side effects due to their strong agonist activities. Pseudoginsenoside F11 (p-F11) is an ocotillol-type ginsenoside isolated from Panax quinquefolium L. (American ginseng). In this study, we found that p-F11 activates PPARγ with modest adipogenic activity. In addition, p-F11 promotes adiponectin oligomerization and secretion in 3T3-L1 adipocytes. We also found that p-F11 inhibits obesity-linked phosphorylation of PPARγ at Ser-273 by Cdk5. Therefore, p-F11 is a novel partial PPARγ agonist, which might have the potential to be developed as a new PPARγ-targeted therapeutics for type 2 diabetes. PMID:24454336

  15. Cultured 3T3L1 adipocytes dispose of excess medium glucose as lactate under abundant oxygen availability

    PubMed Central

    Sabater, David; Arriarán, Sofía; Romero, María del Mar; Agnelli, Silvia; Remesar, Xavier; Fernández-López, José Antonio; Alemany, Marià

    2014-01-01

    White adipose tissue (WAT) produces lactate in significant amount from circulating glucose, especially in obesity;Under normoxia, 3T3L1 cells secrete large quantities of lactate to the medium, again at the expense of glucose and proportionally to its levels. Most of the glucose was converted to lactate with only part of it being used to synthesize fat. Cultured adipocytes were largely anaerobic, but this was not a Warburg-like process. It is speculated that the massive production of lactate, is a process of defense of the adipocyte, used to dispose of excess glucose. This way, the adipocyte exports glucose carbon (and reduces the problem of excess substrate availability) to the liver, but the process may be also a mechanism of short-term control of hyperglycemia. The in vivo data obtained from adipose tissue of male rats agree with this interpretation. PMID:24413028

  16. Sp1 mediates repression of the resistin gene by PPAR{gamma} agonists in 3T3-L1 adipocytes

    SciTech Connect

    Chung, S.S.; Choi, H.H.; Cho, Y.M.; Lee, H.K.; Park, K.S. . E-mail: kspark@snu.ac.kr

    2006-09-15

    Resistin is an adipokine related to obesity and insulin resistance. Expression of the resistin gene is repressed by the treatment of peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}) agonists, thiazolidinediones (TZDs). In this study, we investigated the mechanism by which TZDs inhibit the resistin gene expression. Resistin gene expression was decreased by TZD in fully differentiated 3T3-L1 adipocytes, which was abolished after treatment of cycloheximide (a protein synthesis inhibitor). TZD could not repress the expression of the resistin gene in the presence of mithramycin A (an Sp1 binding inhibitor). Sp1 binding site of the resistin promoter (-122/-114 bp) was necessary for the repression. Further investigation of the effect of TZDs on the modification of Sp1 showed that the level of O-glycosylation of Sp1 was decreased in this process. These results suggest that PPAR{gamma} activation represses the expression of the resistin gene by modulating Sp1 activity.

  17. Membrane voltage, resistance, and channel switching in isolated mouse fibroblasts (L cells): a patch-electrode analysis.

    PubMed Central

    Hosoi, S; Slayman, C L

    1985-01-01

    The whole-cell patch-electrode technique of Fenwick, Marty & Neher (1982) has been applied to single suspension-cultured mouse fibroblasts. Seals in the range of 10-50 G omega were obtained without special cleaning of the cell membranes. Rupture of the membrane patch inside the electrode was accompanied by a shift of measured potential into the range -10 to -25 mV, but in most cases with little change in the recorded resistance. The latter fact implied that the absolute resistance of the cell membrane must be in the same range as the seal resistance and the recorded potential is a poor measure of actual cell membrane potential. Steady-state current-voltage curves (range -160 mV to +80 mV) were generated before and after rupture of the membrane patch, and the difference between these gave (zero-current) membrane potentials of -50 to -75 mV, which represents a leak-corrected estimate of the true cell-membrane potential. The associated slope conductivity of the cell membrane was 5-15 microS/cm2 (assumed smooth-sphere geometry, cells 13-15 microns in diameter) and was K+-dominated. With 0.1 mM (or more) free Ca2+ filling the patch electrode, membrane potentials in the range -60 to -85 mV were observed following patch rupture, with associated slope conductivities of 200-400 microS/cm2, also K+-dominated. Similar voltages and conductivities were observed at the peak of pulse-induced 'hyperpolarizing activation' (Nelson, Peacock, & Minna, 1972), and the two phenomena probably reflect the behaviour of Ca2+-activated K+ channels. Both the pulse-induced conductance and the Ca2+-activated conductance spontaneously decayed, the latter over periods of 5-15 min following patch rupture. Sr2+, Ba2+, and Co2+ could also activate the putative K+ channels, but only Sr2+ really mimicked Ca2+. Co2+ and Ba2+ activated with a delay of several minutes following patch rupture, and deactivated quickly with a small decrease of conductance and a large decrease of membrane potential. Evidently

  18. Depletion of mitoferrins leads to mitochondrial dysfunction and impairment of adipogenic differentiation in 3T3-L1 preadipocytes.

    PubMed

    Chen, Y-C; Wu, Y-T; Wei, Y-H

    2015-01-01

    Dysregulation of iron homeostasis is a potential risk factor for type 2 diabetes mellitus (T2DM) and insulin resistance. Iron transported into mitochondria by mitoferrins is mainly utilized for the biosynthesis of iron-sulfur clusters, heme, and other cofactors. Recent studies revealed that mitochondrial dysfunction leads to impaired adipogenesis and insulin insensitivity in adipocytes. However, it is unknown whether mitochondrial iron import and iron status affect the biogenesis and function of mitochondria during adipogenic differentiation. In this study, we used double knockdown of mitoferrin 1 and mitoferrin 2 (Mfrn1/2) to investigate the role of mitochondrial iron homeostasis in mitochondrial bioenergetic function and adipogenic differentiation. The results showed that depletion of Mfrn1/2 in 3T3-L1 preadipocytes impaired the biosynthesis of iron-sulfur proteins in mitochondria due to a decrease in mitochondrial iron content. This was associated with a decrease in mitochondrial oxygen consumption rate and intracellular ATP level in adipocytes with Mfrn1/2 knockdown. Remarkably, Mfrn1/2 deficiency reduced the expression of adipogenic genes and lipid production during adipogenic differentiation. Moreover, insulin-induced glucose uptake and Akt phosphorylation at the Ser473 residue were decreased concurrently in adipocytes differentiated from 3T3-L1 preadipocytes after knockdown of Mfrn1/2. These findings suggest that dysregulation of mitochondrial iron metabolism elicited by knockdown of Mfrn1/2 results in mitochondrial dysfunction, which culminates in the compromise of differentiation and insulin insensitivity of adipocytes. This scenario may explain the recent findings that iron deficiency or alterations in iron metabolism are associated with the pathogenesis of T2DM.

  19. St. John's wort promotes adipocyte differentiation and modulates NF-κB activation in 3T3-L1 cells.

    PubMed

    Hatano, Tomoko; Sameshima, Yuka; Kawabata, Mami; Yamada, Shizuo; Shinozuka, Kazumasa; Nakabayashi, Toshikatsu; Mizuno, Hideya

    2014-01-01

    St. John's wort (SJW), or Hypericum perforatum, is a perennial herb that has been used in the treatment of depression in several countries. Though its therapeutic effect on depression has been extensively studied, its influence on metabolic syndrome is yet to be fully characterized. Therefore, we investigated the effect of SJW extract on adipocyte differentiation and its anti-inflammatory effects by using 3T3-L1 preadipocytes. Oil Red O staining indicated that SJW promotes adipocyte differentiation, while immunoblots indicated that SJW increases the expression of peroxisome proliferator activated receptor γ (PPARγ), a nuclear receptor regulating adipocyte differentiation, and adiponectin, an anti-inflammatory adipokine. Furthermore, the anti-inflammatory activity of SJW was demonstrated by its inhibition of the activation of nuclear factor-κB (NF-κB), an inflammatory transcription factor. Stimulation of mature 3T3-L1 adipocytes by tumor necrosis factor-α (TNF-α) decreased the expression of the NF-κB inhibitor IκBα, and increased its phosphorylation. Treatment with SJW further decreased the TNF-α-induced perturbation in IκBα expression and phosphorylation, which indicated that SJW mediated the inhibition of NF-κB activation. In addition, SJW decreased the TNF-α-induced increase in the mRNA levels of pro-inflammatory adipokines, interleukin-6 (IL-6), and monocyte chemoattractant protein-1 (MCP-1). Collectively, our results indicate that SJW treatment could promote adipocyte differentiation probably through its anti-inflammatory activity, which in turn suggests that SJW has the potential to minimize the risk factors of metabolic syndrome.

  20. Low-Dose Bisphenol-A Impairs Adipogenesis and Generates Dysfunctional 3T3-L1 Adipocytes.

    PubMed

    Ariemma, Fabiana; D'Esposito, Vittoria; Liguoro, Domenico; Oriente, Francesco; Cabaro, Serena; Liotti, Antonietta; Cimmino, Ilaria; Longo, Michele; Beguinot, Francesco; Formisano, Pietro; Valentino, Rossella

    2016-01-01

    Environmental endocrine disruptors (EDCs), including bisphenol-A (BPA), have been recently involved in obesity and diabetes by dysregulating adipose tissue function. Our aim was to examine whether prolonged exposure to low doses of BPA could affect adipogenesis and adipocyte metabolic functions. Therefore, 3T3-L1 pre-adipocytes were cultured for three weeks with BPA 1 nM to mimic human environmental exposure. We evaluated BPA effect on cell proliferation, differentiation, gene expression and adipocyte metabolic function. BPA significantly increased pre-adipocyte proliferation (p<0.01). In 3T3-L1 adipocytes differentiated in the presence of BPA, the expression of Peroxisome proliferator-activated receptor gamma (PPARγ), Fatty Acid Binding Protein 4/Adipocyte Protein 2 (FABP4/AP2) and CCAAT/enhancer binding protein (C/EBPα) was increased by 3.5, 1.5 and 3 folds, respectively. Mature adipocytes also showed a significant increase in lipid accumulation (p<0.05) and alterations of insulin action, with significant reduction in insulin-stimulated glucose utilization (p<0.001). Moreover, in mature adipocytes, mRNA levels of Leptin, interleukin-6 (IL6) and interferon-γ (IFNγ) were significantly increased (p<0.05). In conclusion, BPA prolonged exposure at low doses, consistent with those found in the environment, may affect adipocyte differentiation program, enhancing pre-adipocyte proliferation and anticipating the expression of the master genes involved in lipid/glucose metabolism. The resulting adipocytes are hypertrophic, with impaired insulin signaling, reduced glucose utilization and increased pro-inflammatory cytokine expression. Thus, these data supported the hypothesis that BPA exposure, during critical stages of adipose tissue development, may cause adipocyte metabolic dysfunction and inflammation, thereby increasing the risk of developing obesity-related diseases.