Regulation of the cfos serum response element by C/EBPbeta.

Serum response element binding protein (SRE BP) is a novel binding factor present in nuclear extracts of avian and NIH 3T3 fibroblasts which specifically binds to the cfos SRE within a region overlapping and immediately 3' to the CArG box. Site-directed mutagenesis combined with transfection experiments in NIH 3T3 cells showed that binding of both serum response factor (SRF) and SRE BP is necessary for maximal serum induction of the SRE. In this study, we have combined size fractionation of the SRE BP DNA binding activity with C/EBPbeta antibodies to demonstrate that homodimers and heterodimers of p35C/EBPbeta (a transactivator) and p20C/EBPbeta (a repressor) contribute to the SRE BP complex in NIH 3T3 cells. Transactivation of the SRE by p35C/EBPbeta is dependent on SRF binding but not ternary complex factor (TCF) formation. Both p35C/EBPbeta and p20C/EBPbeta bind to SRF in vitro via a carboxy-terminal domain that probably does not include the leucine zipper. Moreover, SRE mutants which retain responsiveness to the TCF-independent signaling pathway bind SRE BP in vitro with affinities that are nearly identical to that of the wild-type SRE, whereas mutant SRE.M, which is not responsive to the TCF-independent pathway, has a nearly 10-fold lower affinity for SRE BP. We propose that C/EBPbeta may play a role in conjunction with SRF in the TCF-independent signaling pathway for SRE activation.

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Loss of the amino-terminal helix-loop-helix domain of the vav proto-oncogene activates its transforming potential

Molecular and Cellular Biology ◽

10.1128/mcb.11.4.1912-1920.1991 ◽

1991 ◽

Vol 11 (4) ◽

pp. 1912-1920

Author(s):

S Katzav ◽

J L Cleveland ◽

H E Heslop ◽

D Pulido

Keyword(s):

Leucine Zipper ◽

Ectopic Expression ◽

Cdna Clones ◽

3T3 Cells ◽

Amino Terminal ◽

Helix Loop Helix ◽

Transforming Activity ◽

Nih 3T3 ◽

Nih 3T3 Cells

vav, a novel human oncogene, was originally generated in vitro by replacement of its normal 5' coding sequences with sequences from pSV2neo DNA, cotransfected as a selectable marker (S. Katzav, D. Martin-Zanca, and M. Barbacid, EMBO J. 8:2283-2290, 1989). The vav proto-oncogene is normally expressed in cells of hematopoietic origin. To determine whether the 5' rearrangement of vav or its ectopic expression in NIH 3T3 cells contributes to its transforming potential, we isolated murine and human proto-vav cDNA clones as well as human genomic clones corresponding to the 5' end of the gene. Normal proto-vav was poorly transforming in NIH 3T3 cells, whereas truncation of its 5' end greatly enhanced its transforming activity. The relative failure of full-length proto-vav cDNA clones to transform NIH 3T3 cells indicates that the transforming activity of vav is not simply due to ectopic expression. Analysis of the predicted amino terminus of the vav proto-oncogene shows that it contains a helix-loop-helix domain and a leucine zipper motif similar to that of myc family proteins, though it lacks a basic region that is usually found adjacent to helix-loop-helix domains. Loss of the helix-loop-helix domain of proto-vav, either by truncation or by rearrangement with pSV2neo sequences, activates its oncogenic potential.

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Loss of the amino-terminal helix-loop-helix domain of the vav proto-oncogene activates its transforming potential.

Molecular and Cellular Biology ◽

10.1128/mcb.11.4.1912 ◽

1991 ◽

Vol 11 (4) ◽

pp. 1912-1920 ◽

Cited By ~ 121

Author(s):

S Katzav ◽

J L Cleveland ◽

H E Heslop ◽

D Pulido

Keyword(s):

Leucine Zipper ◽

Ectopic Expression ◽

Cdna Clones ◽

3T3 Cells ◽

Amino Terminal ◽

Helix Loop Helix ◽

Transforming Activity ◽

Nih 3T3 ◽

Nih 3T3 Cells

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Marine Collagen Peptides Promote Cell Proliferation of NIH-3T3 Fibroblasts via NF-κB Signaling Pathway

Molecules ◽

10.3390/molecules24224201 ◽

2019 ◽

Vol 24 (22) ◽

pp. 4201 ◽

Cited By ~ 5

Author(s):

Fei Yang ◽

Shujie Jin ◽

Yunping Tang

Keyword(s):

Cell Proliferation ◽

Growth Factor ◽

Signaling Pathway ◽

3T3 Cells ◽

Promote Cell Proliferation ◽

Collagen Peptides ◽

Iκb Kinase ◽

Nih 3T3 ◽

Nih 3T3 Cells

Marine collagen peptides (MCPs) with the ability to promote cell proliferation and migration were obtained from the skin of Nibea japonica. The purpose of MCPs isolation was an attempt to convert the by-products of the marine product processing industry to high value-added items. MCPs were observed to contain many polypeptides with molecular weights ≤ 10 kDa and most amino acid residues were hydrophilic. MCPs (0.25–10 mg/mL) also exhibited 2, 2-diphenyl-1-picrylhydrazyl (DPPH), hydroxyl, superoxide anion, and 2′-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) radical scavenging activities. Furthermore, MCPs promoted the proliferation of NIH-3T3 cells. In vitro scratch assays indicated that MCPs significantly enhanced the scratch closure rate and promoted the migration of NIH-3T3 cells. To further determine the signaling mechanism of MCPs, western blotting was used to study the expression levels of nuclear factor kappa-B (NF-κB) p65, IκB kinase α (IKKα), and IκB kinase β (IKKβ) proteins of the NF-κB signaling pathway. Our results indicated protein levels of NF-κB p65, IKKα and IKKβ increased in MCPs-treated NIH-3T3 cells. In addition, MCPs increased the expression of epidermal growth factor (EGF), fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), and transforming growth factor (TGF-β) in NIH-3T3 cells. Therefore, MCPs, a by-product of N. japonica, exhibited potential wound healing abilities in vitro.

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Transactivation of Platelet-Derived Growth Factor Receptor α by the GTPase-Deficient Activated Mutant of Gα12

Molecular and Cellular Biology ◽

10.1128/mcb.26.1.50-62.2006 ◽

2006 ◽

Vol 26 (1) ◽

pp. 50-62 ◽

Cited By ~ 17

Author(s):

Rashmi N. Kumar ◽

Ji Hee Ha ◽

Rangasudhagar Radhakrishnan ◽

Danny N. Dhanasekaran

Keyword(s):

Growth Factor ◽

Cell Growth ◽

Signaling Pathway ◽

Growth Factor Receptor ◽

Platelet Derived Growth Factor ◽

Dominant Negative Mutant ◽

Dependent Manner ◽

3T3 Cells ◽

Nih 3T3 ◽

Nih 3T3 Cells

ABSTRACT The GTPase-deficient, activated mutant of Gα12 (Gα12Q229L, or Gα12QL) induces neoplastic growth and oncogenic transformation of NIH 3T3 cells. Using microarray analysis, we have previously identified a role for platelet-derived growth factor receptor α (PDGFRα) in Gα12-mediated cell growth (R. N. Kumar et al., Cell Biochem. Biophys. 41:63-73, 2004). In the present study, we report that Gα12QL stimulates the functional expression of PDGFRα and demonstrate that the expression of PDGFRα by Gα12QL is dependent on the small GTPase Rho. Our results indicate that it is cell type independent as the transient expression of Gα12QL or the activation of Gα12-coupled receptors stimulates the expression of PDGFRα in NIH 3T3 as well as in human astrocytoma 1321N1 cells. Furthermore, we demonstrate the presence of an autocrine loop involving PDGF-A and PDGFRα in Gα12QL-transformed cells. Analysis of the functional consequences of the Gα12-PDGFRα signaling axis indicates that Gα12 stimulates the phosphatidylinositol 3-kinase (PI3K)-AKT signaling pathway through PDGFR. In addition, we show that Gα12QL stimulates the phosphorylation of forkhead transcription factor FKHRL1 via AKT in a PDGFRα- and PI3K-dependent manner. Since AKT promotes cell growth by blocking the transcription of antiproliferative genes through the inhibitory phosphorylation of forkhead transcription factors, our results describe for the first time a PDGFRα-dependent signaling pathway involving PI3K-AKT-FKHRL1, regulated by Gα12QL in promoting cell growth. Consistent with this view, we demonstrate that the expression of a dominant negative mutant of PDGFRα attenuated Gα12-mediated neoplastic transformation of NIH 3T3 cells.

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Induction of β3-Integrin Gene Expression by Sustained Activation of the Ras-Regulated Raf–MEK–Extracellular Signal-Regulated Kinase Signaling Pathway

Molecular and Cellular Biology ◽

10.1128/mcb.21.9.3192-3205.2001 ◽

2001 ◽

Vol 21 (9) ◽

pp. 3192-3205 ◽

Cited By ~ 94

Author(s):

Douglas Woods ◽

Holly Cherwinski ◽

Eleni Venetsanakos ◽

Arun Bhat ◽

Stephan Gysin ◽

...

Keyword(s):

Cell Surface ◽

Signaling Pathway ◽

Erk Signaling ◽

Integrin Expression ◽

3T3 Cells ◽

Extracellular Signal ◽

Integrin Gene ◽

Β3 Integrin ◽

Nih 3T3 ◽

Nih 3T3 Cells

ABSTRACT Alterations in the expression of integrin receptors for extracellular matrix (ECM) proteins are strongly associated with the acquisition of invasive and/or metastatic properties by human cancer cells. Despite this, comparatively little is known of the biochemical mechanisms that regulate the expression of integrin genes in cells. Here we demonstrate that the Ras-activated Raf–MEK–extracellular signal-regulated kinase (ERK) signaling pathway can specifically control the expression of individual integrin subunits in a variety of human and mouse cell lines. Pharmacological inhibition of MEK1 in a number of human melanoma and pancreatic carcinoma cell lines led to reduced cell surface expression of α6- and β3-integrin. Consistent with this, conditional activation of the Raf-MEK-ERK pathway in NIH 3T3 cells led to a 5 to 20-fold induction of cell surface α6- and β3-integrin expression. Induced β3-integrin was expressed on the cell surface as a heterodimer with αv-integrin; however, the overall level of αv-integrin expression was not altered by Ras or Raf. Raf-induced β3-integrin was observed in primary and established mouse fibroblast lines and in mouse and human endothelial cells. Consistent with previous reports of the ability of the Raf-MEK-ERK signaling pathway to induce β3-integrin gene transcription in human K-562 erythroleukemia cells, Raf activation in NIH 3T3 cells led to elevated β3-integrin mRNA. However, unlike immediate-early Raf targets such as heparin binding epidermal growth factor and Mdm2, β3-integrin mRNA was induced by Raf in a manner that was cycloheximide sensitive. Surprisingly, activation of the Raf-MEK-ERK signaling pathway by growth factors and mitogens had little or no effect on β3-integrin expression, suggesting that the expression of this gene requires sustained activation of this signaling pathway. In addition, despite the robust induction of cell surface αvβ3-integrin expression by Raf in NIH 3T3 cells, such cells display decreased spreading and adhesion, with a loss of focal adhesions and actin stress fibers. These data suggest that oncogene-induced alterations in integrin gene expression may participate in the changes in cell adhesion and migration that accompany the process of oncogenic transformation.

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The Role of Coconut Oil to Increase Expression of MMP-9, PDGF-BB, and TGF-β1 in NIH-3T3 Cell Line

Open Access Macedonian Journal of Medical Sciences ◽

10.3889/oamjms.2019.492 ◽

2019 ◽

Vol 7 (22) ◽

pp. 3733-3736

Author(s):

Dian Ika Perbina Meliala ◽

Jansen Silalahi ◽

Yuandani Yuandani ◽

Linda Margata ◽

Denny Satria

Keyword(s):

Healing Process ◽

Coconut Oil ◽

Wound Healing Process ◽

3T3 Cells ◽

Virgin Coconut Oil ◽

Nih3t3 Cells ◽

Nih 3T3 ◽

Tgf Β1 ◽

Nih 3T3 Cells

AIM: The objective of the study was to evaluate protein expression in NIH 3T3 cells that are treated with virgin coconut oil (VCO) and hydrolysed of virgin coconut oil (HVCO) in vitro. METHODS: Coconut oil used in this study was virgin coconut oil (VCO) and VCO hydrolysed by Rhizomucor miehei (HVCO). NIH 3T3 cells (5x105 cells/well) were seeded in nine wells and incubated for overnight, then divided into three groups. Each group consisted of three wells. Group one without treatment, group two added VCO, and group three added HVCO and then incubated for overnight. One well in each group was added MMP-9, PDGF-BB, and TGF-β1 and incubated one hour. Finally, expressions of MMP-9, PDGF-BB, and TGF-β1 were detected using immunocytochemistry method. RESULTS: The results of the study showed that VCO and HVCO increased protein expressions of MMP-9, PDGF-BB, and TGF-β1. Percentage of MMP-9 expressions treated by VCO increased from 2.89 ± 0.07 to 28.16 ± 0.34, PDGF-BB from 28.11 ± 0.13 to 48.53 ± 0.49, and TGF-β1 from 4.19 ± 0.08 to 18.41 ± 0.54. Percentage of MMP-9 expressions treated by HVCO increased from 2.89 ± 0.07 to 55.40 ± 0.94, PDGF-BB from 28.11 ± 0.13 to 61.65 ± 0.42, and TGF-β1 from 4.19 ± 0.08 to 36.35 ± 0.67. CONCLUSION: VCO and HVCO increase the expression of MMP-9, PDGF-BB, dan TGF-β1 in NIH3T3 cells and therefore, coconut oil active in the wound healing process. HVCO is more than active than VCO.

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Endoplasmic Reticulum Stress Is a Determinant of Retrovirus-Induced Spongiform Neurodegeneration

Journal of Virology ◽

10.1128/jvi.77.23.12617-12629.2003 ◽

2003 ◽

Vol 77 (23) ◽

pp. 12617-12629 ◽

Cited By ~ 59

Author(s):

Derek E. Dimcheff ◽

Srdjan Askovic ◽

Audrey H. Baker ◽

Cedar Johnson-Fowler ◽

John L. Portis

Keyword(s):

Endoplasmic Reticulum ◽

Transmissible Spongiform Encephalopathy ◽

Viral Envelope ◽

Spongiform Encephalopathy ◽

Envelope Gene ◽

Viral Envelope Protein ◽

3T3 Cells ◽

Nih 3T3 ◽

Nih 3T3 Cells

ABSTRACT FrCasE is a mouse retrovirus that causes a fatal noninflammatory spongiform neurodegenerative disease with pathological features strikingly similar to those induced by transmissible spongiform encephalopathy (TSE) agents. Neurovirulence is determined by the sequence of the viral envelope protein, though the specific role of this protein in disease pathogenesis is not known. In the present study, we compared host gene expression in the brain stems of mice infected with either FrCasE or the avirulent virus F43, differing from FrCasE in the sequence of the envelope gene. Four of the 12 disease-specific transcripts up-regulated during the preclinical period represent responses linked to the accumulation of unfolded proteins in the endoplasmic reticulum (ER). Among these genes was CHOP/GADD153, which is induced in response to conditions that perturb endoplasmic reticulum function. In vitro studies with NIH 3T3 cells revealed up-regulation of CHOP as well as BiP, calreticulin, and Grp58/ERp57 in cells infected with FrCasE but not with F43. Immunoblot analysis of infected NIH 3T3 cells demonstrated the accumulation of uncleaved envelope precursor protein in FrCasE- but not F43-infected cells, consistent with ER retention. These results suggest that retrovirus-induced spongiform neurodegeneration represents a protein-folding disease and thus may provide a useful tool for exploring the causal link between protein misfolding and the cytopathology that it causes.

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Transformation of NIH 3T3 cells by cotransfection with c-src and nuclear oncogenes.

Molecular and Cellular Biology ◽

10.1128/mcb.7.10.3582 ◽

1987 ◽

Vol 7 (10) ◽

pp. 3582-3590 ◽

Cited By ~ 15

Author(s):

D Shalloway ◽

P J Johnson ◽

E O Freed ◽

D Coulter ◽

W A Flood

Keyword(s):

3T3 Cells ◽

Focus Formation ◽

Adenovirus E1a ◽

Rous Sarcoma ◽

Cellular Homolog ◽

Sarcoma Virus ◽

Nih 3T3 ◽

Nih 3T3 Cells

pp60c-src, the cellular homolog of the Rous sarcoma virus transforming protein, does not completely transform cells even when present at high levels, but has been shown to be involved in polyomavirus-induced transformation when activated by polyomavirus middle T (pmt)-antigen binding. Here we show that cotransfection, but not solo transfection, of expression plasmids for c-src and either adenovirus E1A, v-myc, c-myc, or the 5' half of polyomavirus large T (pltN) antigen into NIH 3T3 cells induces anchorage-independent growth, enhanced focus formation, and, for pltN cotransfection, tumorigenicity in adult NFS mice. Enhancement of transformation was not observed with polyomavirus small t (pst) antigen. Cotransfection of c-src with pltN induced modification of pp60c-src that altered its electrophoretic mobility and in vivo phosphorylation state and stimulated its in vitro kinase activity. Similar alterations were not seen after c-src-E1A cotransfection, suggesting that at least two different mechanisms of enhancement are involved.

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