A Novel Laminin-Binding Form of Syndecan-1 (Cell Surface Proteoglycan) Produced by Syndecan-1 cDNA-Transfected NIH-3T3 Cells

1994 ◽  
Vol 215 (1) ◽  
pp. 180-188 ◽  
Author(s):  
Markku Salmivirta ◽  
Markku Mali ◽  
Jyrki Heino ◽  
Jorma Hermonen ◽  
Markku Jalkanen
Keyword(s):  
Cell Surface ◽  
3T3 Cells ◽  
Nih 3T3 ◽  
Cell Surface Proteoglycan ◽  
Laminin Binding ◽  
Nih 3T3 Cells

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

2001 ◽  
Vol 21 (9) ◽  
pp. 3192-3205 ◽  
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.

scholarly journals Mechanism of activation of the ret proto-oncogene by multiple endocrine neoplasia 2A mutations.

1995 ◽  
Vol 15 (3) ◽  
pp. 1613-1619 ◽  
Author(s):  
N Asai ◽  
T Iwashita ◽  
M Matsuyama ◽  
M Takahashi
Keyword(s):  
Cell Surface ◽  
Multiple Endocrine Neoplasia ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
3T3 Cells ◽  
Endocrine Neoplasia ◽  
Transforming Activity ◽  
Nih 3T3 ◽  
Men 2A ◽  
Nih 3T3 Cells

Transforming activity of the c-ret proto-oncogene with multiple endocrine neoplasia (MEN) 2A mutations was investigated by transfection of NIH 3T3 cells. Mutant c-ret genes driven by the simian virus 40 or cytomegalovirus promoter induced transformation with high efficiencies. The 170-kDa Ret protein present on the cell surface of transformed cells was highly phosphorylated on tyrosine and formed disulfide-linked homodimers. This result indicated that MEN 2A mutations induced ligand-independent dimerization of the c-Ret protein on the cell surface, leading to activation of its intrinsic tyrosine kinase. In addition to the MEN 2A mutations, we further introduced a mutation (lysine for asparaginic acid at codon 300 [D300K]) in a putative Ca(2+)-binding site of the cadherin-like domain. When c-ret cDNA with both MEN 2A and D300K mutations was transfected into NIH 3T3 cells, transforming activity drastically decreased. Western blot (immunoblot) analysis revealed that very little of the 170-kDa Ret protein with the D300K mutation was expressed in transfectants while expression of the 150-kDa Ret protein retained in the endoplasmic reticulum was not affected. This result also demonstrated that transport of the Ret protein to the plasma membrane is required for its transforming activity.

scholarly journals Expression and function of chicken integrin beta 1 subunit and its cytoplasmic domain mutants in mouse NIH 3T3 cells.

1990 ◽  
Vol 110 (1) ◽  
pp. 175-184 ◽  
Author(s):  
Y Hayashi ◽  
B Haimovich ◽  
A Reszka ◽  
D Boettiger ◽  
A Horwitz
Keyword(s):  
Cell Surface ◽  
Cytoplasmic Domain ◽  
Alpha Subunit ◽  
Immunofluorescent Staining ◽  
Wild Type ◽  
3T3 Cells ◽  
Chimeric Receptors ◽  
Focal Contacts ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

Chicken integrin beta 1 cDNA and its site-directed mutants were cloned into a mammalian expression vector and introduced into mouse NIH 3T3 cells. Stable transfectants expressing the chicken beta 1 subunit or its site-directed mutants were identified by immunostaining with antibodies specific for the chicken integrin beta 1 subunit. The chicken beta 1 proteins were expressed predominately in the endoplasmic reticulum of transfectants and to a lesser degree in the plasma membrane. Immunoblots and immunoprecipitations, using anti-chicken integrin antibodies, revealed three different sizes of the chicken subunit (90, 95, and 120 kD) and a mouse 140-kD alpha subunit. Immunoprecipitations of the cell surface receptors showed only two peptides, an 120-kD beta 1 and an 140-kD alpha subunit. Antibodies perturbing mouse and chicken integrin-specific cell adhesions were used to demonstrate that the chimeric receptors functioned in adhesion to both laminin and fibronectin. Immunofluorescent staining with antibodies specific for either the chicken or mouse receptors showed that both the wild type and the chimeric receptors localized in focal contacts. Several mutations in the cytoplasmic domain were synthesized and used in the transfection experiments. In one mutant the tyrosine (Tyr 788) in the consensus sequence for phosphorylation was replaced by a phenylalanine. In another the lysine (Lys 757) at the end of the membrane spanning region was replaced by a leucine. Both of these mutants formed dimers with mouse alpha subunits, participated in adhesion, localized in focal contacts, and displayed biological properties indistinguishable from the wild-type transfection. In contrast, mutants containing deletions greater than 5-15 amino acids nearest the carboxyl end in the cytoplasmic domain neither promoted adhesion nor localized in focal contacts. They did, however, form heterodimers that were expressed on the cell surface.

scholarly journals Overexpression of protein kinase C-delta and -epsilon in NIH 3T3 cells induces opposite effects on growth, morphology, anchorage dependence, and tumorigenicity.

1993 ◽  
Vol 268 (9) ◽  
pp. 6090-6096 ◽  
Author(s):  
H. Mischak ◽  
J.A. Goodnight ◽  
W. Kolch ◽  
G. Martiny-Baron ◽  
C. Schaechtle ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
3T3 Cells ◽  
Growth Morphology ◽  
Protein Kinase C Delta ◽  
Kinase C ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

Metabolism of n-6 fatty acids by NIH-3T3 cells transfected with the ras oncogene

1994 ◽  
Vol 139 (1) ◽  
pp. 71-81 ◽  
Author(s):  
R. J. de Antueno ◽  
R. C. Cantrill ◽  
Y-S. Huang ◽  
G. W. Ells ◽  
M. Elliot ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Ras Oncogene ◽  
3T3 Cells ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

scholarly journals Retinoic Acid Down-regulation of Fibronectin and Retinoic Acid Receptor Proteins in NIH-3T3 Cells

1996 ◽  
Vol 271 (11) ◽  
pp. 6502-6508 ◽  
Author(s):  
Giorgio Scita ◽  
Nadine Darwiche ◽  
Eileen Greenwald ◽  
Miriam Rosenberg ◽  
Katerina Politi ◽  
...  
Keyword(s):  
Retinoic Acid ◽  
Retinoic Acid Receptor ◽  
3T3 Cells ◽  
Down Regulation ◽  
Acid Receptor ◽  
Receptor Proteins ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

Investigation of activin A in inflammatory responses of the testis and its role in the development of testicular fibrosis

2019 ◽  
Vol 34 (8) ◽  
pp. 1536-1550 ◽  
Author(s):  
A Christine Kauerhof ◽  
Nour Nicolas ◽  
Sudhanshu Bhushan ◽  
Eva Wahle ◽  
Kate A Loveland ◽  
...  
Keyword(s):  
Protein Expression ◽  
Activin A ◽  
Collagen I ◽  
3T3 Cells ◽  
Qrt Pcr ◽  
3T3 Fibroblasts ◽  
Total Collagen ◽  
Severity Of The Disease ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

Abstract STUDY QUESTION Does activin A contribute to testicular fibrosis under inflammatory conditions? SUMMARY ANSWER Our results show that activin A and key fibrotic proteins are increased in human testicular biopsies with leukocytic infiltrates and impaired spermatogenesis and in murine experimental autoimmune orchitis (EAO) and that activin A stimulates fibrotic responses in peritubular cells (PTCs) and NIH 3T3 fibroblasts. WHAT IS KNOWN ALREADY Fibrosis is a feature of EAO. Activin A, a regulator of fibrosis, was increased in testes of mice with EAO and its expression correlated with severity of the disease. STUDY DESIGN, SIZE, DURATION This is a cross-sectional and longitudinal study of adult mice immunized with testicular homogenate (TH) in adjuvant to induce EAO, collected at 30 (n = 6), 50 (n = 6) and 80 (n = 5) days after first immunization. Age-matched mice injected with adjuvant alone (n = 14) and untreated mice (n = 15) were included as controls. TH-immunized mice with elevated endogenous follistatin, injected with a non-replicative recombinant adeno-associated viral vector carrying a gene cassette of follistatin (rAAV-FST315; n = 3) or vector with an empty cassette (empty vector controls; n = 2) 30 days prior to the first immunization, as well as appropriate adjuvant (n = 2) and untreated (n = 2) controls, were also examined. Human testicular biopsies showing focal inflammatory lesions associated with impaired spermatogenesis (n = 7) were included. Biopsies showing intact spermatogenesis without inflammation, from obstructive azoospermia patients, served as controls (n = 7). Mouse primary PTC and NIH 3T3 fibroblasts were stimulated with activin A and follistatin 288 (FST288) to investigate the effect of activin A on the expression of fibrotic markers. Production of activin A by mouse primary Sertoli cells (SCs) was also investigated. PARTICIPANTS/MATERIALS, SETTING, METHODS Testicular RNA and protein extracts collected from mice at days 30, 50 and 80 after first immunization were used for analysis of fibrotic marker genes and proteins, respectively. Total collagen was assessed by hydroxyproline assay and fibronectin; collagen I, III and IV, α-smooth muscle actin (α-SMA) expression and phosphorylation of suppressor of mothers against decapentaplegic (SMAD) family member 2 were measured by western blot. Immunofluorescence was used to detect fibronectin. Fibronectin (Fn), αSMA (Acta2), collagen I (Col1a2), III (Col3a1) and IV (Col4a1) mRNA in PTC and NIH 3T3 cells treated with activin A and/or FST288 were measured by quantitative RT-PCR (qRT-PCR). Activin A in SC following tumour necrosis factor (TNF) or FST288 stimulation was measured by ELISA. Human testicular biopsies were analysed by qRT-PCR for PTPRC (CD45) and activin A (INHBA), hydroxyproline assay and immunofluorescence. MAIN RESULTS AND THE ROLE OF CHANCE Production of activin A by SC was stimulated by 25 and 50 ng/ml TNF (P < 0.01, P < 0.001, respectively) as compared to untreated cells. INHBA mRNA was increased in human testicular biopsies with leukocytic infiltrates and impaired spermatogenesis, compared with control biopsies (P < 0.05), accompanied by increased total collagen (P < 0.01) and fibronectin deposition. Total testicular collagen (P < 0.0001) and fibronectin protein expression (P < 0.05) were also increased in EAO, and fibronectin expression was correlated with the severity of the disease (r = 0.9028). In animals pre-treated with rAAV-FST315 prior to immunization with TH, protein expression of fibronectin was comparable to control. Stimulation of PTC and NIH 3T3 cells with activin A increased fibronectin mRNA (P < 0.05) and the production of collagen I (P < 0.001; P < 0.01) and fibronectin (P < 0.05). Moreover, activin A also increased collagen IV mRNA (P < 0.05) in PTC, while αSMA mRNA (P < 0.01) and protein (P < 0.0001) were significantly increased by activin A in NIH 3T3 cells. LARGE SCALE DATA N/A. LIMITATIONS, REASONS FOR CAUTION A limited number of human testicular specimens was available for the study. Part of the study was performed in vitro, including NIH 3T3 cells as a surrogate for testicular fibroblasts. WIDER IMPLICATIONS OF THE FINDINGS Resident fibroblasts and PTC may contribute to the progression of testicular fibrosis following inflammation, and activin A is implicated as a key mediator of this process. STUDY FUNDING/COMPETING INTEREST(S) This work was supported by the National Health and Medical Research Council of Australia, the Victorian Government’s Operational Infrastructure Support Program and the International Research Training Group between Justus Liebig University (Giessen) and Monash University (Melbourne) (GRK 1871/1–2) on `Molecular pathogenesis on male reproductive disorders’ funded by the Deutsche Forschungsgemeinschaft and Monash University. The authors declare no competing financial interests.

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