Overexpression of ubiquitin-specific peptidase 15 in systemic sclerosis fibroblasts increases response to transforming growth factor β

Abstract Objective Ubiquitination of proteins leads to their degradation by the proteasome, and is regulated by ubiquitin ligases and substrate-specific ubiquitin-specific peptidases (USPs). The ubiquitination process also plays important roles in the regulation of cell metabolism and cell cycle. Here, we found that the expression of several USPs is increased in SSc tenosynovial and skin biopsies, and we demonstrated that USP inhibition decreases TGF-β signalling in primary fibroblast cell lines. Methods High-density transcriptomic studies were performed using total RNA obtained from SSc tenosynovial samples. Confirmatory immunostaining experiments were performed on tenosynovial and skin samples. In vitro experiments were conducted in order to study the influence of USP modulation on responses to TGF-β stimulation. Results Tenosynovial biopsies from SSc patients overexpressed known disease-associated gene pathways: fibrosis, cytokines and chemokines, and Wnt/TGF-β signalling, but also several USPs. Immunohistochemistry experiments confirmed the detection of USPs in the same samples, and in SSc skin biopsies. Exposure of primary fibroblast cell lines to TGF-β induced USP gene expression. The use of a pan-USP inhibitor decreased SMAD3 phosphorylation, and expression of COL1A1, COL3A1 and fibronectin gene expression in TGF-β-stimulated fibroblasts. The effect of the USP inhibitor resulted in increased SMAD3 ubiquitination, and was blocked by a proteasome inhibitor, thereby confirming the specificity of its action. Conclusion Overexpression of several USPs, including USP15, amplifies fibrotic responses induced by TGF-β, and is a potential therapeutic target in SSc.

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Gene expression profiling in wild-type and metallothionein mutant fibroblast cell lines

Biological Research ◽

10.4067/s0716-97602006000100015 ◽

2006 ◽

Vol 39 (1) ◽

Cited By ~ 5

Author(s):

ÁNGELA D ARMENDÁRIZ ◽

FELIPE OLIVARES ◽

RODRIGO PULGAR ◽

ALEX LOGUINOV ◽

VERÓNICA CAMBIAZO ◽

...

Keyword(s):

Gene Expression ◽

Gene Expression Profiling ◽

Cell Lines ◽

Expression Profiling ◽

Fibroblast Cell ◽

Wild Type ◽

Fibroblast Cell Lines

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In-vitro examination of the biocompatibility of fibroblast cell lines on alloplastic meshes and sterilized polyester mosquito mesh

Hernia ◽

10.1007/s10029-016-1550-2 ◽

2016 ◽

Vol 21 (3) ◽

pp. 407-416 ◽

Cited By ~ 2

Author(s):

R. Wiessner ◽

T. Kleber ◽

N. Ekwelle ◽

K. Ludwig ◽

D.-U. Richter

Keyword(s):

Cell Lines ◽

Fibroblast Cell ◽

In Vitro Examination ◽

Fibroblast Cell Lines

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27 CLONING OF ADULT PIGS USING SCRIPTAID TREATMENT AND PREOVULATORY EMBRYO TRANSFER

Reproduction Fertility and Development ◽

10.1071/rdv23n1ab27 ◽

2011 ◽

Vol 23 (1) ◽

pp. 120

Author(s):

M. Albornoz ◽

C. Colato ◽

N. El-Beyrouthi ◽

F. Mellano ◽

A. Mellano ◽

...

Keyword(s):

Embryo Transfer ◽

Cell Lines ◽

Fibroblast Cell ◽

Cloning Efficiency ◽

Cell Stage ◽

Expected Time ◽

Reconstructed Embryos ◽

Adult Cell ◽

Skin Biopsies

There is growing interest in the use of swine in biomedical research. Cloning from cultured somatic cells (SCNT) has been the preferred method to generate genetically modified swine models. In a recent report, swine cloning efficiency was increased by treatment of reconstructed embryos with the inhibitor of deacetylase enzymes Scriptaid (Zhao et al. 2010 Cel. Reprog. 12, 75). Also, the timing of SCNT-embryo transfer with respect to the recipient’s expected time of ovulation was shown to affect cloning efficiency, whereas preovulatory embryo transfer resulted in a higher rate of cloned piglets born compared to postovulatory embryo transfer (Petersen et al. 2008 Cloning Stem Cells 10, 355). Therefore, our objective was to combine Scriptaid treatment and preovulatory embryo transfer in the same protocol for swine cloning. Cumulus–oocyte complexes aspirated from 3- to 6-mm diameter follicles were matured in vitro under standard conditions (Martinez Diaz et al. 2010 Cel. Reprog. 12, 85) and used as host oocytes for SCNT. Fibroblast cell lines were established from skin biopsies collected from 2 adult boars and cultured in DMEM supplemented with 10% FBS and 1% antibiotics. Oocytes were micromanipulated in Tyrode’s lactate-pyruvate-HEPES medium supplemented with 7.5 μg mL–1 cytochalasin B (CB) and electrically fused using a single DC pulse of 1.6 kV cm–1 for 70 μs. Activation was performed using ionomycin (15 μM/5 min) followed by exposure to CB (7.5 μg mL–1) and cyclohexemide (10 μg mL–1) for 5 h in porcine zygote medium (PZM-3; Yoshioka et al. 2002 Biol. Reprod. 66, 112). Reconstructed embryos were exposed to 500 nM Scriptaid for 10 to 12 h starting after ionomycin treatment. Oocytes were then washed and cultured in PZM-3 medium until transfer. Peripubertal recipient gilts were synchronized by oral administration of altrenogest (Regu-Mate®; 20 mg day–1) for 12 days, followed by 1.000 IU eCG injected on the last day of altrenogest treatment and 500 IU hCG 72 h later. 1-cell stage embryos were transferred into the oviduct after ∼20 h from hCG injection or 22 h before the expected ovulation time. Pregnancy was confirmed and monitored by ultrasonography and parturition was induced by injecting PGF2α at Day 115 of pregnancy. A total of 840 reconstructed embryos were transferred into 10 gilts [average 84 (range 60–110) embryos/gilt]. 4 gilts (40%) were detected to be pregnant 4 weeks after transfer, and 2 (20%) delivered 1 (1100 g) and 2 (950 and 850 g) healthy cloned piglets. The number of embryos transferred to these 2 gilts was 85 and 70. These results confirm that Scriptaid treatment and preovulatory embryo transfer can be applied in the same cloning protocol to produce cloned piglets from adult cell lines. To our knowledge, these are the first cloned pigs produced in Latin America.

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Dual Role for Transforming Growth Factor β-Dependent Signaling in Trypanosoma cruzi Infection of Mammalian Cells

Infection and Immunity ◽

10.1128/iai.68.4.2077-2081.2000 ◽

2000 ◽

Vol 68 (4) ◽

pp. 2077-2081 ◽

Cited By ~ 37

Author(s):

Belinda S. Hall ◽

Miercio A. Pereira

Keyword(s):

Gene Expression ◽

Growth Factor ◽

Trypanosoma Cruzi ◽

Host Cell ◽

Cell Lines ◽

Mammalian Cells ◽

Transforming Growth Factor ◽

Growth Arrest ◽

Transforming Growth Factor Β

ABSTRACT Expression of functional transforming growth factor β (TGF-β) receptors (TβR) is required for the invasion of mammalian cells by the protozoan parasite Trypanosoma cruzi. However, the precise role of this host cell signaling complex in T. cruzi infection is unknown. To investigate the role of the TGF-β signaling pathway, infection levels were studied in the mink lung epithelial cell lines JD1, JM2, and JM3. These cells express inducible mutant TβR1 proteins that cannot induce growth arrest in response to TGF-β but still transmit the signal for TGF-β-dependent gene expression. In the absence of mutant receptor expression, trypomastigotes invaded the cells at a low level. Induction of the mutant receptors caused an increase in infection in all three cell lines, showing that the requirement for TGF-β signaling at invasion can be divorced from TGF-β-induced growth arrest. TGF-β pretreatment of mink lung cells expressing wild-type TβR1 caused a marked enhancement of infection, but no enhancement was seen in JD1, JM2, and JM3 cells, showing that the ability of TGF-β to stimulate infection is associated with growth arrest. Likewise, expression of SMAD7 or SMAD2SA, inhibitors of TGF-β signaling, did not block infection by T. cruzi but did block the enhancement of infection by TGF-β. Taken together, these results show that there is a dual role for TGF-β signaling in T. cruzi infection. The initial invasion of the host cell is independent of both TGF-β-dependent gene expression and growth arrest, but TGF-β stimulation of infection requires a fully functional TGF-β signaling pathway.

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Use of gene expression and alternative splicing signatures to discriminate breast cancer stem cells from fibroblasts.

Journal of Clinical Oncology ◽

10.1200/jco.2012.30.15_suppl.1057 ◽

2012 ◽

Vol 30 (15_suppl) ◽

pp. 1057-1057

Author(s):

David T. Weaver ◽

Irina M Shapiro ◽

Alan G Derr ◽

Daniel Paterson ◽

Jonathan A Pachter

Keyword(s):

Breast Cancer ◽

Gene Expression ◽

Stem Cells ◽

Alternative Splicing ◽

Cell Lines ◽

Basal Cell ◽

Triple Negative ◽

Fibroblast Cell ◽

Breast Cancer Patients ◽

Fibroblast Cell Lines

1057 Background: Tumors frequently contain cancer stem cells (CSCs) or tumor-initiating subpopulations, with an ability to self-renew and regenerate all cell types within the tumor. Basal-like breast cancers exhibit features of CSCs, including expression of surface markers, even though these cells are rare. Given the role of CSCs in the recurrence and spread of cancer, there is an urgent need to develop new therapeutic agents that target CSCs. Development of CSC-targeted drugs will be greatly facilitated by biomarkers that can identify CSCs to aid in patient selection and determination of drug response. Defining the CSCs in tumors is complicated by the high mesenchymal nature of fibroblasts. Analysis of gene expression and alternative splicing patterns in CSCs that are not observed in fibroblasts may provide valuable new CSC-specific markers. Methods: Alternative splicing and gene expression microarray strategies were used to identify selected exons and differentially expressed genes between 10 Basal human breast cancer cell lines and a combination of 12 Luminal and 3 fibroblast cell lines. Q-PCR analysis was conducted to determine candidate CSC gene differential expression between Basal, Luminal, and Fibroblast cells lines. Results: Expression levels of 11 genes were higher and 24 genes were lower in the Basal cell lines versus Luminal or fibroblastic cell lines. Comparison of Basal cell lines to the Luminal/Fibroblast cell lines identified 36 cassette exons that were included, and 26 that were excluded in Basal cell lines. Also, 19 genes were upregulated in Basal cell lines compared to the other groups as detected by Q-PCR. Interestingly, the 19-multigene model defined the Triple Negative Breast Cancer patients that were Likely to Recur under standard chemotherapy with a p = 1.90e-03 and AUC 0.723. Conclusions: Gene and exon marker sets distinguish CSC versus fibroblasts and may be instructive in identifying patients that recur early in Triple Negative Breast Cancer. The CSC-associated RNA signatures identified here will be further refined to develop new CSC-specific diagnostic markers to stratify breast cancer patients and monitor response to novel CSC-targeted therapies.

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