Establishment of a matrix‐associated transepithelial resistance invasion assay to precisely measure the invasive potential of synovial fibroblasts

Wunrau, C; Schnaeker, Eva‐Maria; Freyth, Katharina; Pundt, Noreen; Wendholt, Doreen; Neugebauer, Katja; Hansen, Uwe; Pap, Thomas; Dankbar, Berno

doi:10.1002/art.24782

Cited by 13 publications

(9 citation statements)

References 14 publications

(14 reference statements)

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“…Investigation of synovial fibroblast invasion in vitro was performed using the recently established matrix associated transepithelial resistance invasion (MATRIN) assay as described (24). This is a highly sensitive electrophysiological technique that is based on the …”

Section: Cell Invasion Assaymentioning

confidence: 99%

Epigenetics and rheumatoid arthritis: The role of SENP1 in the regulation of MMP-1 expression

Maciejewska-Rodrigues¹,

Karouzakis²,

Strietholt³

et al. 2010

Journal of Autoimmunity

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The aggressive phenotype of RA synovial fibroblasts (RASF) is characterised by the increased expression of matrix metalloproteinase (MMP)-1 as well as the small ubiquitin like modifier (SUMO)-1 and decreased expression of SUMO-specific protease SENP1. Since we showed an increased activity of acetyltransferases in this autoimmune disease, we wanted to analyse whether this affects the expression of MMP-1 and can be reversed by the reconstitution of SENP1.In RASF, the acetylation of histone H4 was significantly increased in the distal region of the MMP-1 promoter by 274 ± 36% compared to OASF. Most interestingly, overexpression of SENP1 in RASF decreased acetylation specifically in this region by 51 ± 0.5% and globally by 73 ± 11%. Furthermore, the overexpression of SENP1 resulted in a downregulation of MMP-1 at both the mRNA (58  7%) and protein levels (28 ± 6%), significantly reduced the invasiveness of RASF (from 34  9% to 2  2%) and led to an accumulation of histone deacetylase 4 (HDAC4) on the MMP-1 promoter (197 ± 36%). Interestingly, SENP1 failed to modulate the expression of MMP-1 in the cells silenced for HDAC4. This is the first study linking the SUMOylation pathway and the production of MMP-1 to an epigenetic control mechanism mediated through histone acetylation which has a functional consequence for the invasiveness of RASF.

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Section: Cell Invasion Assaymentioning

confidence: 99%

Epigenetics and rheumatoid arthritis: The role of SENP1 in the regulation of MMP-1 expression

Maciejewska-Rodrigues¹,

Karouzakis²,

Strietholt³

et al. 2010

Journal of Autoimmunity

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“…As expected, lentiviral transduced cells produced persistently high levels of TIMP3. In order to approve activity of lentivirally expressed TIMP3, we used our previously established invasion assay (MATRIN, [28]) to assess whether TIMP3 is able to inhibit MMP-mediated cell invasion. In accordance with studies demonstrating an inhibitory effect of TIMP3 on the invasion of different invasive cancer cells [19], lentiviral overexpression of TIMP3 significantly inhibited the invasiveness of Cal78 cells, indicating the expression of functional active TIMP3.…”

Section: Discussionmentioning

confidence: 99%

“…For the determination of the invasiveness of transduced Cal78 cells the cell-based matrix-associated transepithelial resistance invasion (MATRIN) assay was performed as described previously [28]. In short, an epithelial MDCK–C7 cell monolayer that develops a high transepithelial electrical resistance (TEER) was grown on the reverse side of filter cups (Heidelberg, Germany) within six-well dishes.…”

Section: Methodsmentioning

confidence: 99%

Cell Surface-Bound TIMP3 Induces Apoptosis in Mesenchymal Cal78 Cells through Ligand-Independent Activation of Death Receptor Signaling and Blockade of Survival Pathways

et al. 2013

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BackgroundThe matrix metalloproteinases (MMPs) and their endogenous regulators, the tissue inhibitor of metalloproteinases (TIMPs 1–4) are responsible for the physiological remodeling of the extracellular matrix (ECM). Among all TIMPs, TIMP3 appears to play a unique role since TIMP3 is a secreted protein and, unlike the other TIMP family members, is tightly bound to the ECM. Moreover TIMP3 has been shown to be able to induce apoptotic cell death. As little is known about the underlying mechanisms, we set out to investigate the pro-apoptotic effect of TIMP3 in human mesenchymal cells.Methodology/Principal FindingsLentiviral overexpression of TIMP3 in mesenchymal cells led to a strong dose-dependent induction of ligand-independent apoptosis as reflected by a five-fold increase in caspase 3 and 7 activity compared to control (pLenti6/V5-GW/lacZ) or uninfected cells, whereas exogenous TIMP3 failed to induce apoptosis. Concordantly, increased cleavage of death substrate PARP and the caspases 3 and 7 was observed in TIMP3 overexpressing cultures. Notably, activation of caspase-8 but not caspase-9 was observed in TIMP3-overexpressing cells, indicating a death receptor-dependent mechanism. Moreover, overexpression of TIMP3 led to a further induction of apoptosis after stimulation with TNF-alpha, FasL and TRAIL. Most interestingly, TIMP3-overexpression was associated with a decrease in phosphorylation of cRaf, extracellular signal-regulated protein kinase (Erk1/2), ribosomal S6 kinase (RSK1) and Akt and serum deprivation of TIMP3-overexpressing cells resulted in a distinct enhancement of apoptosis, pointing to an impaired signaling of serum-derived survival factors. Finally, heparinase treatment of heparan sulfate proteoglycans led to the release of TIMP3 from the surface of overexpressing cells and to a significant decrease in apoptosis indicating that the binding of TIMP3 is necessary for apoptosis induction.ConclusionThe results demonstrate that exclusively cell surface-bound endogenous TIMP3 induces apoptosis in mesenchymal Cal78 cells through ligand-independent activation of death receptor signaling and blockade of survival signaling pathways.

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“…The invasive process has been studied in vitro in a transwell system with Matrigel, a method that was designed for the study of metastasis (Tolboom et al, 2002). More recently, a matrix-associated transepithelial resistance invasion (MATRIN) assay was developed to measure the rate of invasiveness from the breakdown of the electrical resistance generated by an epithelial monolayer (Wunrau et al, 2009). This system provides a means of directly assessing the participation of a particular factor in the ability of cells to scatter through the matrix.…”

Section: Hurdles In the Study Of Rheumatoid Synovial Fibroblasts' Feamentioning

confidence: 99%

Innate Mechanisms of Synovitis – Fibrin Deposits Contribute to Invasion

Sánchez‐Pernaute¹,

Gabucio²,

Jüngel³

et al. 2012

Rheumatoid Arthritis - Etiology, Consequences and Co-Morbidities

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Establishment of a matrix‐associated transepithelial resistance invasion assay to precisely measure the invasive potential of synovial fibroblasts

Cited by 13 publications

References 14 publications

Epigenetics and rheumatoid arthritis: The role of SENP1 in the regulation of MMP-1 expression

Epigenetics and rheumatoid arthritis: The role of SENP1 in the regulation of MMP-1 expression

Cell Surface-Bound TIMP3 Induces Apoptosis in Mesenchymal Cal78 Cells through Ligand-Independent Activation of Death Receptor Signaling and Blockade of Survival Pathways

Innate Mechanisms of Synovitis – Fibrin Deposits Contribute to Invasion

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