Arthritis & Rheumatism, Volume 60,
October 2009 Abstract Supplement
The 2009 ACR/ARHP Annual Scientific Meeting
Philadelphia October 16-21, 2009.
Up-Regulation of the Histone Methyltransferase EZH2 by TNF- in Synovial Fibroblasts Is Mediated through NF-KB/JNK-Activity and Enhances Cell Migration
Trenkmann1, Michelle, Brock2, Matthias, Gay1, Renate E., Kolling3, Christoph, Speich2, Rudolf, Michel1, Beat A., Gay1, Steffen
Center of Experimental Rheumatology, Zurich Center of Integrative Human Physiology (ZIHP), University of Zurich, Zurich, Switzerland
Department of Internal Medicine, University Hospital Zurich, Zurich, Switzerland
Schulthess Clinic, Zurich, Switzerland
Previously we reported that the histone methyltransferase Enhancer of Zeste homologue 2 (EZH2) is up-regulated in rheumatoid arthritis (RA) compared to osteoarthritis (OA) synovial fibroblasts (SF) and is further induced by TNF-a. Beside its function in epigenetic gene silencing, EZH2 has also been shown to play a role in actin polymerization and therefore might contribute to cell migration. Here we addressed the pathways by which TNF-a regulates EZH2 and investigated the impact of EZH2 on migration of SF.
RASF and OASF were pre-treated with the IKK-2 inhibitor sc-514 (50 mM), JNK Inhibitor II (20 mM), ERK Inhibitor II (10 mM) or p38 inhibitor SB203580 (10 mM) for 1h and stimulated with TNF-a (10 ng/ml) (n=4 each). After 48h, expression of EZH2 mRNA was quantified by real-time PCR. The effects of the kinase inhibitors were calculated for each individual experiment as compared to TNF-a-stimulation alone. For a reportergene assay, RASF were transfected with a luciferase vector containing the EZH2 promoter or the EZH2 promoter with a mutated binding site for the transcription factor E2F. Luciferase activity was measured upon stimulation with TNF-a for 24h (n=7). For migration analysis, RASF and OASF (n=5 each) were transfected with a vector containing the coding sequence of EZH2 and cell migration was measured using a modified fluorescence Boyden chamber assay.
Stimulation with TNF-a induced the expression of EZH2 by 4±1.4-fold in RASF and 4.4±2-fold in OASF. The inhibitors for IKK-2 and JNK had both a strong effect on the expression of EZH2 by reducing the TNF-a-mediated up-regulation by 105±14% (IKK2) and 47±17% (JNK) in RASF and 86±17% and 81±4% in OASF (p<0.05 for each). The ERK inhibitor decreased the induction of EZH2 by 77±18% in OASF (p<0.05) while the result in RASF was not significant (26±64%). Inhibition of p38 did not affect the expression of EZH2.
To detect upstream regulators of EZH2, a reportergene assay was performed employing a mutated E2F binding site. TNF-a increased the activity of the EZH2 promoter in RASF by 1.54±0.7-fold whereas mutation of the E2F binding site abolished this effect (0.98±0.3-fold, p<0.05). Moreover, SF transfected with EZH2 revealed an increased migratory activity as compared to the mock control (RASF: 398±191 vs. 351±156 RFU, p<0.09; OASF: 420±95 vs. 286±68 RFU, p<0.04).
Our data emphasize that the TNF-a induced up-regulation of EZH2 in SF is mediated through the NF-kB and JNK pathways and that the EZH2 promoter activity is directly regulated by the transcription factor E2F. Enhancing their migratory activity through the induction and overexpression of EZH2 might contribute to the aggressive phenotype of RASF.
To cite this abstract, please use the following information:
Trenkmann, Michelle, Brock, Matthias, Gay, Renate E., Kolling, Christoph, Speich, Rudolf, Michel, Beat A., et al; Up-Regulation of the Histone Methyltransferase EZH2 by TNF- in Synovial Fibroblasts Is Mediated through NF-KB/JNK-Activity and Enhances Cell Migration [abstract]. Arthritis Rheum 2009;60 Suppl 10 :37