Abstract
Matrix metalloproteinase (MMP)-13 has a pivotal, rate-limiting function in cartilage remodeling and degradation due to its specificity for cleaving type II collagen. The proximal MMP13 promoter contains evolutionarily conserved E26 transformation-specific sequence binding sites that are closely flanked by AP-1 and Runx2 binding motifs, and interplay among these and other factors has been implicated in regulation by stress and inflammatory signals. Here we report that ELF3 directly controls MMP13 promoter activity by targeting an E26 transformation-specific sequence binding site at position -78 bp and by cooperating with AP-1. In addition, ELF3 binding to the proximal MMP13 promoter is enhanced by IL-1β stimulation in chondrocytes, and the IL-1β-induced MMP13 expression is inhibited in primary human chondrocytes by siRNA-ELF3 knockdown and in chondrocytes from Elf3(-/-) mice. Further, we found that MEK/ERK signaling enhances ELF3-driven MMP13 transactivation and is required for IL-1β-induced ELF3 binding to the MMP13 promoter, as assessed by chromatin immunoprecipitation. Finally, we show that enhanced levels of ELF3 co-localize with MMP13 protein and activity in human osteoarthritic cartilage. These studies define a novel role for ELF3 as a procatabolic factor that may contribute to cartilage remodeling and degradation by regulating MMP13 gene transcription.
Highlights
Research, Cambridge, Massachusetts 02140, the **Harvard School of Dental Medicine, Boston, Massachusetts 02115, and the ‡‡Department of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, New York 11794-5215
E74-like factor 3 (ELF3) Is a Novel Regulator of MMP13 Expression in Human and Murine Primary Chondrocytes—ELF3 expression is induced by inflammatory cytokines, including IL-1, in different cell types in an NF-B (p65/p50)-dependent manner [22, 23, 27], and in previous work, we showed that ELF3 mediates the antianabolic actions of IL-1 in chondrocytes by binding to the COL2A1 promoter and suppressing its activity [27]
We initially screened for genes whose expression depended on ELF3 for their response to IL-1 by performing a TaqMan low density array screen of RNAs isolated from primary human chondrocytes transfected with siRNA-ELF3 or non-targeting siRNA oligonucleotides, which showed that MMP13 was among the genes that were differentially regulated by ELF3 in response to IL-1 stimulation
Summary
Cell Culture—Human primary chondrocytes were isolated, as described [28], from articular cartilage obtained from intact regions of femoral condyles of OA patients undergoing total knee replacement, with approval by the Institutional Review Board and patient consent. Human immortalized T/C-28a2 chondrocytes were cultured in DMEM/Ham’s F-12 containing 10% FBS, as described previously [27]. SiRNA Transfection—Human primary or immortalized chondrocytes were transfected, as described previously [28]. The nontargeting control siRNA (Dharmacon) or siRNAs against ELF3 (Dharmacon) were transfected at a final concentration of 50 nM using Lipofectamine PLUS reagents in serum-free medium. Before treatments with IL-1, cells were incubated in serum-free medium overnight and challenged with the cytokine for the indicated times. Membranes were blocked with 5% nonfat milk in Tris-buffered saline with 0.1% Tween 20 for 1 h at room temperature and incubated with primary antibodies against ELF3 (Abcam), phospho-p38, phospho-SAPK/JNK, phospho-ERK1/2, total p38, total SAPK/JNK, or total ERK1/2 (Cell Signaling); -tubulin (Abcam) was used as loading control. The sense primer for each construct included an artificial SacI site and the antisense
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