E74-like factor 3 (ELF3) impacts on matrix metalloproteinase 13 (MMP13) transcriptional control in articular chondrocytes under proinflammatory stress

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.

FIGURE 1.

ELF3 regulates IL-1β-induced MMP13 gene expression in human primary articular chondrocytes. Chondrocytes were isolated from articular cartilage from the femoral condyles of five OA patients undergoing total knee replacement, and cultures at passage 1 were examined for the relative levels of (A) ELF3 and (B) MMP13 mRNA in response to IL-1β stimulation for 2–24 h. The same primary chondrocytes were transfected with 50 nm siRNA oligonucleotides against ELF3 (siELF3) or non-targeting siRNA (siCONTROL). Knockdown efficiency was assessed at 72 h by immunoblotting (C) and RT-qPCR (D) at 72 h post-transfection, the cells were stimulated with 1 ng/ml IL-1β for 24 h, and cellular MMP13 mRNA levels were analyzed by RT-qPCR (E). The values were normalized to GAPDH and are shown as mean ± S.E. (error bars). *, p < 0.05; **, p < 0.01; ***, p < 0.001; ELF3 Western blots were reprobed for β-tubulin as a loading reference control.

FIGURE 2.

Effects of ELF3 on MMP13 expression in mouse primary chondrocytes. Mouse primary chondrocytes were isolated from articular cartilage from wild-type 5–6-day-old C57BL/6 mice and incubated with 1 ng/ml IL-1β for the indicated times. IL-1β-induced ELF3 (A) and MMP13 (B) mRNA levels were analyzed by RT-qPCR. C, mouse primary chondrocytes isolated from articular cartilage from wild-type (WT) and Elf3 knock-out (KO) mice were incubated with 1 ng/ml IL-1β for the indicated times. Total RNAs were isolated, and MMP13 mRNA was analyzed by RT-qPCR. Each value was normalized to GAPDH in the same sample and shown as mean ± S.E. (error bars); *, p < 0.05; ***, p < 0.001.

FIGURE 3.

ELF3 protein levels co-localize with increased MMP13 protein levels and activity in human OA cartilage. Sections of knee articular cartilage obtained from OA patients undergoing total knee replacement (n = 10) were Safranin O-stained (a, b, and c) and subjected to evaluation according to the OARSI cartilage OA histopathology grading system. Representative sections are shown. Successive sections were subjected to immunohistochemical staining using antibodies against ELF3 (a.1, b.1, and c.1), MMP13 (a.2, b.2, and c.2) and C1,2C (a.3, b.3, and c.3). Isotype-matched IgG was used as negative control (not shown). Squares indicate areas selected for higher magnification photomicrographs. The arrows indicate some of the areas with positive immunostaining. Note the more diffuse, matrix C1,2C-positive immunostaining with increased OARSI score. Original magnifications were 40× (a, b, and c) and 100× (a.1, b.1, c.1, a.2, b.2, c.2, a.3, b.3, and c.3).

FIGURE 4.

ELF3 transactivates MMP13 and enhances the AP-1-driven MMP13 activation. Schematic representation (not scaled) of the MMP13-luciferase reporter constructs utilized in this study (A). T/C-28a2 cells were co-transfected with 325 ng of the −1602/+20 bp MMP13 promoter and 12.5, 25, or 50 ng of expression vector encoding human ELF3 (B); 25 ng of expression vector encoding human ELF3, alone or in co-transfection with 25 (+) or 50 (++) ng of ELF5 or EHF expression vector (C); or 25 ng of each expression vector encoding ELF3, c-Fos, and c-Jun alone or together (D). Luciferase activities are shown as fold-change; protein input was used to normalize luciferase activities in D. *, p < 0.05; ***, p < 0.001. Error bars, S.E.

FIGURE 5.

ELF3 binds to the proximal MMP13 promoter and transactivates MMP13 via an evolutionarily conserved proximal EBS. T/C-28a2 cells were co-transfected with 325 ng of luciferase reporter constructs spanning −1602/+20, −1007/+20, or −273/+20 bp of the MMP13 promoter along with 25 ng of ELF3 expression vector (A) or wild type −1602/+20 bp or Δ−231/−39 MMP13 reporter construct and 25 ng of expression vector encoding ELF3, RUNX2, c-Fos, or c-Jun (B). Luciferase activities in B were normalized to the protein input. C, T/C-28a2 cells were transfected with ELF3-FLAG or the empty pCDNA3.1 expression vectors for 18 h. Chromatin was cross-linked and enzymatically sheared, and after reverse cross-linking of the DNA-protein complexes, the precleared lysates were incubated overnight at 4 °C with antibodies against FLAG (+) or normal rabbit IgG (−) (top) or against ELF3 (+) or normal rabbit IgG (−) (bottom). The human MMP13 promoter region was PCR-amplified using primers spanning from −157 to −38 bp, and the PCR products were resolved on a 2.5% agarose gel. Data are representative of two independent experiments performed in duplicate. D, T/C-28a2 cells were co-transfected with 25 ng of the ELF3 expression vector and 325 ng of the wild-type −267/+27 bp MMP13 promoter sequence or sequences containing point mutations of the proximal A, B1, or B2 ETS binding sites. *, p < 0.05. Error bars, S.E. IP, immunoprecipitation.

FIGURE 6.

IL-1β enhances the endogenous ELF3 binding to the proximal MMP13 promoter. A, after overnight incubation in serum-free medium, T/C-28a2 cells were incubated with 1 ng/ml IL-1β for 2 h. After stimulation, the chromatin was cross-linked and enzymatically sheared, and after reverse cross-linking of the DNA-protein complexes, the precleared lysates were incubated with antibodies against ELF3 (+) or normal IgG (−) overnight at 4 °C. The human MMP13 promoter region was PCR-amplified using primers spanning from −157 to −38 bp, and the PCR products were resolved on a 2.5% agarose gel. B, ELF3 protein levels were analyzed by Western blotting using cell lysates prepared from T/C-28a2 cells stimulated with vehicle or 1 ng/ml IL-1β for 2 h. C, T/C-28a2 cells were transfected with 50 nm siRNA oligonucleotides against ELF3 (siELF3) or non-targeting siRNA (siCONTROL). At 72 h post-transfection, cells were stimulated with 1 ng/ml IL-1β for 6 h. Total RNA was isolated, and MMP13 mRNA was analyzed by RT-qPCR. Each value was normalized to GAPDH in the same sample and shown as mean ± S.E. *, p < 0.05. IP, immunoprecipitation.

FIGURE 7.

MEK/ERK overexpression enhances the ELF3-driven MMP13 promoter activation. T/C-28a2 cells were co-transfected with 325 ng of the −1602/+20 bp MMP13 promoter, 25 ng of ELF3 expression vector, and (A) 25 ng of each expression vector encoding ERK1 and MEK1; (B) 25 ng of each expression vector encoding JNK and MKK7; (C) 25 ng of each expression vector encoding p38 and MKK6; (D) increasing amounts (0, 12.5 ng, 25 ng, and 50 ng) of expression vector encoding MKP1, and (E) 25 ng of expression vector encoding MEK1 or ERK1 and 50 ng of expression vector encoding MKP1; or (C) treated with vehicle (DMSO) or the indicated concentrations of U0126 at 4 h after transfection, followed by 20 h of incubation after addition of the inhibitor. Luciferase activities are shown as fold-change with untreated controls set at 1.0. *, p < 0.05; **, p < 0.01; ***, p < 0.001. Error bars, S.E.

FIGURE 8.

MEK/ERK signaling is required for the IL-1β-induced ELF3 binding to the proximal MMP13 promoter. After overnight incubation in serum-free medium, T/C-28a2 cells were pretreated with vehicle (DMSO) or 2.5 μm U0126 for 45 min before incubation with 1 ng/ml IL-1β in the presence of inhibitor. At 2 h after IL-1β stimulation, chromatin was cross-linked and enzymatically sheared, and after reverse cross-linking of the DNA-protein complexes, the precleared lysates were incubated with antibodies against ELF3 or normal IgG overnight at 4 °C. The ELF3 binding to the human MMP13 promoter region was analyzed using primers spanning from −157 to −38 bp of the promoter region by quantitative PCR (A) and conventional PCR (B), with PCR products resolved on a 2.5% agarose gel. GAPDH gene-specific primers were used as a negative control. *, p < 0.05. Error bars, S.E.

FIGURE 9.

Effects of ELF3 on MEK/ERK-dependent MMP13 expression in mouse primary chondrocytes. Mouse primary chondrocytes were isolated from articular cartilage from wild type (WT) C57BL/6 and Elf3 knock-out (KO) 5–6-day-old mice. A, after overnight incubation in serum-free medium, cells were pretreated with vehicle (DMSO) or the indicated concentrations of U0126 for 45 min before incubation with 1 ng/ml IL-1β in the presence of the inhibitor. At 24 h after IL-1β stimulation, total RNAs were isolated, and MMP13 mRNA was analyzed by RT-qPCR. Each value was normalized to GAPDH in the same sample and shown as mean ± S.E. (error bars). B, phospho-ERK1/2 (P-ERK1/2) and total ERK1/2 (ERK1/2) protein levels were analyzed by Western blotting using cell lysates prepared from wild type and Elf3 knock-out primary chondrocytes stimulated with 1 ng/ml IL-1β for the indicated times after overnight incubation in serum-free conditions. Western blots were reprobed for β-tubulin as a loading reference control. *, p < 0.05 versus the IL-1β-stimulated controls.