ESE-1 is a potent repressor of type II collagen gene (COL2A1) transcription in human chondrocytes

Abstract

The epithelium-specific ETS (ESE)-1 transcription factor is induced in chondrocytes by interleukin-1beta (IL-1beta). We reported previously that early activation of EGR-1 by IL-1beta results in suppression of the proximal COL2A1 promoter activity by displacement of Sp1 from GC boxes. Here we report that ESE-1 is a potent transcriptional suppressor of COL2A1 promoter activity in chondrocytes and accounts for the sustained, NF-kappaB-dependent inhibition by IL-1beta. Of the ETS factors tested, this response was specific to ESE-1, since ESE-3, which was also induced by IL-1beta, suppressed COL2A1 promoter activity only weakly. In contrast, overexpression of ETS-1 increased COL2A1 promoter activity and blocked the inhibition by IL-1beta. These responses to ESE-1 and ETS-1 were confirmed using siRNA-ESE1 and siRNA-ETS1. In transient cotransfections, the inhibitory responses to ESE-1 and IL-1beta colocalized in the -577/-132 bp promoter region, ESE-1 bound specifically to tandem ETS sites at -403/-381 bp, and IL-1-induced binding of ESE-1 to the COL2A1 promoter was confirmed in vivo by ChIP. Our results indicate that ESE-1 serves a potent repressor function by interacting with at least two sites in the COL2A1 promoter. However, the endogenous response may depend upon the balance of other ETS factors such as ETS-1, and other IL-1-induced factors, including EGR-1 at any given time. Intracellular ESE-1 staining in chondrocytes in cartilage from patients with osteoarthritis (OA), but not in normal cartilage, further suggests a fundamental role for ESE-1 in cartilage degeneration and suppression of repair.

Fig. 1

Potential ESE-1 binding sites in the COL2A1 promoter. The structure of the COL2A1 regulatory region spanning –577 to +3,426 bp is shown at top with potential ETS binding site indicated with arrows. The previously identified Sp1, Egr-1/Sp1, E-box, and TATA-box sites are represented schematically within the –577/+125 bp promoter. The oligonucleotides that were used for EMSAs are listed below with putative ETS binding sites underlined (left) and the corresponding mutant oligonucleotides (right).

Fig. 2

The suppression of COL2A1 gene expression by IL-1β requires NF-κB and is associated with NF-κB-dependent induction of ESE-1 mRNA. A: Cultures of the immortalized human chondrocyte cell line, C-28/I2,were infected with adenovirus expressing IκB (adv-IkB) or, as a null control, adv-βgal, incubated for 4 h in serum-containing medium, changed to serum-free medium containing 1% Nutridoma, and treated with IL-1β (500 pg/ml) for 18 h. Total RNA was extracted and analyzed by RT-PCR using specific primers to detect COL2A1, ESE1, and GAPDH mRNAs. B: Primary cultures of human adult articular chondrocytes at day 5 after isolation from the cartilage were treated with IL-1β for 18h, and ESE1 and COL2A1 mRNA were analyzed by real-time RT-PCR. C: The C-28/I2 cells were cultured in serum-containing culture medium for 5 days, and confluent cultures were changed to serum-free medium containing 1% Nutridoma 24 h before treatment with IL-1β for the times indicated. Emetine (10 μg/ml) was also added to the indicated cultures. Total RNA extracts were analyzed for ESE1 mRNA expression by real-time PCR.

Fig. 3

ESE-1 decreases COL2A1 promoter activity and enhances IL-1β-dependent inhibition. The C-28/I2 chondrocytes were cotransfected with pGL2B or with pGL2-COL2 together with pCI empty vector or with (A) increasing amounts of pCI-ESE1 (50, 100, and 200 ng/ml), (B) pCI-ESE1 (50 ng/ml) in the absence or presence of increasing amounts of dominant-negative (DN)-ESE1. C: The C-28/I2 cells were cotransfected with pGL2-COL2 and pcDNA3.1 (empty vector) or pcDNA-ESE1 together with siRNA-Ctrl (control) or siRNA-ESE1. Incubations were for 24 h followed by treatment with IL-1β (500 pg/ml) for a further 18 h.

Fig. 4

Comparison of ESE-1 with other ETS family members. C-28/I2 cells were cotransfected with pGL2-COL2 and pCI or expression plasmids encoding (A) various ETS family members (100 ng/well in 12-well plates) or (B) ESE-1 (100 ng/ml) or ETS-1 (50 and 100 ng/ml). C: The C-28/I2 cells were cotransfected with pGL2-COL2 and pcDNA3.1 (empty vector) or pcDNA-ETS1 together with siRNA-Ctrl (control) or siRNA-ETS1. The cells were incubated for 24 h prior to the addition of IL-1β for a further 18 h, cell harvest, and luciferase assay.

Fig. 5

IL-1β induces ESE-1 and ESE-3 coordinately with the suppression of COL2A1 mRNA but ESE-1 inhibits COL2A1 promoter activity more effectively than ESE-3. A: The C-28/I2 cells were cultured as describe in Figure 2C and treated with IL-1β and cycloheximide alone or together for the times indicated. Total RNA extracts were analyzed by semi-quantitative RT-PCR for COL2A1, ESE1, ESE2, ESE3, and GAPDH mRNAs. COX2 and NOS2 mRNAs were also assayed as positive controls. B: The C-28/I2 cells were cotransfected with pGL2-COL2 together with pCI empty vector or increasing amounts (25, 50, 100, 150, and 200 ng/ml) of pCI-ESE1 or pCI-ESE3.

Fig. 6

ESE-1 decreases activity of the COL2A1 promoter containing the –577/–132 bp region. A: The C-28/I2 cells were cotransfected with pGL2 constructs containing COL2A1 promoter sequences, –577/+125 bp, –403/+125 bp, –131/+125 bp, and –90/+125 bp, together 200 ng of pCI (filled bar) or 50, 100, or 200 ng/ml of pCI-ESE1. B: The cells were cotransfected with pGL2-COL2 and pcDNA3.1 (empty vector) or expression plasmids encoding ESE-1 (100 ng/ml), DN-ESE1 (50 ng/ml), or DN-ESE1 and DN-Egr1, each at 50 ng/ml. The cultures were then incubated for 24 h to permit expression of recombinant proteins. Some cultures were then treated with IL-1β (empty bar) and the incubations continued for a further 18h. All cultures were harvested at the same time for luciferase assay of reporter gene expression. The results show the means ± SD derived from three separate transfection experiments, each done in triplicate.

Fig. 7

ESE-1 binds to ETS sites E2 and E3 within –403/–381 bp. A: End-labeled double-stranded oligonucleotides containing the ESE-1 consensus (NOS2 promoter) or the E2, E3, and E4 oligonucleotides or mutants (see Fig. 1) were incubated with in vitro translated protein made with empty vector (v) or pCI-ESE1 (E). Competitor oligonucleotides (at 50× and 100× in adjacent lanes) and antibody were added as indicated and as described in the Methods. Similar background binding of in vitro-translated vector or ESE-1 has been reported on other promoter sequences (Brown et al., 2004; Wang et al., 2004; Grall et al., 2005). Note that the third lane of each set shows binding to mutant (mt) E2, E3, and E4 probes. B: Chromatin immunoprecipitations (ChIP) were performed using C-28/I2 cells transfected with pcDNA3-FLAG empty vector (EV) or pcDNA3-FLAG-ESE1 or -DN-ESE1 (left), or they were treated with IL-1β for 6 h (right). Chromatin proteins were crosslinked to DNA with formaldehyde and purified nucleoprotein complexes were immunoprecipitated using anti (α)-FLAG or α-ESE1 antibody. The precipitated DNA fractions were analyzed by PCR for the presence of the COL2A1 promoter region encompassing the ESE-1 binding sites. The input DNA was used as a positive control for the PCR and water was used as negative control (not shown).

Fig. 8

Immunolocalization of ESE-1 in chondrocytes in vivo associated with osteoarthritis but not in normal articular cartilage and induction of ESE-1 mRNA by IL-1β in primary human articular chondrocytes. Samples of articular cartilage from individuals without apparent joint destruction (normal; a and c) and from patients with osteoarthritis (OA; b and d) were subjected to immunohistochemical staining using an antibody against human ESE-1 or isotype-matched IgG as negative control. Positive staining for ESE-1 appears as a brown color in the cytoplasm and nuclei of chondrocytes in this representative sample of OA cartilage. (Original magnifications, 40×). [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

Fig. 9

Mechanism of suppression of COL2A1 promoter activity by IL-1β-induced transcription factors.