TWIST1 induces MMP3 expression through up-regulating DNA hydroxymethylation and promotes catabolic responses in human chondrocytes

Abstract

The objective was to investigate the levels of TWIST1 in normal and OA cartilage and examine its role in regulating gene expression in chondrocytes. Human cartilage tissues and chondrocytes were obtained at autopsy from normal knee joints and from OA-affected joints at the time of total knee arthroplasty. TWIST1 expression was increased in human OA knee cartilage compared to normal knee cartilage. TWIST1 induced matrix metalloproteinase 3 (MMP3) expression without direct binding to MMP3 promoter and increased the 5-hydroxymethylcytosine (5hmC) level at the MMP3 promoter. The effect of TWIST1 on expression of TET family (TET1, 2 and 3) was measured in stable TWIST1 transfected TC28 cells, and TET1 expression was up-regulated. TWIST1 dependent upregulation of Mmp3 expression was suppressed in Tet triple KO fibroblast derived from mouse ES cells. Increased TWIST1 expression is a feature of OA-affected cartilage. We identified a novel mechanism of catabolic reaction where TWIST1 up-regulates MMP3 expression by enriching 5hmC levels at the MMP3 promoter via TET1 induction. These findings implicate TWIST1 as an important factor regulating OA related gene expression. Clarifying epigenetic mechanisms of 5hmC induced by TWIST1 is a critical molecule to understanding OA pathogenesis.

Figure 1. TWIST1 is highly expressed in human OA cartilage.

(A) TWIST1 gene expression in cartilage tissues from 7 normal donors and 8 OA donors were analyzed by real-time qRT-PCR. TWIST1 gene expression levels in OA cartilage tissues are relative to normal cartilage tissues and normalized to GAPDH. Values are the mean ± SEM ratio. (B) Immunohistochemical staining for TWIST1 in human normal and OA cartilage tissue. **P < 0.01. Left scale bars, 500 μm. Right scale bar, 200 μm. (C) The TWIST1 expression changes after IL-1β and TNFα stimulation in human primary chondrocyte were confirmed by real-time PCR. Values are the mean ± SEM. *P < 0.05. (D) The TWIST1 expression changes after 12 hours IL-1β and TNFα stimulation in human primary chondrocyte were confirmed by Western blotting.

Figure 2. TWIST1 regulates MMP3 expression.

(A) Immunohistochemistry analysis of human cartilage tissues. Cartilage sections were obtained from normal and OA-affected cartilages. Sections were stained with MMP3 antibody. Left scale bars, 500 μm. Right scale bar, 200 μm. (B) MMP3 expression was detected by real-time qRT-PCR in human chondrocytes from 3 donors after Ad-GFP or Ad-TWIST1 infection at 72 hours. Values are the mean ± SEM of three independent experiments for each donor. (C) Relative MMP3 expression level in TC28 after Ad-GFP or Ad-TWIST1 infection. Values are the mean ± SEM. *P < 0.05, ***P < 0.001.

Figure 3. MMP expression in human chondrocytes after Ad-TWIST1 infection.

Normal human chondrocytes from 3 donors were infected Ad-GFP or Ad-TWIST1 for 72 hours. MMP expression was analyzed by real-time qRT-PCR. Values are the mean ± SEM of three independent experiments for each donor. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 4. DNA methylation and 5hmC status in the MMP3 promoter.

(A) Immunohistochemistry analysis of human cartilage tissues. Cartilage sections were obtained from normal and OA-affected cartilages. Sections were stained with 5hmC anti body. Left scale bars, 500 μm. Right scale bar, 200 μm. (B) Bisulfite genomic sequencing results of the MMP3 promoter regions in TC28 cells after Ad-GFP or Ad-TWIST1 infection at 72 hours. Black, methylated; white, unmethylated. (C) To detect locus-specific 5hmC and 5mC status at CCGG site in MMP3 promoter, restriction enzyme digestion and quantitative polymerase chain reaction were performed. *P < 0.05. (D) TET1, TET2, and TET3 expressions were detected by real-time qRT-PCR in human chondrocytes after Ad-GFP or Ad-TWIST1 infection at 72 hours. Values are the mean ± SEM of three independent experiments. *P < 0.05.

Figure 5. Effect of TWIST1 on TET1 expression in TC28 cells.

(A) Schematic representation of the human TWIST1 protein. (B) Myc-tagged TWIST1 or TWIST1-ΔbHLH stably transfected TC28 cells were generated. Myc expression was analyzed by Western blotting. (C) TET1, 2, and 3 gene expression levels in TWIST1 stably transfected TC28 cells (TC28-TWIST1) were determined by quantitative PCR. Stable TWIST1-ΔHLH transfected TC28 cells (TC28-TWIST1-ΔHLH) were used as control. (D) Western blot analysis of TET 1, 2 and 3 expression in TC28-TWIST1 and TC28-TWIST1-ΔHLH cells. All gels for western blot analysis were run under the same experimental conditions, and western blot images were cropped with a grey cropping line. (E) Immunohistochemistry analysis of human cartilage tissues. Cartilage sections were obtained from normal and OA-affected cartilages. Sections were stained with TET1 anti body. Scale bar, 200 μm.

Figure 6. MMP3 expression change after TWIST1 overexpression under TET knock down.

(A) The siRNA for TET1 was transfected to human primary chondrocytes, and after 36 hours, cells were infected with Ad-GFP or Ad-TWIST1 and cultured for 72 hours. The MMP3 gene expression was determined by real-time qRT-PCR. (B) Fibroblasts were derived from Tet1, 2 and 3 triple knock out mice embryonic stem cells, and were transfected Ad-GFP or Ad-TWIST1 for 72 hours. Mmp3 gene expression was determined by quantitative PCR. *P < 0.05.