Mesenchyme-specific knockout of ESET histone methyltransferase causes ectopic hypertrophy and terminal differentiation of articular chondrocytes

Abstract

The exact molecular mechanisms governing articular chondrocytes remain unknown in skeletal biology. In this study, we have found that ESET (an ERG-associated protein with a SET domain, also called SETDB1) histone methyltransferase is expressed in articular cartilage. To test whether ESET regulates articular chondrocytes, we carried out mesenchyme-specific deletion of the ESET gene in mice. ESET knock-out did not affect generation of articular chondrocytes during embryonic development. Two weeks after birth, there was minimal qualitative difference at the knee joints between wild-type and ESET knock-out animals. At 1 month, ectopic hypertrophy, proliferation, and apoptosis of articular chondrocytes were seen in the articular cartilage of ESET-null animals. At 3 months, additional signs of terminal differentiation such as increased alkaline phosphatase activity and an elevated level of matrix metalloproteinase (MMP)-13 were found in ESET-null cartilage. Staining for type II collagen and proteoglycan revealed that cartilage degeneration became progressively worse from 2 weeks to 12 months at the knee joints of ESET knock-out mutants. Analysis of over 14 pairs of age- and sex-matched wild-type and knock-out mice indicated that the articular chondrocyte phenotype in ESET-null mutants is 100% penetrant. Our results demonstrate that expression of ESET plays an essential role in the maintenance of articular cartilage by preventing articular chondrocytes from terminal differentiation and may have implications in joint diseases such as osteoarthritis.

Keywords: Arthritis; Articular cartilage; Cartilage biology; Chondrocytes; Differentiation; Epigenetics; Gene Knockout; Histone methylation.

FIGURE 1.

ESET genomic structure and expression in articular chondrocytes. a, diagrams of ESET protein domains and the floxed ESET allele. a. a., amino acids. b, ESET protein expression at the knee joint was assessed by an anti-ESET antibody in wild-type and ESET-null mice. DAPI counterstaining of nuclei was used for tissue outline. Arrows indicate strong staining for ESET protein in subchondral bone marrow cells. c, coronal sections of 2-week-old knee joints were stained by H&E for cell morphology and by Safranin O for proteoglycans (red). Scale bar: 400 μm.

FIGURE 2.

Characterization of articular chondrocytes in ESET-null embryos. a, sagittal sections of knee joints from E18.5 embryos were stained by H&E or with an anti-matrilin-1 antibody. DAPI counterstaining of nuclei was used for tissue outline. The images were merged to show the absence of matrilin-1 in articular cartilage. b, sagittal sections of developing knee from E15.5 embryos were stained by H&E or with a rabbit antibody to show similar ERG expression within future joints in both wild-type and ESET-null embryos. A total of three wild-type and three knock-out embryos were examined, and results from a typical experiment are shown. Genotypes of the embryos are indicated on the left side. Scale bars: 400 μm (a), 200 μm (b).

FIGURE 3.

Articular chondrocyte hypertrophy and proliferation in 1-month-old ESET-null mice. a, 1-month-old knee joints were stained by H&E and Safranin O for detailed morphological analysis. b, 1-month-old knee sections were stained with an anti-type II collagen antibody. The type II collagen network was stained red, and nuclei were stained blue by DAPI. c, anti-type X collagen antibody specifically stains hypertrophic chondrocytes (red) in 1-month-old knee sections. d, anti-PCNA antibody detects cells positive for the proliferation marker in 1-month-old knee sections. Arrows in a–d indicate locations of hypertrophic or proliferative chondrocytes near the superficial zone in ESET-null mice. A total of three wild-type and five knock-out mice were examined, and images from representative experiments are shown. Genotypes of the mice are indicated on the top. Scale bar: 50 μm.

FIGURE 4.

Apoptosis of articular chondrocytes in 1-month-old ESET-null mice. a, 1-month-old knee sections were stained with an anti-active caspase 3 antibody, and nuclei were stained blue by DAPI. b, TUNEL assay was performed on knee sections to measure DNA fragmentation in situ. Arrows in a indicate early stage apoptotic chondrocytes (red), and arrows in b designate late-stage apoptotic nuclei (green). A total of three wild-type and five knock-out mice were examined, and representative experiments are shown. Genotypes of the mice are indicated on the top.

FIGURE 5.

Terminal differentiation of articular chondrocytes in 3-month-old ESET-null mice. a, a significant decrease of the type II collagen network was evident when the joints were stained with an anti-type II collagen antibody. b, the knee joints were stained by Safranin O to assess changes in articular cartilage. Lower panels represent enlargements of the selected areas in the top panels. Negative staining within the superficial zone in the knock-out indicates loss of proteoglycans. c, positive staining (red) for MMP-13 in the articular cartilage was found only in ESET-null mice but not in wild-type controls. d, alkaline phosphatase activity was nearly undetectable in wild-type articular cartilage, but ESET-null chondrocytes close to the articular surface all stained positive for alkaline phosphatase activity. Lower panels represent enlargements of the selected areas in the top panels. A total of three wild-type and three knock-out mice were examined, and representative experiments are shown. Scale bar: 400 μm.

FIGURE 6.

Degradation of articular cartilage in 12-month-old ESET-null mice. a, knee sections from 12-month-old mice were stained with an anti-type II collagen antibody. The type II collagen network was stained red, and nuclei were stained blue by DAPI. b, 12-month-old knee joints were stained by Safranin O for proteoglycans. Note the significant loss of type II collagen and proteoglycans at the joint in ESET-null mice. A total of five wild-type and five knock-out mice were examined, and images from representative experiments are shown. Genotypes of the mice are indicated on the top. Scale bar: 400 μm.