Aging-related loss of the chromatin protein HMGB2 in articular cartilage is linked to reduced cellularity and osteoarthritis

Abstract

Osteoarthritis (OA) is the most common joint disease and typically begins with an aging-related disruption of the articular cartilage surface. Mechanisms leading to the aging-related cartilage surface degeneration remain to be determined. Here, we demonstrate that nonhistone chromatin protein high-mobility group box (HMGB) protein 2 is uniquely expressed in the superficial zone (SZ) of human articular cartilage. In human and murine cartilage, there is an aging-related loss of HMGB2 expression, ultimately leading to its complete absence. Mice genetically deficient in HMGB2 (Hmgb2(-/-)) show earlier onset of and more severe OA. This is associated with a profound reduction in cartilage cellularity attributable to increased cell death. These cellular changes precede glycosaminoglycan depletion and progressive cartilage erosions. Chondrocytes from Hmgb2(-/-) mice are more susceptible to apoptosis induction in vitro. In conclusion, HMGB2 is a transcriptional regulator specifically expressed in the SZ of human articular cartilage and supports chondrocyte survival. Aging is associated with a loss of HMGB2 expression and reduced cellularity, and this contributes to the development of OA.

Fig. 1.

HMGB2 expression in human articular cartilage. (A) Microarray analysis of HMG family genes in normal and OA cartilage. RNA was isolated from 9 normal donors (26.0 ± 8.6 years; OA grade I) and 13 donors with OA (77.3 ± 8.8 years; OA grades III–IV). Total RNA was applied to U95A Affymetrix DNA arrays in duplicate. Statistically significant differences between normal and OA groups are indicated (*, P < 0.05; †, P < 0.01). y axis: % change in gene expression in OA cartilage compared with normal cartilage. (B–E) Immunohistochemical analysis of human knee articular cartilage sections. Normal tissue, donor aged 27 years (B); mild OA tissue, donor aged 85 years (C); and OA tissue, donor aged 71 years (D and E) stained with anti-HMGB2 antibody (B–D) or anti-HMGB1 antibody (E). Images shown are representative of normal (19.0 ± 5.1 years, Mankin score = 0, n = 6), mild OA (68.8 ± 8.6 years; Mankin score, 3.0 ± 0.6; n = 6), and OA (69.0 ± 9.8 years; Mankin score, 7.1 ± 0.7; n = 6) donors. (Magnification: ×400.)

Fig. 2.

HMGB2 expression in normal young mouse knee joints. Immunostaining for HMGB2 in knee joints from neonatal C57BL/6J mice (A and B) and in situ hybridization for HMGB2 at 7 days (P7) after birth (C). (D and F) Between 1 and 2 months of age, there is a reduction in the thickness of the articular cartilage as seen on safranin O stains (bars). HMGB2 is expressed throughout articular cartilage at 1 month (E) and becomes more restricted to the superficial and upper middle zones at 2 months (G). (H–K) High-magnification pictures of cartilage of 2-month-old mice. HMGB2 is also expressed in the superficial layer of cells in menisci (I) and in synovial lining (K, arrows) from 2-month-old mice. (H and J) Safranin O staining of adjacent sections. AC, articular cartilage; M, meniscus. (Magnification: A, C–G, ×100; B, H–K, ×400.)

Fig. 3.

Aging-related reduction in HMGB2 expression in mouse joints. Knee joints from 6-, 9-, and 12-month-old C57BL/6J mice were analyzed by safranin O staining (A, D, G) or for HMGB2 expression by immunohistochemistry (IHC; B, E, H). HMGB2-positive cells are significantly decreased at 9 months (E) and almost completely absent in articular cartilage at 12 months (H). (C, F, I) High-magnification pictures of the central area of the femoral condyle at 6, 9, and 12 months. HMGB2-positive cells are found in the bone marrow without apparent age-related differences. (J) Quantification of SZ cells in cartilage in 4-, 6-, 9-, and 12-month-old mice. Cells were counted on safranin O-stained sections. Cell counting was performed in 3 microscopic fields as shown in A, and summed scores are shown. (K) Quantification of HMGB2-positive cells in the adjacent sections. Knee joints from C57BL/6J WT mice aged 4 months (n = 7), 6 months (n = 7), 9 months (n = 9), and 12 months (n = 7) were examined. Statistically significant differences are indicated (*, P < 0.05; †, P < 0.01). (Magnification: A, B, D, E, G, and H, ×100; C, F, and I, ×400.)

Fig. 4.

Aging-related joint pathology in Hmgb2^−/− mice. Safranin O staining of knee joints from 4-, 6-, and 9-month-old WT mice (A, C, E) and Hmgb2^−/− mice (B, D, F) with high-magnification pictures (×400) of the articular cartilage. (D) Joints from 6-month-old Hmgb2^−/− mice exhibit reduced safranin O staining in the medial femoral condyles and tibial plateaus. There is also a profound reduction in cellularity (arrowhead). (F) Hmgb2^−/− mice at 9 months of age show structural cartilage defects (arrowhead); this is associated with synovial hyperplasia (arrow). (Magnification: A–F, ×100.) Quantification of histopathological changes in C57BL/6J WT and Hmgb2^−/− mice. (G) Pathological changes in knee joints were quantified by a modified Mankin scoring system. C57BL/6J WT mice develop mild OA changes at 9 months, whereas in Hmgb2^−/− mice on the same background, this appears at 6 months. (H) Modified semiquantitative scoring system for WT and Hmgb2^−/− mice at 4 and 6 months. The disruption of the cartilage surface in Hmgb2^−/− mice occurs at 6 months. Statistically significant differences in scores between WT and Hmgb2^−/− mice at each time point are indicated (*, P < 0.05; †, P < 0.01).

Fig. 5.

Apoptosis and MMP expression. Adjacent sections of knee joints from 6- and 9-month-old WT and Hmgb2^−/− mice were stained with safranin O and by immunohistochemistry for PARP-p85, MMP-3, and MMP-13. (B) Articular cartilage from 6-month-old Hmgb2^−/− mice shows diffuse safranin O reduction with a large number of PARP-p85 chondrocytes (F, arrows), particularly in the nonmeniscus-covered areas of cartilage surface. In WT knee joints from 6-month-old mice, safranin O staining is intact (A); only a few chondrocytes are positive for PARP p85 (E). (G and H) At 9 months, PARP-p85 staining is almost absent in the Hmgb2^−/− mice, whereas some chondrocytes are positive for PARP-p85 in WT mice (G, arrows). (C and D) Corresponding safranin O stain shows that articular cartilage is depleted of glycosaminoglycans and cell density is profoundly reduced in Hmgb2^−/− mice compared with WT mice. (I and J) MMP-3 is expressed by a higher number of chondrocytes from 6-month-old Hmgb2^−/− mice (J, arrows) compared with WT mice. (K and L) MMP-13 expression is not detected at 6 months (data not shown), but at 9 months, staining intensity is greater in Hmgb2^−/− mice (L, arrows) than in WT mice. Representative pictures from PARP-p85 staining (n = 4 each) and MMP-3 and MMP-13 staining (n = 2–4 each) are shown. AC, articular cartilage; M, meniscus. (Magnification: A–L, ×400.)

Fig. 6.

Localization of HMGB2 and PRG4/SZP in murine cartilage. (A) Double staining by immunofluorescence shows colocalization of HMGB2 and PRG4/SZP in articular cartilage and meniscus at 2 months. PRG4/SZP is localized in the cytosol, which is distinct from HMGB2 expression in the nuclei (arrows). Nuclei were labeled by using Hoechst 33258 dye. AC, articular cartilage; M, meniscus. (Magnification: ×400.) (B) PRG4/SZP expression in knee joints as visualized by in situ hybridization. At 2 months, PRG4/SZP-positive cells are found in the SZ and synovium in both WT and Hmgb2^−/− strains without apparent difference. At 6 months, PRG4/SZP-positive cells are significantly reduced in the cartilage in Hmgb2^−/− mice (arrowheads), whereas PRG4/SZP expression in synovium is higher in Hmgb2^−/− mice (arrows) than in WT mice. (Magnification: ×100.) (C) PRG4/SZP and HMGB2 expression in cultured mouse chondrocytes. Chondrocytes were isolated from articular cartilage of 5-day-old WT and Hmgb2^−/− mice and cultured for 3 days until analysis. Western blotting shows similar PRG4/SZP and HMGB1 expression in WT and Hmgb2^−/− chondrocytes. HMGB2 expression is absent in chondrocytes from Hmgb2^−/− mice. (D) Apoptosis assays for articular chondrocytes from WT and Hmgb2^−/− mice. After culture in control media (Con) or treatment with CD95 antibody (1 μg/mL) and proteasome inhibitor MG132 (20 μM) for 12–14 h, chondrocytes were labeled with annexin V or propidium iodide (PI) and analyzed by flow cytometry. Three independent experiments with chondrocytes from three different littermate mice were performed. Statistically significant differences between Hmgb2^−/− and WT chondrocytes are indicated (*, P < 0.05).