Involvement of ERCC1 in the pathogenesis of osteoarthritis through the modulation of apoptosis and cellular senescence

Abstract

DNA damage is a cause of age related pathologies, including osteoarthritis (OA). Excision repair cross complementation group 1 (ERCC1) is an endonuclease required for DNA damage repair. In this study we investigated the function of ERCC1 in chondrocytes and its association with the pathophysiology of OA. ERCC1 expression in normal and osteoarthritic cartilage was assessed, as were changes in ERCC1 expression in chondrocytes under catabolic stress. Inhibiting ERCC1 in chondrocytes under interleukin-1β stimulation using small interfering RNA (siRNA) was also evaluated. Finally, cellular senescence and apoptosis were examined in relation to ERCC1 function. ERCC1 expression was decreased in OA cartilage and increased within 4 h of exposure to interleukin (IL)-1β, but decreased after 12 h. The inhibition of ERCC1 by siRNA increased the expression of matrix metallopeptidase 13 and decreased collagen type II. ERCC1 inhibition also increased the number of apoptotic and senescent cells. The inhibition of ERCC1 in chondrocytes increased their expression of OA related proteins, apoptosis, cellular senescence, and hypertrophic-like changes which suggest that ERCC1 is critical for protecting human chondrocytes (HCs) from catabolic stresses and provides insights into the pathophysiology of OA and a potential target for its treatment. (191)

Keywords: ERCC1; apoptosis; interleukin (IL)-1β; osteoarthritis; senescence.

Figure 1

Expression of ERCC1 in murine AC and HC. (A) Representative image of murine cartilage from normal and OA induced knee joints 8 weeks after DMM surgery, stained with toluidine blue. Boxed areas in toluidine blue stained images (scale bar = 100 m) are shown in higher magnification (scale bar = 20µm) and immunostained for ERCC1 (green). (B) Graph indicating the maximum OA and summed OA scores. Summed OA scores were calculated from all four quadrants and eight sections from each knee. Error bars indicate the SD (***p < 0.001). (C) Quantitative RT-PCR to measure ERCC1 expression in murine cartilage from normal and OA induced knees. Error bars indicate the SD (*p < 0.05). (D) Shown is a representative image of an immunoblot to measure the protein level of ERCC1 in HC under IL-1β stress at each time point (hours, h). (E) Quantification of the expression ratios of ERCC1 to β-actin. Error bars indicate the SD (***p < 0.001).

Figure 2

Effect of ERCC1 inhibition on HC under IL-1β stimulation. (A) Shown is a representative image of an immunoblot to measure the protein level of ERCC1 after transfection of control siRNA or ERCC1 siRNA. (B) Plotted is the ratio of ERCC1 to β-actin. Error bars indicate the SD (***p < 0.001). (C) Shown is a representative image of an immunoblot to measure the protein levels of MMP13 and COL2A1 after ERCC1 inhibition and stimulation with IL-1β. (D,E) Quantification of the expression ratio of MMP13 (D) or COL2A1 (E) to β-actin. Error bars indicate the SD. **p < 0.01, not significant (NS).

Figure 3

Effect of ERCC1 inhibition on cell death in HC under IL-1β stimulation. (A) Representative images of TUNEL staining after inhibition of ERCC1 under IL-1β stimulation. HC were immunostained for TUNEL (green) and DAPI (blue). Scale bar = 100µm. (B) Quantification of %TUNEL positive cells was calculated as the percent of cells (DAPI) expressing TUNEL (green). Error bars indicate the SD. ***p <0.001. (C) Representative image of an immunoblot to measure the protein level of full length and cleaved PARP after inhibition of ERCC1 when stimulated with IL-1β. (D) Quantification of the expression ratios of cleaved PARP to β-actin was calculated. Error bars indicate the SD. ***p < 0.001, not significant (NS).

Figure 4

Effect of ERCC1 inhibition on cellular senescence in HC under IL-1β stimulation. (A) Plotted is the average of SA-β-gal-positive cells. Error bars indicate the SD (***p < 0.001). (B) Shown is a representative image of an immunoblot measuring the expression levels of p16 and IL-6 in HC. Quantification of the expression ratios of p16(C) and IL-6 (D) to β-actin were calculated. Error bars indicate the SD. *p < 0.05, ***p < 0.001, not significant (NS).