The involvement and possible mechanism of NR4A1 in chondrocyte apoptosis during osteoarthritis

Abstract

Osteoarthritis (OA) is a joint disease caused by the breakdown of joint cartilage and underlying bone, and places great burdens to daily life of patients. Nuclear orphan receptor nuclear receptor subfamily 4, group A, member 1 (NR4A1) is vital for cell apoptosis, but little is known about its role in OA. This study aims to reveal the expression and function of NR4A1 during OA chondrocyte apoptosis. NR4A1 expression by qRT-PCR and western blot, and chondrocyte apoptosis by TUNEL assay were detected in normal and OA joint cartilage. NR4A1 was located in cartilage sections by immunohistofluorescence. Chondrocytes from normal joint cartilage were cultured in vitro for interleukin 6 (IL6) or tumor necrosis factor (TNF) treatment and si-NR4A1 transfection, after which the possible mechanism involving NR4A1 was analyzed. Results showed that NR4A1 expression and chondrocyte apoptosis were significantly elevated in OA cartilage (P < 0.05 and P < 0.01). NR4A1 was located in nuclei of normal cartilage chondrocytes, but was translocated to mitochondria and co-located with B-cell lymphoma 2 in OA chondrocytes. NR4A1 expression in cultured chondrocytes could be promoted by both IL6 and TNF treatment. si-NR4A1 partly reduced TNF-induced cell apoptosis. Inhibiting p38 by SB203580 could decrease TNF-induced NR4A1 to some extent, while inhibiting JNK could not. So NR4A1 is likely to facilitate OA chondrocyte apoptosis, which is associated with p38 MAPK and mitochondrial apoptosis pathway. This study provides a potential therapeutic target for OA treatment and offers information for regulatory mechanisms in OA.

Keywords: NR4A1; Osteoarthritis; mitochondrial apoptosis pathway; p38; tumor necrosis factor.

Figure 1

Apoptotic chondrocytes (blue-green) in cartilage sections of OA patients and non-OA patients (control) using TUNEL assay and the significant difference in apoptotic cell (FITC-positive) number between the two groups. The experiments are repeated five times. Bar indicates 20 μm. The nuclei are stained by DAPI (blue). **P < 0.01. OA, osteoarthritis. FITC, fluorescein isothiocyanate.

Figure 2

NR4A1 expression is promoted during OA. A. NR4A1 mRNA level detected by qRT-PCR. B. NR4A1 protein level detected by western blot and the histogram drawn based on repeated results. The experiments are repeated five times. *P < 0.05. OA, osteoarthritis. NR4A1, nuclear subfamily 4, group A, member 1.

Figure 3

Localization of NR4A1 in normal and OA patients. A. NR4A1 (green) is translocated to cytoplasm and co-localized with PHB-marked mitochondria (red) in OA patients. B. NR4A1 (red) is translocated to cytoplasm and co-localized with BCL2 (green) in OA patients. Immunohistofluorescence is performed in normal and OA cartilage sections and in each section NR4A1 and PHB or BCL2 are marked with the specific primary antibodies and then the secondary antibodies labelled by Alexa Fluor 647 (red) or 488 (green). DAPI stains nuclei. The experiments are repeated five times. Bar indicates 10 μm. OA, osteoarthritis. NR4A1, nuclear receptor subfamily 4, group A, member 1. PHB, prohibitin. BCL2, B-cell lymphoma 2. merge, pictures showing green, red and blue signals at the same time.

Figure 4

IL6 and TNF induce NR4A1 in chondrocytes. The cultured chondrocytes from normal joint cartilage are treated with IL6 or TNF for 12 or 24 h, after which western blot is performed to examine NR4A1 expression. GAPDH is an internal reference. The experiments are repeated five times. Both IL6 and TNF induce NR4A1 protein expression in a time-dependent manner based on results from the two time points. NR4A1, nuclear receptor subfamily 4, group A, member 1. IL6, interleukin 6. TNF, tumor necrosis factor.

Figure 5

NR4A1 is involved in TNF-induced chondrocyte apoptosis. A. si-NR4A1 partly reduces the TNF-induced up-regulation of cleaved PARP1, as indicated by western blot. GAPDH is an internal reference. B. TUNEL assay shows that the apoptotic cell percent of each group exhibits similar apoptosis changes to western blot results. The experiments are repeated five times. Cells are first treated by TNF for 12 h and then transfected with si-NR4A1. Scramble si-NR4A1 is transfected as control. *P < 0.05. **P < 0.01. ***P < 0.001. NR4A1, nuclear receptor subfamily 4, group A, member 1. TNF, tumor necrosis factor. PARP1, poly (ADP-ribose) polymerase 1.

Figure 6

p38 MAPK is involved in the up-regulation of NR4A1 by TNF. Western blot is used to detect NR4A1 protein level in treated cells. GAPDH is an internal reference. The experiments are repeated five times. The cultured chondrocytes are treated with TNF for 12 h and then SB203580 (the specific inhibitor of p38) or SP600125 (the specific inhibitor of JNK) for 24 h. SP600125 does not affect the up-regulation of NR4A1 by TNF, while SB203580 inhibits the promotive effects of TNF on NR4A1. NR4A1, nuclear receptor subfamily 4, group A, member 1. TNF, tumor necrosis factor.