PADI4 Epigenetically Suppresses p21 Transcription and Inhibits Cell Apoptosis in Fibroblast-like Synoviocytes from Rheumatoid Arthritis Patients

Abstract

Rheumatoid arthritis (RA) is characterized by synovial lining hyperplasia, which involves abnormal growth of fibroblast-like synoviocytes (FLSs). This study aimed to investigate the function and molecular mechanism of peptidylarginine deiminase type 4 (PADI4) in FLSs isolated from RA patients (RA-FLSs). FLSs were isolated from RA patients and transfected with small interfering RNAs (siRNAs) or PADI4 overexpression plasmid. FLSs were treated by Adriamycin (ADR) to induce apoptosis, and apoptotic cells were detected by flow cytometry. The expression of PADI4, p53 and p21 was detected by qRT-PCR and Western blot analysis. The recruitment of PADI4 and histone H3 arginine modifications to p21 promoter was measured by chromatin immunoprecipitation. The results showed that knockdown of PADI4 promoted the apoptosis of RA-FLSs and the expression of p53 and p21. Ectopic expression of PADI4 inhibited ADR-induced apoptosis of RA-FLSs, and down-regulated the expression of p53 and p21. In RA-FLSs, global H3 citrullination (CitH3) and H3 arginine 17 methylation levels were dynamically changed by PADI4 and ADR treatment. PADI4 and H3 could bind p21 promoter region to regulate p21 expression. In conclusion, PADI4 contributes to the pathogenesis of RA by protecting FLSs from apoptosis. PADI4 suppresses p21 transcription through altering histone H3 arginine modifications on p21 promoter region. Our study provides new insight into the anti-apoptotic role of PADI4 in RA development.

Keywords: PADI4; cell apoptosis; histone arginine citrullination; p21.

Figure 1

PADI4 is highly expressed and inhibited the apoptosis of RA-FLSs. (A) Immunohistochemistry staining of PADI4 in arthritic synovial tissues from RA and OA patients. Original magnification: x200. Images represent PADI4 and p21 antibodies staining, IgG control staining (Negative) and HE staining. (B) Quantitative RT-PCR analysis of PADI4 mRNA level in RA and OA FLSs. Data are presented after normalization by GAPDH. (C) Left: flow cytometry analysis of apoptotic RA and OA FLSs after ADR treatment (Upper left, untreated OA; Upper right, ADR treated OA; Lower left, untreated RA; Lower right, ADR treated RA). Right: quantification of apoptotic cells. *, P<0.05, ** P<0.01.

Figure 2

Knockdown of PADI4 promotes the apoptosis and increases the expression of p53 and p21 in RA-FLSs. (A) RA-FLSs were transfected with siRNAs and the levels of p21 and p53 were evaluated by Western blot analysis (left) and qRT-PCR (right). (B) Flow cytometry analysis of apoptotic RA-FLSs after PADI4 knockdown for 72 h. (C) Western blot and qRT-PCR analyses of PADI4, p53 and p21 in RA-FLSs (R1 and R2) after treatment with ADR (1 μg/ml) for 24 h. *, P<0.05, ** P<0.01.

Figure 3

Ectopic expression of PADI4 inhibits the apoptosis and decreases p53 and p21 expression in RA-FLSs. (A) RA-FLSs were transfected with overexpression plasmids and PAID4 protein was evaluated by Western blot analysis. (B & C) RA-FLSs were transfected with PADI4 overexpression plasmid and 24 h later treated with ADR. mRNA and protein levels of PADI4, p53 and p21 were measured by qRT-PCR (B) and Western blot analysis (C). (D & E) Flow cytometry analysis of apoptotic RA-FLSs after PADI4 overexpression alone (D) and in combination with ADR treatment (E). *P<0.05, **P<0.01.

Figure 4

PADI4 modulates the citrullination/methylation of histone H3 in RA-FLSs. (A) Western blot analysis of the levels of CitH3 (citrullination of H3R2, R8 and R17), H3R17me2, H4R3me and total histone H3 in RA-FLSs after transfection with PADI4 siRNAs. (B) Western blot analysis of the levels of CitH3, H3R17me2 and total histone H3 in RA-FLSs with ADR treatment. (C) Western blot analysis of the levels of CitH3, H3R17me2 and total histone H3 in PADI4 overexpressing RA-FLSs after ADR treatment.

Figure 5

Recruitment of PADI4 and H3 to p21 promoter during apoptosis of RA-FLSs. (A) Schematic diagram of the primer l location on p21 distal promoter region. (B) ChIP assays to examine histone H3R17me2, PADI4 and CitH3 binding signals on the p21 promoter. (C) ChIP assays to examine histone H3R17me, PADI4 and CitH3 binding signals on the p21 promoter in RA-FLS with ADR treatment.

Figure 6

Model of the repressive role of PADI4 in p21 transcription. PADI4 suppresses p21 transcription through the deposition of repressive mark CitH3 (for example, H3R17cit) and inhibition of the active mark H3R17me2 on p21 promoter region (A). ADR induced PDK4 down-regulation or siRNAs mediated PADI4 depletion promotes p21 transcription (B). H3R17cit: repressive histone H3R17 citrullination; H3R17me2: active histone H3R17 di-methylation.