Transcription factor NFAT5 promotes macrophage survival in rheumatoid arthritis
Susanna Choi, Sungyong You, Donghyun Kim, Soo Youn Choi, H. Moo Kwon, Hyun-Sook Kim, Daehee Hwang, Yune-Jung Park, Chul-Soo Cho, Wan-Uk Kim
Susanna Choi, Sungyong You, Donghyun Kim, Soo Youn Choi, H. Moo Kwon, Hyun-Sook Kim, Daehee Hwang, Yune-Jung Park, Chul-Soo Cho, Wan-Uk Kim
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Research Article Immunology Inflammation

Transcription factor NFAT5 promotes macrophage survival in rheumatoid arthritis

Abstract

Defective apoptotic death of activated macrophages has been implicated in the pathogenesis of rheumatoid arthritis (RA). However, the molecular signatures defining apoptotic resistance of RA macrophages are not fully understood. Here, global transcriptome profiling of RA macrophages revealed that the osmoprotective transcription factor nuclear factor of activated T cells 5 (NFAT5) critically regulates diverse pathologic processes in synovial macrophages including the cell cycle, apoptosis, and proliferation. Transcriptomic analysis of NFAT5-deficient macrophages revealed the molecular networks defining cell survival and proliferation. Proinflammatory M1-polarizing stimuli and hypoxic conditions were responsible for enhanced NFAT5 expression in RA macrophages. An in vitro functional study demonstrated that NFAT5-deficient macrophages were more susceptible to apoptotic death. Specifically, CCL2 secretion in an NFAT5-dependent fashion bestowed apoptotic resistance to RA macrophages in vitro. Injection of recombinant CCL2 into one of the affected joints of Nfat5+/– mice increased joint destruction and macrophage infiltration, demonstrating the essential role of the NFAT5/CCL2 axis in arthritis progression in vivo. Moreover, after intra-articular injection, NFAT5-deficient macrophages were more susceptible to apoptosis and less efficient at promoting joint destruction than were NFAT5-sufficient macrophages. Thus, NFAT5 regulates macrophage survival by inducing CCL2 secretion. Our results provide evidence that NFAT5 expression in macrophages enhances chronic arthritis by conferring apoptotic resistance to activated macrophages.

Authors

Susanna Choi, Sungyong You, Donghyun Kim, Soo Youn Choi, H. Moo Kwon, Hyun-Sook Kim, Daehee Hwang, Yune-Jung Park, Chul-Soo Cho, Wan-Uk Kim

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Figure 4

NFAT5 regulates the survival and proliferation of RAW 264.7 macrophages.

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NFAT5 regulates the survival and proliferation of RAW 264.7 macrophages....
(A) Increased apoptotic death in NFAT5-deficient RAW 264.7 macrophages. The degree of apoptosis was assessed in NFAT5 shRNA–transfected (NFAT5 KD) RAW 264.7 macrophages versus control vector–transfected cells (Control) 24 hours after treatment with CHX (0.5 μg/ml), TG (100 nM), or SNP (100 μM) using flow cytometry for FITC–annexin V and PI. Data are representative of 3 independent experiments. (B and C) Decreased viability of NFAT5-deficient RAW 264.7 macrophages. NFAT5 KD RAW 264.7 macrophages (1 × 105 cells) were treated with CHX (0–4 μg/ml), SNP (0–1,200 μM), or TG (0–500 nM) for 24 hours. RAW 264.7 macrophages stably transduced with the NFAT5 decoy were treated with CHX (0.5 μg/ml), SNP (1.2 mM), or TG (16 nM) for 24 hours. Cell viability was determined by MTT assay. Data represent the mean ± SD of 3 independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.001 versus control vector–transfected RAW 264.7 cells, by 1-way ANOVA with Tukey’s post-hoc test. (D and E) Reduction of RAW 264.7 cell proliferation due to NFAT5 deficiency. RAW 264.7 macrophages (1 × 104 cells) stably transfected with NFAT5 shRNA (NFAT5 KD) or stably transduced with NFAT5 decoy were cultured for 96 hours in RPMI 1640 containing 10% FBS. Viable cells were manually counted after trypan blue staining (D). Cell proliferation was determined by BrdU assay following a 12- or 24-hour incubation with 10% FBS (E). Data represent the mean ± SD. *P < 0.05 and **P < 0.001 versus control vector–transfected cells, by 1-way ANOVA with Tukey’s post-hoc test.