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. 2016 Feb 29:9:993-1000.
doi: 10.2147/OTT.S92907. eCollection 2016.

The pro-apoptotic effects of TIPE2 on AA rat fibroblast-like synoviocytes via regulation of the DR5-caspase-NF-κB pathway in vitro

Chunyan Shi  1 , Shifeng Zhang  2 , Shifu Hong  3 , Jinglong Pang  3 , Yeletai Yesibulati  3 , Ping Yin  4 , Guohong Zhuang  3
Affiliations

The pro-apoptotic effects of TIPE2 on AA rat fibroblast-like synoviocytes via regulation of the DR5-caspase-NF-κB pathway in vitro

Chunyan Shi et al. Onco Targets Ther. .

Abstract

TIPE2, also known as TNFAIP8L2, a member of the tumor necrosis factor-alpha-induced protein-8 (TNFAIP8) family, is known as an inhibitor in inflammation and cancer, and its overexpression induces cell death. We examined the role of TIPE2 with respect to adjuvant arthritis (AA)-associated pathogenesis by analyzing the TIPE2 regulation of death receptor (DR5)-mediated apoptosis in vitro. The results showed that TIPE2 was detected in normal fibroblast-like synoviocytes (FLSs), but scarcely observed in AA-FLSs. Therefore, recombinant MIGR1/TIPE2(+/+) and control MIGR1 lentivirus vectors were transfected to AA-FLSs, which were denoted as TIPE2(+/+)-FLSs and MIGR1-FLSs, respectively. Our results showed that TIPE2(+/+)-FLSs were highly susceptible to ZF1-mediated apoptosis, and ZF1 was our own purification of an anti-DR5 single chain variable fragment antibody. Under the presence of TIPE2, the expression of DR5 was significantly increased compared with that of the MIGR1-FLS group. In contrast, the level of phosphorylated nuclear factor-kappa B (pNF-κB) was lower in the TIPE2(+/+)-FLS group treated with ZF1, whereas the activity of caspase was higher. Moreover, the rate of apoptosis in the TIPE2(+/+)-FLS group, which was pretreated with caspase inhibitor Z-VAD-FMK, was significantly decreased. In contrast, the apoptosis occurrence in the MIGR1-FLS group increased significantly with the pretreatment of the NF-κB inhibitor Bay. These results indicated that TIPE2 increased the apoptosis of AA-FLSs by enhancing DR5 expression levels, thereby promoting the activation of caspase and inhibiting the activation of NF-κB in AA-FLSs. TIPE2 might potentially act as a therapeutic target for rheumatoid arthritis.

Keywords: DR5; TIPE2; adjuvant arthritis; fibroblast-like synoviocytes; rheumatoid arthritis.

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Figures

Figure 1
Figure 1
Overexpression of TIPE2 in AA-FLSs. Notes: PCR and Western blot analyses of TIPE2 expression in freshly harvested RAW 264.7 cells, normal FLSs, AA-FLSs, and AA-FLSs stably transfected with the lentivirus vector MIGR1/TIPE2+/+ plasmid and the control vector MIGR1 plasmid. TIPE2 expression in RAW 264.7 cells was used as a positive control. (A) RT-PCR analysis of TIPE2 mRNA. GAPDH was used as a loading control, whereas H2O was used as the background control. (B) Quantification of mRNA expression by quantitative real-time PCR. Data are mean ± SD (n=3), ***P<0.001 vs the normal FLS group. (C) Whole cell lysates were prepared. An aliquot (30 μg) of each whole cell lysate was fractionated on a 12% SDS gel, and TIPE2 was analyzed by Western blotting. β-Actin was used as a loading control. (D) Grayscale analysis of Figure 1C, ***P<0.001 vs the normal FLS group. (E) mRNA levels of TNFAIP8 family members were determined by RT-PCR using total RNA isolated from cell preparations. GAPDH was used as a loading control, whereas H2O was used as the background control. (F) Quantification of mRNA expression by quantitative real-time PCR. Data are mean ± SD (n=3), ***P<0.001 vs the MIGR1-FLS group. (G) Whole cell lysates were prepared. An aliquot (30 μg) of each whole cell lysate was fractionated on a 12% SDS gel, and TIPE2 and β-actin were analyzed by Western blotting. (H) Grayscale analysis of Figure 1G, ***P<0.001 vs the MIGR1-FLS group. Abbreviations: AA, adjuvant arthritis; FLS, fibroblast-like synoviocyte; GAPDH, glyceraldehyde-phosphate dehydrogenase; mRNA, messenger RNA; RT-PCR, reverse transcription polymerase chain reaction; SD, standard deviation; SDS, sodium dodecyl sulfate.
Figure 2
Figure 2
Overexpression of TIPE2 enhanced susceptibility to DR5-mediated apoptosis. Notes: (A) Growth inhibition of AA-FLSs. The cells were stimulated with the indicated amounts of plate-bound ZF1 for 12 hours or 0.1 mg/mL of matrine for different time periods (0, 4, 8, 12, 24, and 48 hours) prior to the MTT assay. Mouse IgG (concentration 0.0125–0.2 mg/mL) on FLSs was used as the homotype control. The data shown are the mean from three parallel experiments (mean ± SD), ***P<0.001 vs the IgG group. (B) Flow cytometry detection of apoptosis with annexin V/PI in AA-FLSs stimulated with ZF1 at concentrations of 0, 0.025, 0.05, and 0.1 mg/mL. (C) The percentages of apoptotic cells (Q2 + Q3) (shown in Figure 2B) are presented as the mean ± SD of three independent experiments, **P<0.01; ***P<0.001. Abbreviations: AA, adjuvant arthritis; DR, death receptor; FLS, fibroblast-like synoviocyte; MTT, 20 μL of a 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide; PI, propidium iodide; SD, standard deviation; DR, death receptor; APC, allophycocyanin; IgG, intravenous gamma globulin.
Figure 3
Figure 3
TIPE2 overexpression enhanced DR5 and caspase expression and downregulated the activation of NF-κB. Notes: (A) The FLSs were stimulated with 0.1 mg/mL ZF1 in ZF1 groups or PBS in no ZF1 groups for 6 hours. The total protein was extracted, and the expressions of apoptosis-related and intrinsic mitochondrial pathway-related proteins and pNF-κB-p105/p50 proteins were analyzed by Western blot assay. (B) Flow cytometry detection of apoptosis with annexin V/PI in AA-FLSs stimulated with ZF1. Before stimulation with 0.04 mg/mL ZF1, the TIPE2+/+-FLS group was pretreated with caspase inhibitor Z-VAD-FMK and the MIGR1-FLS group was pretreated with the NF-κB inhibitor Bay. (C) The FLSs were stimulated with 0.1 mg/mL ZF1 in ZF1 groups or without ZF1 in PBS groups for 6 hours. The total protein was extracted and the expressions of TIPE2 and DR5 proteins were analyzed using Western blot assay. (D) TIPE2 upregulated the expression of DR5 on FLSs. Flow cytometry detection of DR5 expression with anti-DR5 and mouse IgG-APC in AA-FLSs stimulated with 0.1 mg/mL ZF1 or without ZF1 for 12 hours was carried out. (E) TIPE2 up-regulated the expression of DR5 on FLSs. The data are presented as the mean ± SD of three independent experiments, ***P<0.001. Data are representative of three independent experiments, which showed similar results. Abbreviations: AA, adjuvant arthritis; DR, death receptor; FLS, fibroblast-like synoviocyte; NF-κB, nuclear factor-kappa B; PBS, phosphate-buffered saline; PI, propidium iodide; SD, standard deviation; APC, Allophycocyanin.
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