Arginase II inhibited lipopolysaccharide-induced cell death by regulation of iNOS and Bcl-2 family proteins in macrophages

Abstract

Arginase II catalyzes the conversion of arginine to urea and ornithine in many extrahepatic tissues. We investigated the protective role of arginase II on lipopolysaccharide-mediated apoptosis in the macrophage cells. Adenoviral gene transfer of full length of arginase II was performed in the murine macrophage cell line RAW264.7. The role of arginase II was investigated with cell viability, cytoplasmic histone-associated DNA fragmentation assay, arginase activity, nitric oxide production, and Western blot analysis. Arginase II is localized in mitochondria of macrophage cells, and the expression of arginase II was increased by lipopolysaccharide (LPS). LPS significantly increased cell death which was inhibited by AMT, a specific inducible nitric oxide synthase (iNOS) inhibitor. In contrast, LPS-induced cell death and nitric oxide production were increased by 2-boronoethyl-L-cysteine, a specific inhibitor of arginase. Adenoviral overexpression of arginase II significantly inhibited LPS-induced cell death and cytoplasmic histone-associated DNA fragmentation. LPS-induced iNOS expression and poly ADP-ribose polymerase cleavage were significantly suppressed by arginase II overexpression. Furthermore, arginase II overexpression resulted in a decrease in the Bax protein level and the reverse induction of Bcl-2 protein. Our data demonstrated that inhibition of NO production by arginase II may be due to arginine depletion as well as iNOS suppression though its reaction products. Moreover, arginase II plays a protective role of LPS-induced apoptosis in RAW264.7 cells.

Fig. 1.

Lipopolysaccharide (LPS) induced arginase II expression and activity in RAW264.7 cells. (A) Mitochondrial localization of arginase II. Arginase II (green, left), mitotracker (red, middle), and merged images (right). Note that fluorescent images for arginase II was similar with mitotracker suggesting the mitochondrial localization of arginase II. (B) Co-induction of arginase II and iNOS by LPS in RAW264.7 cells. Cells were exposed with LPS (300 ng/ml) or AMT (10 μM), an inhibitor of iNOS for 18 h. Western blot for arginase II and iNOS proteins was performed. (C) Effect of AMT on LPS-induced nitric oxide production in RAW264.7 cells. Cells were exposed to AMT for 18 h. Data are presented as mean ± SEM (n = 3). ^*P < 0.05 vs basal, ^#P < 0.05 vs LPS. (D) Effect of AMT on LPS-arginase activity in RAW264.7 cells. Cells were exposed to AMT for 18 h. Data are presented as mean ± SEM (n = 3). ^*P < 0.05 vs basal, ^#P < 0.05 vs LPS.

Fig. 2.

Arginase inhibition increased LPS-induced cell death in RAW264.7 cells. (A) Effect of arginase inhibition on LPS-induced arginase activity. Cells were treated with LPS (300 ng/ml) in the presence or absence of BEC, arginase inhibitor for 18 h. Data are presented as mean ± SEM (n = 3). ^*P < 0.05 vs basal, ^#P < 0.05 vs. LPS. (B) Effect of arginase inhibition on LPS-induced nitrite production. Cells were treated with LPS (300 ng/ml) in the presence or absence of BEC, arginase inhibitor for 18 h. Data are presented as mean ± SEM (n = 5). ^*P < 0.05 vs basal, ^#P < 0.05 vs LPS. (C) Effect of arginase inhibition on LPS-induced cell death. Cells were treated with LPS (300 ng/ml) in the presence or absence of BEC, arginase inhibitor for 18 h. Data are presented as mean ± SEM (n = 5). ^*P < 0.05 vs basal, ^#P < 0.05 vs LPS.

Fig. 3.

Arginase II overexpression inhibited LPS-induced cell death in RAW264.7 cells. (A) Typical Western blot for arginase II (left) and arginase activity (right) in AdArgII-trasfected cells. Total adenoviral multiplicity of infection (100 MOI) was balanced with Adβgal. Data are presented as mean ± SEM (n = 3). ^*P < 0.05 vs. Adβgal. (B) Arginase II overexpression significantly inhibited LPS-induced cell death, which was assessed with propridium iodide staining, compared with Adβgal-transfected cells. Data are presented as mean ± SEM (n = 5). ^*P < 0.05 vs LPS-treated Adβgal-transfected cells. (C) Effect of arginase II expression on apoptosis induction in RAW264.7 cells assessed by quantitation of cytoplasmic histone-associated DNA fragmentation. Data are presented as mean ± SEM (n = 3). ^*P < 0.05 vs basal, ^#P < 0.05 vs LPS in Adβgal-transfected cells.

Fig. 4.

Arginase II overexpression inhibited LPS-induced iNOS expression in RAW264.7 cells. (A) Typical immunoblot for iNOS and arginase II protein using lysates from RAW264.7 cells treated with 300 ng/ml LPS for 18 h. (B) Effect of arginase II overexpression on LPS-induced iNOS expression Data are presented as mean ± SEM (n = 3). ^*P < 0.05 vs LPS in Adβgal-transfected cells. (C) Effect of arginase II overexpression on the nitric oxide production. Nitrite production was measured in supernatant of cultured medium using Griess reagent. Data are presented as mean ± SEM (n = 3). ^*P < 0.05 vs LPS in Adβgal-transfected cells.

Fig. 5.

Arginase II overexpression inhibited mitochondrial apoptosis protein and apoptosis in RAW264.7 cells. (A) Immunoblotting for cleavaged PARP, Bax, and Bcl-2 proteins in RAW264.7 cells. LPS (300 ng/ml) or AMT (10 μM) was treated for 18 h in Adβgal or AdArgII-transfected cells. (B) Summarized data for Fig. 5A. Data are presented as mean ± SEM (n = 4). ^*P < 0.05 vs LPS in Adβgal-transfected cells. ^#P < 0.05 vs. LPS in Adβgal-transfected cells.