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GADD34 inhibits activation-induced apoptosis of macrophages through enhancement of autophagy.

Ito S, Tanaka Y, Oshino R, Aiba K, Thanasegaran S, Nishio N, Isobe K - Sci Rep (2015)

Bottom Line: When LPS stimulation was combined with tyrosine/cysteine-deprivation, a deficiency of GADD34 enhanced cell activation signaling such as Src-family, Erk1/2, p38 MAPK and Akt.LC3-II was increased by LPS stimulation combined with tyrosine/cysteine-deprivation.Defective GADD34 reduced LC3-II and autophagosome formation induced by LPS-stimulation and tyrosine/cysteine-deprivation compared with that seen in wild-type macrophages.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, Nagoya University Graduate School of Medicine, 65 Turumai-cho, Showa-ku, Nagoya, Aichi, 466-8550, Japan.

ABSTRACT
Autophagy is a common physiological function in all eukaryotes. The process is induced by depletion of nutrients including amino acids. GADD34 is expressed following DNA damage, ER stresses and amino acid deprivation. Here, we investigated the effects of GADD34 on autophagy and cell activation in macrophages. The deprivation of tyrosine and cysteine markedly induced the expression of GADD34 in macrophages. LPS stimulation combined with tyrosine/cysteine-deprivation initially activated macrophages, but then shifted to cell death in late phase of stimulation. When LPS stimulation was combined with tyrosine/cysteine-deprivation, a deficiency of GADD34 enhanced cell activation signaling such as Src-family, Erk1/2, p38 MAPK and Akt. In the late phase of stimulation, a deficiency of GADD34 increased apoptosis more than that in wild-type macrophages. Further we found that mTOR-S6K signaling was highly enhanced in GADD34-deficient macrophages compared with wild-type cells when cells were treated by LPS combined with tyrosine/cysteine-deprivation. LC3-II was increased by LPS stimulation combined with tyrosine/cysteine-deprivation. Defective GADD34 reduced LC3-II and autophagosome formation induced by LPS-stimulation and tyrosine/cysteine-deprivation compared with that seen in wild-type macrophages. These results indicates that GADD34 enhances autophagy and suppresses apoptosis stimulated by LPS combined with amino acid deprivation through regulation of mTOR signaling pathway in macrophages.

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Loss of GADD34 increased cell activation signaling induced by LPS with Tyr/Cys-deprivation.(a). GADD34-deficient or control RAW 264.7 cells were treated with LPS (1 μg/mL) with Tyr/Cys-deprivation for the indicated times (0–24 h). Cell lysates were immunoblotted with the indicated antibodies. Graph shows the relative expression as means ± SE of three independent experiments. (b). Cell lysates of GADD34-deficient or control RAW 264.7 cells were subjected to immunoprecipitation (IP) using anti-GADD34 antibody. Immunoprecipitates were immunoblotted (IB) with anti-Lyn and anti-GADD34 antibodies. Data are representative of three independent experiments. The original immunoblots are presented in Supplementary Figure 4. *p < 0.05, **p < 0.01, *** p < 0.001.
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f4: Loss of GADD34 increased cell activation signaling induced by LPS with Tyr/Cys-deprivation.(a). GADD34-deficient or control RAW 264.7 cells were treated with LPS (1 μg/mL) with Tyr/Cys-deprivation for the indicated times (0–24 h). Cell lysates were immunoblotted with the indicated antibodies. Graph shows the relative expression as means ± SE of three independent experiments. (b). Cell lysates of GADD34-deficient or control RAW 264.7 cells were subjected to immunoprecipitation (IP) using anti-GADD34 antibody. Immunoprecipitates were immunoblotted (IB) with anti-Lyn and anti-GADD34 antibodies. Data are representative of three independent experiments. The original immunoblots are presented in Supplementary Figure 4. *p < 0.05, **p < 0.01, *** p < 0.001.

Mentions: We next analyzed the effect of GADD34 on cell activation induced by LPS combined with Tyr/Cys-deprivation in macrophage. Src family of kinases is one of important cell activation signaling. The phosphorylation of Tyr 416 in the Src-family activation kinase domain was already high in GADD34-deficient cells in the absence of stimulation (0 h). High levels were maintained by LPS stimulation combined with Tyr/Cys-deprivation (Fig. 4a). The phosphorylation of Erk and p38 mitogen-activated protein (MAP) kinase was upregulated by LPS with Tyr/Cys-deprivation. This upregulation was higher in GADD34-deficient cells than in control cells. Next we analyzed the interaction of GADD34 with Lyn, a Src-kinase family member. Immunoprecipitation with anti-GADD34 antibody confirmed the binding of GADD34 to Lyn in macrophages after LPS stimulation combined with Tyr/Cys-deprivation (Fig. 4b). These results suggested that during the activation of macrophages induced by LPS combined with Tyr/Cys-deprivation, GADD34 might regulate MAP kinase through modulating the phosphorylation of a Src family kinase, Lyn and thereby suppress cell activation.


GADD34 inhibits activation-induced apoptosis of macrophages through enhancement of autophagy.

Ito S, Tanaka Y, Oshino R, Aiba K, Thanasegaran S, Nishio N, Isobe K - Sci Rep (2015)

Loss of GADD34 increased cell activation signaling induced by LPS with Tyr/Cys-deprivation.(a). GADD34-deficient or control RAW 264.7 cells were treated with LPS (1 μg/mL) with Tyr/Cys-deprivation for the indicated times (0–24 h). Cell lysates were immunoblotted with the indicated antibodies. Graph shows the relative expression as means ± SE of three independent experiments. (b). Cell lysates of GADD34-deficient or control RAW 264.7 cells were subjected to immunoprecipitation (IP) using anti-GADD34 antibody. Immunoprecipitates were immunoblotted (IB) with anti-Lyn and anti-GADD34 antibodies. Data are representative of three independent experiments. The original immunoblots are presented in Supplementary Figure 4. *p < 0.05, **p < 0.01, *** p < 0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4321179&req=5

f4: Loss of GADD34 increased cell activation signaling induced by LPS with Tyr/Cys-deprivation.(a). GADD34-deficient or control RAW 264.7 cells were treated with LPS (1 μg/mL) with Tyr/Cys-deprivation for the indicated times (0–24 h). Cell lysates were immunoblotted with the indicated antibodies. Graph shows the relative expression as means ± SE of three independent experiments. (b). Cell lysates of GADD34-deficient or control RAW 264.7 cells were subjected to immunoprecipitation (IP) using anti-GADD34 antibody. Immunoprecipitates were immunoblotted (IB) with anti-Lyn and anti-GADD34 antibodies. Data are representative of three independent experiments. The original immunoblots are presented in Supplementary Figure 4. *p < 0.05, **p < 0.01, *** p < 0.001.
Mentions: We next analyzed the effect of GADD34 on cell activation induced by LPS combined with Tyr/Cys-deprivation in macrophage. Src family of kinases is one of important cell activation signaling. The phosphorylation of Tyr 416 in the Src-family activation kinase domain was already high in GADD34-deficient cells in the absence of stimulation (0 h). High levels were maintained by LPS stimulation combined with Tyr/Cys-deprivation (Fig. 4a). The phosphorylation of Erk and p38 mitogen-activated protein (MAP) kinase was upregulated by LPS with Tyr/Cys-deprivation. This upregulation was higher in GADD34-deficient cells than in control cells. Next we analyzed the interaction of GADD34 with Lyn, a Src-kinase family member. Immunoprecipitation with anti-GADD34 antibody confirmed the binding of GADD34 to Lyn in macrophages after LPS stimulation combined with Tyr/Cys-deprivation (Fig. 4b). These results suggested that during the activation of macrophages induced by LPS combined with Tyr/Cys-deprivation, GADD34 might regulate MAP kinase through modulating the phosphorylation of a Src family kinase, Lyn and thereby suppress cell activation.

Bottom Line: When LPS stimulation was combined with tyrosine/cysteine-deprivation, a deficiency of GADD34 enhanced cell activation signaling such as Src-family, Erk1/2, p38 MAPK and Akt.LC3-II was increased by LPS stimulation combined with tyrosine/cysteine-deprivation.Defective GADD34 reduced LC3-II and autophagosome formation induced by LPS-stimulation and tyrosine/cysteine-deprivation compared with that seen in wild-type macrophages.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, Nagoya University Graduate School of Medicine, 65 Turumai-cho, Showa-ku, Nagoya, Aichi, 466-8550, Japan.

ABSTRACT
Autophagy is a common physiological function in all eukaryotes. The process is induced by depletion of nutrients including amino acids. GADD34 is expressed following DNA damage, ER stresses and amino acid deprivation. Here, we investigated the effects of GADD34 on autophagy and cell activation in macrophages. The deprivation of tyrosine and cysteine markedly induced the expression of GADD34 in macrophages. LPS stimulation combined with tyrosine/cysteine-deprivation initially activated macrophages, but then shifted to cell death in late phase of stimulation. When LPS stimulation was combined with tyrosine/cysteine-deprivation, a deficiency of GADD34 enhanced cell activation signaling such as Src-family, Erk1/2, p38 MAPK and Akt. In the late phase of stimulation, a deficiency of GADD34 increased apoptosis more than that in wild-type macrophages. Further we found that mTOR-S6K signaling was highly enhanced in GADD34-deficient macrophages compared with wild-type cells when cells were treated by LPS combined with tyrosine/cysteine-deprivation. LC3-II was increased by LPS stimulation combined with tyrosine/cysteine-deprivation. Defective GADD34 reduced LC3-II and autophagosome formation induced by LPS-stimulation and tyrosine/cysteine-deprivation compared with that seen in wild-type macrophages. These results indicates that GADD34 enhances autophagy and suppresses apoptosis stimulated by LPS combined with amino acid deprivation through regulation of mTOR signaling pathway in macrophages.

Show MeSH
Related in: MedlinePlus