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GIT2 deficiency attenuates concanavalin A-induced hepatitis in mice.

Hao YE, He DF, Yin RH, Chen H, Wang J, Wang SX, Zhan YQ, Ge CH, Li CY, Yu M, Yang XM - FEBS Open Bio (2015)

Bottom Line: G protein-coupled receptor kinase interactor 2 (GIT2) is a signaling scaffold protein involved in regulation of cytoskeletal dynamics and the internalization of G protein-coupled receptors (GPCRs).However, the functions of GIT2 in T cells have not yet been determined.Our results suggested that GIT2 plays an important role in T-cell function in vivo and in vitro.

View Article: PubMed Central - PubMed

Affiliation: Southern Medical University, Guangzhou, Guangdong Province, China ; State Key Laboratory of Proteomics, Beijing Proteome Research Center, Beijing Institute of Radiation Medicine, Beijing 100850, China.

ABSTRACT
G protein-coupled receptor kinase interactor 2 (GIT2) is a signaling scaffold protein involved in regulation of cytoskeletal dynamics and the internalization of G protein-coupled receptors (GPCRs). The short-splice form of GIT2 is expressed in peripheral T cells and thymocytes. However, the functions of GIT2 in T cells have not yet been determined. We show that treatment with Con A in a model of polyclonal T-lymphocyte activation resulted in marked inhibitions in the intrahepatic infiltration of inflammatory cells, cytokine response and acute liver failure in Git2 (-/-) mice. CD4(+) T cells from Git2 (-/-) mice showed significant impairment in proliferation, cytokine production and signal transduction upon TCR-stimulated activation. Our results suggested that GIT2 plays an important role in T-cell function in vivo and in vitro.

No MeSH data available.


Related in: MedlinePlus

GIT2 depletion attenuated Con A-induced immunological hepatic injuries. (A) Genomic PCR using genomic DNA from the mouse tail of Git2+/+, Git2+/−, and Git2−/− mice. (B) GIT2 expression in thymocytes, splenic CD4+ T cells and liver tissues were analyzed using mouse GIT2 antibody. (C) Representative photographs of spleens in Git2+/+ and Git2−/− mice. (D) Total splenic cell numbers from Git2+/+ and Git2−/− mice (n = 6). Graphs in figures show mean ± s.e.m., *P < 0.05. (E) Serum ALT and AST levels from Git2+/+ and Git2−/− mice (n = 3) were determined at the indicated time points following 15 mg/kg Con A injection. (F and G) 24 h after 15 mg/kg Con A injection, mice were sacrificed and the liver tissues were stained with H&E for histopathological and morphological analysis. Scare bar, 50 μm (F). The percentage of necrotic area was quantitated using ImageJ software, and values are the mean ± SD of five fields of measurements (G). (H) TUNEL staining was performed on liver sections from Git2+/+ and Git2−/− mice with 15 mg/kg Con A treatment. The TUNEL-positive cells are shown by arrows, and values are the mean ± SD of five fields of measurements. Scare bar, 50 μm. The results were representative of three independent experiments and error bars represent standard deviations (*P < 0.05, **P < 0.01, ***P < 0.001).
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f0005: GIT2 depletion attenuated Con A-induced immunological hepatic injuries. (A) Genomic PCR using genomic DNA from the mouse tail of Git2+/+, Git2+/−, and Git2−/− mice. (B) GIT2 expression in thymocytes, splenic CD4+ T cells and liver tissues were analyzed using mouse GIT2 antibody. (C) Representative photographs of spleens in Git2+/+ and Git2−/− mice. (D) Total splenic cell numbers from Git2+/+ and Git2−/− mice (n = 6). Graphs in figures show mean ± s.e.m., *P < 0.05. (E) Serum ALT and AST levels from Git2+/+ and Git2−/− mice (n = 3) were determined at the indicated time points following 15 mg/kg Con A injection. (F and G) 24 h after 15 mg/kg Con A injection, mice were sacrificed and the liver tissues were stained with H&E for histopathological and morphological analysis. Scare bar, 50 μm (F). The percentage of necrotic area was quantitated using ImageJ software, and values are the mean ± SD of five fields of measurements (G). (H) TUNEL staining was performed on liver sections from Git2+/+ and Git2−/− mice with 15 mg/kg Con A treatment. The TUNEL-positive cells are shown by arrows, and values are the mean ± SD of five fields of measurements. Scare bar, 50 μm. The results were representative of three independent experiments and error bars represent standard deviations (*P < 0.05, **P < 0.01, ***P < 0.001).

Mentions: Heterozygous GIT2-knockout mice were bred to produce Git2+/+ controls and homozygous GIT2-knockout (Git2−/−) mice. Positive founder mice were identified by RT-PCR and Western bolt analyses (Fig. 1A and B). GIT2 was completely absent from the total thymocytes, splenic CD4+ T cells and liver tissues, as determined using an antibody against the mouse GIT2. In agreement with previous reports [13], adult Git2−/− mice showed no gross phenotypic abnormalities but often developed splenomegaly (Fig. 1C and D) and an increased CD4+/CD8+ ratio in the liver MNCs (Table 1). To examine the effects of GIT2 on immune-mediated hepatitis, we applied the Con A-induced hepatitis model in six- to eight-week-old male Git2+/+ and Git2−/− mice. We observed that the serum AST and ALT levels after the injection of 15 mg/kg Con A were significantly lower in Git2−/− mice compared with Git2+/+ mice (Fig. 1E). Histological analyses of liver tissue sections obtained 24 h after the administration of 15 mg/kg Con A indicated that Git2−/− mice were less sensitive to Con A-induced hepatic injury (Fig. 1F). Liver tissue sections of Git2−/− mice showed several solitary areas of necrotic tissue characterized by standard morphologic criteria (Fig. 1F – d–f), and the majority of hepatocytes were not affected. However, liver tissue sections of Git2+/+ mice showed widespread areas of necrosis (Fig. 1F – a) with extensive infiltration of mononuclear cells within the liver lobules (Fig. 1F – b) and around the central veins and portal tracts (Fig. 1F – c), indicating an ongoing inflammatory process. Consistent with these findings, the percentage of liver tissue with necrotic damage was markedly lower in Git2−/− mice (Fig. 1G). The livers of Git2−/− mice also demonstrated a significant decrease in hepatocyte apoptosis 24 h after treatment with 15 mg/kg Con A (Fig. 1H): the liver of Git2+/+ mice showed 37% hepatocyte apoptosis, whereas only 13% apoptosis was observed in the Git2−/− mice. Taken together, these data indicated that mice deficient in GIT2 appeared highly resistant to liver injuries induced by Con A.


GIT2 deficiency attenuates concanavalin A-induced hepatitis in mice.

Hao YE, He DF, Yin RH, Chen H, Wang J, Wang SX, Zhan YQ, Ge CH, Li CY, Yu M, Yang XM - FEBS Open Bio (2015)

GIT2 depletion attenuated Con A-induced immunological hepatic injuries. (A) Genomic PCR using genomic DNA from the mouse tail of Git2+/+, Git2+/−, and Git2−/− mice. (B) GIT2 expression in thymocytes, splenic CD4+ T cells and liver tissues were analyzed using mouse GIT2 antibody. (C) Representative photographs of spleens in Git2+/+ and Git2−/− mice. (D) Total splenic cell numbers from Git2+/+ and Git2−/− mice (n = 6). Graphs in figures show mean ± s.e.m., *P < 0.05. (E) Serum ALT and AST levels from Git2+/+ and Git2−/− mice (n = 3) were determined at the indicated time points following 15 mg/kg Con A injection. (F and G) 24 h after 15 mg/kg Con A injection, mice were sacrificed and the liver tissues were stained with H&E for histopathological and morphological analysis. Scare bar, 50 μm (F). The percentage of necrotic area was quantitated using ImageJ software, and values are the mean ± SD of five fields of measurements (G). (H) TUNEL staining was performed on liver sections from Git2+/+ and Git2−/− mice with 15 mg/kg Con A treatment. The TUNEL-positive cells are shown by arrows, and values are the mean ± SD of five fields of measurements. Scare bar, 50 μm. The results were representative of three independent experiments and error bars represent standard deviations (*P < 0.05, **P < 0.01, ***P < 0.001).
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f0005: GIT2 depletion attenuated Con A-induced immunological hepatic injuries. (A) Genomic PCR using genomic DNA from the mouse tail of Git2+/+, Git2+/−, and Git2−/− mice. (B) GIT2 expression in thymocytes, splenic CD4+ T cells and liver tissues were analyzed using mouse GIT2 antibody. (C) Representative photographs of spleens in Git2+/+ and Git2−/− mice. (D) Total splenic cell numbers from Git2+/+ and Git2−/− mice (n = 6). Graphs in figures show mean ± s.e.m., *P < 0.05. (E) Serum ALT and AST levels from Git2+/+ and Git2−/− mice (n = 3) were determined at the indicated time points following 15 mg/kg Con A injection. (F and G) 24 h after 15 mg/kg Con A injection, mice were sacrificed and the liver tissues were stained with H&E for histopathological and morphological analysis. Scare bar, 50 μm (F). The percentage of necrotic area was quantitated using ImageJ software, and values are the mean ± SD of five fields of measurements (G). (H) TUNEL staining was performed on liver sections from Git2+/+ and Git2−/− mice with 15 mg/kg Con A treatment. The TUNEL-positive cells are shown by arrows, and values are the mean ± SD of five fields of measurements. Scare bar, 50 μm. The results were representative of three independent experiments and error bars represent standard deviations (*P < 0.05, **P < 0.01, ***P < 0.001).
Mentions: Heterozygous GIT2-knockout mice were bred to produce Git2+/+ controls and homozygous GIT2-knockout (Git2−/−) mice. Positive founder mice were identified by RT-PCR and Western bolt analyses (Fig. 1A and B). GIT2 was completely absent from the total thymocytes, splenic CD4+ T cells and liver tissues, as determined using an antibody against the mouse GIT2. In agreement with previous reports [13], adult Git2−/− mice showed no gross phenotypic abnormalities but often developed splenomegaly (Fig. 1C and D) and an increased CD4+/CD8+ ratio in the liver MNCs (Table 1). To examine the effects of GIT2 on immune-mediated hepatitis, we applied the Con A-induced hepatitis model in six- to eight-week-old male Git2+/+ and Git2−/− mice. We observed that the serum AST and ALT levels after the injection of 15 mg/kg Con A were significantly lower in Git2−/− mice compared with Git2+/+ mice (Fig. 1E). Histological analyses of liver tissue sections obtained 24 h after the administration of 15 mg/kg Con A indicated that Git2−/− mice were less sensitive to Con A-induced hepatic injury (Fig. 1F). Liver tissue sections of Git2−/− mice showed several solitary areas of necrotic tissue characterized by standard morphologic criteria (Fig. 1F – d–f), and the majority of hepatocytes were not affected. However, liver tissue sections of Git2+/+ mice showed widespread areas of necrosis (Fig. 1F – a) with extensive infiltration of mononuclear cells within the liver lobules (Fig. 1F – b) and around the central veins and portal tracts (Fig. 1F – c), indicating an ongoing inflammatory process. Consistent with these findings, the percentage of liver tissue with necrotic damage was markedly lower in Git2−/− mice (Fig. 1G). The livers of Git2−/− mice also demonstrated a significant decrease in hepatocyte apoptosis 24 h after treatment with 15 mg/kg Con A (Fig. 1H): the liver of Git2+/+ mice showed 37% hepatocyte apoptosis, whereas only 13% apoptosis was observed in the Git2−/− mice. Taken together, these data indicated that mice deficient in GIT2 appeared highly resistant to liver injuries induced by Con A.

Bottom Line: G protein-coupled receptor kinase interactor 2 (GIT2) is a signaling scaffold protein involved in regulation of cytoskeletal dynamics and the internalization of G protein-coupled receptors (GPCRs).However, the functions of GIT2 in T cells have not yet been determined.Our results suggested that GIT2 plays an important role in T-cell function in vivo and in vitro.

View Article: PubMed Central - PubMed

Affiliation: Southern Medical University, Guangzhou, Guangdong Province, China ; State Key Laboratory of Proteomics, Beijing Proteome Research Center, Beijing Institute of Radiation Medicine, Beijing 100850, China.

ABSTRACT
G protein-coupled receptor kinase interactor 2 (GIT2) is a signaling scaffold protein involved in regulation of cytoskeletal dynamics and the internalization of G protein-coupled receptors (GPCRs). The short-splice form of GIT2 is expressed in peripheral T cells and thymocytes. However, the functions of GIT2 in T cells have not yet been determined. We show that treatment with Con A in a model of polyclonal T-lymphocyte activation resulted in marked inhibitions in the intrahepatic infiltration of inflammatory cells, cytokine response and acute liver failure in Git2 (-/-) mice. CD4(+) T cells from Git2 (-/-) mice showed significant impairment in proliferation, cytokine production and signal transduction upon TCR-stimulated activation. Our results suggested that GIT2 plays an important role in T-cell function in vivo and in vitro.

No MeSH data available.


Related in: MedlinePlus