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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 Increased Protein Stability of αPIX. (A) Purified splenic T cells from the Git2+/+ and Git2−/− mice were analyzed for the expression of PAK, PIX, Rac1, and CDC42 by western-blot. Antibodies against the GAPDH served as a control. (B) Cell homogenates from the indicated tissues from the Git2+/+ and Git2−/− mice were resolved by SDS–PAGE and immunoblotted with a polyclonal anti-PIX antibody. (C) Expression analysis of αPIX or βPIX in indicated tissues by real-time PCR using primers recognizing either αPIX or βPIX. (D) Purified splenic T cells from the Git2+/+ and Git2−/− mice were cultured on 24-well plates with or without 50 μg/ml of cycloheximide (CHX) for the indicated times. (E) Purified splenic T cells from the Git2−/− mice were cultured on 24-well plates and infected with pBPLV-GIT2 or mock virus. 24 h later, the cells were treated with or without 50 μg/ml of cycloheximide (CHX) for the indicated times. αPIX and GIT2 expression were analyzed by Western blotting. Antibodies against the GAPDH served as a control. For (D) & (E), the immunoblot bands were scanned for densitometry analysis with the value obtained from control cells set as 1 (bottom). The results were representative of three independent experiments and error bars represent standard deviations (*P < 0.05).
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f0035: GIT2 Increased Protein Stability of αPIX. (A) Purified splenic T cells from the Git2+/+ and Git2−/− mice were analyzed for the expression of PAK, PIX, Rac1, and CDC42 by western-blot. Antibodies against the GAPDH served as a control. (B) Cell homogenates from the indicated tissues from the Git2+/+ and Git2−/− mice were resolved by SDS–PAGE and immunoblotted with a polyclonal anti-PIX antibody. (C) Expression analysis of αPIX or βPIX in indicated tissues by real-time PCR using primers recognizing either αPIX or βPIX. (D) Purified splenic T cells from the Git2+/+ and Git2−/− mice were cultured on 24-well plates with or without 50 μg/ml of cycloheximide (CHX) for the indicated times. (E) Purified splenic T cells from the Git2−/− mice were cultured on 24-well plates and infected with pBPLV-GIT2 or mock virus. 24 h later, the cells were treated with or without 50 μg/ml of cycloheximide (CHX) for the indicated times. αPIX and GIT2 expression were analyzed by Western blotting. Antibodies against the GAPDH served as a control. For (D) & (E), the immunoblot bands were scanned for densitometry analysis with the value obtained from control cells set as 1 (bottom). The results were representative of three independent experiments and error bars represent standard deviations (*P < 0.05).

Mentions: Because GIT2 regulates diverse cellular functions through interacting with various proteins, we analyzed the protein levels of GIT2 partners, such as PAK, PIX, Rac1 and Cdc42. The results showed similar expression of PAK, Rac1, or Cdc42; except for the reduction of PIX expression in GIT2-deficient T cells (Fig. 7A). The reduction in PIX protein was further confirmed in the thymus, spleens, and lymph nodes of Git2−/− mice using an anti-PIX antibody that recognizes both αPIX and βPIX (Fig. 7B). This effect of GIT2 on the PIX protein levels was found to not be due to changes in their transcription because GIT2 deficiency did not significantly alter the abundance of PIX mRNA in the thymus and spleens cells (Fig. 7C). Moreover, compared with control cells, a clear reduction in PIX stability was observed in GIT2-deficient T cells. In contrast, the PIX half-life was higher than 36 h in wild-type GIT2 cells and was shortened to ∼8–10 h in GIT2-deficient T cells (Fig. 7D). To demonstrate that the reduction in the PIX protein levels was a direct consequence of GIT2 deficiency, we reestablished the wild-type GIT2 expression levels in GIT2-deficient T cells using a lentivirus-based approach. The expression of full-length GIT2 led to a significant increase in the PIX steady-state protein levels (Fig. 7E).


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 Increased Protein Stability of αPIX. (A) Purified splenic T cells from the Git2+/+ and Git2−/− mice were analyzed for the expression of PAK, PIX, Rac1, and CDC42 by western-blot. Antibodies against the GAPDH served as a control. (B) Cell homogenates from the indicated tissues from the Git2+/+ and Git2−/− mice were resolved by SDS–PAGE and immunoblotted with a polyclonal anti-PIX antibody. (C) Expression analysis of αPIX or βPIX in indicated tissues by real-time PCR using primers recognizing either αPIX or βPIX. (D) Purified splenic T cells from the Git2+/+ and Git2−/− mice were cultured on 24-well plates with or without 50 μg/ml of cycloheximide (CHX) for the indicated times. (E) Purified splenic T cells from the Git2−/− mice were cultured on 24-well plates and infected with pBPLV-GIT2 or mock virus. 24 h later, the cells were treated with or without 50 μg/ml of cycloheximide (CHX) for the indicated times. αPIX and GIT2 expression were analyzed by Western blotting. Antibodies against the GAPDH served as a control. For (D) & (E), the immunoblot bands were scanned for densitometry analysis with the value obtained from control cells set as 1 (bottom). The results were representative of three independent experiments and error bars represent standard deviations (*P < 0.05).
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4556731&req=5

f0035: GIT2 Increased Protein Stability of αPIX. (A) Purified splenic T cells from the Git2+/+ and Git2−/− mice were analyzed for the expression of PAK, PIX, Rac1, and CDC42 by western-blot. Antibodies against the GAPDH served as a control. (B) Cell homogenates from the indicated tissues from the Git2+/+ and Git2−/− mice were resolved by SDS–PAGE and immunoblotted with a polyclonal anti-PIX antibody. (C) Expression analysis of αPIX or βPIX in indicated tissues by real-time PCR using primers recognizing either αPIX or βPIX. (D) Purified splenic T cells from the Git2+/+ and Git2−/− mice were cultured on 24-well plates with or without 50 μg/ml of cycloheximide (CHX) for the indicated times. (E) Purified splenic T cells from the Git2−/− mice were cultured on 24-well plates and infected with pBPLV-GIT2 or mock virus. 24 h later, the cells were treated with or without 50 μg/ml of cycloheximide (CHX) for the indicated times. αPIX and GIT2 expression were analyzed by Western blotting. Antibodies against the GAPDH served as a control. For (D) & (E), the immunoblot bands were scanned for densitometry analysis with the value obtained from control cells set as 1 (bottom). The results were representative of three independent experiments and error bars represent standard deviations (*P < 0.05).
Mentions: Because GIT2 regulates diverse cellular functions through interacting with various proteins, we analyzed the protein levels of GIT2 partners, such as PAK, PIX, Rac1 and Cdc42. The results showed similar expression of PAK, Rac1, or Cdc42; except for the reduction of PIX expression in GIT2-deficient T cells (Fig. 7A). The reduction in PIX protein was further confirmed in the thymus, spleens, and lymph nodes of Git2−/− mice using an anti-PIX antibody that recognizes both αPIX and βPIX (Fig. 7B). This effect of GIT2 on the PIX protein levels was found to not be due to changes in their transcription because GIT2 deficiency did not significantly alter the abundance of PIX mRNA in the thymus and spleens cells (Fig. 7C). Moreover, compared with control cells, a clear reduction in PIX stability was observed in GIT2-deficient T cells. In contrast, the PIX half-life was higher than 36 h in wild-type GIT2 cells and was shortened to ∼8–10 h in GIT2-deficient T cells (Fig. 7D). To demonstrate that the reduction in the PIX protein levels was a direct consequence of GIT2 deficiency, we reestablished the wild-type GIT2 expression levels in GIT2-deficient T cells using a lentivirus-based approach. The expression of full-length GIT2 led to a significant increase in the PIX steady-state protein levels (Fig. 7E).

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