The chemokine CCL5 regulates glucose uptake and AMP kinase signaling in activated T cells to facilitate chemotaxis - PubMed (original) (raw)

Clinical Trial

. 2012 Aug 24;287(35):29406-16.

doi: 10.1074/jbc.M112.348946. Epub 2012 Jul 10.

Affiliations

Clinical Trial

The chemokine CCL5 regulates glucose uptake and AMP kinase signaling in activated T cells to facilitate chemotaxis

Olivia Chan et al. J Biol Chem. 2012.

Abstract

Recruitment of effector T cells to sites of infection or inflammation is essential for an effective adaptive immune response. The chemokine CCL5 (RANTES) activates its cognate receptor, CCR5, to initiate cellular functions, including chemotaxis. In earlier studies, we reported that CCL5-induced CCR5 signaling activates the mTOR/4E-BP1 pathway to directly modulate mRNA translation. Specifically, CCL5-mediated mTOR activation contributes to T cell chemotaxis by initiating the synthesis of chemotaxis-related proteins. Up-regulation of chemotaxis-related proteins may prime T cells for efficient migration. It is now clear that mTOR is also a central regulator of nutrient sensing and glycolysis. Herein we describe a role for CCL5-mediated glucose uptake and ATP accumulation to meet the energy demands of chemotaxis in activated T cells. We provide evidence that CCL5 is able to induce glucose uptake in an mTOR-dependent manner. CCL5 treatment of ex vivo activated human CD3(+) T cells also induced the activation of the nutrient-sensing kinase AMPK and downstream substrates ACC-1, PFKFB-2, and GSK-3β. Using 2-deoxy-d-glucose, an inhibitor of glucose uptake, and compound C, an inhibitor of AMPK, experimental data are presented that demonstrate that CCL5-mediated T cell chemotaxis is dependent on glucose, as these inhibitors inhibit CCL5-mediated chemotaxis in a dose-dependent manner. Altogether, these findings suggest that both glycolysis and AMPK signaling are required for efficient T cell migration in response to CCL5. These studies extend the role of CCL5 mediated CCR5 signaling beyond lymphocyte chemotaxis and demonstrate a role for chemokines in promoting glucose uptake and ATP production to match energy demands of migration.

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Figures

FIGURE 1.

FIGURE 1.

CCL5 induces phosphorylation of proteins in the AMPK signaling pathway. A, the Full Moon BioSystems AMPK signaling phospho-specific antibody array includes six replicates (vertical columns) of phospho-specific antibodies and their non-phospho pairs, targeted against proteins in the AMPK signaling pathway. Biotinylated protein lysates were added to microscope slide chambers and fluorescence from Cy3-streptavidin was measured with the Axon GenePix 400A microarray scanner. B, the extent of protein phosphorylation (mean fluorescence intensity, MFI) was normalized within each slide and compared between untreated control and cells treated with 10 n

m

CCL5 for 10 min. The data are represented as fold CCL5-induction relative to untreated controls. Phosphorylated signaling intermediates associated with metabolism are indicated (red arrows).

FIGURE 2.

FIGURE 2.

CCL5 activates the energy-sensing kinase AMPK and the downstream substrate GSK-3β resulting in an increased intracellular ATP levels. A, activated PB T cells were either left untreated or treated with 10 n

m

CCL5 for the indicated times. Cells were harvested and protein lysates resolved by SDS-PAGE and immunoblotted with anti-phospho-AMPKα (Thr-172) or anti-phospho-GSK-3β (Ser-9) antibodies. Membranes were stripped and reprobed for loading. Relative phosphorylation is shown as signal intensity over loading control. Data are representative of two independent experiments. B, activated PB T cells were either treated with dimethyl sulfoxide or 10 μ

m

compound C for 1 h before treatment with 10 n

m

CCL5 for the indicated times. Intracellular ATP was measured using a bioluminescent assay. Data are representative of two independent experiments. *, p < 0.01; **, p < 0.05. C, activated PB T-cells were pretreated with dimethyl sulfoxide (DMSO; carrier control), oligomycin (1 μ

m

), or 2-deoxy-glucose (10 m

m

) for 30 min and then stimulated with IL-2 (20 ng/ml) or CCL5 (10 n

m

) for 30 min. Intracellular ATP levels were then measured using a bioluminescent assay. Data are representative of two independent experiments. *, p < 0.01.

FIGURE 3.

FIGURE 3.

CCL5-mediated glucose uptake is mTOR-dependent. A, activated PB T cells were either left untreated, treated with 20 ng/ml IL-2, or the indicated doses of CCL5 for 2 h. In parallel, cells were pretreated with 10 m

m

of 2-DG for 1 h prior to treatment with 10 n

m

CCL5. At time 0, 2 μCi/rxn of 2-deoxy-

d

-[3H] glucose was added to the cultures. Reactions were quenched and radioactivity measured with a liquid scintillation counter. Data are representative of three independent studies. B, cells were pretreated with either dimethyl sulfoxide (DMSO; carrier) or 50 n

m

of rapamycin for 1 h prior to treatment with 10 n

m

CCL5. Tritiated glucose uptake was measured as in A. Data are representative of two independent studies. C, cells were pretreated with CCR5 antagonist, TAK-779 for 1 h prior to treatment with 10 n

m

CCL5. Tritiated glucose uptake was measured as in A.

FIGURE 4.

FIGURE 4.

CCL5 prolongs cell surface expression of GLUT-1 and CD98. Activated PB T cells were either left untreated or treated with 10 n

m

CCL5 or 20 ng/ml IL-2 for 24 h. Cells were fixed with 2% paraformaldehyde and stained for cell surface GLUT-1 (A and C) or CD98 (B and D) expression and analyzed by FACS.

FIGURE 5.

FIGURE 5.

Glucose uptake and AMPK signaling are required for efficient CCL5-mediated chemotaxis. A, activated PB T cells were either left untreated or pretreated with 2-DG at the doses indicated for 1 h. A total of 1 × 105 cells in 100 μl of chemotaxis buffer were then placed in the upper chamber of Transwell chambers. CCL5-mediated chemotaxis was measured using 10 n

m

CCL5. Data are presented as % migration, with the number of migrated cells at 10 n

m

CCL5 taken as 100%. Data are representative of three independent experiments. B, activated PB T cells were pre-treated with either dimethyl sulfoxide (DMSO; carrier) or different doses of compound C for 1 h. CCL5-mediated chemotaxis was measured as described in A. Data are representative of three independent experiments. *, p < 0.01.

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