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Inhibition of G-protein βγ signaling enhances T cell receptor-stimulated interleukin 2 transcription in CD4+ T helper cells.

Yost EA, Hynes TR, Hartle CM, Ott BJ, Berlot CH - PLoS ONE (2015)

Bottom Line: Blocking Gβγ also enhanced TCR-stimulated increases in nuclear localization of nuclear factor of activated T cells 1 (NFAT1), NFAT transcriptional activity, and levels of intracellular Ca2+.Potentiation of IL-2 transcription required continuous Gβγ inhibition during at least two days of TCR stimulation, suggesting that induction or repression of additional signaling proteins during T cell activation and differentiation might be involved.The potentiation of TCR-stimulated IL-2 transcription that results from blocking Gβγ in CD4+ T helper cells could have applications for autoimmune diseases.

View Article: PubMed Central - PubMed

Affiliation: Weis Center for Research, Geisinger Clinic, Danville, Pennsylvania, 17822-2623, United States of America.

ABSTRACT
G-protein-coupled receptor (GPCR) signaling modulates the expression of cytokines that are drug targets for immune disorders. However, although GPCRs are common targets for other diseases, there are few GPCR-based pharmaceuticals for inflammation. The purpose of this study was to determine whether targeting G-protein βγ (Gβγ) complexes could provide a useful new approach for modulating interleukin 2 (IL-2) levels in CD4+ T helper cells. Gallein, a small molecule inhibitor of Gβγ, increased levels of T cell receptor (TCR)-stimulated IL-2 mRNA in primary human naïve and memory CD4+ T helper cells and in Jurkat human CD4+ leukemia T cells. Gβ1 and Gβ2 mRNA accounted for >99% of Gβ mRNA, and small interfering RNA (siRNA)-mediated silencing of Gβ1 but not Gβ2 enhanced TCR-stimulated IL-2 mRNA increases. Blocking Gβγ enhanced TCR-stimulated increases in IL-2 transcription without affecting IL-2 mRNA stability. Blocking Gβγ also enhanced TCR-stimulated increases in nuclear localization of nuclear factor of activated T cells 1 (NFAT1), NFAT transcriptional activity, and levels of intracellular Ca2+. Potentiation of IL-2 transcription required continuous Gβγ inhibition during at least two days of TCR stimulation, suggesting that induction or repression of additional signaling proteins during T cell activation and differentiation might be involved. The potentiation of TCR-stimulated IL-2 transcription that results from blocking Gβγ in CD4+ T helper cells could have applications for autoimmune diseases.

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Disrupting Gβγ signaling enhances TCR-stimulated IL-2 transcription without affecting IL-2 mRNA stability.(A) Gβ1 siRNA does not increase stability of IL-2 mRNA. After three days of TCR stimulation with plate-bound anti-CD3 and soluble anti-CD28 and treatment with Gβ1 siRNA or NT siRNA, Jurkat cells were incubated for the indicated times with 10 μg/mL of Actinomycin D to inhibit transcription, and the rate of IL-2 mRNA degradation was measured. In both cases, the rates of IL-2 mRNA degradation fit a single exponential. Data represent means ± SD from triplicate determinations from a single experiment representative of 5 experiments. (B) Gallein increases IL-2 promoter activity in a luciferase reporter assay. Jurkat cells were stimulated with plate-bound anti-CD3 and soluble anti-CD28 in the presence or absence of gallein for three days following nucleofection with the indicated plasmids. Data represent means ± SD from triplicate determinations from a single assay representative of 8 assays.
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pone.0116575.g005: Disrupting Gβγ signaling enhances TCR-stimulated IL-2 transcription without affecting IL-2 mRNA stability.(A) Gβ1 siRNA does not increase stability of IL-2 mRNA. After three days of TCR stimulation with plate-bound anti-CD3 and soluble anti-CD28 and treatment with Gβ1 siRNA or NT siRNA, Jurkat cells were incubated for the indicated times with 10 μg/mL of Actinomycin D to inhibit transcription, and the rate of IL-2 mRNA degradation was measured. In both cases, the rates of IL-2 mRNA degradation fit a single exponential. Data represent means ± SD from triplicate determinations from a single experiment representative of 5 experiments. (B) Gallein increases IL-2 promoter activity in a luciferase reporter assay. Jurkat cells were stimulated with plate-bound anti-CD3 and soluble anti-CD28 in the presence or absence of gallein for three days following nucleofection with the indicated plasmids. Data represent means ± SD from triplicate determinations from a single assay representative of 8 assays.

Mentions: Disrupting Gβγ signaling could enhance TCR-stimulated increases in IL-2 mRNA levels by increasing IL-2 transcription and/or IL-2 mRNA stability. To determine whether inhibition of Gβγ signaling increased IL-2 mRNA stability, we measured the half-life of IL-2 mRNA in Jurkat cells stimulated with plate-bound anti-CD3 antibodies and soluble anti-CD28 antibodies for three days and then treated with Actinomycin D to inhibit transcription. Gβ1 siRNA did not increase the stability of IL-2 mRNA (Fig. 5A). In the presence of Gβ1 siRNA, the t1/2 of IL-2 mRNA (11.91 min, SE = 0.58, N = 5) was the same as in the presence of NT siRNA (11.35 min, SE = 0.56, N = 5).


Inhibition of G-protein βγ signaling enhances T cell receptor-stimulated interleukin 2 transcription in CD4+ T helper cells.

Yost EA, Hynes TR, Hartle CM, Ott BJ, Berlot CH - PLoS ONE (2015)

Disrupting Gβγ signaling enhances TCR-stimulated IL-2 transcription without affecting IL-2 mRNA stability.(A) Gβ1 siRNA does not increase stability of IL-2 mRNA. After three days of TCR stimulation with plate-bound anti-CD3 and soluble anti-CD28 and treatment with Gβ1 siRNA or NT siRNA, Jurkat cells were incubated for the indicated times with 10 μg/mL of Actinomycin D to inhibit transcription, and the rate of IL-2 mRNA degradation was measured. In both cases, the rates of IL-2 mRNA degradation fit a single exponential. Data represent means ± SD from triplicate determinations from a single experiment representative of 5 experiments. (B) Gallein increases IL-2 promoter activity in a luciferase reporter assay. Jurkat cells were stimulated with plate-bound anti-CD3 and soluble anti-CD28 in the presence or absence of gallein for three days following nucleofection with the indicated plasmids. Data represent means ± SD from triplicate determinations from a single assay representative of 8 assays.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4309538&req=5

pone.0116575.g005: Disrupting Gβγ signaling enhances TCR-stimulated IL-2 transcription without affecting IL-2 mRNA stability.(A) Gβ1 siRNA does not increase stability of IL-2 mRNA. After three days of TCR stimulation with plate-bound anti-CD3 and soluble anti-CD28 and treatment with Gβ1 siRNA or NT siRNA, Jurkat cells were incubated for the indicated times with 10 μg/mL of Actinomycin D to inhibit transcription, and the rate of IL-2 mRNA degradation was measured. In both cases, the rates of IL-2 mRNA degradation fit a single exponential. Data represent means ± SD from triplicate determinations from a single experiment representative of 5 experiments. (B) Gallein increases IL-2 promoter activity in a luciferase reporter assay. Jurkat cells were stimulated with plate-bound anti-CD3 and soluble anti-CD28 in the presence or absence of gallein for three days following nucleofection with the indicated plasmids. Data represent means ± SD from triplicate determinations from a single assay representative of 8 assays.
Mentions: Disrupting Gβγ signaling could enhance TCR-stimulated increases in IL-2 mRNA levels by increasing IL-2 transcription and/or IL-2 mRNA stability. To determine whether inhibition of Gβγ signaling increased IL-2 mRNA stability, we measured the half-life of IL-2 mRNA in Jurkat cells stimulated with plate-bound anti-CD3 antibodies and soluble anti-CD28 antibodies for three days and then treated with Actinomycin D to inhibit transcription. Gβ1 siRNA did not increase the stability of IL-2 mRNA (Fig. 5A). In the presence of Gβ1 siRNA, the t1/2 of IL-2 mRNA (11.91 min, SE = 0.58, N = 5) was the same as in the presence of NT siRNA (11.35 min, SE = 0.56, N = 5).

Bottom Line: Blocking Gβγ also enhanced TCR-stimulated increases in nuclear localization of nuclear factor of activated T cells 1 (NFAT1), NFAT transcriptional activity, and levels of intracellular Ca2+.Potentiation of IL-2 transcription required continuous Gβγ inhibition during at least two days of TCR stimulation, suggesting that induction or repression of additional signaling proteins during T cell activation and differentiation might be involved.The potentiation of TCR-stimulated IL-2 transcription that results from blocking Gβγ in CD4+ T helper cells could have applications for autoimmune diseases.

View Article: PubMed Central - PubMed

Affiliation: Weis Center for Research, Geisinger Clinic, Danville, Pennsylvania, 17822-2623, United States of America.

ABSTRACT
G-protein-coupled receptor (GPCR) signaling modulates the expression of cytokines that are drug targets for immune disorders. However, although GPCRs are common targets for other diseases, there are few GPCR-based pharmaceuticals for inflammation. The purpose of this study was to determine whether targeting G-protein βγ (Gβγ) complexes could provide a useful new approach for modulating interleukin 2 (IL-2) levels in CD4+ T helper cells. Gallein, a small molecule inhibitor of Gβγ, increased levels of T cell receptor (TCR)-stimulated IL-2 mRNA in primary human naïve and memory CD4+ T helper cells and in Jurkat human CD4+ leukemia T cells. Gβ1 and Gβ2 mRNA accounted for >99% of Gβ mRNA, and small interfering RNA (siRNA)-mediated silencing of Gβ1 but not Gβ2 enhanced TCR-stimulated IL-2 mRNA increases. Blocking Gβγ enhanced TCR-stimulated increases in IL-2 transcription without affecting IL-2 mRNA stability. Blocking Gβγ also enhanced TCR-stimulated increases in nuclear localization of nuclear factor of activated T cells 1 (NFAT1), NFAT transcriptional activity, and levels of intracellular Ca2+. Potentiation of IL-2 transcription required continuous Gβγ inhibition during at least two days of TCR stimulation, suggesting that induction or repression of additional signaling proteins during T cell activation and differentiation might be involved. The potentiation of TCR-stimulated IL-2 transcription that results from blocking Gβγ in CD4+ T helper cells could have applications for autoimmune diseases.

Show MeSH
Related in: MedlinePlus