Gallein enhances TCR-stimulated transcriptional activity of NFAT.

<p>(A) Major TCR-stimulated pathways leading to IL-2 transcription that could be inhibited by Gβγ. Interactions between the TCR and peptide-major histocompatibility complex (MHC) lead to recruitment of CD4 and its associated kinase, p56-Lck, which phosphorylates tyrosine residues in the cytoplasmic tails of the TCR subunits, leading to recruitment and phosphorylation of the tyrosine kinase, ZAP-70. CD28 costimulation provides an additional signal that is needed for complete T cell activation and regulation of IL-2 production [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116575#pone.0116575.ref088" target="_blank">88</a>]. ZAP-70 and p56-Lck then phosphorylate and activate numerous downstream target proteins, including phospholipase C-γ (PLC-γ), leading to Ras activation, Ca²⁺ increases, cytoskeletal rearrangements, and ultimately, activation of transcription factors that bind to the IL-2 promoter and increase IL-2 transcription. (B-C) Gallein increases transcriptional activity of NFAT, but not AP-1 or NFκB. Jurkat cells expressing reporter plasmids for AP-1, NFAT, or NFκB, were stimulated with plate-bound anti-CD3 and soluble anti-CD28 in the presence or absence of gallein for three days. (B) Data from TCR-stimulated cells expressing the indicated reporter plasmids represent means ± SE from 7 experiments. ***, <i>p</i> < 0.001. (C) Data from cells expressing empty vector (pGL3) or the NFAT reporter plasmid represent means ± SD from triplicate determinations in a single experiment representative of 7 experiments. (D) Gallein does not affect mRNA levels of NFAT1, NFAT2, or NFAT4. Portions of the Jurkat cells used for the luciferase assays that measured activity at the NFAT ARRE-2 site were used to measure mRNA levels of NFAT1, NFAT2, and NFAT4 by qPCR. Data represent means ± SE from 7 experiments. (E) Gallein does not cause detectable changes in protein levels of NFAT1 or NFAT2. Jurkat cells were stimulated or not with plate-bound anti-CD3 and soluble anti-CD28 in the absence or presence of gallein or fluorescein. The blot of NFAT1 used lysates from cells stimulated for three days and the blot of NFAT2 used lysates from cells stimulated for two days. Similar results were obtained from cells stimulated for one, two, or three days, and in a second independent experiment.</p

Gallein potentiates TCR-stimulated increases in nuclear localization of NFAT1 and intracellular Ca²⁺.

<p>(A) Quantitation of the ratio of nuclear to cytoplasmic NFAT1-GFP and GFP-NFAT2 in basal and stimulated Jurkat cells in the presence or absence of gallein. Cells were stimulated with plate-bound anti-CD3 and soluble anti-CD28 for three days. Data represent means ± SE from 130–162 cells for each condition. ***, <i>p</i> < 0.001. (B) Representative images of NFAT1-GFP in basal and stimulated cells in the presence or absence of gallein. In the lower of the two rows of stimulated cells the nuclear borders are outlined in white. (C) Gallein potentiates TCR-stimulated increases in intracellular Ca²⁺ after three days of TCR stimulation. Relative Ca²⁺ levels were determined using R-GECO-mCerulean as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116575#sec002" target="_blank">Materials and Methods</a>. Data represent the means ± SE from > 320 cells for each stimulated condition and > 200 cells for each unstimulated condition. **, <i>p</i> < 0.01.</p

Disrupting Gβγ signaling enhances TCR-stimulated IL-2 transcription without affecting IL-2 mRNA stability.

<p>(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.</p

siRNA directed at Gβ₁ but not Gβ₂ enhances TCR-stimulated IL-2 mRNA increases in Jurkat cells.

<p>Expression of Gβ mRNAs in primary naïve (A) and memory (B) human CD4⁺ T cells grown in TH1 or TH2-promoting conditions, and Jurkat T cells (C) treated with Gβ₁ siRNA (si β₁) or NT siRNA (si NT). The primary cells and, where indicated, the Jurkat cells, were stimulated with plate-bound anti-CD3 and soluble anti-CD28 for three days in the presence of the indicated siRNAs. Values represent means ± SE (N = 3). (D) Representative immunoblot (left) and quantification (right) of the effects of Gβ₁ and Gβ₂ siRNAs on the protein levels of Gβ₁ and Gβ₂ in Jurkat cells. Jurkat cells were treated with the indicated siRNAs for 3 days. Values represent means ± SE (N = 3). (E) Gβ₁ siRNA but not Gβ₂ siRNA potentiated TCR-stimulated IL-2 mRNA increases in Jurkat cells. Jurkat cells were stimulated with plate-bound anti-CD3 and soluble anti-CD28 for 3 days in the presence of the indicated siRNAs. Values represent the means ± SE from 17 experiments. The data were normalized to the values for TCR-stimulated cells treated with NT siRNA. (F and G) A second Gβ₁ siRNA also potentiated TCR-stimulated IL-2 mRNA increases. Values represent the means ± SE from 7 experiments. The data were normalized to the values for TCR-stimulated cells treated with NT siRNA. (F) Gβ₁ mRNA was measured in stimulated cells. All mRNA levels were determined by qPCR. **, <i>p</i> < 0.01; ***, <i>p</i> < 0.001.</p

Potentiation of IL-2 mRNA and Ca²⁺ increases by gallein requires two days of TCR stimulation.

<p>(A) IL-2 levels peaked within 24 hours of TCR stimulation and then decreased over the next 48 hours. Jurkat cells were stimulated with plate-bound anti-CD3 and soluble anti-CD28 antibodies and IL-2 mRNA levels were determined by qPCR at the indicated times. Data represent the means ± SD from a single experiment that is representative of three such experiments. (B) TCR-stimulated IL-2 mRNA increases and (C) activity at the minimal 300-bp IL-2 promoter were not potentiated by gallein until after 2–3 days of TCR stimulation. IL-2 promoter activity in (C) was determined in luciferase assays using the same cells in which IL-2 mRNA was measured in (B). Jurkat cells were stimulated with plate-bound anti-CD3 and soluble anti-CD28 antibodies in the presence or absence of gallein for the indicated times. Data points represent the means ± SE of 8 experiments. *, <i>p</i> < 0.05; **, <i>p</i> < 0.01; ***, <i>p</i> < 0.001. (D) IL-2 secretion was not increased by gallein after one day of TCR stimulation. Jurkat cells were stimulated with plate-bound anti-CD3 and soluble anti-CD28 antibodies for one day in the absence or presence of gallein or fluorescein, and IL-2 secreted into the media was quantified by ELISA. Data points represent the means ± SE of 6 experiments. (E) Intracellular Ca²⁺ was not increased by gallein after one day of TCR stimulation. Relative Ca²⁺ levels were determined using R-GECO-mCerulean as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116575#sec002" target="_blank">Materials and Methods</a>. Data represent the means ± SE from > 330 cells for each condition.</p

Expression of Gγ subunit mRNAs in primary human CD4⁺ T cells and Jurkat cells.

<p>Levels of Gγ mRNAs in unstimulated primary naïve and memory human CD4⁺ T cells (A), stimulated primary naïve and memory human CD4⁺ T cells grown in conditions promoting TH1 or TH2 differentiation (B), and Jurkat cells (C) were determined by qPCR. The stimulated cells were stimulated with plate-bound anti-CD3 and soluble anti-CD28 for 3 days. Values represent the means ± SE from three determinations.</p