Phosphorylation changes in ERK but not Akt or Bad correlate with TCR/CD3 inhibition of glucocorticoid-induced apoptosis. Immunoblot analysis was performed on 2B4.11 (Fas-negative) cells treated for 1 h with 0.1% DMSO or 100 μM PD98059 and then for 2 h with 0.1 μM dex and/or 0.5 μg/ml anti-CD3. Immunoblots were performed with antibodies specific for ERK1/2, phospho-ERK1/2, Bcl-2, GR, Akt, phosphoserine 473-Akt, Bad, phosphoserine 136-Bad, and phosphoserine 112-Bad. An 8-h time point is shown for GR.

TCR/CD3, activated Ras, and MEK1 each alter transcriptional activation by GR. Cells from the 2B4.11 (Fas-negative) line were transfected with CMV-β-gal, GR-dependent luciferase reporter, and pSG5 (vector), RasV12, or MEKR4F and then treated with 0.1 μM dex with or without anti-CD3ɛ for 8 h. Luciferase activity was normalized to β-gal activity. Fold induction with dex alone was 6.7 ± 0.25 for TAT₃ luciferase and 26.9 ± 0.80 for (MMTV) long terminal repeat luciferase. Transcription activation with dex alone was set as 100%. Results are representative of three separate experiments.

PI3-kinase and Akt fail to inhibit dex-induced apoptosis. (A) Cells from the 2B4.11 (Fas-negative) line were transfected with CMV-β-gal and one of the following constitutively active mutants: p110CAAX, p110K227E, v-Akt, or 5, 10, or 15 μg of RasV12 C40 (PI3-kinase activator); cells were then treated with 0.1 μM dex or vehicle control for 30 h. β-gal activity was measured in cell extracts. Results are representative of three experiments. (B) Akt is activated in cells expressing constitutively activated PI3-kinase mutants. Phosphorylated (activated) Akt and total Akt protein were analyzed by immunoblot.

TCR/CD3, Ras, and Ras-activated kinase pathways prevent dex-induced apoptosis. (A) Viability measured by MTT assay (gray bars) of 2B4.11 (Fas-negative) cells treated with vehicle control (0.001% ethanol), 0.1 μM dex, or anti-CD3ɛ (2C11) for 24 h and expressed as percentage of vehicle-treated cell viability. Survival of cells (black bars) transfected with 5 μg each of RasV12 or vector, pSG5, and CMV-β-gal is shown as measured by flow cytometry as β-gal activity per live cell. Percentage of survival = [percentage of FDG-positive/PI-negative cells with dex/percentage of FDG-positive/PI-negative cells plus vector alone without dex] × 100. (B) TCR/CD3 operates through multiple signaling pathways. Pathways were investigated for their role in TCR/CD3 antagonism of GR-mediated apoptosis by using activated or dominant-negative mutants of pathway components or specific inhibitory compounds. (C) RasV12 rescues S49 murine thymoma cells from dex-induced apoptosis. Survival of cells transfected with 1, 5, or 10 μg each of RasV12 or vector control, pSG5, and CMV-β-gal, treated with 0.1 μM dex for 30 h as measured as β-gal activity in cell extracts is shown. Percentage of survival in the presence of dex = [β-gal units with dex/β-gal units without dex] × 100. (D) RasV12 derivatives that selectively activate Raf or Ral.GDS inhibit dex-induced apoptosis. Cells from the 2B4.11 (Fas-negative) line transfected with 10 μg of vector; RasV12 or the point mutants RasV12 S35 or RasV12 G37; and 5 μg of CMV-β-gal were treated with 0.1 μM dex for 24 h, and β-gal activity was assayed in cell extracts. Data are representative of three independent experiments with triplicates for each data point, and the error bars throughout indicate standard deviation.

The MEK/ERK pathway confers protection from dex-induced apoptosis. (A) Constitutively active MEK1 inhibits dex-induced death, and inhibition of MEK1 blocks TCR/CD3 rescue from GR-mediated apoptosis. Viability of 2B4.11 (Fas-negative) cells transfected with CMV-β-gal and vector (pSG5), constitutively active MEK1 (MEK R4F), and dominant-negative MEK221A or RasV12 and then treated as indicated with 0.5 μg/ml anti-CD3ɛ, 0.1 μM dex, 100 μM PD98059, or 0.1% DMSO (vehicle control) for 30 h was assayed by β-gal activity in cell extracts. Results are representative of three experiments with triplicates for each data point. (B) An inhibitor of MEK1 activation, PD98059, blocks TCR/CD3-mediated rescue from glucocorticoid-induced apoptosis. TUNEL assay of 2B4.11 (Fas-negative) cells treated for 24 h with 0.1 μM dex or vehicle control (0.001% ethanol) and 0.5 μg/ml 2C11 (anti-CD3ɛ), 100 μM PD98059 (stippled bars), or vehicle control (0.1% DMSO, black bars). Percentage of apoptosis = percentage of TUNEL-positive cells determined by flow cytometry (30,000 events collected per sample). Data are the average of two independent experiments. (C) TCR/CD3-activated MEK rescues primary mouse T cells from dex-induced apoptosis. Dex dose-response of splenic cell cultures treated with 0.1% DMSO alone (open squares), 100 μM PD98059 alone (open triangles), 1 μg/ml anti-CD3 and DMSO (filled squares), or anti-CD3 and PD98059 (filled triangles) for 24 h. Cells were stained with anti-CD4-PE, anti-CD8-FITC, and PI and analyzed by flow cytometry (30,000 events collected per sample). Percentage of viable cells was determined as described in Materials and Methods. Viability of CD4⁺CD8⁻ cells (Left) and CD4⁻CD8⁺ cells (Right). Data are the average values of two independent experiments.