PMC

A scheme of how mammalian target of rapamycin may be involved in the development of steroid resistance. HDAC2 = histone deacetylase-2; mTORC1 = mammalian target of rapamycin complex 1; P = phosphorylated; PI3Kδ = phosphoinositide-3-kinase-δ; S6K = p70 S6 kinase.

c-Jun knockdown improved corticosteroid sensitivity. (A) c-Jun small interfering RNA (siRNA) was transfected into U937 cells, using GenomONE-Neo EX (Cosmo Bio USA, Carlsbad, CA). The effect of c-Jun siRNA on expression of c-Jun was measured by Western blotting. (B) Eight hours after transfection with c-Jun siRNA corticosteroid sensitivity was measured, using the rate of inhibition of tumor necrosis factor-α–induced CXCL8 at 10⁻⁷ M dexamethasone. *P < 0.05; **P < 0.01 (compared with negative control [NC] if not indicated otherwise). CSE = cigarette smoke extract; NT = not treated.

Rapamycin (RM) improved steroid sensitivity in U937 cells, as well as in BEAS2B cells. (A) Rate of inhibition of tumor necrosis factor-α–induced CXCL8 release by various concentrations of RM (0–100 nM) in U937 cells. (B) Corticosteroid sensitivities after pretreatment with various concentrations of RM (5 and 20 nM). (C) U937 cells were treated with RM for 2 hours and incubated with cigarette smoke extract (CSE) for another 2 hours. Cells were washed with phosphate-buffered saline and seeded in starvation medium, and corticosteroid sensitivity was measured on the basis of the rate of CXCL8 inhibition by dexamethasone (Dex). (D) Corticosteroid sensitivity in BEAS2B cells was also measured, based on the rate of inhibition of IL-1β–induced granulocyte–macrophage colony–stimulating factor by dexamethasone. **P < 0.01. NT = not treated.

Effect of cigarette smoke extract (CSE) on phosphoinositide-3-kinase/Akt and mammalian target of rapamycin complex 1/p70 S6 kinase (S6K) activities in U937 cells. (A and B) U937 cells were treated with CSE at various time points (2.5 to 30 min), and (A) phosphorylated Akt (p-Akt) levels and (B) phosphorylated S6K (p-S6K) levels were determined by Western blotting. (C and D) After pretreatment with 20 nM rapamycin (RM) for 2 hours, cells were stimulated with CSE for up to 5 minutes. (C) p-Akt and (D) p-S6K levels were calculated. *P < 0.05; **P < 0.01 (compared with NT if not indicated otherwise). NT = not treated; t-Akt = total Akt; t-S6K = total S6K.

Mammalian target of rapamycin complex 1 (mTORC1)/p70 S6 kinase (S6K) activity and expression of c-Jun in peripheral lung tissue from healthy volunteers (HVs), smoking volunteers (SVs), and patients with chronic obstructive pulmonary disease GOLD category 1 (C1) and category 2 (C2). (A) mTORC1/S6K activity was calculated on the basis of phosphorylated S6K (p-S6K) expression and plotted individually. (B) c-Jun expression was also measured by Western blotting. (C and D) Correlations between mTORC1/S6K activity and (C) FEV₁% predicted and between mTORC1/S6K activity and (D) c-Jun expression were analyzed by Spearman correlation test. *P < 0.05; **P < 0.01.

Expression of c-Jun in peripheral blood mononuclear cell samples from healthy volunteers (HVs), smoking volunteers (SVs), and patients with chronic obstructive pulmonary disease (COPD). (A) c-Jun expression was measured by Western blotting and plotted individually. (B–D) Correlations between c-Jun expression and (B) mammalian target of rapamycin complex 1/p70 S6 kinase (S6K) activity and between c-Jun and (C and D) corticosteroid sensitivity were analyzed by Spearman correlation test. *P < 0.05. Dex-IC₃₀ = dexamethasone concentration inducing 30% inhibition; E_max = percent inhibition at maximal concentration; p-S6K = phosphorylated S6K.

Rapamycin (RM) and phosphoinositide-3-kinase-δ (PI3Kδ) inhibitor IC87114 (IC) decrease c-Jun expression. (A) U937 cells were incubated in the medium at various time points after cigarette smoke extract (CSE) stimulation with or without RM pretreatment for 2 hours. c-Jun expression was measured by Western blotting. (B) c-Jun expression in U937 cells, which were incubated with 4 or 20 nM RM, at various time points. (C and D) Cells were treated with 10 μM IC before CSE stimulation, and (C) PI3K/Akt activity and (D) mammalian target of rapamycin complex 1/p70 S6 kinase (S6K) activity were calculated. (E and F) c-Jun expression after (E) treatment with 5 or 10 μM IC, and (F) CSE stimulation with 10 μM IC pretreatment. *P < 0.05; **P < 0.01 (comparison of activities with and without RM [or IC] pretreatment). NT = not treated; p-Akt = phosphorylated Akt; p-S6K = phosphorylated S6K; t-Akt = total Akt; t-S6K = total S6K.

Effect of rapamycin (RM) treatment of peripheral blood mononuclear cell (PBMC) samples from healthy volunteers (HVs), smoking volunteers (SVs), and patients with chronic obstructive pulmonary disease (COPD). (A) PBMCs were incubated with 20 nM RM for 2 hours, and mammalian target of rapamycin complex 1/p70 S6 kinase (S6K) activity was calculated. (B) CXCL8 release caused by overnight tumor necrosis factor (TNF)-α stimulation with or without RM pretreatment. (C and D) PBMCs were treated with RM for 4 hours before measuring corticosteroid sensitivity. *P < 0.05; **P < 0.01; ***P < 0.001 (compared with NT if not indicated otherwise). Dex-IC₃₀ = dexamethasone concentration inducing 30% inhibition; E_max = percent inhibition at maximal concentration; NT = not treated.

Rapamycin (RM) decreased c-Jun stability. (A–C) U937 cells were stimulated with cigarette smoke extract (CSE) at various time points (30 min and 2 h) with or without RM pretreatment. c-Jun N-terminal kinase (JNK) activity was evaluated as (A) phosphorylated JNK (p-JNK) level, and the phosphorylation status of (B) c-Jun Ser-63 and (C) c-Jun Ser-73 was measured. (D) Quantitative reverse transcriptase–polymerase chain reaction of c-Jun after CSE stimulation with or without RM. (E) c-Jun protein stability was measured with cycloheximide (CHX) at 100 μg/ml. In the presence of CHX, the cells were treated with RM and CSE, using the same procedure as described previously (RM was added at –2 h, and CSE was added at 0 h). Statistical analysis was performed between the CSE and RM+CSE groups. (F) Proteasome inhibitor MG132 was added at a concentration of 4 μM. Again, the cells were treated with CSE for 2 hours with RM pretreatment. *P < 0.05; **P < 0.01 (compared with NT if not indicated). NT = not treated; t-c-Jun = total c-Jun; t-JNK = total JNK.

Analysis of peripheral blood mononuclear cell (PBMC) samples from healthy volunteers (HVs), smoking volunteers (SVs), and patients with chronic obstructive pulmonary disease (COPD). (A and B) PBMCs were seeded in the presence of various concentrations of dexamethasone (Dex) for 1 hour before overnight stimulation with tumor necrosis factor-α at 1 ng/ml. CXCL8 expression in supernatant was measured by ELISA. The rate of inhibition of CXCL8 by dexamethasone was calculated, and corticosteroid sensitivity was measured as (A) the concentration inducing 30% inhibition (IC₃₀) and (B) percent inhibition at maximal concentration (E_max), and plotted individually. (C) Mammalian target of rapamycin complex 1 (mTORC1)/p70 S6 kinase (S6K) activity was evaluated as phosphorylated S6K (p-S6K). (D–F) Correlation of mTORC1/S6K activity with (D) FEV₁% predicted, (E) log(IC₃₀-Dex), and (F) E_max was analyzed by Spearman correlation test. *P < 0.05; **P < 0.01; ***P < 0.001 (compared with NT if not indicated otherwise). NT = not treated.