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.2009 Aug;20(16):3690-9.
doi: 10.1091/mbc.e08-10-1068. Epub 2009 Jul 1.

Proteasome inactivation promotes p38 mitogen-activated protein kinase-dependent phosphatidylinositol 3-kinase activation and increases interleukin-8 production in retinal pigment epithelial cells

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Proteasome inactivation promotes p38 mitogen-activated protein kinase-dependent phosphatidylinositol 3-kinase activation and increases interleukin-8 production in retinal pigment epithelial cells

Alexandre F Fernandes et al. Mol Biol Cell.2009 Aug.

Abstract

Oxidative stress and inflammation are implicated in the pathogenesis of many age-related diseases. We have demonstrated previously that oxidative inactivation of the proteasome is a molecular link between oxidative stress and overexpression of interleukin (IL)-8. Here, we elucidated a novel signaling cascade that leads to up-regulation of IL-8 in response to proteasome inactivation. The sequence of events in this cascade includes proteasome inactivation, activation of mitogen-activated protein kinase kinase (MKK)3/MKK6, activation of p38 mitogen-activated protein kinase (MAPK), epidermal growth factor receptor phosphorylation, phosphatidylinositol 3-kinase (PI3K) activation and increased IL-8 expression. Blocking any of these signaling pathways abolished the up-regulation of IL-8 induced by proteasome inhibition. Although Akt is also activated in response to proteasome inactivation, we found that the PI3K-dependent up-regulation of IL-8 is independent of 3-phosphoinositide-dependent protein kinase (PDK)1 and Akt. Inhibition of PDK1 and Akt with chemical inhibitors or expression of constitutive active Akt had little effects on IL-8 expression in response to proteasome inactivation. In contrast, inhibition of interleukin 2-inducible T cell kinase, a kinase downstream of PI3K, significantly reduced the expression and secretion of IL-8 in response to proteasome inactivation. Together, these data elucidate a novel signaling network that leads to increased IL-8 production in response to proteasome inactivation.

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Figures

Figure 1.
Figure 1.
Proteasome inhibition up-regulates IL-8 production in a PI3K-dependent manner. ARPE-19 cells were cultured in the presence or absence of epoxomicin, LY294002, and epoxomicin plus LY294002 for 8 h. (A) Levels of mRNA for IL-8 were assessed by real-time RT-PCR analysis. GAPDH mRNA was used as a control to normalize the total mRNA levels. (B) IL-8 protein levels in the medium were determined by ELISA. The results are the mean ± SD of three independent experiments. p< 0.05 and **p< 0.01 compared with the control;#p < 0.001 and§p = 0.05 compared with epoxomicin alone.
Figure 2.
Figure 2.
p38 MAPK activation precedes Akt activation after proteasome inactivation in RPE. (A) ARPE-19 cells were cultured in the presence or absence of MG132, a proteasome inhibitor, for 0, 2, 4, and 8 h. (B) ARPE-19 cells were cultured in the presence or absence of epoxomicin and epoxomicin plus SB203580 for 8 h. (C) ARPE-19 cells were cultured in the presence or absence of epoxomicin, LY294002, and epoxomicin plus LY294002 for 8 h. Levels of endogenous phospho-p38 MAPK, total p38 MAPK, phospho-Akt, total Akt, and actin were detected by Western blot by using polyclonal (to phosphorylated and total p38 MAPK and phosphorylated and total Akt) and monoclonal antibodies (to actin). The figures are representative of three independent experiments with similar results.
Figure 3.
Figure 3.
The EGFR is involved in the IL-8 production in response to proteasome inhibition. (A) ARPE-19 cells were cultured in the presence of 0, 1, 5, and 10% FBS for 8 h. (B) ARPE-19 cells were cultured in the presence of different growth factors (EGF, PDGF, and insulin) in serum-free medium for 8 h. (C and D) ARPE-19 cells were cultured in the presence or absence of epoxomicin, AG1478, and epoxomicin plus AG1478 in serum-free medium for 8 h. Levels of mRNA for IL-8 were assessed by real-time RT-PCR analysis (C). GAPDH mRNA was used as a control to normalize the total mRNA levels. IL-8 protein levels in the medium were determined by ELISA (A, B, and D). The results are the mean ± SD of three independent experiments. *p< 0.05 and **p< 0.01 compared with the control;$p < 0.05 and##p < 0.09 compared with epoxomicin alone.
Figure 4.
Figure 4.
EGFR phosphorylation upon proteasome inhibition is p38 MAPK dependent. ARPE-19 cells were cultured in the presence or absence of epoxomicin (A and C) and epoxomicin plus SB203580 (B and D) in serum-free medium for 8 h. Levels of endogenous phospho-EGFR (Tyr1045 and Tyr1068), total EGFR, and actin were detected by Western blot using polyclonal (to phosphorylated and total EGFR) and monoclonal antibodies (to actin). (A and B) EGFR phosphorylation at Tyr1045 position is shown. (C and D) EGFR phosphorylation at Tyr1068 position is shown. The figures are representative of three independent experiments with similar results.
Figure 5.
Figure 5.
MKK3 and MKK6 activation increases IL-8 production in an EGFR- and PI3K-dependent manner. ARPE-19 cells were transfected with an empty vector (CONT) or cotransfected with vectors encoding constitutively active (MKK3 + MKK6) and mutant (Mut MKK3+Mut MKK6) forms of MKK3 and MKK6. After 40 h of transfection, ARPE-19 cells were incubated in fresh serum-free medium for 8 h (A and B). Alternatively, ARPE-19 cells were incubated in the presence of LY294002 or AG1478 in serum-free medium for 8 h (C and D). Levels of mRNA for IL-8 were assessed by real-time RT-PCR analysis (A and C). GAPDH mRNA was used as a control to normalize the total mRNA levels. IL-8 protein levels in the medium were determined by ELISA (B and D). The results are the mean ± SD of three independent experiments. **p< 0.01 compared with the control;&p < 0.01 compared with MKK3 + MKK6 alone.
Figure 6.
Figure 6.
Up-regulation of IL-8 in response to proteasome inhibitors is independent of activation of Akt. (A–C) ARPE-19 cells were cultured in the presence or absence of epoxomicin, Akt X, and epoxomicin plus Akt X (A and B) or Akt IV and epoxomicin plus Akt IV (C) for 8 h. (D) ARPE-19 cells were transfected with an empty vector (CONT) or vectors encoding a constitutively active (Myr-Akt) and a mutant (Mut Akt) form of Akt. After 40 h of transfection, ARPE-19 cells were incubated in fresh serum-free medium for 8 h. Levels of mRNA for IL-8 were assessed by real-time RT-PCR analysis (B). GAPDH mRNA was used as a control to normalize the total mRNA levels. IL-8 protein levels in the medium were determined by ELISA (A, C, and D). The results are the mean ± SD of three independent experiments. **p< 0.01 compared with the control.
Figure 7.
Figure 7.
Itk regulates IL-8 production upon proteasome inhibition. ARPE-19 cells were cultured in the presence or absence of MG132, BX912, MG132 plus BX912 (A), or BMS509744 and MG132 plus BMS509744 (B and C) for 8 h. Levels of mRNA for IL-8 were assessed by real-time RT-PCR analysis (B). GAPDH mRNA was used as a control to normalize the total mRNA levels. IL-8 protein levels in the medium were determined by ELISA (A and C). The results are the mean ± SD of three independent experiments. **p< 0.01 compared with the control;#p < 0.001 and##p = 0.08 compared with MG132 alone.
Figure 8.
Figure 8.
Schematic model of a possible molecular mechanism regulating IL-8 production after prolonged proteasome inhibition. In this model, the impairment of proteasome activity will result in the activation of MKK3 and MKK6, the upstream kinases involved in the activation of p38 MAPK. Activation of p38 MAPK will result in EGFR phosphorylation. This phosphorylation allows docking proteins to bind the receptor, leading to the activation of PI3K. The PI3K then activates Itk, as well as other unidentified effectors, which in turn will lead to an increase in IL-8 gene expression. Akt can also be activated by PI3K, but it may only play a minor role on IL-8 production. Alternatively, p38 MAPK may regulate IL-8 production through other mechanisms, such as stabilization of its mRNA.
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