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Redox-regulated pathway of tyrosine phosphorylation underlies NF-κB induction by an atypical pathway independent of the 26S proteasome.

Cullen S, Ponnappan S, Ponnappan U - Biomolecules (2015)

Bottom Line: Results from these studies demonstrate that phospho-IκBα (Tyr-42) is not subject to proteasomal degradation in a murine stromal epithelial cell line, confirming results previously reported.Correspondingly, proteasome inhibition had no discernable effect on the key signaling intermediaries, Src and ERK1/2, involved in the phospho-tyrosine mechanisms regulating PV-mediated activation of NF-κB.Strikingly, our studies demonstrate that proteasome inhibition can potentiate oxidative stress associated with PV-stimulation without impacting kinase activation, however, other cellular implications for this increase in intracellular oxidation remain to be fully delineated.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA. cullensarahjane@gmail.com.

ABSTRACT
Alternative redox stimuli such as pervanadate or hypoxia/reoxygenation, induce transcription factor NF-κB by phospho-tyrosine-dependent and proteasome-independent mechanisms. While considerable attention has been paid to the absence of proteasomal regulation of tyrosine phosphorylated IκBα, there is a paucity of information regarding proteasomal regulation of signaling events distinct from tyrosine phosphorylation of IκBα. To delineate roles for the ubiquitin-proteasome pathway in the phospho-tyrosine dependent mechanism of NF-κB induction, we employed the proteasome inhibitor, Aclacinomycin, and the phosphotyrosine phosphatase inhibitor, pervanadate (PV). Results from these studies demonstrate that phospho-IκBα (Tyr-42) is not subject to proteasomal degradation in a murine stromal epithelial cell line, confirming results previously reported. Correspondingly, proteasome inhibition had no discernable effect on the key signaling intermediaries, Src and ERK1/2, involved in the phospho-tyrosine mechanisms regulating PV-mediated activation of NF-κB. Consistent with previous reports, a significant redox imbalance leading to the activation of tyrosine kinases, as occurs with pervanadate, is required for the induction of NF-κB. Strikingly, our studies demonstrate that proteasome inhibition can potentiate oxidative stress associated with PV-stimulation without impacting kinase activation, however, other cellular implications for this increase in intracellular oxidation remain to be fully delineated.

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PV-mediated activation of NF-κB involves oxidative stress. (A) ILU-18 cells were washed and briefly incubated in 1X Hank’s Balanced Salt Solution (HBSS), prior to incubation with 10 μM H2DCF-DA for 30 min at 37 °C in the dark. At the end of the incubation, cells were washed and resuspended in 1X HBSS. Intracellular reactive oxygen species (ROS) generation was detected following addition of PV (100 µM) or H2O2 (200 µM), as described in the methods section. (B) ILU-18 cells were either untreated or treated with NAC (20 mM) for 2 h. After media replacement, cells were selectively treated with PV (100 μM) for 20 min. At the end of incubation, cells were washed and cytosolic lysates prepared. As controls, cell lysates were made from ILU-18 cells left untreated or treated with 20 mM NAC alone for 140 min. Lysates, equalized for 30 μg protein, were resolved using SDS-PAGE, followed by Western blotting using an antibody specific to Phospho-Tyrosine residues. Molecular weights derived from standards are indicated in kDa. (C) Cytosolic lysates (30 µg), obtained as in (B), were resolved using SDS-PAGE, followed by Western blotting using a phospho-specific antibody recognizing IκBα (Tyr-42). After stripping, the blot was re-probed with antibody to β-actin to demonstrate equal protein loading. (D) ILU-18 cells were either untreated or treated with NAC (20 mM) for 2 h. After media replacement, cells were selectively treated with PV (100 μM) for 18 h. Additionally, ILU-18 cells were treated with 100 μM H2O2 for 18 h or subjected to treatment with 200 μM BSO for 24 h. Lysates obtained were evaluated for luciferase acitivity, as described previously. Data obtained from at least four independent experiments are presented; values represent data means ± standard error. ** Denotes significant difference at p < 0.001, between treatment groups.
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biomolecules-05-00095-f004: PV-mediated activation of NF-κB involves oxidative stress. (A) ILU-18 cells were washed and briefly incubated in 1X Hank’s Balanced Salt Solution (HBSS), prior to incubation with 10 μM H2DCF-DA for 30 min at 37 °C in the dark. At the end of the incubation, cells were washed and resuspended in 1X HBSS. Intracellular reactive oxygen species (ROS) generation was detected following addition of PV (100 µM) or H2O2 (200 µM), as described in the methods section. (B) ILU-18 cells were either untreated or treated with NAC (20 mM) for 2 h. After media replacement, cells were selectively treated with PV (100 μM) for 20 min. At the end of incubation, cells were washed and cytosolic lysates prepared. As controls, cell lysates were made from ILU-18 cells left untreated or treated with 20 mM NAC alone for 140 min. Lysates, equalized for 30 μg protein, were resolved using SDS-PAGE, followed by Western blotting using an antibody specific to Phospho-Tyrosine residues. Molecular weights derived from standards are indicated in kDa. (C) Cytosolic lysates (30 µg), obtained as in (B), were resolved using SDS-PAGE, followed by Western blotting using a phospho-specific antibody recognizing IκBα (Tyr-42). After stripping, the blot was re-probed with antibody to β-actin to demonstrate equal protein loading. (D) ILU-18 cells were either untreated or treated with NAC (20 mM) for 2 h. After media replacement, cells were selectively treated with PV (100 μM) for 18 h. Additionally, ILU-18 cells were treated with 100 μM H2O2 for 18 h or subjected to treatment with 200 μM BSO for 24 h. Lysates obtained were evaluated for luciferase acitivity, as described previously. Data obtained from at least four independent experiments are presented; values represent data means ± standard error. ** Denotes significant difference at p < 0.001, between treatment groups.

Mentions: While the precise mechanisms responsible for PV-mediated induction of NF-κB remain to be fully explored, it is known that the redox imbalance accompanying pervanadate stimulation plays a significant role in both tyrosine phosphorylation of IκBα and NF-κB transcriptional activity [12]. To assess if PV acts as a potent pro-oxidant in ILU-18 cells, we measured the generation of reactive oxygen species (ROS) by employing the oxidant-sensing fluorescent probe, H2DCF-DA. Compared to the induction of ROS by the exogenous addition of H2O2, PV treatment was more potent at inducing intracellular DCF fluorescence (Figure 4A). Thus, exposure to PV induces ROS generation, as primarily reported in Jurkat and Ramos cells [26].


Redox-regulated pathway of tyrosine phosphorylation underlies NF-κB induction by an atypical pathway independent of the 26S proteasome.

Cullen S, Ponnappan S, Ponnappan U - Biomolecules (2015)

PV-mediated activation of NF-κB involves oxidative stress. (A) ILU-18 cells were washed and briefly incubated in 1X Hank’s Balanced Salt Solution (HBSS), prior to incubation with 10 μM H2DCF-DA for 30 min at 37 °C in the dark. At the end of the incubation, cells were washed and resuspended in 1X HBSS. Intracellular reactive oxygen species (ROS) generation was detected following addition of PV (100 µM) or H2O2 (200 µM), as described in the methods section. (B) ILU-18 cells were either untreated or treated with NAC (20 mM) for 2 h. After media replacement, cells were selectively treated with PV (100 μM) for 20 min. At the end of incubation, cells were washed and cytosolic lysates prepared. As controls, cell lysates were made from ILU-18 cells left untreated or treated with 20 mM NAC alone for 140 min. Lysates, equalized for 30 μg protein, were resolved using SDS-PAGE, followed by Western blotting using an antibody specific to Phospho-Tyrosine residues. Molecular weights derived from standards are indicated in kDa. (C) Cytosolic lysates (30 µg), obtained as in (B), were resolved using SDS-PAGE, followed by Western blotting using a phospho-specific antibody recognizing IκBα (Tyr-42). After stripping, the blot was re-probed with antibody to β-actin to demonstrate equal protein loading. (D) ILU-18 cells were either untreated or treated with NAC (20 mM) for 2 h. After media replacement, cells were selectively treated with PV (100 μM) for 18 h. Additionally, ILU-18 cells were treated with 100 μM H2O2 for 18 h or subjected to treatment with 200 μM BSO for 24 h. Lysates obtained were evaluated for luciferase acitivity, as described previously. Data obtained from at least four independent experiments are presented; values represent data means ± standard error. ** Denotes significant difference at p < 0.001, between treatment groups.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4384113&req=5

biomolecules-05-00095-f004: PV-mediated activation of NF-κB involves oxidative stress. (A) ILU-18 cells were washed and briefly incubated in 1X Hank’s Balanced Salt Solution (HBSS), prior to incubation with 10 μM H2DCF-DA for 30 min at 37 °C in the dark. At the end of the incubation, cells were washed and resuspended in 1X HBSS. Intracellular reactive oxygen species (ROS) generation was detected following addition of PV (100 µM) or H2O2 (200 µM), as described in the methods section. (B) ILU-18 cells were either untreated or treated with NAC (20 mM) for 2 h. After media replacement, cells were selectively treated with PV (100 μM) for 20 min. At the end of incubation, cells were washed and cytosolic lysates prepared. As controls, cell lysates were made from ILU-18 cells left untreated or treated with 20 mM NAC alone for 140 min. Lysates, equalized for 30 μg protein, were resolved using SDS-PAGE, followed by Western blotting using an antibody specific to Phospho-Tyrosine residues. Molecular weights derived from standards are indicated in kDa. (C) Cytosolic lysates (30 µg), obtained as in (B), were resolved using SDS-PAGE, followed by Western blotting using a phospho-specific antibody recognizing IκBα (Tyr-42). After stripping, the blot was re-probed with antibody to β-actin to demonstrate equal protein loading. (D) ILU-18 cells were either untreated or treated with NAC (20 mM) for 2 h. After media replacement, cells were selectively treated with PV (100 μM) for 18 h. Additionally, ILU-18 cells were treated with 100 μM H2O2 for 18 h or subjected to treatment with 200 μM BSO for 24 h. Lysates obtained were evaluated for luciferase acitivity, as described previously. Data obtained from at least four independent experiments are presented; values represent data means ± standard error. ** Denotes significant difference at p < 0.001, between treatment groups.
Mentions: While the precise mechanisms responsible for PV-mediated induction of NF-κB remain to be fully explored, it is known that the redox imbalance accompanying pervanadate stimulation plays a significant role in both tyrosine phosphorylation of IκBα and NF-κB transcriptional activity [12]. To assess if PV acts as a potent pro-oxidant in ILU-18 cells, we measured the generation of reactive oxygen species (ROS) by employing the oxidant-sensing fluorescent probe, H2DCF-DA. Compared to the induction of ROS by the exogenous addition of H2O2, PV treatment was more potent at inducing intracellular DCF fluorescence (Figure 4A). Thus, exposure to PV induces ROS generation, as primarily reported in Jurkat and Ramos cells [26].

Bottom Line: Results from these studies demonstrate that phospho-IκBα (Tyr-42) is not subject to proteasomal degradation in a murine stromal epithelial cell line, confirming results previously reported.Correspondingly, proteasome inhibition had no discernable effect on the key signaling intermediaries, Src and ERK1/2, involved in the phospho-tyrosine mechanisms regulating PV-mediated activation of NF-κB.Strikingly, our studies demonstrate that proteasome inhibition can potentiate oxidative stress associated with PV-stimulation without impacting kinase activation, however, other cellular implications for this increase in intracellular oxidation remain to be fully delineated.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA. cullensarahjane@gmail.com.

ABSTRACT
Alternative redox stimuli such as pervanadate or hypoxia/reoxygenation, induce transcription factor NF-κB by phospho-tyrosine-dependent and proteasome-independent mechanisms. While considerable attention has been paid to the absence of proteasomal regulation of tyrosine phosphorylated IκBα, there is a paucity of information regarding proteasomal regulation of signaling events distinct from tyrosine phosphorylation of IκBα. To delineate roles for the ubiquitin-proteasome pathway in the phospho-tyrosine dependent mechanism of NF-κB induction, we employed the proteasome inhibitor, Aclacinomycin, and the phosphotyrosine phosphatase inhibitor, pervanadate (PV). Results from these studies demonstrate that phospho-IκBα (Tyr-42) is not subject to proteasomal degradation in a murine stromal epithelial cell line, confirming results previously reported. Correspondingly, proteasome inhibition had no discernable effect on the key signaling intermediaries, Src and ERK1/2, involved in the phospho-tyrosine mechanisms regulating PV-mediated activation of NF-κB. Consistent with previous reports, a significant redox imbalance leading to the activation of tyrosine kinases, as occurs with pervanadate, is required for the induction of NF-κB. Strikingly, our studies demonstrate that proteasome inhibition can potentiate oxidative stress associated with PV-stimulation without impacting kinase activation, however, other cellular implications for this increase in intracellular oxidation remain to be fully delineated.

Show MeSH
Related in: MedlinePlus