Redox-Regulated Pathway of Tyrosine Phosphorylation Underlies NF-κB Induction by an Atypical Pathway Independent of the 26S Proteasome

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.

Recent studies regarding "bortezomib resistance" have highlighted the biological significance of mechanisms of NF-țB induction, which are resistant to proteasome inhibition [18,19]. Hence, the in vivo relevance of the phospho-tyrosine-dependent mechanism of NF-țB induction may ultimately be defined due to its distinction as a proteasome-independent mechanism of NF-țB activation. Based on this prevision, we sought to investigate how proteasome inhibition affects facets of phospho-tyrosine-dependent NF-țB signaling, both related and unrelated to tyrosine phosphorylation of IțBĮ.
While considerable attention has been paid to the absence of proteasomal regulation of tyrosine phosphorylated IțBĮ, there is a paucity of information regarding proteasomal regulation of signaling events distinct from tyrosine phosphorylation of IțBĮ. Further, this includes an incomplete understanding of the role for ubiquitin-like modifiers, such as NEDD8 and SUMO, in the signaling events of the atypical NF-țB pathway. To delineate unexplored roles for the ubiquitin-proteasome pathway in the phospho-tyrosine dependent mechanism of NF-țB induction, we employed the proteasome inhibitor, Aclacinomycin, and the phosphotyrosine phosphatase inhibitor, pervanadate. Results from these studies demonstrate that phospho-IțBĮ (Tyr-42) is not subject to proteasomal degradation in a murine stromal epithelial cell line, confirming results previously reported in HeLa and Jurkat cell lines [7,12]. 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-țB. 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-țB in this cell type. Strikingly, our studies demonstrate that proteasome inhibition can potentiate oxidative stress associated with PV-stimulation; however, the cellular implications for this increase in intracellular oxidation remain to be delineated. In particular, this study highlights a regulatory mechanism underlying the inhibition of tyrosine phosphatases, a concomitant activation of tyrosine kinases accompanying cellular oxidation, and a significant role for proteasome inhibition in the potentiation of these responses.

Pervanadate Stimulation Induces Tyrosine Phosphorylation of IțBĮ But Not Its Proteolytic Degradation
TNFĮ-mediated activation of NF-țB induction has been demonstrated to invoke serine phosphorylation of the inhibitory IțB proteins followed by ubiquitination and degradation via the 26S proteasome pathway [5]. In contrast, NF-țB activation by pervanadate involves tyrosine phosphorylation of IțBĮ and is not contigent upon proteasomal degradation of IțBĮ [6,7]. To test whether PV-mediated activation of NF-țB occurs by a proteasomal-independent mechanism in a murine stromal cell line, we subjected ILU-18 cells to short-term activation with TNFĮ or PV and then tested cytosolic lysates by immunoblotting with an antibody recognizing IțBĮ. While IțBĮ is no longer detected in response to TNFĮ treatment, IțBĮ remains in the cytosol following short-term PV treatment, indicating absence of IțBĮ degradation in PV-induced NF-țB ( Figure 1A). At the end of treatment, cytosolic lysates were obtained and 30ȝg protein from each lysate was resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Resolved proteins were detected by Western blotting using antibody to nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha (IțBĮ) and enhanced chemiluminescence (ECL). The blot was stripped and re-probed with antibody to ȕ-actin to ensure equal protein loading. (B) ILU-18 cells were treated with PV (100 ȝM) for 20 min, with or without prior treatment with Aclacinomycin [Acla] (0.25 ȝM) for 2 h. At the end of incubation, cells were washed and cytosolic lysates prepared. As controls, cell lysates were made from ILU-18 cells either left untreated or treated for 140 min with 0.25 ȝM Aclacinomycin alone. Lysates, equalized for 30 ȝg protein, were resolved using SDS-PAGE, followed by Western blotting using a phospho-specific antibody recognizing IțBĮ (Tyr-42) or (Tyr-305). After stripping, the blot was re-probed with antibody to ȕ-actin to demonstrate equal protein loading.
To elucidate whether PV-mediated NF-țB activation occurs by a mechanism involving tyrosine phosphorylation, we next assessed if both in the absence and presence of proteasome inhibitor (Aclacinomycin), IțBĮ undergoes tyrosine phosphorylation [20]. By employing immunoblotting with an antibody recognizing phospho-IțBĮ (Tyr42 or Tyr305), we now demonstrate that PV induces tyrosine phosphorylation of IțBĮ at Tyr-42 and Tyr305, both in the absence and presence of proteasome inhibitor ( Figure 1B). Overall, these results demonstrate that in response to PV stimulation, tyrosine phosphorylation of IțBĮ occurs, independently of proteasome catalytic function.

Pretreatment with Inhibitors of Src and MEK but not p38, PI3K, JNK and IKK Complex Interfere with PV-Induced NF-țB Activity
Pervanadate mediates both the coordinated inhibition of cellular tyrosine phosphatases as well as the activation of cellular tyrosine kinases. Kinase families, which are prone to activation by PV, include the Src and Syk family of tyrosine kinases and the family of serine/threonine specific kinases, MAPK [12,15,21,22]. To delineate if these kinases are involved in NF-țB-dependent transcription induced by PV, we employed a luciferase reporter assay, which had previously been shown to be dependent on PV-mediated NF-țB activity. Pretreatment with Piceatannol, the resveratrol metabolite and tyrosine kinase inhibitor, resulted in a significant reduction in PV-induced NF-țB-dependent luciferase activity, thus indicating an important role for the Src/Syk family of tyrosine kinases in PV-mediated, NF-țB-dependent luciferase expression ( Figure 2A). Furthermore, the mitogen-activated protein kinase kinase (MEK) inhibitor, PD98059, substantially interfered with PV-mediated NF-țB activity, indicating that MEK also contributes to pervanadate-mediated NF-țB activation ( Figure 2A).
To elucidate which family of tyrosine kinases is principally involved in PV-dependent NF-țB activation, we subjected ILU-18 cells to short-term activation with pervanadate (20 min) and then examined Syk and phospho-Syk by immunoblotting with antibodies recognizing Syk & tyrosine-phosphorylated Syk. Though Syk was detected, tyrosine phosphorylation of Syk was not observed following PV treatment in ILU-18 cells ( Figure 2B). In contrast, we confirmed that c-Src is indeed phosphorylated following PV stimulation in this cell line by employing an antibody to phospho-Src ( Figure 3). Given the PV-mediated phosphorylation of c-Src, we sought to determine if Src kinase was necessary in PV-dependent NF-țB activity. Employing a selective inhibitor of Src, we pretreated cells with Src Kinase Inhibitor I, before subjecting them to PV-stimulation. As depicted in Figure 2C, pretreatment with Src Kinase Inhibitor I significantly inhibited PV-induced NF-țB activity, thus demonstrating the involvement of Src kinase in PV-induced activation of NF-țB. Since c-Src kinase has been implicated in the tyrosine phosphorylation of IțBĮ, we evaluated if in the ILU-18 cell line, Src kinase plays a role in the tyrosine phosphorylation of IțBĮ at tyrosine 42. Pretreatment with Src Kinase Inhibitor I abrogated PV-dependent, tyrosine phosphorylation of IțBĮ (Tyr-42), as determined by immunoblotting with an antibody recognizing phospho-IțBĮ (Tyr-42) ( Figure 2D). Taken together, these results demonstrate that c-Src contributes to PV-mediated NF-țB activation by phosphorylating IțBĮ at tyrosine 42.
As JNK, p38, and PI3K subfamilies have been connected with stress conditions conducive for inactivation of tyrosine phosphatases [23], we next tested their involvement in PV-mediated NF-țB signaling by employing inhibitors to the kinase activity of each of these pathways ( Figure 2E). Unlike MEK and the Src family kinases, the involvement of JNK, p38, and PI3K activity was found not to be critical in pervanadate-mediated NF-țB activation. As IKK complex activation following activation of PKD has been demonstrated to be necessary in H2O2-induced NF-țB responses in epithelial cells [24], we next tested the role of IKK in PV-induced NF-țB activity. Employing a cell permeable IKK Nemo-binding peptide (IKK-NBD) as an inhibitor of IKK complex formation, we demonstrate that pretreatment with IKK-NBD did not inhibit pervanadate-mediated luciferase expression, indicating that luciferase expression is independent of the IKK complex. TNFĮ induced IL-6 expression was, however, dependent on IKK ( Figure 2F). Thus, pretreatment with Src and MEK inhibitors, but not JNK, p38, PI3K, and IKK complex inhibitors, modulated pervanadate-induced luciferase expression, providing evidence that Src and MEK kinases are key signaling intermediaries in PV-mediated NF-țB induction. At the end of 18 h, lysates were obtained and luciferase activity was determined employing the Promega luciferase assay kit. Fold induction represents a ratio of luciferase activity obtained in treatment-induced to that of untreated cells. Data represents mean ± standard error obtained from four independent experiments, following normalization. ** Denotes significant difference at p < 0.004, between treatment groups. (B) ILU-18 cells were treated with PV (100 ȝM) for 20 min, with or without prior treatment with Aclacinomycin (0.25 ȝM) for 2 h. 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 140 min with 0.25 ȝM Aclacinomycin alone. Lysates, equalized for 30 ȝg protein, were resolved using SDS-PAGE, followed by Western blotting using antibodies for phospho-Syk (Tyr-352) and, as a control, Syk. (C) ILU-18 cells were treated with 100 μM pervanadate for 18 h, with or without pretreatment for 6 h with 2 μM Src Kinase Inhibitor I (SKI-1). As controls, cells were either left untreated or subjected to treatment with 2 μM Src Kinase Inhibitor I for 24 h [25]. As described in (A), a luciferase assay was performed. Normalized data are presented as % activity with fold induction values from PV-treated cells set at 100%. Data represents mean ± S.E. from duplicates derived from two independent experiments. (D) ILU-18 cells were treated with 100 μM pervanadate for 20 min, with or without pretreatment for 4 h with 5 μM Src Kinase Inhibitor I (SKI-1). 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 260 min with 5 μM Src Kinase Inhibitor I alone. Lysates, equalized for 30 ȝg protein, were resolved using SDS-PAGE, followed by Western blotting using a phospho-specific antibody recognizing IțBĮ (Tyr-42). The blot was stripped and re-probed with antibody to ȕ-actin to ensure equal protein loading. (E) ILU-18 cells were treated with 100 μM pervanadate, 100nM SB23580 (SB.-p38 Kinase inhibitor), 10 μM Wortmannin (WRT-PI3K inhibitor), or 40 nM JNK Inhibitor for 18 h. ILU-18 cells were also pretreated for 1 h with 40 nM JNK Inhibitor or for 30 min with either 100nM SB23580 or 10 μM Wortmannin followed by activation with 100 μM pervanadate for 18 h. Fold induction was determined as in (A). Data represents mean ± S.E. from four independent experiments. (F) ILU-18 cells were either pretreated with 10 μM IKK-NBD for 1 h or left untreated. After 1 h, cells were either treated with 100 μM PV or 20 ng/mL TNFĮ for 18 h, yielding the following treatments: Untreated, PV, IKK-NBD+PV, TNFĮ, IKK-NBD+TNFĮ. Fold induction was determined as in (A). Data represents mean ± S.E. from duplicates derived from two independent experiments. ** Denotes significant difference at p < 0.001, between treatment groups.

Proteasome Inhibition Does Not Alter PV-Mediated Activation of Src or MEK Kinases
As PV represents a proteasome-independent mechanism of NF-țB induction, we assessed the effect of proteasome inhibition on the signaling pathways identified in PV-induced NF-țB activity, e.g., Src and MEK kinases. To determine the effect of proteasome inhibition on the activation of the Src and ERK 1/2 kinases, we subjected ILU-18 cells to short-term activation with PV, both in the absence or presence of proteasome inhibitor, and then tested cytosolic lysates by immunoblotting with antibodies recognizing phosphorylated c-Src and ERK 1/2. As depicted in Figure 3, both in the absence or presence of proteasome inhibitor, pervanadate induced the phosphorylation of c-Src and ERK 1/2. Thus, loss in proteasomal catalytic activity does not interfere with the phospho-tyrosine dependent mechanisms that regulate PV-mediated induction of NF-țB.

PV-Mediated Activation of NF-țB Involves Oxidative Stress
While the precise mechanisms responsible for PV-mediated induction of NF-țB remain to be fully explored, it is known that the redox imbalance accompanying pervanadate stimulation plays a significant role in both tyrosine phosphorylation of IțBĮ and NF-țB 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].
To ascertain the functional significance of redox imbalance, we determined if cellular tyrosine phosphorylation induced by PV was dependent upon intracellular oxidation. By measuring oxidative stress with the H2DCF-DA probe, we determined that pretreatment of ILU-18 cells with the antioxidant, N-acetylcysteine (NAC), significantly impaired the pro-oxidant effects of PV [25]. Based on this observation, we pretreated ILU-18 cells with NAC to boost nonenzymatic detoxification of reactive electrophiles and free radicals and then measured PV-dependent phospho-tyrosine accumulation by immunoblotting with an anti-phosphotyrosine antibody. As depicted in Figure 4B, pretreatment with NAC inhibited the induction of cellular tyrosine phosphorylation by PV. Additionally, NAC pretreatment blocked the tyrosine phosphorylation of IțBĮ (Tyr-42) accompanying PV-stimulation ( Figure 4C). Taken together, these results demonstrate that the generation of ROS and subsequent intracellular oxidation by PV plays a prominent role in tyrosine phosphorylation of cellular proteins, including IțBĮ.
To further support the contention that NF-țB activation by PV occurs as a result of oxidative stress, we evaluated the effect of NAC pretreatment on PV-mediated NF-țB activity. As expected, the ROS scavenging effects of NAC offset Pervanadate-mediated oxidative stress, resulting in levels of luciferase activity on a par with background levels ( Figure 4D). This reversal by NAC demonstrates that Pervanadate is inducing luciferase expression by the generation of ROS.  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 ȝg 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 IțBĮ (Tyr-42). After stripping, the blot was re-probed with antibody to ȕ-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 ȝM) for 18 h. Additionally, ILU-18 cells were treated with 100 ȝM H2O2 for 18 h or subjected to treatment with 200 ȝM 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.
Having established that Pervanadate induces NF-țB activation by oxidative stress, we next tested whether perturbations in the redox balance were sufficient to induce NF-țB activity. For this, NF-țB-dependent luciferase expression was analyzed using Buthionine-sulfoximine (BSO) treatment. BSO mediates its pro-oxidant effects by inhibiting the enzyme responsible for synthesis of the primary intracellular thiol and ROS scavenger, glutathione (GSH). BSO treatment of ILU-18 cells for 24 h resulted in only a two-fold induction of luciferase activity ( Figure 4D). These BSO results would likely have been higher if BSO was co-administered with a pro-oxidant, thereby more potently disrupting the redox balance and activating NF-țB. Similarly, H2O2 did not induce NF-țB-dependent luciferase activity ( Figure 4D). This finding is consistent with our observations that in ILU-18 cells, H2O2 was neither a potent pro-oxidant ( Figure 4A) nor an effective inducer of cellular tyrosine phosphorylation [25]. Collectively, these results indicate that a significant redox imbalance leading to the activation of tyrosine kinases, as occurs with Pervanadate, is required for the induction of NF-țB in this cell type.

Proteasome Inhibition Potentiates Oxidative Stress Associated with PV-Stimulation
While proteasome inhibition has no effect on the phospho-tyrosine dependent mechanisms that regulate PV-mediated induction of NF-țB (Figure 3), it remains unclear how proteasome inhibition impacts the intracellular redox balance, which is also critically necessary for PV-mediated induction of NF-țB. By measuring generation of ROS with the H2DCF-DA probe, we observed that pretreatment with proteasome inhibitors potentiated oxidative stress generated by pervanadate stimulation ( Figure 5A). ILU-18 cells treated with proteasome inhibitor alone induced intracellular oxidation near background levels, thereby affirming that proteasome inhibition does not independently induce oxidative stress ( Figure 5A). Despite the amplification in intracellular ROS generation, pretreatment with proteasome inhibitor did not intensify cellular tyrosine phosphorylation accompanying PV treatment ( Figure 5B). Additionally, pretreatment with proteasome inhibitor did not interfere with redox-dependent tyrosine phosphorylation induced by PV ( Figure 5B). Thus, while loss in proteasomal catalytic activity enhances PV-mediated oxidative stress, this effect appears to have no discernable impact on redox-dependent tyrosine phosphorylation by PV.
For an assessment of the long-term implications of proteasome inhibition on PV-induced ROS generation, we measured intracellular glutathione (GSH) following 18 h of PV stimulation. As GSH participates in the enzymatic conversion of H2O2 to H2O, depletion of this antioxidant is indicative of oxidative stress within the cell. Both in the absence and presence of proteasome inhibitor, GSH levels declined significantly after 18 h exposure to PV. Yet, despite pretreatment with proteasome inhibitor, PV-associated loss in functional GSH was nearly equivalent; in other words, proteasomal inhibition did not result in an elevation in GSH depletion ( Figure 5C). In agreement with ROS data, treatment with proteasome inhibitor alone had no effect on GSH levels ( Figure 5C), indicating that loss in proteasomal catalytic activity alone is not sufficient to induce intracellular oxidation in ILU-18 cells. Overall, these results show for the first time that proteasome inhibition can potentiate oxidative stress associated with PV-stimulation; however, the cellular source and implications for this increase in intracellular oxidation have to yet to be determined.

Discussion
For a subset of NF-țB signaling pathways, proteasome inhibition is ineffective at blocking NF-țB induction because these pathways rely on proteasome-independent mechanisms for releasing NF-țB from its inhibitor IțBĮ. For instance, the atypical pathway of NF-țB induction entails upstream signaling mechanisms, which culminate in tyrosine phosphorylation of IțBĮ and subsequent dissociation from NF-țB without the involvement of the proteasome. Considerable attention has been paid to the absence of proteasome-mediated degradation of IțBĮ within this subset of NF-țB signaling pathways; however, the effect of proteasome inhibition on additional upstream signaling events in these pathways remains unexplored. For this reason, we delineated the effect of proteasome inhibition on facets of phospho-tyrosine-dependent NF-țB induction, both related and unrelated to tyrosine phosphorylation of IțBĮ.
We demonstrate in a murine bone marrow-derived stromal cell line that, upon short-term activation with PV, cytosolic IțBĮ undergoes tyrosine phosphorylation but not proteasome-mediated degradation-in keeping with data reported from previous studies employing HeLa and Jurkat cell lines [7,12]. Additionally, as in HeLa and Jurkat cell lines, phosphorylation of IțBĮ at Tyr-42 is regulated by the tyrosine kinase, c-Src. Based on immunoblotting, we demonstrate that c-Src assumes its active form, regardless of proteasome inhibition. Since these results analyzed Src kinase activation following short-term exposure to PV, we cannot exclude the possibility that after prolonged exposure to PV, a discrepancy in Src kinase activity may ensue from the combined treatment of proteasome inhibitor and PV, as compared to PV treatment alone. This stems from studies describing the active form of Src as a preferential target for degradation by the proteasome [27,28]. The intriguing possibility that proteasome down-regulates PV-dependent Src activity introduces the idea of the proteasome as a key negative regulator of signaling associated with the atypical NF-țB pathway. At the present time, there is no evidence in the literature, which either supports or refutes this hypothesis. However, stabilization of Src by the chaperone, Hsp 90, has recently been shown to be an essential mechanism in the Src-regulated phosphorylation of IțBĮ Y42 accompanying PV stimulation [29]. This emphasizes that mechanisms associated with the regulation of Src have a profound impact on PV-mediated induction of NF-țB. Accordingly, Src represents a "lynch pin" in the induction of NF-țB by PV, and as such, constitutes a prime target for negative regulators of the atypical NF-țB pathway.
Whereas c-Src kinase phosphorylates IțBĮ at Tyr-42 [12], the tyrosine kinase, c-Abl, has been implicated in the phosphorylation of IțBĮ at Tyr-305 [30]. The precise role of phospho-IțBĮ (Tyr-305) in the atypical NF-țB signaling pathway remains elusive because the stimuli generally associated with atypical NF-țB responses have yet to be vetted as inducers of phospho-IțBĮ (Tyr-305). Thus far, overexpression of either a constitutively-active c-Abl protein or Hepatitis C virus nonstructural protein 5A (NS5A) has been conclusively shown to induce the tyrosine phosphorylation of IțBĮ at Tyr-305 [30,31]. Due to discrepancies in the findings between these studies, tyrosine phosphorylation of IțBĮ Y305 appears to modulate both its stability as well as its ability to simultaneously undergo tyrosine phosphorylation at Tyr-42, in a stimulus-dependent manner. To our knowledge, PV has not been characterized as an inducer of phospho-IțBĮ (Tyr-305) but as we demonstrate here, PV can indeed induce tyrosine phosphorylation of IțBĮ at both Tyr-42 and Tyr-305 residues. Without rigorous, biochemical experimentation, we are unable to definitively elucidate whether PV induces pools of IțBĮ which are uniquely phosphorylated at each tyrosine residue (Tyr-42 or Tyr-305), dually modified at each tyrosine residue (Tyr-42 and Tyr-305), or a combination of both. Given that Tyr-42/-305 phospho-IțBĮ has been demonstrated to undergo calpain-mediated degradation in NS5A overexpressing cells, it is noteworthy that PV-induced phospho-IțBĮ does not undergo any degradation in ILU-18 cells. Further, pretreatment with the calpain inhibitor, E64D, prior to PV-treatment did not interfere with NF-țB activity [25]. Nevertheless, our demonstration that PV is capable of inducing tyrosine phosphorylation of IțBĮ at both tyrosine residues provides the assurance that PV can be used to evaluate the crosstalk not only between these two tyrosine phosphorylation sites but also the potential crosstalk between tyrosine phosphorylation and sumoylation.
Strikingly, modification of IțBĮ by either tyrosine phosphorylation or sumoylation confers similar outcomes in terms of the stability. Since IțBĮ undergoes modification by SUMO-1 at the same lysine residue targeted for ubiquitination (K21), sumoylated IțBĮ is resistant to ubiquitin-mediated degradation by the proteasome [32]. Accordingly, modification of IțBĮ by SUMO-1 has emerged as the prototypical example of the stabilizing effect of sumoylation. Similarly, tyrosine phosphorylation has been found to protect IțBĮ from the cytokine-induced serine phosphorylation which precedes ubiquitin-mediated degradation by the proteasome [33]. Despite the overlap in function between these two modifications, the possibility that IțBĮ is dually modified by SUMO-1 and tyrosine phosphorylation remains unexplored.
Finally, the objective of this study was to understand the effect of proteasome inhibition on facets of phospho-tyrosine-dependent NF-țB signaling, both related and unrelated to tyrosine phosphorylation of IțBĮ. While proteasome inhibition had no discernable effect on the early signaling events accompanying PV-mediated NF-țB induction, proteasome inhibition did potentiate oxidative stress associated with PV-stimulation. The cellular implications for this increase in intracellular oxidation are uncertain in this cell type, however, there is mounting evidence that proteasome inhibition, in combination with oxidative stress, has appreciable effects on cellular viability, clearance of oxidatively damaged proteins, detoxification capacity of antioxidant enzymes, and inflammation in other cell types [34][35][36][37]. In neuronal cells, both acute and chronic inhibition of proteasome activity alters mitochondria homeostasis, resulting in overproduction of reactive oxygen species (ROS) by dysfunctional mitochondria [37,38]. Contrary to these reports, in our system, proteasome inhibition alone did not induce an increase in ROS. Instead, proteasome inhibition had an additive effect on cellular oxidation induced by PV. We investigated the possibility that the combination of proteasome inhibition and oxidative stress unduly stressed detoxification capacity of GSH but despite pretreatment with proteasome inhibitor, PV-associated loss in functional GSH was nearly equivalent. However, a potential effect of proteasome inhibition on additional antioxidants, such as catalase and superoxide dismutase remains to be tested. In fact, this possibility is supported by a recent study delineating the effect of pretreatment with proteasome inhibitor on liver ischemia/reperfusion (I/R). In this study, Alexandrova et al. demonstrated that compared to I/R alone, the combination of proteasome inhibition and I/R disproportionately stressed the activities of the antioxidant enzymes, catalase and superoxide dismutase [39].
In summary, the results presented here reiterate that early signaling events associated with atypical pathway of NF-țB induction are proteasome-independent. While mechanisms and biological significance of IțBĮ tyrosine-phosphorylation in protein stability remain unclear, these studies highlight the potential for an intriguing interplay between redox regulation, tyrosine phosphorylation, and the atypical mechanism of NF-țB induction.

Measurement of Intracellular Oxidation Using H2DCF-DA
ILU-18 cells (1 × 10 6 /mL) were washed with 1X Hank's Balanced Salt Solution (HBSS) before resuspending the cells in 1 mL HBSS and equilibrating at 37 °C for 15 min. At the end of equilibration, cells were treated with 10 ȝM H2DCF-DA and incubated further for 30 min at 37 °C in the dark. After washing the cells with 1X HBSS, cells were resuspended in 1 mL of 1X HBSS. ROS generation was monitored following addition of PV (100 ȝM) or H2O2 (200 ȝM) and detected using a LS-50 spectrofluorometer (Perkin Elmer Corporation, Norwalk, CT, USA) at excitation wavelength of 480 nm and emission wavelength of 525 nm.

Measurement of Intracellular GSH
Intracellular GSH levels were assayed as described previously [40]. Briefly, 100 ȝL of ice-cold lysis buffer was added to ILU-18 cells (1 × 10 6 ), incubated on ice for 10 min, and centrifuged at 12,000× g for 10 min. To this preparation, 2 ȝL of 25 mM monochlorobimane (MCB) and 50 U/mL glutathione-S-transferase (GST) were added and incubated at 37 °C for 30 min. At the end of incubation, fluorescence was measured by employing a fluorescence plate reader (Perkin Elmer Corporation, Norwalk, CT, USA) with an excitation wavelength of 340 nm and emission wavelength of 465 nm. Values obtained in the absence of GST and MCB served as negative controls.

Luciferase Reporter Assay
ILU-18 cells, with or without pretreatment, were activated with pervanadate (100 ȝM) for 18 h at 37 °C. Additionally, ILU-18 cells were subjected to treatment with H2O2 (100 ȝM) for 18 h or 200 ȝM Buthionine-sulfoximine (BSO) for 24 h at 37 °C. Utilizing a Luciferase Reporter assay kit (Promega, Madison, WI, USA), lysates were prepared with Reporter lysis buffer by repeated freezing and thawing. Protein content was determined by Bio-Rad assay. Per the supplier's protocol [41], cell extract (50 ȝg protein) was combined with 100 ȝL of the luciferase assay reagent at room temperature and analyzed by luminometer.

Statistical Analyses
Differences between means of the data generated in the study were analyzed using Student's t-test. Differences were considered significant, if p < 0.05.

Conclusions
The results presented here demonstrate that while proteasome inhibition can potentiate oxidative stress associated with pervanadate stimulation, it fails to influence the induction of NF-țB by the atypical pathway. Thus, the regulation of IțBĮ and upstream signaling pathway intermediates in the atypical pathway of NF-țB induction, are also proteasome-independent.