eIF2α-Independent Inhibition of TNF-α-Triggered NF-κB Activation by Salubrinal

Abstract

Salubrinal is a selective inhibitor of cellular complexes that dephosphorylate eukaryotic translation initiation factor 2α (eIF2α). In previous reports, salubrinal was shown to have the potential to inhibit the activation of nuclear factor-κB (NF-κB) by several stimuli. However, the effects of salubrinal on NF-κB signaling are largely unknown. In this study, we investigated whether and how salubrinal affects NF-κB activation induced by tumor necrosis factor (TNF)-α and interleukin (IL)-1β. We found that salubrinal selectively blocked TNF-α- but not IL-1β-induced activation of NF-κB. This inhibitory effect occurred upstream of transforming growth factor (TGF)-β-activated kinase 1 (TAK1). Further experiments revealed that salubrinal blocked TNF-α-triggered NF-κB activation independent of its action on eIF2α because knockdown of eIF2α by small interfering RNA (siRNA) did not reverse the inhibitory effect of salubrinal on NF-κB. Moreover, guanabenz, a selective inhibitor of the regulatory subunit of protein phosphatase (PP) 1, also preferentially inhibited TNF-α-triggered activation of NF-κB. These findings raise the possibility that salubrinal may selectively block TNF-α-triggered activation of the NF-κB pathway through inhibition of the PP1 complex.

Salubrinal is a small molecule that inhibits the dephosphorylation of eukaryotic translation initiation factor 2α (eIF2α) through the suppression of protein phosphatase (PP) 1 catalytic subunits, resulting in sustained phosphorylation of eIF2α on Ser51. Through this effect, salubrinal protects cells from endoplasmic reticulum (ER) stress-mediated apoptosis.¹⁾ It has been shown that salubrinal influences several signaling pathways involved in apoptosis, autophagy and bone homeostasis.^2–4) The nuclear factor-κB (NF-κB) pathway is also a target of salubrinal. Recently, Huang et al. reported that salubrinal protected against β-amyloid-triggered neuronal cell death and microglial activation through the suppression of NF-κB activation.⁵⁾ Moreover, Hamamura et al. suggested that salubrinal reduced the inflammatory cytokine-driven expression of matrix metalloproteinase 13 via blockade of the p38 mitogen-activated protein kinase pathway and the NF-κB pathway.⁶⁾ Currently, however, the mechanism by which salubrinal regulates NF-κB remains largely unknown.

The Rel/NF-κB family consists of five different members, namely, p50, p52, p65 (RelA), RelB and c-Rel, and functions as a transcription factor through the formation of homo- or heterodimers.⁷⁾ NF-κB plays crucial roles in the regulation of inflammation, immune responses and apoptosis.⁸⁾ In unstimulated cells, NF-κB is retained in the cytoplasm through binding to inhibitors of NF-κB (IκBs). However, in response to several stimuli such as inflammatory cytokines, oxidative stress, ultraviolet light and bacterial components, IκB kinase (IKK) complex including IKKα, IKKβ and IKKγ (also called NF-κB essential modulator; NEMO) is rapidly activated, resulting in phosphorylation and ubiquitin proteasome-mediated degradation of IκBα. Released NF-κB rapidly translocates to the nucleus and binds to the κB enhancer element, leading to the transcription of inflammatory target genes.⁷⁾

Salubrinal inhibits two regulatory subunits of protein phosphatase 1: growth arrest and DNA damage-inducible protein (GADD34) (also called phosphoprotein phosphatase regulatory subunit 15A) and constitutive repressor of eIF2α (also called phosphoprotein phosphatase regulatory subunit 15B).¹⁾ Previous reports suggested that PPs such as PP1 and PP2A are involved in regulation of the NF-κB pathway.^9,10) Mitsuhashi et al. suggested that PP1 is recruited to the tumor necrosis factor (TNF) receptor (TNFR) 1 complex in response to TNF-α stimuli and involved in positive regulation of NF-κB.¹¹⁾ These findings prompted us to speculate that salubrinal might affect the activation of NF-κB through blockade of PP1 complex activity. Therefore, we evaluated whether i) salubrinal suppresses activation of NF-κB by TNF-α or interleukin (IL)-1β; ii) induction of eIF2α phosphorylation by salubrinal is involved in its suppressive effect on NF-κB; and iii) another GADD34–PP1 complex inhibitor also inhibits NF-κB activation by TNF-α or IL-1β.

Here, we show that salubrinal suppresses TNF-α- but not IL-1β-triggered activation of NF-κB upstream of the transforming growth factor (TGF)-β-activated kinase 1 (TAK1)/TAK1-binding protein (TAB) 1 complex. Our data indicate that the induction of eIF2α phosphorylation by salubrinal is not involved in its suppressive effect. In addition, an inhibitor of the GADD34–PP1 complex also preferentially inhibits the activation of NF-κB by TNF-α, implying that salubrinal selectively blocks the TNF-α–NF-κB pathway through the suppression of PP1 complex activity.

MATERIALS AND METHODS

Reagents

Salubrinal was purchased from Tocris Bioscience (Ellisville, MO, U.S.A.). Recombinant human IL-1β and TNF-α were from R&D Systems (Minneapolis, MN, U.S.A.). Guanabenz acetate salt was from Sigma-Aldrich Japan (Tokyo, Japan).

Cells and Stable Transfectants

Cells of the rat renal tubular epithelial cell line NRK-52E were obtained from American Type Culture Collection (Manassas, VA, U.S.A.). Cells of the rat mesangial cell line SM43 were established as described previously.¹²⁾ Murine podocytes were kindly provided by Dr. Karlhans Endlich.¹³⁾ Cells of the murine mammary epithelial cell line NMuMG were kindly provided by Dr. Keiji Miyazawa (University of Yamanashi, Yamanashi, Japan). These cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM)/Ham’s F-12 (Gibco-BRL, Gaithersburg, MD, U.S.A.) supplemented with 5% fetal bovine serum (FBS). The media for the culture of NMuMG cells were also supplemented with 10 µg/mL insulin.

Using electroporation, NRK-52E cells were stably transfected with pNFκB-Luc (Panomics, Fremont, CA, U.S.A.), which introduces a luciferase gene under the control of the κB site, and NRK/κB-Luc cells were established.

Transient Transfection

Using electroporation, NRK/κB-Luc cells were transiently transfected with pCMV2-FLAG-TAK1 and pcDNA-HA-TAB1 (provided by Dr. Hiroaki Sakurai; University of Toyama, Toyama, Japan)¹⁴⁾ in combination or pcDNA3.1 as a control. After 48 h, the cells were then treated with salubrinal for 8 h and subjected to luciferase assay to evaluate NF-κB activity.

Using HiPerFect Transfection Reagent (Qiagen, Tokyo, Japan), NRK-52E cells were transiently transfected with eIF2α small interfering RNA (siRNA), PP1α siRNA (MISSION siRNA, Sigma) or control siRNA (MISSION siRNA Universal Negative Control, Sigma). After 72 h, the cells were treated with salubrinal for 8 h, exposed to TNF-α and subjected to Western blot analysis.

Luciferase Assay

Using Luciferase Assay System (Promega, Madison, WI, U.S.A.), luciferase activity was evaluated according to the manufacturer’s protocol. Its activity (relative light unit) was normalized by the number of viable cells counted using a formazan assay, and fold changes compared with the control (−) were plotted in graphs.

Northern Blot Analysis

Total RNA was extracted by a single-step method, and Northern blot analysis was performed as described previously.¹⁵⁾ cDNAs for monocyte chemoattractant protein 1 (MCP-1)¹⁶⁾ were used to prepare radio-labeled probes. The expression of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a loading control.

Western Blot Analysis

Western blot analysis was performed as described previously.¹⁵⁾ Briefly, cells were lysed in Radio-Immunoprecipitation Assay (RIPA) buffer (25 mM Tris–HCl [pH 7.5], 150 mM sodium chloride (NaCl), 1% Triton-X, 1% sodium dodecyl sulfate (SDS), 50 mM sodium fluoride (NaF), 20 mM N-ethylmaleimide and 1 mM sodium orthovanadate (Na₃VO₄)) with protease inhibitor cocktail (Nacalai Tesque, Kyoto, Japan), and cell lysates were then mixed with sample buffer containing dithiothreitol and bromophenol blue. After boiling for 5 min, samples were subjected to Western blot analysis. Immunoreactive proteins were visualized using the Chemi-Lumi One L (Nacalai Tesque). Primary antibodies were those against phospho-eIF2α (Ser51), phospho-IKKα/β (Ser176/Ser177), IKKβ, phospho-IκBα (Ser32), phospho-p65 (Ser536), p65, receptor-interacting protein (RIP) 1, TAB2, phospho-TAK1 (Thr187), TAK1 and TNFR-associated factor (TRAF) 2 from Cell Signaling Technology (Beverly, MA, U.S.A.); eIF2α, IκBα, TNFR1 and PP1α from Santa Cruz Biotechnology (Santa Cruz, CA, U.S.A.); cellular inhibitor of apoptosis (c-IAP) 1 from Abcam Inc. (Cambridge, MA, U.S.A.); β-actin from Sigma-Aldrich Japan; and lamin B1 from Invitrogen (Carlsbad, CA, U.S.A.).

Fractionation of Cytoplasmic and Nuclear Proteins

Cells were lysed with a ProteoExtract Subcellular Proteome Extraction Kit Reagent (Merck KGaA, Darmstadt, Germany) to separate cytoplasmic and nuclear fractions, according to the manufacturer’s protocol. After fractionation, nuclear extracts were subjected to Western blot analysis of p65. As a loading control, levels of lamin B1 were used.

Formazan Assay

The number of viable cells was assessed by a formazan assay using Cell Counting Kit-8 (Dojindo Laboratory, Kumamoto, Japan).

Statistical Analysis

In reporter assays and formazan assay, experiments were performed in quadruplicate, and data are expressed as mean±standard error (S.E.). Statistical analysis was performed using the non-parametric Mann–Whitney U test to compare data in different groups. A p value <0.05 was considered to indicate a statistically significant difference.

RESULTS

Suppression of TNF-α-Triggered NF-κB Activation by Salubrinal

To determine the effective concentration for the induction of eIF2α phosphorylation, NRK-52E cells were treated with several concentrations of salubrinal and subjected to Western blot analysis of phospho-eIF2α. Salubrinal induced eIF2α phosphorylation at concentrations as low as 5 µM. The maximal induction was at 50 µM (Fig. 1A). Therefore, 50 µM salubrinal was chosen for the subsequent experiments.

Fig. 1. Inhibition of TNF-α- but Not IL-1β-Induced Activation of NF-κB by Salubrinal

(A) NRK-52E cells were treated with several concentrations of salubrinal (Sal) for 8 h and subjected to Western blot analysis of phospho-eIF2α and total eIF2α; relative intensity compared with the control is shown. (B) NRK/κB-Luc cells were pretreated with or without salubrinal for 6 h, exposed to 10 ng/mL TNF-α or 10 ng/mL IL-1β for 6 h and subjected to luciferase assay. Activity of luciferase was normalized by the number of viable cells estimated by formazan assay, and fold change compared with the control is shown. Assay was performed in quadruplicate, and data are presented as means±S.E. Statistical analysis was performed using the non-parametric Mann–Whitney U test to compare data in different groups. An asterisk indicates a statistically significant difference, p<0.05, TNF-α versus Sal+TNF-α, IL-1β versus Sal+IL-1β. N.S., not statistically significant. (C) NRK-52E cells were exposed to TNF-α or IL-1β in the absence or presence of pretreatment with salubrinal for 8 h and subjected to fractionation of cytoplasmic and nuclear proteins. Nuclear protein was subjected to Western blot analysis of p65. The level of lamin B1 is shown at the bottom as a loading control. (D) Cells were treated with salubrinal for 8 h, stimulated with TNF-α or IL-1β for up to 3 h and subjected to Northern blot analysis of MCP-1. Expression of GAPDH is shown at the bottom as a loading control. T1, T3: TNF-α for 1 h, 3 h; IL1, IL3: IL-1β for 1 h, 3 h.

We next examined whether pre-exposure of cells to salubrinal affected inflammatory cytokine-triggered NF-κB activation. Reporter assay showed that salubrinal markedly suppressed the activation of NF-κB by TNF-α, but not IL-1β (Fig. 1B). Consistent with this result, salubrinal only suppressed the TNF-α-triggered nuclear translocation of p65 and expression of MCP-1, a NF-κB-dependent gene (Figs. 1C, D), suggesting that salubrinal selectively blocks the activation of NF-κB triggered by TNF-α.

Involvement of Molecular Events Upstream of TAK1/TAB1 Complex in Inhibition of NF-κB Activation by Salubrinal

To examine the mechanisms involved in the effect of salubrinal, we examined the influence of salubrinal on the phosphorylation of IKKα/β, IκBα and p65, as well as the degradation of IκBα. The exposure of cells to TNF-α or IL-1β caused rapid phosphorylation of IKKα/β, IκBα and p65 and degradation of IκBα. In the presence of salubrinal, however, the event induced by TNF-α was effectively blocked, while the event induced by IL-1β was not inhibited (Figs. 2A, B). Similar blocking effects were also observed in NMuMG cells, murine podocytes and SM43 cells (Supplementary Figures S1A–C).

Fig. 2. Suppression of TNF-α-Triggered Activation of the NF-κB Signaling by Salubrinal Upstream of TAK1/TAB1 Complex

(A–D) NRK-52E cells were treated with salubrinal for 8 h, exposed to TNF-α (A, C) or IL-1β (B, D) for the indicated time periods and subjected to Western blot analysis of the indicated molecules. (E) NRK/κB-Luc cells were transiently transfected with empty vector or with pCMV2-FLAG-TAK1 and pcDNA-HA-TAB1 in combination (1 : 1). After 48 h, the cells were then treated with salubrinal for 8 h and subjected to luciferase assay. Fold change compared with the control is shown. N.S., not statistically significant.

Given that the signaling events downstream of TAK1/TAB1 are shared by TNF-α and IL-1β in the activation of NF-κB,¹⁷⁾ we examined the effect of salubrinal on TAK1 phosphorylation. Pretreatment with salubrinal significantly suppressed the phosphorylation of TAK1 by TNF-α. However, this inhibitory effect was modest in IL-1β-stimulated cells (Figs. 2C, D). For further confirmation of the effect of salubrinal on TAK1/TAB1 complex activity, NRK/κB-Luc cells were transiently transfected with pCMV2-FLAG-TAK1 and pcDNA-HA-TAB1 in combination or with empty vector as a control. The transfected cells were exposed to salubrinal and subjected to luciferase assay. As shown in Fig. 2E, overexpression of TAK1 and TAB1 clearly induced the activation of NF-κB. However, salubrinal did not suppress this activation (Fig. 2E), indicating that it did not affect the activity of the TAK1/TAB1 complex. These findings suggest that salubrinal inhibits the TNF-α-induced activation of NF-κB through interference with molecular events upstream of the TAK1/TAB1 complex.

eIF2α Phosphorylation-Independent Blockade of NF-κB Activation by Salubrinal

ER stress causes activation of the unfolded protein response (UPR) to maintain ER homeostasis. The UPR consists of three branches including the protein kinase-like ER kinase (PERK)–eIF2α pathway, the activating transcription factor 6 pathway and the inositol-requiring enzyme 1–X-box-binding protein 1 pathway.¹⁸⁾ It is known that ER stress and the UPR have the potential to regulate NF-κB positively or negatively, depending on the cellular context.¹⁹⁾ In particular, our recent report demonstrated that the PERK–eIF2α pathway is important for ER stress-mediated inhibition of NF-κB.²⁰⁾ We therefore examined whether the induction of eIF2α phosphorylation is involved in the suppressive effect of salubrinal on NF-κB. Interestingly, short-term treatment (1 h) with salubrinal did not inhibit activation of the NF-κB pathway by TNF-α or IL-1β (Figs. 3A, B) and nuclear translocation of p65 (Fig. 3C), although it induced eIF2α phosphorylation. These results suggest that salubrinal might inhibit the activation of NF-κB independently of the induction of eIF2α phosphorylation. To confirm this, NRK/κB-Luc cells were transfected with control siRNA or eIF2α siRNA (Fig. 3D, bottom), and the cells were then treated with salubrinal, exposed to TNF-α and subjected to luciferase assay. In accordance with a previous report,²¹⁾ knockdown of eIF2α markedly suppressed TNF-α-induced activation of NF-κB (Fig. 3D, top). Because a previous report showed that increased phosphorylation of protein kinase R and eIF2α by TNF-α contributes to NF-κB activation and cytokine production,²²⁾ it is conceivable that the knockdown of eIF2α should decrease the effect of TNF-α on NF-κB activation. However, knockdown of eIF2α did not reverse the suppressive effect of salubrinal on NF-κB (Fig. 3D, top). Consistent with this result, Western blot analysis also showed that knockdown of eIF2α did not restore salubrinal-mediated inhibition of the NF-κB pathway (Fig. 3E). These data indicate that salubrinal suppresses TNF-α-triggered activation of NF-κB independently of the induction of eIF2α phosphorylation.

Fig. 3. Non-involvement of eIF2α Phosphorylation in the Suppressive Effect of Salubrinal on NF-κB

(A, B) NRK-52E cells were treated with salubrinal for 1 h, exposed to TNF-α (A) or IL-1β (B) for up to 60 min and subjected to Western blot analysis of the indicated molecules. (C) Cells were treated with salubrinal for 1 h, exposed to TNF-α for 30 min and subjected to fractionation of cytoplasmic and nuclear proteins. Nuclear proteins were subjected to Western blot analysis of p65. (D) NRK/κB-Luc cells were transiently transfected with control siRNA or eIF2α siRNA. After 72 h, the cells were then treated with salubrinal for 6 h, exposed to TNF-α for 6 h, and subjected to luciferase assay (top) and Western blot analysis of phospho-eIF2α and total eIF2α (bottom). Relative values (%) of Sal+TNF-α/TNF-α are shown. Asterisks indicate statistically significant differences, p<0.05. (E) NRK-52E cells were transiently transfected with control siRNA or eIF2α siRNA. After 72 h, the cells were then treated with salubrinal for 8 h, exposed to TNF-α for 5 min and subjected to Western blot analysis of the indicated molecules.

Preferential Inhibition of TNF-α-Triggered NF-κB Activation by Guanabenz

Recently, Tsaytler et al. demonstrated that guanabenz, an α₂-adrenergic receptor agonist,²³⁾ directly bound to the GADD34–PP1 complex and prolonged the phosphorylation of eIF2α in ER stress conditions without affecting the basal level of eIF2α phosphorylation.²⁴⁾ To elucidate the role of the GADD34–PP1 complex in activation of the NF-κB pathway, NRK-52E cells were stimulated with TNF-α or IL-1β in the presence or absence of guanabenz, and the effect on NF-κB activity was assessed. As shown in Fig. 4A, guanabenz preferentially blocked TNF-α-induced NF-κB activation, as revealed by promoter activity assay (Fig. 4A). Western blot analysis also showed that guanabenz exerted more potent suppression of TNF-α-induced phosphorylation of IKKα/β and IκBα, as well as degradation of IκBα, compared with its effect on IL-1β (Fig. 4B). Surprisingly, guanabenz itself also induced eIF2α phosphorylation (Fig. 4B). This effect of guanabenz might be related to its suppressive effect on the basal activity of GADD34-PP1 complex. Moreover, konckdown of PP1α, a subfamily member of PP1, by siRNA clearly attenuated the TNF-α-triggered phosphorylation of IKKα/β (Supplementary Figure S2), implying that the PP1 complex (GADD34–PP1 complex) might be involved in activation of the TNF-α–NF-κB pathway in NRK-52E cells.

Fig. 4. Preferential Suppression of TNF-α-Induced Activation of NF-κB by Guanabenz

(A) NRK/κB-Luc cells were treated with 200 µM guanabenz (Gua) for 6 h, exposed to TNF-α or IL-1β for 6 h and subjected to luciferase assay. Asterisks indicate statistically significant differences, p<0.05. (B) NRK-52E cells were treated with guanabenz for 8 h, exposed to TNF-α or IL-1β for up to 15 min and subjected to Western blot analysis of the indicated molecules.

Taken together, the present data suggest that salubrinal might selectively suppress activation of the TNF-α–NF-κB pathway through inhibition of the PP1 complex independent of its action on eIF2α phosphorylation.

DISCUSSION

Previous papers showed that salubrinal has the potential to suppress the activation of NF-κB by several stimuli.^5,6) However, more detailed effects of salubrinal on the NF-κB signaling have not been elucidated. In the present study, we demonstrated that salubrinal selectively inhibited TNF-α- but not IL-1β-triggered activation of NF-κB through interfering with a molecular event upstream of the TAK1/TAB1 complex. Moreover, we found that the suppressive effect of salubrinal on NF-κB was independent of its action on eIF2α phosphorylation. Because guanabenz, a GADD34–PP1 complex inhibitor, also preferentially blocked TNF-α-induced activation of NF-κB, it is conceivable that the effect of salubrinal might occur through inhibition of the PP1 complex.

It is currently unknown which molecules are involved as targets of salubrinal in the activation pathway of NF-κB. When TNF-α binds to TNFR, TRAF2, c-IAP and RIP1 are recruited to TNFR, leading to the generation of K63 ubiquitin chains on RIP1.²⁵⁾ K63-linked polyubiquitin chains further recruit complexes consisting of TAB1, TAB2/3 and TAK1, resulting in auto-phosphorylation of TAK1.²⁶⁾ It is known that the induction of eIF2α phosphorylation attenuates global cellular protein synthesis.²⁷⁾ Therefore we tested whether salubrinal affects the basal level of essential molecules for the activation of NF-κB by TNF-α. We actually did not detect an obvious alteration in most of proteins upstream of TAK1, including TNFR1, c-IAP1, RIP1 and TAB2, after the treatment of cells with salubrinal for a long period, even at high concentrations (Supplementary Figure S3A). However, the expression level of TRAF2 was slightly decreased (Supplementary Figure S3A). Of note, repression of TRAF2 attenuates the TNF-α-triggered activation of NF-κB.¹⁰⁾ Therefore, slight reduction of TRAF2 expression might also partially contributes to suppressive effects of salubrinal on the activation of NF-κB by TNF-α. On the other hand, salubrinal significantly abrogated TNF-α-induced ubiquitin-like ladder bands of RIP1 (Supplementary Figure S3B), indicating that it might at least suppress events upstream of RIP1 modification.

According to our current investigation, long-term treatment with salubrinal (at least for several hours) was required to elicit the suppressive effect on NF-κB, implying that secondary effects of salubrinal might block the activation of NF-κB by TNF-α. Salubrinal quickly induced the phosphorylation of eIF2α within 1 h (Fig. 3A). However, induction of downstream targets such as CCA AT/enhancer-binding protein-homologous protein and CCA AT/enhancer-binding protein β²⁰⁾ were occurred 3 to 6 h after the treatment (our unpublished data). Because, in this work, we have shown that the suppressive effect of salubrinal is independent of its action on eIF2α, salubrinal may induced phosphorylation of some molecules through suppression of GADD34–PP1 complex, leading to up-regulation of some inhibitory molecules against the TNF-α–NF-κB pathway in the later time point.

In the present paper, we found that salubrinal exerted a more selective inhibition on TNF-α–NF-κB pathway in comparison with guanabenz. The mechanisms behind the selective inhibition are presently unclear. Guanabenz has been reported not only to inhibit PP1, but also to function as α₂-adrenergic receptor agonist.²³⁾ Several reports suggested that adrenergic receptor agonists blocked the activation of NF-κB,^28,29) suggesting that guanabenz might suppress the activation of NF-κB through both PP1 inhibition and adrenergic receptor activation. Another PP1 inhibitor tautomycetin also has PP1-independent biological actions. It inactivated serine–threonine kinase Akt (also called as protein kinase B), which is reported to be required for NF-κB activation by TNF,³⁰⁾ through a PP1-independent pathway.³¹⁾ Thus, tautomycetin might also suppress the activation of NF-κB independently of PP1 inhibition. Based on these previous reports, guanabenz and tautomycetin might have pleiotropic potential for NF-κB suppression. In the case of salubrinal, it did not cause inactivation of Akt.³²⁾ In addition, inhibition of PP1 with calyculin A, an active-site inhibitor of PP1 catalysis, induced threonine phosphorylation on many proteins whereas salubrinal did not induced them. Proteomics analysis revealed that salubrinal treatment only affected three or four proteins,¹⁾ suggesting that salubrinal is distinct from other PP1 inhibitors. The specific effects of salubrinal on certain proteins might be the reason why it selectively inhibited TNF-α–NF-κB pathway.

In the current study, we demonstrated a novel suppressive effect of salubrinal on the TNF-α–NF-κB pathway. Our novel findings that salubrinal selectively inhibited TNF-α- but not IL-1β-triggered activation of NF-κB can have important clinical implications that salubrinal might be useful for TNF-α-dominant inflammatory diseases. However, the action of salubrinal on NF-κB in vivo has not been tested. Toward efficient therapies for inflammatory diseases by using salubrinal, further investigations will be required. Elucidation of the therapeutic utility of salubrinal will be our next line of investigation.

Acknowledgments

We thank Dr. Karlhans Endlich, Dr. Keiji Miyazawa and Dr. Hiroaki Sakurai for providing us with experimental materials. We are also grateful to Dr. Masanori Kitamura for helpful discussions and Mr. Tatsuya Yoshitomi for technical assistance. This work was supported by a Research Fellowship for Young Scientists from the Japan Society for the Promotion of Science (to S. Nakajima).

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

The online version of this article contains supplementary materials.

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