Isopanduratin A Inhibits Tumor Necrosis Factor (TNF)- α -Induced Nuclear Factor κ B Signaling Pathway by Promoting Extracellular Signal-Regulated Kinase-Dependent Ectodomain Shedding of TNF Receptor 1 in Human Lung Adenocarcinoma A549 Cells

α -Induced Nuclear Factor κ B Signaling Pathway by Promoting Extracellular Signal-Regulated Kinase-Dependent Ectodomain Shedding of TNF Receptor 1 Abstract: Tumor necrosis factor α (TNF- α ) induces the nuclear factor κ B (NF- κ B) signaling pathway via TNF receptor 1 (TNF-R1). We recently reported that isopanduratin A inhibited the TNF- α -induced NF- κ B signaling pathway in human lung adenocarcinoma A549 cells. In the present study, we found that isopanduratin A did not inhibit the interleukin-1 α -induced NF- κ B signaling pathway in A549 cells. Isopanduratin A down-regulated the expression of TNF-R1 in these cells. We also revealed that isopanduratin A down-regulated the cell surface expression of TNF-R1 by promoting the cleavage of TNF-R1 into its soluble forms. TAPI-2, an inhibitor of TNF- α -converting enzyme, suppressed the inhibitory activity of isopanduratin A against the TNF- α -induced activation of NF- κ B. The mitogen-activated protein (MAP) kinase/extracellular signal-regulated kinase (ERK) kinase inhibitor U0126, but not the p38 MAP kinase inhibitor SB203580, blocked the ectodomain shedding of TNF-R1 induced by isopanduratin A. Consistent with this result, isopanduratin A induced the rapid phosphorylation of ERK, but not p38 MAP kinase. Isopanduratin A also promoted the phosphorylation of eukaryotic initiation factor 2 α (eIF2 α ). The present results indicate that isopanduratin A inhibits TNF- α -induced NF- κ B signaling pathway by promoting ERK-dependent ectodomain shedding of cell surface TNF-R1, and also decreases cellular TNF-R1 levels through the phosphorylation of eIF2 α in A549 cells.


Introduction
Inflammatory cytokines, such as tumor necrosis factor α (TNF-α), are produced by activated macrophages, and stimulate other types of cells to provoke inflammatory responses [1]. TNF-α induces intracellular signaling pathways, one of which leads to the activation of the transcription factor nuclear factor κB (NF-κB) [2,3]. Upon engaging with TNF-α, TNF receptor 1 (TNF-R1) recruits adaptor proteins to form a membrane-proximal complex, which is required for the activation of inhibitor of NF-κB (IκB) kinase [2,3]. IκBα is associated with NF-κB subunits in the cytosol and is rapidly phosphorylated by IκB kinase in response to a TNF-α stimulation, leading to its ubiquitination and degradation by proteasomes [4,5]. NF-κB subunits, typically composed of RelA (also known as p65) and p50, translocate to the nucleus and activate many of the genes that are essential for inflammatory responses [6,7].
Panduratins are characterized by cyclohexene chalcones and belong to a family of flavonoids [22]. Panduratin derivatives have been reported to exert diverse biological effects, including anti-cancer and anti-inflammatory activities [22][23][24][25]. Isopanduratin A has been shown to exhibit anti-bacterial activity against Streptococcus mutans and acnecausing microorganisms [26,27], inhibitory activities toward melanin biosynthesis and tyrosinase [28], suppressive effects on TNF-α-induced cytotoxicity and aminopeptidase N [29], and α-glucosidase and pancreatic lipase inhibitory activities [30]. We previously identified eight new cyclohexene chalcones and seven known compounds, including isopanduratin A, in the rhizome extracts of Boesenbergia pandurata, a medicinal plant belonging to the Zingiberaceae family, as agents that exhibited potent cytotoxic activities in the human pancreatic cancer line PANC-1 [31]. Consistent with previous findings [32], we showed that isopanduratin A exhibited anti-cancer activity towards PANC-1 and the human hepatocellular carcinoma cell line HepG2 [31,33]. In addition to these anti-cancer activities, we recently reported that isopanduratin A inhibited the TNF-α-induced NF-κBdependent signaling pathway in the human lung adenocarcinoma cell line A549 [34]. 4-Hydroxypanduratin A has also been shown to inhibit the TNF-α-induced NF-κB-dependent signaling pathway [34]. Consistent with our studies, panduratin A and its derivatives inhibited the NF-κB signaling pathway in different cell lines [35][36][37][38]. However, the molecular targets of panduratin derivatives in the NF-κB signaling pathway remain unclear. In the present study, we elucidated the mechanisms by which isopanduratin A inhibited the TNF-α-induced NF-κB signaling pathway.

Cell Culture
A549 cells (JCRB0076) were obtained from the National Institutes of Biomedical Innovation, Health, and Nutrition JCRB Cell Bank (Osaka, Japan) and maintained in RPMI 1640 medium (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with heat-inactivated fetal calf serum (Sigma-Aldrich, St. Louis, MO, USA) and penicillinstreptomycin mixed solution (Nacalai Tesque, Kyoto, Japan) as previously described [39,40]. RPMI 1640 medium without fetal calf serum was used in experiments to examine the phosphorylation of ERK, p38 MAP kinase, and c-Jun N-terminal kinase (JNK).

Isopanduratin A Inhibited IκB Degradation Induced by TNF-α, but Not IL-1α
In NF-κB signaling pathways, TNF-α and IL-1α induce the formation of different membrane-proximal complexes, which converge to activate IκB kinases as the main target [2,3]. To clarify whether isopanduratin A inhibits the converged NF-κB signaling pathway, A549 cells were pretreated with isopanduratin A, followed by TNF-α or IL-1α. Consistent with our previous findings [34], isopanduratin A inhibited IκBα degradation induced by TNF-α ( Figure 1B). By contrast, IκBα degradation induced by IL-1α was not inhibited by isopanduratin A ( Figure 1B). These results indicated that isopanduratin A selectively inhibited the early NF-κB signaling pathway induced by TNF-α in A549 cells.

Isopanduratin A Decreased the Expression of TNF-R1
A549 cells have been shown to express TNF-R1, but not TNF-R2 [16]. We investigated the expression of TNF-R1 at the protein level by Western blotting. The treatment with

Western Blotting
Cells were washed with phosphate-buffered saline (PBS) and then lysed with Triton X-100 lysis buffer containing the protease inhibitor cocktail cOmplete TM (Sigma-Aldrich) and phosphatase inhibitor cocktail (Nacalai Tesque). Nuclear and cytoplasmic fractions were prepared as reported in previous studies [19,41]. Cell lysates were centrifuged (15,300× g, 5 min) and separated into supernatants (cytoplasmic fractions) and pellets. The pellets were washed with Triton X-100 lysis buffer, sonicated, and collected as nuclear fractions. The culture medium was processed to precipitate proteins by chloroform/methanol as previously described [16,19]. Western blotting was performed according to a previously described method [39,40]. Proteins were separated by SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes, followed by blocking with skimmed milk. Membranes were then reacted serially with primary antibodies and peroxidase-conjugated anti-mouse IgG or anti-rabbit IgG antibodies (Jackson ImmunoResearch, West Grove, PA, USA), and subjected to a chemiluminescence reaction. Blots were analyzed by Image-Quant LAS 4000 mini (GE Healthcare Japan, Tokyo, Japan) and Amersham Imager 680 (GE Healthcare Japan).

Transfection
The pCR3 expression vector encoding C-terminal FLAG-tagged human full-length TNF-R1 was previously described [42]. A549 cells were transfected with the pCR3 expression vector encoding TNF-R1-FLAG by the lipofection method using HilyMax transfection reagent (Dojindo, Kumamoto, Japan). Transfected A549 cells were then treated with compounds.

Flow Cytometry
Cells were harvested and then stained with either an anti-TNF-R1 antibody (16803) or an isotype control antibody (MOPC-21). Cells were washed and stained with a phycoerythrinlabeled anti-mouse IgG antibody (Jackson ImmunoResearch). Flow cytometric analysis was performed using FACSCalibur (BD Biosciences) as previously described [43]. Data were analyzed by FlowJo software (Tomy Digital Biology, Tokyo, Japan).

Statistical Analysis
Data were analyzed by a one-way ANOVA and Tukey's post-hoc test for multiple comparisons.

Isopanduratin A Inhibited IκB Degradation Induced by TNF-α, but Not IL-1α
In NF-κB signaling pathways, TNF-α and IL-1α induce the formation of different membrane-proximal complexes, which converge to activate IκB kinases as the main target [2,3]. To clarify whether isopanduratin A inhibits the converged NF-κB signaling pathway, A549 cells were pretreated with isopanduratin A, followed by TNF-α or IL-1α. Consistent with our previous findings [34], isopanduratin A inhibited IκBα degradation induced by TNF-α ( Figure 1B). By contrast, IκBα degradation induced by IL-1α was not inhibited by isopanduratin A ( Figure 1B). These results indicated that isopanduratin A selectively inhibited the early NF-κB signaling pathway induced by TNF-α in A549 cells.

Isopanduratin A Decreased the Expression of TNF-R1
A549 cells have been shown to express TNF-R1, but not TNF-R2 [16]. We investigated the expression of TNF-R1 at the protein level by Western blotting. The treatment with isopanduratin A for 1 h markedly decreased bands reactive to the anti-TNF-R1 monoclonal antibody (clone H-5) in a dose-dependent manner (Figure 2A). Allantopyrone A is a natural metabolite produced by the endophytic fungus Allantophomopsis lycopodiana KS-97 and contains two α,β-unsaturated carbonyl moieties [44], which are reactive to cysteinyl thiol groups. We previously showed that allantopyrone A, which directly bound to TNF-R1, possibly via cysteine residues, reduced the TNF-R1 reactivity of H-5, which appeared to decrease TNF-R1 bands on Western blots [42]. To exclude this possibility, we transfected A549 cells with an expression vector encoding C-terminal FLAG-tagged TNF-R1. Isopanduratin A also decreased the expression of TNF-R1-FLAG in a dose-dependent manner ( Figure 2B). Collectively, these results indicated that isopanduratin A reduced the amount of TNF-R1 in A549 cells.
inyl thiol groups. We previously showed that allantopyrone A, which directly bound to TNF-R1, possibly via cysteine residues, reduced the TNF-R1 reactivity of H-5, which appeared to decrease TNF-R1 bands on Western blots [42]. To exclude this possibility, we transfected A549 cells with an expression vector encoding C-terminal FLAG-tagged TNF-R1. Isopanduratin A also decreased the expression of TNF-R1-FLAG in a dose-dependent manner ( Figure 2B). Collectively, these results indicated that isopanduratin A reduced the amount of TNF-R1 in A549 cells.  [42]. TNF-R1-FLAG (%) is shown as the mean ± S.E. of three independent experiments. *** p < 0.001.

Isopanduratin A Induced the Ectodomain Shedding of TNF-R1
TACE has the ability to cleave membrane-bound TNF-R1, and its soluble form is released into culture medium [8,9]. The time-course experiment showed that soluble TNF-R1 was steadily augmented 40-60 min after the exposure to isopanduratin A ( Figure 3A). The amount of membrane-bound TNF-R1 in the cell lysate was conversely decreased by isopanduratin A ( Figure 3A). TAPI-2, N-(R)-[2-(hydroxyaminocarbonyl)methyl]-4methypentanoyl-L-t-butyl-alanyl-L-analnine, 2-aminoethyl amide, is a small molecule inhibitor of TACE. TAPI-2 has been shown to block the TACE-dependent cleavage of TNF-R1 in A549 cells [16,[18][19][20]. TAPI-2 decreased the augmentation of soluble TNF-R1 in isopanduratin A-treated A549 cells ( Figure 3B). In contrast, TAPI-2 did not markedly restore the decreased amount of membrane-bound TNF-R1 in the cell lysate of isopanduratin A-treated cells ( Figure 3B). These results indicated that isopanduratin A induced the TACE-dependent cleavage of membrane-bound TNF-R1 into its soluble form. An additional TACE-independent mechanism was also suggested to be responsible for the reduction induced in membrane-bound TNF-R1 in the cell lysate by isopanduratin A.
BioChem 2021, 1, FOR PEER REVIEW 6 dependent cleavage of membrane-bound TNF-R1 into its soluble form. An additional TACE-independent mechanism was also suggested to be responsible for the reduction induced in membrane-bound TNF-R1 in the cell lysate by isopanduratin A. . Cell lysates and culture medium were analyzed by Western blotting. The amount of TNF-R1 was normalized to that of γ1-actin. The amounts of soluble TNF-R1 and membrane TNF-R1 in control A549 cells were set to 1-fold and 100%, respectively. Soluble TNF-R1 (fold) in medium and membrane TNF-R1 (%) in cell lysates are shown as the mean ± S.E. of three independent experiments. * p < 0.05, ** p < 0.01, and *** p < 0.001.
We then evaluated the cell surface expression of TNF-R1 by flow cytometry. In comparison with the control (Figure 4(Aa)), isopanduratin A markedly decreased the expression of cell surface TNF-R1 (Figure 4(Ab)). TAPI-2 alone up-regulated the expression of cell surface TNF-R1 (Figure 4(Ac)), suggesting that TNF-R1 was constitutively processed by TACE. TAPI-2 reversed the expression of cell surface TNF-R1 in isopanduratin Atreated A549 cells (Figure 4(Ad)). These results were confirmed by the quantitation of cell surface TNF-R1 ( Figure 4B). These results clearly indicated that isopanduratin A induced the ectodomain shedding of TNF-R1. . Cell lysates and culture medium were analyzed by Western blotting. The amount of TNF-R1 was normalized to that of γ1-actin. The amounts of soluble TNF-R1 and membrane TNF-R1 in control A549 cells were set to 1-fold and 100%, respectively. Soluble TNF-R1 (fold) in medium and membrane TNF-R1 (%) in cell lysates are shown as the mean ± S.E. of three independent experiments. * p < 0.05, ** p < 0.01, and *** p < 0.001.
We then evaluated the cell surface expression of TNF-R1 by flow cytometry. In comparison with the control (Figure 4Aa

Isopanduratin A Inhibited the TNF-α-Induced NF-κB Signaling Pathway by Inducing the Ectodomain Shedding of TNF-R1
Consistent with our previous findings [34], isopanduratin A inhibited the nuclear translocation of RelA in A549 cells stimulated with TNF-α ( Figure 5A, Lanes 3 and 4). The pretreatment with TAPI-2 markedly suppressed the isopanduratin A-induced inhibition of the nuclear translocation of RelA ( Figure 5A, Lanes 4 and 6). TAPI-2 also restored TNFα-induced IκBα degradation in isopanduratin A-treated A549 cells ( Figure 5B, Lanes 4 and 6). These results indicated that isopanduratin A inhibited the TNF-α-induced the NF-κB signaling pathway by inducing the ectodomain shedding of TNF-R1.

Isopanduratin A Inhibited the TNF-α-Induced NF-κB Signaling Pathway by Inducing the Ectodomain Shedding of TNF-R1
Consistent with our previous findings [34], isopanduratin A inhibited the nuclear translocation of RelA in A549 cells stimulated with TNF-α ( Figure 5A, Lanes 3 and 4). The pretreatment with TAPI-2 markedly suppressed the isopanduratin A-induced inhibition of the nuclear translocation of RelA ( Figure 5A, Lanes 4 and 6). TAPI-2 also restored TNFα-induced IκBα degradation in isopanduratin A-treated A549 cells ( Figure 5B, Lanes 4 and 6). These results indicated that isopanduratin A inhibited the TNF-α-induced the NF-κB signaling pathway by inducing the ectodomain shedding of TNF-R1.

Isopanduratin A Induced the Ectodomain Shedding of TNF-R1 by ERK Activation
The catalytic activity of TACE was previously shown to be activated by the phosphorylation of the Thr 735 residue in its cytoplasmic tail by ERK and p38 MAP kinase [10][11][12][13].
To elucidate the involvement of ERK and/or p38 MAP kinase, A549 cells were pretreated with these kinase inhibitors, followed by isopanduratin A. The MAP kinase/ERK kinase (MEK) inhibitor U0126 diminished the amount of soluble TNF-R1 in the culture medium of isopanduratin A-treated A549 cells ( Figure 6, Lane 3). In contrast, the p38 MAP kinase inhibitor SB203580 and the JNK inhibitor SP600125 did not markedly affect the augmentation of soluble TNF-R1 in isopanduratin A-treated A549 cells (Figure 6, Lanes 4 and 5). The decrease observed in membrane-bound TNF-R1 in the cell lysate by isopanduratin A was not markedly reversed by U0126 (Figure 6, Lane 3). These results clearly indicated that isopanduratin A induced the ERK-dependent ectodomain shedding of TNF-R1. BioChem 2021, 1, FOR PEER REVIEW 8

Isopanduratin A Induced the Ectodomain Shedding of TNF-R1 by ERK Activation
The catalytic activity of TACE was previously shown to be activated by the phosphorylation of the Thr 735 residue in its cytoplasmic tail by ERK and p38 MAP kinase [10][11][12][13]. To elucidate the involvement of ERK and/or p38 MAP kinase, A549 cells were pretreated with these kinase inhibitors, followed by isopanduratin A. The MAP kinase/ERK kinase (MEK) inhibitor U0126 diminished the amount of soluble TNF-R1 in the culture medium of isopanduratin A-treated A549 cells ( Figure 6, Lane 3). In contrast, the p38 MAP kinase inhibitor SB203580 and the JNK inhibitor SP600125 did not markedly affect the augmentation of soluble TNF-R1 in isopanduratin A-treated A549 cells (Figure 6, Lanes 4 and 5). The decrease observed in membrane-bound TNF-R1 in the cell lysate by isopanduratin A was not markedly reversed by U0126 ( Figure 6, Lane 3). These results clearly indicated that isopanduratin A induced the ERK-dependent ectodomain shedding of TNF-R1.

Isopanduratin A Induced the Rapid Phosphorylation of ERK, but Not p38 MAP Kinase or JNK
We investigated whether isopanduratin A induced the activation of ERK, p38 MAP kinase, and JNK. Deoxynivalenol, a translation inhibitor, was used as a positive control because it was previously shown to promote the phosphorylation of ERK, p38 MAP kinase, and JNK in A549 cells [19,21]. Consistent with these findings, phosphorylated bands of ERK, p38 MAP kinase, and JNK were clearly detectable in deoxynivalenol-treated A549 cells ( Figure 7B). In contrast, the phosphorylation of ERK was augmented by isopanduratin A within 20 min and maintained for up to 120 min, while it induced the phosphorylation of p38 MAP kinase and JNK and at later time points ( Figure 7A). These results indicate that isopanduratin A induced the rapid phosphorylation of ERK.

Isopanduratin A Induced the Rapid Phosphorylation of ERK, but Not p38 MAP Kinase or JNK
We investigated whether isopanduratin A induced the activation of ERK, p38 MAP kinase, and JNK. Deoxynivalenol, a translation inhibitor, was used as a positive control because it was previously shown to promote the phosphorylation of ERK, p38 MAP kinase, and JNK in A549 cells [19,21]. Consistent with these findings, phosphorylated bands of ERK, p38 MAP kinase, and JNK were clearly detectable in deoxynivalenol-treated A549 cells ( Figure 7B). In contrast, the phosphorylation of ERK was augmented by isopanduratin A within 20 min and maintained for up to 120 min, while it induced the phosphorylation of p38 MAP kinase and JNK and at later time points ( Figure 7A). These results indicate that isopanduratin A induced the rapid phosphorylation of ERK. The amounts of phospho-ERK, total ERK, phospho-p38 MAP kinase, total p38 MAP kinase, phospho-JNK, and total JNK were normalized to that of β-actin. The amounts of phospho-ERK, phospho-p38 MAP kinase, and phospho-JNK in control A549 cells are set to 1-fold. The amounts of total ERK, total p38 MAP kinase, and total JNK in control A549 cells are set to 100%. Phospho-ERK (fold), phospho-p38 MAP kinase (fold) phospho-JNK (fold), total ERK (%), total p38 MAP kinase (%), and total JNK (%) are shown as the mean ± S.E. of three independent experiments. * p < 0.05, ** p < 0.01, and *** p < 0.001. The amounts of phospho-ERK, total ERK, phospho-p38 MAP kinase, total p38 MAP kinase, phospho-JNK, and total JNK were normalized to that of β-actin. The amounts of phospho-ERK, phospho-p38 MAP kinase, and phospho-JNK in control A549 cells are set to 1-fold. The amounts of total ERK, total p38 MAP kinase, and total JNK in control A549 cells are set to 100%. Phospho-ERK (fold), phospho-p38 MAP kinase (fold) phospho-JNK (fold), total ERK (%), total p38 MAP kinase (%), and total JNK (%) are shown as the mean ± S.E. of three independent experiments. * p < 0.05, ** p < 0.01, and *** p < 0.001.
We also investigated the selectivity of protein kinase inhibitors on ERK phosphorylation. The isopanduratin A-induced phosphorylation of ERK in A549 cells was markedly reduced by U0126 or U0126 plus SB203580 (Figure 8, Lanes 3 and 6), while it was barely affected by SB203580 or SP600125 (Figure 8, Lanes 4 and 5). These results confirmed that U0126 inhibited the isopanduratin A-induced activation of ERK.
BioChem 2021, 1, FOR PEER REVIEW 11 We also investigated the selectivity of protein kinase inhibitors on ERK phosphorylation. The isopanduratin A-induced phosphorylation of ERK in A549 cells was markedly reduced by U0126 or U0126 plus SB203580 (Figure 8, Lanes 3 and 6), while it was barely affected by SB203580 or SP600125 (Figure 8, Lanes 4 and 5). These results confirmed that U0126 inhibited the isopanduratin A-induced activation of ERK. Figure 8. U0126 inhibited ERK phosphorylation induced by isopanduratin A. A549 cells were treated with or without U0126, SB203580, SP600125, or U0126 plus SB203580 for 1 h, and incubated with (+) or without (−) isopanduratin A (50 µM) for 1 h in the presence or absence of U0126, SB203580, SP600125, or U0126 plus SB203580 (10 µM each). Cell lysates were analyzed by Western blotting. The amounts of phospho-ERK and total ERK were normalized to that of β-actin. The amounts of phospho-ERK in isopanduratin A-treated A549 cells and total ERK in control A549 cells were set to 100%. Phospho-ERK (%) and total ERK (%) are shown as the mean ± S.E. of three independent experiments. * p < 0.05, ** p < 0.01, and *** p < 0.001.

Isopanduratin A Promoted eIF2α Phosphorylation
The inhibition of the ectodomain shedding of TNF-R1 by TAPI-2 or U0126 efficiently rescued the TNF-α-induced NF-κB signaling pathway, while it did not reverse the decrease in TNF-R1 in isopanduratin A-treated A549 cells (Figures 3B and 6). These data suggested that TNF-R1 is down-regulated in a manner independent of ectodomain shedding in isopanduratin A-treated A549 cells. eIF2 plays an essential role in the transport and entry of Met-tRNAi Met to the ribosome small subunit during the initiation of translation [45,46]. The initiation step of translation is inhibited by Ser51 phosphorylation of the α subunit of eIF2 [45,46]. We found that isopanduratin A augmented the phosphorylation of eIF2α in a dose-dependent manner (Figure 9). In contrast, isopanduratin A did not affect the cellular level of eIF2α under the same conditions (Figure 9). These results indicated that isopanduratin A promoted eIF2α phosphorylation. Figure 8. U0126 inhibited ERK phosphorylation induced by isopanduratin A. A549 cells were treated with or without U0126, SB203580, SP600125, or U0126 plus SB203580 for 1 h, and incubated with (+) or without (−) isopanduratin A (50 µM) for 1 h in the presence or absence of U0126, SB203580, SP600125, or U0126 plus SB203580 (10 µM each). Cell lysates were analyzed by Western blotting. The amounts of phospho-ERK and total ERK were normalized to that of β-actin. The amounts of phospho-ERK in isopanduratin A-treated A549 cells and total ERK in control A549 cells were set to 100%. Phospho-ERK (%) and total ERK (%) are shown as the mean ± S.E. of three independent experiments. * p < 0.05, ** p < 0.01, and *** p < 0.001.

Isopanduratin A Promoted eIF2α Phosphorylation
The inhibition of the ectodomain shedding of TNF-R1 by TAPI-2 or U0126 efficiently rescued the TNF-α-induced NF-κB signaling pathway, while it did not reverse the decrease in TNF-R1 in isopanduratin A-treated A549 cells (Figures 3B and 6). These data suggested that TNF-R1 is down-regulated in a manner independent of ectodomain shedding in isopanduratin A-treated A549 cells. eIF2 plays an essential role in the transport and entry of Met-tRNA i Met to the ribosome small subunit during the initiation of translation [45,46]. The initiation step of translation is inhibited by Ser51 phosphorylation of the α subunit of eIF2 [45,46]. We found that isopanduratin A augmented the phosphorylation of eIF2α in a dose-dependent manner (Figure 9). In contrast, isopanduratin A did not affect the cellular level of eIF2α under the same conditions (Figure 9). These results indicated that isopanduratin A promoted eIF2α phosphorylation. Figure 9. Isopanduratin A promoted eIF2α phosphorylation. A549 cells were treated with the indicated concentrations of isopanduratin A for 1 h. Cell lysates were analyzed by Western blotting. The amounts of phospho-elF2α and total elF2α were normalized to that of γ1-actin. The amounts of phospho-eIF2α and total eIF2α are set to 1-fold and 100%, respectively. Phospho-elF2α (fold) and total eIF2α (%) are shown as the mean ± S.E. of three independent experiments. * p < 0.05, ** p < 0.01, and *** p < 0.001.

Discussion
Panduratin derivatives have been shown to exhibit anti-cancer activity through the induction of apoptosis and cell cycle arrest [36,47,48]. Panduratin derivatives have also been reported to exert anti-inflammatory effects [35][36][37][38]. Consistent with these findings, we previously showed that isopanduratin A inhibited TNF-α-induced IκBα phosphorylation and subsequent degradation at the early NF-κB signaling pathway in A549 cells [34]. The present results demonstrated that isopanduratin A selectively induced the ectodomain shedding of TNF-R1 via the ERK-dependent activation of TACE, thereby decreasing the cell surface expression of TNF-R1. Moreover, we showed that isopanduratin A did not affect IL-1α-induced IκBα degradation in A549 cells. These results clearly indicated that the inhibition of the TNF-α-induced NF-κB signaling pathway by isopanduratin A was due to a decrease in cell surface TNF-R1 expression and subsequent TNF-α unresponsiveness. Isopanduratin A also reduced the cellular amount of TNF-R1 by preventing translation via the phosphorylation of eIF2α. A schematic model for the biological activity of isopanduratin A on the ectodomain shedding of TNF-R1 and TNF-α-induced NF-κB activation is shown in Figure 10. The amounts of phospho-elF2α and total elF2α were normalized to that of γ1-actin. The amounts of phospho-eIF2α and total eIF2α are set to 1-fold and 100%, respectively. Phospho-elF2α (fold) and total eIF2α (%) are shown as the mean ± S.E. of three independent experiments. * p < 0.05, ** p < 0.01, and *** p < 0.001.

Discussion
Panduratin derivatives have been shown to exhibit anti-cancer activity through the induction of apoptosis and cell cycle arrest [36,47,48]. Panduratin derivatives have also been reported to exert anti-inflammatory effects [35][36][37][38]. Consistent with these findings, we previously showed that isopanduratin A inhibited TNF-α-induced IκBα phosphorylation and subsequent degradation at the early NF-κB signaling pathway in A549 cells [34]. The present results demonstrated that isopanduratin A selectively induced the ectodomain shedding of TNF-R1 via the ERK-dependent activation of TACE, thereby decreasing the cell surface expression of TNF-R1. Moreover, we showed that isopanduratin A did not affect IL-1α-induced IκBα degradation in A549 cells. These results clearly indicated that the inhibition of the TNF-α-induced NF-κB signaling pathway by isopanduratin A was due to a decrease in cell surface TNF-R1 expression and subsequent TNF-α unresponsiveness. Isopanduratin A also reduced the cellular amount of TNF-R1 by preventing translation via the phosphorylation of eIF2α. A schematic model for the biological activity of isopanduratin A on the ectodomain shedding of TNF-R1 and TNF-α-induced NF-κB activation is shown in Figure 10.
Panduratin A has been reported to inhibit the TNF-α-induced nuclear translocation of NF-κB subunits (i.e., RelA and p50) in A549 cells [36,37]. Panduratin A and isopanduratin A were also reported to inhibit TNF-α-induced cytotoxicity in L929 cells [29]. L929 cells undergo necroptosis in response to TNF-α [49]. In addition, panduratin A suppressed TNF-α-dependent muscle atrophy in rat skeletal muscle L6 cells [50]. Collectively, these findings indicated that panduratin A and isopanduratin A inhibited TNF-α-dependent cellular processes in different cell lines. This is consistent with our present result showing that isopanduratin A reduced TNF-α responsiveness by promoting the ectodomain shedding of TNF-R1 via TACE activity in A549 cells. TNF-R1 is mostly expressed in normal and transformed cells [51]. TACE is broadly expressed in various somatic tissues [8,52]. Therefore, it is conceivable that the cell surface expression of TNF-R1 is broadly regulated via TACE-dependent ectodomain shedding in various cell types.
BioChem 2021, 1, FOR PEER REVIEW Figure 10. A schematic model for the biological activity of isopanduratin A. TAC role in the ectodomain shedding of TNF-R1, and cleaves cell surface TNF-R1 into TACE is activated by the phosphorylation of its C-terminal cytoplasmic tail. Iso duces the phosphorylation of ERK and eIF2α. Phosphorylated ERK mediates the and activation of TACE, which down-regulates cell surface TNF-R1 expression. phorylated eIF2α blocks translation, thereby down-regulating cellular TNF-R1 exp κB heterodimer composed of RelA and p50 is associated with IκBα in the cytoso with TNF-α, IκBα is phosphorylated and undergoes ubiquitination and proteaso leading to the liberation and subsequent nuclear translocation of RelA and p50. inhibits TNF-α-induced NF-κB activation by down-regulating cell surface TNF-R1 Panduratin A has been reported to inhibit the TNF-α-induced nucle of NF-κB subunits (i.e., RelA and p50) in A549 cells [36,37]. Panduratin A ratin A were also reported to inhibit TNF-α-induced cytotoxicity in L929 cells undergo necroptosis in response to TNF-α [49]. In addition, pan pressed TNF-α-dependent muscle atrophy in rat skeletal muscle L6 cells [5 these findings indicated that panduratin A and isopanduratin A inhibited ent cellular processes in different cell lines. This is consistent with our pres ing that isopanduratin A reduced TNF-α responsiveness by promoting shedding of TNF-R1 via TACE activity in A549 cells. TNF-R1 is mostly ex mal and transformed cells [51]. TACE is broadly expressed in various [8,52]. Therefore, it is conceivable that the cell surface expression of TN regulated via TACE-dependent ectodomain shedding in various cell type We showed that isopanduratin A promoted the ectodomain sheddin TNF-R1. Nevertheless, isopanduratin A still decreased the cellular amo even when A549 cells were treated together with TAPI-2 to block the cleav quent release of membrane-bound TNF-R1. These results suggested that i reduced the de novo protein synthesis of TNF-R1. The phosphorylation o is well known to prevent a step in the initiation of translation and there protein synthesis [45,46]. eIF2α was phosphorylated in response to v stresses [53,54]. We found that isopanduratin A up-regulated the level of Figure 10. A schematic model for the biological activity of isopanduratin A. TACE plays a critical role in the ectodomain shedding of TNF-R1, and cleaves cell surface TNF-R1 into its soluble form. TACE is activated by the phosphorylation of its C-terminal cytoplasmic tail. Isopanduratin A induces the phosphorylation of ERK and eIF2α. Phosphorylated ERK mediates the phosphorylation and activation of TACE, which down-regulates cell surface TNF-R1 expression. In contrast, phosphorylated eIF2α blocks translation, thereby down-regulating cellular TNF-R1 expression. The NF-κB heterodimer composed of RelA and p50 is associated with IκBα in the cytosol. Upon engaging with TNF-α, IκBα is phosphorylated and undergoes ubiquitination and proteasomal degradation, leading to the liberation and subsequent nuclear translocation of RelA and p50. Isopanduratin A inhibits TNF-α-induced NF-κB activation by down-regulating cell surface TNF-R1 expression.
We showed that isopanduratin A promoted the ectodomain shedding of cell surface TNF-R1. Nevertheless, isopanduratin A still decreased the cellular amount of TNF-R1, even when A549 cells were treated together with TAPI-2 to block the cleavage and subsequent release of membrane-bound TNF-R1. These results suggested that isopanduratin A reduced the de novo protein synthesis of TNF-R1. The phosphorylation of eIF2α at Ser51 is well known to prevent a step in the initiation of translation and thereby block global protein synthesis [45,46]. eIF2α was phosphorylated in response to various cellular stresses [53,54]. We found that isopanduratin A up-regulated the level of phospho-eIF2α (Ser 51) within 1 h. Consistent with this, panduratin A has also been reported to up-regulate phospho-eIF2α (Ser51) in human malignant melanoma A375 cells for 24 h [55]. Therefore, panduratin derivatives appear to up-regulate eIF2α phosphorylation at least in different cell lines; however, their kinetics may differ between their structures and the cell types used.
Several translation inhibitors have the ability to induce the activation of MAP kinase superfamily members via a ribotoxic stress response mediated by ribosomes [14,15,56,57]. Among MAP kinase superfamily members, ERK and p38 MAP kinase phosphorylate the Thr 735 residue of TACE and activate its catalytic activity [10][11][12][13]. We previously showed that the ectodomain shedding of TNF-R1 was induced by acetoxycycloheximide, cytotrienin A, deoxynivalenol, and irciniastatin A [16][17][18][19][20][21]. We previously reported that the ectodomain shedding of TNF-R1 induced by three translation inhibitors (i.e., acetoxycycloheximide, cytotrienin A, or deoxynivalenol) was decreased to some extent by a single treatment with U0126 or SB203580, but was completely inhibited by a combined treatment of U0126 and SB203580 in A549 cells [17][18][19]. These studies reveal that ERK and p38 MAP kinase both play essential roles in the TACE-dependent TNF-R1 ectodomain shedding induced by those translation inhibitors in A549 cells. In contrast, the ectodomain shedding of TNF-R1 induced by irciniastatin A was inhibited by U0126, but not by SB203580, largely due to the fact that irciniastatin A induced activation of ERK, but not p38 MAP kinase in A549 cells [21]. These findings indicate that irciniastatin A uniquely induces TNF-R1 ectodomain shedding in a manner that is solely dependent on ERK. In this study, we showed that isopanduratin A promoted the ectodomain shedding of TNF-R1, which was sensitive to U0126, but not SB203580. Isopanduratin A induced the rapid activation of ERK, but not p38 MAP kinase. Thus, our present study reveals that isopanduratin A promotes the ERK-dependent ectodomain shedding of TNF-R1 and thereby inhibits the TNF-α-induced signaling pathway in A549 cells.

Conclusions
In conclusion, we revealed that isopanduratin A promoted the ectodomain shedding of TNF-R1 via the ERK-dependent activation of TACE. Moreover, isopanduratin A rapidly up-regulated the phosphorylation of eIF2α (Ser51). To date, panduratin derivatives have been shown to exert various biological effects, such as anti-cancer and anti-inflammatory activities. The present results showing that isopanduratin A is linked to ERK and eIF2α kinase provide insights into the mechanisms of actions of panduratin derivatives on various cellular activities.