The Ubiquitin-like Protein MNSFβ Regulates ERK-MAPK Cascade*

MNSFβ is a ubiquitously expressed member of the ubiquitin-like family that has been implicated in various biological functions. Previous studies have demonstrated that MNSFβ covalently binds to intracellular proapoptotic protein Bcl-G in mitogen-activated murine T cells. In this study, we further investigated the intracellular mechanism of action of MNSFβ in macrophage cell line, Raw 264.7 cells. We present evidence that MNSFβ·Bcl-G complex associates with ERKs in non-stimulated Raw 264.7. We found that MNSFβ·Bcl-G directly bound to ERKs and inhibited ERK activation by MEK1. In Raw 264.7 cells treated with MNSFβ small interfering RNA (siRNA) lipopolysaccharide (LPS)-induced ERK1/2 activation was enhanced and LPS-induced JNK and p38 activation was unaffected. SiRNA-mediated knockdown of MNSFβ increased tumor necrosis factor α (TNFα) expression at mRNA and protein levels in LPS-stimulated Raw 264.7 cells. Finally, we found that transfection with MNSFβ expression construct resulted in a significant inhibition of LPS-induced ERK activation and TNFα production. Co-transfection experiments with MNSFβ and Bcl-G greatly enhanced this inhibition. Collectively, these findings indicate that MNSFβ might be implicated in the macrophage response to LPS.

The covalent attachment of ubiquitin to proteins is an important cellular function that is required for protein degradation, DNA repair, cell cycle control, stress response, transcriptional regulation, signal transduction, and vesicular traffic (1)(2)(3)(4)(5)(6). The attachment of a single ubiquitin polypeptide (monoubiquitination) is important for cellular regulation (7). Polyubiquitination targets proteins for destruction by the proteasome. Polyubiquitin chains are formed via isopeptide bond linkages between the C-terminal Gly-76 of ubiquitin and the side chain -NH 2 from Lys-48 of another. In addition to ubiquitin, it is evident that several ubiquitin-like proteins have been found to be covalently or noncovalently attached to target proteins (8 -12). Interestingly, small ubiquitin-like modifier 1 (SUMO-1) 2 conjugation of IB occurs on the same residues used for ubiquitination, thus making the protein resistant to proteasome-mediated degradation and consequently inhibiting NFB activation (13).
Monoclonal nonspecific suppressor factor (MNSF), a lymphokine produced by murine T cell hybridoma, possesses pleiotrophic anti-gen-nonspecific suppressive functions (14). We have cloned a cDNA encoding a subunit of MNSF, which was termed MNSF␤ (15). MNSF␤ cDNA encodes a protein of 133 amino acids consisting of a ubiquitin-like protein (36% identity with ubiquitin) fused to the ribosomal protein S30. The ubiquitin-like moiety of MNSF␤ shows MNSF-like biologic activity without cytotoxic action (16). Interferon ␥ (IFN␥) is involved in the mechanism of action of MNSF␤. We have demonstrated that Ubi-L specifically binds to cell surface receptors on mitogen-activated lymphocytes and the T helper type 2 clone, the D.10 cells (17).
We have also shown that MNSF␤ covalently conjugates to acceptor proteins and forms MNSF␤ adducts including 33.5-kDa protein in concanavalin A-and IFN␥-stimulated D.10 cells (18). Recently, we found that this MNSF␤ adduct consists of 8.5-kDa ubiquitin-like protein and Bcl-2-like protein (19), murine orthologue of previously cloned human BCL-G gene product with proapoptotic function (20). The BCL-G gene is a proapoptotic p53 target gene (21). Murine Bcl-G mRNA was highly expressed in testis and significantly in spleen (19).
In this study, we investigated the intracellular mechanism of action of MNSF␤ in murine macrophage cell line, Raw 264.7 cells. We observed that MNSF␤ siRNA increased ERK activation and TNF␣ production by LPS-stimulated Raw 264.7 cells. We will show that the MNSF␤ is implicated in the regulation of the ERK-MAPK cascade.
Immunoprecipitation-Immunoprecipitation was performed with a horseradish peroxidase-conjugated antibody that recognizes native rabbit IgG (TrueBlot TM , eBioscience, San Diego, CA) according to the manufacturer's instructions. RIPA buffer (50 mM Tris, 1% Nonidet P-40, 0.25% deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, pH 7.4, containing 1 g/ml each of the protease inhibitors aprotinin, leupeptin, and pepstatin) extracts of Raw cells were precleared with 50 l of anti-rabbit IgG beads for 1 h on ice. Subsequently, 5 g of primary antibody to MNSF␤ or ERK1 was added to precleared lysates and incubated on ice for additional 1 h. Samples were then incubated overnight at 4°C with 50 l of anti-rabbit IgG bead. The beads were washed with five times with RIPA buffer, and immunoprecipitates were released from the beads by 10 min boiling in NuPAGE LDS sample buffer (Invitrogen) buffer. Immunoblotting was carried out with anti-ERK1 or anti-Bcl-G antibody. A rabbit IgG TrueBlot was employed as a second antibody.
siRNA directed against 5Ј-GGACTCGACGCAATGGCGTCA-3Ј was the negative control. Cells were treated with siRNA according to the instructions provided with the RNAiFect TM transfection reagent (Qiagen, Inc.). Raw 264.7 cells (1.2 ϫ 10 5 ) were treated with 3 g of siRNA in RPMI 1640 medium supplemented with 10% FBS in the presence of the RNAiFect TM transfection reagent. After a 48-h incubation at 37°C, the medium containing the mixture of RNAiFectTM and siRNA was replaced by Dulbecco's modified Eagle's medium that contained 10% FBS and cells were incubated for a further 24 h.
Reverse Transcription (RT)-PCR-RT-PCR was performed for 30 cycles as described previously (19). The PCR primers used to detect mRNA are as follows: MNSF␤, CGCCCAGGAACTACACACC (sense) and GCCTGC-TACTTCCAGAGTGG (antisense) (222 bp); Bcl-G, CCCAAGCTCTC-CAGAACAAG (sense) and CTGAGCTCGGATCTCCTTTG (antisense) (213 bp). All short amplified PCR products were isolated and sequenced to verify their identity. PCR products were separated in 2% agarose gel electrophoresis and stained with ethidium bromide. In some experiments, signals were quantitated by densitometry and optical densities for MNSF␤ and Bcl-G were normalized to the corresponding values for glyceraldehyde-3-phosphate dehydrogenase.
Mutagenesis and Transfection-Mutant MNSF␤ (G76A) was generated by replacing the codon for glycine 76 with the codon for alanine by utilizing QuikChange site-directed mutagenesis (Stratagene). cDNAs encoding MNSF␤ and Bcl-G were subcloned into the vector pcDNA3.1(ϩ) (Invitrogen Corp.). Transient DNA transfections were conducted using Lipofectamine Plus reagent (Invitrogen) with the protocol provided by the manufacturer and 8 g of plasmid DNA per 6-well plate.
Quantification of Cytokines-Murine cytokines were measured using sandwich ELISA (R&D System, Minneapolis, MN). The lower limits of detection for the cytokines were TNF␣, 10 pg/ml; RANTES (regulated on activation normal T cell expressed and secreted), 20 pg/ml.
Electrophoretic Mobility Shift Assay (EMSA)-Nuclear extracts were prepared following the methods of Dignam et al. (22). Protein-DNA complexes were detected using biotin end-labeled double-stranded DNA probes. The sequence for the NFB site was: GGGGACTTTCCC. Oligonucleotides were labeled in a reaction using terminal deoxynucleotide transferase and biotin-14-dCTP (Pierce). The binding reaction was performed using the LightShift kit according to the manufacture's instructions (Pierce). The reaction products were separated on a 5% polyacrylamide gel in 0.5% Tris borate-EDTA, transferred onto a nylon membrane, and fixed on the membrane by UV cross-linking. The biotin-labeled probe was detected using chemiluminescence (LightShift kit; Pierce).

MNSF␤ Complex Is Associated with
ERKs-It has been reported that ubiquitin-like protein, ISG15, covalently binds to ERK1 (8). Thus, first we addressed whether MNSF␤ could associate with the MAPK family including ERK, JNK, and p38 MAPK. Cell lysates were prepared from non-stimulated Raw 264.7 cells and immunoprecipitated with antibod-FIGURE 2. A, MNSF␤⅐Bcl-G directly associates with ERK and regulates its activity. GST pulldown assay: purified MNSF␤⅐Bcl-G was incubated with GST or GST-ERK2 bound to GSH-Sepharose as described under "Experimental Procedures." Bound proteins were separated by SDS-PAGE and detected by immunoblotting with anti-MNSF␤ antibody (top panel). Western blot analysis for GST was performed to confirm equal loading of proteins (bottom panel). The peptide competition assay was carried out by using peptide for the MEK dual phosphorylation site of ERK2. The data represent one of three independent experiments yielding similar results. B, peptide affinity chromatography. For affinity chromatography on peptide columns, synthetic peptides derived from ERK2 were coupled to N-hydroxysuccinimide-activated agarose columns. MNSF␤⅐Bcl-G was incubated with peptide columns, washed extensively, and eluted. The eluates were subjected to SDS-PAGE and detected by immunoblotting with anti-MNSF␤ antibody. The flow-through (FT) and elusion material (Elu) are indicated. Lane C, MNSF␤⅐Bcl-G as a control. C, MEK kinase assay. Activated GST-MEK1 was incubated with unactivated GST-ERK2 in a kinase assay buffer. To evaluate the effect of MNSF␤⅐Bcl-G complex on ERK activation, unphosphorylated GST-ERK2 was preincubated with the MNSF␤ adduct (0.5 and 2 g) for 1 h at 4°C prior to addition of activated GST-MEK1. The reaction mixture was immunoblotted with using anti-phospho-ERK1/2 antibody. The data represent one of three independent experiments that gave similar results.

TABLE 1 Assignments of peptide fragments from a Staphylococcus V8 protease digest of the 33.5-kDa MNSF␤ adduct
The 33.5-kDa MNSF␤ adduct was digested by V8 protease and subjected to MALDI-MS analysis. The data in the second column are the mass values obtained experimentally, whereas the results in the third column are those calculated from the V8 protease fragmentation of the gene products of Bcl-G and MNSF␤. The fourth column indicates the number of the first and last amino acid of the identified Bcl-G and MNSF␤ peptides, whereas the fifth shows the corresponding amino acid sequences. The amino acid sequences derived from MNSF␤ are shown in italic.

Protein
Mass ies directed against MNSF␤, and associated proteins were analyzed by Western blot analysis by using anti-ERK1, anti-JNK, and anti-p38 antibodies. As shown in Fig. 1A, MNSF␤ associated with ERKs, but not with other members of the MAPK family, under non-stimulated conditions. It should be noted that anti-ERK1 polyclonal antibodies recognize both the 44-kDa ERK1 and the 42-kDa ERK2. Immunoprecipitation of cell lysates with normal IgG followed by Western blot analysis revealed no detectable association of ERKs, indicating the specificity of MNSF␤ with ERKs. In addition, converse immunoprecipitation with anti-ERK1 antibody and immunoblot analysis with anti-MNSF␤ antibody confirmed the association between ERKs and the MNSF␤ adduct (Fig. 1B).
It should be pointed out that anti-MNSF␤ antibody does not recognize free 8.5-kDa MNSF␤ in murine T helper type 2 clone, D.10 cells (18). In Raw macrophages as well as D.10 cells, anti-MNSF␤ antibody recognized several bands including a band of 33.5-kDa protein (Fig. 1A). We have demonstrated that Bcl-G, a novel proapoptotic member of the Bcl-2 family, is post-translationally modified by MNSF␤ (19). Thus, it seemed likely that the 33.5-kDa MNSF␤ adduct was the MNSF␤⅐Bcl-G complex. To determine this, we carried out Western blot analysis with anti-Bcl-G antibody. As depicted in Fig. 1B, antibody directed against Bcl-G reacted with the 33.5-kDa MNSF␤ adduct, indicative of the covalent interaction of MNSF␤ and Bcl-G as described (19). To confirm the interaction between MNSF␤ and Bcl-G in Raw cells, MALDI-TOF was performed. The 33.5-kDa MNSF␤ adduct was purified to homogeneity from Raw cell lysates by a combination of ion exchange chromatography, anti-MNSF␤ affinity chromatography, and hydroxylapatite chromatography as described (19). The purified MNSF␤ adduct was digested by V8 protease and subjected to MALDI-MS analysis. Table 1 shows the peptide masses of observed by MALDI-TOF mass fingerprinting of 33.5-kDa MNSF␤ adduct purified from Raw cells. The resulting sets of peptide masses were then used to search the NCBI data base for potential matches, confirming the MNSF␤ adduct as MNSF␤⅐Bcl-G complex. MNSF␤ may conjugate to Bcl-G with a linkage between the C-terminal Gly-74 and Lys-110, as described (19). These results indicate that covalent MNSF␤⅐Bcl-G complex can specifically associate with ERKs in unstimulated Raw macrophages.
MNSF␤⅐Bcl-G Directly Binds to ERK2-We next investigated the nature of the binding of MNSF␤⅐Bcl-G to ERKs. Purified MNSF␤⅐Bcl-G complex was incubated with GST or GST-ERK2 bound to GSH-Sepharose. The bead matrices were extensively washed before eluting bound proteins off of the bead matrices. MNSF␤⅐Bcl-G complex was separated by SDS-PAGE and detected by immunoblotting with anti-MNSF␤ antibody. As shown in Fig. 2A, MNSF␤⅐Bcl-G bound to GST-ERK2 but not to GST. Excess peptide for the MEK dual phosphorylation site of ERK2 inhibited 50 -60% this association, compared with a control peptide (mapping at the C terminus of ERK2), indicating that MNSF␤⅐Bcl-G might bind to near the phosphorylation site. To confirm these results, we carried out peptide affinity chromatography. MNSF␤⅐Bcl-G was incubated with peptides derived from ERK2 immobilized on agarose columns. Bound and eluted MNSF␤⅐Bcl-G was resolved by SDS-PAGE and immunoblotted with anti-MNSF␤ antibody. As can be seen in Fig.  2B, MNSF␤⅐Bcl-G bound to a column of immobilized the competitive peptide but not of control peptide, albeit this binding was not complete.
MNSF␤⅐Bcl-G Inhibits ERK Activation-To investigate whether ERK function is directly modified by MNSF␤⅐Bcl-G, we carried out MEK kinase assay. GST-MEK1 activated with c-Raf was employed in this assay. Activated GST-MEK1 was incubated with unphosphorylated GST-ERK2 in the presence or absence of MNSF␤⅐Bcl-G as described under "Experimental Procedures." The reaction mixture was immunoblotted with using anti-phospho-ERK1/2 antibody. As depicted in Fig.   2C, ERK activation by MEK1 was significantly inhibited in the presence of MNSF␤⅐Bcl-G. These observations were consistent with the results of GST pulldown experiments showing that MNSF␤⅐Bcl-G directly binds to near the phosphorylation site of ERKs ( Fig. 2A).
MNSF␤ siRNA Increases LPS-stimulated TNF␣ Production by Raw Cells-It has been reported that ERK pathway is involved in the regulation of TNF␣ production (23)(24)(25)(26). Because our data suggested that covalent MNSF␤⅐Bcl-G complex affects ERK activation, it seemed likely that inhibition of MNSF␤ expression would result in increased or decreased TNF␣ synthesis. Raw cells were transfected with scramble siRNA or siRNA directed against MNSF␤. After 72 h of siRNA transfection, Raw cells were stimulated with 100 ng/ml of LPS for 4 h. Then the concentration of TNF␣ in the supernatant was determined by ELISA. Production of the TNF␣ in LPS-stimulated Raw cells transfected with MNSF␤ siRNA-437 was significantly (over 2-fold) up-regulated, compared with the cells with scramble siRNA (Fig. 3A). RT-PCR analysis demonstrated that MNSF␤ siRNA-437, but not control scramble siRNA, specifically reduced the expression of MNSF␤ but not glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Fig. 3C). Western blot analysis demonstrated that MNSF␤ siRNA-437 reduced complex formation of MNSF␤ with Bcl-G (33.5-kDa MNSF␤ adduct) (Fig. 3D, lane  3). Like MNSF␤ siRNA, Bcl-G siRNA also inhibited the complex formation of MNSF␤ with Bcl-G (Fig. 3D, lane 4). We also determined whether Bcl-G siRNA would affect TNF␣ production by LPS-stimulated Raw cells. As can be seen in Fig. 3A, Bcl-G siRNA caused a significantly increased TNF␣ production, although the effect was less than that seen with MNSF␤ siRNA. We did not observe a synergistic effect of transfection with both siRNA. To explore the RNAi effect at the mRNA level, we performed RT-PCR on total RNA isolated from siRNA-transfected Raw cells. MNSF␤ siRNA-437 up-regulated 60% TNF␣ expression (data not shown). We also investigated whether MNSF␤ siRNA would affect RANTES, a member of C-C chemokine superfamily, production by LPS-stimulated Raw cells. MNSF␤ siRNA-437 caused a significantly increased RANTES production (Fig. 3B), indicative of the involvement of MNSF␤ in RANTES production by LPS-stimulated Raw cells.
MNSF␤⅐Bcl-G Association with ERK Is Decreased in a LPS-dependent Manner-To determine whether the interaction of MNSF␤⅐Bcl-G with ERK could be altered by treatment with LPS, Raw cells were stimulated with 100 ng/ml of LPS followed by immunoprecipitation with antibody against ERK1 or IgG and Western blot analysis to detect MNSF␤⅐Bcl-G complex. The binding of MNSF␤ adduct with ERKs was decreased at both 15 and 45 min post-LPS stimulation (Fig. 4, upper panel). This blot was subsequently re-probed to ensure equal loading of ERKs from each condition (Fig. 4, middle panel). Control immunoprecipitates with normal rabbit IgG did not demonstrate an association of MNSF␤ adduct with ERKs (Fig. 4, bottom panel). Taken together, these results, which were seen in three independent experiments, demonstrated that LPS treatment results in decreased interaction of MNSF␤⅐Bcl-G and ERKs.
MNSF␤ Inhibits LPS-induced ERK Activation-LPS is known to activate three major MAPKs, ERK1/2, p38, and JNK, which play an impor-tant role in LPS-induced cellular effects. We determined whether MNSF␤ is involved in these pathway by treating cells with an MNSF␤specific siRNA. In Raw 264.7 cells, LPS-stimulated ERK1/2 phosphorylation peaked at 20 min after LPS stimulation and was detected slowly over 1 h (Fig. 5A). Phosphorylation of p38 and JNK also peaked at 20 min and returned quickly to basal levels (Fig. 5, B and C). In Raw 264.7 cells treated with MNSF␤ siRNA-437 LPS-induced ERK1/2 phosphorylation was enhanced (Fig. 5A), and LPS-induced p38 and JNK phosphorylation were unaffected (Fig. 5, B and C). LPS-induced phosphorylation of all three MAPKs was unaffected in cells transfected with control siRNA. Taken together with the results of protein interaction experiments (Fig.  2), MNSF␤ may mediate negative regulation of the ERK-MAPK cascade.
MNSF␤ Inhibits LPS-induced TNF␣ Production-The role of MNSF␤ in regulating LPS-induced TNF␣ production was addressed in transfection studies. As can be seen in Fig. 6A, transfection with pcDNA3.1-MNSF␤ resulted in a significant inhibition of LPS-induced TNF␣ production. In contrast to MNSF␤, Bcl-G alone did not show any inhibitory effect. However, co-transfection with MNSF␤ and Bcl-G greatly decreased the TNF␣ production. Unlike co-transfection with wild-type MNSF␤, co-transfection of a mutant MNSF␤ (G74A) and Bcl-G did not result in a decrease in the TNF␣ production. These results suggest that Bcl-G may enhance and/or stabilize the inhibitory activity of MNSF␤ by complex formation. Further supporting this idea is the observation that overexpression of mutant MNSF␤ (G74A) fails to form a MNSF␤⅐Bcl-G complex (Fig. 6B, lane 3). We also determined whether ERK activation is affected by transfection with pcDNA3.1-MNSF␤. Raw cells co-transfected with MNSF␤ and Bcl-G significantly decreased the ERK1/2 phosphorylation at 20 -40 min (Fig. 6C). These results are in good accordance with findings by siRNA experiments (Fig. 5).
MNSF␤ Inhibits LPS-induced NFB Activation-NFB is involved in the LPS signaling cascade leading to TNF␣ production. Gel shift assay was performed to determine whether MNSF␤ is relevant to LPS-induced NFB activation. Raw cells were transfected with either siRNA directed against MNSF␤ or scramble siRNA. After 72 h of siRNA transfection, Raw cells were treated with 100 ng/ml LPS for 60 min. Then EMSA was performed as described under "Experimental Procedures." NFB activation in LPS-stimulated Raw cells transfected with MNSF␤ siRNA-437 was slightly enhanced, compared with the cells with scramble siRNA (Fig. 7). We also examined whether Bcl-G siRNA could affect LPS-induced NFB activation in Raw cells. Bcl-G siRNA treatment did not affect NFB activation induced by LPS (Fig. 7).
We have previously demonstrated that Bcl-G, a novel proapoptotic member of Bcl-2 family, is covalently modified by MNSF␤ in concanavalin A-stimulated D.10 G4.1 cells, a murine T helper clone type 2 (19). In this study, we showed that MNSF␤ covalently binds to Bcl-G in unstimulated Raw 264.7 macrophage cell line. Thus, the mechanism of modification of Bcl-G by MNSF␤ may differ in each cell type. We also demonstrated that MNSF␤ conjugates to Bcl-G with a linkage between the C-terminal Gly-74 and Lys-110 as previously described in D.10 cells (19). We presented data showing that this isopeptide bond is important for MNSF␤⅐Bcl-G interaction in Raw cells (Fig. 6).
We showed that MNSF␤⅐Bcl-G directly binds to ERK inhibiting ERK phosphorylation by MEK (Figs. 1 and 2). This association is decreased in a LPS-dependent manner (Fig. 4). Interestingly, blocking peptide for the phosphorylation site on ERK2 inhibited this association, suggesting that this phosphorylation site is critical for the association with the MNSF␤ adduct (Fig. 2). It cannot be ruled out that another molecule(s) might be implicated in the complex formation in vivo. This is an intriguing possibility that needs to be carefully addressed experimentally. Shin et al. (30) mentioned that the p21-activated kinase 2 (PAK2) directly binds to ERKs. The formation of a multimeric complex consisting of ERK/ PAK2/PAK-interacting exchange factor (PIX) is required for fibroblast growth factor-induced neurite outgrowth (30). In addition, complex formation among ERK, 14-3-3⑀, and heat shock factor 1 during stress is evident (31).
We presented the evidence that the MNSF␤ adduct directly regulates ERK activation (Fig. 2). It should be pointed out that MNSF␤ is an aggregable polypeptide (14,15). Even recombinant MNSF␤ has a tendency to form aggregation (15). Thus, it might be inferred that Bcl-G functions as a stabilizer of this aggregable polypeptide. Indeed, knockdown experiments using Bcl-G siRNA showed a significant increased TNF␣ production (Fig. 3). In addition, transfection experiments using a point mutation showed that covalent interaction between MNSF␤ and Bcl-G enhances the inhibitory activity (Fig. 6). Interestingly, ubiquitinrelated BAG-1 (Bcl-2-associated athanogene-1) interacts with antiapoptotic protein, Bcl-2, and enhances the anti-apoptotic activity of Bcl-2 (32). Thus, it is possible that MNSF␤ may regulate the pro-apoptotic protein, Bcl-G. We are currently investigating whether MNSF␤ is involved in the mechanism of apoptosis.
Like MNSF␤, other ubiquitin-like proteins are also implicated in MAPK pathway. The ubiquitin-related BAG-1 described above binds to and activates the kinase Raf-1 (33). Modification of Smad4 with the ubiquitin-like protein SUMO-1 is enhanced by TGF␤-induced activation of the p38 MAP kinase pathway (34). Recently, Malakhov et al. (8) demonstrated that ubiquitin-like protein ISG15 modifies three key regulators of signal transduction, phospholipase C␥1, Jak1, and ERK1. ISG15 conjugates to these target proteins via an isopeptide bond in a manner similar to ubiquitin and other ubiquitin-like proteins. In contrast to ISG15, MNSF␤ non-covalently binds to ERK. In addition, MNSF␤ failed to bind to Jak1 (data not shown). It might be inferred that ubiquitin-like proteins are differentially involved in the regulation of the ERK-MAPK cascade. Interestingly, MNSF␤, ISG15, and FAT10, a ubiquitin-like protein involved in apoptosis, are induced by IFN␥ (16,(35)(36)(37)(38).
It is well known that NFB and MAPK signaling proteins are activated by LPS. Many reports have demonstrated that inhibition of both NFB and ERK-MAPK signaling affects LPS-mediated TNF␣ production in Raw cells (23,25,26). In this study, we demonstrated that MNSF␤ affects ERK-MAPK cascade rather than NFB signaling (Figs.

5-7)
. However, we observed that MNSF␤ siRNA was effective in suppressing LPS-induced NFB activation, albeit its effect was weak. To clarify the role of MNSF␤ in LPS-induced NFB activation, careful experiments must be carried out.