Kelch-like Protein 21 (KLHL21) Targets IκB Kinase-β to Regulate Nuclear Factor κ-Light Chain Enhancer of Activated B Cells (NF-κB) Signaling Negatively*

Activation of IKKβ is the key step in canonical activation of NF-κB signaling. Extensive work has provided insight into the mechanisms underlying IKKβ activation through the identification of context-specific regulators. However, the molecular processes responsible for its negative regulation are not completely understood. Here, we identified KLHL21, a member of the Kelch-like gene family, as a novel negative regulator of IKKβ. The expression of KLHL21 was rapidly down-regulated in macrophages upon treatment with proinflammatory stimuli. Overexpression of KLHL21 inhibited the activation of IKKβ and degradation of IκBα, whereas KLHL21 depletion via siRNA showed the opposite results. Coimmunoprecipitation assays revealed that KLHL21 specifically bound to the kinase domain of IKKβ via its Kelch domains and that this interaction was gradually attenuated upon TNFα treatment. Furthermore, KLHL21 did not disrupt the interaction between IKKβ and TAK1, TRAF2, or IκBα. Also, KLHL21 did not require its E3 ubiquitin ligase activity for IKKβ inhibition. Our findings suggest that KLHL21 may exert its inhibitory function by binding to the kinase domain and sequestering the region from potential IKKβ inducers. Taken together, our data clearly demonstrate that KLHL21 negatively regulates TNFα-activated NF-κB signaling via targeting IKKβ, providing new insight into the mechanisms underlying NF-κB regulation in cells.

tory factors (e.g. TNF␣ and IL-1␤), trigger specific signaling pathways that lead to the activation of NF-B. Most of these diverse pathways converge on the activation of the IB kinase (IKK) complex, which phosphorylates specific serine residues on the classical NF-B inhibitors, IB␣, IB␤, and IB⑀. Phosphorylated IB proteins are targeted for ubiquitination and rapid degradation via the ubiquitin-proteasome pathway, resulting in the nuclear translocation and transcriptional activity of NF-B proteins (2,3).
KLHL21 is a member of the Kelch-like (KLHL) gene family (16). At present, 42 KLHL genes have been identified. They encode a group of highly conserved proteins that generally possess a BTB/POZ (Bric-a-brac-Tramtrack-broad complex/poxvirus and zinc finger) domain, a BACK domain, and 5-6 Kelch motifs (16). Members of this protein family have been reported to be involved in a number of cellular and molecular processes, such as inflammatory responses, oxidative stress responses, cytokinesis, embryonic development, and lymphogenesis (11,12,(17)(18)(19). For example, Keap1 (also known as KLHL19) was recently found to interact specifically with IKK␤. It promotes the degradation of IKK␤ via autophagic (11) or 26S proteasome (12) pathways and inhibits the phosphorylation of IKK␤ in response to extracellular stimuli (11,12).
A previous study demonstrated that KLHL21 is required for successful cytokinesis by regulating translocation of the chromosomal passenger complex from chromosomes to the spindle midzone during anaphase (20). Mechanistically, KLHL21 interacts with Cul3 to mediate the ubiquitination of aurora B kinase in vitro (20). However, the functions of KLHL21 in other cellular processes remain unclear. Here, we demonstrate that KLHL21 is a novel negative regulator of IKK␤. Exogenous expression of KLHL21 suppressed TNF␣-stimulated IKK␤ activation, resulting in reduced NF-B signaling, whereas depletion of KLHL21 led to enhanced IKK␤ activity. This inhibitory function of KLHL21 was independent of its E3 ubiquitin ligase activity. Thus, our results reveal a novel mechanism by which IKK␤ is regulated in cells.

Results
Cells Exposed to Proinflammatory Stimuli Show Down-regulated Klhl21 Expression-To identify novel genes regulated by LPS (specific agonist of TLR4 receptor) treatment, we performed a comprehensive search of the NCBI Gene Expression Omnibus (GEO) database. The expression of Klhl21 was consistently down-regulated in RAW264.7 monocytic macrophage cells upon LPS treatment (GEO accession number GDS1043) (21). Additional analyses revealed that Klhl21 expression is down-regulated in LPS-treated dendritic cells (GEO accession numbers GDS2216 and GDS883) (22,23) and peripheral bloodderived monocytes (GEO accession number GDS2856) (24), CpG oligonucleotide-treated bone marrow-derived dendritic cells (GEO accession number GDS1315) (25), and Kaposi sarcoma-associated herpesvirus-infected primary human dermal endothelial cells (GEO accession number GDS940) (26). Taken together, these findings suggest that down-regulation of Klhl21 may be a general consequence of the inflammatory response and that KLHL21 might contribute to the regulation of related cellular processes.
We confirmed that LPS treatment induced rapid down-regulation of Klhl21 in RAW264.7 cells (Fig. 1A). KLHL21 mRNA levels were lowest by 4 h after treatment, representing Ͻ10% of the levels observed in untreated cells, before gradually increasing and reaching their peak (about 70% of the levels of untreated cells) around 24 h. However, levels cannot be completely restored even extending LPS treatment to 48 h (Fig. 1A). We observed a similar Klhl21 expression trend in LPS-treated bone marrow-derived macrophages (Fig. 1B). KLHL21 protein abundance also appeared to be temporally regulated, decreasing dramatically in RAW264.7 cells at 1 h after LPS treatment, followed by a steady increase (Fig. 1C). Further analysis revealed that Klhl21 mRNA levels were down-regulated in RAW264.7 cells exposed to Pam2CSK4 (TLR2/6 agonist), Pam3CSK4 (TLR1/2 agonist), or ODN1826 (TLR9 agonist) (Fig. 1, D and  E). Treatment with TNF␣ also resulted in reduced expression of Klhl21 mRNA and protein (Fig. 1F). These results indicate that rapid down-regulation of Klhl21 may be a general phenomenon in response to extracellular proinflammatory stimuli in macrophages.
KLHL21 Inhibits Activation of the NF-B Signaling Pathway-To investigate the potential roles of KLHL21 in the inflammatory process, we first assessed its impact on NF-B signaling. KLHL21 was coexpressed with known inducers of NF-B signaling in HEK293T cells carrying an NF-B luciferase reporter. Coexpression of KLHL21 and TRAF2 (upstream reg-ulator of the TNFR1 signaling pathway), TRAF6 (upstream regulator of the TLRs and IL-1R signaling pathways), TBK1, or the IKK␤ (SS/EE) constitutively active mutant (Ser-177/181 to Glu mutations) resulted in significant inhibition of NF-B activation ( Fig. 2A). Conversely, KLHL21 had no effect on p65 (RelA)mediated NF-B activity ( Fig. 2A). Similar findings were obtained in HeLa cells (Fig. 2B). Coexpression of KLHL21 and the IKK␤ (SS/EE) mutant suppressed luciferase expression driven by the mouse NFKBIA (encoding IB␣, a NF-B transcriptional target) promoter (Fig. 2C). Moreover, overexpression of KLHL21 inhibited TNF␣-induced NF-B-luciferase activity in HEK293T cells and IL-1␤-induced NF-B-luciferase activity in HeLa cells, respectively (Fig. 2, D and E). These results strongly imply that KLHL21 might function downstream of IKK␤ and upstream of p65 to inhibit NF-B activation.
In resting cells, NF-B is retained in the cytoplasm when bound to inhibitory IB proteins. The ubiquitin-dependent degradation of IBs, especially IB␣, releases bound NF-B and leads to its nuclear translocation (2). Given our observation that KLHL21 inhibits the activation of transcription factor NF-B, we analyzed its effect on the nuclear translocation of p65/RelA in HEK293T cells upon TNF␣ treatment. Overexpression of KLHL21 suppressed p65/RelA nuclear translocation after 10 and 30 min of TNF␣ treatment, but nuclear p65/RelA reached levels similar to control cells by 60 min posttreatment (Fig. 3A). These results reveal that overexpressing KLHL21 inhibits the early phase of NF-B/p65 nuclear translocation after TNF␣ treatment, thereby attenuating NF-B activation.
We then examined the effects of KLHL21 on IB␣ degradation. In control cells, TNF␣ treatment induced rapid degradation of IB␣, followed by its reaccumulation (Fig. 3B, left). In contrast, overexpression of KLHL21 suppressed the degradation and reaccumulation of IB␣ (Fig. 3B, right). To corroborate these findings, we introduced chemosynthesized siRNA to knock down KLHL21 in HEK293T cells and confirmed reduced mRNA (Fig. 3C) and protein (Fig. 3D) levels. KLHL21 depletion did not affect IB␣ protein in resting cells, but it enhanced the degradation of TNF␣-induced IB␣ (Fig. 3D).
Prior phosphorylation of both Ser-32 and Ser-36 is required for the ubiquitin-dependent degradation of IB␣ protein (2). Therefore, we analyzed the consequences of KLHL21 overexpression on TNF␣-induced phosphorylation of IB␣. In response to TNF␣ treatment, the amount of phosphorylated IB␣ (Ser-32) increased substantially in control cells but was significantly suppressed in cells overexpressing KLHL21 (Fig.  3E). In contrast, knocking down KLHL21 with specific siRNAs enhanced the phosphorylation of IB␣ in HEK293T cells upon TNF␣ treatment (Fig. 3F). Our findings that KLHL21 suppressed TNF␣-induced phosphorylation and ubiquitin-dependent degradation of IB␣ as well as the IKK␤ (SS/EE) mutantinduced activation of NF-B further suggest that KLHL21 might act directly on the IKK complex and inhibit its activity.
KLHL21 Directly Interacts with IKK Complex-To identify potential interacting partners of KLHL21 in the NF-B signaling pathway, FLAG-tagged KLHL21 protein was transiently expressed in HEK293T cells. Putative interactions between KLHL21 and endogenous IKK␤, IB␣, and p65 were examined by coimmunoprecipitation assays. The interaction between KLHL21 and Cullin3 was included as a positive control (20). Only IKK␤ specifically interacted with KLHL21 ( Fig. 4A). An interaction between endogenous IKK␤ and KLHL21 was also observed in HEK293T and RAW264.7 cells (Fig. 4B), suggesting a physiologically relevant role for this association. KLHL21 and IKK␤ demonstrated a direct interaction by in vitro GST pulldown assays (Fig. 4C) and colocalized in HeLa cells (Fig. 4D). Furthermore, interaction between KLHL21 with IKK␣ and NEMO (other components of the IKK complex) was also detected in HEK293T cells (Fig. 4E), demonstrating that KLHL21 specifically interacts with the IKK complex in cells.
To define the nature of the KLHL21-IKK␤ interaction under inflammatory conditions, we performed coimmunoprecipitation assays in TNF␣-treated HEK293T cells and found that their interaction was gradually attenuated upon TNF␣ treatment, significantly reduced after 10 min (Fig. 4F). It led to the release of IKK␤, which can then be activated by its upstream inducers subsequently. It is consistent with the above findings that overexpression of KLHL21 inhibited the early phase of p65 nuclear translocation after TNF treatment. Taken together, these findings demonstrate that KLHL21 directly interacts with the IKK complex under both transient and endogenous circumstances and that TNF␣-mediated immune responses may perturb this interaction to activate NF-B signaling.
KLHL21 Binds to the KD of IKK␤ via Its Kelch Domains-IKK␤ contains an N-terminal KD, a ubiquitin-like domain, an elongated, ␣-helical scaffold/dimerization domain, and a C-terminal NEMO-binding domain (27). To identify the domain of IKK␤ through which the interaction with KLHL21 occurs, we generated five deletion mutants of mouse IKK␤ and examined their abilities to interact with wild-type KLHL21 (Fig. 5A). Similar to wild-type IKK␤, deletion mutants M3 (containing KD only), M4 (lacking the scaffold/dimerization domain and NEMO-binding domain), and M5 (lacking the NEMO-binding domain) were efficiently coprecipitated with KLHL21 (Fig. 5A). In contrast, deletion of the N-terminal KD (mutant M1) dramatically reduced the ability of KLHL21 to coisolate with IKK␤. The interaction between KLHL21 and IKK␤ was completely abolished when both KD and ubiquitin-like domains were deleted (mutant M2) (Fig. 5A). These results indicate that the KD of IKK␤ is critical for association with KLHL21.
Next, to identify the domain of KLHL21 responsible for interaction with IKK␤, we generated four deletion mutants (⌬1, ⌬2, ⌬3, and ⌬4) of mouse KLHL21 (Fig. 5B). Coimmunoprecipitation assays revealed that deletion mutants ⌬3 and ⌬4 (both containing Kelch domains) were coisolated with IKK␤. Deletion of the Kelch domains of KLHL21 (mutants ⌬1 and ⌬2) abolished the interaction with IKK␤. These results revealed that the C-terminal Kelch domains were required for KLHL21 to interact with IKK␤. Point mutant KLHL21M (D114A/L115A/Q117A), which is unable to bind CUL3 ( Fig. 5C) (20), was also coisolated with IKK␤ ( Fig. 5B). This finding reveals that the BTB domain of KLHL21 is unnecessary for association with IKK␤.  KEAP1, another member of the KLHL gene family, binds to a unique (E/A)TGE motif in the KD of IKK␤ via its Kelch domains and promotes the degradation of IKK␤ (11,12). To test whether KLHL21 also binds to this unique motif, Glu-39 of the mouse IKK␤ motif was mutated (E39A). Consistent with previous reports, the E39A mutation dramatically reduced the interaction between IKK␤ and KEAP1 (11, 12), but it had no effect on the interaction between KLHL21 and IKK␤ (Fig. 5D).
These results imply that KLHL21 is unlikely to compete with KEAP1 for binding to this unique motif within the KD of IKK␤ in cells.
KLHL21 Negatively Regulates TNF␣-induced IKK␤ Activation-Activation of the IKK complex, especially via phosphorylation of both Ser-177 and Ser-181 within the KD of IKK␤, is the key step driving stimulation of NF-B signaling. Given our observations that KLHL21 binds to the KD of IKK␤ and inhibits   the activation of NF-B signaling, we hypothesized that KLHL21 may regulate the phosphorylation of IKK␤. Overexpression of IKK␤ in HEK293T cells resulted in its autophosphorylation, which was dramatically suppressed when KLHL21 was coexpressed (Fig. 6A). Interestingly, the KLHL21M mutant functioned similarly to wild-type KLHL21 (Fig. 6A)  ( Fig. 6B). Conversely, siRNA-mediated depletion of KLHL21 resulted in enhanced phosphorylation of IKK␣/␤ (Ser-177) in response to TNF␣ treatment without affecting total endogenous IKK␤ protein levels (Fig. 6C).
To rule out off-target effects, we performed a rescue experiment where HEK293T cells were cotransfected with siRNA specifically targeting human KLHL21 and mCherry-tagged wild-type or enzymatically inactive mutant of mouse KLHL21 construct. Consistent with above findings, knocking down KLHL21 promoted the phosphorylation of IKK␤ and IB␣ and the degradation of IB␣ in HEK293T cells exposed to TNF␣ (Fig. 6D, lane 4), whereas coexpression of either WT KLHL21 or E3 ligase activity-inactive mutant KLHL21M reversed these effects (Fig. 6D, lanes 6 and 8). This result further confirmed our observation that KLHL21 negatively regulates TNF␣-induced IKK␤ activation, which is independent of its E3 ligase activity.  KLHL21 interacts with Cul3 and mediates the ubiquitination of aurora B kinase in vitro (20). Therefore, we next tested whether KLHL21 plays a role in the ubiquitination of IKK␤. In the presence of the proteasome inhibitor MG132, long-term exposure of the nitrocellulose membrane revealed smeared bands of polyubiquitinated IKK␤ in HEK293T cells (Fig. 6E). Overexpression of either wild-type KLHL21 or the KLHL21M mutant failed to enhance the ubiquitination of IKK␤ (Fig. 6E). Furthermore, exogenously expressed KLHL21 did not significantly affect endogenous levels of IKK␤ protein (Fig. 6B). These results further demonstrate that KLHL21 is unlikely to target IKK␤ endogenously through its E3 ubiquitin ligase function.
Given our observations that KLHL21 efficiently inhibited TBK1-, TRAF2-, and TRAF6-induced activation of NF-B ( Fig.  2A), we next tested whether KLHL21 can bind directly to TBK1, TRAF2, or TRAF6. Coimmunoprecipitation results demonstrated that KLHL21 did not bind to TBK1, TRAF2, or TRAF6 in HEK293T cells (Fig. 7A). Upon TNF␣ stimulation, TRAF2 rapidly recruits the IKK complex to TNFR1 in cells, which leads to TAK1-mediated phosphorylation and activation of IKK␣ and IKK␤, subsequently resulting in the phosphorylation and proteasomal degradation of IB␣. To examine whether KLHL21 binding to IKK␤ interferes with any step of this process, we cotransfected KLHL21 and IKK␤ with TRAF2, TAK1, or the undegradable IB␣ mutant (S32A/S36A) in HEK293T cells. Overexpression of KLHL21 failed to disrupt the interaction between IKK␤ and TRAF2, TAK1, or IB␣ (Fig. 7, B-D), further suggesting that KLHL21 inhibits activation of the NF-B signaling pathway by directly targeting IKK␤.
KLHL21 Differentially Modulates the Expression of NF-B Target Genes-The above data clearly reveal that KLHL21 suppresses NF-B signaling by directly targeting IKK␤ to inhibit its activation. Next, the effects of KLHL21 on the expression of endogenous NF-B target genes NFKBIA, DUSP1, NFKBIZ, CXCL1, TNFAIP3, and IL-8 were assessed in TNF␣-treated HEK293T cells. Overexpressing KLHL21 significantly suppressed TNF␣-induced transcription of the immediate early response genes, such as NFKBIA, DUSP1, and NFKBIZ (which peaked at 30 min after TNF␣ treatment) but, unexpectedly, enhanced the relative late phase expression of CXCL1, IL-8, and TNFAIP3 (which peaked at 2 h or even later after TNF␣ treatment) (Fig. 8A). Conversely, siRNA-mediated knockdown of KLHL21 resulted in marked up-regulation of NFKBIA and down-regulation of IL-8 transcript levels (2 and 4 h) but had no effect on TNFAIP3 expression (Fig. 8B). Similarly, KLHL21 depletion in LPS-treated RAW264.7 macrophages (Fig. 8C) was associated with up-regulated Nfkbia and down-regulated Tnfaip3 and Il-1b transcript levels (Fig. 8D).
To clarify the discrepancy that overexpressing KLHL21 inhibited the activation of IKK␤ yet enhanced the expression of some relative late phase NF-B target genes, such as CXCL1, IL-8, and TNFAIP3, we then analyzed the nuclear duration of p65 in HEK293T cells upon TNF␣ treatment. By 2 and 4 h after TNF␣ treatment, nuclear levels of p65 were dramatically reduced in control cells compared with KLHL21-overexpressing cells (Fig. 8E). These findings suggest that KLHL21 overexpression prolongs the duration of p65 in the nucleus, which may partly account for the up-regulation of IL-8 and TNFAIP3 by 4 h after TNF␣ treatment.

Discussion
Here, we demonstrate that KLHL21 specifically binds to and negatively regulates the activation of IKK␤ in resting cells. The interaction between KLHL21 and IKK␤ is gradually attenuated in cells upon TNF␣ treatment. This leads to the release of IKK␤, which can then be activated, suggesting that IKK␤ activity is finely tuned by KLHL21 under inflammatory conditions. The expression of KLHL21 is also dynamically regulated in macrophages treated with diverse proinflammatory stimuli, high-   lighting its potentially important function in the appropriate activation of NF-B signaling. KLHL21 was previously reported to interact with Cul3 and to mediate the ubiquitination of aurora B in vitro (20). However, exogenous expression of KLHL21 had no effect on the ubiquitination states of endogenous IKK␤ or its general translation.
The KLHL21M mutant, which is unable to interact with Cul3, functioned in a similar manner to wild-type KLHL21 by suppressing the autophosphorylation of IKK␤. These findings demonstrate that KLHL21 negatively regulates IKK␤ independently of its E3 ubiquitin ligase activity. It is not uncommon for an E3 ubiquitin ligase to carry out alternative cellular functions independent of its traditional enzymatic activity (8,28,29). For example, the E3 ubiquitin ligase CUEDC2 inhibits the activity of IKK by recruiting phosphatase PP1 to the IKK complex (8). Furthermore, KLHL21 did not interfere with the interaction between IKK␤ and TRAF2, TAK1, or IB␣. KLHL21 probably inhibits IKK␤ by masking the KD residues to be phosphorylated. Given that KLHL21 also suppressed the activity of the IKK␤ SS/EE mutant, it is plausible that it may also sterically block access to the activated KD from IKK␤ substrates. However, we cannot exclude the possibility that KLHL21 may negatively regulate IKK␤ by recruiting other unidentified proteins. Future studies are necessary to clarify this hypothesis.
KEAP1 was recently demonstrated to bind specifically to a unique (E/A)TGE motif in the KD of IKK␤ through its Kelch domains (11,12). KEAP1 promotes the degradation of IKK␤ via the autophagic (11) or 26S proteasome (12) pathways and inhibits the phosphorylation of IKK␤ in response to extracellular stimuli. Consistent with previous reports, mutation of the second glutamate to alanine (E39A) in this motif dramatically suppressed the interaction between IKK␤ and KEAP1 (11,12) but failed to disrupt the IKK␤-KLHL21 interaction. These findings imply that KLHL21 may bind to other motifs within the KD of IKK␤ and probably does not compete with KEAP1 for binding to IKK␤ endogenously. This possibility further confirms that the interaction between KLHL21 and IKK␤ is specific.
IKK␤-mediated phosphorylation and degradation of IB␣ lead to the nuclear translocation of NF-B and the induced expression of its target genes, including NFKBIA (encoding IB␣ protein). The IB␣ protein then translocates to the nucleus and terminates the activity of NF-B, thereby generating a negative feedback loop for NF-B signaling and acting as an internal timer for the first phase of NF-B activity. Reduced IB␣ expression results in longer retention of NF-B in the nucleus and enhanced expression of its late phase target genes (30,31). In this study, overexpression of KLHL21 in TNF␣stimulated HEK293T cells resulted in the inhibition of IKK␤ activation, leading to reduced phosphorylation and subsequent ubiquitin-dependent degradation of IB␣. However, the interaction between KLHL21 and IKK␤ was dynamically attenuated upon TNF␣ treatment, which led to the release of IKK␤ and its delayed activation. Thus, overexpression of KLHL21 is only sufficient to inhibit TNF␣-induced nuclear translocation of p65 in the early phase, thus leading to reduced expression of IB␣ (an immediate early response gene). It results in prolonged nuclear retention of NF-B, which potentially accounts, at least in part, for the enhanced expression of TNFAIP3, CXCL1, and IL-8 in the relatively late phase (at 2 and 4 h post-TNF␣ treatment). But we also cannot exclude the possibility that KLHL21 may differentially regulate the expression of those NF-B target genes through targeting other unidentified components in NF-B signaling pathway. More detailed studies are needed to elucidate its mechanism.
Attenuated interactions between IKK␤ and its regulators in response to extracellular stimuli have also been reported in many other cellular contexts (8,9,13). However, the differential regulation of the expression of NF-B target genes appears to be unique to KLHL21. This information extends our understanding of NF-B signaling regulation. Differential modula-tion of NF-B target gene expression by KLHL21 may also reflect a unique function of IKK␤ in NF-B signaling. IKK␤ deficiency was recently shown to affect NF-B target gene expression selectively (32). CCL2, TNFAIP3, and TNF demonstrated normal or slightly reduced expression levels, whereas CCL5, CXCL3, and CXCL10 had markedly reduced expression levels in IKK␤-deficient human primary fibroblasts in response to TNF␣ treatment (32).
Interestingly, KLHL21 expression was reported in the NCBI GEO database to be up-regulated in synovial macrophages from patients with rheumatoid arthritis (RA), a chronic inflammatory joint disease (GEO accession number GDS3192) (33). TNF␣ levels are elevated in synovial fluid from RA patients in proportion to cartilage destruction (34). As a master proinflammatory cytokine, TNF␣ induces the expression of other cytokines, such as IL-8, IL-1␤, and IL-6, all of which are also up-regulated in the synovium of RA patients (34). Based on our finding that overexpression of KLHL21 leads to the up-regulation of IL-8 and TNFAIP3 in response to prolonged TNF␣ treatment, KLHL21 up-regulation in synovial macrophages from RA patients may also play a role in the induction of IL-8, IL-1␤, and other proinflammatory cytokines. Further detailed studies are needed to decipher the potential relationship between KLHL21 and RA development.
In conclusion, we have identified KLHL21 as a novel negative regulator of IKK␤. KLHL21 specifically binds to the KD of IKK␤ through its Kelch domains and inhibits IKK␤ activation. The rapid down-regulation of KLHL21 in macrophages treated with diverse proinflammatory stimuli and the dynamically attenuated interaction between IKK␤ and KLHL21 after TNF␣ stimulation suggest that IKK␤ activation is tightly controlled by KLHL21 under physiological conditions (Fig. 9). Protein A/G-agarose beads were purchased from Sangon Biotech (Shanghai, China). TRIzol reagent, polyclonal goat anti-rabbit Alexa-488 antibody, and Lipofectamine RNAiMAX were obtained from Life Technologies, Inc. LPS (from Escherichia coli 0111:B4) and phosphatase inhibitor mixture were obtained from Sigma-Aldrich. The Dual-Luciferase Reporter Assay system was purchased from Promega (Madison, WI). FastStart Universal SYBR Green Master (Rox) and EDTA-free complete protease inhibitors were purchased from Roche Diagnostics GmbH (Mannheim, Germany). SuperSignal chemiluminescence reagents and NE-PER nuclear and cytoplasmic extraction reagents were obtained from Pierce. ReverTra Ace quantitative RT-PCR kit was purchased from Toyobo Life Science (Osaka, Japan). Pam3CSK4 and pam2CSK4 were obtained from Invivogen (San Diego, CA). Recombinant human and mouse TNF␣, IL-1␤, and M-CSF were purchased from Pepro-Tech (Rocky Hill, NJ).

Materials
Cell Culture and Stimulation-RAW264.7 cells and human embryonic kidney (HEK) 293T cell lines were cultured in DMEM (Life Technologies) supplemented with 10% fetal bovine serum in a humidified atmosphere of 5% CO 2 at 37°C. Bone marrow-derived macrophages were prepared as adherent cultures using procedures described previously (35). In selected experiments, cells were treated with 100 ng/ml LPS, 100 ng/ml pam3CSK4, and 100 ng/ml pam2CSK4, 5 M ODN1826, or 20 ng/ml TNF␣ for NF-B activation respectively.
cDNAs and Site-directed Mutagenesis-Total RNA was extracted from RAW264.7 cells and reverse transcribed into cDNA. It was used to amplify the cDNAs of mouse Klhl21, Traf2, Traf6, Tbk1, Ikk␤, p65 (RelA), and Keap1. These cDNAs were subcloned into pcDNA3 expression plasmids with appropriate 3Ј tag 3ϫFLAG (3F), Glu-Glu (EE), or mCherry (MC), respectively, as specified. Point mutations were introduced in the expression plasmids using the QuikChange site-directed mutagenesis protocol (Stratagene) and verified by DNA sequencing.
Luciferase Assay-Luciferase reporter plasmid pNFB-TAluc, which contains 6ϫB binding sites and a minimal TA promoter, was obtained from Beyotime Biotechnology (Jiangsu, China). HEK293T cells were transfected with 0.2 g of pNFB-TA-luc plus 0.02 g of Renilla reporter pRL-TK as an internal control and various amounts of Klhl21, Traf2, Traf6, Tbk1, Ikk␤ (SS/EE), and p65 (RelA) expression vectors, respectively. 24 h after the transfection, the cells were harvested and subjected to the Dual-Luciferase Reporter Assay system according to the manufacturer's recommendations.
Quantitative Real-time PCR Analysis-Total RNA was extracted by using TRIzol reagent according to the manufacturer's instruction. RNA was reverse transcribed by using the ReverTra Ace quantitative RT-PCR kit. For detection and quantification, PCRs were performed using the FastStart Universal SYBR Green Master (Rox) (Roche Diagnostics) and the 7500 real-time PCR system (Applied Biosystems). The primers for quantitative PCR are listed in Table 1.
Coimmunoprecipitation, GST Pull-down, and Immunoblotting-Cells transfected with various plasmids as specified were incubated for 24 h before analysis. Cell lysate preparation, immunoprecipitation, and immunoblotting analysis were done as described previously (36). Nuclear proteins were isolated using NE-PER nuclear and cytoplasmic extraction reagents according to the manufacturer's instructions (Pierce). For detection of ubiquitinated proteins in vivo, transfected cells were treated with 5 M MG132 for 16 h before collection. Then the cells were rapidly lysed by boiling in a buffer containing 2% SDS, 150 mM NaCl, 10 mM Tris-HCl, and 1 mM DTT. This will inactivate cellular ubiquitin hydrolases and therefore preserve ubiquitin-protein conjugates present in cells before lysis. For immunoprecipitation, these lysates were diluted 5-fold in ΙκB α FIGURE 9. Proposed model illustrating how KLHL21 modulates the NF-B signaling pathway via targeting IKK␤. In resting cells, KLHL21 protein specifically binds to IKK␤ and inhibits its activation, which leads to the suppression of NF-B signaling. Their interaction is gradually attenuated in response to TNF␣ treatment, which leads to the release of IKK␤ and the activation of the NF-B signaling pathway. This results in the up-regulation of those immediate early response genes, including NFKBIA (encoding IB␣). Up-regulation of IB␣ leads to the termination of NF-B signaling and down-regulation of its relative late phase target genes, such as CXCL1, IL-8, and IL-1B. Furthermore, the expression of KLHL21 is rapidly down-regulated in macrophages upon extracellular proinflammatory stimuli treatment, which also releases its inhibitory effects on IKK␤ activation. buffer lacking SDS and incubated with anti-FLAG antibody. Immunoprecipitated proteins were analyzed by immunoblotting with anti-FLAG and anti-ubiquitin antibodies. The blots were developed using Supersignal chemiluminescence reagents, and images were captured with an Eastman Kodak Co. Image Station 1000. pGEX4T plasmid containing the C-terminal BACK and Kelch domains of mouse KLHL21 (amino acids 134 -597) was introduced into E. coli BL21 (DE3) strain, and expression of the GST fusion protein was induced by the addition of 0.1 M isopropyl ␤-D-1-thiogalactopyranoside at 25°C for 4 h. Whole cell lysates of HEK293T and RAW264.7 were incubated with GST-KLHL21 protein purified with glutathione-Sepharose 4B for 4 h at 4°C. The beads were washed four times with cell lysis buffer, and the bound proteins were analyzed by Western blotting with anti-IKK␤ antibody.
RNA Interference and Rescue-Chemosynthesized siRNAs CGTCCATGAATCAGGTACA (siRNA 1), GCATCTTCCG-CCAGTTCAT (siRNA 2), and GCTGGGCAATGACAT-CTAC (siRNA 3) targeting human KLHL21; siRNAs GGTAC-GACAACACCTTTGA (siRNA 4) and GCTGAACAGTGGT-AGCAAT (siRNA 5) targeting mouse KLHL21; and TTCTCC-GAACGTGTCACGT (negative control siRNA) were all obtained from GenePharma (Shanghai, China). To achieve efficient knockdown and limit off-target effects, a pool of siRNA oligonucleotides was used by mixing the different siRNAs at equal concentration. The siRNAs were transfected at the specified concentrations by Lipofectamine RNAiMax. For siRNA rescue experiments, HEK293T cells were cotransfected with chemosynthesized siRNA 3, which does not target mouse KLHL21 mRNA, and mCherry-tagged wild type or enzymatically inactive mutant (KLHL21M) of mouse KLHL21 construct by using jetPRIME (PolyPlus) according to the manufacturer's protocol. At 24 h post-transfection, the cells were treated with TNF␣ or LPS and harvested to analyze the expression of KLHL21 and IB␣ protein by Western blotting or the mRNA level of KLHL21, NFKBIA, TNFAIP3, and IL-8 by real-time PCR as specified.
Fluorescent Confocal Microscopy-HeLa cells were grown on poly-L-lysine-coated glass coverslips and transfected C-terminal mCherry-tagged KLHL21 by using jetPRIME (PolyPlus) according to the manufacturer's protocol. At 24 h post-transfection, the cells were fixed with 4% formaldehyde for 15 min, followed by blocking with 5% normal goat serum for 1 h. Anti-IKK␤ antibody was used for detecting endogenous IKK␤. Goat anti-rabbit Alexa Fluor 488 was used as secondary antibody. Confocal microscopy images were obtained using Zeiss LSM 700 confocal microscope (Carl Zeiss) with the excitation source at 488 and 587 nm for Alexa Fluor 488 and mCherry, respectively. The acquired images were processed in ZEN 2012.
Statistical Analysis-Unless otherwise indicated, experiments were performed in triplicate and repeated at least three times. The figures show the results from one representative experiment. The intensity of the protein bands was determined by densitometry using ImageJ. All data are presented as means Ϯ S.E. Data sets were tested using two-tailed Student's t test. Statistical analyses were carried out using Origin version 8 software (OriginLab). Differences were considered statistically significant for values of p Ͻ 0.05.