FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2048657955> ?p ?o ?g. }

• W2048657955 endingPage "33948" @default.
• W2048657955 startingPage "33943" @default.
• W2048657955 abstract "Post-translational modification and degradation of proteins by the ubiquitin-proteasome system are key regulatory events in cellular responses to various stimuli. The NF-κB signaling pathway is controlled by the ubiquitin-mediated proteolysis. Although mechanisms of ubiquitination in the NF-κB pathway have been extensively studied, deubiquitination-mediated regulation of the NF-κB signaling remains poorly understood. The present studies show that a deubiquitinating enzyme, USP11, specifically regulates IκB kinase α (IKKα) among the NF-κB signaling molecules. Knocking down USP11 attenuates expression of IKKα in the transcriptional, but not the post-translational, level. However, down-regulation of USP11 dramatically enhances NF-κB activity in response to tumor necrosis factor-α, indicating that IKKα does not require activation of NF-κB. Instead, knock down of USP11 or IKKα is associated with abrogation of p53 expression upon exposure to tumor necrosis factor-α. In concert with these results, silencing of USP11 is associated with transcriptional attenuation of the p53-responsive genes, such as p21 or Bax. Importantly, the ectopic expression of IKKα into cells silenced for USP11 restores p53 expression, demonstrating that USP11 functions as an upstream regulator of an IKKα-p53 signaling pathway. Post-translational modification and degradation of proteins by the ubiquitin-proteasome system are key regulatory events in cellular responses to various stimuli. The NF-κB signaling pathway is controlled by the ubiquitin-mediated proteolysis. Although mechanisms of ubiquitination in the NF-κB pathway have been extensively studied, deubiquitination-mediated regulation of the NF-κB signaling remains poorly understood. The present studies show that a deubiquitinating enzyme, USP11, specifically regulates IκB kinase α (IKKα) among the NF-κB signaling molecules. Knocking down USP11 attenuates expression of IKKα in the transcriptional, but not the post-translational, level. However, down-regulation of USP11 dramatically enhances NF-κB activity in response to tumor necrosis factor-α, indicating that IKKα does not require activation of NF-κB. Instead, knock down of USP11 or IKKα is associated with abrogation of p53 expression upon exposure to tumor necrosis factor-α. In concert with these results, silencing of USP11 is associated with transcriptional attenuation of the p53-responsive genes, such as p21 or Bax. Importantly, the ectopic expression of IKKα into cells silenced for USP11 restores p53 expression, demonstrating that USP11 functions as an upstream regulator of an IKKα-p53 signaling pathway. NF-κB is an inducible transcription factor that controls the expression of a number of proteins involved in the regulation of cell survival and immune response (1Karin M. Lin A. Nat. Immunol. 2002; 3: 221-227Crossref PubMed Scopus (2470) Google Scholar). NF-κB is activated by a bewildering array of stimuli, including biological agents such as tumor necrosis factor α (TNFα), 3The abbreviations used are: TNFα, tumor necrosis factor α; IκB, inhibitor of NF-κB; IKK, IκB kinase; siRNA, small interfering RNA; HA, hemagglutinin; RT, reverse transcription. interleukin-1, bacterial endotoxin, and phorbol esters and cytotoxic stimuli such as chemotherapeutic agents, ultraviolet light, oxidative stress, and ionizing radiation (2Ghosh S. Karin M. Cell. 2002; 109: S81-S96Abstract Full Text Full Text PDF PubMed Scopus (3300) Google Scholar, 3Li Q. Verma I.M. Nat. Rev. Immunol. 2002; 2: 725-734Crossref PubMed Scopus (3353) Google Scholar). The NF-κB signaling pathway is enzymatically controlled by ubiquitination-deubiquitination (4Chen Z.J. Nat. Cell Biol. 2005; 7: 758-765Crossref PubMed Scopus (1026) Google Scholar). These post-translational modifications are critical for “on-off” switch in the signaling cascade. For example, one member of IκB, designated as IκBα, is phosphorylated in response to TNFα or interleukin-1 and is thereby subjected to ubiquitination and degradation by the 26 S proteasome (5Verma I.M. Stevenson J.K. Schwarz E.M. Van Antwerp D. Miyamoto S. Genes Dev. 1995; 9: 2723-2735Crossref PubMed Scopus (1665) Google Scholar). The NF-κB essential modifier (NEMO, also known as IKKγ) is a key to activating IκB kinase (IKK) complex and is polyubiquitinated by TRAF6 (6Kanayama A. Seth R.B. Sun L. Ea C.K. Hong M. Shaito A. Chiu Y.H. Deng L. Chen Z.J. Mol. Cell. 2004; 15: 535-548Abstract Full Text Full Text PDF PubMed Scopus (703) Google Scholar). In contrast, deubiquitinating mechanisms that target to ubiquitinated NF-κB signaling molecules are largely unclear. Certain insights have been derived from the finding that CYLD was identified by its association with NEMO and through systematic screening for deubiquitinating enzymes that impede NF-κB signaling (7Brummelkamp T.R. Nijman S.M. Dirac A.M. Bernards R. Nature. 2003; 424: 797-801Crossref PubMed Scopus (835) Google Scholar, 8Kovalenko A. Chable-Bessia C. Cantarella G. Israel A. Wallach D. Courtois G. Nature. 2003; 424: 801-805Crossref PubMed Scopus (857) Google Scholar, 9Trompouki E. Hatzivassiliou E. Tsichritzis T. Farmer H. Ashworth A. Mosialos G. Nature. 2003; 424: 793-796Crossref PubMed Scopus (808) Google Scholar). Overexpression of CYLD represses NF-κB activation in response to various stimuli, including TNFα. Conversely, inactivation of CYLD by RNA interference increases inducible NF-κB activity. These findings thus suggest that CYLD acts as a negative regulator of the NF-κB signaling pathway. Little is otherwise known about direct deubiquitinating enzymes targeting to ubiquitinating molecules that are involved in NF-κB signaling. A recent study mapped a protein interaction network for TNFα/NF-κB pathway components by means of an integrated approach, including tandem affinity purification, liquid chromatography tandem mass spectrometry, network analysis, and directed functional perturbation studies using RNA interference (10Bouwmeester T. Bauch A. Ruffner H. Angrand P.O. Bergamini G. Croughton K. Cruciat C. Eberhard D. Gagneur J. Ghidelli S. Hopf C. Huhse B. Mangano R. Michon A.M. Schirle M. Schlegl J. Schwab M. Stein M.A. Bauer A. Casari G. Drewes G. Gavin A.C. Jackson D.B. Joberty G. Neubauer G. Rick J. Kuster B. Superti-Furga G. Nat. Cell Biol. 2004; 6: 97-105Crossref PubMed Scopus (880) Google Scholar). This study identified numerous previously unknown interactors, including a deubiquitinating enzyme, USP11. However, the role for USP11 in the NF-κB signaling pathway remains obscure. Our recent studies demonstrated that IKKα, but not IKKβ, is activated and translocates into the nucleus in response to oxidative stress (11Yamaguchi T. Miki Y. Yoshida K. Cell. Signal. 2007; 19: 2088-2097Crossref PubMed Scopus (35) Google Scholar). Upon exposure to oxidative stress, IKKα activation does not contribute to NF-κB activation; instead, nuclear IKKα regulates the transcription activity of the p53 tumor suppressor, indicating that the IKKα → p53 signaling pathway is associated with the cellular response to oxidative stress. In this study, we show that USP11 regulates IKKα expression in a transcriptional level. Importantly, down-regulation of IKKα does not contribute to NF-κB activation in response to TNFα. Instead, USP11-mediated regulation of IKKα functions to control p53 transcription activity. These findings support a novel mechanism in which the deubiquitinating enzyme USP11 contributes to TNFα-induced IKKα → p53 signaling pathway. Cell Culture—Human MOLT-4 and HL-60 leukemia cells were cultured in RPMI 1640 medium supplemented with human U2OS osteosarcoma cells, and MCF-7 mammary gland carcinoma cells were grown in Dulbecco's modified Eagle's medium containing 10% heat-inactivated fetal bovine serum, penicillin (100 units/ml), streptomycin (100 μg/ml), and l-glutamine (2 mm). Cells were treated with 50 ng/ml TNFα (Pepro-Tech EC) or 5 μm MG132 (carbobenzoxy-l-leucyl-l-leucyl-l-leucinal; Nacalai Tesque). Plasmids—Constructs of IKKα and IKKβ are described elsewhere (12Nakano H. Shindo M. Sakon S. Nishinaka S. Mihara M. Yagita H. Okumura K. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3537-3542Crossref PubMed Scopus (473) Google Scholar). IKKγ cDNA was amplified by PCR from a human fetal brain cDNA library and cloned into the pcDNA3-FLAG vector as described elsewhere (13Yoshida K. Liu H. Miki Y. J. Biol. Chem. 2006; 281: 5734-5740Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). USP11 cDNA (14Ideguchi H. Ueda A. Tanaka M. Yang J. Tsuji T. Ohno S. Hagiwara E. Aoki A. Ishigatsubo Y. Biochem. J. 2002; 367: 87-95Crossref PubMed Scopus (72) Google Scholar) was cloned into the pcDNA3-FLAG vector. An RNA interference-resistant form of USP11 was constructed by introducing silent mutations using PCR-based site-directed mutagenesis. The sequences of oligonucleotide primers are as follows: 5′-CATACCGATTCaATaGGgCTAGTATTGCGC-3′ and 5′-GCGCAATACTAGcCCtATtGAATCGGTATG-3′. Lowercase letters represent silent mutations. Mutations were confirmed by sequencing. HA-ubiquitin plasmid has been reported previously (15Kawabata M. Inoue H. Hanyu A. Imamura T. Miyazono K. EMBO J. 1998; 17: 4056-4065Crossref PubMed Scopus (249) Google Scholar). Cell transfection was performed as described (16Yoshida K. Komatsu K. Wang H.G. Kufe D. Mol. Cell. Biol. 2002; 22: 3292-3300Crossref PubMed Scopus (88) Google Scholar, 17Taira N. Nihira K. Yamaguchi T. Miki Y. Yoshida K. Mol. Cell. 2007; 25: 725-738Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar). The total DNA concentration was kept constant by inclusion of an empty vector. siRNA Transfections—siRNA duplexes (siRNAs) were synthesized and purified by Invitrogen (Stealth Select RNA interference). Transfection of siRNAs was performed using Lipofectamine RNAiMAX (Invitrogen). Immunoprecipitation and Immunoblot Analysis—Cell lysates were prepared as described elsewhere (18Yoshida K. Weichselbaum R. Kharbanda S. Kufe D. Mol. Cell. Biol. 2000; 20: 5370-5380Crossref PubMed Scopus (54) Google Scholar, 19Yoshida K. Kharbanda S. Kufe D. J. Biol. Chem. 1999; 274: 34663-34668Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar) and cleared by centrifugation at 12,000 × g for 15 min. Soluble proteins were incubated with anti-FLAG M2 affinity gel (Sigma-Aldrich) for 2 h at 4°C. The immune complexes were washed three times with lysis buffer. Cell lysates or immunoprecipitates were separated by SDS-PAGE and transferred to nitrocellulose filters, which were then incubated with anti-FLAG, anti-HA (Roche Applied Science), anti-USP11 (Santa Cruz Biotechnology), anti-IKKα (MBL International Corp.), IKKβ (Cell Signaling Technology), anti-IKKγ (Santa Cruz Biotechnology), anti-RelA/p65 (Santa Cruz Biotechnology), anti-RelB (Santa Cruz Biotechnology), anti-p100 (Santa Cruz Biotechnology), anti-IκBα (Santa Cruz Biotechnology), anti-Ets-1 (Santa Cruz Biotechnology), anti-PCNA (Santa Cruz Biotechnology), or anti-tubulin (Sigma-Aldrich). The antigen-antibody complexes were visualized by chemiluminescence (PerkinElmer Life Sciences). RT-PCR Analysis for Gene Expression—Total cellular RNA was extracted using the RNeasy kit (Qiagen). First-stand cDNA synthesis and the following PCR reactions were performed with 500 ng of total RNA using SuperScript one-step RT-PCR system (Invitrogen) according to the manufacturer's protocol. The reaction products were resolved on a 2% agarose gel. Reporter Gene Assays—293 cells stably transfected with pNF-κB-luc and pTK-hyg (Panomics) were transfected with a variety of siRNAs followed by the treatment with TNFα. The luciferase activity was determined with a Bright-Glo luciferase assay system (Promega) according to the manufacturer's protocol. Chromatin Immunoprecipitation Assays—Cells were harvested and washed with chilled phosphate-buffered saline once followed by incubation in 1% formaldehyde for 15 min at room temperature for chromatin cross-linking. The cells were then collected and washed with chilled phosphate-buffered saline again. After centrifugation, the cell pellets were resuspended in SDS lysis buffer (1% SDS, 10 mm EDTA, 50 mm Tris-HCl, pH 8.0, 1 mm dithiothreitol, 1 mm phenylmethylsulfonyl fluoride, 1 mm Na3VO4, 10 μg/ml aprotinin, 1 μg/ml leupeptin, and 1 μg/ml pepstatin A), and the lysates were sonicated to obtain DNA fragments 200–500 bp in length. After centrifugation, 50 μl of the supernatant was used as an input, and the remainder was diluted 2–2.5-fold in washing buffer (50 mm Tris-HCl, pH 7.6, 150 mm NaCl, 0.1% Nonidet P-40, and protease inhibitors as described above). This diluted fraction was subjected to immunoprecipitation with 2 μg of indicated antibodies for 2 h to overnight at 4 °C with rotation. The immunocomplexes were collected with 30 μl of protein A-Sepharose beads (Santa Cruz Biotechnology) for 1–2 h at 4 °C with rotation. The beads were then pelleted by centrifugation and washed sequentially with 300 μl of the following buffers: wash buffer I (500 mm NaCl, 0.1% SDS, 2 mm EDTA, and 20 mm Tris-HCl, pH 8.0), wash buffer II (250 mm LiCl, 1 mm EDTA, 10 mm Tris-HCl, pH 8.0, and 1% deoxycholate), and then twice with Tris-EDTA buffer. Precipitated chromatin complexes were removed from the beads by shaking with 150 μl of elution buffer (1% SDS and 0.1 m NaHCO3) for 15 min, and this step was repeated. All the eluates were collected, and then the cross-linking was reversed by adding NaCl to a final concentration of 200 mm. The mixture was allowed to stand overnight at 65 °C. The remaining proteins were digested with the extraction buffer (50 mm Tris-HCl, pH 6.8, 10 mm EDTA, and 40 μg/ml proteinase K) for 1 h at 45 °C. DNA was recovered by phenol/chloroform/isoamyl alcohol (25/24/1) extraction and precipitated with 0.1 volume of 3 m sodium acetate and 2.5 volumes of ethanol. PCR amplification was performed in chromatin immunoprecipitated fragments using oligonucleotide pairs as described elsewhere (20Gu L. Zhu N. Findley H.W. Woods W.G. Zhou M. J. Biol. Chem. 2004; 279: 52141-52149Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). Subcellular Fractionation—Subcellular fractionation was performed as described previously (21Yoshida K. Wang H.G. Miki Y. Kufe D. EMBO J. 2003; 22: 1431-1441Crossref PubMed Scopus (133) Google Scholar, 22Yoshida K. Yamaguchi T. Shinagawa H. Taira N. Nakayama K.I. Miki Y. Mol. Cell. Biol. 2006; 26: 3414-3431Crossref PubMed Scopus (41) Google Scholar, 23Yoshida K. Yamaguchi T. Natsume T. Kufe D. Miki Y. Nat. Cell Biol. 2005; 7: 278-285Crossref PubMed Scopus (206) Google Scholar). Purity of the fractions was monitored by immunoblot analysis with anti-PCNA and anti-tubulin. USP11 Is Associated with IKKα Expression—Previous studies have implicated a number of deubiquitinating enzymes in several signaling cascades, including the NF-κB pathway. In particular, a recent study suggested a potential involvement of USP11 in NF-κB signaling. To determine whether USP11 affects the expression of NF-κB signalosome, U2OS cells were transfected with a scramble siRNA or a siRNA that targets USP11. We examined the expression of seven gene products that are directly involved in the NF-κB signaling pathway (Fig. 1A). Among these products, the expression of IKKα was significantly reduced in cells silenced for USP11. To exclude the possibility that knocking down USP11 is associated with off-target effects, siRNAs that target different USP11 sequences (USP11 siRNA-a and siRNA-b) were transfected into U2OS cells. Transfection of USP11 siRNA-a or siRNA-b resulted in similar levels of attenuated IKKα expression, indicating that USP11 specifically down-regulates IKKα expression (see Fig. 4A). To confirm the regulation of IKKα expression by USP11, U2OS cells were transfected with the USP11 siRNA followed by the transfection of the FLAG-USP11 mutant that is resistant for the USP11 siRNA by introducing silent mutations. The results demonstrated that IKKα expression was restored by forced expression of USP11 into U2OS cells silenced for USP11 (Fig. 1B). To investigate whether suppression of IKKα by USP11 silencing is regulated by the ubiquitin-proteasome pathway, FLAG-tagged IKK families were transfected into U2OS cells together with HA-tagged ubiquitin. Previous studies demonstrated that IKKγ is degraded by the ubiquitin-proteasome system (4Chen Z.J. Nat. Cell Biol. 2005; 7: 758-765Crossref PubMed Scopus (1026) Google Scholar). In concert with these results, overexpression of IKKγ was sufficient for substantial ubiquitination, regardless of TNFα treatment (Fig. 1C). By contrast, there was little if any ubiquitination on IKKα or IKKβ, indicating that IKKα and IKKβ function independently of ubiquitination-mediated regulation. These results thus suggest that USP11 regulates IKKα by a ubiquitin-independent manner. Another possibility is that regulation of IKKα by USP11 is mediated by undefined molecules that are controlled by the ubiquitin-deubiquitin mechanism. To further define USP11-mediated regulation of NF-κB signalosome, total RNA from U2OS cells in the presence or absence of USP11 was subjected to RT-PCR analysis. Knock down of USP11 specifically attenuated IKKα expression, suggesting that regulation of IKKα by USP11 occurs at the transcriptional level (Fig. 2A). In this context, a previous study showed that IKKα is positively regulated by a transcription factor, Ets-1 (20Gu L. Zhu N. Findley H.W. Woods W.G. Zhou M. J. Biol. Chem. 2004; 279: 52141-52149Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). Thus, it is plausible that USP11 affects Ets-1 by the ubiquitin-deubiquitin mechanism to control IKKα expression. To examine this possibility, U2OS cells were transfected with the scramble siRNA or USP11 siRNA followed by treatment with or without TNFα. The expression level of Ets-1 remained unchanged even in USP11-silencd cells, clearly indicating that Ets-1 is not targeted by USP11, at least post-translational level (Fig. 2B).FIGURE 4USP11 controls IKKα → p53 signaling in response to TNFα. A, U2OS cells transfected with various siRNAs were analyzed by immunoblotting (IB) with the indicated antibodies. B, MCF-7 cells were treated with TNFα for the indicated times. Cell lysates were analyzed by immunoblotting with the indicated antibodies. C, MCF-7 cells transfected with scramble siRNA or USP11 siRNA were treated with TNFα for the indicated times and then analyzed by Western blotting and RT-PCR. D, MCF-7 cells transfected with scramble siRNA (S) or USP11 siRNA (U) were pretreated with MG132 followed by treatment with TNFα for 8 h. Cell lysates were analyzed by immunoblotting with the indicated antibodies. E, MCF-7 cells transfected as indicated were left untreated or treated with TNFα for 8 h. Cell lysates were analyzed by immunoblotting with the indicated antibodies.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 2USP11 regulates IKKα expression at the transcriptional level. A, U2OS cells transfected with scramble siRNA or USP11 siRNA were treated with TNFα. Total RNA was analyzed by semiquantitative RT-PCR using specific primers targeting as indicated. B, U2OS cells were transfected as described above and left untreated (C) or treated with TNFα for 1 h (T). Cell lysates were analyzed by immunoblotting (IB) with the indicated antibodies. C, U2OS cells were transfected as described above and left untreated or treated with TNFα for 8 h. Chromatin immunoprecipitation assays were performed by using anti-Ets-1. Immunoprecipitated chromatin was analyzed by PCR using primer sequences containing IKKα promoter region.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To further define the USP11-independent regulation of Ets-1, we performed chromatin immunoprecipitation assays with anti-Ets-1 antibody. As reported previously (20Gu L. Zhu N. Findley H.W. Woods W.G. Zhou M. J. Biol. Chem. 2004; 279: 52141-52149Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar), Ets-1 occupied IKKα promoter, and this occupancy was increased after TNFα stimulation in U2OS cells (Fig. 2C). Importantly, the finding that Ets-1 occupancy on the IKKα promoter in cells silenced for USP11 was comparable with that in control cells indicates that Ets-1 transcriptional activity is regulated in an USP11-independent matter (Fig. 2C). Similar results were obtained in MCF-7 cells (data not shown). Taken together, these findings support an essential role for USP11 in the regulation of IKKα expression at a transcriptional level. Knocking Down USP11 Enhances TNFα-induced NF-κB Activation—To assess the effects of USP11 on the activity of NF-κB, 293 cells stably transfected with the luciferase-reporter vector containing NF-κB response elements were silenced for USP11 or RelA/p65. As shown previously, silencing of RelA/p65 was associated with pronounced inhibition of NF-κB activity in response to TNFα (Fig. 3A). By contrast, knocking down USP11 enhanced TNFα-induced NF-κB activation (Fig. 3B). To further define the involvement of USP11 in NF-κB activation, U2OS cells were transfected with the scramble siRNA or USP11 siRNA followed by TNFα treatment. Immunoblot analysis of whole cell lysates demonstrated substantial attenuation of resynthesized IκBα in the absence of USP11 (Fig. 3B). In accordance with this result, immunoblotting of nuclear lysates with anti-RelA/p65 revealed that at 2 h after TNFα treatment, 73% of nuclear RelA/p65 was exported from the nucleus in cells transfected with the scramble siRNA (Fig. 3B). In contrast, 83% of RelA/p65 remained in the nucleus in cells silenced for USP11 at the same time point (Fig. 3B), indicating that nuclear export of RelA/p65 was abrogated in cells silenced for USP11 (Fig. 3B). These findings provide a potential model in which, in USP11-silencing cells, RelA/p65 remains in the nucleus because of attenuation of resynthesized IκBα, resulting in sustained activation of NF-κB. In addition, given the data from reporter assays that imply enhancement of NF-κB activity in the absence of USP11, the confirmed results that IKKα expression is down-regulated by silencing USP11 further suggested that NF-κB activation occurs independently of IKKα under our experimental conditions (Fig. 3B). Taken together, these findings indicate that USP11 negatively regulates NF-κB activity in response to TNFα by a yet undefined mechanism. USP11 Controls the IKKα → p53 Signaling in Response to TNFα—Our recent study showed that the protein kinase C δ → IKKα signaling pathway stabilizes and activates p53 in response to oxidative stress (11Yamaguchi T. Miki Y. Yoshida K. Cell. Signal. 2007; 19: 2088-2097Crossref PubMed Scopus (35) Google Scholar). These finding led us to examine whether USP11 controls p53 through IKKα upon exposure to TNFα. As shown previously, knocking down USP11 in U2OS cells was associated with attenuation of IKKα expression (Fig. 4A). More importantly, steady-state levels of p53 expression were also down-regulated in the absence of USP11 (Fig. 4A). Although U2OS cells express p53 at low but detectable levels (13Yoshida K. Liu H. Miki Y. J. Biol. Chem. 2006; 281: 5734-5740Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar), p53 expression is little if any in unstressed MCF-7 cells. In this regard, we examined the effect of TNFα on p53 expression by MCF-7 cells. In concert with accumulating studies (24Ladiwala U. Li H. Antel J.P. Nalbantoglu J. J. Neurochem. 1999; 73: 605-611Crossref PubMed Scopus (57) Google Scholar, 25Gotlieb W.H. Watson J.M. Rezai A. Johnson M. Martinez-Maza O. Berek J.S. Am. J. Obstet. Gynecol. 1994; 170 (discussion 1128–1130): 1121-1128Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 26Donato N.J. Perez M. J. Biol. Chem. 1998; 273: 5067-5072Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 27Jeoung D.I. Tang B. Sonenberg M. J. Biol. Chem. 1995; 270: 18367-18373Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar), p53 was stabilized after treatment of cells with TNFα for 8 h, and its expression was sustained at least 24 h following TNFα treatment (Fig. 4B). In these experimental conditions, TNFα-induced p53 expression was completely abrogated in cells silenced for USP11 (Fig. 4C). In concert with this result, mRNA expression of p53-responsive genes, such as Bax and p21, were also attenuated in the absence of USP11 (Fig. 4C). By contrast, mRNA of p53 was constant regardless of USP11 expression, suggesting that USP11 regulates p53 via a post-translational, not a transcriptional, mechanism (Fig. 4C). To further define whether regulation of p53 by USP11 is associated with ubiquitin-proteasome system, MCF-7 cells transfected with scramble siRNA or USP11 siRNA were pretreated with proteasome inhibitor, MG132, followed by treatment with TNFα. Immunoblot analysis with anti-p53 revealed that, even in MG132-treated cells, knocking down USP11 reduced the expression levels of p53, suggesting that regulation of p53 by USP11 occurs independently of ubiquitin-proteasome proteolysis (Fig. 4D). To determine whether USP11 regulation of p53 involves IKKα, MCF-7 cells were transfected with scramble siRNA, USP11 siRNA, or IKKα siRNA and then treated with or without TNFα. The results demonstrated that p53 expression was substantially attenuated in USP11- or IKKα-deficient cells (Fig. 4E). Importantly, the ectopic expression of IKKα in USP11-silenced cells restored p53 expression (Fig. 4E), suggesting the possibility that effect of USP11 on p53 expression is regulated exclusively through IKKα. In this regard, recent studies have demonstrated that IKKα specifically translocates into the nucleus and phosphorylates histone H3 (28Anest V. Hanson J.L. Cogswell P.C. Steinbrecher K.A. Strahl B.D. Baldwin A.S. Nature. 2003; 423: 659-663Crossref PubMed Scopus (471) Google Scholar, 29Yamamoto Y. Verma U.N. Prajapati S. Kwak Y.T. Gaynor R.B. Nature. 2003; 423: 655-659Crossref PubMed Scopus (520) Google Scholar). Moreover, IKKα forms a complex with transcription coactivators such as AIB1/SRC-3 to modulate gene expression (30Park K.J. Krishnan V. O'Malley B.W. Yamamoto Y. Gaynor R.B. Mol. Cell. 2005; 18: 71-82Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar). Other studies have suggested that IKKα is recruited to the chromatin to derepress the silencing mediator for retinoic acid and thyroid hormone receptor (SMRT) (31Hoberg J.E. Popko A.E. Ramsey C.S. Mayo M.W. Mol. Cell. Biol. 2006; 26: 457-471Crossref PubMed Scopus (160) Google Scholar, 32Hoberg J.E. Yeung F. Mayo M.W. Mol. Cell. 2004; 16: 245-255Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar). Taken together, these findings indicate a pivotal role for nuclear IKKα as a regulator of transcription factors in response to TNFα. Given the previous finding that IKKα also stabilizes p53 by Ser-20 phosphorylation (11Yamaguchi T. Miki Y. Yoshida K. Cell. Signal. 2007; 19: 2088-2097Crossref PubMed Scopus (35) Google Scholar), these results thus support a mechanism by which USP11 regulates the expression of IKKα, which in turn controls p53 in response to TNFα. Regulation of IKKα → p53 Signaling Pathway by USP11—Previous work showed that protein kinase C δ activates IKKα in response to oxidative stress (11Yamaguchi T. Miki Y. Yoshida K. Cell. Signal. 2007; 19: 2088-2097Crossref PubMed Scopus (35) Google Scholar). Such activation was associated with increased p53 expression, indicating that the protein kinase C δ → IKKα signaling pathway functions as a positive regulator of p53. In this context, it should be clarified whether the protein kinase C δ → IKKα signaling is also involved in TNFα-induced p53 expression. Nevertheless, the present study identified another regulator of IKKα, USP11. Interestingly, despite the fact that USP11 is a deubiquitinating enzyme, USP11 regulation of IKKα was independent of ubiquitin-deubiquitin status. Instead, USP11 controlled IKKα at the transcriptional level by unknown mechanism. Importantly, USP11 also controlled the stabilization and activation of p53 through IKKα regulation at a steady-state level and in response to TNFα. In this regard, there is indeed another possibility that USP11 directly deubiquitinates p53 to stabilize its expression. However, other work demonstrated that overexpression of USP11 has no obvious effect on the levels of p53 ubiquitination or stabilization of p53 (33Li M. Chen D. Shiloh A. Luo J. Nikolaev A.Y. Qin J. Gu W. Nature. 2002; 416: 648-653Crossref PubMed Scopus (802) Google Scholar). The present findings that ectopic expression of IKKα in USP11-depleted MCF-7 cells markedly increased p53 expression also support an indirect role for USP11 in the regulation of p53. Although the precise mechanism by which IKKα controls p53 remains unclear, available evidence indicated that IKKα phosphorylates p53 for its stabilization and activation in response to oxidative stress. In this context, activation of p53 by TNFα may be, at least in part, involved in IKKα-mediated phosphorylation. A recent study has demonstrated that p53 expression in IKKα–/– knock-out mouse embryonic fibroblast is comparable with that in wild type mouse embryonic fibroblast (34Huang W.C. Ju T.K. Hung M.C. Chen C.C. Mol. Cell. 2007; 26: 75-87Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar). The result also demonstrated that p53 expression was substantially high even in the steady-state level, and no increase was observed after TNFα exposure. In this regard, given the present study demonstrating that p53 expression was attenuated in human cancer cells silenced for IKKα (Fig. 4E), there is indeed an apparent discrepancy to be solved in the future work. However, regulation of expression for human p53 is considerably different from that for mouse p53 (35Toledo F. Wahl G.M. Nat. Rev. Cancer. 2006; 6: 909-923Crossref PubMed Scopus (1045) Google Scholar), suggesting the possibility that human IKKα is specifically associated with the regulation of human p53 expression. Obviously, further studies are needed to clarify this issue. In summary, the present studies demonstrate that USP11 controls IKKα by a deubiquitination-independent mechanism. Regulation of IKKα by USP11 is associated with p53 stabilization and activation upon exposure to TNFα (Fig. 5). These findings indicate that USP11 controls the IKKα → p53 signaling pathway in response to TNFα. We thank Dr. Hiroyasu Nakano for FLAG-IKKα and IKKβ plasmids, Dr. Yoshiaki Ishigatsubo for USP11 cDNA, and Dr. Takeshi Imamura for HA-ubiquitin plasmid." @default.
• W2048657955 created "2016-06-24" @default.
• W2048657955 creator A5031695335 @default.
• W2048657955 creator A5059199772 @default.
• W2048657955 creator A5079742854 @default.
• W2048657955 creator A5091623593 @default.
• W2048657955 date "2007-11-01" @default.
• W2048657955 modified "2023-10-07" @default.
• W2048657955 title "The Deubiquitinating Enzyme USP11 Controls an IκB Kinase α (IKKα)-p53 Signaling Pathway in Response to Tumor Necrosis Factor α (TNFα)" @default.
• W2048657955 cites W1499561015 @default.
• W2048657955 cites W1965738579 @default.
• W2048657955 cites W1976382352 @default.
• W2048657955 cites W1979634801 @default.
• W2048657955 cites W1982776595 @default.
• W2048657955 cites W1988376074 @default.
• W2048657955 cites W1993720756 @default.
• W2048657955 cites W1994968768 @default.
• W2048657955 cites W2010495529 @default.
• W2048657955 cites W2017052599 @default.
• W2048657955 cites W2018394492 @default.
• W2048657955 cites W2025476423 @default.
• W2048657955 cites W2029454644 @default.
• W2048657955 cites W2039254532 @default.
• W2048657955 cites W2051599584 @default.
• W2048657955 cites W2052417990 @default.
• W2048657955 cites W2054050890 @default.
• W2048657955 cites W2056402897 @default.
• W2048657955 cites W2057653317 @default.
• W2048657955 cites W2068010171 @default.
• W2048657955 cites W2071198627 @default.
• W2048657955 cites W2077229666 @default.
• W2048657955 cites W2082724854 @default.
• W2048657955 cites W2091695331 @default.
• W2048657955 cites W2095424116 @default.
• W2048657955 cites W2109056181 @default.
• W2048657955 cites W2123738327 @default.
• W2048657955 cites W2124844891 @default.
• W2048657955 cites W2136190597 @default.
• W2048657955 cites W2144934582 @default.
• W2048657955 cites W2146204355 @default.
• W2048657955 cites W2316580498 @default.
• W2048657955 cites W2335925695 @default.
• W2048657955 cites W2470223656 @default.
• W2048657955 cites W2603306439 @default.
• W2048657955 doi "https://doi.org/10.1074/jbc.m706282200" @default.
• W2048657955 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/17897950" @default.
• W2048657955 hasPublicationYear "2007" @default.
• W2048657955 type Work @default.
• W2048657955 sameAs 2048657955 @default.
• W2048657955 citedByCount "65" @default.
• W2048657955 countsByYear W20486579552012 @default.
• W2048657955 countsByYear W20486579552013 @default.
• W2048657955 countsByYear W20486579552014 @default.
• W2048657955 countsByYear W20486579552015 @default.
• W2048657955 countsByYear W20486579552016 @default.
• W2048657955 countsByYear W20486579552017 @default.
• W2048657955 countsByYear W20486579552018 @default.
• W2048657955 countsByYear W20486579552019 @default.
• W2048657955 countsByYear W20486579552020 @default.
• W2048657955 countsByYear W20486579552021 @default.
• W2048657955 countsByYear W20486579552022 @default.
• W2048657955 countsByYear W20486579552023 @default.
• W2048657955 crossrefType "journal-article" @default.
• W2048657955 hasAuthorship W2048657955A5031695335 @default.
• W2048657955 hasAuthorship W2048657955A5059199772 @default.
• W2048657955 hasAuthorship W2048657955A5079742854 @default.
• W2048657955 hasAuthorship W2048657955A5091623593 @default.
• W2048657955 hasBestOaLocation W20486579551 @default.
• W2048657955 hasConcept C100175707 @default.
• W2048657955 hasConcept C102658986 @default.
• W2048657955 hasConcept C104317684 @default.
• W2048657955 hasConcept C17991360 @default.
• W2048657955 hasConcept C181199279 @default.
• W2048657955 hasConcept C184235292 @default.
• W2048657955 hasConcept C185592680 @default.
• W2048657955 hasConcept C203014093 @default.
• W2048657955 hasConcept C25602115 @default.
• W2048657955 hasConcept C2777730290 @default.
• W2048657955 hasConcept C3020084786 @default.
• W2048657955 hasConcept C55493867 @default.
• W2048657955 hasConcept C62478195 @default.
• W2048657955 hasConcept C86803240 @default.
• W2048657955 hasConcept C95444343 @default.
• W2048657955 hasConcept C98274493 @default.
• W2048657955 hasConceptScore W2048657955C100175707 @default.
• W2048657955 hasConceptScore W2048657955C102658986 @default.
• W2048657955 hasConceptScore W2048657955C104317684 @default.
• W2048657955 hasConceptScore W2048657955C17991360 @default.
• W2048657955 hasConceptScore W2048657955C181199279 @default.
• W2048657955 hasConceptScore W2048657955C184235292 @default.
• W2048657955 hasConceptScore W2048657955C185592680 @default.
• W2048657955 hasConceptScore W2048657955C203014093 @default.
• W2048657955 hasConceptScore W2048657955C25602115 @default.
• W2048657955 hasConceptScore W2048657955C2777730290 @default.
• W2048657955 hasConceptScore W2048657955C3020084786 @default.
• W2048657955 hasConceptScore W2048657955C55493867 @default.
• W2048657955 hasConceptScore W2048657955C62478195 @default.
• W2048657955 hasConceptScore W2048657955C86803240 @default.

• next