Heat Shock Protein 27 Association with the IκB Kinase Complex Regulates Tumor Necrosis Factor α-induced NF-κB Activation
2003; Elsevier BV; Volume: 278; Issue: 37 Linguagem: Inglês
10.1074/jbc.m305095200
ISSN1083-351X
AutoresKyu-Jin Park, Richard B. Gaynor, Youn Tae Kwak,
Tópico(s)NF-κB Signaling Pathways
ResumoHeat shock protein 27 (Hsp27) is a ubiquitously expressed member of the heat shock protein family that has been implicated in various biological functions including the response to heat shock, oxidative stress, and cytokine treatment. Previous studies have demonstrated that heat shock proteins are involved in regulating signal transduction pathways including the NF-κB pathway. In this study, we demonstrated that Hsp27 associates with the IκB kinase (IKK) complex and that this interaction was stimulated by tumor necrosis factor α treatment. Phosphorylation of Hsp27 by the kinase mitogen-activated protein kinase-activated protein kinase 2, a downstream substrate of the mitogen-activated protein kinase p38, enhanced the association of Hsp27 with IKKβ to result in decreased IKK activity. Consistent with these observations, treatment of cells with a p38 inhibitor reduced the association of Hsp27 with IKKβ and thus resulted in increased IKK activity. These studies indicate that Hsp27 plays a negative role in down-regulating IKK signaling by reducing its activity following tumor necrosis factor α stimulation. Heat shock protein 27 (Hsp27) is a ubiquitously expressed member of the heat shock protein family that has been implicated in various biological functions including the response to heat shock, oxidative stress, and cytokine treatment. Previous studies have demonstrated that heat shock proteins are involved in regulating signal transduction pathways including the NF-κB pathway. In this study, we demonstrated that Hsp27 associates with the IκB kinase (IKK) complex and that this interaction was stimulated by tumor necrosis factor α treatment. Phosphorylation of Hsp27 by the kinase mitogen-activated protein kinase-activated protein kinase 2, a downstream substrate of the mitogen-activated protein kinase p38, enhanced the association of Hsp27 with IKKβ to result in decreased IKK activity. Consistent with these observations, treatment of cells with a p38 inhibitor reduced the association of Hsp27 with IKKβ and thus resulted in increased IKK activity. These studies indicate that Hsp27 plays a negative role in down-regulating IKK signaling by reducing its activity following tumor necrosis factor α stimulation. The NF-κB pathway is a critical regulator of the expression of genes involved in diverse biological processes including the immune and inflammatory responses and cellular growth and death (1Baeuerle P.A. Baltimore D. Cell. 1996; 87: 13-20Abstract Full Text Full Text PDF PubMed Scopus (2919) Google Scholar, 2Baldwin Jr., A.S. Annu. Rev. Immunol. 1996; 14: 649-683Crossref PubMed Scopus (5552) Google Scholar, 3Maniatis T. Genes Dev. 1999; 13: 505-510Crossref PubMed Scopus (368) Google Scholar). In most cells the NF-κB proteins are sequestered in the cytoplasm bound to a family of inhibitory proteins known as IκB. Exposure of cells to a variety of extracellular stimuli including the cytokines tumor necrosis factor (TNF) 1The abbreviations used are: TNF, tumor necrosis factor; IL, interleukin; Hsp27, heat shock protein 27; MAPK, mitogen-activated protein kinase; MK, MAPK-activated protein kinase; siRNA, small interfering RNA; IKK, IκB kinase; CMV, cytomegalovirus; RSV, Rous sarcoma virus; HA, hemagglutinin; GST, glutathione S-transferase; NEMO, NF-κB essential modulator.1The abbreviations used are: TNF, tumor necrosis factor; IL, interleukin; Hsp27, heat shock protein 27; MAPK, mitogen-activated protein kinase; MK, MAPK-activated protein kinase; siRNA, small interfering RNA; IKK, IκB kinase; CMV, cytomegalovirus; RSV, Rous sarcoma virus; HA, hemagglutinin; GST, glutathione S-transferase; NEMO, NF-κB essential modulator. α and interleukin-1 (IL-1) leads to the activation of the IκB kinase (IKK) complex, resulting in the phosphorylation and ubiquitination of the IκB proteins and their degradation by the proteosome (3Maniatis T. 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For example, although IKKα–/– mice die of severe skin and skeletal abnormalities shortly after birth (18Hu Y. Baud V. Delhase M. Zhang P. Deerinck T. Ellisman M. Johnson R. Karin M. Science. 1999; 284: 316-320Crossref PubMed Scopus (708) Google Scholar, 19Li Q. Lu Q. Hwang J.Y. Buscher D. Lee K.F. Izpisua-Belmonte J.C. Verma I.M. Genes Dev. 1999; 13: 1322-1328Crossref PubMed Scopus (416) Google Scholar, 20Tanaka M. Fuentes M.E. Yamaguchi K. Durnin M.H. Dalrymple S.A. Hardy K.L. Goeddel D.V. Immunity. 1999; 10: 421-429Abstract Full Text Full Text PDF PubMed Scopus (491) Google Scholar), IKKβ–/– mice exhibit embryonic lethality caused by severe liver degeneration as a result of massive hepatic apoptosis (20Tanaka M. Fuentes M.E. Yamaguchi K. Durnin M.H. Dalrymple S.A. Hardy K.L. Goeddel D.V. Immunity. 1999; 10: 421-429Abstract Full Text Full Text PDF PubMed Scopus (491) Google Scholar, 21Li Q. Van Antwerp D. Mercurio F. Lee K.F. Verma I.M. 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A recent study demonstrated that in addition to IKKα, IKKβ, and IKKγ/NEMO, Hsp90 and Cdc37 are also associated with the IKK complex (24Chen G. Cao P. Goeddel D.V. Mol. Cell. 2002; 9: 401-410Abstract Full Text Full Text PDF PubMed Scopus (333) Google Scholar). These results suggested that the heat shock proteins may play a role in regulating the NF-κB pathway. The heat shock proteins (Hsps) can be divided into five major families on the basis of their size, structure, and function and include the Hsp110, Hsp90, Hsp70, and Hsp60 in addition to Hsp27 and other small heat shock proteins (25Lindquist S. Craig E.A. Annu. Rev. Genet. 1988; 22: 631-677Crossref PubMed Scopus (4383) Google Scholar). The expression of these proteins is induced by a wide variety of different physical, chemical, and biological stimuli including oxidative stress, heavy metals, osmotic stress, and heat shock (25Lindquist S. Craig E.A. Annu. Rev. Genet. 1988; 22: 631-677Crossref PubMed Scopus (4383) Google Scholar). 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Biol. Chem. 1999; 274: 18947-18956Abstract Full Text Full Text PDF PubMed Scopus (619) Google Scholar) that associate with the actin cytoskeleton. While investigating the role of Hsp90 and other heat shock proteins on IKK activation, we found that TNFα increased p38-MK2 phosphorylation of Hsp27 to enhance its association with IKKβ and thus decrease IKK activity. Treatment of cells with a specific inhibitor of p38 disrupted the interaction of Hsp27 and IKKβ and resulted in increased IKK activity. These studies indicate that Hsp27 plays a negative role in TNFα-mediated IKK activity and may be involved in down-regulating IKK activity at later times after TNFα stimulation. Cell Lines and Transfection Assay—HeLa cells were obtained from ATCC and maintained in Dulbecco's modified Eagle's medium with 10% (v/v) fetal bovine serum, 100 units/ml streptomycin, and 100 units/ml penicillin (Invitrogen). HeLa cells were transfected with GeneJuice (Novagen) using 1 μg of a CMV expression vector encoding Hsp27, 100 ng of an NF-κB-luciferase reporter (Stratagene, La Jolla, CA), and 100 ng of an RSV-β-galactosidase reporter. At 24 h post-transfection, HeLa cells were stimulated with either human TNFα (20 ng/ml) or IL-1 (5 ng/ml) for 6 h. Antibodies and Reagents—SB203580 (CalBiochem) was dissolved in Me2SO to a final concentration of 1 μm, and recombinant human TNF-α (20 ng/ml) and IL-1 (5 ng/ml) were purchased from Roche Applied Science. Anti-IKKα (catalog number 556532), anti-IKKβ (catalog number 550621), and anti-p38 (catalog number 612168) were obtained from BD Biosciences (San Diego, CA). Rabbit polyclonal anti-Hsp27 (catalog number spa-803) and mouse anti-Hsp27 (catalog number spa-800) were purchased from StressGen (Victoria, Canada). Anti-phospho-Hsp27 (Ser78) (catalog number 05-645) was purchased from Upstate Biotechnology, Inc. Anti-Hsp90 (catalog number sc-1055), anti-HA (catalog number sc-805), and anti-IKKα (sc-7182), as well as goat anti-Hsp70 (catalog number sc-1060) and anti-HSC 70 (catalog number sc-1059) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-IκBα (catalog number 9242), anti-phospho-IκBα (catalog number 9241), and anti-phospho-p38 (catalog number 9211) antibodies were purchased from Cell Signaling Technology. Anti-FLAG M2 and antiactin monoclonal antibodies were purchased from Sigma. Recombinant MAPK-activated protein kinase kinase 2 and Hsp27 proteins were purchased from Upstate Biotechnology, Inc. Plasmids—For the construction of expression vector encoding Hsp27, this cDNA was obtained by reverse transcription-PCR of HeLa cell total RNA and subcloned into the HindIII and XbaI sites of pFLAG-CMV2 (Sigma). The expression vector encoding Hsp27 phosphorylation mutants were constructed using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). All of the constructs were confirmed by DNA sequencing. RNA Oligonucleotides—Small interfering RNA (siRNAs) with two thymidine residues (dTdT) at the 3′ end of the oligonucleotides were designed for Hsp27 (5′-ACG GUC AAG ACC AAG GAU G-3′), HTLV-1 Tax (5′-GAU GGA CGC GUU AUC GGC U-3′) genes, GFP (5′-GGCUACGUCCAGGAGCGCACC-3′), and their complementary RNA oligonucleotides as described (34Elbashir S.M. Harborth J. Lendeckel W. Yalcin A. Weber K. Tuschl T. Nature. 2001; 411: 494-498Crossref PubMed Scopus (8107) Google Scholar) (Dharmacon Research Inc, Lafayette, CO). These RNAs were dissolved in TE (10 mm Tris-Cl, pH 8.0, and 1 mm EDTA) as 200 μm solutions and annealed at room temperature following heating to 95 °C in buffer (30 mm HEPES-KOH, pH 7.9, 100 mm potassium acetate, and 2 mm magnesium acetate). Transfection of siRNAs—Approximately, 1 × 106 cells were plated on 100-mm dishes in medium containing 10% fetal bovine serum to 30–50% confluency, and transfection of the RNA oligonucleotides was performed as described previously (35Surabhi R.M. Gaynor R.B. J. Virol. 2002; 76: 12963-12973Crossref PubMed Scopus (185) Google Scholar, 36Takaesu G. Surabhi R.M. Park K.J. Ninomiya-Tsuji J. Matsumoto K. Gaynor R.B. J. Mol. Biol. 2003; 326: 105-115Crossref PubMed Scopus (317) Google Scholar). Briefly, the cells were transfected using Oligofectamine (Invitrogen), resulting in a final RNA concentration of 20 nm. After 48 h, the cells were treated with either TNF-α (20 ng/ml) or IL-1 (5 ng/ml) for the times indicated and lysed in buffer (20 mm Tris, pH 7.5, 150 mm NaCl, 1 mm EDTA, 30 mm NaF, 2 mm NaPPi, 0.5 mm phenylmethylsulfonyl fluoride, and 0.1% Nonidet P-40 supplemented with protease inhibitor mixture) (Roche Applied Science) and then used in immunoprecipitation and Western blot analysis (37He K.L. Ting A.T. Mol. Cell. Biol. 2002; 22: 6034-6045Crossref PubMed Scopus (166) Google Scholar). Western Blot Analysis and Coimmunoprecipitation—The cell extracts were incubated with 1 μg of Hsp27 antibody for overnight and further incubated for 2 h following the addition of 30 μl of protein A beads (Zymed Laboratories Inc.). The samples were separated by SDS-PAGE, transferred to a nitrocellulose membrane, and detected by Western blot analysis using ECL (Amersham Biosciences) or Super Signal (Pierce). In Vitro Kinase Assays—The cells were incubated with lysis buffer followed by the addition of anti-IKKα antibody. The immunoprecipitates were incubated in kinase reaction buffer (20 mm HEPES, pH 7.4, 1 mm MnCl2, 5 mm MgCl2, 10 mm β-glycerolphosphate, 0.1 mm sodium orthovanadate, 2 mm NaF, and 1 mm dithiothreitol) containing 5 μCi of [γ-32P]ATP at 30 °C for 30 min (38Park K.J. Choi S.H. Lee S.Y. Hwang S.B. Lai M.M. J. Biol. Chem. 2002; 277: 13122-13128Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). Substrates used in these kinase reactions were either GST-IκBα (1 μg) containing wild type (amino acids 1–54) or mutant sequences that in residues 32 and 36 were substituted with alanine or Hsp27 (1 μg). The reaction mixtures were resolved by SDS-PAGE and then detected by autoradiography. Hsp27 Is Associated with the IKK Proteins—First we addressed whether other members of the heat shock protein family in addition to Cdc37 and Hsp90 (24Chen G. Cao P. Goeddel D.V. Mol. Cell. 2002; 9: 401-410Abstract Full Text Full Text PDF PubMed Scopus (333) Google Scholar) could also associate with the IKK complex. HeLa lysates were prepared from non-stimulated HeLa cells and immunoprecipitated with antibodies directed against specific heat shock proteins as indicated, and associated proteins were analyzed by Western blot analysis (Fig. 1). Hsp27 interacted with both IKKα and IKKβ under nonstimulated conditions (Fig. 1, lane 1), whereas Hsp90 interacted with only IKKα (Fig. 1, lane 4). Other family members including Hsp70 and Hsc70 did not interact with either IKKα or IKKβ (Fig. 1, lanes 2 and 3). Immunoprecipitation of these extracts with normal IgG followed by Western blot analysis with the IKK antibodies revealed no detectable association of these proteins, indicating the specificity of IKK interactions with Hsp27 and Hsp90 (Fig. 1, lane 5). Western blot analysis of these extracts showed equivalent expression of these proteins (Fig. 1, bottom panels). These results indicate that Hsp27, like Hsp90, can specifically associate with the IKK complex. Hsp27 Association with IKKβ Is Increased in a TNFα-dependent Manner—To determine whether the interaction of Hsp27 with IKKα and IKKβ could be altered by treatment with either TNFα or IL-1, HeLa cells were stimulated with either TNFα or IL-1 followed by immunoprecipitation with antibody directed against Hsp27 or IgG and Western blot analysis to detect IKKα or IKKβ (Fig. 2). Although both IKKα and IKKβ were associated with Hsp27 under nonstimulated conditions, the binding of Hsp27 with IKKβ was increased at both 5 and 30 min post-TNFα stimulation (Fig. 2A, lanes 2 and 3, second panel) whereas the binding of Hsp27 to IKKα did not change (Fig. 2A, top panel). In contrast to the results with TNFα treatment, the interaction of Hsp27 with IKKα and IKKβ did not change following IL-1 stimulation (Fig. 2B). Control immunoprecipitates with normal rabbit IgG did not demonstrate an association of Hsp27 with the IKKs (Fig. 2). Western blot analysis demonstrated equal expression of these proteins (Fig. 2, A and B, bottom panels). Taken together, these results, which were seen in four independent experiments, demonstrated that TNFα treatment results in increased interaction of Hsp27 with IKKβ. Hsp27 Down-regulates IKK-mediated NF-κB Activation— The role of Hsp27 in regulating TNFα-mediated NF-κB activation was addressed in transfection studies using an NF-κB luciferase reporter (Fig. 3). HeLa cells were cotransfected with an NF-κB-luciferase reporter construct using either a CMV expression vector alone or encoding Hsp27. At 24 h post-transfection, the cells were either left untreated or treated with TNFα for 6 h, and then luciferase activity was assayed. TNFα-treated cells resulted increased NF-κB activity (∼70-fold increase) as compared with the untreated cells, whereas overexpression of Hsp27 dramatically suppressed TNFα-mediated NF-κB activation (Fig. 3A). To determine whether Hsp27-mediated decreases in NF-κB activity were dependent on IKKβ, the effects of Hsp27 on IKKβ-induced NF-κB luciferase activity was tested. IKKβ increased NF-κB luciferase activity, and this increase was dramatically reduced by the expression of Hsp27 (Fig. 3B). To test the specificity the effects of Hsp27 on the NF-κB pathway, HeLa cells were cotransfected a TK-luciferase reporter construct in either the presence or absence of an Hsp27 expression vector (Fig. 3, C and D). In contrast to the results with the NF-κB reporter, Hsp27 did not alter the expression of the TK-luciferase reporter in either the presence of TNFα (Fig. 3C) or IKKβ (Fig. 3D). These results demonstrate that Hsp27 inhibits both TNFα- and IKKβ-mediated NF-κB gene expression likely via its ability to interact with IKKβ. Hsp27 siRNA Increases TNFα-mediated Activation of the NF-κB Pathway—Because our data indicated that Hsp27 overexpression could inhibit IKKβ-mediated NF-κB activation, it seemed likely that inhibition of Hsp27 expression would result in increased TNFα-mediated NF-κB activity. HeLa cells were transfected with Oligofectamine alone or siRNA directed against Hsp27 or GFP as a control. After 48 h of siRNA transfection, an NF-κB luciferase reporter construct was cotransfected along with an RSV-β-galactosidase expression vector. After an additional 24 h, the cells were stimulated with TNFα for 6 h. Hsp27 siRNA but not control or GFP siRNA up-regulated TNFα-mediated NF-κB activation and also slightly increased the basal NF-κB activity (Fig. 4A, top panel), Western blot analysis demonstrated that Hsp27 siRNA, but not control or GFP siRNA, specifically reduced the expression of Hsp27 but not actin (Fig. 4A, bottom panel). In vitro kinase assays were performed to confirm the effects of Hsp27 siRNA on TNFα-induced IKK activity (Fig. 4B). HeLa cells were transfected with Oligofectamine alone or either Hsp27 siRNA or Tax siRNA as a negative control. After 72 h post-transfection of these siRNAs, the cells were treated with TNFα for 10 min, and in vitro kinase assays were performed on the immunoprecipitated IKK complex using as substrates either wild type GST-IκBα (1–54) or mutant GST-IκBα (1–54) containing mutations of serine residues 32 and 36 to alanine. TNFα stimulation increased the level of IKK activity in both Oligofectamine and Tax siRNA transfected cells (Fig. 4B, lane 4), and there was a further increase in TNFα-mediated IKK activity in Hsp27 siRNA transfected cells (Fig. 4B, lane 5). Hsp27 siRNA, but not Tax siRNA, specifically reduced the expression of Hsp27 but not the expression of IKKα, IKKβ, or actin (Fig. 4B, bottom panels). These results demonstrate that the reduction of Hsp27 leads to increased TNFα-mediated IKK activity. Finally, we determined whether inhibition of endogenous Hsp27 expression altered TNFα-mediated IκBα degradation. Hsp27 siRNA was transfected into HeLa cells, and at 72 h post-transfection TNFα was added for the indicated times. Western blot analysis with antibody directed against the IκBα demonstrated that Hsp27 siRNA enhanced TNFα-induced degradation of IκBα as compared with Oligofectamine-treated or GFP siRNA transfected cells (Fig. 4C and data not shown). Taken together, these results indicate that Hsp27 down-regulates TNFα-induced IKK and NF-κB activity. Phosphorylation of Hsp27 Down-regulates TNFα-induced NF-κB Activation by Increasing Its Interaction with IKKβ— Previous reports demonstrated that phosphorylation of serine residues 15, 78, and 82 in Hsp27 is induced in response to heat shock and cytokine treatment (39Landry J. Lambert H. Zhou M. Lavoie J.N. Hickey E. Weber L.A. Anderson C.W. J. Biol. Chem. 1992; 267: 794-803Abstract Full Text PDF PubMed Google Scholar, 40Guesdon F. Freshney N. Waller R.J. Rawlinson L. Saklatvala J. J. Biol. Chem. 1993; 268: 4236-4243Abstract Full Text PDF PubMed Google Scholar, 41Freshney N.W. Rawlinson L. Guesdon F. Jones E. Cowley S. Hsuan J. Saklatvala J. Cell. 1994; 78: 1039-1049Abstract Full Text PDF PubMed Scopus (774) Google Scholar). Therefore, it was important to address whether TNFα-mediated phosphorylation of Hsp27 is involved in regulating IKK activity. HeLa cells were treated with TNFα for the indicated times, and the phosphorylation state of Hsp27 in these extracts was determined Western blot analysis. Hsp27 was maximally phosphorylated at 30 min post-TNFα treatment (Fig. 5A, lane 3, top panel), and this correlated with the peak of TNFα-induced binding of Hsp27 with IKKβ (Fig. 2A). IL-1 stimulated Hsp27 phosphorylation with similar kinetics as seen with TNFα (data not shown and Ref. 41Freshney N.W. Rawlinson L. Guesdon F. Jones E. Cowley S. Hsuan J. Saklatvala J. Cell. 1994; 78: 1039-1049Abstract Full Text PDF PubMed Scopus (774) Google Scholar). In contrast, maximal TNFα-induced IκBα phosphorylation occurred at 5 min post-treatment (Fig. 5A, lane 2, middle panel). To further characterize the role of Hsp27 phosphorylation on regulating its interaction with IKKβ, wild type, or Hsp27 mutants in specific phosphorylation sites at residues 15, 78, and 82 were constructed and assayed following cotransfection into HeLa cells with HA-tagged IKKβ (Fig. 5B). Following immunoprecipitation of these FLAG-tagged Hsp27 proteins, Western blot analysis was then performed to detect HA-tagged IKKβ. Wild type Hsp27 strongly associated with IKKβ, whereas the Hsp27 phosphorylation mutants S15A and S78A/S82A were unable to associate with IKKβ, even though similar amounts of these proteins were present (Fig. 5B, top panel). In contrast, a mutant that substituted aspartate for serine residues 78 and 82 to mimic Hsp27 phosphorylation resulted in similar interactions with IKKβ as wild type Hsp27 (Fig. 5B, top panel). These results indicated that phosphorylation of Hsp27 is involved in mediating its interaction with IKKβ. To determine whether phosphorylation of Hsp27 alters TNFα-mediated NF-κB activation, expression vectors encoding wild type or mutant Hsp27 proteins were analyzed for their effects or NF-κB luciferase activity in the presence and absence of TNFα treatment. Similar to the results seen in Fig. 3A, TNFα induced a ∼70-fold increase of NF-κB activity, whereas wild type Hsp27 inhibited TNFα-induced NF-κB activation (Fig. 5C). However, transfection of the Hsp27 phosphorylation mutants did not suppress TNFα-mediated NF-κB activation (Fig. 5C). There was no effect of the wild type or mutant Hsp27 constructs on the TK-luciferase construct (Fig. 5D). These results suggest that phosphorylation of Hsp27 is important in regulating its ability to modulate the TNFα-mediated NF-κB activity. TNFα-dependent Interaction of Hsp27 with IKKβ Is Disrupted by a p38 Kinase Inhibitor—Because Hsp27 is associated with IKKβ, it is possible that IKK may be involved in the phosphorylation of Hsp27. Baculovirus-purified wild type IKKβ or a kinase defective mutant (SS/AA) was tested in in vitro kinase assays using Hsp27 as the substrate (Fig. 6A). As a positive control, bacterial expressed MK2, which is a direct substrate of p38 MAPKα and β (30Stokoe D. Engel K. Campbell D.G. Cohen P. Gaestel M. FEBS Lett. 1992; 313: 307-313Crossref PubMed Scopus (469) Google Scholar, 31Ludwig S. Engel K. Hoffmeyer A. Sithanandam G. Neufeld B. Palm D. Gaestel M. Rapp U.R. Mol. Cell. Biol. 1996; 16: 6687-6697Crossref PubMed Scopus (153) Google Scholar) and has been directly demonstrated to phosphorylate Hsp27, was utilized (41Freshney N.W. Rawlinson L. Guesdon F. Jones E. Cowley S. Hsuan J. Saklatvala J. Cell. 1994; 78: 1039-1049Abstract Full Text PDF PubMed Scopus (774) Google Scholar). Consistent with previous results (41Freshney N.W. Rawlinson L. Guesdon F. Jones E. Cowley S. Hsuan J. Saklatvala J. Cell. 1994; 78: 1039-1049Abstract Full Text PDF PubMed Scopus (774) Google Scholar), MK2 but not IKKβ was found to directly phosphorylate Hsp27 (Fig. 6A, lanes 2 and 4). These results suggest that TNFα-mediated phosphorylation of Hsp27 was predominantly due to activation of the p38-MK2 cascade pathway. To extend these results, the p38-specific inhibitor SB203580 was utilized to test whether inhibition of the p38 signal pathway prevented TNFα-induced phosphorylation of Hsp27. HeLa cells were treated with either Me2SO or the p38 inhibitor, SB203580 for 1 h prior to TNFα stimulation. Immunoprecipitation assays were performed with Hsp27 antibody followed by Western blot analysis with antibodies directed against either IKKα or IKKβ (Fig. 6B). Following TNFα treatment, Hsp27 was strongly associated with IKKα in both Me2SO- and SB203580-treated HeLa cells (Fig. 6B, lanes 1–8, top panel), whereas its association with IKKβ was increased in Me2SO-but not SB203580-treated cells (Fig. 6B, lanes 1–8, second panel). The decreased association of Hsp27 with IKKβ seen in SB203580-treated cells correlated with decreased Hsp27 phosphorylation (Fig. 6B, third panel). These results indicate that the p38-MK2 cascade pathway is involved in both TNFα-mediated phosphorylation of Hsp27 and its ability to associate with IKKβ. Next, we addressed whether decreased Hsp27 phosphorylation altered IKK activity. IKK activity was enhanced in the SB203580-treated HeLa cells as compared with Me2SO-treated cells (Fig. 6C, top panel, lanes 1–8). However, treatment of cells with SB203580 reduced TNFα-mediated NF-κB luciferase reporter activity likely via inhibition of the MSK-1 kinase, which has been demonstrated to phosphorylate and thus activate p65 transactivation (data not shown and Ref. 42Vermeulen L. De Wilde G. Van Damme P. Berghe W.V. Haegeman G. EMBO J. 2003; 22: 1313-1324Crossref PubMed Scopus (638) Google Scholar). Taken together, these results indicated that p38-MK2 phosphorylation of Hsp27 increases the association of Hsp27 with IKKβ to inhibit of TNFα-mediated IKKβ activity. TNFα is a proinflammatory cytokine that is involved in diverse cellular responses that are dependent on the activation of various inducible transcription factors, among them NF-κB (4Alkalay I. Yaron A. Hatzubai A. Orian A. Ciechanover A. Ben-Neriah Y. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10599-10603Crossref PubMed Scopus (388) Google Scholar). Following exposure of cells to cytokines such as TNFα, the IKKs are activated and stimulate the NF-κB pathway (7Chen Z. Hagler J. Palombella V.J. Melandri F. Scherer D. Ballard D. Maniatis T. Genes Dev. 1995; 9: 1586-1597Crossref PubMed Scopus (1163) Google Scholar, 8Chen Z.J. Parent L. Maniatis T. Cell. 1996; 84: 853-862Abstract Full Text Full Text PDF PubMed Scopus (867) Google Scholar, 9Lee F.S. Hagler J. Chen Z.J. Maniatis T. Cell. 1997; 88: 213-222Abstract Full Text Full Text PDF PubMed Scopus (658) Google Scholar, 10Rothwarf D.M. Zandi E. Natoli G. Karin M. Nature. 1998; 395: 297-300Crossref PubMed Scopus (845) Google Scholar). Recently other proteins in addition to IKKα, IKKβ, and IKKγ/NEMO have been demonstrated to be associated with IKK complex (24Chen G. Cao P. Goeddel D.V. Mol. Cell. 2002; 9: 401-410Abstract Full Text Full Text PDF PubMed Scopus (333) Google Scholar, 43Fu D.X. Kuo Y.L. Liu B.Y. Jeang K.T. Giam C.Z. J. Biol. 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These results suggest that Hsp27 functions as a negative regulator of the NF-κB pathway likely by down-regulating IKK activity following TNFα treatment. Hsp27 is a small heat shock protein that is involved in a variety of biological activities such as chaperone function, preventing apoptosis, and stabilizing the cytoskeleton (33Rogalla T. Ehrnsperger M. Preville X. Kotlyarov A. Lutsch G. Ducasse C. Paul C. Wieske M. Arrigo A.P. Buchner J. Gaestel M. J. Biol. Chem. 1999; 274: 18947-18956Abstract Full Text Full Text PDF PubMed Scopus (619) Google Scholar, 45Mehlen P. Schulze-Osthoff K. Arrigo A.P. J. Biol. Chem. 1996; 271: 16510-16514Abstract Full Text Full Text PDF PubMed Scopus (581) Google Scholar, 46Garrido C. Ottavi P. Fromentin A. Hammann A. Arrigo A.P. Chauffert B. Mehlen P. Cancer Res. 1997; 57: 2661-2667PubMed Google Scholar). Hsp27 is ubiquitously expressed in most human cells (47Bhat S.P. Nagineni C.N. Biochem. Biophys. Res. 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A recent study demonstrated that Hsp90/Cdc37 are components of the high molecular weight IKK complex and regulate its activity via recruitment to the TNFα receptor (24Chen G. Cao P. Goeddel D.V. Mol. Cell. 2002; 9: 401-410Abstract Full Text Full Text PDF PubMed Scopus (333) Google Scholar). These results suggested that heat shock proteins are involved in regulating a critical step in IKK activation and led to our analysis of the roles of these proteins on this process. Our data indicated that Hsp27 is a negative regulator of TNFα-mediated NF-κB activity via its association with IKK. Hsp27 interaction with IKKβ is regulated by TNFα treatment but does not change following IL-1 stimulation, suggesting that Hsp27 is specifically involved in regulation of TNFα-mediated signaling (Fig. 2). Although the Hsp90/Cdc37 complex was previously demonstrated to bind to both IKKα and IKKβ (24Chen G. Cao P. Goeddel D.V. Mol. Cell. 2002; 9: 401-410Abstract Full Text Full Text PDF PubMed Scopus (333) Google Scholar), we observed that Hsp90 interacted with only IKKα (Fig. 1). This discrepancy in our analysis and a previous study could be due to differences in assay conditions. Furthermore, we found that unlike Hsp27 siRNA, which up-regulates TNFα-mediated activation of IKK and NF-κB reporter assays, Hsp90 siRNA down-regulates TNFα-mediated NF-κB activity (data not shown). These results are consistent with the role of Hsp90 as a positive regulator of the NF-κB pathway. Other heat shock family members, Hsp70 and Hsc70, did not associate with IKK complex or regulate NF-κB activity, indicating that not all members of heat shock proteins are involved this process. It is unclear why TNFα treatment increases the association of Hsp27 with IKKβ without changing its association with IKKα. Both IKKα and IKKβ can form heterodimers and homodimers, and dimerization of these kinases is critical for their kinase activity. Hsp27 could independently associate with either IKKα or IKKβ homodimers, and its association with IKKβ could block the formation of an IKKα/β-heterodimer following TNFα stimulation. Further studies will be required to better understand its role as a negative regulator of IKK activity following TNFα treatment. Hsp27 is phosphorylated by stimulation of the p38 MAPK cascade and subsequent activation of MK2 and MK3 in response to different treatments including heat shock, cytokines, growth factors, and peptide hormones (39Landry J. Lambert H. Zhou M. Lavoie J.N. Hickey E. Weber L.A. Anderson C.W. J. Biol. Chem. 1992; 267: 794-803Abstract Full Text PDF PubMed Google Scholar). Phosphorylation of Hsp27 on serine residues 15, 78, and 82 is critical for its biological role in response to environmental stress, heat shock, TNFα, and IL-1 (29Benn S.C. Perrelet D. Kato A.C. Scholz J. Decosterd I. Mannion R.J. Bakowska J.C. Woolf C.J. Neuron. 2002; 36: 45-56Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar, 33Rogalla T. Ehrnsperger M. Preville X. Kotlyarov A. Lutsch G. Ducasse C. Paul C. Wieske M. Arrigo A.P. Buchner J. Gaestel M. J. Biol. Chem. 1999; 274: 18947-18956Abstract Full Text Full Text PDF PubMed Scopus (619) Google Scholar, 52Charette S.J. Lavoie J.N. Lambert H. Landry J. Mol. Cell. Biol. 2000; 20: 7602-7612Crossref PubMed Scopus (369) Google Scholar, 53Benndorf R. Hayess K. Ryazantsev S. Wieske M. Behlke J. Lutsch G. J. Biol. Chem. 1994; 269: 20780-20784Abstract Full Text PDF PubMed Google Scholar). Our analysis demonstrated that phosphorylation of Hsp27 is important for its interaction with IKKβ following TNFα treatment. Although wild type Hsp27 interacts with IKKβ, the Hsp27 phosphorylation mutants S15A and S78A/S82A, but not S78D/S82D, were unable to efficiently bind to IKKβ (Fig. 5B) and could not suppress TNFα-mediated NF-κB activation (Fig. 5C). Finally, treatment with the p38 specific inhibitor SB203580 disrupted the association of Hsp27 with IKKβ in response to TNFα (Fig. 6B). Taken together, these data suggest that p38-MK2-mediated phosphorylation of Hsp27 increases its association with IKKβ to suppress TNFα-mediated IKK activity. The differential ability of Hsp90 and Hsp27 to serve as chaperones that bind the IKK complex can result in both positive and negative effects on TNFα-mediated activation of the NF-κB pathway. Our results indicate that p38-MK2 phosphorylation of Hsp27 increases its association with IKKβ and reduces IKK activity. The mechanism by which Hsp27 reduces IKK activity is unknown, although its ability to prevent IKKβ association with other components of IKK complex may decrease its interactions with the TNF receptor following TNFα treatment. Further studies will be needed to better define the mechanism by which Hsp27 reduces IKK activity and how cross-talk between the p38 and NF-κB pathways is involved in regulating these important signal transduction pathways. We thank Sharon Hill for preparing the manuscript and Alex Herrera for assistance with preparation of the figures.
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