Heat Shock Protein 27 Association with the IκB Kinase Complex Regulates Tumor Necrosis Factor α-induced NF-κB Activation

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. 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. Genes Dev. 1999; 13: 505-510Crossref PubMed Scopus (368) Google Scholar, 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, 5Beg A.A. Finco T.S. Nantermet P.V. Baldwin Jr., A.S. Mol. Cell. Biol. 1993; 13: 3301-3310Crossref PubMed Google Scholar, 6Brown K. Gerstberger S. Carlson L. Franzoso G. Siebenlist U. Science. 1995; 267: 1485-1488Crossref PubMed Scopus (1311) Google Scholar). The IKK complex includes two catalytic subunits, IKKα and IKKβ, in addition to a regulatory subunit IKKγ/NEMO. These proteins are components of a 700–900-kDa complex whose activity is increased by cytokines including TNFα and IL-1 to result in increased IκB phosphorylation (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. 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IKKβ, which has higher activity for IκB than does IKKα, is the dominant kinase involved in cytokine-mediated activation of the NF-κB pathway (14Lee F.S. Peters R.T. Dang L.C. Maniatis T. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9319-9324Crossref PubMed Scopus (354) Google Scholar, 15Mercurio F. Zhu H. Murray B.W. Shevchenko A. Bennett B.L. Li J. Young D.B. Barbosa M. Mann M. Manning A. Rao A. Science. 1997; 278: 860-866Crossref PubMed Scopus (1841) Google Scholar, 16Yin M.J. Christerson L.B. Yamamoto Y. Kwak Y.T. Xu S. Mercurio F. Barbosa M. Cobb M.H. Gaynor R.B. Cell. 1998; 93: 875-884Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar, 17Yin M.J. Yamamoto Y. Gaynor R.B. Nature. 1998; 396: 77-80Crossref PubMed Scopus (1421) Google Scholar). Studies with knock-out mice have confirmed the role of IKKβ as the dominant kinase in regulating cytokine-activation of the NF-κB pathway. 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). In mammalian cells, the small heat shock proteins, which include the AB-crystallins and Hsp27, are oligomeric phosphoproteins whose expression is regulated at both the transcriptional and post-translational levels in a cell type- and tissue-specific manner (26Arrigo AP M.P. Morimoto R. Georgopoulos T.A. The Biology of Heat Shock Proteins and Molecular Chaperones. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1994: 335Google Scholar). The expression of Hsp27 confers cellular resistance to a variety of stimuli including both physical and chemical stress and cytokine treatment (26Arrigo AP M.P. Morimoto R. Georgopoulos T.A. The Biology of Heat Shock Proteins and Molecular Chaperones. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1994: 335Google Scholar, 27Arrigo A.P. Biol. Chem. 1998; 379: 19-26PubMed Google Scholar, 28Mehlen P. Hickey E. Weber L.A. Arrigo A.P. Biochem. Biophys. Res. Commun. 1997; 241: 187-192Crossref PubMed Scopus (198) Google Scholar, 29Benn S.C. 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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 and in with and cells with of a expression Hsp27, of an and of an cells stimulated with TNFα IL-1 for and was in to a of and and IL-1 Science. and and was and as as and and Cell and MAPK-activated protein kinase kinase 2 and Hsp27 proteins the of expression Hsp27, this was by of cell and into the and of The expression Hsp27 phosphorylation the of the confirmed by interfering with two at the of the for Hsp27 and their as J. W. A. Weber K. T. Nature. 2001; PubMed Scopus Google Scholar) These in and as and at following to in and 2 Transfection of cells on in to and of the was as Gaynor R.B. J. 2002; PubMed Scopus Google Scholar, G. J. K. Gaynor R.B. J. Mol. Biol. PubMed Scopus Google Scholar). the cells resulting in a of the cells with IL-1 for the times and in 2 and with inhibitor and used in and K.L. Mol. Cell. Biol. 2002; 22: PubMed Scopus Google Scholar). and cell with of Hsp27 for and for 2 following the addition of of protein A The by to a and by In cells with by the addition of The in kinase 2 and of at for Lee Hwang J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). used in these kinase that in and with Hsp27 The by and by Hsp27 with the IKK we other 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) also associate with the IKK cells and with specific heat shock proteins as and associated proteins by Hsp27 with both IKKα and IKKβ Hsp90 with IKKα family including and with IKKα IKKβ 2 and of these with by with the IKK association of these proteins, the of IKK with Hsp27 and Hsp90 of these expression of these proteins These results indicate that Hsp27, Hsp90, can associate with the IKK Hsp27 with IKKβ in a the interaction of Hsp27 with IKKα and IKKβ be by treatment with TNFα cells stimulated with TNFα IL-1 by with Hsp27 and to IKKα IKKβ both IKKα and IKKβ associated with Hsp27 the of Hsp27 with IKKβ was increased at both and 2 and the of Hsp27 to IKKα In to the results with TNFα the interaction of Hsp27 with IKKα and IKKβ following IL-1 with an association of Hsp27 with the demonstrated expression of these proteins 2, A and these which in demonstrated that TNFα treatment results in increased interaction of Hsp27 with Hsp27 NF-κB The role of Hsp27 in regulating TNFα-mediated NF-κB activation was in studies an NF-κB cells with an a expression the cells with TNFα for and activity was cells resulted increased NF-κB activity as with the cells, of Hsp27 TNFα-mediated NF-κB activation in NF-κB activity on IKKβ, the of Hsp27 on NF-κB activity was IKKβ increased NF-κB and this was reduced by the expression of Hsp27 the the of Hsp27 on the NF-κB cells a in the of an Hsp27 expression and In to the results with the NF-κB Hsp27 the expression of the in the of TNFα IKKβ These results that Hsp27 both and NF-κB expression its to with Hsp27 TNFα-mediated of the NF-κB that Hsp27 NF-κB activation, that of Hsp27 expression result in increased TNFα-mediated NF-κB activity. cells with Hsp27 as a of an NF-κB was with an expression an the cells stimulated with TNFα for Hsp27 TNFα-mediated NF-κB activation and also increased the NF-κB activity demonstrated that Hsp27 siRNA, siRNA, reduced the expression of Hsp27 actin In kinase to the of Hsp27 on IKK activity cells with Hsp27 as a negative of these the cells with TNFα for and in kinase on the IKK complex as of and to TNFα increased the of IKK activity in both and cells and was a in TNFα-mediated IKK activity in Hsp27 cells Hsp27 siRNA, siRNA, reduced the expression of Hsp27 the expression of IKKα, IKKβ, actin These results that the of Hsp27 leads to increased TNFα-mediated IKK activity. we of Hsp27 expression TNFα-mediated Hsp27 was into cells, and at TNFα was for the with the demonstrated that Hsp27 enhanced degradation of as with cells and these results indicate that Hsp27 IKK and NF-κB activity. Phosphorylation of Hsp27 NF-κB by with Previous demonstrated that phosphorylation of and in Hsp27 is induced in response to heat shock and cytokine treatment J. H. M. Hickey E. Weber L.A. J. Biol. Chem. 1992; 267: Full Text PDF PubMed Google Scholar, F. R.J. L. J. J. Biol. Chem. 1993; Full Text PDF PubMed Google Scholar, L. F. E. S. J. J. Cell. Full Text PDF PubMed Scopus Google Scholar). was important to TNFα-mediated phosphorylation of Hsp27 is involved in regulating IKK activity. cells with TNFα for the and the phosphorylation of Hsp27 in these was Hsp27 was phosphorylated at treatment and this with the of of Hsp27 with IKKβ IL-1 stimulated Hsp27 phosphorylation with similar as with TNFα and L. F. E. S. J. J. Cell. Full Text PDF PubMed Scopus Google Scholar). In phosphorylation at 2, the role of Hsp27 phosphorylation on regulating its interaction with IKKβ, Hsp27 in specific phosphorylation at and and following into cells with IKKβ of these Hsp27 proteins, was to Hsp27 associated with IKKβ, the Hsp27 phosphorylation and to associate with IKKβ, similar of these proteins In a that for and to Hsp27 phosphorylation resulted in similar with IKKβ as Hsp27 These results that phosphorylation of Hsp27 is involved in its interaction with phosphorylation of Hsp27 TNFα-mediated NF-κB activation, expression Hsp27 proteins for their NF-κB activity in the and of TNFα treatment. to the results in TNFα induced a of NF-κB Hsp27 NF-κB activation However, of the Hsp27 phosphorylation TNFα-mediated NF-κB activation was of the Hsp27 on the These results that phosphorylation of Hsp27 is important in regulating its to the TNFα-mediated NF-κB activity. of Hsp27 with IKKβ by a p38 Hsp27 is associated with IKKβ, is that IKK may be involved in the phosphorylation of IKKβ a kinase was in in kinase Hsp27 as the substrate a expressed which is a substrate of p38 and (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 demonstrated to Hsp27, was L. F. E. S. J. J. Cell. Full Text PDF PubMed Scopus Google Scholar). Consistent with results L. F. E. S. J. J. Cell. Full Text PDF PubMed Scopus Google Scholar), IKKβ was found to Hsp27 2 and These results that TNFα-mediated phosphorylation of Hsp27 was to activation of the p38-MK2 pathway. these the inhibitor was to of the p38 signal pathway phosphorylation of cells with the p38 for to TNFα stimulation. with Hsp27 by with IKKα IKKβ TNFα Hsp27 was associated with IKKα in both and cells its association with IKKβ was increased in cells The decreased association of Hsp27 with IKKβ in cells with decreased Hsp27 phosphorylation These results indicate that the p38-MK2 pathway is involved in both TNFα-mediated phosphorylation of Hsp27 and its to associate with we decreased Hsp27 phosphorylation IKK activity. IKK activity was enhanced in the cells as with cells However, treatment of cells with reduced TNFα-mediated NF-κB activity of the which has been demonstrated to and thus and L. G. Van P. G. J. 22: PubMed Scopus Google Scholar). these results that p38-MK2 phosphorylation of Hsp27 the association of Hsp27 with IKKβ to of TNFα-mediated IKKβ activity. TNFα is a cytokine that is involved in diverse cellular responses that are on the activation of various NF-κB 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). of cells to cytokines as the are and 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). other proteins in addition to IKKα, IKKβ, and 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, J. Biol. Chem. 278: Full Text Full Text PDF PubMed Scopus Google Scholar, E. F. C. B. A. M. L. R.T. L. J. Immunol. PubMed Scopus Google Scholar). However, is these proteins IKK activity in a negative manner and TNFα-mediated activation of different signal transduction pathway is involved in these In this study, we that the Hsp27 TNFα-mediated NF-κB activity. Hsp27 association with IKKβ is increased in a Hsp27 both and NF-κB activation, and Hsp27 this negative on NF-κB the association of Hsp27 with IKK is by its phosphorylation as demonstrated Hsp27 phosphorylation and the inhibitor These results that Hsp27 functions as a negative regulator of the NF-κB pathway by down-regulating IKK activity following TNFα treatment. Hsp27 is a small heat shock protein that is involved in a variety of biological as and the 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, P. K. Arrigo A.P. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, C. P. A. A. Arrigo A.P. B. P. Res. 1997; Google Scholar). Hsp27 is ubiquitously expressed in most cells Biochem. Biophys. Res. Commun. PubMed Scopus Google Scholar, I. 1993; PubMed Scopus Google Scholar), and its expression is increased to high levels in tumor cells, including and L.A. J.C. 1997; Google Scholar, J. Natl. PubMed Scopus Google Scholar, H. K. H. M. A. S. Cell. Mol. 1995; PubMed Scopus Google Scholar). Hsp70, and Hsp90, Hsp27 is an A recent study demonstrated that are components of the high IKK complex and its activity to the TNFα (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 in IKK activation and to of the of these proteins on this that Hsp27 is a negative regulator of TNFα-mediated NF-κB activity its association with Hsp27 interaction with IKKβ is regulated by TNFα treatment does following IL-1 that Hsp27 is involved in of TNFα-mediated signaling the complex was demonstrated to 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 that Hsp90 with IKKα in and a study be to in we found that Hsp27 siRNA, which TNFα-mediated activation of IKK and NF-κB Hsp90 TNFα-mediated NF-κB activity These results are with the role of Hsp90 as a regulator of the NF-κB pathway. heat shock family and associate with IKK complex NF-κB that of heat shock proteins are involved this is TNFα treatment the association of Hsp27 with IKKβ its association with Both IKKα and IKKβ can and and of these kinases is critical for their kinase activity. Hsp27 associate with IKKα IKKβ and its association with IKKβ the of an following TNFα stimulation. studies be to its role as a negative regulator of IKK activity following TNFα treatment. Hsp27 is phosphorylated by of the p38 MAPK and activation of and in response to different including heat shock, growth and J. H. M. Hickey E. Weber L.A. J. Biol. Chem. 1992; 267: Full Text PDF PubMed Google Scholar). Phosphorylation of Hsp27 on and is critical for its biological role in response to stress, heat shock, and IL-1 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, S.J. H. Landry J. Mol. Cell. Biol. PubMed Scopus Google Scholar, R. K. S. Wieske M. J. Lutsch G. J. Biol. Chem. Full Text PDF PubMed Google Scholar). demonstrated that phosphorylation of Hsp27 is important for its interaction with IKKβ following TNFα treatment. Hsp27 with IKKβ, the Hsp27 phosphorylation and to to IKKβ and TNFα-mediated NF-κB activation treatment with the p38 specific inhibitor disrupted the association of Hsp27 with IKKβ in response to TNFα these that phosphorylation of Hsp27 its association with IKKβ to TNFα-mediated IKK activity. The of Hsp90 and Hsp27 to as that the IKK complex can result in both and negative on TNFα-mediated activation of the NF-κB pathway. results indicate that p38-MK2 phosphorylation of Hsp27 its association with IKKβ and IKK activity. The by which Hsp27 IKK activity is although its to IKKβ association with other components of IKK complex may decrease its with the following TNFα treatment. studies be to the by which Hsp27 IKK activity and the p38 and NF-κB pathways is involved in regulating these important signal transduction for the and for with of the