Nuclear Factor κB-inducing Kinase and IκB Kinase-α Signal Skeletal Muscle Cell Differentiation

Nuclear factor κB (NF-κB)-inducing kinase (NIK), IκB kinase (IKK)-α and -β, and IκBα are common elements that signal NF-κB activation in response to diverse stimuli. In this study, we analyzed the role of this pathway during insulin-like growth factor II (IGF-II)-induced myoblast differentiation. L6E9 myoblasts differentiated with IGF-II showed an induction of NF-κB DNA-binding activity that correlated in time with the activation of IKKα, IKKβ, and NIK. Moreover, the activation of IKKα, IKKβ, and NIK by IGF-II was dependent on phosphatidylinositol 3-kinase, a key regulator of myogenesis. Adenoviral transduction with the IκBα(S32A/S36A) mutant severely impaired both IGF-II-dependent NF-κB activation and myoblast differentiation, indicating that phosphorylation of IκBα at Ser-32 and Ser-36 is an essential myogenic step. Adenoviral transfer of wild-type or kinase-deficient forms of IKKα or IKKβ revealed that IKKα is required for IGF-II-dependent myoblast differentiation, whereas IKKβ is not essential for this process. Finally, overexpression of kinase-proficient wild-type NIK showed that the activation of NIK is sufficient to generate signals that trigger myogenin expression and multinucleated myotube formation in the absence of IGF-II. Nuclear factor κB (NF-κB)-inducing kinase (NIK), IκB kinase (IKK)-α and -β, and IκBα are common elements that signal NF-κB activation in response to diverse stimuli. In this study, we analyzed the role of this pathway during insulin-like growth factor II (IGF-II)-induced myoblast differentiation. L6E9 myoblasts differentiated with IGF-II showed an induction of NF-κB DNA-binding activity that correlated in time with the activation of IKKα, IKKβ, and NIK. Moreover, the activation of IKKα, IKKβ, and NIK by IGF-II was dependent on phosphatidylinositol 3-kinase, a key regulator of myogenesis. Adenoviral transduction with the IκBα(S32A/S36A) mutant severely impaired both IGF-II-dependent NF-κB activation and myoblast differentiation, indicating that phosphorylation of IκBα at Ser-32 and Ser-36 is an essential myogenic step. Adenoviral transfer of wild-type or kinase-deficient forms of IKKα or IKKβ revealed that IKKα is required for IGF-II-dependent myoblast differentiation, whereas IKKβ is not essential for this process. Finally, overexpression of kinase-proficient wild-type NIK showed that the activation of NIK is sufficient to generate signals that trigger myogenin expression and multinucleated myotube formation in the absence of IGF-II. insulin-like growth factors phosphatidylinositol 3-kinase nuclear factor κB IκB kinase NF-κB-inducing kinase glutathione S-transferase green fluorescent protein Dulbecco's modified Eagle's medium phosphate-buffered saline The IGFs1 are the only known growth factors that are crucial to myogenesis (1Florini J.R. Ewton D.Z. Coolican S.A Endocr. Rev. 1996; 17: 481-517PubMed Google Scholar). IGF-I and IGF-II switch on the myogenic program through the IGF-I receptor (2Ewton D.Z. Falen S.L. Florini J.R Endocrinology. 1987; 120: 115-123Crossref PubMed Scopus (158) Google Scholar), activating the expression of myogenic transcription factors, cell cycle arrest, muscle-specific protein expression, and cell fusion to form multinucleated myotubes (3Andrés V. Walsh K. J. Cell Biol. 1996; 132: 657-666Crossref PubMed Scopus (505) Google Scholar, 4Olson E.N. Dev. Biol. 1992; 154: 261-272Crossref PubMed Scopus (377) Google Scholar). PI3K is an essential second messenger for myogenesis (5Kaliman P. Viñals F. Testar X. Palacı́n M. Zorzano A. J. Biol. Chem. 1996; 271: 146-151Abstract Full Text Full Text PDF Scopus (186) Google Scholar, 6Kaliman P. Canicio J. Shepherd P.R. Beeton C.A. Testar X. Palacı́n M. Zorzano A. Mol. Endocrinol. 1998; 12: 66-77Crossref PubMed Scopus (100) Google Scholar, 7Coolican S.A. Samuel D.S. Ewton D.Z. McWade F.J. Florini J.R. J. Biol. Chem. 1997; 272: 6653-6662Abstract Full Text Full Text PDF PubMed Scopus (555) Google Scholar, 8Pinset C. Garcia A. Rousse S. Dubois C. Montarras D. CR Acad. Sci. III. 1997; 320: 367-374Crossref PubMed Scopus (30) Google Scholar, 9Jiang B.-H. Aoki M. Zheng J.Z. Li J. Vogt P.K. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2077-2081Crossref PubMed Scopus (228) Google Scholar). We have recently described a myogenic signaling cascade initiated by IGF-II that leads to biochemical and morphological skeletal muscle cell differentiation and that involves PI3K activation, NF-κB activation, and inducible nitric-oxide synthase expression and activation (10Kaliman P. Canicio J. Testar X. Palacı́n M. Zorzano A. J. Biol. Chem. 1999; 274: 17437-17444Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). In this report, we further analyze the role of the NF-κB-activating signaling cascade in myogenesis. NF-κB transcription factors are key mediators of inflammatory responses, immune system functioning, transformation, oncogenesis, and anti-apoptotic signaling (11Baeuerle P. Baltimore D. Cell. 1996; 87: 13-20Abstract Full Text Full Text PDF PubMed Scopus (2935) Google Scholar, 12Baldwin Jr., A.S. Annu. Rev. Immunol. 1996; 14: 649-683Crossref PubMed Scopus (5592) Google Scholar, 13Verma I.M. Stevenson J.K. Schwartz E.M. Van Antwerp D. Miyamoto S. Genes Dev. 1995; 9: 2723-2735Crossref PubMed Scopus (1665) Google Scholar). NF-κB exists in the cytoplasm in an inactive form by virtue of its association with inhibitory proteins termed IκB (11Baeuerle P. Baltimore D. Cell. 1996; 87: 13-20Abstract Full Text Full Text PDF PubMed Scopus (2935) Google Scholar, 12Baldwin Jr., A.S. Annu. Rev. Immunol. 1996; 14: 649-683Crossref PubMed Scopus (5592) Google Scholar, 13Verma I.M. Stevenson J.K. Schwartz E.M. Van Antwerp D. Miyamoto S. Genes Dev. 1995; 9: 2723-2735Crossref PubMed Scopus (1665) Google Scholar, 14Gilmore T.D. Morin P.J. Trends Genet. 1993; 9: 427-433Abstract Full Text PDF PubMed Scopus (157) Google Scholar, 15Haskill S. Beg A.A. Tompkins S.M. Morris J.S. Yurochko A.D. Sampson-Johannes A. Mondal K. Ralph P. Baldwin Jr., A.S. Cell. 1991; 65: 1281-1289Abstract Full Text PDF PubMed Scopus (586) Google Scholar). NF-κB translocation to the nucleus and activation are most frequently achieved through the signal-induced proteolytic degradation of IκB in the cytoplasm. Two kinases, IKKα and IKKβ, which are contained in a high-molecular-weight multiprotein complex, show inducible IκB kinase activity and play a key role in NF-κB activation by a variety of stimuli (16DiDonato J.A. Hayakawa M. Rothwarf D.M. Zandi E. Karin M. Nature. 1997; 388: 548-554Crossref PubMed Scopus (1917) Google Scholar, 17Mercurio 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: 860-865Crossref PubMed Scopus (1855) Google Scholar, 18Woronicz J.D. Gao X. Cao Z. Rothe M. Goeddel D.V. Science. 1997; 278: 866-869Crossref PubMed Scopus (1068) Google Scholar, 19Zandi E. Karin M. Mol. Cell. Biol. 1999; 19: 4547-4551Crossref PubMed Scopus (307) Google Scholar). Despite their high sequence similarity, IKKα and IKKβ have different regulatory and functional roles. In mice lacking IKKβ, the activation of NF-κB by cytokines is abolished, and mouse embryos die on days 12–13 of gestation due to massive liver apoptosis (20Li Q. Antwerp D.V. Mercurio F. Lee K.F. Verma I.M. Science. 1999; 284: 321-325Crossref PubMed Scopus (857) Google Scholar). In contrast, IKKα is dispensable for pro-inflammatory responses, but plays an essential role in embryonic development. Mice lacking IKKα exhibit defective proliferation and differentiation of epidermal keratinocytes and defective limb and skeletal patterning (21Hu Y. Baud V. Delhase M. Zhang P. Deerinck T. Ellisman M. Johnson R. Karin M. Science. 1999; 284: 316-320Crossref PubMed Scopus (713) Google Scholar, 22Takeda K. Takeuchi O. Tsujimura T. Itami S. Adachi O. Kawai T. Sanjo H. Yoshikawa K. Terada N. Akira S. Science. 1999; 284: 313-316Crossref PubMed Scopus (538) Google Scholar). IKKα and IKKβ are themselves phosphorylated and activated by one or more upstream kinases, like NIK, which is a member of the mitogen-activating protein kinase kinase kinase family (23Malinin N.L. Boldin M.P. Kovalenko A.V. Wallach D. Nature. 1997; 385: 540-544Crossref PubMed Scopus (1166) Google Scholar, 24Ling L. Cao Z. Goeddel D.V. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3792-3797Crossref PubMed Scopus (450) Google Scholar, 25Lee F.S. Peters R.T. Dang L.C. Maniatis T. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9319-9324Crossref PubMed Scopus (359) Google Scholar). We report here that IκBα phosphorylation at Ser-32 and Ser-36 is required for both IGF-II-dependent NF-κB activation and differentiation in L6E9 myoblasts. We show that IKKα is involved in IGF-II-dependent multinucleated myotube formation and muscle-specific gene expression, whereas IKKβ is not essential for these processes. Our data suggest that NIK activation triggers myogenin expression and multinucleated myotube formation in the absence of IGF-II. IGF-II was kindly given by Lilly. The rat skeletal muscle cell line L6E9 was kindly provided by Dr. B. Nadal-Ginard (Harvard University). Low-passage 293 cells were from Microbix (Ontario, Canada). The PI3K inhibitor LY294002 was from BIOMOL Research Labs Inc. (Plymouth Meeting, PA). The NF-κB probe for electrophoretic mobility shift assay was kindly given by Dr. Jean-François Peyron (INSERM U364, Nice, France). The cDNAs encoding FLAG-IKKα, FLAG-IKKα(K44A), FLAG-IKKβ, and FLAG-IKKβ(K44A) were provided by Dr. D. Goeddel (Tularik, Inc.). The cDNA encoding GST-IκBα-(1–54) was provided by Dr. M. Karin (University of California, San Diego, CA). Recombinant adenoviral vectors expressing IκBα(S32A/S36A), kinase-proficient FLAG-tagged NIK, LacZ, and green fluorescent protein (GFP) were provided by Dr. Yibin Wang (University of Maryland, Baltimore, MD). Polyclonal antibodyC38320 raised against caveolin-3 was from Transduction Laboratories (Lexington, KY). Mouse monoclonal antibody MF20, which stains all sarcomeric myosin heavy chain isoforms, and mouse anti-rat myogenin monoclonal antibody F5D were from the Developmental Studies Hybridoma Bank. Polyclonal antibodies against IκBα (C-15), IKKα, IKKβ, and NIK were from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-β-actin (clone AC-15) and anti-FLAG M2 monoclonal antibodies were from Sigma. Rat L6E9 myoblasts were cultured as described previously (5Kaliman P. Viñals F. Testar X. Palacı́n M. Zorzano A. J. Biol. Chem. 1996; 271: 146-151Abstract Full Text Full Text PDF Scopus (186) Google Scholar). Subconfluent myoblasts were differentiated by serum depletion in DMEM plus antibiotics with or without IGF-II (40 nm) in the absence or presence of other compounds, as indicated for each experiment. Cells were photographed after staining the nuclei with Mayer's hemalum solution for microscopy (Merck, Darmstadt, Germany), and cell fusion was quantified by counting nuclei in myotubes from a total of at least 1000 nuclei from 10–20 randomly selected microscope fields for each condition. For adenoviral transduction, subconfluent L6E9 myoblasts were transduced at a multiplicity of 50–100 particles/cell and then cultured for an additional 36 h before inducing differentiation with or without IGF-II. To compare the impact of IKKα and IKKβ on myoblast differentiation, experimental conditions were selected to ensure similar levels of expression of IKKα and IKKβ constructs (data not shown). Recombinant adenoviruses expressing FLAG-tagged versions of either wild-type or mutant IKKα and IKKβ were by as described by and L. Mol. 1995; PubMed Scopus Google Scholar). cDNAs were the and with 293 cells to were and for protein expression after of 293 Recombinant adenoviruses were further in 293 by against and and at Y. S. Jr., J. J. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). were by 293 cells with of the and the on the cells h after was given that a multiplicity of of is required to a at In of the was to the Cells were for at in and with Cell were at for at and of the proteins was and were as described previously (5Kaliman P. Viñals F. Testar X. Palacı́n M. Zorzano A. J. Biol. Chem. 1996; 271: 146-151Abstract Full Text Full Text PDF Scopus (186) Google Scholar). Cells were in and at in solution and at was to and cells were in a at for The the was For antibodies were on protein at for h and in solution and before with the protein for h at The were in the and in kinase and were for at of and GST-IκBα-(1–54) as for NIK kinase in which of NIK was The were analyzed on and revealed by NF-κB DNA-binding activity was analyzed in total cell in and (10Kaliman P. Canicio J. Testar X. Palacı́n M. Zorzano A. J. Biol. Chem. 1999; 274: 17437-17444Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). from a were The NF-κB probe was a the of the gene were for at with of and of and in a of The probe was for a further of at The of the was by a of NF-κB probe not show activity (data not shown). Cells on were for with in in and then as in in and in were with antibodies monoclonal for h at in were with antibodies or for Cells were in and then in medium were a microscope with a IGF-II NF-κB DNA-binding activity as an during L6E9 myoblast differentiation (10Kaliman P. Canicio J. Testar X. Palacı́n M. Zorzano A. J. Biol. Chem. 1999; 274: 17437-17444Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). stimuli that the NF-κB pathway phosphorylation of the NF-κB IκBα at Ser-32 and Ser-36 as a for its degradation E. Karin M. Mol. Cell. Biol. 1999; 19: 4547-4551Crossref PubMed Scopus (307) Google Scholar). for NF-κB activation have described V. A. C. D. B. P. Peyron Cell. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, N. Karin M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: PubMed Scopus Google Scholar). To analyze the required by IGF-II for NF-κB activation during differentiation, L6E9 myoblasts were transduced with an expressing an IκBα mutant with Ser-32 and Ser-36 by mutant NF-κB in phosphorylation and degradation of L6E9 myoblasts differentiated for h with IGF-II an induction of NF-κB DNA-binding activity which was in myoblasts IκBα(S32A/S36A) with an expressing green fluorescent protein were as we analyzed the impact of IκBα phosphorylation on IGF-II-dependent myoblast differentiation. days of IGF-II the expression of muscle-specific proteins as myosin heavy chain and caveolin-3 was in myoblasts the IκBα(S32A/S36A) mutant with cells or cells transduced with whereas the expression of the protein was similar all conditions Moreover, cells the IκBα(S32A/S36A) mutant not to whereas cells showed of the nuclei in myotubes from a total of nuclei randomly data suggest that IGF-II NF-κB activation through a that involves IκBα phosphorylation to trigger skeletal muscle cell differentiation. The data indicating that IκBα phosphorylation was required by IGF-II to myoblast differentiation to analyze the activity and expression of IKKα and IKKβ during this process. L6E9 myoblasts a of IKKα activity after h in differentiation medium The of IKKα activation was with that of IGF-II-dependent NF-κB DNA-binding activation, which we have previously to dependent on PI3K activity (10Kaliman P. Canicio J. Testar X. Palacı́n M. Zorzano A. J. Biol. Chem. 1999; 274: 17437-17444Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). To analyze IGF-II-dependent IKKα activation involves L6E9 myoblast differentiation was with or without IGF-II in the absence or presence of the PI3K inhibitor The PI3K inhibitor the of IKKα to GST-IκBα-(1–54) in response to IGF-II IGF-II LY294002 IKKα protein expression the the of the IGF-II and the activation of IKKα to that IKKα was not the of the phosphorylation to the as IKKα activation by IGF-II was in the presence of indicating that on protein of the GST-IκBα-(1–54) by IKKβ was h after differentiation with IGF-II IKKβ activation by IGF-II was by the PI3K inhibitor LY294002 IGF-II LY294002 modified the of IKKβ expression in myoblasts indicating that in activity are by activation of the kinase in IKKβ protein for IKKα, the activation of IKKβ by IGF-II was by To the activation of by IGF-II is to myogenic differentiation, we adenoviral vectors expressing FLAG-tagged wild-type and or and forms of IKKα and Subconfluent L6E9 myoblasts were transduced with the different adenoviruses as a and 36 h differentiation was by the cells in an and kinase activity of the transduced proteins were h after inducing differentiation with IGF-II. Cells transduced with GST-IκBα-(1–54) phosphorylation activity in with an anti-FLAG monoclonal whereas IκBα phosphorylation activity was in from cells transduced either with or these similar levels of wild-type and forms of IKKα were as by anti-FLAG antibody days of differentiation in the presence of cells as by caveolin-3 and myosin heavy chain expression, with cells transduced with either or The expression of the protein was not by overexpression To analyze the role of IKKβ activity in myoblast differentiation, cells were transduced with or proteins were to similar levels as on anti-FLAG and cells IκBα kinase activity on anti-FLAG In to IKKα, IKKβ not to play an essential role in myoblast differentiation, as cells transduced with skeletal muscle-specific proteins as as cells or cells transduced with or the morphological overexpression of FLAG-IKKβ(K44A) not the of myoblasts to in response to IGF-II of nuclei in myotubes from a total of nuclei randomly with cells total of cells transduced with total of or cells transduced with total of show of nuclei in was only myotubes were with the myotubes in cells transduced with or cells transduced with after days in differentiation only of the nuclei of in cells were in myotubes with the of the nuclei from cells of were in myotubes with these suggest that IKKα plays a role in IGF-II-dependent morphological and biochemical differentiation of skeletal muscle whereas IKKβ is not essential to this process. NIK is a common in the NF-κB signaling and IKKα to a IKKβ for phosphorylation by NIK L. Cao Z. Goeddel D.V. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3792-3797Crossref PubMed Scopus (450) Google Scholar). The kinase activity and protein expression of NIK in L6E9 myoblasts were of NIK activity was after h in differentiation medium for NF-κB DNA-binding activation (10Kaliman P. Canicio J. Testar X. Palacı́n M. Zorzano A. J. Biol. Chem. 1999; 274: 17437-17444Abstract Full Text Full Text PDF PubMed Scopus (113) Google and IKKα and IKKβ the activation of NIK was by indicating that IGF-II PI3K to NIK in myoblasts were in NIK protein expression in response to IGF-II or LY294002 for IKKα and IKKβ, NIK activation by IGF-II was by indicating that protein LY294002 NIK activation only was with IGF-II during the In contrast, LY294002 was during the h or the h of IGF-II or h of differentiation, of NIK activity was suggest that PI3K is most involved in the of the required for NIK activation a upstream of the To the activation of NIK by IGF-II was a key during differentiation, we transduced L6E9 myoblasts with expressing kinase-proficient FLAG-tagged wild-type NIK that a high of activity on anti-FLAG or the was with antibodies in cells transduced with is due to of NIK by the days in the absence of IGF-II myoblasts with from a total of nuclei at were in multinucleated In contrast, transduction with myoblast fusion of nuclei in myotubes from a total of nuclei at showed that cells myogenin in their nuclei The of nuclei expressing myogenin was in cells NIK in cells randomly selected fields were analyzed from each of with each in We a pathway by which IGF-II skeletal muscle cell differentiation through activation of the The activation of the NF-κB cascade IKKα, and IKKβ IκB and NF-κB DNA-binding was h after subconfluent myoblasts in differentiation are during the differentiation program by but in their with the activation of this which of of cells to cytokines or other stimuli. that not a of the phosphorylation an by the that and NIK activation protein The of the by IGF-II to trigger NF-κB cascade activation during myogenesis the to is that IGF-II the of an factor by myoblasts. In this we not expression of of by chain in myoblasts to by IGF-II (data not shown). is that the protein is a kinase that a for IGF-II or for an factor in response to IGF-II. The of NF-κB in myogenic signaling described in and embryonic myoblasts (10Kaliman P. Canicio J. Testar X. Palacı́n M. Zorzano A. J. Biol. Chem. 1999; 274: 17437-17444Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, D.B. J. Biol. Chem. Full Text PDF PubMed Google Scholar, J. 1997; PubMed Scopus Google Scholar), differentiation of skeletal muscle cells in a medium to through a signaling cascade in which NF-κB plays a regulatory role C. C. Baldwin Jr., A.S. Mol. Cell. Biol. 1999; 19: PubMed Google Scholar). We have previously that and inducible nitric-oxide synthase are elements of a common myogenic cascade in which IGF-II through a a in IκBα protein that with a in the of IκBα with NF-κB DNA-binding activation, and (10Kaliman P. Canicio J. Testar X. Palacı́n M. Zorzano A. J. Biol. Chem. 1999; 274: 17437-17444Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). PI3K is a key of myogenesis (5Kaliman P. Viñals F. Testar X. Palacı́n M. Zorzano A. J. Biol. Chem. 1996; 271: 146-151Abstract Full Text Full Text PDF Scopus (186) Google Scholar, 6Kaliman P. Canicio J. Shepherd P.R. Beeton C.A. Testar X. Palacı́n M. Zorzano A. Mol. Endocrinol. 1998; 12: 66-77Crossref PubMed Scopus (100) Google Scholar, 7Coolican S.A. Samuel D.S. Ewton D.Z. McWade F.J. Florini J.R. J. Biol. Chem. 1997; 272: 6653-6662Abstract Full Text Full Text PDF PubMed Scopus (555) Google Scholar, 8Pinset C. Garcia A. Rousse S. Dubois C. Montarras D. CR Acad. Sci. III. 1997; 320: 367-374Crossref PubMed Scopus (30) Google Scholar, 9Jiang B.-H. Aoki M. Zheng J.Z. Li J. Vogt P.K. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2077-2081Crossref PubMed Scopus (228) Google Scholar), and the role of PI3K in NF-κB cascade activation during myogenesis is by data here that activation of both NIK and by IGF-II in myoblasts is by PI3K is known to involved in the activation of NF-κB in like anti-apoptotic growth factor signaling J.A. Nature. 1999; PubMed Scopus Google and immune and inflammatory J.A. D.B. Nature. 1999; PubMed Scopus Google Scholar). in of PI3K not to NIK phosphorylation NIK activation only was with IGF-II during the but not during the h or the data suggest that PI3K is most involved in the of required for NIK activation an upstream in the activation of NIK. In contrast, not a role for in the of a factor in NIK activation by the protein that of the in only through a the stimuli that IKKβ and the that its activity are the stimuli and the for IKKα are IKKα and IKKβ to different and roles. revealed that IKKα is not involved in the activation of NF-κB by pro-inflammatory is involved in (20Li Q. Antwerp D.V. Mercurio F. Lee K.F. Verma I.M. Science. 1999; 284: 321-325Crossref PubMed Scopus (857) Google Scholar, Y. Baud V. Delhase M. Zhang P. Deerinck T. Ellisman M. Johnson R. Karin M. Science. 1999; 284: 316-320Crossref PubMed Scopus (713) Google Scholar, 22Takeda K. Takeuchi O. Tsujimura T. Itami S. Adachi O. Kawai T. Sanjo H. Yoshikawa K. Terada N. Akira S. Science. 1999; 284: 313-316Crossref PubMed Scopus (538) Google Scholar). In this show that IGF-II both IKKα and IKKβ during the differentiation the overexpression of a kinase-deficient mutant of IKKβ not the expression of muscle-specific proteins or the formation of multinucleated The differentiation by a kinase-deficient mutant of IKKα, that IKKβ for IKKα in myogenic skeletal muscle IKKβ, whereas is one of the with the expression levels of IKKα E. Rothwarf D.M. Delhase M. Hayakawa M. Karin M. Cell. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). differentiation in and the and of during myogenesis are in the phosphorylation of the cell cycle regulatory protein in skeletal myoblasts N. J. Cell Biol. 1996; PubMed Scopus Google Scholar, S.M. Proc. Natl. Acad. Sci. U. S. A. 1995; PubMed Scopus Google Scholar). from the cell a of proliferation in which NF-κB is required to expression and E. Rothwarf D.M. Delhase M. Hayakawa M. Karin M. Cell. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, S.M. Proc. Natl. Acad. Sci. U. S. A. 1995; PubMed Scopus Google Scholar, M. D. A. A. C. M. Mol. Cell. Biol. 1999; 19: PubMed Scopus Google Scholar). a in NF-κB activity by a in levels to required to the from the cell cycle that differentiation (3Andrés V. Walsh K. J. Cell Biol. 1996; 132: 657-666Crossref PubMed Scopus (505) Google P. M. P. A. 1996; PubMed Scopus Google Scholar). NF-κB and myogenesis was without that the NF-κB activity after h in differentiation medium was that during a myogenic role P. M. P. A. 1996; PubMed Scopus Google Scholar). this to the with data NF-κB activity was to required by and myoblasts to (10Kaliman P. Canicio J. Testar X. Palacı́n M. Zorzano A. J. Biol. Chem. 1999; 274: 17437-17444Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, J.A. Nature. 1999; PubMed Scopus Google Scholar, J.A. D.B. Nature. 1999; PubMed Scopus Google Scholar). We show here that IGF-II-dependent differentiation triggers a induction of the NF-κB-activating which PI3K activity and of Our data suggest that the activation of NIK and IKKα and the phosphorylation of IκBα at Ser-32 and Ser-36 are key in skeletal muscle differentiation by IGF-II. We Yibin Wang and M. (University of at San Diego, for in and M. and S. of for on microscopy and for

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