Tumor Necrosis Factor-regulated Biphasic Activation of NF-κB Is Required for Cytokine-induced Loss of Skeletal Muscle Gene Products

NF-κB activation is classically defined as a transient response initiated by the degradation of IκB inhibitor proteins leading to nuclear import of NF-κB and culminating with the resynthesis of IκBα and subsequent inactivation of the transcription factor. Although this type of regulation is considered the paradigm for NF-κB activation, other regulatory profiles are known to exist. By far the most common of these is chronic or persistent activation of NF-κB. In comparison, regulation of NF-κB in a biphasic manner represents a profile that is scarcely documented and whose biological significance remains poorly understood. Here we show using differentiated skeletal muscle cells, that tumor necrosis factor (TNF) induces NF-κB activation in a biphasic manner. Unlike the first transient phase, which is terminated within 1 h of cytokine addition, the second phase persists for an additional 24–36 h. Biphasic activation is mediated at both the levels of NF-κB DNA binding and transactivation function, and both phases are dependent on the IKK/26 S proteasome pathway. We find that regulation of the first transient phase is mediated by the degradation and subsequent resynthesis of IκBα, as well as by a TNF-induced expression of A20. Second phase activity correlates with persistent down-regulation of both IκBα and IκBβ proteins, derived from a continuous TNF signal. Finally, we demonstrate that inhibition of NF-κB prior to initiation of the second phase of activity inhibits cytokine-mediated loss of muscle proteins. We propose that the biphasic activation of NF-κB in response to TNF may play a key regulatory role in skeletal muscle wasting associated with cachexia. NF-κB activation is classically defined as a transient response initiated by the degradation of IκB inhibitor proteins leading to nuclear import of NF-κB and culminating with the resynthesis of IκBα and subsequent inactivation of the transcription factor. Although this type of regulation is considered the paradigm for NF-κB activation, other regulatory profiles are known to exist. By far the most common of these is chronic or persistent activation of NF-κB. In comparison, regulation of NF-κB in a biphasic manner represents a profile that is scarcely documented and whose biological significance remains poorly understood. Here we show using differentiated skeletal muscle cells, that tumor necrosis factor (TNF) induces NF-κB activation in a biphasic manner. Unlike the first transient phase, which is terminated within 1 h of cytokine addition, the second phase persists for an additional 24–36 h. Biphasic activation is mediated at both the levels of NF-κB DNA binding and transactivation function, and both phases are dependent on the IKK/26 S proteasome pathway. We find that regulation of the first transient phase is mediated by the degradation and subsequent resynthesis of IκBα, as well as by a TNF-induced expression of A20. Second phase activity correlates with persistent down-regulation of both IκBα and IκBβ proteins, derived from a continuous TNF signal. Finally, we demonstrate that inhibition of NF-κB prior to initiation of the second phase of activity inhibits cytokine-mediated loss of muscle proteins. We propose that the biphasic activation of NF-κB in response to TNF may play a key regulatory role in skeletal muscle wasting associated with cachexia. nuclear factor κB tumor necrosis factor α interleukin lipopolysaccharide interferon γ nuclear localization sequence IkBα super repressor IκB kinase myosin heavy chain differentiation medium electrophoretic mobility shift assay reverse transcription polymerase chain reaction phosphate-buffered saline Rel/NF-κB (NF-κB)1 is a dynamic transcription factor family involved in the regulation of innate immune response, cellular proliferation and differentiation, and cell survival (1Baldwin A.S., Jr. Annu. Rev. Imm. 1996; 14: 649-683Crossref PubMed Scopus (5544) Google Scholar, 2Ghosh S. May M.J. Kopp E.B. Annu. Rev. Immunol. 1998; PubMed Scopus Google Scholar, S. PubMed Scopus Google In this family of and proteins are by a in the which DNA and the nuclear localization (1Baldwin A.S., Jr. Annu. Rev. Imm. 1996; 14: 649-683Crossref PubMed Scopus (5544) Google Scholar, 2Ghosh S. May M.J. Kopp E.B. Annu. Rev. Immunol. 1998; PubMed Scopus Google Scholar, S. PubMed Scopus Google family proteins that a transactivation at and and that and Although in of these proteins the to or the of NF-κB of the In most cells, the of NF-κB in the to the IκB which IκBα, and proteins as which to the of NF-κB and the NF-κB nuclear (1Baldwin A.S., Jr. Annu. Rev. Imm. 1996; 14: 649-683Crossref PubMed Scopus (5544) Google Scholar, 2Ghosh S. May M.J. Kopp E.B. Annu. Rev. Immunol. 1998; PubMed Scopus Google Scholar, S. PubMed Scopus Google of NF-κB is mediated the IκB kinase which to on IκB proteins Annu. Rev. PubMed Scopus Google of these the and subsequent degradation of IκB proteins by the S proteasome Annu. Rev. PubMed Scopus Google loss of NF-κB is to to the to DNA sequence and with the transcription and to expression S. 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Immunol. 1998; PubMed Scopus Google Scholar, S. PubMed Scopus Google family proteins that a transactivation at and and that and Although in of these proteins the to or the of NF-κB of the In most cells, the of NF-κB in the to the IκB which IκBα, and proteins as which to the of NF-κB and the NF-κB nuclear (1Baldwin A.S., Jr. Annu. Rev. Imm. 1996; 14: 649-683Crossref PubMed Scopus (5544) Google Scholar, 2Ghosh S. May M.J. Kopp E.B. Annu. Rev. Immunol. 1998; PubMed Scopus Google Scholar, S. PubMed Scopus Google of NF-κB is mediated the IκB kinase which to on IκB proteins Annu. Rev. PubMed Scopus Google of these the and subsequent degradation of IκB proteins by the S proteasome Annu. Rev. PubMed Scopus Google loss of NF-κB is to to the to DNA sequence and with the transcription and to expression S. PubMed Scopus Google of the by NF-κB is IκBα PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, S. S. 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PubMed Scopus Google Scholar, PubMed Scopus Google with this that the of skeletal muscle with TNF and down-regulation of A.S., Jr. PubMed Scopus Google cytokine-mediated muscle loss in NF-κB that this transcription factor may as a of skeletal muscle on the role of NF-κB in muscle the to the regulation of NF-κB in response to TNF and in differentiated skeletal on to to with TNF to NF-κB to a for muscle differentiated muscle that TNF-induced activation of that the of TNF and to muscle loss from the of NF-κB in the of this we that of with TNF a and biphasic activity of NF-κB. In to the first phase, which the activity of the second phase an additional 24–36 h TNF type of NF-κB activity profile is the by a transient from the chronic or persistent activity of NF-κB. in this differentiated skeletal muscle to the biphasic activation of NF-κB in response to and to biological significance with to muscle of this on that skeletal muscle loss on the of TNF and A.S., Jr. PubMed Scopus Google that NF-κB activity for to muscle A.S., Jr. PubMed Scopus Google NF-κB in muscle we to first these to NF-κB in differentiated Unlike the of TNF and on NF-κB in and cell A.S., Jr. PubMed Google demonstrate that of TNF-induced activity of NF-κB. in the of this we that in skeletal NF-κB activity in a biphasic manner by In to the first phase of NF-κB which the second phase as and to TNF other known of or this biphasic that this regulation on NF-κB may to NF-κB and that NF-κB is an transcription the is as an the by IκB (1Baldwin A.S., Jr. Annu. Rev. Imm. 1996; 14: 649-683Crossref PubMed Scopus (5544) Google Scholar, 2Ghosh S. May M.J. Kopp E.B. Annu. Rev. Immunol. 1998; PubMed Scopus Google Scholar, S. PubMed Scopus Google of by a of to degradation of IκB proteins, and subsequent nuclear of NF-κB. well that activation of by most is a transient response, within by a to levels within h. transient of NF-κB activity is in to the resynthesis of IκBα PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, S. S. PubMed Scopus Google which and NF-κB from the to the PubMed Google Scholar, S. S. 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In the of both IκBα and IκBβ proteins are In addition, of TNF from in the of this second phase and levels of IκB proteins. the that regulation of both IκBα and IκBβ proteins the of the second phase of NF-κB that down-regulation of IκBβ in the of the second phase of NF-κB activity the of degradation this regulatory of an NF-κB inhibitor of IκBβ to to the role of this IκB in the first and second phases of NF-κB. factor the second activation phase may to the expression of A20. in to the biphasic expression with an NF-κB and the IκBα expression to In of that of in to chronic down-regulation of IκBα and persistent activity of NF-κB S. PubMed Scopus Google is to that the persistent second phase of NF-κB activity from the of these remains at this and TNF in skeletal to muscle to TNF we that the biphasic activation of NF-κB is in for both TNF and to the loss of muscle biphasic activation of NF-κB that with TNF levels of TNF are associated with chronic Annu. Rev. of PubMed Scopus Google that the biphasic regulation of NF-κB is to a TNF levels in the as is the in Rev. PubMed Scopus Google that the of TNF to NF-κB that the by these as a of activation of as NF-κB for TNF and for 1996; PubMed Scopus Google in other Immunol. Google these may on a common to the expression of a that may a of skeletal muscle wasting associated in cachexia. of this on that skeletal muscle loss on the of TNF and A.S., Jr. PubMed Scopus Google that NF-κB activity for to muscle A.S., Jr. PubMed Scopus Google NF-κB in muscle we to first these to NF-κB in differentiated Unlike the of TNF and on NF-κB in and cell A.S., Jr. PubMed Google demonstrate that of TNF-induced activity of NF-κB. in the of this we that in skeletal NF-κB activity in a biphasic manner by In to the first phase of NF-κB which the second phase as and to TNF other known of or this biphasic that this regulation on NF-κB may to NF-κB and that NF-κB is an transcription the is as an the by IκB (1Baldwin A.S., Jr. Annu. Rev. Imm. 1996; 14: 649-683Crossref PubMed Scopus (5544) Google Scholar, 2Ghosh S. May M.J. Kopp E.B. Annu. Rev. Immunol. 1998; PubMed Scopus Google Scholar, S. PubMed Scopus Google of by a of to degradation of IκB proteins, and subsequent nuclear of NF-κB. well that activation of by most is a transient response, within by a to levels within h. transient of NF-κB activity is in to the resynthesis of IκBα PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, S. S. PubMed Scopus Google which and NF-κB from the to the PubMed Google Scholar, S. S. PubMed Scopus Google on the cell other activation profiles of NF-κB By far the most common of these profiles is the persistent or activity of NF-κB that NF-κB activity to and S. 1996; PubMed Google a in with In these cells, the persistent activity of NF-κB derived from binding to a of which NF-κB from IκBα binding and NF-κB nuclear of by IκBβ to the persistent activity of NF-κB in with the PubMed Scopus Google to for a persistent the type 1 to and the in chronic NF-κB activity PubMed Scopus Google PubMed Scopus Google In cells, that a for the chronic degradation of IκBα, leading to levels of activity S. Immunol. 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In the of both IκBα and IκBβ proteins are In addition, of TNF from in the of this second phase and levels of IκB proteins. the that regulation of both IκBα and IκBβ proteins the of the second phase of NF-κB that down-regulation of IκBβ in the of the second phase of NF-κB activity the of degradation this regulatory of an NF-κB inhibitor of IκBβ to to the role of this IκB in the first and second phases of NF-κB. factor the second activation phase may to the expression of A20. in to the biphasic expression with an NF-κB and the IκBα expression to In of that of in to chronic down-regulation of IκBα and persistent activity of NF-κB S. PubMed Scopus Google is to that the persistent second phase of NF-κB activity from the of these remains at this and TNF in skeletal to muscle to TNF we that the biphasic activation of NF-κB is in for both TNF and to the loss of muscle biphasic activation of NF-κB that with TNF levels of TNF are associated with chronic Annu. 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