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TAK-1/p38/nNFκB signaling inhibits myoblast differentiation by increasing levels of Activin A.

Trendelenburg AU, Meyer A, Jacobi C, Feige JN, Glass DJ - Skelet Muscle (2012)

Bottom Line: This anti-differentiation effect requires activation of TGF-β-activated kinase (TAK)-1.Surprisingly, the anti-differentiation effect of the cytokines required the transcriptional upregulation of Activin A, which in turn acted through its established signaling pathway: ActRII/ALK/SMAD.In this study, we found an unexpected connection between cytokine and Activin signaling, revealing a new mechanism by which cytokines affect skeletal muscle, and establishing the physiologic relevance of this pathway in the impaired regeneration seen in sarcopenia.

View Article: PubMed Central - HTML - PubMed

Affiliation: Novartis Institutes for Biomedical Research, Forum 1, Novartis Campus, 4056 Basel, Switzerland. anne-ulrike.trendelenburg@novartis.com.

ABSTRACT

Background: Skeletal-muscle differentiation is required for the regeneration of myofibers after injury. The differentiation capacity of satellite cells is impaired in settings of old age, which is at least one factor in the onset of sarcopenia, the age-related loss of skeletal-muscle mass and major cause of frailty. One important cause of impaired regeneration is increased levels of transforming growth factor (TGF)-β accompanied by reduced Notch signaling. Pro-inflammatory cytokines are also upregulated in aging, which led us hypothesize that they might potentially contribute to impaired regeneration in sarcopenia. Thus, in this study, we further analyzed the muscle differentiation-inhibition pathway mediated by pro-inflammatory cytokines in human skeletal muscle cells (HuSKMCs).

Methods: We studied the modulation of HuSKMC differentiation by the pro-inflammatory cytokines interleukin (IL)-1α and tumor necrosis factor (TNF)-α The grade of differentiation was determined by either imaging (fusion index) or creatine kinase (CK) activity, a marker of muscle differentiation. Secretion of TGF-β proteins during differentiation was assessed by using a TGF-β-responsive reporter-gene assay and further identified by means of pharmacological and genetic inhibitors. In addition, signaling events were monitored by western blotting and reverse transcription PCR, both in HuSKMC cultures and in samples from a rat sarcopenia study.

Results: The pro-inflammatory cytokines IL-1α and TNF-α block differentiation of human myoblasts into myotubes. This anti-differentiation effect requires activation of TGF-β-activated kinase (TAK)-1. Using pharmacological and genetic inhibitors, the TAK-1 pathway could be traced to p38 and NFκB. Surprisingly, the anti-differentiation effect of the cytokines required the transcriptional upregulation of Activin A, which in turn acted through its established signaling pathway: ActRII/ALK/SMAD. Inhibition of Activin A signaling was able to rescue human myoblasts treated with IL-1β or TNF-α, resulting in normal differentiation into myotubes. Studies in aged rats as a model of sarcopenia confirmed that this pro-inflammatory cytokine pathway identified is activated during aging.

Conclusions: In this study, we found an unexpected connection between cytokine and Activin signaling, revealing a new mechanism by which cytokines affect skeletal muscle, and establishing the physiologic relevance of this pathway in the impaired regeneration seen in sarcopenia.

No MeSH data available.


Related in: MedlinePlus

Inhibition of human skeletal muscle cell (HuSKMC) differentiation by interleukin (IL)-1α and tumor necrosis factor (TNF)-α is mediated by the transforming growth factor-β-activated kinase (TAK)-1/p38/nuclear factor (NF)κB/Activin A/SMAD2/3 pathway. (A) Analysis of NFκB-luc assay (RGA) from HuSKMC myoblasts treated with IL-1α (1 ng/ml) and TNF-α (1 ng/ml) alone, and in combination with TAK-1 inhibitor (0.1-1 μmol/l) or withaferin A (0.1-1 μmol/l). Data are expressed as relative light units (RLU). Data are means ± SEM from four independent experiments. Differences from IL-1α- and TNF-α-treated HuSKMCs (first column),*P < 0.05. (B) Immunoblotting of phospho-TAK-1, phospho-SEK1/MKK4, phospho-p38MAPK, phospho-c-Jun, phospho-ATF2, phospho-NFκB, phospho-SMAD2 and phospho-SMAD3 of samples from HuSKMCs treated with TNF-α (0.1 to 10 ng/ml) or IL-1α (0.1 to 10 ng/ml) for 15 minutes alone, and in the presence of TAK-1 inhibitor (1 μmol/l) starting at the onset of differentiation. The TAK-1 inhibitor was given 3 hours before IL-1α or TNF-α stimulation. Shown are representative immunoblots. (C) Immunoblotting of phospho-SMAD2, phospho-SMAD3 and pAKT of samples from HuSKMCs treated with TNF-α (0.1 to 10 ng/ml) or IL-1α (0.1 to 10 ng/ml) for 24 hours, alone and in the presence of SB431542 (1 μmol/l), αActA (10 μg/ml), TAK-1 inhibitor (1 μmol/l), SB203580 (10 μmol/l) or withaferin A (300 nmol/l) starting at the onset of differentiation. Inhibitors were given 3 hours before IL-1α or TNF-α stimulation. Shown are representative immunoblots.
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Figure 4: Inhibition of human skeletal muscle cell (HuSKMC) differentiation by interleukin (IL)-1α and tumor necrosis factor (TNF)-α is mediated by the transforming growth factor-β-activated kinase (TAK)-1/p38/nuclear factor (NF)κB/Activin A/SMAD2/3 pathway. (A) Analysis of NFκB-luc assay (RGA) from HuSKMC myoblasts treated with IL-1α (1 ng/ml) and TNF-α (1 ng/ml) alone, and in combination with TAK-1 inhibitor (0.1-1 μmol/l) or withaferin A (0.1-1 μmol/l). Data are expressed as relative light units (RLU). Data are means ± SEM from four independent experiments. Differences from IL-1α- and TNF-α-treated HuSKMCs (first column),*P < 0.05. (B) Immunoblotting of phospho-TAK-1, phospho-SEK1/MKK4, phospho-p38MAPK, phospho-c-Jun, phospho-ATF2, phospho-NFκB, phospho-SMAD2 and phospho-SMAD3 of samples from HuSKMCs treated with TNF-α (0.1 to 10 ng/ml) or IL-1α (0.1 to 10 ng/ml) for 15 minutes alone, and in the presence of TAK-1 inhibitor (1 μmol/l) starting at the onset of differentiation. The TAK-1 inhibitor was given 3 hours before IL-1α or TNF-α stimulation. Shown are representative immunoblots. (C) Immunoblotting of phospho-SMAD2, phospho-SMAD3 and pAKT of samples from HuSKMCs treated with TNF-α (0.1 to 10 ng/ml) or IL-1α (0.1 to 10 ng/ml) for 24 hours, alone and in the presence of SB431542 (1 μmol/l), αActA (10 μg/ml), TAK-1 inhibitor (1 μmol/l), SB203580 (10 μmol/l) or withaferin A (300 nmol/l) starting at the onset of differentiation. Inhibitors were given 3 hours before IL-1α or TNF-α stimulation. Shown are representative immunoblots.

Mentions: Signaling experiments were performed in differentiating HuSKMCs, using either analysis of NFκB activity (Figure 4A) or western blotting (Figure 4B,C) to determine the contributing pathways required for Activin A release. NFκB signaling was assessed by an adenoviral NFκb-Luciferase reporter. The NFκB CAGA-luc activity induced by IL-1α and TNF-α was counteracted by TAK-1 inhibitor and by withaferin A (Figure 4A) indicating that TAK-1 is involved in IL-1α and TNF-α activation of NFκB signaling and, thus is upstream of NFκB. However, TAK-1 inhibitor was less efficacious than withaferin in blocking NFκB signaling, indicating only partial NFκB inhibition by TAK-1.


TAK-1/p38/nNFκB signaling inhibits myoblast differentiation by increasing levels of Activin A.

Trendelenburg AU, Meyer A, Jacobi C, Feige JN, Glass DJ - Skelet Muscle (2012)

Inhibition of human skeletal muscle cell (HuSKMC) differentiation by interleukin (IL)-1α and tumor necrosis factor (TNF)-α is mediated by the transforming growth factor-β-activated kinase (TAK)-1/p38/nuclear factor (NF)κB/Activin A/SMAD2/3 pathway. (A) Analysis of NFκB-luc assay (RGA) from HuSKMC myoblasts treated with IL-1α (1 ng/ml) and TNF-α (1 ng/ml) alone, and in combination with TAK-1 inhibitor (0.1-1 μmol/l) or withaferin A (0.1-1 μmol/l). Data are expressed as relative light units (RLU). Data are means ± SEM from four independent experiments. Differences from IL-1α- and TNF-α-treated HuSKMCs (first column),*P < 0.05. (B) Immunoblotting of phospho-TAK-1, phospho-SEK1/MKK4, phospho-p38MAPK, phospho-c-Jun, phospho-ATF2, phospho-NFκB, phospho-SMAD2 and phospho-SMAD3 of samples from HuSKMCs treated with TNF-α (0.1 to 10 ng/ml) or IL-1α (0.1 to 10 ng/ml) for 15 minutes alone, and in the presence of TAK-1 inhibitor (1 μmol/l) starting at the onset of differentiation. The TAK-1 inhibitor was given 3 hours before IL-1α or TNF-α stimulation. Shown are representative immunoblots. (C) Immunoblotting of phospho-SMAD2, phospho-SMAD3 and pAKT of samples from HuSKMCs treated with TNF-α (0.1 to 10 ng/ml) or IL-1α (0.1 to 10 ng/ml) for 24 hours, alone and in the presence of SB431542 (1 μmol/l), αActA (10 μg/ml), TAK-1 inhibitor (1 μmol/l), SB203580 (10 μmol/l) or withaferin A (300 nmol/l) starting at the onset of differentiation. Inhibitors were given 3 hours before IL-1α or TNF-α stimulation. Shown are representative immunoblots.
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Figure 4: Inhibition of human skeletal muscle cell (HuSKMC) differentiation by interleukin (IL)-1α and tumor necrosis factor (TNF)-α is mediated by the transforming growth factor-β-activated kinase (TAK)-1/p38/nuclear factor (NF)κB/Activin A/SMAD2/3 pathway. (A) Analysis of NFκB-luc assay (RGA) from HuSKMC myoblasts treated with IL-1α (1 ng/ml) and TNF-α (1 ng/ml) alone, and in combination with TAK-1 inhibitor (0.1-1 μmol/l) or withaferin A (0.1-1 μmol/l). Data are expressed as relative light units (RLU). Data are means ± SEM from four independent experiments. Differences from IL-1α- and TNF-α-treated HuSKMCs (first column),*P < 0.05. (B) Immunoblotting of phospho-TAK-1, phospho-SEK1/MKK4, phospho-p38MAPK, phospho-c-Jun, phospho-ATF2, phospho-NFκB, phospho-SMAD2 and phospho-SMAD3 of samples from HuSKMCs treated with TNF-α (0.1 to 10 ng/ml) or IL-1α (0.1 to 10 ng/ml) for 15 minutes alone, and in the presence of TAK-1 inhibitor (1 μmol/l) starting at the onset of differentiation. The TAK-1 inhibitor was given 3 hours before IL-1α or TNF-α stimulation. Shown are representative immunoblots. (C) Immunoblotting of phospho-SMAD2, phospho-SMAD3 and pAKT of samples from HuSKMCs treated with TNF-α (0.1 to 10 ng/ml) or IL-1α (0.1 to 10 ng/ml) for 24 hours, alone and in the presence of SB431542 (1 μmol/l), αActA (10 μg/ml), TAK-1 inhibitor (1 μmol/l), SB203580 (10 μmol/l) or withaferin A (300 nmol/l) starting at the onset of differentiation. Inhibitors were given 3 hours before IL-1α or TNF-α stimulation. Shown are representative immunoblots.
Mentions: Signaling experiments were performed in differentiating HuSKMCs, using either analysis of NFκB activity (Figure 4A) or western blotting (Figure 4B,C) to determine the contributing pathways required for Activin A release. NFκB signaling was assessed by an adenoviral NFκb-Luciferase reporter. The NFκB CAGA-luc activity induced by IL-1α and TNF-α was counteracted by TAK-1 inhibitor and by withaferin A (Figure 4A) indicating that TAK-1 is involved in IL-1α and TNF-α activation of NFκB signaling and, thus is upstream of NFκB. However, TAK-1 inhibitor was less efficacious than withaferin in blocking NFκB signaling, indicating only partial NFκB inhibition by TAK-1.

Bottom Line: This anti-differentiation effect requires activation of TGF-β-activated kinase (TAK)-1.Surprisingly, the anti-differentiation effect of the cytokines required the transcriptional upregulation of Activin A, which in turn acted through its established signaling pathway: ActRII/ALK/SMAD.In this study, we found an unexpected connection between cytokine and Activin signaling, revealing a new mechanism by which cytokines affect skeletal muscle, and establishing the physiologic relevance of this pathway in the impaired regeneration seen in sarcopenia.

View Article: PubMed Central - HTML - PubMed

Affiliation: Novartis Institutes for Biomedical Research, Forum 1, Novartis Campus, 4056 Basel, Switzerland. anne-ulrike.trendelenburg@novartis.com.

ABSTRACT

Background: Skeletal-muscle differentiation is required for the regeneration of myofibers after injury. The differentiation capacity of satellite cells is impaired in settings of old age, which is at least one factor in the onset of sarcopenia, the age-related loss of skeletal-muscle mass and major cause of frailty. One important cause of impaired regeneration is increased levels of transforming growth factor (TGF)-β accompanied by reduced Notch signaling. Pro-inflammatory cytokines are also upregulated in aging, which led us hypothesize that they might potentially contribute to impaired regeneration in sarcopenia. Thus, in this study, we further analyzed the muscle differentiation-inhibition pathway mediated by pro-inflammatory cytokines in human skeletal muscle cells (HuSKMCs).

Methods: We studied the modulation of HuSKMC differentiation by the pro-inflammatory cytokines interleukin (IL)-1α and tumor necrosis factor (TNF)-α The grade of differentiation was determined by either imaging (fusion index) or creatine kinase (CK) activity, a marker of muscle differentiation. Secretion of TGF-β proteins during differentiation was assessed by using a TGF-β-responsive reporter-gene assay and further identified by means of pharmacological and genetic inhibitors. In addition, signaling events were monitored by western blotting and reverse transcription PCR, both in HuSKMC cultures and in samples from a rat sarcopenia study.

Results: The pro-inflammatory cytokines IL-1α and TNF-α block differentiation of human myoblasts into myotubes. This anti-differentiation effect requires activation of TGF-β-activated kinase (TAK)-1. Using pharmacological and genetic inhibitors, the TAK-1 pathway could be traced to p38 and NFκB. Surprisingly, the anti-differentiation effect of the cytokines required the transcriptional upregulation of Activin A, which in turn acted through its established signaling pathway: ActRII/ALK/SMAD. Inhibition of Activin A signaling was able to rescue human myoblasts treated with IL-1β or TNF-α, resulting in normal differentiation into myotubes. Studies in aged rats as a model of sarcopenia confirmed that this pro-inflammatory cytokine pathway identified is activated during aging.

Conclusions: In this study, we found an unexpected connection between cytokine and Activin signaling, revealing a new mechanism by which cytokines affect skeletal muscle, and establishing the physiologic relevance of this pathway in the impaired regeneration seen in sarcopenia.

No MeSH data available.


Related in: MedlinePlus