Figure 2
From: TAK-1/p38/nNFκB signaling inhibits myoblast differentiation by increasing levels of Activin A
Interleukin (IL)-1α and tumor necrosis factor (TNF)-α induced secretion of Activin A via activation of transforming growth factor-β-activated kinase (TAK)-1, p38 and nuclear factor (NF)κB pathways during human skeletal muscle cell (HuSKMC) differentiation. Supernatants (SNs) from HuSKMC myotubes differentiated for 3 days were analyzed in (A,B,D) HEK293Tcells stably transfected with the SMAD-sensitive CAGA-luc reporter in a reporter-gene assay (RGA) to assess occurrence of active TGF-β proteins or (C) with Activin A ELISA. (E) Activin A β-chain mRNA in HuSKMC myotubes differentiated for 6 hours was analyzed by reverse transcriptase (RT)-PCR. HuSKMCs were differentiated in the absence or presence of IL-1α (0.1 ng/ml) or TNF-α (0.1 ng/ml), alone or in combination (A-C) with the neutralizing anti-Activin A antibody (αActA), TAK-1 inhibitor, the p38 inhibitor SB203580 or the NFκB inhibitor withaferin A, or (D) after small interfering (si)RNA treatment. Data are expressed as (A,B,D) relative light units (RLU) or as percentage of control untreated HuSKMCs for (C) Activin A ELISA and (E) RT-PCR. Data are means ± SEM from 3 to 17 independent experiments. Differences from untreated HuSKMCs,*P < 0.05; differences from IL-1α- and TNF-α-treated HuSKMCs, #P < 0.05. (A) SNs were analyzed in CAGA-luc RGA. The soluble TGF-βRIIb/Fc chimera (500 ng/ml; TGF-βRIIb) was added alone to the supernatant to determine the contribution of the TGF-β isoforms (TGF-β1, TGF-β2 and TGF-β3) to CAGA-luc activity or in combination with αActA (10 μg/ml; TGF-βRIIb + αActA) to assess contribution of Activin A. (B) SNs were analyzed in CAGA-luc RGA. To eliminate responses to the TGF-β isoforms, CAGA-luc activity was measured after addition of TGF-βRIIb to the SN. (C) SNs were analyzed with Acitivin A ELISA. (D) SNs were analyzed in CAGA-luc RGA after addition of TGFβRIIb. SN were taken after treatment with siRNA against non-targeting control (siNTC), Activin A β-chain (siActivin A β-chain) or SMAD2 and SMAD3 (siSMAD2/3). (E) Activin A β-chain mRNA were analyzed by RT-PCR. Inhibitors were given 3 hours before IL-1α or TNF-α stimulation.