TNF-alpha acts via p38 MAPK to stimulate expression of the ubiquitin ligase atrogin1/MAFbx in skeletal muscle

Abstract

Atrogin1/MAFbx is an ubiquitin ligase that mediates muscle atrophy in a variety of catabolic states. We recently found that H2O2 stimulates atrogin1/MAFbx gene expression. Since the cytokine tumor necrosis factor-alpha (TNF-alpha) stimulates both reactive oxygen production and general activity of the ubiquitin conjugating pathway, we hypothesized that TNF-alpha would also increase atrogin1/MAFbx gene expression. As with H2O2, we found that TNF-alpha exposure up-regulates atrogin1/MAFbx mRNA within 2 h in C2C12 myotubes. Intraperitoneal injection of TNF-alpha increased atrogin1/MAFbx mRNA in skeletal muscle of adult mice within 4 h. Exposing myotubes to either TNF-alpha or H2O2 also produced general activation of the mitogen-activated protein kinases (MAPKs): p38, ERK1/2, and JNK. The increase in atrogin1/MAFbx gene expression induced by TNF-alpha was not altered significantly by ERK inhibitor PD98059 or the JNK inhibitor SP600125. In contrast, atrogin1/MAFbx up-regulation and the associated increase in ubiquitin conjugating activity were both blunted by p38 inhibitors, either SB203580 or curcumin. These data suggest that TNF-alpha acts via p38 to increase atrogin1/MAFbx gene expression in skeletal muscle.

Figure 1

TNF-α up-regulates atrogin1/MAFbx gene expression in C2C12 myotubes. C2C12 myotubes were incubated with TNF-α or vehicle. Total RNA was extracted and analyzed by Northern blot using a full-length human atrogin1/MAFbx cDNA probe. Representative blot depicts multiple atrogin/ MADbx transcripts (upper band ~6.5 kb, lower band ~2.4 kb). Both increased 2 h after TNF-α exposure and returned to basal levels by 6 h. Ethidium bromide-stained 28S and 18S rRNA are shown as loading controls.

Figure 2

TNF-α up-regulates atrogin1/MAFbx gene expression in mouse gastrocnemius. Mice were IP injected with TNF-α 100 ng/g and gastrocnemius was surgically removed for analysis of the atrogin1/MAFbx mRNA A) A representative Northern blot with multiple transcripts (~6.5, 5, and 2.4 kb) and the loading control are shown. B) Bands representing the atrogin1/MAFbx mRNA were quantified by densitometry and normalized to 18S. Data from 3 mice in each time group were analyzed by ANOVA combined with Tukey’s multiple comparison test. *A difference (P<0.05) from control (0 h).

Figure 3

TNF-α and H₂O₂ increase MAPK activity in C2C12 myotubes. C2C12 myotubes were incubated 30 min with TNF-α 6 ng/mL, H₂O₂ 300 µM, or vehicle (control). Cell lysates were analyzed with Western blot using an antibody against the phosphorylated form of p38, ERK1/2, or JNK, and an antibody against total p38, ERK1/2, or JNK. Arrows indicate samples that were exposed to TNF-α or H₂O₂. Optical density data from 4 independent experiments were analyzed by Student’s t test.

Figure 4

TNF-α and H₂O₂ activate p38 MAPK in mouse diaphragm. Mouse diaphragm was excised and incubated in Krebs-Ringer’s solution bubbled with 95%-5% O₂-CO₂ at room temperature. One hemidiaphragm from each animal was exposed to TNF-α 500 ng/mL or H₂O₂ 300 µM for 30 min (arrows). The contralateral hemidiaphragm was exposed to the buffer only. Protein extracts prepared from the hemidiaphragms were analyzed by Western blot for p38 activation by use of a phosphospecific antibody. Optical density data from 4 hemidiaphragm pairs were analyzed by Student’s t test.

Figure 5

TNF-α effects on p38 MAPK phosphorylation are dose dependent. C2C12 myotubes were exposed to TNF-α at the indicated concentrations for 2 h and protein was extracted for Western blot analysis. Upper panel shows original blots from 3 experiments. Extracts were analyzed for phosphorylated p38 (left) and total p38 (right). Lower panel depicts the averaged ratios of phospho-to-total p38 (±se) at each TNF-α concentration as calculated from densitometry data. TNF-α increased this ratio in a dose-dependent manner; P < 0.001 by ANOVA.

Figure 6

TNF-α effects on atrogin1/MAFbx mRNA levels are dose dependent. C2C12 myotubes were exposed to TNF-α at the indicated concentrations for 2 h and total RNA was extracted for Northern blot analysis. Upper panel is a representative blot showing multiple atrogin1/MAFbx transcripts (left) and ethidium bromide-stained loading controls (right). Lower panel depicts averaged densitometry data from 3 separate experiments showing the dose dependence of atrogin1/MAFbx up-regulation; P < 0.03 by ANOVA.

Figure 7

Relationship between p38 MAPK activation and atrogin1/MAFbx expression. Figure depicts changes in atrogin1/MAFbx mRNA as a function of phospho-p38 levels; each data point represents averaged measurements from 3 experiments in which C2C12 myotubes were exposed to a common TNF-α concentration; filled triangle, 6 pg/mL; open square, 60 pg/mL; filled circle, 600 pg/mL; open triangle, 6,000 pg/mL; filled square, 60,000 pg/mL; regression analysis indicated best fit by a second order polynomial: y = −0.0048x² + 2.0104x − 10.841, R² = 0.9699, P < 0.05 (curve not shown).

Figure 8

p38 MAPK mediates TNF-α up-regulation of atrogin1/MAFbx gene expression. C2C12 myotubes were preincubated with SB203580 5 µM, PD98059 35 µM, SP600125 0.1–10 µM, or DMSO 0.1% (vehicle) for 30 min before 2 h incubation with TNF-α 6 ng/mL. Total RNA was extracted and analyzed by Northern blot for atrogin1/MAFbx mRNA. A, B) Representative blots showing multiple transcripts (bands at ~6.5, 5, 4, and 2.4 kb) and RNA loading. Optical density data are shown in panel C. SP600125 data reflect responses to 10 µM. Data were analyzed using ANOVA and Tukey’s multiple comparison test. Sample sizes are indicated by numbers shown in each bar; *different from control; **different from TNF-α-treated samples (P<0.05).

Figure 9

p38 MAPK mediates TNF-α stimulation of ubiquitin conjugating activity. C2C12 myotubes were preincubated with SB203580, PD98059, or DMSO for 30 min before 6 h incubation with TNF-α. Cell extracts were prepared and analyzed for ubiquitin conjugating activity. Muscle proteins that incorporated ¹²⁵I-Ubiquitin were separated by SDS-PAGE and visualized by autoradiography. Ubiquitin conjugating activity was quantified by densitometry. Data from 5 experiments were analyzed by ANOVA with Tukey’s multiple comparison test. *Different from control (P<0.05).