Suppression of mitochondrial respiration through recruitment of p160 myb binding protein to PGC-1alpha: modulation by p38 MAPK

Abstract

The transcriptional coactivator PPAR gamma coactivator 1 alpha (PGC-1alpha) is a key regulator of metabolic processes such as mitochondrial biogenesis and respiration in muscle and gluconeogenesis in liver. Reduced levels of PGC-1alpha in humans have been associated with type II diabetes. PGC-1alpha contains a negative regulatory domain that attenuates its transcriptional activity. This negative regulation is removed by phosphorylation of PGC-1alpha by p38 MAPK, an important kinase downstream of cytokine signaling in muscle and beta-adrenergic signaling in brown fat. We describe here the identification of p160 myb binding protein (p160MBP) as a repressor of PGC-1alpha. The binding and repression of PGC-1alpha by p160MBP is disrupted by p38 MAPK phosphorylation of PGC-1alpha. Adenoviral expression of p160MBP in myoblasts strongly reduces PGC-1alpha's ability to stimulate mitochondrial respiration and the expression of the genes of the electron transport system. This repression does not require removal of PGC-1alpha from chromatin, suggesting that p160MBP is or recruits a direct transcriptional suppressor. Overall, these data indicate that p160MBP is a powerful negative regulator of PGC-1alpha function and provide a molecular mechanism for the activation of PGC-1alpha by p38 MAPK. The discovery of p160MBP as a PGC-1alpha regulator has important implications for the understanding of energy balance and diabetes.

Figure 1.

p160^MBP interacts with GST-PGC1(200-400) and suppresses PGC-1α-mediated transcription. C2C12 muscle cell nuclear extracts were incubated with bacterially expressed GST fusion proteins on glutathione sepharose beads. PGC(1-200) is GST-PGC1α(1-200) and PGC(200-400) is GST-PGC1α(200-400). The interacting proteins were run on an SDS-PAGE gel and (A) silver stained or (B) Western blotted with antibodies against p160^MBP. (C) p160^MBP or p67^MBP was cotransfected with Gal4-PGC1α or Gal4-PGC1(Δ170-350) and a UAS-luciferase reporter in HIB1B cells. Luciferase activity was measured after 24 h. (Asterisks) P < 0.001, paired t-test. (D) PPARγ and RXRα with or without PGC-1α were cotransfected with a DR-1-luciferase reporter into HIB1B cells. p160^MBP or p67^MBP were also added and luciferase levels were measured after 24 h. All luciferase assays were normalized for transfection efficiencies using β-galactosidase. (Asterisks) P < 0.001, paired t-test.

Figure 2.

PGC-1α binds to amino acids 200-400 and to the C terminus of p160^MBP. (A) Summary of p160^MBP deletion constructs used in this experiment. (B) GST, labeled G, or GST-PGC1(200-400), labeled P, were incubated with in vitro translated ³⁵S-p160^MBP deletions for 1 h. Interacting proteins were run on an SDS-PAGE gel and visualized by autoradiography.

Figure 3.

p160^MBP's interaction with PGC-1α is modulated by p38 MAPK phosphorylation. (A) GST-PGC1(200-400) was phosphorylated in vitro by activated p38 MAPK in the presence of ATP [GST-PGC1(200-400)-P] or mock phosphorylated in the absence of ATP [GST-PGC1(200-400)]. The GST proteins were then incubated with C2C12 nuclear extracts and interacting proteins were visualized by Western blot by using antibodies against p160^MBP or against cdc16 (Santa Cruz Biotechnology). (B) Flag-tagged PGC-1α (f:PGC1α) was cotransfected with myc-tagged p160^MBP (myc-p160^MBP) in BOSC cells and after 24 h treated with the cytokines IL-1α, IL-1β, and TNFα overnight in DMEM + 0.5% BSA. Whole-cell extracts were immunoprecipitated with M2 anti-Flag antibodies (Sigma) and Western blots were conducted with antibodies against myc or PGC-1α (Santa Cruz Biotechnology). The left panel is a Western blot of 5% of the whole-cell extract before immunoprecipitation and the right panel is a Western blot of the immunoprecipitated proteins. (C) Gal4-PGC1α was cotransfected in C2C12 cells with a UAS-luciferase reporter and a vector expressing MKK6, the upstream activator of p38 MAPK. Medium was changed to DMEM + 0.5% BSA after 24 h, and luciferase activity was measured after 48 h. (Asterisks) P < 0.05, paired t-test. (D) Myc-p160^MBP was cotransfected with f:PGC1α, f:PGC1α with the phosphorylation sites mutated to aspartic acid (f:PGC1-3D), or f:PGC1α with selected leucines at L2 and L3 mutated to alanine (f:PGC1-L2/3A). Extracts were immunoprecipitated with anti-Flag antibodies as described earlier. (E) PGC-1α or PGC1-3D were cotransfected with PPARγ, RXR, and DR-1 luciferase reporter in HIB1B cells. Cells were harvested after 24 h and fold activation over PPARγ was graphed. (Asterisks) P < 0.05, paired t-test. (F) Gal4-PGC1 or Gal4-PGC1-L2/3A were cotransfected with a UAS-TATA-luciferase reporter in C2C12 cells, and luciferase activity was measured after 24 h. (Asterisks) P < 0.05, paired t-test.

Figure 4.

p160^MBP suppresses PGC-1α's biological effects in muscle cells. C2C12 myoblasts were coinfected with adenoviral PGC1α and p160^MBP. Adenoviral GFP was used in the control infections. After 48 h, (A) total mitochondrial oxygen consumption and (B) uncoupled respiration were measured in live muscle cells. Uncoupled respiration represents the fraction of mitochondrial respiration that is not coupled to ATP production and was attained through addition of the ATP-synthase inhibitor oligomycin. (Asterisks) P < 0.05, repeat ANOVA, a posteriori Tukey test. (C) Same as in A and B, except instead of using cells for oxygen consumption assays, RNA was harvested and Northern blots were performed. (D) C2C12 myoblasts were coinfected with adenoviral PGC1α and p160^MBP in serum-free media. TNFα, IL-1α, and IL-1β were added at the time of infection and cells were harvested after 24 h. Real-time PCR was used to quantitate RNA levels. (Asterisks) P < 0.05, paired t-test; (n.s.) not statistically significant, paired t-test.

Figure 5.

p160^MBP does not alter PGC-1α protein level or recruitment to DNA. (A) HIB1B brown fat cells were cotransfected with p160^MBP and various versions of PGC-1α. Cell extracts were Western blotted for PGC-1α. (B) C2C12 myotubes were coinfected with adenoviral MEF2C, PGC-1α, and p160^MBP and harvested for RNA and protein. RNA was subjected to real-time PCR analysis to determine the amounts of PGC-1α and myoglobin transcripts. Protein was blotted with antibodies against PGC-1α or the loading control actin. (Asterisk) P < 0.05, paired t-test. (C) Diagram of PGC-1α interaction with MEF2C on the myoglobin promoter; sites for PCR primers for chromatin immunoprecipitation assays are indicated. (D) C2C12 myotubes were coinfected with adenoviral MEF2C, Flag-tagged PGC-1α, and p160^MBP. Chromatin immunoprecipitations were carried out using M2 anti-Flag antibodies. The input and immunoprecipitated DNA were used as templates for PCR with primers flanking the MEF2C site at -130 on the myoglobin promoter. Primers amplifying a region of the GAPDH gene were used as a negative control. (E) Gal4 fusion protein constructs were transfected into C2C12 myoblasts with a UAS-tk-luciferase reporter. The cells were harvested after 24 h and luciferase activity was measured. (F) Similar to E, except 100 μg/mL trichostatin A (TSA) was added after 12 h.

Figure 6.

Schematic representation of p160^MBP repression of PGC-1α on DNA; regulation by p38 MAPK signaling events. p160^MBP binds to PGC-1α and represses transcription of PGC-1α target genes. On cytokine or β-adrenergic signaling, p38 MAPK becomes active and phosphorylates PGC-1α. The phosphates disrupt p160^MBP binding to PGC-1α, leading to increased activation of PGC-1α target genes.