TLR4-Mediated AKT Activation Is MyD88/TRIF Dependent and Critical for Induction of Oxidative Phosphorylation and Mitochondrial Transcription Factor A in Murine Macrophages

Abstract

Mitochondria play a critical role in cell survival and death. Mitochondrial recovery during inflammatory processes such as sepsis is associated with cell survival. Recovery of cellular respiration, mitochondrial biogenesis and function requires coordinated expression of transcription factors encoded by nuclear and mitochondrial genes, including mitochondrial transcription factor A (T-fam) and cytochrome c oxidase (COX, complex IV). LPS elicits strong host defenses in mammals with pronounced inflammatory responses but also triggers activation of survival pathways such as AKT pathway. AKT/PKB is a serine/threonine protein kinase playing an important role in cell survival, protein synthesis, and controlled inflammation in response to TLRs. Hence, we investigated the role of LPS mediated AKT activation in mitochondrial bioenergetics and function in cultured murine macrophages (B6-MCL) and bone marrow derived macrophages. We show that LPS challenge led to increased expression of T-fam and COX subunit I and IV in a time dependent manner through early phosphorylation of the PI3kinase/AKT pathway. PI3K/AKT pathway inhibitors abrogated LPS mediated T-fam and COX induction. Lack of induction was associated with decreased ATP production, increased proinflammatory cytokines (TNF-α), nitric oxide production and cell death. The TLR4 mediated AKT activation and mitochondrial biogenesis required activation of adaptor protein MyD88 and Toll-IL-1R-containing adaptor inducing IFN-β (TRIF). Importantly, using a genetic approach, we show that the AKT1 isoform is pivotal in regulating mitochondrial biogenesis in response to TLR4 agonist.

Rapamycin Induces Mitogen-activated Protein (MAP) Kinase Phosphatase-1 (MKP-1) Expression through Activation of Protein Kinase B and Mitogen-activated Protein Kinase Kinase Pathways

“…Cell Culture-Murine macrophage-like cell line (B6-MCL) was cultured as previously described (22). Briefly, B6-MCL were grown in Iscove's modified Dulbecco's medium supplemented with 10% heat-inactivated fetal calf serum (Invitrogen), antibiotics, sodium pyruvate, nonessential amino acids, and ␤-mercaptoethanol.…”

“…BMDMs from mice were prepared as described previously (22). Macrophages were cultured with Iscove's modified Dulbecco's medium supplemented with 30% L929 supernatant containing macrophage-stimulating factor, glutamine, sodium pyruvate, 10% heat-inactivated FBS, and antibiotics for 5-7 days.…”

Toll-like receptors, signaling adapters and regulation of the pro-inflammatory response by PI3K

“…79 MyD88-deficient macrophages were also found to be defective for Akt phosphorylation through several TLR ligands, as reported by us and others. 21,63,77 Similarly, TRIF signals were required by macrophages for Akt phosphorylation upon LPS stimulation and likely serves as the upstream adaptor allowing RIP-mediated PI3K activation. 63 Together, these data suggest that TLR adaptors may serve to recruit a previously unknown downstream target mediating PI3K activation.…”

“…29,62 Importantly, detection of phosphorylated Akt in macrophages stimulated through TLR4 is largely abrogated in Akt1-deficient cells but not Akt2-deficient cells, suggesting that Akt1 is the most critical isoform utilized by the TLR-PI3K signaling axis. 63 Phosphatase and tensin homolog (PTEN), a 3-phosphatase counterbalancing PI3K by catalyzing conversion of PtdIns(3,4,5)P 3 back into PtdIns(4,5)P 2 , plays a positive role in inducing inflammation in TLR-stimulated cells. PTENdeficient macrophages secreted decreased quantities of Tnf and Il6 upon stimulation with TLR ligands.…”

The AMPK-PPARGC1A pathway is required for antimicrobial host defense through activation of autophagy

“…The mitochondrial membrane potential (ΔΨm) of intact cells was measured as described previously, 61 with modifications. Cells were washed with PBS and trypsinized.…”