Akt inhibition promotes ABCA1-mediated cholesterol efflux to ApoA-I through suppressing mTORC1.

ATP-binding cassette transporter A1 (ABCA1) plays an essential role in mediating cholesterol efflux to apolipoprotein A-I (apoA-I), a major housekeeping mechanism for cellular cholesterol homeostasis. After initial engagement with ABCA1, apoA-I directly interacts with the plasma membrane to acquire cholesterol. This apoA-I lipidation process is also known to require cellular signaling processes, presumably to support cholesterol trafficking to the plasma membrane. We report here that one of major signaling pathways in mammalian cells, Akt, is also involved. In several cell models that express ABCA1 including macrophages, pancreatic beta cells and hepatocytes, inhibition of Akt increases cholesterol efflux to apoA-I. Importantly, Akt inhibition has little effect on cells expressing non-functional mutant of ABCA1, implicating a specific role of Akt in ABCA1 function. Furthermore, we provide evidence that mTORC1, a major downstream target of Akt, is also a negative regulator of cholesterol efflux. In cells where mTORC1 is constitutively activated due to tuberous sclerosis complex 2 deletion, cholesterol efflux to apoA-I is no longer sensitive to Akt activity. This suggests that Akt suppresses cholesterol efflux through mTORC1 activation. Indeed, inhibition of mTORC1 by rapamycin or Torin-1 promotes cholesterol efflux. On the other hand, autophagy, one of the major pathways of cholesterol trafficking, is increased upon Akt inhibition. Furthermore, Akt inhibition disrupts lipid rafts, which is known to promote cholesterol efflux to apoA-I. We therefore conclude that Akt, through its downstream targets, mTORC1 and hence autophagy, negatively regulates cholesterol efflux to apoA-I.

ABCA1 protein enhances Toll-like receptor 4 (TLR4)-stimulated interleukin-10 (IL-10) secretion through protein kinase A (PKA) activation.

BACKGROUND: ABCA1 is known to suppress proinflammatory cytokines. RESULTS: ABCA1 activates PKA and up-regulates anti-inflammatory cytokine IL-10. Elevated PKA transforms macrophages to M2-like phenotype. Disrupting lipid rafts by statins MCD, and filipin recuperates ABCA1 phenotype and likely functions downstream of ABCA1. CONCLUSION: By modulating cholesterol, ABCA1 activates PKA. This generates M2-like macrophages. SIGNIFICANCE: ABCA1 does not simply suppress inflammatory response. It promotes M2-like activation and facilitates resolution. Nonresolving inflammatory response from macrophages is a major characteristic of atherosclerosis. Macrophage ABCA1 has been previously shown to suppress the secretion of proinflammatory cytokine. In the present study, we demonstrate that ABCA1 also promotes the secretion of IL-10, an anti-inflammatory cytokine critical for inflammation resolution. ABCA1(+/+) bone marrow-derived macrophages secrete more IL-10 but less proinflammatory cytokines than ABCA1(-/-) bone marrow-derived macrophages, similar to alternatively activated (M2) macrophages. We present evidence that ABCA1 activates PKA and that this elevated PKA activity contributes to M2-like inflammatory response from ABCA1(+/+) bone marrow-derived macrophages. Furthermore, cholesterol lowering by statins, methyl-ß-cyclodextrin, or filipin also activates PKA and, consequently, transforms macrophages toward M2-like phenotype. Conversely, cholesterol enrichment suppresses PKA activity and promotes M1-like inflammatory response. As the primary function of ABCA1 is cholesterol removal, our results suggest that ABCA1 activates PKA by regulating cholesterol. Indeed, forced cholesterol enrichment in ABCA1-expressing macrophages suppresses PKA activation and elicits M1-like response. Collectively, these findings reveal a novel protective process by ABCA1-activated PKA in macrophages. They also suggest cholesterol lowering in extra-hepatic tissues by statins as an anti-inflammation strategy.

Ht31, a protein kinase A anchoring inhibitor, induces robust cholesterol efflux and reverses macrophage foam cell formation through ATP-binding cassette transporter A1.

Macrophage foam cell is the predominant cell type in atherosclerotic lesions. Removal of excess cholesterol from macrophages thus offers effective protection against atherosclerosis. Here we report that a protein kinase A (PKA)-anchoring inhibitor, st-Ht31, induces robust cholesterol/phospholipid efflux, and ATP-binding cassette transporter A1 (ABCA1) greatly facilitates this process. Remarkably, we found that st-Ht31 completely reverses foam cell formation, and this process is ABCA1-dependent. The reversal is also accompanied by the restoration of well modulated inflammatory response to LPS. There is no detectable toxicity associated with st-Ht31, even when cells export up to 20% cellular cholesterol per hour. Using FRET-based PKA biosensors in live cells, we provide evidence that st-Ht31 drives cholesterol efflux by elevating PKA activity specifically in the cytoplasm. Furthermore, ABCA1 facilitates st-Ht31 uptake. This allows st-Ht31 to effectively remove cholesterol from ABCA1-expressing cells. We speculate that de-anchoring of PKA offers a novel therapeutic strategy to remove excess cholesterol from lipid-laden lesion macrophages.

Cholesterol efflux to apoA-I in ABCA1-expressing cells is regulated by Ca2+-dependent calcineurin signaling.

ATP-binding cassette transporter A1 (ABCA1) is required for the lipidation of apolipoprotein A-I (apoA-I), although molecular mechanisms supporting this process remain poorly defined. In this study, we focused on the role of cytosolic Ca(2+) and its signaling and found that cytosolic Ca(2+) was required for cholesterol efflux to apoA-I. Removing extracellular Ca(2+) or chelating cytosolic Ca(2+) were equally inhibitory for apoA-I lipidation. We provide evidence that apoA-I induced Ca(2+) influx from the medium. We further demonstrate that calcineurin activity, the downstream target of Ca(2+) influx, was essential; inhibition of calcineurin activity by cyclosporine A or FK506 completely abolished apoA-I lipidation. Furthermore, calcineurin inhibition abolished apoA-I binding and diminished JAK2 phosphorylation, an established signaling event for cholesterol efflux to apoA-I. Finally, we demonstrate that neither Ca(2+) manipulation nor calcineurin inhibition influenced ABCA1's capacity to release microparticles or to remodel the plasma membrane. We conclude that this Ca(2+)-dependent calcineurin/JAK2 pathway is specifically responsible for apoA-I lipidation without directly modifying ABCA1 activity.

Proteomic analysis of neuroendocrine peptidergic system disruption using the AtT20 pituitary cell line as a model.

Environmental pollutants may affect the activities of many cellular enzymes. The effect on the proteome of enzymatic inhibitors can be determined using two-dimensional (2D) gel electrophoresis. In neuroendocrine cells, proprotein convertases 1 and 2 (PC1 and PC2) mediate the proteolytic activation of many precursors to peptide hormones and neuropeptides. Enzymatic activities of these calcium-dependent proteinases are readily regulated by chelating agents and by heavy metals ions found in the environment. Such an inhibition could result in a potentially pathological disruption of the peptidergic system. We are interesting in finding out to what extent specific inhibition of these enzymes could affect the proteome of a neuroendocrine cell. To address this question, we used the mouse pituitary AtT20 cell line as a model. We compared the proteomic pattern of control cells to that of cells overexpressing proSAAS, a PC1-specific inhibitor. The comparison was conducted using two-dimensional (2D) gel electrophoresis, mass spectrometric identification of differing proteins and immunoblotting to confirm their identity. The 2D analysis revealed a number of alterations in the proteome of proSAAS-overexpressing cells. Mass spectrometric analysis of tryptic peptides identified two proteins found in more abundance in these cells as proSAAS and Ephrin type A receptor 2.

Artemis phosphorylated by DNA-dependent protein kinase associates preferentially with discrete regions of chromatin.

Artemis is a nuclear phosphoprotein required for genomic integrity whose phosphorylation is increased subsequent to DNA damage. Artemis phosphorylation by the DNA-dependent protein kinase (DNA-PK) and the association of Artemis with DNA-PK catalytic subunit (DNA-PKcs) have been proposed to be crucial for the variable, diversity, joining (V(D)J) reaction, genomic stability and cell survival in response to double-stranded DNA breaks. The exact nature of the effectors of Artemis phosphorylation is presently being debated. Here, we have delimited the interface on Artemis required for its association with DNA-PKcs and present the characterization of six DNA-PK phosphorylation sites on Artemis whose phosphorylation shows dependence on its association with DNA-PKcs and is induced by double-stranded DNA damage. Surprisingly, DNA-PKcs Artemis association appeared to be dispensable in a V(D)J recombination assay with stably integrated DNA substrates. Phosphorylation at two of the sites on Artemis, S516 and S645, was verified in vivo using phosphospecific antibodies. Basal Artemis S516 and S645 phosphorylation in vivo showed a significant dependence on DNA-PKcs association. However, regardless of its association with DNA-PKcs, phosphorylation of Artemis at both S516 and S645 was stimulated in response to the double-stranded DNA-damaging agent bleomycin, albeit to a lesser extent. This suggests that additional factors contribute to promote DNA damage-induced Artemis phosphorylation. Intriguingly, pS516/pS645 Artemis was concentrated in chromatin-associated nuclear foci in naïve cells. These foci were maintained upon DNA damage but failed to overlap with the damage-induced gammaH2AX. These results provide the expectation of a specific role for DNA-PK-phosphorylated Artemis in both naïve and damaged cells.