Role of LXR alpha phosphorylation in macrophage activation and atherogenesis

Atherosclerosis is a disease caused by the build up of fatty material (cholesterol) in blood vessels (arteries) causing them to narrow, which reduces the amount of blood and oxygen delivered to vital organs. Both cholesterol and inflammation play an important role in the initiation and progression of the disease. Activation of the receptor LXRa_decreases atherosclerosis in animal models and its ligands could potentially treat the disease in humans. Macrophages are specialized cells that eat and digest germs, are present in the arteries and are crucial for the progression of the disease. There are two types of macrophages, ?classically? activated and pro-inflammatory and the ?alternatively? activated and anti-inflammatory. Activation of LXRa by specific compounds (ligands) increases the production of certain genes. We have found that one of those genes, called arginase I, is characteristic of the alternative activation state of macrophages. In addition, we have seen that by introducing a specific mutation in the receptor we can also activate it resulting in even more elevated levels of arginase I. We hypothesize that activation of LXRa may also affect the production of other genes so that its activation induces is a switch from the classical to the alternative state of macrophages. This would change the inflammatory status of these cells and could ultimately affect the progression of atherosclerosis. We also believe that the specific mutation in LXRa would influence this activation switch. We have cell lines that express the unmodified LXRa receptor, and cells that express the mutated LXRa. Using these cells we aim to first determine what is the mechanism that explains the induction of arginase 1 in cells that have the mutated receptor. Next we will determine whether the cells with the mutated receptor are in a more alternative state and therefore if they are less inflammatory compared with cells having the unmodified receptor. Finally, to examine that effects in these cells can affect the whole body and the development of atherosclerosis we propose to generate a mouse model that will have the mutated receptor. Then we will investigate if the mutation has an effect on inflammation in these mice and whether it affects the progression of the atherosclerosis compared to mice that have the unmodified receptor in their cells. The proposed work will provide a deeper understanding on how we can regulate the LXRa receptor and could eventually lead to novel therapies for the treatment of atherosclerosis and other inflammatory diseases.

LXRs (a and b) are ligand-activated transcription factors that behave like cholesterol content sensors. Activation of LXRs inhibit the development of atherosclerosis and macrophage LXR activity is required for this inhibition. LXRa activation also reduces inflammation and decreases progression of atherosclerosis and insulin resistance. Therefore LXRs are now considered promising drug development targets for the management of metabolic and inflammatory diseases. Th1 cytokines induce a classical activation state while Th2 cytokines such as IL-4 induce an anti-inflammatory alternative activation program in macrophages. Interestingly, induction of Th2-cell responses affects the development of atherosclerosis. LXR activation inhibits classical macrophage activation. However, the impact of LXRa on the alternative activation status of macrophages and its physiological implications remains unexplored. We previously demonstrated that modulation of LXRa phosphorylation regulates LXRa actions. Our preliminary studies now show that LXRa activation in the macrophage RAW 264.7 cell line induces the expression and IL-4 induction of markers for the alternative (anti-inflammatory) activation, such as arginase I (ArgI). Reduction of LXRa phosphorylation markedly enhances these effects. Thus we hypothesize that changes in LXRa expression and phosphorylation may play a role in macrophage polarization towards the alternative anti-inflammatory state. In turn, this would affect macrophage inflammation and may have important physiological consequences in the development of atherosclerosis. In this proposal we will first aim to elucidate the mechanisms underlying the regulation of ArgI by LXRa. We will characterize ArgI promoter activity, mRNA and protein expression as well as enzymatic activity upon changes in LXRa expression and phosphorylation. RAW-LXRa WT and S198A stable cell lines as well as primary macrophages will be employed. Next, the impact of LXRa expression and phosphorylation on macrophage activation status and inflammation will be investigated by measuring pro- and anti-inflammatory cytokines and markers for each state. Finally, the physiological relevance of LXRa phosphorylation will be investigated by generating mice homozygous for the phosphorylation site mutation. The influence of LXRa phosphorylation on the activation state of resident macrophages from these mice will be examined. Additionally, the impact of LXRa phosphorylation on atherogenesis will be also investigated by crossing these mutant mice with the apoE-deficient mouse atherosclerosis model. Intracellular lipid and macrophage content analysis of the lesions will be performed and the expression of macrophage inflammatory markers within the plaque will be examined. Successful completion of the present proposal will provide a better understanding of the pathophysiological consequences of LXRa transcriptional actions in response to its phosphorylation.