Mitochondrial cholesterol trafficking: impact on inflammatory mediators

Grant English, Janice M Taylor, Asma Riaz, Amanda Gibbon, Ann Graham. Glasgow Caledonian University, Glasgow, United Kingdom.

The impact of Liver X Receptor activation in macrophages, via increased cholesterol delivery to the oxysterol generating enzyme, mitochondrial sterol 27-hydroxylase, on the expression of inflammatory mediators: Toll-like receptor 3 (Tlr3), Toll-like receptor 6 (Tlr6) and lymphotoxin alpha (Lta) genes was considered.

Murine RAW 264.7 macrophages stably transfected with pCMV.5 (empty vector control) and pCMV.5_StarD1 (StAR over-expressing) were challenged for 24h, in the presence of dibutyryl cAMP [(Bu)2 cAMP; 0.3mM], lipopolysaccharide (LPS; 0.1μg/ml), LXR agonist (T0901317; 10μM) and combinations thereof. Following isolation of RNA (Trizol) and cDNA synthesis, Stard1, Tlr3, Tlr6 and Lta gene expression levels were determined by QPCR and expressed as a ratio to the housekeeping gene, Gapdh (Values are the mean ± SEM of three independent experiments; n=3).

Over-expression of mitochondrial cholesterol trafficking protein, steroidogenic acute regulatory protein (StAR), in murine (RAW 264.7) macrophages activates, and induces, expression of Liver X Receptors (LXRs), and significantly alters expression of genes involved in the innate immune response, decreasing expression of Tlr6 (92.1%) and markedly increasing expression of Tlr3 (88.3%) and Ltα (87.9%) mRNA when subject to an inflammatory challenge. The effect of StAR over-expression on the expression of inflammatory factors was mimicked by non-sterol LXR agonist, T901317. Evidence of Tlr6 transrepression was detected in StAR over-expressing cells challenged with LPS and LPS + cAMP whereas Tlr3 expression was induced by endogenous and exogenous LXR agonism suggesting Tlr3 may be an LXR target. The expression of Lta was induced in StAR over-expressing cells and further induced by LPS challenge, suggesting a pro-atherogenic role for StAR in this regard.

Macrophage over-expression of StAR significantly enhances LXR-dependent expression of Tlr3 and Ltα and transrepresses expression of Tlr6. These results should be considered in the development of novel therapeutic strategies for human cardiovascular diseases.