FXR and Anti-HIV Therapy Figure 8. Diagramatic representation of the effect exerted by FXR and PPARa ligands on dyslipidemia and accelerated atherosclerosis caused by the HIV-PI ritonavir. Exposure to ritonavir causes SREBP1c activation in the liver and monocytes. In circulating monocytes ritonavir causes a SREBP1c-dependent induction of CD36. Increased expression of CD36 leads to uptake of ac-LDL and favours the acquisition of an activated phenotype by monocytes and their transformation into foam cells. Activation of FXR, reduces CD36 expression on macrophages by interfering with SREBP1c. Knocking down SREBP1c by siRNAs abrogates the activation of CD36 caused by ritonavir. Activatory pathways are shown in black and inhibitory pathways in red. doi:10.1371/journal.pone.0013238.g008 100 mg/day twice a day are <1 mM, ritonavir accumulates in the peripheral blood mononuclear cells reaching concentrations of <20 mM. Importantly, while CDCA and GW4064 had no effect on basal expression of CD36 both agents were effective in reducing CD36 protein expression induced by 10 mM of ritonavir as measured by flow cytometry. Further on, modulation of CD36 expression was functionally correlated with a reduction of ac-LDL uptake by macrophages. Several of our results indicate that FXR directly antagonizes the effect of ritonavir on CD36 by interacting with SREBP1c. Exposure to ritonavir induces the expression of SREBP1c mRNA as well as its nuclear accumulation. Our results are therefore a variation of previous studies reporting that ritonavir inhibits SREBP1c degrada- tion [9]. The genomic effects of ritonavir could be explained by several mechanisms. In macrophages, HIV PIs activate the unfolded protein response (UPR) which is involved in the regulation of CD36 expression an event that has been involved in foam cell formation [11]. Despite FXR ligands have no effect on post-translational handling of SREBP1c we have found that FXR ligation reduces total SREBP-1c, protein and mRNA levels, by approximately 50% therefore reducing its nuclear translocation. In the present study we have found that CDCA causes a 4–5 fold increase of SHP mRNA and we have documented an inverse correlation between SHP and SREBP-1 expression in RAW264.7 cells exposed to CDCA. These data are consistent with the finding that FXR activation by natural and synthetic agonists represses the expression of SREBP-1c and its lipogenic target genes in mouse primary hepatocytes and in liver [18–20,33]. One important observation we made is that exposure of EAW264.7 cells to CDCA resulted in robust induction in the expression of ABAC1, a gene whose protein product mediates cholesterol efflux from macrophages. The ability of FXR to induce ABCA 1 expression could be of mechanistic relevance in explaining the anti-atherogenic effects of CDCA [44]. Several experimental data link SREBP responsive elements (SRE) to the regulation of CD36 gene expression. Thus, knocking down the expression of SREBP1c greatly attenuated the induction of CD36 caused by ritonavir. However, despite we have identified a putative SRE-binding site in the promoter of mouse CD36 gene, we have been unable to show a functional role for this SRE in RAW264.7 cells exposed to ritonavir, indicating that ritonavir induces CD36 expression by multiple, yet not completely identified, pathways. Despite the present study demonstrates that FXR activation attenuates dyslipidemia caused by ritonavir, this is a preclinical PLoS ONE | www.plosone.org 10 October 2010 | Volume 5 | Issue 10 | e13238