The present work shows that VLR, a human native VLDL- and LDL-rich lipoprotein mix, induces a net global decrease in gene expression that mirrors the previously reported global de novo DNA methylation caused by VLR in THP-1 macrophages . Proteome data independently support these findings, although the effects of VLR were more prominent on protein species than on transcripts. This observation points to VLR affecting a combination of translation repression, post-translational modification regulation and nuclear protein stability. VLR-induced transcript down-regulation includes pathways involved in fundamental macrophage functions such as inflammation, chemotaxis, metal and cation transport. The down-regulation of pro-inflammatory genes is a prominent effect of VLR. Seven pro-inflammatory genes are repressed, in addition to one solute carrier gene (SLC7A2) that participates in macrophage activation by various cytokines . Although apparently in contrast with data showing atherogenic effects of VLDL, these distinct effects of VLR are consistent with existing literature indicating that stimulation by native VLDL per se results in a weak, if any, inflammatory response in cell culture models . For example, VLDL induces inflammatory marker expression only in synergy with cytokines in human aortic endothelial cells and macrophages [35–37]. Similar results were obtained in a side-by-side gene expression analysis in THP-1 macrophages stimulated with oxidised LDL, or oxidised or native chylomicrons remnants. Oxidised LDL and oxidised chylomicrons produced a radically different gene expression pattern from the one induced by native chylomicrons, including a down-regulation of ABCA1 by the latter, resembling the effects of VLR on ABCG1 presented here [9, 38]. Furthermore, native LDL is a poor activator of the NLRP3 inflammasome, a protein complex involved in IL-1beta production, compared with corresponding oxidised versions . Based on our results and supported by evidence discussed above, we conclude that VLR-induced de novo DNA methylation in THP-1 macrophages does not reflect a pro-atherogenic response as our earlier data seemed to indicate , rather it underlies an anti-inflammatory response. It is possible that in normal physiological conditions circulating native VLDL and possibly other lipoproteins buffer macrophage functions by limiting pro-inflammatory gene activity. The observation that exogenous lipoproteins can protect against endotoxin-induced death and decrease plasma TNF in vivo provides ground to this idea . On the other hand, when pro-inflammatory signals are present presumably above a critical threshold, particularly in association with lipoprotein oxidation, or act chronically as in sustained hyperlipidaemia, anti-inflammatory gene regulation mechanisms would be overrun. As a consequence, native lipoproteins cease to exert negative controls on macrophage function and indeed act as an activating switch by synergising with inflammatory factors. Persson and co-workers discussed this basic idea in detail .
For all analyzed genes but ABCG1, VLR-induced silencing is associated with de novo DNA methylation at the corresponding promoter. Interestingly, the non-random distribution of down-regulated genes suggests that VLR regulates gene expression by a coordinated mechanism resembling instructive epigenetic regulation reported in cancer .
In addition, our results are in apparent contrast with published data suggesting that DNA hypermethylation may underlie a pro-inflammatory response in some conditions . Our data complement the latter observations, by showing that DNA hypermethylation is not invariably associated with inflammation. Rather, we suggest that specific epigenetic modifications are imposed on distinct sets of sequences in a stimulus-specific fashion thus inducing specific gene expression patterns, although the resulting global epigenetic parameters - i.e. total genome DNA methylation level - may be similar.
Furthermore, we observed a potentially interesting selectivity in de novo DNA methylation and H4K20 hypermethylation responses between individual lipoprotein species. Our data indicate that the response to VLR must be the sum of activities of factors specific for or present at different amounts in each lipoprotein species. The mechanisms underlying the observed differential effects on histone post-translational modifications are currently unknown and deserve investigation. Clearly, understanding the mechanisms by which lipoproteins modulate chromatin structure in macrophages and other cell types requires a detailed screening of lipoprotein components. The observation that lipoprotein preparations from Sweden or Mexico produced consistent effects in whole genome expression array analysis and its validation, suggests the preliminary conclusion that the factor eliciting the responses described here is a structural lipoprotein component probably not qualitatively or quantitatively affected by diet. Interestingly, recent evidence shows that palmitic acid, an abundant pro-inflammatory fatty acid of endogenous and dietary origin, promotes global DNA hypermethylation in primary human myocytes .
As for the identity of intracellular factors mediating VLDL-induced de novo DNA methylation, the absence of the canonical de novo DNMTs DNMT3A and -3B was unexpected in THP-1 macrophages. Our data indicate that DNMT1 is necessary and sufficient for de novo DNA methylation in response to VLDL, in contrast with its widely accepted role as canonical maintenance DNMT. Interestingly, these observations are supported by our preliminary kinome data showing that VLDL lowers the cellular levels of T410-phosphorylated - i.e. active - PKCzeta, which has been recently shown to inhibit DNMT1 . Previous studies demonstrated that DNMT1 might participate in de novo DNA methylation in cooperation with DNMT3A and -3B [45, 46]. Accordingly, exogenous DNMT1 expression induced de novo methylation of a relatively small number of genes in HEK-293T cells, possibly in cooperation with endogenous DNMT3A/3B . To our knowledge, the only previous example of an independent de novo methylation activity for DNMT1 is the demonstration that this enzyme re-establishes somatic patterns of non-CpG methylation following their erasure in the germline . The present study provides new evidence that DNMT1 can perform de novo DNA methylation independently of maintenance DNMTs. A de novo activity for DNMT1 may be physiologically relevant in tissues in which an age-related decline of DNMT3A/B expression has been documented [49, 50]. Interestingly, DNMT1 might be a specific mediator of de novo DNA methylation in response to pro-inflammatory signals, as IL-6 induces upregulation and nuclear translocation of DNMT1 [51–53]. As for DNMT2, the absence of any effects of this DNMT on DNA methylation confirms previous literature data .
In addition, our data provide genetic evidence on the involvement of the Dicer pathway in epigenetic responses to VLDL. Taken together, the absence of Dicer expression and the lack of any effect of VLDL on miRNA production demonstrate that the latter factors or other small non-coding RNAs generated by Dicer do not mediate VLDL-induced de novo DNA methylation in THP-1 macrophages. These observations indicate that chromatin regulation by Dicer-mediated pathways if present in mammals, is confined to specific cell types rather than being a universal mechanism. Nonetheless, it is possible that Dicer-independent RNA-mediated DNA methylation operates in THP-1 cells as reported in plants .