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    • Here we investigated on epigenetic regulatory mechanisms of

    2022-05-21

    Here, we investigated on epigenetic regulatory mechanisms of ALX/FPR2 expression. Epigenetic changes, that include phosphorylation, acetylation, methylation and ubiquitination of DNA and histone proteins, occur in a variety of diseases [21] and are being recognized as key targets for personalized medicine. We reasoned that epigenetic modifications could be responsible for variable ALX/FPR2 levels in different cell types and that the analysis of these changes could provide knowledge for novel pharmacology. To test this hypothesis, we examined epigenetic modifications of histone proteins along the ALX/FPR2 gene, comparing profiles in PMN, that express high ALX/FPR2 transcript and protein and MDA-MB231 cells, a breast cancer cell line expressing low ALX/FPR2 mRNA and protein (Fig. 1). We focused on select acetylated and methylated lysine residues on the H3 histone, namely K27, which, when tri-methylated or acetylated, respectively represses or stimulates transcription [22], and on K4, which in its tri-methylated form sustains gene transcription [23]. As expected, we found that PMN exhibited a great abundance of H3K27ac over H3K27me3 (~100:1 ratio) in all of the examined regions of the ALX/FPR2 gene (Fig. 2). This pattern was completely reversed in MDA-MB231 cells, which showed a ~1:2 ratio (Fig. 2B). Also, PMN displayed a great prevalence of H3K4me3 over H3K27me3 (~15:1 to 80:1), whereas H3K27me3 predominated over H3K4me3 in MDA-MB231 L 012 sodium salt sale (Fig. 2B). The observation that the ALX/FPR2 gene undergoes transcription silencing in the MDA-MB231 breast cancer cell line is consistent with recent data that show downregulation of ALX/FPR2 expression in human laryngeal squamous cell carcinoma [24], suggesting a role for this receptor in cancer. Proliferative or anti-proliferative actions of ALX/FPR2 agonists on cancer cells, depending on the agonist and the cell type have been described [25], [26]. On the other hand, beneficial immunomodulatory signaling through ALX/FPR2 in colon cancer and in experimental lung carcinoma has been reported [27], [28], suggesting that ALX/FPR2 may have protective roles in some selected neoplasms by regulating the host response to cancer cells. This implies that challenging cancer cells with ALX/FPR2 agonists in vitro may not necessarily provide indication on the pathobiology of this receptor in cancer, which can be quite complex and tumor type-related and, therefore, requires further studies. The direct relationship between the asset of H3 protein and ALX/FPR2 expression was confirmed by experiments with acetylating and/or demethylating agents. For these experiments, we selected 5-aza-dC, a known demethylating agent, largely used in anti-cancer therapy; TSA [27], a histone deacetylase (HDAC) inhibitor [28] and CTPB, which activates p300 histone acetyltransferase activity [29]. Comparing data from different experimental settings, we observed that 5-aza-dC increased significantly the relative abundance of H3K4me3. The combination 5-aza-dC plus CTPB gave the strongest increment in H3K27ac and H3K4me as well as in ALX/FPR2 mRNA and protein (Fig. 3 A and C). This was paralleled by a ~3 fold increase in luciferase activity of the ALX/FPR2 promoter (Fig. 3B). On the other hand, H3K27me3 was not significantly affected by treatments, suggesting that H3K27ac and H4K4me3 are preeminent to unlock ALX/FPR2 expression in MDA-MB231 cells. These results, in particular those with 5-aza-dC, are consistent with our previous data [16] and indicate that the accumulation of H3K27ac and H3K4me3 unlocks chromatin-dependent transcriptional repression of ALX/FPR2 expression in MDA-MB231 cells. Whether this represents a general mechanism and, therefore, whether acetylating and/or demethylating agents upregulate ALX/FPR2 in cell types, expressing low levels of this receptor, other than MDA-MB231 cells, remains to be determined, although in our experimental setting PAEC, which express more abundant ALX/FPR2 mRNA levels than MDA-MB231 but lower compared to PMN responded as well as MDA-MB231 (Fig. 4). It is, however, remarkable that these agents gave a considerable increase in ALX/FPR2 protein, which in the appropriate pathophysiological settings can burst up agonist-induced responses. Indeed, this GPCR conveys bioactions from endogenous anti-inflammatory and pro-resolution mediators, such as LXA4, 15-epi-LXA4, RvD1, AnxA1, which may serve as templates for novel immunoresolving therapeutics [30]. These agonists are produced during inflammation and the potency of their cellular bioactions depend on receptor availability and expression level. Thus, molecules that increase ALX/FPR2 expression may be useful in clinical settings characterized by deficient resolution of the inflammatory response [14]. In this respect, data with PAEC (Fig. 4) are particularly relevant since LXA4 exerts a number of protective, anti-inflammatory bioactions on the vascular endothelium [31].