80 Chapter 3 homeostasis (53) and it has been shown that alterations in this process can lead to changes in macrophage function, especially in generation of reactive oxygen species (54). It has been reported that the activity of the TNFα signaling pathway is controlled by ubiquitination and that inhibiting protein ubiquitination leads to increased inflammatory activity in macrophages (54–56). Since ubiquitination and acetylation are mutually exclusive post-translational modifications, meaning that they share the same modification site, i.e. the epsilon amino group of lysine (57,58), it is conceivable that changing protein acetylation patterns results in a modified ubiquitination pattern. The balance between protein acetylation and protein ubiquitination plays an important role in regulating protein stability, since an acetylated lysine residue cannot be ubiquitinated, therefore enhancing protein stability. For acetylated lysine residues to be ubiquitinated, they need to be deacetylated by KDAC and this process is blocked by treatment with the inhibitors. This could be one of the reasons why many more proteins (403 versus 20) were differentially expressed in macrophages pre-treated with MS275 compared to those pre-treated with RGFP966, since MS275 has a broader activity spectrum than RGFP966. While pathway enrichment provided insights into possible biological mechanisms, specific proteins that were strongly upregulated after MS275 and LPS treatment may give further insights into particular responses. The top 3 most upregulated proteins in alveolar-like macrophages pre-treated with MS275 compared to LPSonly treatment, were INCA1 (Inhibitor Of CDK, Cyclin A1 Interacting Protein 1), JAM-A (junctional adhesion molecule A), and MMP12 (macrophage metalloelastase 12). The function of INCA1 is still relatively unknown, but it has been reported to inhibit the activity of cyclin-dependent kinases and cell proliferation (59). However, no clear connection to the lung (60) has been described. JAM-A was shown to have an important role in inflammatory processes, since it is a transmembrane domain protein that is necessary for diapedesis at inflammatory sites (61). A recent publication by Kiss et al. demonstrated that JAM-A expression increased in tumors during monocyte-to-macrophage differentiation (62) indicating that it may play a role in macrophage-mediated inflammation. Furthermore, JAM-A was shown to play a key role in maintaining lung epithelial barrier function (63), making this protein a potential candidate for further investigations in the context of macrophage-epithelial communication. MMP12 is an endopeptidase that plays a crucial role in smoke-induced inflammation in COPD by contributing to alveolar tissue destruction and subsequent emphysema development (64). Its higher expression after KDAC inhibitor treatment is therefore somewhat puzzling. However, MMP12 has been reported to have a dual role by being associated with both pro-inflammatory (65,66) and anti-inflammatory (65,66) effects, reflecting the
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