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  • The second part of this study evaluated the role of


    The second part of this study evaluated the role of CK1 in the eFABP4 cellular uptake in endothelial cells. By blocking CK1 expression using a specific siRNA, we corroborated that the presence of CK1 was fundamental for FABP4 cellular uptake and internalization in endothelial cells, thus decreased membrane expression of CK1 is associated with the decreased presence of exogenous FABP4 in the membrane, cytoplasm, and nucleus. We also evaluated the functional implication of the CK1-FABP4 interaction, and we observed that the inhibition of FABP4 internalization was related to decreased activation and nuclear translocation of the NRF2 transcription factor, although we did not observe effects on nuclear translocation of the p65 subunit of NF-κB in non-disturbed endothelial cells. Interestingly, previous studies reported that administration of a FABP4 inhibitor, the BMS309403, decreased the formation of the eFABP4-CK1 complex and that fatty acids would play an important role for FABP4 functionality [14]. Through the analysis of how different fatty imidazoline receptors types affect the internalization of eFABP4 in endothelial cells, we determined that eFABP4 nuclear translocation was fatty-acid-dependent. Our results corroborated the ligand-dependency of FABP4 nuclear translocation, so although FABP4 can bind numerous ligands, only specific compounds can activate its nuclear translocation [31]. Specifically, we found that although no differences attributed to the FA type were reported in eFABP4 expression in the cytoplasm, only eFABP4-transporting palmitate significantly increased eFABP4 nuclear translocation. It should be noted that elevated circulating concentrations of saturated free fatty acids, including palmitate, are implicated in obesity-associated inflammation and insulin resistance in endothelial cells [32]. Therefore, the next step was to assess whether knocking down CK1 or HK-mediated CK1 blocking would affect the endocytosis of palmitate-transporting eFABP4, in a metabolically stressed cell context. Using these approaches, we determined that eFABP4 cellular uptake is also mediated by CK1 in palmitate-stressed endothelial cells. Treating cells with eFABP4-transporting palmitate increased NRF2 expression in the cytoplasm and by blocking the expression of CK1, the phosphorylation and nuclear translocation of NRF2 were inhibited. Furthermore, we corroborated the role of FABP4 in the regulation of lipid-mediated processes, so when eFABP4 is pre-incubated with palmitate, the activation and nuclear translocation of the p65 subunit of NF-κB was increased compared with the non-FABP4 pre-incubated cells. In addition, by CK1-silencing, the nuclear translocation of p65 was significantly reduced. This pro-inflammatory effect of eFABP4 was not observed in the absence of palmitate. In unstressed cells, NRF2 and NF-κB transcription factors are maintained latent in cytoplasmic complexes, oxidative stress or pro-inflammatory stimuli, respectively, trigger the dissociation of these complexes leading to their nuclear import promoting the expression of downstream target genes. Both transcriptions factors are highly sensitive to their triggering stimulus, therefore, the decrease in the expression and activation of both transcription factors observed in CK1-silenced cells might be reflecting that cells are receiving less pro-oxidative and pro-inflammatory stimuli, that is, the less the cells are triggered, the less is the response to the triggering stimulus. Therefore, eFABP4 regulates the cellular response to oxidative stress in non-disturbed or metabolically stressed endothelial cells. In addition, when cells are metabolically stressed using palmitate, eFABP4 enhances the pro-inflammatory effects induced by palmitate per se, possibly due to increased transport inside the cell. Moreover, these eFABP4-mediated effects were dependent on CK1 expression.
    Conclusions In conclusion, we demonstrated that CK1 is necessary for eFABP4 cellular uptake in endothelial cells and that the main effect of silencing CK1 is the blockade of eFABP4 cellular uptake, decreasing subsequently not only the effects mediated by eFABP4 per se but also the FABP4-transported lipid-mediated processes. Therefore, by targeting the CK1-FABP4 interaction in the membrane of endothelial cells, the endocytosis of eFABP4 is hindered in both, non-disturbed or metabolically stressed cells, offering a potential target for the prevention of eFABP4-mediated endothelial dysfunction and therefore a new therapeutic strategy for preventing cardiovascular diseases associated with obesity and diabetes. Further studies are necessary to completely characterize the eFABP4 cellular uptake and endocytosis mechanism as well as to study the regulation of other proteins involved in the cellular uptake of fatty acids, including CD36 and the fatty acid transport protein family (FATP), when CK1-FABP4 interaction is blocked.