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  • AZ505 On the other hand the IL promoter activity induced

    2019-11-30

    On the other hand, the IL-8 promoter activity induced by the signalling cascade of the CysLT1-G300S receptor is higher than that of the WT receptor. The chemokine IL-8 is mainly involved in the recruitment and activation of neutrophils and we have previously shown that this chemokine gene is a target for the transcription factors activated by the CysLT1 receptor, by the presence of binding sites for NF-κB, NF-IL-6 and AP-1, via the NF-κB and MAPKinase pathways [24], [33]. It is interesting to note that, although the ligand of predilection of CysLT1 is LTD4, there is also a difference in the responses induced by LTC4, between the WT and the variant CysLT1-G300S receptors. Our results indicate that LTD4-induced signalling is stronger with the variant CysLT1-G300S than with the CysLT1-WT receptor, due to a higher response at the same low concentrations (Fig. 4). Taken together, our results let us speculate that the enhanced IL-8 promoter transactivation through the variant CysLT1-G300S may be the result of a synergic activation of PLCβ and the MAPK/Erk pathway, leading to a more efficient downstream signalling cascade. Using Genomatix software MatInspector [41], we predicted that the human IL-13 promoter had STAT, GATA, AP-1 and NF-κB binding sites. In addition to the fact that IL-13 is a Th2 cytokine involved in AZ505 and allergic rhinitis [34], [42], and that it up-regulates CysLT1 receptor expression [14], [16], [34], [43], it was also shown that CysLT1 activation induces IL-13 production in type 2 Innate Lymphoid Cells [44]. Since the IL-13 promoter shares several binding sites with the IL-8 promoter, we speculated that IL-13 promoter transactivation could also be induced by CysLT1 activation by cysLTs in HEK293-cells. Indeed, whereas both LTD4 and LTC4 induced IL-13 promoter activation, suboptimal concentrations of LTD4 induced a higher response through CysLT1-G300S variant, (Fig. 4 and Fig. 5). Many mutations in the structure of G-protein-coupled receptors have been studied and were shown to have different signalling potential from the WT receptors. For example, the receptor variant due to a mutation in the DRY motif of the CXCR2 receptor produces more IPs and exhibited more active signalling [45]. Our laboratory has shown that the variant CysLT2-M201V has a reduced signalling in response to both LTC4 and LTD4, compared to the CysLT2-WT receptor [46]. Finally, since the signalling is different at many levels between the variants and the WT receptor, it would be plausible to speculate that the binding capacity of the receptors could be affected as well. Even though only one concentration of ligand was tested, our findings suggest a greater specific binding of LTD4 to the variant CysLT1-G300S than to the CysLT1-I206S variant or the WT receptors (Fig. 6). This could be due to a higher affinity of the binding sites on CysLT1-G300S associated with differences in structure and conformation of the variant, thus explaining the higher signal transduction, despite the same constitutive cell surface expression of the variant receptor. Mutations that alter ligand binding and affinity have been reported. We have shown that a mutation in the C-tail of the BLT1 receptor increased the number of binding sites and cellular signalling [47]. In the β2-adrenergic receptor, mutations in the amino-acid structure alter the dynamics and conformation of the receptor and the ligand-binding capacity [48]. However, further experiments need to be carried out to determine the binding affinity of the cysLTs LTD4 and LTC4 to the CysLT1 receptor variants by conducting saturation and/or competition binding assays. On the other hand, in addition to the signalling cascade, it is known that the receptor CysLT1 may form homo-dimers at the cell surface [49], [50]. It is possible that dimerization may naturally occur between the variants and the WT receptor. This could add an important element to the signalisation and induce alterations in the pathways, the effectors and the combined functional response. Up to now, the dynamic structure of the CysLT1 receptor is not fully understood, and such studies will be important in order to determine the functional activity of the receptor.