Mitogen-activated protein kinase phosphatase-2 (MKP-2) is a type 1 nuclear dual specificity phosphatase which known to play key roles in cellular function through dephosphorylation of the MAPKs (ERK, JNK and p38). MKP-2 has recently been shown to play a significant role in the immune response following parasite and bacterial infections. However, the effect of MKP-2 deletion on a number of other key macrophages functions including phagocytosis, motility and proliferation is yet to be studied. This thesis therefore utilised a novel DUSP4 gene knockout mouse and investigated the effect of cellular MKP-2 removal in bone marrow derived macrophage (BMDMs) using a number of approaches.Results obtained from macrophage characterisation experiments demonstrated that following LPS stimulation MKP-2 deleted BMDMs showed enhanced JNK activation as opposed to other MAPKs, this effect was found to correlate with enhanced endothelin-1 (EDN1) expression at both gene and protein synthesis level. This is the first study to reveal that EDN1 expression is negatively regulated by MKP-2. MKP-2 deletion also resulted in different kinetic profiles for phagocytosis and migration which was also differed in M1 and M2 stimulated cultures. MKP-2-/- macrophages showed more phagocytic activity but less motility upon LPS activation, this effect was reversed when cells were pre-treated with IL-4 which gave less phagocytic activity but more motility. Also MKP-2 deletion reduced macrophage migration towards C5a suggesting a new role for MKP-2 gene in regulating macrophage motility.Loss of MKP-2 also resulted in decreased macrophage proliferation activity.Finally, a metabolomics profile was established for both MKP-2+/+ and MKP-2-/- macrophages stimulated by different agents. MKP-2 deletion resulted in enhanced production of metabolites associated with glycolysis and the pentose phosphate pathway during the time at which MAPKs were upregulated indicative of a tight correlation between signalling and metabolic changes that underlie macrophage functions. In contrast, the proline and arginine pathway was downregulated in MKP-2 deleted macrophages. This was confirmed by studying nitric oxide production which was downregulated in MKP-2-/- macrophages upon LPS challenge which further correlated with changes in citrulline and ornithine. Collectively, this is the first study to investigate and determine the role of MKP-2 gene in macrophage functions; deletion or inhibition of MKP-2 in macrophages may be a therapeutically desirable approach.
|Date of Award||1 Oct 2017|
- University Of Strathclyde
|Supervisor||Robin Plevin (Supervisor) & Andrew Paul (Supervisor)|