Examination of potential mechanisms linking AMPK to inhibition of IL-6 signalling

Considerable recent evidence supports the role of AMP-activated protein kinase (AMPK) as an anti-inflammatory mediator, yet the mechanisms of its anti-inflammatory actions are only starting to be unravelled. Inappropriate cytokine stimulated Janus kinase (JAK)-signal transducer and activator of transcription (STAT) signalling is a key feature of many pro-inflammatory events, including atherogenesis. Previous unpublished studies in our group have investigated whether AMPK modifies cytokine stimulation of JAK-STAT signalling in HUVECs. These preliminary investigations demonstrated that pre-treatment of HUVECs with AMPK activator, A769662, significantly inhibits both sIL-6Rα/IL-6 and IFN-α stimulation of STAT3 Tyr705 phosphorylation in HUVECs. IFN-α activates STATs via an IFNα/β receptor 1 (IFNAR1/IFNAR2) complex which is distinct from the sIL-6Rα/IL-6/gp130 complex. The studies in this thesis therefore tested the hypothesis that AMPK was exerting its inhibitory effects at one or more common signalling loci downstream of IFNAR1/IFNAR2 and gp130 at a post-receptor level. First, it was investigated whether AMPK exerts its inhibitory effects on JAK-STAT signalling via a known regulator of JAK or STAT, or an AMPK downstream target known to either directly or indirectly impact on JAK-STAT signalling. A combination of genetic and pharmacological approaches was utilised to assess the role of each of the following AMPK targets: TC-PTP, SHP2, eNOS, PKCλ, SIRT1, CPT1 and mTOR. It was demonstrated that activation of AMPK in HUVECs inhibited sIL-6Rα/IL-6 stimulated STAT3 Tyr705 phosphorylation via a mechanism independent of TC-PTP, eNOS, PKC, SIRT1 and mTOR. Furthermore, inhibition of mTOR and eNOS reduced sIL-6Rα/IL-6 stimulated STAT3 Tyr705 phosphorylation, independent of AMPK activation by A769662. Next, it was investigated whether AMPK acts directly on a signalling component of the JAK-STAT pathway. Specifically, it was hypothesised that AMPK could directly phosphorylate serine or threonine residues within JAK to inhibit IL-6 signalling. siRNA-mediated downregulation of JAK isoforms demonstrated that IL-6 induced STAT3 Tyr705 phosphorylation predominantly via JAK1 in human umbilical vein endothelial cells (HUVECs). In vitro kinase assays of JAK1-derived peptides demonstrated that AMPK can directly phosphorylate two residues, Ser515 and Ser518, within the JAK1 SH2 domain. Subsequently, a GST- 14-3-3 pull down assay of cell lysates produced from A769662 treated JAK1- defcient U4C cells transiently expressing either wild type or S515A/S518A double mutant JAK1 demonstrated that pharmacological activation of AMPK promotes 14-3-3 binding of JAK1 via a mechanism requiring Ser515 and Ser518. Furthermore, mutation of Ser515 and Ser518 abolishes the ability of AMPK to inhibit JAK-STAT signalling by an IL-6 trans-signalling complex and from a constitutively active Val658Phe-mutated JAK1. In this study it is proposed that AMPK phosphorylation of JAK1 at Ser515 and Ser518 inhibits IL-6 stimulated JAK1 phosphorylating STAT3 by interfering with the ability of JAK1 to interact and phosphorylate the GP130 receptor and /or STAT3 and STAT1. Therefore, AMPK phosphorylation of JAK1 could potentially be a novel regulatory mechanism that could be developed as a therapy for treating chronic inflammatory diseases such as atherosclerosis.