EVALUATION OF THE ROLE OF TGF-β ACTIVATED KINASE 1 IN THE PATHOGENESIS OF RHEUMATIC DISEASES

Paul Michael Panipinto

doi:10.7273/000007196

Rheumatic diseases are a collection of diseases that involve pain and inflammation, often in the joints or periarticular tissues. These progressive diseases are often accompanied by increasing loss of function of the affected tissues, creating debilitating effects for patients afflicted by them. The pathogenesis of rheumatic diseases is nearly always driven by dysregulated inflammation – events that should be transient, but instead occur in an extended or chronic state. This is mediated by signaling molecules called cytokines and chemokines which are the natural communication system of the body, and which in normal circumstances, both upregulate and resolve inflammatory cycles. Several signaling cascades are responsible for the involvement of cytokines in these diseases, but two of the major pathways are the mitogen activated protein kinase (MAPK) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathways. These two critical pathways start from a hierarchically upstream common signaling protein called transforming growth factor beta activated kinase-1 (TAK-1). Because of its central location at the nexus of these pathways, TAK-1 has been a promising pharmacological target for many years. The complex nature and dynamic functions of TAK-1 have prevented quick progress toward an inhibitory therapeutic. However, as our research and the research of other groups progressed over the last decade, the findings consistently pointed to TAK-1 modulation as a potential non-immunosuppressive therapeutic option to treat rheumatic diseases. This work looks at two rheumatic diseases in particular – gout and rheumatoid arthritis (RA) – to examine the role of TAK-1 and several experimental TAK-1 inhibitors. Gout is primarily a metabolic disease in which serum urate exceeds homeostatic levels and deposits in the joints in crystal structures. This causes an autoinflammatory reaction in tissue resident macrophages which rely on TAK-1 to initiate a gout flare – a painful, transient inflammatory state caused by the overproduction of the cytokine interleukin-1 beta (IL-1β), activated by the NOD-, LRR- and pyrin-containing domain-3 (NLRP3) inflammasome. Both the production of the inactive pro-form of IL-1β and the activation of it depend on the activity of TAK-1, however, TAK-1 modulation has not been explored as a therapeutic option for gout. RA has much more heterogenous origins and a wider variety of circulating inflammatory cytokines, however, many of these rely on either TAK-1-mediated NF-κB or MAPK activation, making TAK-1 an attractive therapeutic target. Here we examined the inhibitory potential of the natural product, pentagalloyl glucose (PGG), a hydrolysable tannin found abundantly in more than 70 natural products and marketed in the supplement market as a cure for inflammation. Our in vitro experiments demonstrate the mechanisms by which PGG suppreses inflammation in a cell culture model of gout – particularly its unexpected inhibition of macrophage priming and NLRP3 activation. To examine the broader effect of TAK-1 inhibition on the THP-1 proteome, we performed untargeted phosphoproteomics on unstimulated, monosodium urate (MSU) activated, and MSU+inhibitor-treated macrophages (including PGG) to identify key phosphorylation events that occur during homeostatic, disease, and pharmacologically treated states. Two major results can be credited to these phosphoproteomic experiments – 1) this is the first data set addressing the entire gout kinome in these cells that are widely used for research, and 2) there is an interdependence between the Rho- GTPase and inflammatory cycles that may be individually targeted to treat gout. Furthermore, this work demonstrates the in vivo efficacy of PGG, reducing the overall inflammation in a murine model of gout and decreasing the time-to-resolution for these animals. Finally, we examined takinib – a putative inhibitor of TAK-1, which was untested in gout, but demonstrated efficacy in reducing inflammation in tumor necrosis factor-alpha (TNF-α) treated T lymphocytes. Here we found that rather than inhibiting TAK-1, takinib induced its kinase activity while conversely lowering cytokine concentrations in vitro. Evaluation of the mechanisms identified that takinib inhibited a signal transducer and activator of transcription-3 (STAT3) pathway, providing clarity about the mechanism of action of takinib in RA arthritis synovial fibroblasts, a key mediator of inflammation and RA progression. Overall, these results provide a strong scientific rationale for TAK-1 inhibition as a potential therapeutic for inflammation in gout and RA that is also inhibits the NLRP3 inflammasome and provide rationale for investigating the Rho/MAPK/ERK pathway as a therapeutic mechanism in gout.

EVALUATION OF THE ROLE OF TGF-β ACTIVATED KINASE 1 IN THE PATHOGENESIS OF RHEUMATIC DISEASES

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