Tumor necrosis factor-stimulated gene-6 (TSG-6) is a protein that is expressed in many inflammatory contexts e.g. arthritic joints, where it acts as an endogenous protector of tissues from the damaging effects of inflammation. There is evidence that this occurs via multiple mechanisms; for example, TSG-6 suppresses neutrophil extravasation by interacting with CXCL8 and inhibiting the activity of this chemokine. In this regard, recent work has identified TSG-6 as a broad-spectrum chemokine-binding protein and pluripotent regulator of chemokine function; thus, it could modulate the migration of other leukocyte populations. In the context of arthritic disease, TSG-6 has been shown to protect against cartilage damage in murine models of inflammatory arthritis, and recent in vitro studies have revealed that it acts directly on chondrocytes to inhibit cytokine-induced expression of aggrecanase and collagenase enzymes that are associated with cartilage degradation in osteoarthritis (OA). Furthermore, TSG-6 suppresses bone resorption by osteoclasts in vitro and its isolated Link module domain (Link_TSG6) has been found to reduce bone loss in a mouse model of osteoporosis. Thus, TSG-6 represents an attractive therapeutic target for arthritic diseases such as OA; however, the mechanisms underlying its protective activities are not fully understood. TSG-6 is known to interact with at least seven bone morphogenetic proteins (BMPs), including BMP-2 and BMP-7, which are important mediators of bone and cartilage maintenance; it also binds to receptor activator of NF-ÎºB ligand (RANKL), a key activator of bone resorption. The aim of this study was to fully characterise these protein-protein interactions and to identify reagents and suitable in vitro models to investigate their functional consequences. Surface plasmon resonance (SPR) experiments furthered our understanding of TSG-6 interactions with BMP-2, BMP-7 and RANKL, revealing that TSG-6 binds with high affinity to these proteins and that these interactions are mediated primarily via the Link module of TSG-6. Analysis of a panel of Link_TSG6 single-site mutants by SPR shed light on the location of the BMP-binding site(s) on TSG-6, indicating that BMP-2 and BMP-7 likely interact with a common surface on the Link module that appears to be distinct from previously characterised binding sites for other TSG-6 ligands e.g. heparin and hyaluronic acid. This work was extended to include chemokines from both the CC and CXC subfamilies, where solid phase binding assays provided evidence that the heparin- and chemokine-binding sites on Link_TSG6 do not seem to overlap. Furthermore, preliminary data from SPR experiments with CCL2 and CXCL6 (a newly identified TSG-6-binding chemokine) suggest that these chemokines might share an interaction surface on the Link module of TSG-6, which might overlap with the BMP-binding site identified in this study. Finally, evaluation of in vitro models of articular cartilage has identified the chondrocyte pellet culture system as the most suitable model to investigate if/how TSG-6's interactions with BMP-2 and/or BMP-7 contribute to its effects on chondrocyte gene expression. Future studies in this model system will be carried out using mutants of Link_TSG6 with impaired/enhanced affinities for BMPs, identified here by SPR analyses as reagents for studying the functional consequences of the TSG-6/BMP interactions.
|Date of Award||1 Aug 2018|
- The University of Manchester
|Supervisor||Anthony Day (Supervisor) & Caroline Milner (Supervisor)|
Investigation of the interactions of TSG-6 with BMP-2, BMP-7 and RANKL; towards understanding the protective effects of TSG-6 in arthritis
Valdambrini, E. (Author). 1 Aug 2018
Student thesis: Phd