Signaling regulating active TG2 release from cells: implications for osteoarthritis.

Objectives: Although altered joint mechanics is considered the main risk for osteoarthritis (OA), disease development is linked to inflammatory processes as well. Importantly, OA can affect the temporomandibular joint, causing facial pain, headaches and limiting mouth mobility. Transglutaminase 2 (TG2) is potentially an important player in OA pathogenesis, as its extracellular actions promote aberrant chondrocyte hypertrophy and cartilage calcification. However, TG2 release from cells occurs through a non-conventional and currently unknown process. We hypothesize that ATP secretion from immune cells, and damaged or mechanically stimulated chondrocytes drives TG2 release in OA via P2X7 receptor (P2X7R) activation. The aim of this study was to investigate whether TG2 externalization is mediated by activation of P2X7R.
Methods: Cell models were differentiated THP-1 monocytes and HEK293 cells expressing P2X7R. TG2 secretion was quantified using Western blotting. Release of vesicles was analyzed by nanoparticle tracking. Pharmacological agents were used to determine the role of Ca²⁺ signaling and P2X7R-dependent pore formation in TG2 secretion. Finally, TG2 externalization was monitored during IL-1β/oncostatin-M treatment of 3D cartilage constructs.
Results: In macrophage-like cells TG2 release was dependent on P2X7R. In HEK293 cells P2X7R activation induced time-dependent release of TG2 but not other cytoplasmic proteins. TG2 release was independent of inflammasome formation, occurred through a mechanism distinct from microvesicle shedding and was not due to apoptosis. Significantly, P2X7R-dependent membrane pore formation but not the initial ion flux or membrane depolarization led to TG2 secretion. IL-1β/oncostatin-M treated cartilage displayed enhanced TG2 expression and activity, thus demonstrating the importance of inflammatory signaling in regulation of TG2 function.
Conclusions: We demonstrate that P2X7R-dependent membrane pore formation is regulating TG2 release from cells. Our data indicates that P2X7R has a central role in in controlling TG2 externalization and downstream events, especially in the context of inflammation. This identifies a new avenue for therapeutic intervention.