External GTP-bound transglutaminase 2 is a molecular switch for chondrocyte hypertrophic differentiation and calcification.

Chondrocyte maturation to hypertrophy, associated with up-regulated transglutaminase 2 (TG2) expression, mediates not only physiologic growth plate mineralization but also pathologic matrix calcification and dys-regulated matrix repair in osteoarthritic articular cartilage. TG2-/- mouse chondrocytes demonstrate markedly inhibited progression to hypertrophic differentiation in response to both retinoic acid and the chemokine CXCL1. Here, our objectives were to test if up-regulated TG2 alone is sufficient to promote chondrocyte hypertrophic differentiation and to identify TG2 molecular determinants and potential downstream signals involved. TG2 activities, regulated by nucleotides and calcium, include cross-linking of cartilage matrix proteins, binding of fibronectin, and hydrolysis of GTP and ATP. Following transfection of TG2 site-directed mutants into chondrocytic cells, we observed that wild type TG2, and TG catalytic site and fibronectin-binding mutants promoted type X collagen expression and matrix calcification consistent with chondrocyte hypertrophic differentiation. In contrast, transfected mutants of TG2 GTP binding (K173L) and externalization (Y274A) sites did not stimulate chondrocyte hypertrophy. Recombinant TG2 treatment of bovine cartilage explants demonstrated that extracellular TG2 induced hypertrophy more robustly in the GTP-bound state, confirming an essential role of TG2 GTP binding. Finally, TG2 treatment induced type X collagen in a beta1 integrin-mediated manner, associated with rapid phosphorylation of both Rac1 and p38 kinases that were inhibited by mutation of the TG2 GTP binding site. In conclusion, externalized GTP-bound TG2 serves as a molecular switch for differentiation of chondrocytes to a hypertrophic, calcifying phenotype in a manner that does not require either TG2 transamidation activity or fibronectin binding.