UC San Diego

Transglutaminases (TG) are a group of enzymes which catalyze the transamidation or "crosslinking" of proteins. This generates a covalent, protease resistant bond between two proteins that relies upon the presence and binding of Ca²⁺ to the TG. Of the nine known human isozymes, tissue transglutaminase (TG2) is ubiquitously expressed and most well characterized. A second TG extensively examined is Factor XIIIA (FXIIIA), the tissue form of the blood clotting zymogen FXIII. Both enzymes have other defined functions in addition to the ability to transamidate proteins. In cartilage, FXIIIA and TG2 have an increased expression and activity during aging and osteoarthritis (OA). TG2 is also highly expressed in atherosclerotic lesions and important in artery remodeling. To understand the consequences of excess TG2 in OA, a detailed molecular structure function study was performed to examine its role in mediating the progression of chondrocyte hypertrophic differentiation. TG2 is active as a TG in the presence of Ca²⁺ and a functional GTPase when bound by GTP. Using site -directed mutagenesis, external GTP-bound TG2 (Lys¹⁷⁴) was required for an effective induction of chondrocyte differentiation, primarily evidenced by increased expression of type X collagen and calcification. The second TG in cartilage, FXIIIA, was found to also induce chondrocyte hypertrophy, but required TG2 to do so. Site- directed mutagenesis studies revealed the requirement of FXIIIA to bind Ca²⁺ to induce type X collagen, with no reliance upon its catalytic activity. Extracellular FXIIIA bound to alpha1 integrin, an integrin subunit localized to hypertrophic chondrocytes in OA. This binding was critical for FXIIIA to mobilize TG2, phosphorylate p38 MAP kinase and induce chondrocyte differentiation. In response to injury, aortic smooth muscle cells (SMC) can differentiate through an osteoblastic / chondrocytic mechanism which leads to matrix calcification. To understand the role of TG2 during this process, TG2-/- SMC were examined. These experiments determined that TG2 promoted calcification of SMC, requiring its catalytic activity. Additionally TG2 can directly regulate the expression of osteopontin, a calcification inhibitor. Collectively the research in this dissertation describes that both TG2 and FXIIA have critical roles in mediating the progression of events that occurs during pathologic cell differentiation