Unraveling the novel role of Transglutaminase1 in osteoclastogenesis and bone remodeling

Osteoclasts are multinucleated, large bone-resorbing cells derived from monocyte-macrophage precursors and are a major drug target cell for osteoporosis. Our research and that of others have demonstrated that transglutaminase enzyme activity from all or one of the three TGs; TG2, FXIII-A, and TG1, regulate osteoclast differentiation and bone remodeling. In vivo and in vitro studies on Tgm2–/–; F13a1–/– and Tgm2–/– mice revealed increased osteoclastogenesis and a significant loss of bone mass. However, the role of TG1 in osteoclast and bone remain poorly understood. First, I report that the increased osteoclastogenesis in Tgm2–/– can be reversed by a TG inhibitor, NC9, highlighting the involvement of TG1, whose activity was increased in the absence of TG2, while FXIII-A activity remained unchanged. Moreover, my findings show that aspartate proteases like Cathepsin D activate TG1. Inhibition of osteoclastogenesis and reduction in TG1 activity were observed in Tgm2–/– osteoclasts treated with aspartate protease inhibitor, Pepstatin A. Despite the important role of TG1 in osteoclastogenesis, the in vivo role of TG1 in bone has not been explored as global TG1 deletion in mice leads to postnatal lethality prior to any observable bone remodeling phenotype. To overcome this, we developed an osteoclast-specific TG1 knockout mouse using a novel Tgm1flx/flx mouse and osteoclast-specific Cathepsin K-Cre driver mouse resulting in the Tgm1-/- CTSK (Tgm1CKO) mouse model. I report that the successful deletion of TG1 in osteoclasts from Tgm1CKO mice resulted in impaired osteoclastogenesis. This impairment was evidenced by significantly smaller osteoclasts with a 60% decrease in the number of nuclei per cell and a significant reduction in Dcstamp expression, a cell fusion marker, compared to controls. However, differentiation itself was not affected. This data would predict an increase in bone mass in vivo. However, in vivo skeletal phenotyping of Tgm1CKO mice, conducted using dual x-ray absorptiometry (DEXA), µComputed Tomography (CT) scans, and Three-Point-Bending tests revealed a significant reduction in bone mineral density, trabecular bone parameters, and femur strength in the appendicular skeleton of 10-week-old Tgm1CKO mice. This deterioration worsened with age, affecting both axial and appendicular bones in 5-month-old mice compared to littermate controls, suggesting a critical role of TG1 in maintaining bone mass and integrity. Ablation of TG1 from osteoclast did not affect cortical bone. I also conducted proteomics to identify specific substrates of TG1 in osteoclast precursors using K5-Hitomi-peptide analysis. Many of the identified proteins are known to be involved in cytoskeletal dynamics. In summary, this thesis provides additional information supporting the potential role of TGs in osteoclastogenesis and assigns a novel role for TG1 in osteoclastogenesis and bone remodeling

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