Transglutaminase 1 – a novel regulator of osteoclastogenesis and bone remodeling: insights from in vitro and in vivo studies
Sahar Ebrahimi Samani1, Hideki Tatsukawa2, Kyotaka Hitomi2, Mari T Kaartinen1,3,4
1Division of Experimental Medicine, Faculty of Medicine and Health Sciences, McGill University, Montreal, QC, Canada, 2Department of Basic Medicinal Sciences, Graduate School of Pharmaceutical Sciences, Nagoya, University, Japan, 3Faculty of Dental Medicine and Oral Health Sciences, McGill University, Montreal, QC, Canada, 4Centre for Bone and Periodontal Research, 3Faculty of Dental Medicine and Oral Health Sciences, McGill University, Montreal, QC, Canada
Osteoclasts are multinucleated, large bone resorbing cells and a major drug target cell for osteoporosis. We and others have shown that transglutaminase enzyme activity from all or one of the three TGs; TG2, FXIII-A and TG1, regulates osteoclast differentiation and bone remodeling. The role of TG1 in bone has not been explored as the global TG1 deletion in mice leads to postnatal lethality prior to any observable bone remodeling phenotype. In this study, we have developed an osteoclast-specific TG1 knockout mouse using a novel Tgm1flx/flx mouse and osteoclast-specific Cathepsin K-Cre driver mouse resulting in Tgm1-/-CTSK mouse model. In vitro osteoclastogenesis was assessed by differentiating bone marrow macrophages (BMMs) from Tgm1-/-CTSK and controls with RANKL and M-CSF for 6 days. A significant decrease of TG1 protein and mRNA level was seen upon RANKL treatment, confirming a successful deletion of TG1 in osteoclasts. Measuring osteoclast marker, TRAP levels in supernatant media showed a significant reduction in TG1-deficient osteoclasts, accompanied by a significant decrease in osteoclast size, number of nuclei and actin ring formation. qPCR analysis of differentiation marker, Ctsk, and cell fusion marker, Dcstamp, showed a significant decrease in Tgm1 knockout osteoclasts supporting defective osteoclastogenesis. This data would predict that in vivo, Tgm1-/-CTSK mice may have increased bone mass. In vivo skeletal phenotyping and assessment of tibial trabecular bone parameters of 10-week-old and 5-month-old Tgm1-/-CTSK mice using dual x-ray absorptiometry (DEXA) and Computed Tomography (CT) scans. Femoral biomechanical strength was analyzed with Three-Point-Bending test. Surprisingly, 10-week-old knockout mice exhibited significantly reduced bone mineral density and reduced trabecular thickness of tibia, along with decreased femur strength compared to controls. Similarly, 5-month-old knockout mice demonstrated a significant decrease in bone mass and bone mineral density, with significant alterations in trabecular bone parameters including reduction in trabecular number, thickness and increased porosity compared controls. Further investigation is needed to explore if changes in expression or activity of other transglutaminases or pro-osteoclastogenesis factors in marrow cells contribute to this unexpected phenotype. Bone formation may also be affected. Our study assigns a novel role for TG1 in osteoclastogenesis.