Transglutaminase activity regulates differentiation, migration and fusion of osteoclasts via affecting actin dynamics

Abstract

Osteoclasts, bone resorbing cells, derive from monocyte/macrophage cell lineage. Increased osteoclast activity is responsible for bone destruction in diseases such as osteoporosis, periodontitis and rheumatoid arthritis. Transglutaminases (TGs), protein crosslinking enzymes, were recently found involved in osteoclastogenesis in vivo, however their mechanisms of action have remained unknown. In this study, we have investigated the role of TG activity in osteoclastogenesis in vitro using four TG inhibitors, NC9, Z006, T101, and monodansyl cadaverine. Our results showed that all TG inhibitors were capable of blocking the entire osteoclastogenesis process. The most potent of the inhibitors, NC9 when added to cultures at different phases of osteoclastogenesis, inhibited differentiation, migration, and fusion of pre-osteoclasts as well as resorption activity of mature osteoclasts. Further investigation into the mechanisms revealed that NC9 increased RhoA levels and blocked podosome belt formation suggesting that TG activity regulates actin dynamics in pre-osteoclasts. The inhibitory effect of NC9 on osteoclastogenesis as well as podosome belt formation was completely reversed with a Rho-family inhibitor Exoenzyme C3. Microtubule architecture, acetylation, and detyrosination of α-tubulin were not affected. Finally, we demonstrated that macrophages and osteoclasts expressed mRNA of three TGs:TG1, TG2, and Factor XIII-A which were all differentially regulated in these cells during differentiation. Immunofluoresence microscopic analysis showed that all three enzymes co-localized to podosomes in osteoclasts. Taken together, our data suggests that TG activity regulates differentiation, migration and fusion of osteoclasts via affecting actin dynamics and that this may involve contribution from all three TG enzymes.

Proteolytic and nonproteolytic activation mechanisms result in conformationally and functionally different forms of coagulation factor XIII A

“…Perhaps the most studied is the role of FXIIIA in cross‐linking the fibrin network, making it more mechanically stable and resistant to fibrinolysis. As physiological knowledge on FXIII expands, it becomes apparent that beyond serving as a key player of the blood coagulation system, FXIIIA functions in wound healing, bone tissue dynamics, signaling, and other areas .…”

Coagulation Factor XIIIA: biochemical properties underlying physiological function.

“…As observed in the SEC experiments, the Y283C substitution did not significantly affect the size of the FXIIIA Perhaps the most studied is the role of FXIIIA in crosslinking the fibrin network. As physiological knowledge on FXIII expands, it becomes apparent that beyond serving as a key player of the blood coagulation system, FXIIIA functions in would healing, bone tissue dynamics, and even signaling processes (2)(3)(4)(5)21,48,49,54,130).…”

“…FXIIIA in the ECM has recently begun to receive considerable attention, and a few published studies outline the role of FXIIIA in bone tissue dynamics (47,49,54,55,57). In an early study, Kaartinen and coworkers proposed that a 37 kDa proteolytic fragment was a major FXIIIA form in the bone ECM (47).…”

“…However, a work examining the maintaince of bone mass and remodeling activity of bone cells, revealed that together with TG2, FXIIIA was a negative regulator of osteoclastogenesis. Absence of TG2 and FXIIIA led to overexpression of osteoclastogenesis-promoting transglutaminase 1, whose inhibition likely reduced bone loss in the pathogenesis of arthritis (49,54).…”

“…Plasma membrane-associated FXIIIA promotes secretion of COL1 by osteoblasts (56). Extracellularly, FXIIIA and TG2 coordinate COL1 matrix deposition thereby promoting bone mineralization (49,51,54,56,57).…”