The Role of Transglutaminase 2 in RANKL-Induced Osteoclast Differentiation

Abstract

Transglutaminase 2 (TG2) is a multifunctional protein that can perform functions as transglutaminase, protein kinase, cell surface adhesion mediator, G protein, protein disulfide isomerase and isopeptidase depending on stimuli. Several studies have shown the effect of transglutaminase 2 on bone formation. TG2 has been reported to be required for fibronectin and type I collagen matrix deposition. However, functions of TG2 in bone metabolism still remain unclear and TG2 has not yet been examined in osteoclastogenesis. Therefore, this study was carried out to clarify whether TG2 regulates osteoclastogenesis. First, I found that TG2, among TG family members, was selectively expressed in osteoclast precursors and pre-fusion osteoclasts (pOCs). I next used TG2 siRNA and TG2 knockout mice to investigate the role of TG2 in osteoclast differentiation. I confirmed that reduction of TG2 increased formation of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells (MNCs). Western blot and real-time PCR analyses demonstrated that the TG2 deficiency significantly increased the expression of osteoclastogenic transcription factors such as c-fos and nuclear factor of activated T cell c1 (NFATc1). Moreover, reduction of TG2 augmented the activation of MAPKs, ERK, JNK and p38, and NF-B signaling pathways by RANKL. In addition, I found that the TG2 deficiency increased nuclear translocation of NFATc1 and p65. Consequently, TG2 deficiency resulted in a potent increase of sealing zone formation and bone resorption activity. In contrast, TG2 overexpression suppressed osteoclast formation and expression of osteoclastogenic genes in RAW264.7 cells. B lymphocyte induced maturation protein 1 (Blimp1) has been shown to be a positive regulator of osteoclastogenesis and to be induced by the activation of NF-B signaling pathway. As TG2 was reported to suppress Blimp1 expression, I investigated the interrelationship between TG2 and Blimp1 in osteoclast differentiation. I found that reduction of TG2 increased Blimp1 expression in BMMs and pOCs. Furthermore, the augmentation of osteoclastogenesis by TG2 knockdown was attenuated by Blimp1 knockdown. In addition, TG2 knockout mice exhibited lower bone mass compared to wildtype mice. Bone histomorphometry revealed higher number and surface area of osteoclasts in TG2 knockout mice. In conclusion, these results indicate that TG2 plays an inhibitory role in osteoclast differentiation and function via Blimp1.