Protease-Activated Receptor 1 Deletion Causes Enhanced Osteoclastogenesis in Response to Inflammatory Signals through a Notch2-Dependent Mechanism

Abstract

We found that protease-activated receptor 1 (PAR1) was transiently induced in cultured osteoclast precursor cells. Therefore, we examined the bone phenotype and response to resorptive stimuli of PAR1-deficient (knockout [KO]) mice. Bones and bone marrow-derived cells from PAR1 KO and wild-type (WT) mice were assessed using microcomputed tomography, histomorphometry, in vitro cultures, and RT-PCR. Osteoclastic responses to TNF-a (TNF) challenge in calvaria were analyzed with and without a specific neutralizing Ab to the Notch2-negative regulatory region (N2-NRR Ab). In vivo under homeostatic conditions, there were minimal differences in bone mass or bone cells between PAR1 KO and WT mice. However, PAR1 KO myeloid cells demonstrated enhanced osteoclastogenesis in response to receptor activator of NF-kB ligand (RANKL) or the combination of RANKL and TNF. Strikingly, in vivo osteoclastogenic responses of PAR1 KO mice to TNF were markedly enhanced. We found that N2-NRR Ab reduced TNF-induced osteoclastogenesis in PAR1 KO mice to WT levels without affecting WT responses. Similarly, in vitro N2-NRR Ab reduced RANKL-induced osteoclastogenesis in PAR1 KO cells to WT levels without altering WT responses. We conclude that PAR1 functions to limit Notch2 signaling in responses to RANKL and TNF and moderates osteoclastogenic response to these cytokines. This effect appears, at least in part, to be cell autonomous because enhanced osteoclastogenesis was seen in highly purified PAR1 KO osteoclast precursor cells. It is likely that this pathway is involved in regulating the response of bone to diseases associated with inflammatory signals.

F2r negatively regulates osteoclastogenesis through inhibiting the Akt and NFκB signaling pathways

“…Interestingly, Jastrzebski et.al. detected the p65 expression in cytoplasmic and nuclear under RANKL (30 ng/ml) stimulated 15 min, and found no significant differences between control and F2r-defcienct cell [45]. In our study, F2r knockdown increased p65 and IKBα phosphorylation level under RANKL (10 ng/ml) stimulated at 5 min and 10 min, indicating that F2r responds to RANKL signaling at early stages of osteoclastogenesis.…”

“…al. demonstrated that F2r deletion causes enhanced osteoclastogenesis [45]. However, the functions of F2r for osteoclast acidification, differentiation, activation, and bone resorption are still unknown; and without detailed mechanism studies, the mechanisms underlying the role of F2r in osteoclastogenesis remains unclear.…”

Protease Activated Receptors and Arthritis

“…The authors showed an increase in urokinase plasminogen activating factor production following LPS treatment, and it is believed that subsequent plasmin production activated PAR-1 and reduced osteoclast formation [40]. These data were supported by another study showing that osteoclastogenesis was enhanced in PAR-1 knockout mice treated with TNFalpha, indicating that PAR-1 acts as an inhibitor of osteoclast maturation in inflammatory joint diseases [54]. In a model of tibial damage, bone repair was found to be inhibited in PAR-1 knockout mice while thrombin improved bone marrow stromal cell proliferation in a PAR-1-dependent manner [24,55].…”

Mechanisms of Bone Remodeling Disorder in Hemophilia

“…[62][63][64] Tudpor et al also reported an increased bone volume in Par1-knockout mice, which contrasted with the data of Aronovich et al 45,61 Furthermore, another group found that the difference was slight and although the bone mass was decreased in Par1-knockout mice, deficiency of PAR-1 resulted in osteoclastogenesis via the Notch receptor 2 (NOTCH2) signaling pathway, and overexpression of PAR-1 significantly inhibited the osteoclastogenic response. 65 In addition to its effects on osteoblasts, Sivagurunathan et al 66 reported that thrombin inhibited osteoclastogenesis. Furthermore, they found that thrombin inhibited osteoclast differentiation of different organs in Par-1 knockout mice, which suggested that the PAR-1 pathway was not involved in thrombin-induced downregulation of osteoclastogenesis.…”