Thrombin inhibits osteoclast differentiation through a non-proteolytic mechanism

Abstract

Thrombin stimulates expression of interleukin 6 and cyclooxygenase 2 by osteoblasts, both of which enhance osteoblast-mediated osteoclast differentiation by increasing the ratio of receptor activator of nuclear factor kB ligand (RANKL) expression to that of osteoprotegerin (OPG) in osteoblasts. We hypothesised that thrombin would also increase this ratio and thereby stimulate osteoclast differentiation in mixed cultures of osteoblastic cells and osteoclast precursors. In primary mouse osteoblasts, but not in bone marrow stromal cells, thrombin increased the ratio of RANKL to OPG expression. Thrombin inhibited differentiation of osteoclasts, defined as tartrate-resistant acid phosphatase (TRAP)-positive cells with three or more nuclei, in mouse bone marrow cultures treated with osteoclastogenic hormones; this effect was not mediated by the major thrombin receptor, protease-activated receptor 1, nor did it require thrombin's proteolytic activity. Thrombin also caused a decrease in the number of TRAP-positive cells with fewer than three nuclei. Thrombin (active or inactive) also inhibited osteoclast differentiation and bone resorption, respectively, in cultures of mouse spleen cells and human peripheral blood mononuclear cells induced to undergo osteoclastogenesis by treatment with RANKL and macrophage colony-stimulating factor. Osteoclast differentiation in spleen cells was inhibited when they were exposed to thrombin from days 0 to 3 or 3 to 5 of culture but not days 5 to 7 when most fusion occurred. Thrombin inhibited expression of RANK by spleen cells. These observations indicate that, although thrombin stimulates production of osteoclastogenic factors by osteoblastic cells, it inhibits the early stages of RANKL-induced osteoclast differentiation through a direct effect on osteoclast precursors that does not require thrombin's proteolytic activity.

Regulation of long‐term repopulating hematopoietic stem cells by EPCR/PAR1 signaling

“…139 Enhanced HSC mobilization and osteoclast activation during inflammation presumably involves TF activity in osteoclasts to locally produce thrombin, which releases HSCs from their niches. On the other hand, thrombin inhibits osteoclast differentiation through a nonproteolytic mechanism, 140 which may be a regulatory mechanism to shut down the inflammatory response. PAR1, the major thrombin receptor, also plays a role in bone repair.…”

Mannan-Binding Lectin Attenuates Inflammatory Arthritis Through the Suppression of Osteoclastogenesis

“…The involvement of MBL in the processes of coagulation cascade initiation is extensive studied (10). Notably, thrombin inhibits the early stages of RANKL-induced osteoclast differentiation through a direct effect on osteoclast precursors (39). Low MBL level caused by a genetic variation has been reported to be disturbed the particular process during bone healing (40).…”

The effect of oral dabigatran etexilate on bone density, strength, and microstructure in healthy mice

“…The effect of thrombin and the thrombin receptor in relation to bone have been investigated in both in vitro and in vivo experiments ( Pagel et al, 2006 , Sivagurunathan et al, 2013 , Wienen et al, 2007 ). In vitro studies of cell cultures have shown that thrombin can increase the expression of RANKL relatively to OPG mediated by IL6 and PGE 2 , thereby enhancing osteoclast activation and bone degeneration ( Kozawa et al, 1997 , Pagel et al, 2009 ).…”

“…Thrombin also plays a role in direct cleavage of the bone protein osteopontin ( Senger et al, 1994 ), which is primarily produced by osteoblasts ( Haylock and Nilsson, 2006 ), and is important for anchoring osteoclasts to the mineral matrix of bones ( Reinholt et al, 1990 , Senger et al, 1994 , Sivagurunathan et al, 2013 ).…”