LOX Fails to Substitute for RANKL in Osteoclastogenesis

Abstract

Osteoclasts are the exclusive bone-resorbing cells that have a central role in bone homeostasis as well as bone destruction in cancer and autoimmune disease. Both mouse and human genetic studies have clearly proven that receptor activator of NF-kB ligand (RANKL; encoded by the Tnfsf11 gene) and its receptor RANK are essential for osteoclastogenesis. Although there have been several reports on RANKL-independent osteoclastogenesis, previous studies have never provided in vivo evidence showing RANKL can be substituted by other molecules using RANKL-or RANK-deficient genetic backgrounds. Thus, to date, there is no clear evidence of RANKL-independent osteoclastogenesis and no molecule has ever been proven capable of inducing osteoclast differentiation more efficiently than RANKL. Recently, lysyl oxidase (LOX), the enzyme that mediates collagen cross-linking, has been shown to induce human osteoclasts in the absence of RANKL and has a stronger osteoclastogenic activity than RANKL. Here, we investigated the effect of LOX on osteoclast differentiation using RANKL-and RANK-deficient cells to strictly explore RANKL-independent osteoclastogenesis. CD14 þ human peripheral blood cells as well as osteoclast precursor cells derived from wild-type, RANKL-and RANK-deficient mice were treated with RANKL and/or LOX in short-term (3 days) or long-term (3 weeks) experimental settings. LOX treatment alone did not result in the formation of tartrate-resistant acid phosphatase (TRAP) þ cells or resorption pits in either short-term or long-term culture. In combination with RANKL, long-term treatment with LOX synergistically promoted osteoclastogenesis in cells derived from wild-type mice; however, this was abrogated in RANKL-deficient cells. Long-term treatment with LOX stimulated RANKL expression in mouse bone marrow stromal cells via the production of reactive oxygen species (ROS). Furthermore, LOX injection failed to rescue the phenotype of RANKL-deficient mice. These results suggest that LOX has the ability to induce RANKL expression on stromal cells; however, it fails to substitute for RANKL in osteoclastogenesis.

Regulation of Breast Cancer-Induced Osteoclastogenesis by MacroH2A1.2 Involving EZH2-Mediated H3K27me3

“…Consistent with previous observations (Cox et al, 2015; Tsukasaki et al, 2017), rLOX-enhanced osteoclast formation increased the rate of bone resorption, stimulating the formation of resorption pits (Figure 4A). To further characterize the involvement of LOX in osteo- clastogenesis and investigate the effect of rLOX treatment in vivo, we injected rLOX into the periosteal regions of female mouse calvaria once daily for 10 days.…”

“…The osteoclastogenic function of LOX seems to be dependent on LOX-mediated collagen cross-linking, because the expression of LOX wild-type, but not LOX oxidase-dead mutant, in LOX-depleted cells restored CM-induced osteoclast differentiation rates. Notably, and consistent with recent studies (Cox et al, 2015; Tsukasaki et al, 2017), our investigation demonstrated that rLOX can recapitulate the osteoclastogenic properties in vitro and stimulate bone resorption in vivo. These findings underscore the importance of mH2A1.2 in the regulation of LOX expression and also suggest that accurate targeting of mH2A1.2 within the LOX gene locus may be vital for the control of breast cancer-induced osteoclastogenesis and its related physiological responses.…”

“…Because LOX is known to facilitate the dissemination of cancer cells in the bone marrow, formation of mature osteoclasts, and generation of osteolytic lesions (Cox et al, 2015; Reynaud et al, 2017; Tsukasaki et al, 2017), we decided to examine whether LOX is responsible for the major osteoclastogenic activity of CMs and behaves as a primary downstream target for mH2A1.2. Accordingly, we transfected MDA-MB-468 cells with specific shRNAs against LOX and mH2A1 alone or together (Figure 3A).…”

Iguratimod, A Synthetic Disease Modifying Anti- Rheumatic Drug (Sdmard), and Various Dmards Suppress Joint Destruction. The Pathophysiological Mechanisms of the Inhibition of Bone/Cartilage Destruction

“…Recently, possible RANKL-independent osteoclastogenesis has been reported by combinations of IL-6 and IL-11 [68], and TNF-α and IL-6 [69], and by lysyl oxidase (LOX) [70]. However, several investigators indicate that there is no clear evidence of RANKL-independent osteoclastogenesis or factor that replaces RANKL [71,72].…”

Osteoimmunology — Bidirectional dialogue and inevitable union of the fields of bone and immunity —

“…It has been suggested that the membrane-bound form functions more efficiently than the soluble form, but there was no clear in vivo data to support this difference. 3),43), 44) To address this question, we generated mice in which the cleavage site of RANKL was deleted. 39), 45) The mice had no detectable soluble form of RANKL but a normal level of membranebound RANKL.…”