WNT16 is Robustly Increased by Oncostatin M in Mouse Calvarial Osteoblasts and Acts as a Negative Feedback Regulator of Osteoclast Formation Induced by Oncostatin M

Abstract

Background Bone loss is often observed adjacent to inflammatory processes. The WNT signaling pathways have been implicated as novel regulators of both immune responses and bone metabolism. WNT16 is important for cortical bone mass by inhibiting osteoclast differentiation, and we have here investigated the regulation of WNT16 by several members of the pro-inflammatory gp130 cytokine family. Methods The expression and regulation of Wnt16 in primary murine cells were studied by qPCR, scRNAseq and in situ hybridization. Signaling pathways were studied by siRNA silencing. The importance of oncostatin M (OSM)-induced WNT16 expression for osteoclastogenesis was studied in cells from Wnt16 -deficient and wild-type mice. Results We found that IL-6/sIL-6R and OSM induce the expression of Wnt16 in primary mouse calvarial osteoblasts, with OSM being the most robust stimulator. The induction of Wnt16 by OSM was dependent on gp130 and OSM receptor (OSMR), and downstream signaling by the SHC1/STAT3 pathway, but independent of ERK. Stimulation of the calvarial cells with OSM resulted in enhanced numbers of mature, oversized osteoclasts when cells were isolated from Wnt16 deficient mice compared to cells from wild-type mice. OSM did not affect Wnt16 mRNA expression in bone marrow cell cultures, explained by the finding that Wnt16 and Osmr are expressed in distinctly different cells in bone marrow, nor was osteoclast differentiation different in OSM-stimulated bone marrow cell cultures isolated from Wnt16 −/- or wild-type mice. Furthermore, we found that Wnt16 expression is substantially lower in cells from bone marrow compared to calvarial osteoblasts. Conclusion These findings demonstrate that OSM is a robust stimulator of Wnt16 mRNA in calvarial osteoblasts and that WNT16 acts as a negative feedback regulator of OSM-induced osteoclast formation in the calvarial bone cells, but not in the bone marrow.

Stimulation of Osteoclast Formation by Oncostatin M and the Role of WNT16 as a Negative Feedback Regulator

“…We recently discovered a novel mechanism whereby OSM-induced osteoclast differentiation can be balanced. We identified OSM and IL-6 as very strong stimulators of Wnt16 expression in primary mouse calvarial osteoblasts [ 14 ]. Human large-scale genome-wide association studies have identified the WNT16 locus as the strongest determinant of cortical bone mass and susceptibility to forearm fractures [ 110 , 111 ].…”

“…Mice with the Wnt16 gene deleted have increased numbers of osteoclasts [ 112 , 113 ], while mice with osteoblastic overexpression of Wnt16 have decreased osteoclast numbers [ 114 ]. Moreover, in vitro studies using cultures of human monocytes, mouse bone marrow macrophages and mouse spleen cells showed a direct inhibitory effect of WNT16 on RANKL-induced osteoclast differentiation [ 14 , 112 ]. In primary calvarial bone cell cultures containing osteoblasts and osteoclast progenitors, OSM, in addition to inducing Rankl mRNA expression and osteoclast differentiation, increased the expression of Wnt16 mRNA [ 14 ].…”

“…Moreover, in vitro studies using cultures of human monocytes, mouse bone marrow macrophages and mouse spleen cells showed a direct inhibitory effect of WNT16 on RANKL-induced osteoclast differentiation [ 14 , 112 ]. In primary calvarial bone cell cultures containing osteoblasts and osteoclast progenitors, OSM, in addition to inducing Rankl mRNA expression and osteoclast differentiation, increased the expression of Wnt16 mRNA [ 14 ]. We further demonstrated that OSM-induced Wnt16 mRNA expression in calvarial bone cells was mediated by the OSMR/gp130/Shc1/STAT3 signaling pathway.…”

“…We further demonstrated that OSM-induced Wnt16 mRNA expression in calvarial bone cells was mediated by the OSMR/gp130/Shc1/STAT3 signaling pathway. In cultures of calvarial bone cells from Wnt16 − / − mice, OSM-induced osteoclastogenesis was enhanced, showing that WNT16 acted as a negative feedback regulator of osteoclast differentiation induced by OSM [ 14 ]. Interestingly, this negative feedback mechanism could not be seen in whole-bone-marrow cell cultures containing stromal cells and osteoclast progenitors.…”

“…The present overview will describe how bone mass is regulated by OSM in physiological and pathological conditions. Although bone mass is dependent on both bone resorption and bone formation, the overview will mainly describe effects of OSM on bone resorption, including our recent finding of the role of OSM-stimulated osteoblast-derived WNT16 acting in a negative feedback loop to restrict the stimulatory effect of OSM on osteoclast formation [ 14 ].…”