Title: Genetic Evidence for RANKL-independent Osteoclast Formation in Cherubism Mice
Henry Hoffman (Presenter)
University of Missouri-Kansas City
Mizuho Kittaka, University of Missouri-Kansas City
Tetsuya Yoshimoto, University of Missouri-Kansas City
Yasuyoshi Ueki, University of Missouri-Kansas City
Objectives: Osteoclasts are large, multinucleated, tartrate-resistant acid phosphatase-positive (TRAP+) cells responsible for bone resorption. Several mediators are involved in the mechanisms of osteoclast differentiation. While RANKL has been regarded as the indispensable inducer of osteoclastogenesis, our previous studies showed that in vitro, a gain-of-function (GoF) mutation in Sh3bp2, which is responsible for the auto-inflammatory disease cherubism, could induce osteoclast differentiation by TNF-α alone. This study was conducted to demonstrate RANKL-independent osteoclastogenesis in the mouse model of cherubism.
Methods: Two inflammatory mouse models were created on a RANKL knockout (Rankl−/−) background. First, a Sh3bp2 GoF knock-in (KI) homozygote (Sh3bp2KI/KI) and second a Sh3bp2 GoF knock-in heterozygote (Sh3bp2KI/+) with transgenic human TNF-α gene (hTNFtg). MicroCT imaging was used to calculate the BV/TV of mouse femurs. TRAP-stained histological sections were used to identify osteoclasts. Quantitative PCR was used to analyze mRNA from femur to examine levels of osteoclast marker gene expression. T-test was used to analyze the Sh3bp2KI/KI data and one-way ANOVA with Tukey’s post-hoc analysis was used for the Sh3bp2KI/+/hTNFtg model.
Results: Seventy-five percent of Sh3bp2KI/KI/Rankl−/− mice that were histologically examined (n=4) exhibited multinucleated TRAP+ cells and showed a significant decrease in BV/TV (n=19) and an increase in expression of osteoclast marker genes compared to control Sh3bp2+/+/Rankl−/− mice (n=14). Sh3bp2KI/+/hTNFtg/Rankl−/− mice (n=6) showed a significant increase in osteoclast marker gene expression compared to Sh3bp2+/+/hTNFtg/Rankl−/− (n=9) and Sh3bp2+/+/non-hTNFtg/Rankl−/− mice (n=14). However TRAP+ cells were not observed in Sh3bp2KI/+/hTNFtg/Rankl−/− mice and no significant difference was found in BV/TV.
Conclusions: RANKL-independent osteoclastogenesis is possible in vivo in mice with a homozygous GoF mutation in Sh3bp2. These mice exhibited TRAP+ multinucleated cells, lower BV/TV, and increased expression of osteoclast marker genes. In contrast, partial osteoclast differentiation may have occurred in Sh3bp2KI/+/hTNFtg/Rankl−/− mice, although full maturity was not reached. Supported by UMKC SOD Summer Scholars program.