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Dominant negative mutations in CLCN7, which encodes a homodimeric chloride channel needed for matrix acidification by osteoclasts, cause Albers-Schonberg disease (also known as autosomal dominant osteopetrosis type 2). More than 25 different CLCN7 mutations have been identified in patients affected with Albers-Schonberg disease, but only one mutation (Clcn7G213R) has been introduced in mice to create an animal model of this disease. Here we describe a mouse with a different osteopetrosis-causing mutation (Clcn7F318L). Compared to Clcn7+/+ mice, 12-week-old Clcn7F318L/+ mice have significantly increased trabecular bone volume, consistent with Clcn7F318L acting as a dominant negative mutation. Clcn7F318L/F318L and Clcn7F318L/G213R mice die by 1month of age and resemble Clcn7 knockout mice, which indicate that p.F318L mutant protein is non-functional and p.F318L and p.G213R mutant proteins do not complement one another. Since it has been reported that treatment with interferon gamma (IFN-G) improves bone properties in Clcn7G213R/+ mice, we treated Clcn7F318L/+ mice with IFN-G and observed a decrease in osteoclast number and mineral apposition rate, but no overall improvement in bone properties. Our results suggest that the benefits of IFN-G therapy in patients with Albers-Schonberg disease may be mutation-specific.

Mechanical signal transduction in bone tissue begins with load-induced activation of several cellular pathways in the osteocyte population. A key pathway that participates in mechanotransduction is Wnt/Lrp5 signaling. A putative downstream mediator of activated Lrp5 is the nucleocytoplasmic shuttling protein β-catenin (βcat), which migrates to the nucleus where it functions as a transcriptional co-activator. We investigated whether osteocytic βcat participates in Wnt/Lrp5-mediated mechanotransduction by conducting ulnar loading experiments in mice with or without chemically induced βcat deletion in osteocytes. Mice harboring βcat floxed loss-of-function alleles (βcat(f/f)) were bred to the inducible osteocyte Cre transgenic (10)(kb)Dmp1-CreERt2. Adult male mice were induced to recombine the βcat alleles using tamoxifen, and intermittent ulnar loading sessions were applied over the following week. Although adult-onset deletion of βcat from Dmp1-expressing cells reduced skeletal mass, the bone tissue was responsive to mechanical stimulation as indicated by increased relative periosteal bone formation rates in recombined mice. However, load-induced improvements in cross sectional geometric properties were compromised in recombined mice. The collective results indicate that the osteoanabolic response to loading can occur on the periosteal surface when β-cat levels are significantly reduced in Dmp1-expressing cells, suggesting that either (i) only low levels of β-cat are required for mechanically induced bone formation on the periosteal surface, or (ii) other additional downstream mediators of Lrp5 might participate in transducing load-induced Wnt signaling.

The time-varying electromagnetic field (EMF) has been widely studied as one of the exogenous stimulation methods for improving bone healing. Our previous study showed that osteogenic differentiation of adipose-derived stem cells was accelerated by a 45-Hz EMF, whereas a 7.5-Hz EMF inhibited osteogenic marker expression. Accordingly, we hypothesized that each negative and positive condition for the osteogenic differentiation could inversely influence osteoclast formation and differentiation. Here, we demonstrated that osteoclast formation, differentiation, and activity can be regulated by altering the frequency of the electromagnetic stimulation, such as 7.5 (negative for osteogenic differentiation) and 45 Hz (positive for osteogenic differentiation). A 45 Hz EMF inhibited osteoclast formation whereas a 7.5-Hz EMF induced differentiation and activity. Osteoclastogenic markers, such as NFATc1, TRAP, CTSK, MMP9, and DC-STAMP were highly expressed under the 7.5-Hz EMF, while they were decreased at 45 Hz. We found that the 7.5-Hz EMF directly regulated osteoclast differentiation through ERK and p38 MAPK activation, whereas the EMF at 45 Hz suppressed RANKL-induced phosphorylation of IκB. Additionally, actin ring formation with tubules and bone resorptive activity were enhanced at 7.5 Hz through increased integrin β3 expression. However, these were inhibited at 45 Hz. Although many questions remain unanswered, our study indicates that osteoclast formation and differentiation were controllable using physical tools, such as an EMF. It will now be of great interest to study the ill-defined correlation between electromagnetic conditions and osteoclast activities, which eventually could lead to determining the therapeutic characteristics of an EMF that will treat bone-related diseases.