Characterization of the Bone Phenotype in ClC-7-Deficient Mice

Abstract

Mice deficient in the chloride channel ClC-7, which is likely involved in acidification of the resorption lacuna, display severe osteopetrosis. To fully characterize the osteopetrotic phenotype, the phenotypes of osteoclasts and osteoblasts were evaluated. ClC-7(-/-) mice and their corresponding wild-type littermates were killed at 4-5 weeks of age. Biochemical markers of bone resorption (CTX-I), osteoclast number (TRAP5b), and osteoblast activity (ALP) were evaluated in serum. Splenocytes were differentiated into osteoclasts using M-CSF and RANKL. Mature osteoclasts were seeded on calcified or decalcified bone slices, and CTX-I, Ca(2+), and TRAP were measured. Acidification rates in membrane vesicles from bone cells were measured using acridine orange. Osteoblastogenesis and nodule formation in vitro were investigated using calvarial osteoblasts. ClC-7(-/-) osteoclasts were unable to resorb calcified bone in vitro. However, osteoclasts were able to degrade decalcified bone. Acid influx in bone membrane vesicles was reduced by 70% in ClC-7(-/-) mice. Serum ALP was increased by 30% and TRAP5b was increased by 250% in ClC-7(-/-) mice, whereas the CTX/TRAP5b ratio was reduced to 50% of the wild-type level. Finally, evaluation of calvarial ClC-7(-/-) osteoblasts showed normal osteoblastogenesis. In summary, we present evidence supporting a pivotal role for ClC-7 in acidification of the resorption lacuna and evidence indicating that bone formation and bone resorption are no longer balanced in ClC-7(-/-) mice.

Dissociation of Bone Resorption and Bone Formation in Adult Mice with a Non-Functional V-ATPase in Osteoclasts Leads to Increased Bone Strength

“…The biomarker measurements were conducted in samples from all mice, and for the samples collected during the first 12 weeks on pooled data from both experiments as described in the Methods section. doi:10.1371/journal.pone.0027482.g004 degradation markers, these data suggest that it is the remaining calcified cartilage in the bones of young osteopetrotic mice that is the basis of the poor bone strength [51]. However, the gained bone was notably devoid of woven bone, a phenomenon observed in classical osteopetrosis, and thus the increase in lamellar bone volume is likely to also contribute the increased bone strength observed in the adult osteopetrotic mice.…”

Genetics in Endocrinology: Autosomal dominant osteopetrosis revisited: lessons from recent studies

“…Along the same line, data from in vitro experiments show that allowing osteoclasts to resorb bone facilitates bone formation by osteoblasts in the resorbed areas (82,83). Furthermore, studies indicate that osteoclasts deposit TRACP on the bone surface leading to recruitment of osteoblasts (84), a finding that correlates with the high levels of TRACP found on the resorbed bone surfaces in ADO patients and other acid secretion-deficient systems (41,85,86) (Fig. 3).…”

“…Studies in the aged ovariectomized rat model using these inhibitors have shown that bone resorption is lowered, while osteoclast numbers and bone formation are maintained (63,73), thereby mimicking the phenotype of the ADO patients and ClC-7-deficient mice (11,41,85,86). Treatment with these inhibitors also resulted in increased BMD and bone strength (63,73), thereby underlining the potential of these molecules.…”

Odanacatib in the treatment of postmenopausal women with low bone mineral density: Three-year continued therapy and resolution of effect

“…Since catK is expressed primarily in osteoclasts, inhibition of this enzyme to reduce bone resorption is being explored as a pharmacologic treatment for osteoporosis. (2)(3)(4)(5)(6)(7) Recent advances have led to the discovery of drugs that selectively inhibit either acid secretion (8)(9)(10) or catK activity. (2)(3)(4)(5)11,12) Some early catK inhibitors proved to have crossreactivity with other cathepsins, (5,13,14) and cutaneous adverse events were reported for one.…”