Medicine, Indiana University School of Medicine, IN 46202, USA.
Medicine, Indiana University School of Medicine, IN 46202, USA.
Bone. 2021 Dec;153:116160. doi: 10.1016/j.bone.2021.116160. Epub 2021 Aug 28.
Autosomal Dominant Osteopetrosis type II (ADO2) is a bone disease of impaired osteoclastic bone resorption that usually results from heterozygous missense mutations in the chloride channel 7 (CLCN7) gene. We created mouse models of ADO2 by introducing a knock-in (p.G213R) mutation in the Clcn7 gene, which is analogous to one of the common mutations (G215R) found in humans. The mutation leads to severe osteopetrosis and lethality in homozygous mice but produces substantial phenotypic variability in heterozygous mice on different genetic backgrounds that phenocopy the human disease of ADO2. ADO2 is an osteoclast-intrinsic disease, and lysosomal enzymes and proteins are critical for osteoclast activity. Chloroquine (CQ) is known to affect lysosomal trafficking, intracellular signaling and the lysosomal and vesicular pH, suggesting it might improve ADO2 osteoclast function. We tested this hypothesis in cell culture studies using osteoclasts derived from wild-type (WT or ADO2) and ADO2 heterozygous (ADO2) mice and found that CQ and its metabolite desethylchloroquine (DCQ), significantly increased ADO2 osteoclasts bone resorption activity in vitro, whereas bone resorption of ADO2 osteoclasts was increased only by DCQ. In addition, we exploited our unique animal model of ADO2 on 129 background to identify the effect of CQ for the treatment of ADO2. Female ADO2 mice at 8 weeks of age were treated with 5 doses of CQ (1, 2.5, 5, 7.5 and 10 mg/kg BW/day) via drinking water for 6 months. Bone mineral density and bone micro-architecture were analyzed by longitudinal in vivo DXA and micro-CT at baseline, 3 and 6 months. Serum bone biomarkers (CTX, TRAP and P1NP) were also analyzed at these time points. CQ treatment at the doses tested failed to produce any significant changes of aBMD, BMC (whole body, femur and spine) and trabecular BV/TV (distal femur) in ADO2 mice compared to the control group (water only). Further, levels of bone biomarkers were not significantly changed due to CQ treatment in these mice. Our findings indicate that while CQ increased osteoclast activity in vitro, it did not improve the osteopetrotic bone phenotypes in ADO2 heterozygous mice.
常染色体显性遗传骨硬化症 2 型（ADO2）是一种破骨细胞骨吸收受损的骨骼疾病，通常由氯离子通道 7（CLCN7）基因的杂合错义突变引起。我们通过在 Clcn7 基因中引入敲入（p.G213R）突变创建了 ADO2 的小鼠模型，该突变类似于在人类中发现的常见突变之一（G215R）。该突变导致纯合子小鼠发生严重的骨硬化症和致死性，但在不同遗传背景的杂合子小鼠中产生了大量表型变异性，这些表型模拟了 ADO2 的人类疾病。ADO2 是一种破骨细胞内在疾病，溶酶体酶和蛋白对于破骨细胞的活性至关重要。氯喹（CQ）已知会影响溶酶体运输、细胞内信号转导以及溶酶体和囊泡的 pH 值，这表明它可能改善 ADO2 破骨细胞的功能。我们在使用来自野生型（WT 或 ADO2）和 ADO2 杂合子（ADO2）小鼠的破骨细胞进行的细胞培养研究中检验了这一假设，结果发现 CQ 和其代谢物去乙基氯喹（DCQ）显著增加了体外 ADO2 破骨细胞的骨吸收活性，而 ADO2 破骨细胞的骨吸收仅被 DCQ 增加。此外，我们利用我们在 129 背景下的独特 ADO2 动物模型来确定 CQ 治疗 ADO2 的效果。8 周龄的雌性 ADO2 小鼠通过饮用水接受 5 剂 CQ（1、2.5、5、7.5 和 10mg/kgBW/天）治疗，为期 6 个月。在基线、3 个月和 6 个月时通过纵向体内 DXA 和微 CT 分析骨矿物质密度和骨微结构。还在这些时间点分析了血清骨生物标志物（CTX、TRAP 和 P1NP）。与对照组（仅水）相比，在测试剂量下，CQ 治疗未能使 ADO2 小鼠的 aBMD、BMC（全身、股骨和脊柱）和小梁 BV/TV（股骨远端）产生任何显著变化。此外，由于 CQ 治疗，这些小鼠的骨生物标志物水平没有显著变化。我们的研究结果表明，尽管 CQ 在体外增加了破骨细胞的活性，但它并没有改善 ADO2 杂合子小鼠的成骨过度的骨骼表型。
