Autosomal Dominant Osteopetrosis type II (ADO2) is a rare bone disease of impaired osteoclastic bone resorption caused by heterozygous missense mutations in the chloride channel 7 (CLCN7). Adenylate cyclase, which catalyzes the formation of cAMP, is critical for lysosomal acidification in osteoclasts. We found reduced cAMP levels in ADO2 osteoclasts compared to wild-type (WT) osteoclasts, leading us to examine whether regulating cAMP would improve ADO2 osteoclast activity. Although forskolin, a known activator of adenylate cyclase and cAMP levels, negatively affected osteoclast number, it led to an overall increase in ADO2 and WT osteoclast resorption activity in vitro. Next, we examined cAMP hydrolysis by the phosphodiesterase 4 (PDE4) proteins in ADO2 versus WT osteoclasts. QPCR analysis revealed higher expression of the three major PDE4 subtypes (4a, 4b, 4d) in ADO2 osteoclasts compared in WT, consistent with reduced cAMP levels in ADO2 osteoclasts. In addition, we found that the PDE4 antagonists, rolipram and roflumilast, stimulated ADO2 and WT osteoclast formation in a dose-dependent manner. Importantly, roflumilast and rolipram displayed a concentration-dependent increase in osteoclast resorption activity which was greater in ADO2 than WT osteoclasts. Moreover, treatment with roflumilast rescued cAMP levels in ADO2 OCLs. The key findings from our studies demonstrate that osteoclasts from ADO2 mice exhibit reduced cAMP levels and PDE4 inhibition rescues cAMP levels and ADO2 osteoclast activity dysfunction in vitro. The mechanism of action of PDE4 inhibitors and their ability to reduce the high bone mass of ADO2 mice in vivo are currently under investigation. Importantly, these studies advance the understanding of the mechanisms underlying the ADO2 osteoclast dysfunction which is critical for the development of therapeutic approaches to treat clinically affected ADO2 patients.

常染色体显性骨石症 II 型 (ADO2) 是一种罕见的骨病，由氯离子通道 7 (CLCN7) 杂合错义突变引起的破骨细胞骨吸收受损。腺苷酸环化酶可催化 cAMP 的形成，对于破骨细胞中的溶酶体酸化至关重要。我们发现与野生型 (WT) 破骨细胞相比，ADO2 破骨细胞中的 cAMP 水平降低，这促使我们研究调节 cAMP 是否会改善 ADO2 破骨细胞活性。尽管毛喉素（一种已知的腺苷酸环化酶和 cAMP 水平激活剂）会对破骨细胞数量产生负面影响，但它导致 ADO2 和 WT 破骨细胞体外吸收活性总体增加。接下来，我们检查了 ADO2 与 WT 破骨细胞中磷酸二酯酶 4 (PDE4) 蛋白对 cAMP 的水解作用。QPCR 分析显示，与 WT 相比，ADO2 破骨细胞中三种主要 PDE4 亚型（4a、4b、4d）的表达较高，这与 ADO2 破骨细胞中 cAMP 水平降低一致。此外，我们发现 PDE4 拮抗剂咯利普兰和罗氟司特以剂量依赖性方式刺激 ADO2 和 WT 破骨细胞形成。重要的是，罗氟司特和咯利普兰显示破骨细胞吸收活性呈浓度依赖性增加，ADO2 中的破骨细胞吸收活性高于 WT 破骨细胞。此外，罗氟司特治疗可恢复 ADO2 OCL 中的 cAMP 水平。我们研究的主要发现表明，ADO2 小鼠的破骨细胞表现出 cAMP 水平降低，PDE4 抑制可在体外挽救 cAMP 水平和 ADO2 破骨细胞活性功能障碍。PDE4抑制剂的作用机制及其降低ADO2小鼠体内高骨量的能力目前正在研究中。重要的是，这些研究增进了对 ADO2 破骨细胞功能障碍潜在机制的理解，这对于开发治疗临床受影响的 ADO2 患者的治疗方法至关重要。