Department of Biomedical Sciences and Comprehensive Care, Indiana University School of Dentistry, 1121 West Michigan Street, DS266, Indianapolis, IN, 46202, USA.
Department of Medicine, Indiana University School of Medicine, Indianapolis, IN, 46202, USA.
Calcif Tissue Int. 2024 Apr;114(4):430-443. doi: 10.1007/s00223-024-01191-7. Epub 2024 Mar 14.
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.
常染色体显性遗传骨硬化症 2 型（ADO2）是一种罕见的骨骼疾病，由氯离子通道 7（CLCN7）的杂合错义突变引起破骨细胞的骨吸收受损。腺苷酸环化酶可催化 cAMP 的形成，对于破骨细胞溶酶体酸化至关重要。我们发现 ADO2 破骨细胞中的 cAMP 水平低于野生型（WT）破骨细胞，这促使我们研究调节 cAMP 是否会改善 ADO2 破骨细胞活性。虽然 forskolin 是已知的腺苷酸环化酶和 cAMP 水平的激活剂，但其对破骨细胞数量产生负面影响，但它导致 ADO2 和 WT 破骨细胞在体外的吸收活性总体增加。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，ADO2 破骨细胞中三种主要 PDE4 亚型（4a、4b、4d）的表达高于 WT，这与 ADO2 破骨细胞中 cAMP 水平降低一致。此外，我们发现 PDE4 拮抗剂罗利普兰和罗氟司特以剂量依赖性方式刺激 ADO2 和 WT 破骨细胞的形成。重要的是，罗氟司特和罗利普兰在浓度依赖性方式下增加破骨细胞吸收活性，在 ADO2 中比 WT 破骨细胞中更为显著。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降低的 cAMP 水平，PDE4 抑制可挽救 cAMP 水平和 ADO2 破骨细胞功能障碍体外。PDE4 抑制剂的作用机制及其在体内降低 ADO2 小鼠高骨量的能力目前正在研究中。重要的是，这些研究加深了对 ADO2 破骨细胞功能障碍机制的理解，这对于开发治疗临床上受影响的 ADO2 患者的治疗方法至关重要。
