ClC-7 Regulates the Pattern and Early Development of Craniofacial Bone and Tooth

Abstract

Human <i>CLCN7</i> encodes voltage-gated chloride channel 7 (ClC-7); mutations of <i>CLCN7</i> lead to osteopetrosis which is characterized by increased bone mass and impaired osteoclast function. In our previous clinical practice, we noticed that osteopetrosis patients with <i>CLCN7</i> mutations had some special deformities in craniofacial morphology and tooth dysplasia. It is unclear whether these phenotypes are the typical features of <i>CLCN7</i> involved osteopetrosis and whether ClC-7 could regulate the development of craniofacial bone and tooth in some signaling pathways. <b>Methods</b>: First, we collected 80 osteopetrosis cases from the literature and compared their craniofacial and dental phenotypes. Second, four osteopetrosis pedigrees with <i>CLCN7</i> mutations were recruited from our clinic for gene testing and clinical analysis of their craniofacial and dental phenotypes. Third, we used a zebrafish model with <i>clcn7</i> morpholino treatment to detect the effects of ClC-7 deficiency on the development of craniofacial and dental phenotypes. General observation, whole mount alcian blue and alizarin red staining, whole mount <i>in situ</i> hybridization, scanning electron microscope observation, lysoSensor staining, Q-PCR and western blotting were performed to observe the <i>in vivo</i> characteristics of craniofacial bone and tooth changes. Fourth, mouse marrow stromal cells were further primarily cultured to detect ClC-7 related mRNA and protein changes using siRNA, Q-PCR and western blotting. <b>Results:</b> Over 84% of osteopetrosis patients in the literature had some typical craniofacial and tooth phenotypes, including macrocephaly, frontal bossing, and changes in shape and proportions of facial skeleton, and these unique features are more severe and frequent in autosomal recessive osteopetrosis than in autosomal dominant osteopetrosis patients. Our four pedigrees with <i>CLCN7</i> mutations confirmed the aforementioned clinical features. <i>clcn7</i> knockdown in zebrafish reproduced the craniofacial cartilage defects and various dental malformations combined the decreased levels of <i>col10a1</i>, <i>sp7</i>, <i>dlx2b</i>, <i>eve1</i>, and <i>cx43</i>. Loss of <i>clcn7</i> function resulted in lysosomal storage in the brain and jaw as well as downregulated cathepsin K (CTSK). The craniofacial phenotype severity also presented a dose-dependent relationship with the levels of ClC-7 and CTSK. ClC-7/CTSK further altered the balance of TGF-β/BMP signaling pathway, causing elevated TGF-β-like Smad2 signals and reduced BMP-like Smad1/5/8 signals in <i>clcn7</i> morphants. SB431542 inhibitor of TGF-β pathway partially rescued the aforementioned craniofacial bone and tooth defects of <i>clcn7</i> morphants. The ClC-7 involved CTSK/BMP and SMAD changes were also confirmed in mouse bone marrow stromal cells. <b>Conclusion:</b> These findings highlighted the vital role of <i>clcn7</i> in zebrafish craniofacial bone and tooth development and mineralization, revealing novel insights for the causation of osteopetrosis with <i>CLCN7</i> mutations. The mechanism chain of ClC-7/CTSK/ TGF-β/BMP/SMAD might explain the typical craniofacial bone and tooth changes in osteopetrosis as well as pycnodysostosis patients.