Deletion Of TMCO1 Gene Caused Osteoblast Dysplasia In Mice

TMCO1 (Transmembrane and Coiled - Coil Domains - 1) is a highly conserved protein locating on the endoplasmic reticulum membrane. It has been shown that TMCO1’s function is to sense the concentration of calcium ion in the endoplasmic reticulum and actively maintain the calcium homeostasis. Patients who have TMCO1 gene mutation suffer from an autosomal recessive hereditary disease - craniofacial thoracic deformity (Cerebro - Facio - Thoracic dysplasia, CFT dysplasia). Patients often exhibit the abnormal craniofacial bone and brisket bone, accompanied with severe cognitive disorder. Consanguineous marriages is a cause of CFT dysplasia. Because the affected areas are mainly craniofacial bone, spine and ribs, the disease is named the craniofacial thoracic deformity. Patients with mutation of TMCO1 gene show symptom of skull closing early, but there are also cases of incomplete closure.This study is the extension and expansion on the previous research in our laboratory. The CT images showed that the craniofacial bone abnormality of mice with deleted TMCOl gene is due to uncomplete cranial suture. Mice carrying deleted TMCO1 gene show total incomplete closure of herringbone, sagittal and crowns, and with thinning of the calvarium of uncomplete cranial suture. However it is unclear that the abnormal bone of TMCO1 gene defect mice is due to deficiency of osteogenesis or overactivity of osteoclasts. We chose a TMCO1 konckout model mouse in this study.To detect the differences of trabecular number, osteoblast and osteoclast in hind leg femur bone of mice in different ages, we carried out the experiment of the bone tissue sectioning HE staining, and immunohistochemical staining, then we isolated osteoblasts from harnpan of different genotype newborn mice and cultured them in the inducing medium. Moreover, we assessed the differences of cell proliferation activity by CCK8 kit and the ability of osteoblast forming calcium nodules by alizarin red staining. The expression level differences of osteogenesis specificity genes (such as ALP, RUNX2, Osteocalcin, BSP, the Type of alpha 1/2, Hprt, BMP4) were also evaluated by RT-qPCR. The calcium ion concentration in the osteoblast endoplasmic reticulum of different genotype mice osteoblasts were detected by the Nikon Inverted Microscope-based calcium imaging system. After a careful design of experiments and a series of tests, we got the following conclusions:TMCO1 genetic delection mice exhibited lower birth rate and slower growth speed compared with wildtype mice in the same litter. The cell proliferation activity assay by CCK8 kit showed that, osteoblasts isolated from skull of TMCO1 gene defective newborn mice manifested distinctly slow proliferation. The ability to form mineralized nodules in vitro of TMCO1 gene knockout osteoblasts is decreased. HE staining of bone tissue slices showed that the bone trabecular number is significantly less than the wildtype mice. The above results indicated that there were defects in osteoblasts in the TMCO1 knokout mice. Detection of calcium ion concentration in endoplasmic reticulum(ER) of osteoblasts showed calcium overload in the TMCO1 genetic defect mice. Therefore the skeletal dysplasia is likely owing to the ER calcium overload in the TMCO1 genetic defect mice.