Abstract
Caspases are well known proteases in the context of inflammation and apoptosis. Recently, novel roles of pro-apoptotic caspases have been reported, including findings related to the development of hard tissues. To further investigate these emerging functions of pro-apoptotic caspases, the in vivo localisation of key pro-apoptotic caspases (-3,-6,-7,-8, and -9) was assessed, concentrating on the development of two neighbouring hard tissues, cells participating in odontogenesis (represented by the first mouse molar) and intramembranous osteogenesis (mandibular/alveolar bone). The expression of the different caspases within the developing tissues was correlated with the apoptotic status of the cells, to produce a picture of whether different caspases have potentially distinct, or overlapping non-apoptotic functions. The in vivo investigation was additionally supported by examination of caspases in an osteoblast-like cell line in vitro. Caspases-3,-7, and -9 were activated in apoptotic cells of the primary enamel knot of the first molar; however, caspase-7 and -8 activation was also associated with the non-apoptotic enamel epithelium at the same stage and later with differentiating/differentiated odontoblasts and ameloblasts. In the adjacent bone, active caspases-7 and -8 were present abundantly in the prenatal period, while the appearance of caspases-3,-6, and -9 was marginal. Perinatally, caspases-3 and -7 were evident in some osteoclasts and osteoblastic cells, and caspase-8 was abundant mostly in osteoclasts. In addition, postnatal activation of caspases-7 and -8 was retained in osteocytes. The results provide a comprehensive temporo-spatial pattern of pro-apoptotic caspase activation, and demonstrate both unique and overlapping activation in non-apoptotic cells during development of the molar tooth and mandibular/alveolar bone. The importance of caspases in osteogenic pathways is highlighted by caspase inhibition in osteoblast-like cells, which led to a significant decrease in osteocalcin expression, supporting a role in hard tissue cell differentiation.
Highlights
Tooth development proceeds via stages referred to as placode, bud, cap, and bell (Tucker and Sharpe, 2004), and is completed by differentiation of two main cell types, mesenchymal-derived odontoblasts producing dentin and epithelial-derived ameloblasts secreting enamel (e.g. Lesot et al, 2014)
Alveolar bone was already apparent at E15, surrounding the molar tooth germ and forming a functional component with the mandibular bone (Figures 2A, 3A)
The initiator caspase of the intrinsic pathway, caspase-9, has previously been reported to be essential for apoptosis in the primary enamel knot (PEK) (Setkova et al, 2007), and caspase-9 deficiency was shown to result in reduced apoptosis (Kuida et al, 1998)
Summary
Tooth development proceeds via stages referred to as placode, bud, cap, and bell (Tucker and Sharpe, 2004), and is completed by differentiation of two main cell types, mesenchymal-derived odontoblasts producing dentin and epithelial-derived ameloblasts secreting enamel (e.g. Lesot et al, 2014). Pro-apoptotic caspases are classified as initiators (in mouse these include caspases2,-8, and -9) or executors (caspases-3,-6, and -7). These caspases are engaged in both major pathways of apoptosis; extrinsic (receptor mediated) and intrinsic (mitochondrial). Beyond pro-apoptotic caspases, inflammatory caspases (caspases-1, -11, and -12) have been identified in the mouse along with two additional caspases (caspases-14 and -16), which do not fit in either category (reviewed in Shalini et al, 2015). This investigation focused on the pro-apoptotic caspases with the exception of caspase-2, the position of which in the apoptotic pathway is unclear (Fava et al, 2012)
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