Structural and Morphometric Comparison of the Molar Teeth in Pre-eruptive Developmental Stage of PACAP-Deficient and Wild-Type Mice

Abstract

Pituitary adenylate cyclase activating polypeptide (PACAP) is a pleiotropic neuropeptide with widespread distribution. It plays pivotal role in neuronal development. PACAP-immunoreactive fibers have been found in the tooth pulp, and recently, it has been shown that PACAP may also play a role in the regeneration of the periodontium after luxation injuries. However, there is no data about the effect of endogenous PACAP on tooth development. Ectodermal organogenesis including tooth development is regulated by different members of bone morphogenetic protein (BMP), fibroblast growth factor (FGF), hedgehog (HH), and Wnt families. There is also a growing evidence to support the hypothesis that PACAP interacts with sonic hedgehog (SHH) receptor (PTCH1) and its downstream target (Gli1) suggesting its role in tooth development. Therefore, our aim was to study molar tooth development in mice lacking endogenous PACAP. In this study morphometric, immunohistochemical and structural comparison of molar teeth in pre-eruptive developmental stage was performed on histological sections of 7-day-old wild-type and PACAP-deficient mice. Further structural analysis was carried out with Raman microscope. The morphometric comparison of the 7-day-old samples revealed that the dentin was significantly thinner in the molars of PACAP-deficient mice compared to wild-type animals. Raman spectra of the enamel in wild-type mice demonstrated higher diversity in secondary structure of enamel proteins. In the dentin of PACAP-deficient mice higher intracrystalline disordering in the hydroxyapatite molecular structure was found. We also obtained altered SHH, PTCH1 and Gli1 expression level in secretory ameloblasts of PACAP-deficient mice compared to wild-type littermates suggesting that PACAP might play an important role in molar tooth development and matrix mineralization involving influence on SHH signaling cascade.

PACAP and VIP signaling in chondrogenesis and osteogenesis

“…We found that addition of PACAP elevated the deposition of inorganic matrix components in the ECM of UMR-106 cells [16]. Moreover, an altered mineralization was detected during tooth formation of PACAP deficient mice [94], suggesting a yet unknown connection between PACAP and Ca 2+ release of osteoblasts, ameloblasts and/or odontoblasts. As a possible mechanism for PACAP induced extracellular Ca 2+ accumulation during osteogenesis, calcitonin gene-related protein was proved to effect on osteoclast function [95] and the presence of PACAP decreased the matrix-resorption and consequent Ca-release by these cells [96,97].…”

Role of PACAP and VIP Signalling in Regulation of Chondrogenesis and Osteogenesis

“…We have reported that the inorganic matrix components of UMR-106 cell line can be elevated by PACAP addition [ 14 ]. Moreover, an altered mineralization was detected during tooth formation of PACAP defi cient mice [ 10 ], suggesting a yet unknown connection between PACAP and mineralized tissue formation. As a possible mechanism for PACAP induced extracellular Ca 2+ accumulation during osteogenesis, calcitonin gene-related peptide was proven to infl uence osteoclast function [ 105 ] and the presence of PACAP decreased the matrix-resorption and consequent Ca-release by these cells [ 90 , 106 ].…”

“…20.2 ). In PACAP KO mice, enhanced SHH signalling was detected during tooth development [ 10 ]. On the contrary, exogenous administration of PACAP elevated the expression of SHH and a more pronounced nuclear presence of Gli1 was found in rat UMR-106 cells [ 14 ].…”

“…The N-terminal region of the polypeptide is evolutionary conserved and shows a high homology with that of VIP [ 5 ]. In the last decade, increasing amount of evidence has emerged regarding the important roles of PACAP in peripheral organs such as uterus [ 8 ], ovary [ 9 ], and teeth [ 10 ]; moreover, its presence has been proven in human milk [ 11 ]. Nonetheless, only sporadic data exist about its function in skeletal elements [ 12 -15 ].…”

“…The elevated activity of PP2B regulates NFAT4 being responsible for the augmented matrix production. PACAP has a connection with the IHH signalling pathway by decreasing the activity of Gli transcription factors alterations have been found in tooth formation of PACAP KO mice [ 10 ]. The complex phenotypic changes raise the possibility of multiple cross talk of PACAP signalling with developmental pathways connected to various morphogenes, as well as certain compensatory mechanisms of PACAP signalling cascades.…”