Response of immortalized murine cementoblasts/periodontal ligament cells to parathyroid hormone and parathyroid hormone-related protein in vitro
Introduction
A healthy periodontium, consisting of periodontal ligament, cementum, gingival tissue, and alveolar bone, is essential for maintaining the structural and functional integrity of the dentition. The formation of the periodontium involves a series of precisely coordinated biological events that include the degradation or possible transformation of epithelial root-sheath cells; the subsequent formation of cementum and a functional periodontal ligament; the continued apical elongation of the tooth root; and the coronal eruption of teeth. Existing knowledge suggests that these events require a complex cascade of exchanges between specific cells and their local environment (Bosshardt and Selvig, 1997, Ten Cate, 1997) including cell–cell, cell–matrix, and cell–diffusible factor interactions (Thesleff et al., 1996). In an attempt to define the interactions of cells within the periodontal region with their local environment, the studies presented here focused on characterizing the responsiveness of a murine cell population, containing both cementoblasts and periodontal ligament cells, to PTH and PTHrP in vitro.
PTH is synthesized by the parathyroid gland. It is an essential systemic regulator required for maintaining calcium homeostasis, partially via direct actions on one of its major target organs, bone. PTHrP has been isolated and cloned as a result of its PTH-like actions in patients suffering from hypercalcaemia of malignancy (Moseley et al., 1987, Suva et al., 1987). It is synthesized by a variety of normal tissues, including bone, cartilage and mammary gland (Moseley and Gillespie, 1995), where it is suggested to have an important role in regulating their development. PTH and PTHrP share considerable homology in their N-terminal amino acids, especially the first 13 amino acids, but differ from each other beyond this region. In osteoblasts, both PTH and PTHrP can bind to the PTH-1 receptor (Jüppner et al., 1991, Okano et al., 1994), elevate cAMP production (Abou-Samra et al., 1992), and induce gene expression of the leucine zipper transcription factor c-fos (Lee et al., 1994, Lee et al., 1995, Pearman et al., 1996). Several additional pieces of evidence indicate that, besides targeting bone, PTH and PTHrP may also regulate the activities of periodontal tissues. These findings include: (1) PTH and parathyroid gland extract enhance tooth eruption and orthodontic tooth movement (Schneider et al., 1972, Davidovitch et al., 1972); (2) tissues within the developing periodontium synthesize PTHrP and possess PTH-binding sites (Beck et al., 1995, Tenorio and Hughes, 1996); and (3) certain strains of PTHrP-deficient mice exhibit failure of tooth eruption and ankylosis of cementum with surrounding alveolar bone (Schipani et al., 1997, Philbrick et al., 1998). In order to investigate the function of PTH and PTHrP in periodontal development, controlled in vitro analyses of their interactions with cells of the periodontium can be advantageous. Such efforts have been hampered by lack of suitable and reliable cell lines representing cell types associated with periodontal tissues in situ.
Our laboratory has established an immortalized murine cell population from the root surface of the first mandibular molars of day-41 CD-1 mice (vaginal plug=day 0, newborns were usually born on day 19) by enzymatic digestion of the molars, which released cells that were immortalized immediately with SV-40 large T antigen (D'Errico et al., 1997, D'Errico et al., 1999, MacNeil et al., 1998). Importantly, as previously published, specific cautions were taken to prevent contamination of the cementoblast/periodontal ligament population with osteoblasts associated with surrounding alveolar bone. These included a dissection technique designed to separate tooth germs from the surrounding alveolar bone, and in situ hybridization using an osteocalcin probe to confirm that osteocalcin-positive cells within the cementblast/periodontal ligament population were associated with root surface, but not with the surrounding tissues/ligament cells removed during this procedure (D'Errico et al., 1997, MacNeil et al., 1998). Cells in the resultant cultures expressed gene markers associated with cementoblasts in situ, including bone sialoprotein and osteocalcin, as well as those shared by both cementoblasts and periodontal ligament cells, such as type I and XII collagens, and alkaline phosphatase, indicating that this population contains both cementoblasts and periodontal ligament cells (D'Errico et al., 1997). Therefore, this population was termed cementoblast/periodontal ligament cells. The heterogeneous cell composition of this population reflects the physiological condition in vivo, where multiple cell phenotypes exist and interact with each other. Given the diverse nature of cells within the periodontium, characterization of this population for its responsiveness to PTH/PTHrP actions constitutes the first and a very important step towards defining the function of PTH/PTHrP in regulating the activities of the periodontium. The present study was designed to determine (1) whether cementoblast/periodontal ligament cells contain functional PTH-1 receptors, and (2) how PTHrP affects the proliferation and gene expression of cementoblast/periodontal ligament cells, as well as biomineralization mediated by these cells in vitro.
Section snippets
Cell culture
Cementoblast/periodontal ligament cells were obtained from mandibular first molars of day-41 CD-1 mice (Charles River Labs, Cambridge, MA, USA). In order to maintain cell phenotypes and promote cell proliferation, cells were immortalized with wild-type SV-40 large T antigen immediately after isolation (D'Errico et al., 1999). Day-41 mice were selected because of our previous findings that gene markers for bone sialoprotein and osteocalcin were expressed by cementoblasts but not periodontal
Expression of PTH-1 receptor mRNA by cementoblast/periodontal ligament cells
To determine whether the periodontium contains cells that are targets for PTH/PTHrP, the expression of PTH-1 receptor mRNA in these cells was evaluated via northern blot analysis. As shown in Fig. 1, the cells expressed PTH-1 receptor mRNA. As reported previously (McCauley et al., 1994) and reproduced here, the positive control, ROS 17/2.8 cells, also expressed PTH-1 receptor mRNA. Human periodontal ligament cells did not express PTH-1 receptor mRNA.
Elevation of cAMP by PTH/PTHrP
Elevation of cAMP is a characteristic
Discussion
Accumulating evidence indicates that there exist multiple receptors with different binding affinities for PTH/PTHrP, such as the PTH-1 receptor with an equal binding affinity for both (Jüppner et al., 1991) and the PTH-2 receptor with selective binding to PTH (Usdin et al., 1995). In addition, various spliced forms of the PTH-1 receptor with altered ability to transduce PTH/PTHrP signals have been described (Usdin et al., 1995, Joun et al., 1997). The presence of these different forms of
Acknowledgements
We are grateful to Dr. Hen-Li Chen and Mrs. Amy Koh-Paige for their technical assistance in preparing human periodontal ligament cells and performing the cAMP stimulation assay, respectively. This work was supported by NIDR/NIH Grants DE09532, DE05683, and DK53904 and the Block Grant awarded by Rackham School of Graduate Studies, at the University Michigan, Ann Arbor, Michigan.
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