Characterization of Parathyroid Hormone Receptor 1 in Periodontal Ligament Cells

Abstract

In addition to the classic catabolic effects, it is now widely accepted that parathyroid hormone (PTH) exerts anabolic effects on bone, when administered intermittently. As a result of the regenerative characteristic, Teriparatide (Forsteo® Europe, Forteo® U.S.A., Eli Lilly) which is a recombinant PTH (1-34), was recently approved for treatment of osteoporosis in the USA and the Europe. The dual actions of PTH are mediated primarily through PTH receptor 1 (PTH1R), which is a class II G protein-coupled receptor. PTH1R can activate diverse signaling pathways, including cAMP/PKA and PLC/PKC pathways (Vilardaga et al., 2011).<br /> Periodontitis is an inflammatory disease, which manifests clinically as loss of supporting periodontal tissues. Accumulating evidences<em> in vivo </em>and<em> vitro</em> indicate that the intermittent PTH administration exerts anabolic effects on periodontal ligament (PDL) tissue and alveolar bone (Nohutcu et al., 1995; Ouyang et al., 2000; Barros et al., 2003; Lossdörfer et al., 2005, 2006b). Understanding the physiology of PTH1R is crucial to promote the regenerative effect of PTH. PTH1R has been exclusively studied in kidney and bone cells. However, the knowledge on PTH1R characteristics and physiology in PDL cells is still in its infancy.<br /> In this study, we characterized the PTH1R in PDL cells, in terms of its cellular localization, binding affinity, density, signal transduction and gene regulation, and compared these characteristics with those of PTH1R in human osteosarcoma cell line (MG63) and Human Embryonic Kidney 293 cells (HEK293). In the second part, we transplanted human PDL cells into immunodeficient nude mice and <em>evaluated in vivo</em> the regenerative capacity of PDL cells upon intermittent hPTH (1-34) administration. <br /> PTH1R mRNA and protein were detected in PDL, MG63 and HEK293 cells. Like other GPCRs, PTH1R was found on the plasma membrane and in the cytoplasm of the three cell lines, while they were to some extent also present in the nuclei of PDL and MG63 cells. Binding characteristics of PTH1R were cell type specific in the examined three cell lines, with PDL cells demonstrating a low binding affinity (Kd=1030±10 nM) and a relative high number of receptors (3.03±0.57 million receptors/cell). Dexamethason and 1,25-dihydroxyvitamin D3 increased the expression level of PTH1 mRNA in PDL cells (12-fold and 14-fold of the corresponding control group, respectively), whereas the effect of hPTH (1-34) on receptor mRNA expression was depended on the mode of its administration. The response of cAMP in MG63 and HEK293 cells was additive with growing concentration of hPTH (1-34), while it was concentration dependent in PDL cells. However, in all three cell lines, we observed a cross-talk between the cAMP/PKA and PLC/PKC signaling pathways, which were regulated oppositely at a given concentration of hPTH (1-34). The results of the <em>in vivo</em> experiments proved that the implanted human PDL cells not only survived, but also were able to develop a bone/cementum like tissue which closely resembles natural bone or cementum and this capacity was significantly enhanced by intermittent PTH administration.