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Parathyroid Hormone Protects against Periodontitis-associated Bone Loss
1 Department of Morphology, Division of Histology, School of Dentistry at Piracicaba, University of Campinas, Av. Limeria, 901, 13414-903 Piracicaba SP, Brazil; Correspondence: *corresponding author, sbarros{at}fop.unicamp.br
Parathyroid hormone (PTH) functions as a major mediator of bone remodeling and as an essential regulator of calcium homeostasis. In addition to the well-established catabolic effects (activation of bone resorption) of PTH, it is now recognized that intermittent PTH administration has anabolic effects (promotion of bone formation). The aim of this study was to investigate whether intermittent administration of PTH in rodents would block the alveolar bone loss observed in rats when a ligature model of periodontitis is used. Morphometric analysis showed that intermittent PTH administration (40 µg/kg) was able to protect the tooth site from periodontitis-induced bone resorption. In addition, there was a significant reduction in the number of inflammatory cells at the marginal gingival area in sections obtained from animals receiving PTH compared with control animals. These findings demonstrated that intermittent PTH administration was able to protect against periodontitis-associated bone loss in a rodent model.
Key Words: parathyroid hormone • periodontitis • anabolism
Parathyroid hormone (PTH) functions as a major mediator of bone remodeling and as an essential regulator of calcium homeostasis, producing several distinct and independent effects on the bone remodeling process, resulting in both bone formation (anabolic activity) and bone resorption (catabolic activity), e.g., continuous infusion of PTH decreases bone mass by stimulating osteoclast activity, while intermittent administration increases bone mass by stimulating osteoblast differentiation (Neer et al., 2001). The impressive and relevant results obtained in clinical trials (Neer et al., 2001; Horwitz et al., 2003) resulted in approval by the US Food & Drug Administration (FDA) for FORTEO® (PTH) to treat osteoporosis.
Various actions of PTH on cells of the osteoblast lineage affect the process of remodeling. PTH modulates cell morphology, proliferation, and matrix gene/protein expression (Canalis et al., 1989). These effects of PTH on bone metabolism are mediated by its binding to G-protein-coupled receptors (GPCR) on stromal and osteoblastic cells (Swarthout et al., 2001). GPCR stimulation of osteoblast-mediated bone formation is typically followed by osteoclast-mediated bone resorption, and different signaling pathways are thought to be involved in mediating these two actions. It has been suggested that the kinetics of GPCR activation/deactivation may determine whether GPCR stimulation is anabolic or catabolic. Two key factors secreted by osteoblasts, T-lymphocytes, and stromal cells—osteoprotegerin (OPG) and receptor activator of NF
Osteoclast activation requires induction of RANKL, a member of the tumor necrosis factor (TNF) ligand family—also called ODF/ TRANCE/OPGL—that stimulates the differentiation of osteoclast progenitors of the monocyte/macrophage lineage into osteoclasts in the presence of macrophage colony-stimulating factor (M-CSF). Osteoclast precursors that express RANK (receptor activator of NF Lipopolysaccharides (LPS)/endotoxin, biologically active pro-inflammatory factors found in the cell walls of Gram-negative bacteria, have been identified as factors involved in stimulating bone resorption. Analysis of current data indicates that prolonged or excessive production of such pro-inflammatory factors represents an important etiologic factor in bone loss associated with chronic inflammatory diseases, such as periodontitis. Therefore, the aim of the present study was to determine whether or not intermittent administration of PTH (1-34), in rats subjected to periodontal disease, would result in protection against periodontitis-associated bone loss. As described here, PTH administration proved to protect animals from periodontitis-associated bone loss when compared with placebo-treated animals.
Animals Twenty male Wistar rats, aged 4 wks at the beginning of this study, were housed in the animal research facility at the University of Campinas. They were maintained in plastic cages and fed ad libitum with standard rat diet. The experimental procedures were approved by the Institutional Animal Research Committee at the University of Campinas (São Paulo, Brazil).
Experimental Design
Histomorphometric Analysis
Quantification of Inflammatory Cells
Statistical Analysis
Histological analysis of sections obtained from ligated teeth, placebo group (Fig. 1A  ), indicated significant bone loss in the molar teeth furcation area when compared with the contralateral unligated teeth from PTH- or placebo-treated animals (Fig. 1B) or with ligated teeth from the PTH-treated group (Fig. 1C). Such findings were noted in all 10 samples. In marked contrast, the ligated molars in the animals that received intermittent PTH (40 µg/kg) showed no bone loss within the furcation area in comparison with placebo-treated ligated teeth (Fig. 1Bvs . 1A), a finding observed in sections from all 10 molars. Statistical analysis showed no significant difference in histomorphometric measurements in sections obtained from ligated teeth, PTH-treated animals, compared with unligated molars from either the placebo- or PTH-treated groups (Fig. 1D). In the furcation area from placebo-treated ligated teeth, multinucleated osteoclasts were observed (Figs. 2A, 2B), and the periodontal ligament fibers were disarranged (Fig. 2C). These findings were not seen in tissues from ligated and unligated PTH-treated animals or from the placebo-treated unligated group.
Next, to determine whether differences in bone loss correlated with differences in inflammatory activity, we measured the number of inflammatory cells present in the marginal gingival tissue. The data demonstrated more intense inflammation associated with samples from the placebo-treated ligated group, consisting mainly of polymorphonuclear leukocytes (Fig. 3A ), when compared with samples obtained from PTH-treated animals, regardless of the presence of ligature, or with placebo-treated unligated animals, where the inflammatory infiltrate was minimal (Fig. 3B). Statistical analysis showed that the extent of inflammatory cells around ligated teeth was significantly greater in animals from the placebo-treated ligated group when compared with the placebo-treated unligated sites or with ligated PTH-treated sites (Fig. 3C).
Although PTH has been reported either to increase (catabolic effect) or to decrease (anabolic effect) osteoclastogenesis via OPG and RANKL, respectively (Simonet et al., 1997), to date, no in vivo studies have focused on the effect of intermittent PTH administration on bone loss associated with periodontal diseases. Previous studies of periodontal disease using a rodent model similar to the one used here have shown that the resulting bone loss is related to the presence of bacteria (Rovin et al., 1966). Here, using a ligature-induced periodontitis model in rodents, we show that PTH may prevent or reverse the impact of LPS and other inflammatory-associated factors on the periodontium. Since PTH receptors are present on osteoblasts and not osteoclasts, these results suggest that this protective effect is mediated through the regulation of osteoblast activity, e.g., secretion of RANKL and OPG, although PTH may also block other LPS factors involved in activation of osteoclast function. At a clinical level, analysis of the data provided here suggests that PTH, currently approved for use in treatment of osteoporosis, may also prove useful for treatment of other bone-associated diseases such as periodontal disease. In fact, bisphosphonates, another drug approved for treatment of osteoporosis, have been evaluated for use in treatment of periodontal diseases, with promising results (El-Shinnawi and El-Tantawy, 2003). Bisphosphonates are bone-sparing agents, while PTH is a bone-forming agent, and thus, PTH provides a new approach for treating bone-loss-related diseases. PTH acts through dual signaling pathways in bone cells, with the osteoblast being the principal target. In the osteoblast, the type I PTH/PTH-related peptide receptor is coupled to both the adenylate cyclase activating G-protein-coupled protein (Gs) (cAMP/protein kinase A pathway) and the phospholipase C-activating Gq protein (Morley et al., 1997). Activaction of the Gs-mediated pathway stimulates the production of 3,5-cyclic adenosine monophosphate (cAMP), and there is evidence that this pathway is important for bone formation (Burgering et al., 1993). PTH receptor activation of the Gq-coupled pathway results in phospholipase C-mediated phosphatidylinositol hydrolysis, producing diacylglycerol (DAG), an activator of protein kinase C (PKC) isozymes (Jouishomme et al., 1994). DAG can also derive from phospholipase D (PLD)-catalyzed hydrolysis of phosphatidylcholine, and PTH stimulation of PLD activity provides another potential pathway for PKC activation in osteoblasts (Cornish et al., 1999). The dual functionality of PTH may derive from its ability to stimulate both adenylate cyclase (Spurney et al., 2002) and phospholipase C (Civitelli et al., 1998). Overall, the activation profile of PTH in bone cells leads to induction of several growth factor genes, including those for IGF-1, IGF-2, and TGF-β. In addition, IGFBP-1, -4, and -5 are induced by PTH, as are IGFBP protease-3 and -5 (Canalis et al., 1989; Linkhart and Mohan, 1989; Morley et al., 1997). On a cellular level, PTH enhances the recruitment of pre-osteoblasts from marrow stromal cells and induces maturation of lining osteoblasts, increasing collagen synthesis. Expression of skeletal IGF-1 is markedly enhanced in situ by PTH administration (Linkhart and Mohan, 1989). Notwithstanding these observations, the underlying molecular physiology accounting for the true anabolic effect of PTH remains unknown. In addition, it is uncertain why intermittent, low-dose PTH administration differs so drastically in its effect on bone cells from chronic sustained PTH treatment in which catabolic effects at cortical sites predominate (Goltzman, 1999). Evidence has emerged that PTH reduces osteoblastic apoptosis, prolonging osteoblast survival and possibly potentiating its differentiated function in collagen synthesis (Jilka et al., 1999). In addition, the anabolic effect of PTH has been demonstrated in clinical trials, and there is a suggestion that the kinetics of activation/deactivation may determine whether G-protein-coupled receptor stimulation is catabolic or anabolic (Chen et al., 2002).
Osteoblast-mediated bone formation is often linked to osteoclast bone resorption. This is due, at least in part, to a balance between RANKL and OPG (Martin and Ng, 1994), both known to be released from osteoblast precursors, mature osteoblasts, PDL cells, and T-lymphocytes (Zou and Bar-Shavit, 2002). Agonists of several GPCR systems have been shown to modulate OPG and RANKL production (Swarthout et al., 2002; Zou and Bar-Shavit, 2002). Inhibition of RANKL function via the decoy receptor OPG has been shown to reduce alveolar bone destruction significantly, as reported by Teng et al.(2000). Additional studies have focused specifically on elucidating the mechanism of osteoclast generation and control in the microenvironment of the periodontium and have shown that both OPG and RANKL are present within the periodontium (Teng et al., 2000). However, it is not clear how bacteria- or endotoxin-induced bone resorption occurs. Analysis of existing data indicates that pro-inflammatory factors, e.g., cytokines, known to promote osteoclast activity, act through osteoblasts, stromal bone-lining cells, or T-lymphocytes (Teng et al., 2000). These pro-inflammatory cytokines have been shown to act synergistically with RANKL to promote osteoblast-mediated bone resorption (Chiang et al., 1999), although more recent studies suggest that factors such as LPS and TNF
LPS produced by various periodontopathogens such as P. gingivalis and Actinobacillus actinomycetemcomitans induce a local inflammatory response that ultimately leads to periodontal bone resorption (Fletcher et al., 2001), but the exact mechanisms by which such micro-organisms induce bone resorption are unclear. In the periodontium, LPS may promote an inflammatory reaction through the induction of several cytokines, including IL-1, IL-6, TNF Taken together, these results suggest that intermittent administration of PTH may protect against bone resorption associated with periodontitis. At the clinical level, the anabolic effect of PTH on bone metabolism may represent an attractive approach for stimulating bone formation, and thus improve the outcome of periodontal therapy.
This work was supported by the National Council for Scientific and Technologic Development-Brazil and by NIDCR (NIH DE-09532) (to MJS). Received for publication December 27, 2002. Revision received June 30, 2003. Accepted for publication July 16, 2003.
Journal of Dental Research, Vol. 82, No. 10,
791-795 (2003)
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ABSTRACT
INTRODUCTION
β ligand (RANKL)—have been linked to anabolic and catabolic activity, respectively (
may have direct effects on osteoclasts (