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Role of the Activation of the Nuclear Enzyme Poly(ADP-Ribose) Polymerase in the Pathogenesis of Periodontitis1 Institute of Human Physiology and Clinical Experimental Research, and 2 Department of Anatomy, Semmelweis University, 78/A Üllöi út, Budapest, Hungary, 1082; and 3 Inotek Pharmaceuticals Corporation, Beverly, MA, USA; Correspondence: *corresponding author, Lohinai{at}elet2.sote.hu
We have investigated the role of the activation of nuclear poly(ADP-ribose) polymerase (PARP) enzyme, a mediator of downstream nitric oxide toxicity, using a combined approach of pharmacological inhibition and genetic disruption in a ligature-induced-periodontitis model in rats and mice. Immunohistochemical analysis revealed significantly increased poly(ADP-ribose) nuclear staining (indicative of PARP activation) in the subepithelial connective tissue of the ligated side compared with the non-ligated side. Ligation-induced periodontitis resulted in marked plasma extravasation in the gingivomucosal tissue and led to alveolar bone destruction compared with the non-ligated side, as measured by the Evans blue technique and by videomicroscopy, respectively. PARP inhibition with PJ34, as well as genetic PARP-1 deficiency, significantly reduced the extravasation and the alveolar bone resorption of the ligated side compared with controls. Thus, PARP activation contributes to the development of periodontal injury. Inhibition of PARP may represent a novel host response modulatory approach for the therapy of periodontitis.
Key Words: poly(ADP-ribose) polymerase • DNA breaks • nitric oxide • peroxynitrite • inflammation • periodontal disease • gingiva • gut • knockout • rat • mice
We have previously demonstrated that nitric oxide (NO) and peroxynitrite (the reactive reaction product of NO and superoxide) participate in the pathogenesis of periodontitis (Lohinai et al., 1998, 2001b; Lohinai and Szabo, 1998). We have proposed that bacteria of the oral cavity trigger the inducible NO synthase (iNOS) up-regulation in periodontal tissues (Lohinai et al., 1998, 2001b; Lohinai and Szabo, 1998). The consequent chronic locally high production of NO, peroxynitrite, and their derivatives, which presumably serve as a mechanism for combating periodontal pathogens, may also contribute to host tissue damage, causing breakdown of the periodontal attachment apparatus (Lohinai et al., 1998, 2001b; Lohinai and Szabo, 1998). Much of the NO/peroxynitrite-mediated downstream tissue toxicity can be attributed to excessive activation of poly(adenosine 5'-diphosphate-ribose) polymerase (PARP). PARP is one of the most abundant nuclear proteins of eukaryotic cells functioning as a DNA nick-sensor enzyme (see, for review, Virag and Szabo, 2002). Upon binding to DNA breaks, activated PARP cleaves NAD+ into nicotinamide and ADP-ribose, and polymerizes the latter onto nuclear acceptor proteins, including histones, transcription factors, and PARP itself. Poly(ADP-ribos)ylation can dramatically affect the function of the target protein. PARP activation has been implicated in the regulation of various cellular processes, such as DNA repair, cell differentiation, gene expression, and cell death. However, massive oxidative and nitrosative stress-induced severe DNA damage, and consequent over-activation of PARP, initiates an energy-consuming futile intracellular metabolic cycle by producing extended chains of ADP-ribose on nuclear proteins and results in substantial depletion of its reaction substrate, the NAD+ stores. The rapid decrease of NAD+ slows the rate of glycolysis and mitochondrial respiration, leading to energy collapse, cell dysfunction, and, ultimately, cell necrosis. This mechanism, known as the PARP "suicide hypothesis", has been proposed to occur in a wide range of pathophysiological conditions associated with reactive species-induced stress, such as arthritis, colitis and other forms of inflammation, diabetes, or ischemia-reperfusion injury (Berger and Berger, 1986; Virag and Szabo, 2002). PARP has also been shown to participate in the up-regulation of a variety of pro-inflammatory mediators in various disease conditions, via activation of nuclear factor (NF)-kappaB and other nuclear transcription factors (see, for review, Virag and Szabo, 2002). On the other hand, PARP inhibition therapy represents an effective novel approach to the treatment of a variety of diseases (see, for review, Cosi, 2002; Virag and Szabo, 2002). Therefore, we tested the role of PARP in murine models of ligature-induced periodontitis. The aims of this study were to: (i) test whether PARP becomes activated in periodontitis; (ii) investigate whether pharmacological inhibition of PARP with a potent PARP inhibitor, PJ34 (Mabley et al., 2001; Soriano et al., 2001; Jagtap et al., 2002), can limit inflammation and associated alveolar bone loss; and (iii) determine if disruption of the PARP-1 gene, the major isoform of the PARP family, can alter the outcome of periodontitis.
Experimental Protocol All protocols were approved by the local Institutional Ethics Committee. Wistar rats (male, 250-300 g; n = 24) were anesthetized with 35 mg/kg pentobarbital. A 2-0 silk ligature was placed around the 1st bottom molar on the animal’s left side (Lohinai et al., 1998). Animals recovered, were given food and water ad libitum, and were treated with PARP inhibitor PJ34 (N-(6-oxo-5,6-dihydro-phenanthridin-2-yl)-N,N-dimethylacetamide), 0 or 10 mg/kg/day i.p. in two divided doses for eight days. At Day 8, the gingivomucosal tissues encircling the 1st mandibular molars were removed on both sides and processed for poly(ADP-ribose) immunohistochemistry (n = 2 x 3) or for plasma extravasation measurement by the Evans blue technique (n = 2 x 9). The mandible was also harvested for measurements of alveolar bone loss (n = 2 x 9). Mice deficient in the predominant isoform of PARP (PARP-1) and their wild-type littermates (male, 20-26 g; n = 2 x 8), the colony originally derived from Dr. Z.Q. Wang’s laboratory (Wang et al., 1995), were subjected to the same ligature (4-0) placement as the rats. At Day 15, the tissues were harvested; the subsequent procedures were identical to those described above.
Biological Events The vascular permeability was measured by the Evans blue technique in rats as described in Lohinai et al. (1998). For mice, the procedure was slightly modified: The Evans blue was injected into the right superficial jugular vein instead of the femoral vein, and 4 mL saline wash was administered transcardially instead of via the abdominal aorta. Alveolar bone resorption was expressed as the linear distance on the lingual surface from the cemento-enamel junction at the mediolingual root in the rat and the distal root in mice of the first lower molar to the alveolar crest on both sides; resorption was measured by videomicroscopy as previously published (Lohinai et al., 1998).
Data Analysis
Immunohistochemical analysis revealed a significantly increased poly(ADP-ribose) nuclear staining in the subepithelial connective tissue of the ligated side compared with the contralateral (control) side (11.0 + 4.7 vs. 2.0 + 1.2 in 10 randomly selected 10,000-µm2 connective tissue areas of all animals, n = 3, p < 0.05, respectively) and unstained epithelium in both sides in rats (Figs. 1, 2  ). Most of the immunoreactive nuclei were located mainly beneath the epithelium and more distally, as well as around or within the blood vessels. Some of the nuclei showed very high immunoreactivity, whereas others were stained only moderately. The shapes of the stained nuclei varied: round, oval, triangular, reniform, segmented, or irregular. Based on their form, size, and location, they can be identified as fibroblasts, monocytes/histiocytes, lymphocytes, PMNs, and mast or endothelial cell types (Figs. 2a, 2b, 2c, 2d, 2e, 2f, 2g). Outside the nucleus, the cytoplasm of the mast cells showed a pale immunoreactivity as well (Figs. 2d, 2e). The PARP activation was attenuated by treatment of PJ34. Rats treated with PJ34 have less and lighter poly(ADP-ribose) nuclear staining around the ligature (Fig. 1).
Ligation increased Evans blue extravasation in the gingivomucosal tissue compared with the right (control) side in vehicle-treated rats (p < 0.05). PARP inhibition with PJ34 prevented the vascular permeability increase of the inflamed gingivomucosal tissue compared with the vehicle administration, as evidenced by decreased Evans blue extravasation (p < 0.05) or the non-significant difference in comparison with the rat’s own right side (Fig. 3 ). Ligation enhanced alveolar bone loss compared with the right side in both groups (p < 0.05). However, PARP inhibition reduced the alveolar bone loss as well compared with vehicle administration (p < 0.05, Fig. 3).
Ligation also induced Evans blue extravasation in the gingivomucosal tissue compared with the right side in both mouse groups (p < 0.05). However, genetic PARP deletion diminished vascular permeability of the inflamed gingivomucosal tissue compared with the wild-type control, as evidenced by decreased Evans blue extravasation (p < 0.05, Fig. 4 ). Ligation increased alveolar bone loss compared with the right side in both groups as well (p < 0.05). However, genetic PARP deletion also attenuated alveolar bone loss when compared with the response seen in the wild-type control animals after ligation (p < 0.05, Fig. 4).
PARP activation can be visualized by immunohistochemistry, with antibodies that recognize poly(ADP-ribose), the enzymatic product of PARP. In our studies, massive PARP activation was localized to the areas of the most severe gingivomucosal injury in fibroblasts, monocytes/histiocytes, lymphocytes, PMNs, and mast and endothelial cell types identified according to the characteristics of the cell nuclei. The pale reactivity of the cytoplasm of the mast cells can be explained as non-specific reaction because of their endogenous peroxidase activity (although the activity of cytoplasmically located minor PARP isoforms cannot be excluded). It is also interesting to note that the PMNs stained for poly(ADP-ribose), because it has been postulated that the PMNs are the only mammalian cells that do not contain PARP, presumably to protect themselves from their own frequently generated high-oxidant production. We found some poly(ADP-ribose) immunoreactivity in the control gingivomucosal connective tissue as well, which reinforced our previous hypothesis that, even in healthy gingivomucosal tissue, there is a subclinical spontaneous inflammation caused by the oral flora (Lohinai et al., 1998, 2001a,b). The reduction of poly(ADP-ribose) staining in PJ34-treated animals directly demonstrates that the dosing of the PARP inhibitor is sufficient to suppress PARP activation in the periodontal tissues. PARP appears to play a role in the development of acute exudative vasculitis of the inflamed gingivomucosal tissue. This is supported by the following observations: (1) The endothelial cells were highly reactive for poly(ADP-ribose), and (2) both pharmacological PARP inhibition as well as the genetic ablation of the PARP gene reduced the extravasation induced by the ligature. In previous studies, increased PARP activation was found in endothelial dysfunction associated with endotoxin shock or diabetes. This vascular dysfunction was prevented or even reversed by PARP inhibition (Szabo et al., 1997; Soriano et al., 2001; Jagtap et al., 2002). PARP is also involved in company with other factors in alveolar bone resorption in periodontitis, since PARP inhibition and lack of activity due to genetic deletion were protective in our experimental models. There are no direct data in the literature on the potential role of PARP in bone degradation or resorption, although it is clear that pro-inflammatory mediators play an important role in this process, and PARP inhibition is known to result in a generalized down-regulation of the inflammatory response (reviewed in Virag and Szabo, 2002). It is also noteworthy that recent work demonstrates that a superoxide dismutase mimetic markedly reduced PARP activity and also attenuated the focal bone resorption associated with experimental arthritis (Salvemini et al., 2001). The results of our study suggest that PARP is involved in the progression of the periodontal inflammatory process and that inhibition of PARP decreases the various symptoms of periodontal injury. Recent studies identified several novel isoforms and related spliced forms of PARP with poly(ADP-ribos)ylating capabilities (Cosi, 2002; Virag and Szabo, 2002). The transgenic mice used in this study, lacking the functional gene for PARP-1, provided a unique opportunity for definition of the role of the major PARP isoform in periodontitis. Using the murine ligament model of periodontal disease, we found that the absence of a functional PARP-1 gene resulted in a significant prevention of periodontal injury. Results from our and most pharmacological studies could be reproduced by the use of PARP-1-deficient animals/cells and may suggest that PARP-1 is the major target of PARP inhibitors in inflammations; the other isoforms of PARP probably have only minor roles (Oliver et al., 1999; Virag and Szabo, 2002). The physiological role of PARP-1 has been much debated this last decade. PARP-1 has been implicated in the regulation of a diverse array of biological processes, such as chromatin structure, DNA repair, replication (proliferation, cell cycle), protein degradation, and cell death (necrosis and apoptosis). A recent concept (Virag and Szabo, 2002) is that PARP-1 activated by mild genotoxic stimuli facilitates DNA repair, and that cells survive without the risk of passing on mutated genes. More severe DNA damage induces apoptotic cell death, during which caspases inactivate PARP-1. This pathway may conserve energy needed for the apoptotic process and allows cells with irreparable DNA damage to become eliminated in a safe way, while the third route is induced by extensive DNA breakage usually triggered by a massive degree of oxidative and nitrosative stress (hydroxyl radical, peroxynitrite, nitroxyl anion). Overactivation of PARP, as described in the "suicide theory" above, causes cellular energetic catastrophy, inhibits the apoptotic machinery, and leads to necrotic cell death. There is compelling evidence in the literature that, in active periodontitis, the third route, necrosis, is the common feature, e.g., the majority of neutrophils lose their viability within periodontal pockets by necrosis and not by apoptosis (Crawford et al., 2000). Furthermore, the severity of periodontal inflammation or the loss of periodontal attachment is associated with increased levels of cytoplasmic enzymes (e.g., aspartate aminotransferase, lactate dehydrogenase, creatine kinase) and potassium in gingival crevicular fluid, because they spill from the cytoplasm into the extracellular environment as a result of cell death (Bang et al., 1973; Atici et al., 1998; Lindhe et al., 1998). During necrosis, the cell content leaks out, liberating dangerous proteases (and other toxic factors as well) into tissues, contributing directly to periodontal injury. The effects of the NAD+-level alterations on periodontal status also support the validity of the PARP-related suicide pathway. Epidemiological studies showed that, in endemic low-niacin (nicotinic acid, the precursor of NAD+) intake areas, gingivitis and other forms of oral inflammation have high incidence (Buzina, 1976). Furthermore, in cases of experimental niacin deficiency by long-term dietary deprivation, among other symptoms, stomatitis was developed, highlighted by necrotizing gingivitis, periodontitis, and glossitis (Dreizen et al., 1977). In contrast, NAD+-containing multivitamin supplementation was beneficial to the gingival state (Cheraskin and Ringsdorf, 1969). Similarly, nicotinate paste ameliorated experimental gingivitis (Taguchi et al., 1989). The PARP-related activation of NF-kappaB, activator protein-1, and mitogen-activated protein kinases and the expression of adhesion molecules (ICAM-1, E-selectin) may also participate in pathogenesis, because all these factors have already been described as significant contributors of periodontitis (Yoneda et al., 1997; Le Page et al., 1998; Lindhe et al., 1998; Sugita et al., 1998; Oliver et al., 1999; Darveau et al., 2002; Virag and Szabo, 2002). For example, NF-kappaB is a key transcription factor involved in the generation of chemokines and enzymes (e.g., iNOS, inducible cyclooxygenase, collagenase) in immunostimulated cells, and PARP inhibition suppresses the induction of these mediators via the inhibition of NF-kappaB activation (Ehrlich et al., 1995; Yoneda et al., 1997; Le Page et al., 1998; Virag and Szabo, 2002). Because PARP promotes several interrelated pathways—e.g., production of pro-inflammatory mediators and infiltration of inflammatory cells by adhesion molecules as well—therefore, more and more oxygen- and nitrogen-centered free radicals/oxidants are generated to attack the invading microbes, which in turn, through severe DNA damage of the host cells, further activate PARP. Thus, the positive feedback cycles of the local host response may excessively exaggerate the inflammatory cascade beyond what can be considered controllable by the body’s own defense system, and therefore may become detrimental to the periodontal tissue. Our data are also in good agreement with recent studies in a variety of models of experimental inflammation (Mabley et al., 2001; Virag and Szabo, 2002). However, the results of the present work contradict the studies by Hussain’s group. Cultured fibroblasts derived from diseased gingival sites displayed reduced PARP activity compared with healthy controls (Hussain et al., 1994). Gingival biopsy from adult periodontitis and healthy periodontal tissue reflected similar patterns of enzyme activity (Ghani et al., 1996). The reasons for the discrepancy in results are puzzling. It is conceivable that the decreased ex vivo PARP activity may be related to either an earlier increase in PARP activation followed by auto-ADP-ribosylation of the PARP enzyme, which causes auto-inactivation, or to massive cleavage and inactivation of PARP by caspases during the apoptotic process, followed by the release of the cellular content via post-apoptotic necrosis (Wu et al., 2001; Cosi, 2002; Virag and Szabo, 2002). It is important to point out that our current murine models were designed to mimic the acute to subacute phases of periodontal inflammation. It is also possible that different types of changes in PARP activity (up- vs. down-regulation) may occur in the acute vs. chronic phases of periodontal inflammatory diseases. In conclusion, our results suggest that PARP activation plays a crucial role in the pathogenesis of acute periodontal injury. We propose that, in periodontitis, the oxidative and nitrosative species are inducers of DNA strand breaks, which trigger PARP overactivation (Lohinai et al., 1998, 2001b; Lohinai and Szabo, 1998). This process generates an energy-consuming futile cellular cycle leading to cell dysfunction and, ultimately, necrosis. Furthermore, PARP triggers, via positive feedback cycles, the amplification of various inflammatory mediators as well. Inhibition of PARP may represent a novel host response modulatory approach for the treatment of periodontal disease.
This study was supported by Hungarian Research Grants OTKA (#F-20469, F-030448), ETT (#152/2000), by the Bolyai Research Foundation, by an Eötvös Scholarship, by BLUE & Blue Co. (Hungary), and by a Grant from the US NIH, #1 R43 DE13625. The authors are grateful to Ágnes Gara and Éva Burka for their invaluable assistance. The results of the current paper were presented in preliminary form at the 79th General Session of the IADR, Chiba, Japan, 2001 [J Dent Res 80(Spec Iss B):769, 2001]. Received for publication December 27, 2002. Revision received June 9, 2003. Accepted for publication September 15, 2003.
Journal of Dental Research, Vol. 82, No. 12,
987-992 (2003) This article has been cited by other articles:
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