A p38 Selective Mitogen-Activated Protein Kinase Inhibitor Prevents Periodontal Bone Loss

In the oral microbial environment, Gram-negative bacterial derived lipopolysaccharide (LPS) can initiate inflammatory bone loss as seen in periodontal diseases. p38 Mitogen-activated protein kinase (MAPK) signaling is critical to inflammatory cytokine and LPS-induced cytokine expression, which may contribute toward periodontal bone loss. The purpose of this proof-of-principle study was to evaluate the ability of an orally active p38 MAPK inhibitor (SD-282) to reduce periopathogenic LPS-induced alveolar bone loss in an experimental rat model. Five groups of Sprague-Dawley rats received one of the following treatments: LPS injected to the palatal gingiva adjacent to the maxillary molars three times per week for 8 weeks, LPS plus two doses of SD-282 (15 or 45 mg/kg) twice daily by oral gavage, or control groups given drug vehicle (1% polyethylene glycol) or SD-282 (45 mg/kg) only. Baseline and 8-week alveolar bone loss was assessed by microcomputed tomography ( CT) and histological examination. LPS induced severe bone loss over this time period, whereas control groups were unchanged from baseline measurements. Both doses of SD-282 showed significant protection from LPSinduced bone loss. Bone area and volumetric analysis of maxillas by CT indicated significant loss of bone volume with LPS treatment, which was blocked with the p38 inhibitor. Histological examination indicated significantly fewer tartate-resistant acid phosphatase-positive osteoclasts and a significant decrease in interleukin (IL)-6, IL-1 , and tumor necrosis factor expression in p38 inhibitor-treated groups compared with LPS groups by immunostaining. Results from this in vivo study suggest that orally active p38 MAPK inhibitors can reduce LPS-induced inflammatory cytokine production and osteoclast formation and protect against LPS-stimulated alveolar bone loss. Periodontal disease initiation and progression occurs as a consequence of the host immune inflammatory response to oral pathogens. Periodontal pathogen-derived lipopolysaccharide (LPS) is considered a key factor in the development of chronic inflammation leading to bone loss, the main hallmark feature of periodontitis. LPS-induced alveolar bone loss results from a local host response in gingival tissues through recruitment of inflammatory cells, generation of prostanoids and cytokines, elaboration of proteolytic enzymes, and activation of osteoclasts resulting in alveolar bone destruction and eventual tooth loss (Baker, 2000; Madianos et al., 2005). Activated monocytes, macrophages, and fibroblasts all produce cytokines, such as TNF, IL-1 , prostaglandin E2, and IL-6, within periodontal lesions (Lee et al., 1995; Reddi et al., 1996), and have all been found to be significantly elevated in diseased periodontal sites compared with healthy or inactive sites. Multiple inflammatory signals can modulate receptor activator of NFB ligand (RANKL), RANK, or osteoprotegerin (OPG)—three novel members of the TNF ligand and receptor superfamilies, which modulate osteoclastogenesis (Aubin and Bonnelye, 2000; Hofbauer and Heufelder, 2001). For osteoclastogenesis to occur, RANKL must bind to its cognate receptor, RANK, a receptor on the cell surface of osteoclasts and osteoclast precursors, to stimulate proliferation and differentiation This work was supported by Scios, Inc., and National Institutes of Health Grant P30AR46024. Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. doi:10.1124/jpet.106.112466. ABBREVIATIONS: LPS, lipopolysaccharide; PD, periodontal disease; OC, osteoclast; ABC, alveolar bone crest; CEJ, cementoenamel junction; CT, microcomputed tomography; SD-282, indole-5-carboxamide (ATP competitive inhibitor of p38 kinase; TRAP, tartate-resistant acid phosphatase; TNF, tumor necrosis factor; IL, interleukin; MAPK, mitogen-activated protein kinase; NF, nuclear factor; RANK, receptor activator of NFB; RANKL, receptor activator of NFB ligand; OPG, osteoprotegerin; ROI, region of interest; P-p38, phosphorylated p38; BAF, bone area fraction; BVF, bone volume fraction. 0022-3565/07/3201-56–63$20.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 320, No. 1 Copyright © 2007 by The American Society for Pharmacology and Experimental Therapeutics 112466/3163565 JPET 320:56–63, 2007 Printed in U.S.A. 56 at A PE T Jornals on Sptem er 3, 2017 jpet.asjournals.org D ow nladed from of cells from the monocyte/macrophage lineage to form the functional osteoclasts. OPG, a soluble decoy receptor produced by osteoblasts, marrow stromal cells, and other cells, profoundly modifies the effects of RANKL by inhibiting RANKL/RANK interaction (Simonet et al., 1997) and has shown promising results for the treatment of bone-related diseases (Kostenuik et al., 2004). Within the diseased periodontal tissues, activated osteoclasts are an integral component of bone destruction (Assuma et al., 1998; Crotti et al., 2003). One of the major intracellular pathways activated by environmental stimuli, including periopathogenic LPS, is the mitogen-activated protein kinase (MAPK) pathway. MAPKs are divided into three major subgroups: the extracellular signal-regulated kinases 1/2, c-jun N-terminal kinases, and p38. Mitogens and growth factors primarily activate extracellular signal-regulated kinases 1/2, whereas the proinflammatory cytokines IL-1 and TNFand cell stress-inducing factors, such as LPS, heat shock, osmotic shock, ultraviolet radiation, and oxygen radicals, chiefly activate c-jun N-terminal kinases and p38. The three MAPKs control the activation of many transcription factors, including AP-1 (homodimer or heterodimer of the proteins c-fos and c-jun), NFB, or CAAT-enhancer-binding protein. MAPKs, most notably p38, can activate NFB. Aggregatibacter actinomycetemcomitans (formerly known as Actinobacillus actinomycetemcomitans) is highly associated with localized aggressive periodontitis (Slots and Ting, 1999). Within periodontal resident cell types, including tissue macrophages and other periodontal cells, MAPKs are activated by A. actinomycetemcomitans LPS (Patil et al., 2006). p38 MAPK, most notably its p38 isoform, is activated mainly within cells involved in the inflammatory process. Activation of p38 induces synthesis of proinflammatory cytokines, such as tumor necrosis factor (TNF), IL-1, IL-6, and IL-8, either via direct activation of gene transcription or via mRNA stabilization (Adams et al., 2001; Hoffmann et al., 2002; Kirkwood et al., 2003; Patil et al., 2004). p38 MAPK stabilizes mRNA via the enzyme substrate MAPK-activated protein kinase 2, which may act on one or more proteins capable of binding to mRNA (Holtmann et al., 1999). In addition, p38 MAPK controls the synthesis of other compounds, including chemokines, metalloproteinases, and prostaglandins (Lee et al., 2000). Recently, our research group has shown that IL-1 and TNF-induced RANKL expression in bone marrow stromal cells requires p38 signaling for maximal expression (Rossa et al., 2006). Collectively, these data suggest that p38 inhibitors may be beneficial to target bacterial induced alveolar bone loss—the hallmark of periodontitis. Recently, a p38 -specific inhibitor, SD-282, has been shown to be efficacious in reducing and reversing bone and cartilage destruction in an experimental arthritis model (Medicherla et al., 2006). The purpose of the present in vivo study was to determine whether this orally active p38 inhibitor can prevent alveolar bone loss initiated by A. actinomycetemcomitans LPS in an experimental rat model. Materials and Methods LPS Preparation. A . actinomycetemcomitans strain Y4 (serotype B) was grown in brain heart infusion media at 37°C, 5% CO2. LPS was extracted by the hot phenol-water method as described previously (Wilson and Hamilton, 1992). The bacteria were sequentially treated with lysozyme, DNase, RNase, and proteases to extract and isolate the lipopolysaccharide. The LPS used in the present study contained 0.001% nucleic acid by spectrophotometry and ca. 0.7% protein by BCA protein assay. The absence of protein in the LPS preparations was confirmed by polyacrylamide gel electrophoresis of extract samples and subsequent staining with silver nitrate