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The health benefits of polyunsaturated fatty acids (PUFAs) present in fish oil have been long known, but the cellular and molecular mediators of their actions are just being discovered (Serhan, 2007). Resolvin E1 (5S,12R,18R-trihydroxy-eicosapentaenoic acid, RvE1) is an oxygenated product of eicosapentaenoic acid (EPA), one of the main dietary essential ω-3 PUFAs (Serhan et al., 2000; Arita et al., 2005a). RvE1 was first described in inflammatory exudates of the murine dorsal pouch (Serhan et al., 2000). RvE1 acts by reducing neutrophil infiltration and by promoting phagocyte removal, thus hastening inflammation resolution (Arita et al., 2005a; Schwab et al., 2007; Serhan, 2007). RvE1 reduces leukocyte infiltration into the peritoneum and protects from experimental colitis (Arita et al., 2005c). In experimental periodontal disease, topical RvE1 prevents Porphyromonas gingivalis-induced periodontal inflammation and alveolar bone loss (Hasturk et al., 2006). The question arising from the latter observation is whether the bone-sparing action is entirely mediated through RvE1's anti-inflammatory and pro-resolution actions, or whether RvE1 directly acts on bone cells. Other lipid PUFA derivatives such as prostaglandin E2 (PGE2) and leukotriene B4 (LTB4) act directly on osteoclast (OC) in addition to their pro-inflammatory actions (Raisz, 2005).
Dietary intake of PUFA can also influence bone metabolism, as increasing the ratio of ω-6/ω-3 PUFA in the diet leads to increased arachidonic acid/EPA ratio in the bone, increased PGE2 production and decreased bone formation in rats (Watkins et al., 2000). Treatment of bone marrow cultures with EPA, the precursor of RvE1, decreases OC formation in vitro, implying direct lipid action on OC (Sun et al., 2003). In the oral cavity, EPA-enriched diet reduces OC activity and alveolar bone resorption during orthodontic movement of rat teeth (Iwami-Morimoto et al., 1999). However, the molecular mechanisms of PUFA on bone cells are not known. In this study, we explore the potential of RvE1 to directly influence OC differentiation and bone resorption.
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RvE1 was initially discovered in resolving inflammatory exudates and identified as a potent regulator of resolution of acute inflammation (Serhan et al., 2000; Arita et al., 2005a). RvE1 acts by reducing neutrophil infiltration and by promoting phagocyte removal, thus hastening inflammation resolution (Arita et al., 2005a; Schwab et al., 2007; Serhan, 2007). OC-mediated bone resorption is often a consequence of inflammatory disease. Results presented here support a novel function for RvE1 in directly modulating OC differentiation and consequently bone resorption.
Several lipid mediators have been shown to regulate bone metabolism, including prostaglandins and leukotrienes. PGE2 stimulates OC differentiation through prostanoid receptors EP4 and EP2 by inducing RANKL production and inhibiting osteoprotegerin expression (Suzawa et al., 2000; Nukaga et al., 2004; Liu et al., 2005). Direct binding of PGE2 to OC has been proposed, but remains controversial, as differentiated OC do not express EP4 and EP2 receptors (Sakuma et al., 2000; Kobayashi et al., 2005). LTB4 increases bone resorption in vivo as well as in vitro in calvarial organ cultures and isolated OCs (Garcia et al., 1996). High- and low-affinity LTB4 binding sites have been identified on avian OCs, suggesting a direct cellular action (Flynn et al., 1999). On the other hand, bone protection in pathological inflammation has been ascribed to EPA (Sun et al., 2003). However, the molecular mechanism of the action of EPA and its metabolites on OC remains unclear. Data presented here show that direct administration of EPA to OC cultures in nanomolar doses results in increased OC formation, whereas administration of RvE1 in similar doses inhibits OC differentiation, suggesting that the bone sparing by EPA observed in vivo may not be a direct action of EPA on OC, but rather the action of a metabolic derivative of EPA such as RvE1. Significantly higher EPA doses were required for direct inhibition of OC growth, an observation consistent with EPA cytotoxicity demonstrated in other cell types such as breast cancer cells (Schley et al., 2005). In acute inflammation, RvE1 is produced from EPA in the presence of ASA through oxygenation by acetylated cyclooxygenase in endothelial cells, resulting in the production of 18-HEPE. 18-HEPE is further oxygenated by 5-lipoxygenase-like activity found in leukocytes to generate RvE1 (Serhan et al., 2000; Arita et al., 2005a). An alternative pathway also exists in bacteria-rich organs such as the gut or the oral cavity where microbial cytochrome P450 monooxygenase can generate 18-HEPE and initiate RvE1 production in the absence of acetylated cyclooxygenase (Arita et al., 2005b). LC-MS/MS analysis presented here shows that in the presence of EPA and ASA primary OC cultures are capable of generating the first intermediate, 18-HEPE. For further processing of 18-HEPE into RvE1, 5-LO activity is necessary, supplied here by activated neutrophils. The observation that co-incubation of OC with neutrophils results in RvE1 generation is the first evidence that transcellular biosynthetic pathways between OC and neutrophils exist in vitro and that locally produced RvE1 may have a significant function in vivo in moderating inflammation-induced bone resorption (Serhan et al., 2000; Hasturk et al., 2006).
OC differentiation from myelomonocytic progenitor cells is induced by the coordinated actions of M-CSF and RANKL (Boyle et al., 2003). M-CSF is a permissive factor necessary in early cell-fate determination, whereas RANKL is the final mediator of many physiological regulators of bone resorption (such as parathyroid hormone, calcitonin and calcitriol) and is an obligatory factor for OC differentiation and survival (Boyle et al., 2003). RANKL induces the fusion of mononucleated OC precursors to form a giant polykarion, which consequently forms an actin ring to seal off the ruffled membrane active in bone resorption. RvE1 decreases RANKL-induced OC growth and dentin disc resorption to a similar degree, suggesting that RvE1 downregulates the development of functional OC, but OC that do develop can function normally. Several lines of evidence suggest that RvE1 primarily affects OC differentiation: (1) the number of small OC precursors increase, whereas the number of larger OC decrease in the presence of RvE1, suggesting a partial blockade of OC fusion, (2) time course experiments show decreased OC growth at the earliest observable stages of polykarion development and (3) the nuclear translocation of the p50 subunit of NF-κB, a cardinal transcription factor in OC differentiation, is decreased. Interestingly, a primary action of RvE1 on leukocytes is inhibition of chemotaxis (Arita et al., 2005c; Schwab et al., 2007). OCs are also highly motile (Chellaiah et al., 2000); however, molecular mechanisms regulating OC mobility are just beginning to be discovered (Yu et al., 2004). As both RvE1 and LTB4 are powerful modulators of chemotaxis in leukocytes, further studies are warranted to evaluate the potential of RvE1 and LTB4 in regulating mononuclear OC precursor migration and fusion.
OC cell death does not appear to be influenced by RvE1. OC die by apoptosis upon RANKL withdrawal (Boyle et al., 2003). OC covered area rapidly declined in vitro in the absence of RANKL and in 8 h, most OC disappeared from the culture dish in our experiments. RvE1 did not alter the course of cell death or the rate of apoptosis as determined by OC integrity and nuclear morphology. However, RvE1 treatment markedly decreased the phosphorylation of Akt, an intracellular mediator generally associated with cell survival. Phosphorylated Akt (the active form of the kinase) promotes cell survival in many cell types by phosphorylating BAD, caspase-9 and AFX (Datta et al., 1999). In OC, however, the function of Akt is debatable. siRNA-induced gene silencing experiments indicate that Akt is not required for OC survival, but it is critical for OC differentiation as it mediates RANKL-induced NF-κB activation (Sugatani and Hruska, 2005). Moreover, pharmacological inhibition of the Akt phosphorylating mediator PI3 kinase results in suppression of OC fusion (Hotokezaka et al., 2006). Thus, downregulation of Akt phosphorylation by RvE1 may mediate its inhibitory actions on OC growth.
Two cell surface receptors denoted ChemR23 and BLT1 bind and mediate the intracellular actions of RvE1 in leukocytes (Arita et al., 2005a, 2007). In this study, we found that both ChemR23 and BLT1 mRNA are expressed by cultured OC. Receptor-binding studies using radiolabelled RvE1 demonstrated specific RvE1 binding on cell membrane preparations of OC that could be specifically displaced with unlabelled RvE1 as well as with the BLT1 ligand LTB4. In contrast, the ChemR23 peptide ligand chemerin was unable to compete with [3H]RvE1-specific binding on OC. Chemerin competes with RvE1-specific binding to ChemR23 in monocytes (Arita et al., 2005a); however, as this competition was not observed in OC, it is likely that the BLT1 is the main site of RvE1 actions in OC culture. Inhibition of the BLT1 receptor with U75302 reversed RvE1-mediated inhibition of OC growth, providing functional evidence for BLT1's critical function in mediating Rv1's action on OC. BLT1 is also a receptor for LTB4, a pro-inflammatory arachidonic acid derivative that is known to enhance bone resorption in vitro and in vivo (Garcia et al., 1996). LTB4 is specifically implicated in promoting OC fusion (Flynn et al., 1999). As RvE1 decreases only the number of large OC, it is plausible that RvE1 and LTB4 have counter-regulatory functions on the BLT1 receptor in determining OC growth and function.
In summary, the results presented here indicate that RvE1 blocks OC differentiation and bone resorption in vitro, suggesting a bone-sparing action distinct from RvE1's known anti-inflammatory and pro-resolution actions. RvE1 interfered with the late phase of OC differentiation corresponding to the formation of the polykarion. RvE1 bound to the BLT1 receptor and attenuated the nuclear translocation or the p50 subunit of NF-κB as well as the phosphorylation of Akt. OC can generate RvE1 by transcellular biosynthesis through interactions with leukocyte lipoxygenases, providing a local mechanism for RvE1 production in sites of inflammatory bone resorption (Figure 6). RvE1 attenuated OC growth at nanomolar concentrations, therefore, in conditions where inflammation is present along with potential bone resorption, such as rheumatoid arthritis (Walsh et al., 2005), post-menopausal osteoporosis (Raisz, 2005) or periodontitis (Hasturk et al., 2007), RvE1 may be beneficial not only in resolving local inflammation but also as a novel bone-preserving factor.
Figure 6. Proposed transcellular biosynthetic pathway of resolvin E1 (RvE1) generation by osteoclast (OC) and neutrophils. Acetylsalicylic acid (ASA) acetylates COX-2, which in turn converts eicosapentaenoic acid (EPA) into 18R-hydroxy-eicosapentaenoic acid (18-HEPE), the first metabolic intermediate of RvE1. 18-HEPE is further oxygenated by activated leukocytes through 5-lipoxygenase and subsequent reactions resulting in bioactive RvE1. In turn, the locally generated RvE1 may act on OC to inhibit OC growth, as well as on leukocytes to mediate resolution of inflammation.
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