Molecular Mechanism of the Bifunctional Role of Lipopolysaccharide in Osteoclastogenesis

Lipopolysaccharide (LPS), a common bacteria-derived product, has long been recognized as a key factor implicated in periodontal bone loss. However, the precise cellular and molecular mechanisms by which LPS induces bone loss still remains controversial. Here, we show that LPS inhibited osteoclastogenesis from freshly isolated osteoclast precursors but stimulated osteoclast formation from those pretreated with RANKL in vitro in tissue culture dishes, bone slices, and a co-culture system containing osteoblasts, indicating that RANKL-mediated lineage commitment is a prerequisite for LPS-induced osteoclastogenesis. Moreover, the RANKL-mediated lineage commitment is long term, irreversible, and TLR4-dependent. LPS exerts the dual function primarily by modulating the expression of NFATc1, a master regulator of osteoclastogenesis, in that it abolished RANKL-induced NFATc1 expression in freshly isolated osteoclast precursors but stimulated its expression in RANKL-pretreated cells. In addition, LPS prolonged osteoclast survival by activating the Akt, NF-κB, and ERK pathways. Our current work has not only unambiguously defined the role of LPS in osteoclastogenesis but also has elucidated the molecular mechanism underlying its complex functions in osteoclast formation and survival, thus laying a foundation for future delineation of the precise mechanism of periodontal bone loss.LPS,² a common bacteria-derived product, has long been recognized as a key factor implicated in the development of chronic periodontitis. LPS plays an important role in periodontitis by initiating a local host response in gingival tissues that involves recruitment of inflammatory cells, production of prostanoids and cytokines, elaboration of lytic enzymes and activation of osteoclast formation and function to induce bone loss (1-3).Osteoclasts, the body''s sole bone-resorbing cells, are multinucleated giant cells that differentiate from cells of hematopoietic lineage upon stimulation by two critical factors: the macrophage/monocyte colony-forming factor (M-CSF) and the receptor activator of NF-κB ligand (RANKL) (4-6). RANKL exerts its effects on osteoclast formation and function by binding to its receptor, RANK (receptor activator of NF-κB) expressed on osteoclast precursors and mature osteoclasts (7-9). RANKL also has a decoy receptor, osteoprotegerin, which inhibits RANKL action by competing with RANK for binding RANKL (10, 11).RANK is a member of the tumor necrosis factor receptor (TNFR) family (12). Members of the TNFR family lack intrinsic enzymatic activity, and hence they transduce intracellular signals by recruiting various adaptor proteins including TNF receptor-associated factors (TRAFs) through specific motifs in the cytoplasmic domain (13, 14). It has been established that RANK contains three functional TRAF-binding sites (³⁶⁹PFQEP³⁷³, ⁵⁵⁹PVQEET⁵⁶⁴, and ⁶⁰⁴PVQEQG⁶⁰⁹) that, redundantly, play a role in osteoclast formation and function (15, 16). Collectively, through these functional TRAF-binding motifs, RANK activates six major signaling pathways, NF-κB, JNK, ERK, p38, NFATc1, and Akt, which play important roles in osteoclast formation, function, and/or survival (15, 17-19). In particular, NFATc1 has been established as a master regulator of osteoclast differentiation (20-22).The involvement of osteoclasts in the pathogenesis of periodontal bone loss is supported by observations that osteoclasts are physically present and functionally involved in bone resorption in periodontal tissues (23-27). RANKL and RANK knockout mice develop osteopetrosis and show failure in tooth eruption due to a lack of osteoclasts (24, 25, 28). Moreover, op/op mice, in which a mutation in the coding region of the M-CSF gene generates a stop codon that leads to premature termination of translation of M-CSF mRNA, also show osteopetrosis and failure in tooth eruption due to a defect in osteoclast development (26, 27).Whereas the role of osteoclasts in periodontal disease associated alveolar bone destruction has been well established, the precise role of LPS in osteoclastogenesis still remains controversial. The vast majority of the previous studies demonstrated that LPS stimulates osteoclastogenesis. This is consistent with the role that LPS, a well recognized pathogenic factor in periodontitis, presumably plays in periodontal bone loss (29-33). However, two previous studies demonstrated, surprisingly, that LPS plays bifunctional roles in osteoclastogenesis in that although it inhibits osteoclast formation from normal osteoclast precursors, it reverses to promote osteoclastogenesis from osteoclast precursors pretreated with RANKL (34, 35). Given that this finding is inconsistent with the presumed role of LPS as a pathogenic factor in periodontal bone loss and lacks careful and further validation, the prevalent view is still that LPS stimulates osteoclastogenesis (1-3). Importantly, if LPS indeed has a dual function in osteoclastogenesis, the molecular mechanism by which LPS exerts a dual effect on osteoclastogenesis need to be further elucidated.In the present work, using various in vitro assays, we have demonstrated independently that LPS inhibits osteoclastogenesis from normal osteoclast precursors but promotes the development of osteoclasts from RANKL-pretreated cells in tissue culture dishes and bone slices in single-cell and co-culture settings, confirming the two previous observations that LPS play a bifunctional role in osteoclastogenesis (34, 35). Moreover, we have further shown that the RANKL-mediated lineage commitment is long term and irreversible in LPS-mediated osteoclastogenesis. More importantly, we have revealed that LPS inhibits osteoclastogenesis by suppressing NFATc1 expression and JNK activation while it prolongs osteoclast survival by activating the Akt, NF-κB, and ERK pathways. These studies have not only unambiguously and precisely defined the role of LPS in osteoclastogenesis but, more importantly, may also lead to a paradigm shift in future investigation of the molecular mechanism of periodontal bone loss.     

Impact of riding in a coercively obtained Rollkur posture on welfare and fear of performance horses

Rollkur, the usually coercively obtained hyperflexion of the horse's neck, is employed as a training method by some dressage riders; however, its use is controversial as it may cause discomfort and adversely affect the horse's welfare. The objectives of this study were to determine: (1) if horses showed differences in stress, discomfort and fear responses as measured by heart rate and behaviour when ridden in Rollkur (R) obtained by pressure on the reins compared to regular poll flexion (i.e. with the nose-line being at or just in front of the vertical; N), and (2) if they showed a preference between the two riding styles when given the choice. Fifteen riding horses were ridden 30 times through a Y-maze randomly alternating between sides. Riding through one arm of the Y-maze was always followed by a short round ridden in R, whereas riding through the other arm was followed by a short round ridden in N. Immediately after the conditioning phase, horses were again repeatedly ridden into the maze; however, riders left it to the horse to decide which arm of the maze to enter. During R, horses moved slower and showed more often behavioural signs of discomfort, such as tail-swishing, head-tossing or attempted bucks (P < 0.05), and 14 of the 15 horses chose significantly (P < 0.05) more often the maze-arm associated with N rather than R. Subsequently, eight of the horses were also subjected to two fear tests following a short ride in N as well as a ride in R. During R, horses tended to react stronger (P = 0.092) to the fear stimuli and to take longer (P = 0.087) to approach them. These findings indicate that a coercively obtained Rollkur position may be uncomfortable for horses and that it makes them more fearful and therefore potentially more dangerous to ride. Further studies need to assess horses’ reaction to gradual training of Rollkur, as opposed to a coercively obtained hyperflexion, in order to decide whether the practice should be banned.     

Role of mitogen-activated protein kinases in Thy-1-induced T-lymphocyte activation

Thy-1 (CD90) crosslinking by monoclonal antibodies (mAb) in the context of costimulation causes the activation of mouse T-lymphocytes; however, the associated signal transduction processes have not been studied in detail. In this study we investigated the role of mitogen-activated protein kinases (MAPKs) in Thy-1-mediated T-lymphocyte activation using mAb-coated polystyrene microspheres to crosslink Thy-1 and costimulatory CD28 on murine T-lymphocytes. Concurrent Thy-1 and CD28 crosslinking induced DNA synthesis by T-lymphocytes, as well as interleukin (IL)-2 and IL-2 receptor (IL-2R) α chain (CD25) expression. Increased phosphorylation of extracellular signal-regulated kinase (ERK) 1/2, p38 MAPK, and c-Jun N-terminal protein kinase (JNK) was also observed. Pharmacologic inhibition of ERK1/2 or JNK activation inhibited Thy-1-induced DNA synthesis and IL-2 production by T-lymphocytes. p38 MAPK inhibition also decreased DNA synthesis in Thy-1-stimulated T-lymphocytes; however, IL-2 production was increased in these cells. Inhibition of JNK, but not ERK1/2 or p38 MAPK, caused a marked reduction in Thy-1-induced CD25 expression. In addition, inhibition of p38 MAPK or JNK, but not ERK1/2, impaired the growth of IL-2-dependent CTLL-2 T-lymphocytes but did not substantially affect CD25 expression. Finally, exogenous IL-2 reversed the inhibitory effect of ERK1/2 or JNK inhibition on Thy-1-stimulated DNA synthesis by T-lymphocytes but did not substantially reverse JNK inhibition of CD25 expression. Collectively, these results suggest that during Thy-1-induced T-lymphocyte activation, ERK1/2 and JNK promoted IL-2 production whereas p38 MAPK negatively regulated IL-2 expression. JNK signalling was also required for CD25 expression. IL-2R signalling involved both p38 MAPK and JNK in CTLL-2 cells, whereas p38 MAPK was most important for IL-2R signalling in primary T-lymphocytes. MAPKs are therefore essential signalling intermediates for the Thy-1-driven proliferation of mouse T-lymphocytes.     

SMK-1/PPH-4.1–mediated silencing of the CHK-1 response to DNA damage in early C. elegans embryos

Yb regulates the proliferation of both germline and somatic stem cells in the Drosophila melanogaster ovary by activating piwi and hh expression in niche cells. In this study, we show that Yb protein is localized as discrete cytoplasmic spots exclusively in the somatic cells of the ovary and testis. These spots, which are different from all known cytoplasmic structures in D. melanogaster, are evenly electron-dense spheres 1.5 µm in diameter (herein termed the Yb body). The Yb body is frequently associated with mitochondria and a less electron-dense sphere of similar size that appears to be RNA rich. There are one to two Yb bodies/cell, often located close to germline cells. The N-terminal region of Yb is required for hh expression in niche cells, whereas the C-terminal region is required for localization to Yb bodies. The entire Yb protein is necessary for piwi expression in niche cells. A double mutant of Yb and a novel locus show male germline loss, revealing a function for Yb in male germline stem cell maintenance.     

A Self-Scaffolding Model for G Protein Signaling

Activation of heterotrimeric G proteins is generally believed to induce dissociation of Gα and Gβγ subunits, which are then free to bind to and change the catalytic activity of a variety of intracellular enzymes. We have previously found that in cells, Gαq subunits remain complexed with its major effector, phospholipase Cβ1, through the activation cycle. To determine whether this behavior may be operative in other systems, we carried out Förster resonance energy transfer studies and found that eYFP-Gαi and eCFP-Gβγ remain associated after stimulation in HEK293 cells. We also found that the level of Forster resonance energy transfer between Alexa546-phospholipase Cβ2 and eGFP-Gβγ is significant and unchanged upon activation in HEK293 cells, thus showing that these proteins can localize into stable signaling complexes. To understand the basis for this stabilization, we carried out in vitro studies using a series of single-Cys mutants labeled with fluorescence tags and monitored their interaction with Gβγ subunits and changes in their fluorescence properties and accessibility upon activation and Gβγ binding. Our studies suggest a significant change in the orientation between G protein subunits upon activation that allows the G proteins to remain complexed while activating effectors.     

Plasma membrane glutathione transporters and their roles in cell physiology and pathophysiology

Reduced glutathione (GSH) is critical for many cellular processes, and both its intracellular and extracellular concentrations are tightly regulated. Intracellular GSH levels are regulated by two main mechanisms: by adjusting the rates of synthesis and of export from cells. Some of the proteins responsible for GSH export from mammalian cells have recently been identified, and there is increasing evidence that these GSH exporters are multispecific and multifunctional, regulating a number of key biological processes. In particular, some of the multidrug resistance-associated proteins (Mrp/Abcc) appear to mediate GSH export and homeostasis. The Mrp proteins mediate not only GSH efflux, but they also export oxidized glutathione derivatives (e.g., glutathione disulfide (GSSG), S-nitrosoglutathione (GS-NO), and glutathione-metal complexes), as well as other glutathione S-conjugates. The ability to export both GSH and oxidized derivatives of GSH, endows these transporters with the capacity to directly regulate the cellular thiol-redox status, and therefore the ability to influence many key signaling and biochemical pathways. Among the many processes that are influenced by the GSH transporters are apoptosis, cell proliferation, and cell differentiation. This report summarizes the evidence that Mrps contribute to the regulation of cellular GSH levels and the thiol-redox state, and thus to the many biochemical processes that are influenced by this tripeptide.     

Proliferation of Ty3/gypsy-like retrotransposons in hybrid sunflower taxa inferred from phylogenetic data

Background  

Long terminal repeat (LTR) retrotransposons are a class of mobile genetic element capable of autonomous transposition via an RNA intermediate. Their large size and proliferative ability make them important contributors to genome size evolution, especially in plants, where they can reach exceptionally high copy numbers and contribute substantially to variation in genome size even among closely related taxa. Using a phylogenetic approach, we characterize dynamics of proliferation events of Ty3/gypsy-like LTR retrotransposons that led to massive genomic expansion in three Helianthus (sunflower) species of ancient hybrid origin. The three hybrid species are independently derived from the same two parental species, offering a unique opportunity to explore patterns of retrotransposon proliferation in light of reticulate evolutionary events in this species group.     

Role of mycorrhizal networks and tree proximity in ectomycorrhizal colonization of planted seedlings

The importance of mycorrhizal network (MN)-mediated colonization under field conditions between trees and seedlings was investigated. We also determined the combined influences of inoculum source and distance from trees on the ectomycorrhizal (EM) community of seedlings. On six sites, we established trenched plots around 24 residual Pseudotsuga menziesii var. glauca trees and then planted seedlings at four distances (0.5, 1.0, 2.5, and 5.0 m) from the tree into four mesh treatments that served to restrict inoculum access (i.e., planted into mesh bags with 0.5, 35, 250 μm pores or directly into soil). Ectomycorrhizal communities were identified after two growing seasons using morphological and molecular techniques. Mesh treatments had no effect on seedling mycorrhizal colonization, richness, or diversity, suggesting that MN-mediated colonization, was not an essential mechanism by which EM communities were perpetuated to seedlings. Instead, wind-borne and soil inoculum played an important role in seedling colonization. The potential for MNs to form in these forests was not dismissed, however, because trees and seedlings shared 83 % of the abundant EM. Seedlings furthest from trees had a simpler EM community composition and reduced EM richness and diversity compared to seedlings in closer proximity.