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.     

The Cytoskeletal Protein ��-Actinin Regulates Acid-sensing Ion Channel 1a through a C-terminal Interaction

The acid-sensing ion channel 1a (ASIC1a) is widely expressed in central and peripheral neurons where it generates transient cation currents when extracellular pH falls. ASIC1a confers pH-dependent modulation on postsynaptic dendritic spines and has critical effects in neurological diseases associated with a reduced pH. However, knowledge of the proteins that interact with ASIC1a and influence its function is limited. Here, we show that α-actinin, which links membrane proteins to the actin cytoskeleton, associates with ASIC1a in brain and in cultured cells. The interaction depended on an α-actinin-binding site in the ASIC1a C terminus that was specific for ASIC1a versus other ASICs and for α-actinin-1 and -4. Co-expressing α-actinin-4 altered ASIC1a current density, pH sensitivity, desensitization rate, and recovery from desensitization. Moreover, reducing α-actinin expression altered acid-activated currents in hippocampal neurons. These findings suggest that α-actinins may link ASIC1a to a macromolecular complex in the postsynaptic membrane where it regulates ASIC1a activity.Acid-sensing ion channels (ASICs)² are H⁺-gated members of the DEG/ENaC family (1–3). Members of this family contain cytosolic N and C termini, two transmembrane domains, and a large cysteine-rich extracellular domain. ASIC subunits combine as homo- or heterotrimers to form cation channels that are widely expressed in the central and peripheral nervous systems (1–4). In mammals, four genes encode ASICs, and two subunits, ASIC1 and ASIC2, have two splice forms, a and b. Central nervous system neurons express ASIC1a, ASIC2a, and ASIC2b (5–7). Homomeric ASIC1a channels are activated when extracellular pH drops below 7.2, and half-maximal activation occurs at pH 6.5–6.8 (8–10). These channels desensitize in the continued presence of a low extracellular pH, and they can conduct Ca²⁺ (9, 11–13). ASIC1a is required for acid-evoked currents in central nervous system neurons; disrupting the gene encoding ASIC1a eliminates H⁺-gated currents unless extracellular pH is reduced below pH 5.0 (5, 7).Previous studies found ASIC1a enriched in synaptosomal membrane fractions and present in dendritic spines, the site of excitatory synapses (5, 14, 15). Consistent with this localization, ASIC1a null mice manifested deficits in hippocampal long term potentiation, learning, and memory, which suggested that ASIC1a is required for normal synaptic plasticity (5, 16). ASICs might be activated during neurotransmission when synaptic vesicles empty their acidic contents into the synaptic cleft or when neuronal activity lowers extracellular pH (17–19). Ion channels, including those at the synapse often interact with multiple proteins in a macromolecular complex that incorporates regulators of their function (20, 21). For ASIC1a, only a few interacting proteins have been identified. Earlier work indicated that ASIC1a interacts with another postsynaptic scaffolding protein, PICK1 (15, 22, 23). ASIC1a also has been reported to interact with annexin II light chain p11 through its cytosolic N terminus to increase cell surface expression (24) and with Ca²⁺/calmodulin-dependent protein kinase II to phosphorylate the channel (25). However, whether ASIC1a interacts with additional proteins and with the cytoskeleton remain unknown. Moreover, it is not known whether such interactions alter ASIC1a function.In analyzing the ASIC1a amino acid sequence, we identified cytosolic residues that might bind α-actinins. α-Actinins cluster membrane proteins and signaling molecules into macromolecular complexes and link membrane proteins to the actincytoskeleton (for review, Ref. 26). Four genes encode α-actinin-1, -2, -3, and -4 isoforms. α-Actinins contain an N-terminal head domain that binds F-actin, a C-terminal region containing two EF-hand motifs, and a central rod domain containing four spectrin-like motifs (26–28). The C-terminal portion of the rod segment appears to be crucial for binding to membrane proteins. The α-actinins assemble into antiparallel homodimers through interactions in their rod domain. α-Actinins-1, -2, and -4 are enriched in dendritic spines, concentrating at the postsynaptic membrane (29–35). In the postsynaptic membrane of excitatory synapses, α-actinin connects the NMDA receptor to the actin cytoskeleton, and this interaction is key for Ca²⁺-dependent inhibition of NMDA receptors (36–38). α-Actinins can also regulate the membrane trafficking and function of several cation channels, including L-type Ca²⁺ channels, K⁺ channels, and TRP channels (39–41).To better understand the function of ASIC1a channels in macromolecular complexes, we asked if ASIC1a associates with α-actinins. We were interested in the α-actinins because they and ASIC1a, both, are present in dendritic spines, ASIC1a contains a potential α-actinin binding sequence, and the related epithelial Na⁺ channel (ENaC) interacts with the cytoskeleton (42, 43). Therefore, we hypothesized that α-actinin interacts structurally and functionally with ASIC1a.