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Abstract Background Tau protein forms neurofibrillary tangles and becomes deposited in the brain during Alzheimer’s disease (AD). Tau oligomers are the most reactive species, mediating neurotoxic and inflammatory activity. Microglia are the immune cells in the central nervous system, sense the extracellular Tau via various cell surface receptors. Purinergic P2Y12 receptor can directly interact with Tau oligomers and mediates microglial chemotaxis via actin remodeling. The disease-associated microglia are associated with impaired migration and express a reduced level of P2Y12, but elevate the level of reactive oxygen species and pro-inflammatory cytokines. Results Here, we studied the formation and organization of various actin microstructures such as-podosome, filopodia and uropod in colocalization with actin nucleator protein Arp2 and scaffold protein TKS5 in Tau-induced microglia by fluorescence microscopy. Further, the relevance of P2Y12 signaling either by activation or blockage was studied in terms of actin structure formations and Tau deposits degradation by N9 microglia. Extracellular Tau oligomers facilitate the microglial migration via Arp2-associated podosome and filopodia formation through the involvement of P2Y12 signaling. Similarly, Tau oligomers induce the TKS5-associated podosome clustering in microglial lamella in a time-dependent manner. Moreover, the P2Y12 was evidenced to localize with F-actin-rich podosome and filopodia during Tau-deposit degradation. The blockage of P2Y12 signaling resulted in decreased microglial migration and Tau-deposit degradation. Conclusions The P2Y12 signaling mediate the formation of migratory actin structures like- podosome and filopodia to exhibit chemotaxis and degrade Tau deposit. These beneficial roles of P2Y12 in microglial chemotaxis, actin network remodeling and Tau clearance can be intervened as a therapeutic target in AD.

Mesenchymal migration usually happens on adhesive substrates, while cells adopt amoeboid migration on low/nonadhesive surfaces. Protein‐repelling reagents, e.g., poly(ethylene) glycol (PEG), are routinely employed to resist cell adhering and migrating. Contrary to these perceptions, this work discovers a unique locomotion of macrophages on adhesive–nonadhesive alternate substrates in vitro that they can overcome nonadhesive PEG gaps to reach adhesive regions in the mesenchymal mode. Adhering to extracellular matrix regions is a prerequisite for macrophages to perform further locomotion on the PEG regions. Podosomes are found highly enriched on the PEG region in macrophages and support their migration across the nonadhesive regions. Increasing podosome density through myosin IIA inhibition facilitates cell motility on adhesive–nonadhesive alternate substrates. Moreover, a developed cellular Potts model reproduces this mesenchymal migration. These findings together uncover a new migratory behavior on adhesive–nonadhesive alternate substrates in macrophages. [ABSTRACT FROM AUTHOR]

During immune responses against invading pathogenic bacteria, the cytoskeleton network enables macrophages to implement multiple essential functions. To protect the host from infection, macrophages initially polarize to adopt different phenotypes in response to distinct signals from the microenvironment. The extracellular stimulus regulates the rearrangement of the cytoskeleton, thereby altering the morphology and migratory properties of macrophages. Subsequently, macrophages degrade the extracellular matrix (ECM) and migrate toward the sites of infection to directly contact invading pathogens, during which the involvement of cytoskeleton‐based structures such as podosomes and lamellipodia is indispensable. Ultimately, macrophages execute the function of phagocytosis to engulf and eliminate the invading pathogens. Phagocytosis is a complex process that requires the cooperation of cytoskeleton‐enriched super‐structures, such as filopodia, lamellipodia, and phagocytic cup. This review presents an overview of cytoskeletal regulations in macrophage polarization, ECM degradation, migration, and phagocytosis, highlighting the pivotal role of the cytoskeleton in host defense against infection. [ABSTRACT FROM AUTHOR]

Abstract Mesenchymal migration usually happens on adhesive substrates, while cells adopt amoeboid migration on low/nonadhesive surfaces. Protein‐repelling reagents, e.g., poly(ethylene) glycol (PEG), are routinely employed to resist cell adhering and migrating. Contrary to these perceptions, this work discovers a unique locomotion of macrophages on adhesive–nonadhesive alternate substrates in vitro that they can overcome nonadhesive PEG gaps to reach adhesive regions in the mesenchymal mode. Adhering to extracellular matrix regions is a prerequisite for macrophages to perform further locomotion on the PEG regions. Podosomes are found highly enriched on the PEG region in macrophages and support their migration across the nonadhesive regions. Increasing podosome density through myosin IIA inhibition facilitates cell motility on adhesive–nonadhesive alternate substrates. Moreover, a developed cellular Potts model reproduces this mesenchymal migration. These findings together uncover a new migratory behavior on adhesive–nonadhesive alternate substrates in macrophages.

Background: Tau protein forms neurofibrillary tangles and becomes deposited in the brain during Alzheimer's disease (AD). Tau oligomers are the most reactive species, mediating neurotoxic and inflammatory activity. Microglia are the immune cells in the central nervous system, sense the extracellular Tau via various cell surface receptors. Purinergic P2Y12 receptor can directly interact with Tau oligomers and mediates microglial chemotaxis via actin remodeling. The disease-associated microglia are associated with impaired migration and express a reduced level of P2Y12, but elevate the level of reactive oxygen species and pro-inflammatory cytokines. Results: Here, we studied the formation and organization of various actin microstructures such as-podosome, filopodia and uropod in colocalization with actin nucleator protein Arp2 and scaffold protein TKS5 in Tau-induced microglia by fluorescence microscopy. Further, the relevance of P2Y12 signaling either by activation or blockage was studied in terms of actin structure formations and Tau deposits degradation by N9 microglia. Extracellular Tau oligomers facilitate the microglial migration via Arp2-associated podosome and filopodia formation through the involvement of P2Y12 signaling. Similarly, Tau oligomers induce the TKS5-associated podosome clustering in microglial lamella in a time-dependent manner. Moreover, the P2Y12 was evidenced to localize with F-actin-rich podosome and filopodia during Tau-deposit degradation. The blockage of P2Y12 signaling resulted in decreased microglial migration and Tau-deposit degradation. Conclusions: The P2Y12 signaling mediate the formation of migratory actin structures like- podosome and filopodia to exhibit chemotaxis and degrade Tau deposit. These beneficial roles of P2Y12 in microglial chemotaxis, actin network remodeling and Tau clearance can be intervened as a therapeutic target in AD. [ABSTRACT FROM AUTHOR]

Introduction: The actin cytoskeleton remodels to enable diverse processes essential to immunity, such as cell adhesion, migration and phagocytosis. A panoply of actin-binding proteins regulate these rapid rearrangements to induce actin-based shape changes and to generate force. L-plastin (LPL) is a leukocyte-specific, actin-bundling protein that is regulated in part by phosphorylation of the Ser-5 residue. LPL deficiency in macrophages impairs motility, but not phagocytosis; we recently found that expression of LPL in which the S5 residue is converted to a non-phosphorylatable alanine (S5A-LPL) resulted in diminished phagocytosis, but unimpaired motility.Methods: To provide mechanistic insight into these findings, we now compare the formation of podosomes (an adhesive structure) and phagosomes in alveolar macrophages derived from wild-type (WT), LPL-deficient, or S5A-LPL mice. Both podosomes and phagosomes require rapid remodeling of actin, and both are force-transmitting. Actin rearrangement, force generation, and signaling rely upon recruitment of many actin-binding proteins, including the adaptor protein vinculin and the integrin-associated kinase Pyk2. Prior work suggested that vinculin localization to podosomes was independent of LPL, while Pyk2 was displaced by LPL deficiency. We therefore chose to compare vinculin and Pyk2 co-localization with F-actin at sites of adhesion of phagocytosis in AMs derived from WT, S5A-LPL or LPL−/− mice, using Airyscan confocal microscopy.Results: As described previously, podosome stability was significantly disrupted by LPL deficiency. In contrast, LPL was dispensable for phagocytosis and was not recruited to phagosomes. Recruitment of vinculin to sites of phagocytosis was significantly enhanced in cells lacking LPL. Expression of S5A-LPL impeded phagocytosis, with reduced appearance of ingested bacteria-vinculin aggregates.Discussion: Our systematic analysis of the regulation of LPL during podosome vs. phagosome formation illuminates essential remodeling of actin during key immune processes.

Podosomes are mechanosensitive protrusive actin structures that are prominent in myeloid cells, and they have been linked to vascular extravasation. Recent studies have suggested that podosomes are hierarchically organized and have coordinated dynamics on the cell scale, which implies that the local force generation by single podosomes can be different from their global combined action. Complementary to previous studies focusing on individual podosomes, here we investigated the cell-wide force generation of podosome-bearing ER-Hoxb8 monocytes. We found that the occurrence of focal tractions accompanied by a cell-wide substrate indentation cannot be explained by summing the forces of single podosomes. Instead, our findings suggest that superimposed contraction on the cell scale gives rise to a buckling mechanism that can explain the measured cell-scale indentation. Specifically, the actomyosin network contraction causes peripheral in-plane substrate tractions, while the accumulated internal stress results in out-ofplane deformation in the central cell region via a buckling instability, producing the cell-scale indentation. Hence, we propose that contraction of the actomyosin network, which connects the podosomes, leads to a substrate indentation that acts in addition to the protrusion forces of individual podosomes. [ABSTRACT FROM AUTHOR]

Osteoclasts form special integrin-mediated adhesion structures called sealing zones that enable them to adhere to and resorb bone. Sealing zones consist of densely packed podosomes tightly interconnected by actin fibers. Their formation requires the presence of the hematopoietic integrin regulator kindlin-3 (also known as Fermt3). In this study, we investigated osteoclasts and their adhesion structures in kindlin-3 hypomorphic mice expressing only 5-10% of the kindlin-3 level of wild-type mice. Low kindlin-3 expression reduces integrin activity, results in impaired osteoclast adhesion and signaling, and delays cell spreading. Despite these defects, in vitro-generated kindlin-3-hypomorphic osteoclast-like cells arrange their podosomes into adhesion patches and belts, but their podosome and actin organization is abnormal. Remarkably, kindlin-3-hypomorphic osteoclasts form sealing zones when cultured on calcified matrix in vitro and on bone surface in vivo. However, functional assays, immunohistochemical staining and electron micrographs of bone sections showed that they fail to seal the resorption lacunae properly, which is required for secreted proteinases to digest bone matrix. This results in mild osteopetrosis. Our study reveals a new, hitherto understudied function of kindlin-3 as an essential organizer of integrinmediated adhesion structures, such as sealing zones. [ABSTRACT FROM AUTHOR]

Abstract Background Podosomes are membrane‐bound adhesive structures formed by actin remodeling. They are capable of extracellular matrix (ECM) degradation, which is a prerequisite for cancer cell invasion and metastasis. The signaling mechanism of podosome formation is still unknown in cancer. We previously reported that Nck adaptors regulate directional cell migration and endothelial lumen formation by actin remodeling, while deficiency of Nck reduces cancer metastasis. This study evaluated the role of Nck adaptors in podosome biogenesis and cancer invasion. Methods This study was conducted in vitro using both healthy cells (Human Umbilical Vein Endothelial Cell, 3T3 fibroblasts) and cancer cells (prostate cancer cell line; PC3, breast cancer cell line; MDA‐MB‐231). Confocal and TIRF imaging of cells expressing Green Fluorescence Protein (GFP) mutant under altered levels of Nck or downstream of kinase 1 (Dok1) was used to evaluate the podosome formation and fluorescent gelatin matrix degradation. Levels of Nck in human breast carcinoma tissue sections were detected by immune histochemistry using Nck polyclonal antibody. Biochemical interaction of Nck/Dok1 was detected in podosome forming cells using immune precipitation and far‐western blotting. Results This study demonstrates that ectopic expression of Nck1 and Nck2 can induce the endothelial podosome formation in vitro. Nck silencing by short‐hairpin RNA blocked podosome biogenesis and ECM degradation in cSrc‐Y530F transformed endothelial cells in this study. Immunohistochemical analysis revealed the Nck overexpression in human breast carcinoma tissue sections. Immunoprecipitation and far‐western blotting revealed the biochemical interaction of Nck/p62Dok in podosome forming cells. Conclusions Nck adaptors in interaction with Dok1 induce podosome biogenesis and ECM degradation facilitating cancer cell invasion, and therefore a bona fide target of cancer therapy.

Podosomes are actin-rich adhesion structures formed in a variety of cell types, such as monocytic cells or cancer cells, to facilitate attachment to and degradation of the extracellular matrix (ECM). Previous studies showed that dynamin 2, a large GTPase involved in membrane remodeling and actin organization, is required for podosome function. However, precise roles of dynamin 2 at the podosomes remain to be elucidated. In this study, we identified a BAR (Bin-Amphiphysin-Rvs167) domain protein pacsin 2 as a functional partner of dynamin 2 at podosomes. Dynamin 2 and pacsin 2 interact and co-localize to podosomes in Src-transformed NIH 3T3 (NIH-Src) cells. RNAi of either dynamin 2 or pacsin 2 in NIH-Src cells inhibited podosome formation and maturation, suggesting essential and related roles at podosomes. Consistently, RNAi of pacsin 2 prevented dynamin 2 localization to podosomes, and reciprocal RNAi of dynamin 2 prevented pacsin 2 localization to podosomes. Taking these results together, we conclude that dynamin 2 and pacsin 2 co-operatively regulate organization of podosomes in NIH-Src cells. • Podosomes are actin-rich structures used for degradation of extracellular matrix. • Dynamin 2 was implicated in podosome formation and function in various cells. • Pacsin 2 interacts with dynamin 2 and they co-localize to the podosomes. • Pacsin 2 and dynamin 2 cooperatively regulate formation/maturation of podosomes. [ABSTRACT FROM AUTHOR]

Phagocytosis consists in ingestion and digestion of large particles, a process strictly dependent on actin re-organization. Using synchronized phagocytosis of IgG-coated latex beads (IgG-LB), zymosan or serum opsonized-zymosan, we report the formation of actin structures on both phagocytic cups and closed phagosomes in human macrophages. Their lifespan, size, protein composition and organization are similar to podosomes. Thus, we called these actin structures phagosome-associated podosomes (PAPs). Concomitantly to the formation of PAPs, a transient disruption of podosomes occurred at the ventral face of macrophages. Similarly to podosomes, which are targeted by vesicles containing proteases, the presence of PAPs correlated with the maturation of phagosomes into phagolysosomes. The ingestion of LB without IgG did not trigger PAPs formation, did not lead to podosome disruption and maturation to phagolysosomes, suggesting that these events are linked together. Although similar to podosomes, we found that PAPs differed by being resistant to the Arp2/3 inhibitor CK666. Thus, we describe a podosome subtype which forms on phagosomes where it probably serves several tasks of this multifunctional structure.

Macrophages are one of the major targets of human immunodeficiency virus 1 (HIV-1) and play crucial roles in viral dissemination and persistence during AIDS progression. Here, we reveal the dynamic podosome-mediated entry of HIV-1 into macrophages. Inhibition of podosomes prevented HIV-1 entry into macrophages, while stimulation of podosome formation promoted viral entry. Single-virus tracking revealed the temporal and spatial mechanism of the dynamic podosomemediated viral entry process. The core and ring structures of podosomes played complex roles in viral entry. The HIV coreceptor CCR5 was recruited to form specific clusters at the podosome ring, where it participated in viral entry. The podosome facilitated HIV-1 entry with a rotation mode triggered by dynamic actin. Our discovery of this novel HIV-1 entry route into macrophages, mediated by podosomes critical for cell migration and tissue infiltration, provides a new view of HIV infection and pathogenesis, which may assist in the development of new antiviral strategies. [ABSTRACT FROM AUTHOR]

Osteoclasts are hematopoietic cells attached to the bones containing type I collagen-deposited hydroxyapatite during bone resorption. Two major elements determine the stiffness of bones: regular calcified bone (bone that is resorbable by osteoclasts) and un-calcified osteoid bone (bone that is un-resorbable by osteoclasts). The osteolytic cytokine RANKL promotes osteoclast differentiation; however, the roles of the physical interactions of osteoclasts with calcified and un-calcified bone at the sealing zones and the subsequent cellular signaling remain unclear. In this study, we investigated podosomes, actin-rich adhesion structures (actin-ring) in the sealing zone that participates in sensing hard stiffness with collagen in the physical environment during osteoclast differentiation. RANKL-induced osteoclast differentiation induction was promoted when Raw264.7 cells were cultured on collagen-coated plastic dishes but not on non-coated plastic dishes, which was associated with the increased expression of podosome-related genes and Src. In contrast, when cells were cultured on collagen gel, expression of podosome-related genes and Src were not upregulated. The induction of podosome-related genes and Src requires hard stiffness with RGD-containing substratum and integrin-mediated F-actin polymerization. These results indicate that osteoclasts sense both the RGD sequence and stiffness of calcified collagen through their podosome components regulating osteoclast differentiation via the c-Src pathway. • Podosomes are actin-rich adhesion structures in the sealing zone of osteoclast. • Osteoclasts sense both the RGD and stiffness of calcified collagen by podosomes. • Osteoclast differentiation is associated with Src pathway in podosome. [ABSTRACT FROM AUTHOR]

Prostaglandin E2 (PGE2) is a lipid mediator that modulates the function of myeloid immune cells such as macrophages and dendritic cells (DCs) through the activation of the G protein-coupled receptors EP2 and EP4. While both EP2 and EP4 signaling leads to an elevation of intracellular cyclic adenosine monophosphate (cAMP) levels through the stimulating Gαs protein, EP4 also couples to the inhibitory Gαi protein to decrease the production of cAMP. The receptor-specific contributions to downstream immune modulatory functions are still poorly defined. Here, we employed quantitative imaging methods to characterize the early EP2 and EP4 signaling events in myeloid cells and their contribution to the dissolution of adhesion structures called podosomes, which is a first and essential step in DC maturation. We first show that podosome loss in DCs is primarily mediated by EP4. Next, we demonstrate that EP2 and EP4 signaling leads to distinct cAMP production profiles, with EP4 inducing a transient cAMP response and EP2 inducing a sustained cAMP response only at high PGE2 levels. We further find that simultaneous EP2 and EP4 stimulation attenuates cAMP production, suggesting a reciprocal control of EP2 and EP4 signaling. Finally, we demonstrate that efficient signaling of both EP2 and EP4 relies on an intact microtubule network. Together, these results enhance our understanding of early EP2 and EP4 signaling in myeloid cells. Considering that modulation of PGE2 signaling is regarded as an important therapeutic possibility in anti-tumor immunotherapy, our findings may facilitate the development of efficient and specific immune modulators of PGE2 receptors.

Migrating precursors of epidermal sensory neurons are a relatively rare cell type in developing amphioxus. Serial blockface scanning electron microscopy (SBSEM) was used here to locate these cells and reconstruct them in three dimensions in the context of the surrounding tissues. The reconstructions showed that, in contrast to some earlier reports, these cells are not located in an appreciable subepidermal space while they are crawling dorsally up the flank of the animal. Instead, they migrate while tightly sandwiched between overlying epidermal cells and the underlying basal lamina. The cytoplasmic region at the leading edge of the crawling cell has some characteristics of a large podosome, an F‐actin‐rich organelle with prominent roles in adhesion, matrix degradation and cell migration. The discussion includes a comparison of the precursors of epidermal sensory cells of amphioxus with migrating neurogenic cells from vertebrate placodes. [ABSTRACT FROM AUTHOR]

Prostaglandin E2 (PGE2) is a lipid mediator that modulates the function of myeloid immune cells such as macrophages and dendritic cells (DCs) through the activation of the G protein-coupled receptors EP2 and EP4. While both EP2 and EP4 signaling leads to an elevation of intracellular cyclic adenosine monophosphate (cAMP) levels through the stimulating Gαs protein, EP4 also couples to the inhibitory Gαi protein to decrease the production of cAMP. The receptor-specific contributions to downstream immune modulatory functions are still poorly defined. Here, we employed quantitative imaging methods to characterize the early EP2 and EP4 signaling events in myeloid cells and their contribution to the dissolution of adhesion structures called podosomes, which is a first and essential step in DC maturation. We first show that podosome loss in DCs is primarily mediated by EP4. Next, we demonstrate that EP2 and EP4 signaling leads to distinct cAMP production profiles, with EP4 inducing a transient cAMP response and EP2 inducing a sustained cAMP response only at high PGE2 levels. We further find that simultaneous EP2 and EP4 stimulation attenuates cAMP production, suggesting a reciprocal control of EP2 and EP4 signaling. Finally, we demonstrate that efficient signaling of both EP2 and EP4 relies on an intact microtubule network. Together, these results enhance our understanding of early EP2 and EP4 signaling in myeloid cells. Considering that modulation of PGE2 signaling is regarded as an important therapeutic possibility in anti-tumor immunotherapy, our findings may facilitate the development of efficient and specific immune modulators of PGE2 receptors. [ABSTRACT FROM AUTHOR]

• Re -epithelialization is a critical step in the skin wound healing response. • Keratinocytes deposit extracellular matrix laminin 332 along their path while migrating. • Matrix metalloproteinases-9 and −14 exert their proteolytic activity in epithelial podosomes in primary keratinocytes. • The laminin α3 chain globular domains 4 and 5 recruit and merge syndecan-1 and CD44 in podosomes. • The proteoglycan receptor syndecan-1 regulates epithelial podosome formation and proteolytic activity. Re -epithelialization describes the resurfacing of a skin wound with new epithelium. In response to various stimuli including that of growth factors, cytokines and extracellular matrix (ECM), wound edge epidermal keratinocytes undergo cytoskeleton rearrangements compatible with their motile behavior and develop protrusive adhesion contacts. Matrix metalloproteinases (MMP) expression is crucial for proper cell movement and ECM remodeling; however, their deposition mechanism is unknown in keratinocytes. Here, we show that similar to cytokine IL-1ß, the precursor laminin 332 pro-migratory fragment G45 induces expression of the MMP-9 pro-enzyme, which together with MMP-14, further exerts its proteolytic activity within epithelial podosomes. This event strictly depends on the expression of the proteoglycan receptor syndecan-1 that was found in a ring surrounding the podosome core, co-localised with CD44. Our findings uncover that by directly recruiting both syndecan-1 and CD44, the laminin-332 G45 domain plays a major role in regulating mechanisms underlying keratinocyte / ECM remodeling during wound repair. [ABSTRACT FROM AUTHOR]

At vertebrate neuromuscular junctions (NMJs), the synaptic basal lamina contains different extracellular matrix (ECM) proteins and synaptogenic factors that induce and maintain synaptic specializations. Here, we report that podosome-like structures (PLSs) induced by ubiquitous ECM proteins regulate the formation and remodeling of acetylcholine receptor (AChR) clusters via focal ECM degradation. Mechanistically, ECM degradation is mediated by PLS-directed trafficking and surface insertion of membrane-type 1 matrix metalloproteinase (MT1-MMP) to AChR clusters through microtubule-capturing mechanisms. Upon synaptic induction, MT1-MMP plays a crucial role in the recruitment of aneural AChR clusters for the assembly of postsynaptic specializations. Lastly, the structural defects of NMJs in embryonic MT1-MMP-/- mice further demonstrate the physiological role of MT1-MMP in normal NMJ development. Collectively, this study suggests that postsynaptic MT1-MMP serves as a molecular switch to synaptogenesis by modulating local ECM environment for the deposition of synaptogenic signals that regulate postsynaptic differentiation at developing NMJs.

Actin remodeling plays an essential role in diverse cellular processes such as cell motility, vesicle trafficking or cytokinesis. The scaffold protein and actin nucleation promoting factor Cortactin is present in virtually all actin-based structures, participating in the formation of branched actin networks. It has been involved in the control of endocytosis, and vesicle trafficking, axon guidance and organization, as well as adhesion, migration and invasion. To migrate and invade through three-dimensional environments, cells have developed specialized actin-based structures called invadosomes, a generic term to designate invadopodia and podosomes. Cortactin has emerged as a critical regulator of invadosome formation, function and disassembly. Underscoring this role, Cortactin is frequently overexpressed in several types of invasive cancers. Herein we will review the roles played by Cortactin in these specific invasive structures. [ABSTRACT FROM AUTHOR]

Podosomes and tight junctions (TJs) are subcellular compartments that both exist in endothelial cells and localize at cell surfaces. In contrast to the well‐characterized role of TJs in maintaining cerebrovascular integrity, the specific function of endothelial podosomes remains unknown. Intriguingly, we discovered cross‐talk between podosomes and TJs in human brain endothelial cells. Tight junction scaffold proteins ZO‐1 and ZO‐2 localize at podosomes in response to phorbol‐12‐myristate‐13‐acetate treatment. We found that both ZO proteins are essential for podosome formation and function. Rather than being derived from new protein synthesis, podosomal ZO‐1 and ZO‐2 are relocated from a pre‐existing pool found at the peripheral plasma membrane with enhanced physical interaction with cortactin, a known protein marker for podosomes. Sequestration of ZO proteins in podosomes weakens tight junction complex formation, leading to increased endothelial cell permeability. This effect can be further attenuated by podosome inhibitor PP2. Altogether, our data revealed a novel cellular function of podosomes, specifically, their ability to negatively regulate tight junction and endothelial barrier integrity, which have been linked to a variety of cerebrovascular diseases. [ABSTRACT FROM AUTHOR]

Podosome formation in osteoclasts is an important initial step in osteoclastic bone resorption. Mice lacking c‐Src (c‐Src−/−) exhibited osteopetrosis due to a lack of podosome formation in osteoclasts. We previously identified p130Cas (Crk‐associated substrate [Cas]) as one of c‐Src downstream molecule and osteoclast‐specific p130Cas‐deficient (p130CasΔOCL−/−) mice also exhibited a similar phenotype to c‐Src−/− mice, indicating that the c‐Src/p130Cas plays an important role for bone resorption by osteoclasts. In this study, we performed a cDNA microarray and compared the gene profiles of osteoclasts from c‐Src−/− or p130CasΔOCL−/− mice with wild‐type (WT) osteoclasts to identify downstream molecules of c‐Src/p130Cas involved in bone resorption. Among several genes that were commonly downregulated in both c‐Src−/− and p130CasΔOCL−/− osteoclasts, we identified kinesin family protein 1c (Kif1c), which regulates the cytoskeletal organization. Reduced Kif1c expression was observed in both c‐Src−/− and p130CasΔOCL−/− osteoclasts compared with WT osteoclasts. Kif1c exhibited a broad tissue distribution, including osteoclasts. Knockdown of Kif1c expression using shRNAs in WT osteoclasts suppressed actin ring formation. Kif1c overexpression restored bone resorption subsequent to actin ring formation in p130CasΔOCL−/− osteoclasts but not c‐Src−/− osteoclasts, suggesting that Kif1c regulates osteoclastic bone resorption in the downstream of p130Cas (191 words). Significance of the study: We previously showed that the c‐Src/p130Cas (Cas) plays an important role for bone resorption by osteoclasts. In this study, we identified kinesin family protein 1c (Kif1c), which regulates the cytoskeletal organization, as a downstream molecule of c‐Src/p130Cas axis, using cDNA microarray. Knockdown of Kif1c expression using shRNAs in wild‐type osteoclasts suppressed actin ring formation. Kif1c overexpression restored bone resorption subsequent to actin ring formation in osteoclast‐specific p130Cas‐deficient (p130CasΔOCL−/−) osteoclasts but not c‐Src−/− osteoclasts, suggesting that Kif1c regulates osteoclastic bone resorption in the downstream of p130Cas. [ABSTRACT FROM AUTHOR]

Podosomes are dynamic adhesion structures formed constitutively by macrophages, dendritic cells and osteoclasts and transiently in a wide variety of cells, such as endothelial cells and megakaryocytes. They mediate numerous functions, including cell-matrix adhesion, extracellular matrix degradation, mechanosensing and cell migration. Podosomes present as micron-sized F-actin cores surrounded by an adhesive ring of integrins and integrin-actin linkers, such as talin and vinculin. In this Review, we highlight recent research that has considerably advanced our understanding of the complex architecture-function relationship of podosomes by demonstrating that the podosome ring actually consists of discontinuous nanoclusters and that the actin network in between podosomes comprises two subsets of unbranched actin filaments, lateral and dorsal podosome-connecting filaments. These lateral and dorsal podosomeconnecting filaments connect the core and ring of individual podosomes and adjacent podosomes, respectively. We also highlight recent insights into the podosome cap as a novel regulatory module of actomyosin-based contractility. We propose that these newly identified features are instrumental for the ability of podosomes to generate protrusion forces and to mechanically probe their environment. Furthermore, these new results point to an increasing complexity of podosome architecture and have led to our current view of podosomes as autonomous force generators that drive cell migration. [ABSTRACT FROM AUTHOR]

Background: Podosomes are membrane‐bound adhesive structures formed by actin remodeling. They are capable of extracellular matrix (ECM) degradation, which is a prerequisite for cancer cell invasion and metastasis. The signaling mechanism of podosome formation is still unknown in cancer. We previously reported that Nck adaptors regulate directional cell migration and endothelial lumen formation by actin remodeling, while deficiency of Nck reduces cancer metastasis. This study evaluated the role of Nck adaptors in podosome biogenesis and cancer invasion. Methods: This study was conducted in vitro using both healthy cells (Human Umbilical Vein Endothelial Cell, 3T3 fibroblasts) and cancer cells (prostate cancer cell line; PC3, breast cancer cell line; MDA‐MB‐231). Confocal and TIRF imaging of cells expressing Green Fluorescence Protein (GFP)  mutant under altered levels of Nck or downstream of kinase 1 (Dok1) was used to evaluate the podosome formation and fluorescent gelatin matrix degradation. Levels of Nck in human breast carcinoma tissue sections were detected by immune histochemistry using Nck polyclonal antibody. Biochemical interaction of Nck/Dok1 was detected in podosome forming cells using immune precipitation and far‐western blotting. Results: This study demonstrates that ectopic expression of Nck1 and Nck2 can induce the endothelial podosome formation in vitro. Nck silencing by short‐hairpin RNA blocked podosome biogenesis and ECM degradation in cSrc‐Y530F transformed endothelial cells in this study. Immunohistochemical analysis revealed the Nck overexpression in human breast carcinoma tissue sections. Immunoprecipitation and far‐western blotting revealed the biochemical interaction of Nck/p62Dok in podosome forming cells. Conclusions: Nck adaptors in interaction with Dok1 induce podosome biogenesis and ECM degradation facilitating cancer cell invasion, and therefore a bona fide target of cancer therapy. [ABSTRACT FROM AUTHOR]

The aim of this study was to elucidate the role of the zipper-like structure (ZLS), a podosome-related structure that transiently appears at the cell contact zone, in osteoclast fusion. Live-cell imaging of osteoclasts derived from RAW264.7 cells transfected with EGFP-actin revealed consistent symmetrical retrograde actin flow in the ZLS, but not in the podosome cluster, the podosome ring or the podosome belt. Confocal imaging showed that the distributions of F-actin, vinculin, paxillin and zyxin in the ZLS were different from those in the podosome belt. Thick actin filament bundles running outside the ZLS appeared to recruit non-muscle myosin IIA. The F-actin-rich domain of the ZLS contained actin-related protein 2/3 complex (Arp2/3). Inhibition of Arp2/3 activity disorganized the ZLS, disrupted actin flow, deteriorated cell-cell adhesion and inhibited osteoclast hypermultinucleation. In contrast, ML-7, an inhibitor of myosin light chain kinase, had little effect on the structure of ZLS and promoted osteoclast hypermultinucleation. These results reveal a link between actin flow in the ZLS and osteoclast fusion. Osteoclast fusion was promoted by branched actin elongation and negatively regulated by actomyosin contraction.

Aims: Nrf2 (nuclear factor erythroid 2-like 2) is a transcription factor known to modulate blood vessel formation. Various experimental settings, however, attribute to Nrf2 either stimulatory or repressive influence on angiogenesis. Our findings unveil the mechanism of Nrf2-dependent vessel formation, which reaches beyond transactivation of gene expression and reconciles previous discrepancies. Results: We provide evidence that growth differentiation factor 15 (GDF-15)- and stromal cell-derived factor 1 (SDF-1)-induced angiogenesis strongly depends on the presence of Nrf2 protein but does not rely on its transcriptional activity. Instead, Nrf2 serves as a protein restraining Keap1 (Kelch-like ECH-associated protein 1), its known transcriptional repressor. Angiogenic response is abrogated in Nrf2-deficient endothelial cells but not in cells expressing dominant negative form or Keap1-binding fragment of Nrf2. Deficiency of Nrf2 protein available for Keap1 leads to the overabundance of RhoGAP1 (Rho GTPase-activating protein 1), the protein regulating cell division cycle 42 (Cdc42) activity. This impairs podosome assembly and disrupts actin rearrangements, thereby preventing angiogenesis. Effects of Nrf2 deficiency can be rescued by concomitant knockdown of RhoGAP1 or Keap1. Importantly, in the established murine model of Nrf2 deficiency, the N-terminal fragment of Nrf2 containing Keap1 binding domain is preserved. Thus, this model can be used to characterize Nrf2 as a transcription factor, but not as a Keap1-sequestering protein. Innovation and Conclusion: To date, the significance of Nrf2 in cell function has been ascribed solely to the regulation of transcription. We demonstrate that Nrf2 serves as a protein tethering Keap1 to allow podosome assembly and angiogenesis. Moreover, we emphasize that the new Nrf2 function of a Keap1 scavenger implies revisiting the interpretation of some of the previous data on the Nrf2-Keap1 system. [ABSTRACT FROM AUTHOR]

Successful pregnancy requires normal placentation, which largely depends on the tight regulation of proliferation, invasion, and migration of trophoblast cells. Abnormal functioning of trophoblast cells may cause failure of uterine spiral artery remodeling, which may be related to pregnancy-related disorders, such as preeclampsia. Here, we reported that an actin-binding protein, α-actinin (ACTN)4, was dysregulated in placentas from early onset preeclampsia. Moreover, knockdown of ACTN4 markedly inhibited trophoblast cell proliferation by reducing AKT membrane translocation. Furthermore, E-cadherin regulated ACTN4 and ß-catenin colocalization on trophoblast cell podosomes, and ACTN4 down-regulation suppressed the E-cadherin-induced cell invasion increase via depolymerizing actin filaments. Moreover, loss of ACTN4 recapitulated a number of the features of human preeclampsia. Therefore, our data indicate that ACNT4 plays a role in trophoblast function and is required for normal placental development. [ABSTRACT FROM AUTHOR]

Abstract Macrophage functions in the immune response depend on their ability to infiltrate tissues and organs. The penetration between and within the tissues requires degradation of extracellular matrix (ECM), a function performed by the specialized, endopeptidase- and actin filament- rich organelles located at the ventral surface of macrophage, called the podosomes. Podosome formation requires local inhibition of small GTPase RhoA activity, and depends on Rac 1/Rho guanine nucleotide exchange factor 7, β-PIX and its binding partner the p21-activated kinase (PAK-1). The activity of RhoA and Rac 1 is in turn regulated by mTOR/mTORC2 pathway. Here we showed that a fungus metabolite Fingolimod (FTY720, Gilenya), which is clinically approved for the treatment of multiple sclerosis, down-regulates Rictor, which is a signature molecule of mTORC2 and dictates its substrate (actin cytoskeleton) specificity, down-regulates RhoA, up-regulates PAK-1, and causes amplification of podosomes in mouse peritoneal macrophages. [ABSTRACT FROM AUTHOR]

Abstract Transgelin-1 (SM22α) has been recognized as a smooth muscle marker and a tumor suppressor, but many details of the working mechanisms remain unclear. Transgelin-1 belongs to the calponin family of actin-binding proteins with an N-terminal calponin homology domain (CH-domain) and a C-terminal calponin-like module (CLIK23). Here, we demonstrate that transgelin-1 interacts with actin stress fibers and podosomes in smooth muscle cells via its type-3 CH-domain, while CLIK23 is dispensable for the binding to the actin structures. We further suggest that the EF-hand motif in transgelin-1 contributes to proper folding of the CH-domain and in turn to the interaction with the actin structures. These results are in contrast to the ones reported in in vitro studies that demonstrated CLIK23 was necessary for the transgelin-1–actin binding, while the CH-domain was not. Besides, within cells, transgelin-1 phosphorylation at Ser181 in CLIK23 did not affect its colocalization with the actin structures, while the same phosphorylation was reported in in vitro studies to negatively regulate actin binding. Thus, our results suggest the molecular basis of intracellular interaction between transgelin-1 and actin, distinct from that in vitro. The actin binding capability intrinsic to CLIK23 may not appear within cells probably because of the weaker competition for actin binding compared to other actin binding molecules. Highlights • Transgelin-1 interacts with stress fibers and podosomes via its type-3 CH-domain. • EF-hand affects the transgelin-1–actin interaction cooperatively with CLIK23. • Phosphorylation in CLIK23 does not affect the transgelin-1–actin interaction. • Increased numbers of CLIK23 do not compensate for the deletion of the CH-domain. [ABSTRACT FROM AUTHOR]

The forces that cells, tissues, and organisms exert on the surface of a soft substrate can be measured using Traction Force Microscopy (TFM), an important and well-established technique in Mechanobiology. The usual TFM technique (two-dimensional, 2D TFM) treats only the in-plane component of the traction forces and omits the out-of-plane forces at the substrate interfaces (2.5D) that turn out to be important in many biological processes such as tissue migration and tumour invasion. Here, we review the imaging, material, and analytical tools to perform "2.5D TFM" and explain how they are different from 2D TFM. Challenges in 2.5D TFM arise primarily from the need to work with a lower imaging resolution in the z-direction, track fiducial markers in three-dimensions, and reliably and efficiently reconstruct mechanical stress from substrate deformation fields. We also discuss how 2.5D TFM can be used to image, map, and understand the complete force vectors in various important biological events of various length-scales happening at two-dimensional interfaces, including focal adhesions forces, cell diapedesis across tissue monolayers, the formation of three-dimensional tissue structures, and the locomotion of large multicellular organisms. We close with future perspectives including the use of new materials, imaging and machine learning techniques to continuously improve the 2.5D TFM in terms of imaging resolution, speed, and faithfulness of the force reconstruction procedure. [ABSTRACT FROM AUTHOR]

The role of myosin light chain kinase (MLCK) in inducing podosomes was examined by confocal and electron microscopy. Removal of myosin from the actin core of podosomes using blebbistatin, a myosin inhibitor, resulted in the formation of smaller podosomes. Downregulation of MLCK by the transfection of MLCK small interfering RNA (siRNA) led to the failure of podosome formation. However, ML-7, an inhibitor of the kinase activity of MLCK, failed to inhibit podosome formation. Based on our previous report (Thatcher et al. J.Pharm.Sci. 116 116–127, 2011), we outlined the important role of the actin-binding activity of MLCK in producing smaller podosomes.

Graphical abstract Abstract Cytosolic protein tyrosine phosphatase epsilon (cyt-PTPε) plays a central role in controlling differentiation and function of osteoclasts, whose overactivation causes osteoporosis. Based on our previous study reporting a number of cyt-PTPε inhibitory chemical compounds, we carried out a further and extended analysis of our compounds to examine their effects on cyt-PTPε-mediated dephosphorylation and on osteoclast organization and differentiation. Among five compounds showing target selectivity to cyt-PTPε over three other phosphatases in vitro , two compounds exhibited an inhibitory effect against the dephosphorylation of cellular Src protein, the cyt-PTPε substrate. Moreover, these two compounds caused destabilization of the podosome structure that is necessary for the bone-resorbing activity of osteoclasts, and also attenuated cellular differentiation of monocytes into osteoclasts, without affecting cell viability. Therefore, these findings not only verified anti-osteoclastic effects of our cyt-PTPε inhibitory compounds, but also showed that cyt-PTPε expressed in osteoclasts could be a putative therapeutic target worth considering. [ABSTRACT FROM AUTHOR]

Non-muscle caldesmon (l-CaD) is involved in the regulation of actin cytoskeletal remodeling in the podosome formation, but its function in osteoclastogenesis remains to be determined. In this study, RANKL-induced differentiation of RAW264.7 murine macrophages to osteoclast-like cells (OCs) was used as a model to determine the physiological role of l-CaD and its phosphorylation in osteoclastogenesis. UponRANKL treatment, RAW264.7 cells undergo cell-cell fusion into multinucleate, and TRAP-positive large OCs with a concomitant increase of l-CaD expression. Using gain- and loss-of-function in OC precursor cells followed by RANKL induction, we showed that the expression of l-CaD in response to RANKL activation is an important event for osteoclastogenesis, and bone resorption. To determine the effect of l-CaD phosphorylation in osteoclastogenesis, three decoy peptides of l-CaD were used with, respectively, Ser-to-Ala mutations at the Erk- and Pak1-mediated phosphorylation sites, and Ser-to-Asp mutation at the Erk-mediated phosphorylation sites. Both the former two peptides competed with the C-terminal segment of l-CaD for F-actin binding and accelerated formation of podosome-like structures in RANKL-induced OCs, while the third peptide did not significantly affect the F-actin binding of l-CaD, and decreased the formation of podosome-like structures in OCs. With the experiments using dephosphorylated and phosphorylated l-CaD mutants, we further showed that dephosphorylated l-CaD mutant facilitated RANKL-induced TRAP activity with an increased cell fusion index, whereas phosphorylated l-CaD decreased the TRAP activity and cell fusion. Our findings suggested that both the level of l-CaD expression and the extent of l-CaD phosphorylation play a role in RANKL-induced osteoclast differentiation. [ABSTRACT FROM AUTHOR]

Abstract: Cells shed submicronic extracellular vesicles (EVs), containing a variety of soluble and membrane‐embedded factors, to mediate intercellular communication. EVs show differential immunomodulatory features depending on the cell source and physiological status, endowing these nanosystems with intrinsic immunotherapeutic potential. EVs could be used to mount a potent immune response against tumor cells, by regulating inflammatory cues in tumors and modulating macrophage activation and migratory behavior. On the other part, EVs are loaded with theranostic nanoparticles, such as superparamagnetic iron oxide nanoparticles (SPIONs), to confer them magnetic detection, manipulation, and heating capabilities. However, little is known about the impact that SPION may have on intrinsic immunomodulatory properties of EVs. Here, the effects are characterized on the activation and dynamics of macrophages induced by EVs derived from mouse mesenchymal stem cells and mouse/human endothelial cells, loaded or not with SPION. EVs differently activate macrophages as determined by cytokine secretion, phagocytosis capacity, migratory behavior, and extracellular matrix degradation capacity. SPION loading prove to amplify the immunoregulatory properties of EVs, increasing the macrophage ability to migrate, and degrade extracellular matrix. Altogether, EVs might potentiate natural or induced immune response by modulating macrophage activation/migratory behavior, suggesting their use as nanocarriers in combinatorial therapeutic approaches with SPION. [ABSTRACT FROM AUTHOR]

Osteoclasts form a specialized cell-matrix adhesion structure, known as the "sealing zone", during bone resorption. The sealing zone is a dynamic actin-rich structure that defines the resorption area of the bone. The detailed dynamics and fine structure of the sealing zone have been elusive. Osteoclasts plated on glass do not form a sealing zone, but generate a separate supra-molecular structure called the "podosome belt". Podosomes are integrin-based adhesion complexes involved in matrix adhesion, cell migration, matrix degradation, and mechanosensing. Invadopodia, podosome-like protrusions in cancer cells, are involved in cell invasion into other tissues by promoting matrix degradation. Both podosomes and invadopodia exhibit actin pattern transitions during maturation. We previously found that Arp2/3-dependent actin flow occurs in all observed assembly patterns of podosomes in osteoclasts on glass. It is known that the actin wave in Dictyostelium cells exhibits a similar pattern transition in its evolution. Because of significant advances in our understanding regarding the mechanism of podosomes/invadopodia formation over the last decade, we revisited the structure and function of the sealing zone in this review, highlighting the possible involvement of self-organized actin waves in the organogenesis of the sealing zone. [ABSTRACT FROM AUTHOR]

Bone deficits are frequent in HIV-1-infected patients. We report here that osteoclasts, the cells specialized in bone resorption, are infected by HIV-1 in vivo in humanized mice and ex vivo in human joint biopsies. In vitro, infection of human osteoclasts occurs at different stages of osteoclastogenesis via cell-free viruses and, more efficiently, by transfer from infected T cells. HIV-1 infection markedly enhances adhesion and osteolytic activity of human osteoclasts by modifying the structure and function of the sealing zone, the osteoclast-specific bone degradation machinery. Indeed, the sealing zone is broader due to F-actin enrichment of its basal units (i.e., the podosomes). The viral protein Nef is involved in all HIV-1-induced effects partly through the activation of Src, a regulator of podosomes and of their assembly as a sealing zone. Supporting these results, Nef-transgenic mice exhibit an increased osteoclast density and bone defects, and osteoclasts derived from these animals display high osteolytic activity. Altogether, our study evidences osteoclasts as host cells for HIV-1 and their pathological contribution to bone disorders induced by this virus, in part via Nef. [ABSTRACT FROM AUTHOR]

Endothelial cells (EC) act as leading actors in angiogenesis. Understanding the complex network of signal transduction pathways which regulate angiogenesis might offer insights in the regulation of normal and pathological events, including tumours, vascular, inflammatory and immune diseases. The effects of olive oil and of Blueberry extracts upon the phosphoinositide (PI)-specific phospholipase C (PLC) enzymes were evaluated both in quiescent and inflammatory stimulated human umbilical vein EC (HUVEC) using molecular biology (multiliquid bioanalysis) and immunofluorescence techniques. Oleuropein significantly increased the number of surviving HUVEC compared to untreated controls, suggesting that it favours the survival and proliferation of EC. Our results suggest that Oleuropein might be useful to induce EC proliferation, an important event during angiogenesis, with special regard to wound healing. Blueberry extracts increased the number of surviving HUVEC, although the comparison to untreated controls did not result statistically significant. Lipopolysaccharide (LPS) administration significantly reduced the number of live HUVEC. LPS can also modify the expression of selected PLC genes. Adding Blueberry extracts to LPS treated HUVEC cultures did not significantly modify the variations of PLC expression induced by LPS. Oleuropein increased or reduced the expression of PLC genes, and statistically significant results were identified for selected PLC isoforms. Oleuropein also modified the effects of LPS upon PLC genes' expression. Thus, our results corroborate the hypothesis that Oleuropein owns anti-inflammatory activity. The intracellular localization of PLC enzymes was modified by the different treatments we used. Podosome-like structures were observed in differently LPS treated HUVEC. [ABSTRACT FROM AUTHOR]

Alzheimer's disease (AD) is a progressive neurodegenerative disease that is associated with protein misfolding, plaque accumulation, neuronal dysfunction, synaptic loss, and cognitive decline. The pathological cascade of AD includes the intracellular Tau hyperphosphorylation and its subsequent aggregation, extracellular Amyloid-β plaque formation and microglia-mediated neuroinflammation. The extracellular release of aggregated Tau is sensed by surveilling microglia through the involvement of various cell surface receptors. Among all, purinergic P2Y12R signaling is involved in microglial chemotaxis towards the damaged neurons. Microglial migration is highly linked with membrane-associated actin remodeling leading to the phagocytosis of extracellular Tau species. Here, we studied the formation of various actin structures such as podosome, lamellipodia and filopodia, in response to extracellular Tau monomers and aggregates. Microglial podosomes are colocalized with actin nucleator protein WASP, Arp2 and TKS5 adaptor protein during Tau-mediated migration. Moreover, the P2Y12 receptors were associated with F-actin-rich podosome structures, which signify the potential of Tau aggregates in microglial chemotaxis through the involvement of actin remodeling. [Display omitted] • The extracellular Tau regulates the Arp2 and TKS5-associated podosomes and filopodia formation. • Tau aggregates can act as dual inducers of P2Y12R signaling and the podosomes and filopodia-associated actin remodeling. • The activation of actin remodeling pathways and associated proteins signaling by Tau aggregates evoke new insights of beneficial activation of microglia in disease condition. [ABSTRACT FROM AUTHOR]

Arf GTPase-activating proteins (Arf GAPs) control the activity of ADP-ribosylation factors (Arfs) by inducing GTP hydrolysis and participate in a diverse array of cellular functions both through mechanisms that are dependent on and independent of their Arf GAP activity. A number of these functions hinge on the remodeling of actin filaments. Accordingly, some of the effects exerted by Arf GAPs involve proteins known to engage in regulation of the actin dynamics and architecture, such as Rho family proteins and nonmuscle myosin 2. Circular dorsal ruffles (CDRs), podosomes, invadopodia, lamellipodia, stress fibers and focal adhesions are among the actin-based structures regulated by Arf GAPs. Arf GAPs are thus important actors in broad functions like adhesion and motility, as well as the specialized functions of bone resorption, neurite outgrowth, and pathogen internalization by immune cells. Arf GAPs, with their multiple protein-protein interactions, membrane-binding domains and sites for post-translational modification, are good candidates for linking the changes in actin to the membrane. The findings discussed depict a family of proteins with a critical role in regulating actin dynamics to enable proper cell function.

Proinflammatory cytokine production, cell chemotaxis, and osteoclastogenesis can lead to inflammatory bone loss. Previously, we showed that sphingosine-1-phosphate receptor 2 (S1PR2), a G protein coupled receptor, regulates inflammatory cytokine production and osteoclastogenesis. However, the signaling pathways regulated by S1PR2 in modulating inflammatory bone loss have not been elucidated. Herein, we demonstrated that inhibition of S1PR2 by a specific S1PR2 antagonist (JTE013) suppressed phosphoinositide 3-kinase (PI3K), mitogen-activated protein kinases (MAPKs), and nuclear factor kappa-B (NF-κB) induced by an oral bacterial pathogen, Aggregatibacter actinomycetemcomitans, and inhibited the release of IL-1β, IL-6, TNF-α, and S1P in murine bone marrow cells. In addition, shRNA knockdown of S1PR2 or treatment by JTE013 suppressed cell chemotaxis induced by bacteria-stimulated cell culture media. Furthermore, JTE013 suppressed osteoclastogenesis and bone resorption induced by RANKL in murine bone marrow cultures. ShRNA knockdown of S1PR2 or inhibition of S1PR2 by JTE013 suppressed podosome components, including PI3K, Src, Pyk2, integrin β3, filamentous actin (F-actin), and paxillin levels induced by RANKL in murine bone marrow cells. We conclude that S1PR2 plays an essential role in modulating proinflammatory cytokine production, cell chemotaxis, osteoclastogenesis, and bone resorption. Inhibition of S1PR2 signaling could be a novel therapeutic strategy for bone loss associated with skeletal diseases.