Adhesion, motility and matrix-degrading gene expression changes in CSF-1-induced mouse macrophage differentiation.

Migratory macrophages play critical roles in tissue development, homeostasis and disease, so it is important to understand how their migration machinery is regulated. Whole-transcriptome sequencing revealed that CSF-1-stimulated differentiation of bone marrow-derived precursors into mature macrophages is accompanied by widespread, profound changes in the expression of genes regulating adhesion, actin cytoskeletal remodeling and extracellular matrix degradation. Significantly altered expression of almost 40% of adhesion genes, 60-86% of Rho family GTPases, their regulators and effectors and over 70% of extracellular proteases occurred. The gene expression changes were mirrored by changes in macrophage adhesion associated with increases in motility and matrix-degrading capacity. IL-4 further increased motility and matrix-degrading capacity in mature macrophages, with additional changes in migration machinery gene expression. Finally, siRNA-induced reductions in the expression of the core adhesion proteins paxillin and leupaxin decreased macrophage spreading and the number of adhesions, with distinct effects on adhesion and their distribution, and on matrix degradation. Together, the datasets provide an important resource to increase our understanding of the regulation of migration in macrophages and to develop therapies targeting disease-enhancing macrophages.

Early induction of uncoupling protein-2 in pulmonary macrophages in hyperoxia-associated lung injury.

CONTEXT: High concentrations of inspired oxygen contribute to the pathogenesis of neonatal bronchopulmonary dysplasia and adult acute respiratory distress syndrome. Animal models of hyperoxia-associated lung injury (HALI) are characterized by enhanced generation of reactive oxygen species (ROS) and an adaptive antioxidant response. ROS contribute to pathogenesis, partly through enhancing pro-inflammatory activity in macrophages. Uncoupling protein-2 (UCP2) is an inner mitochondrial membrane protein whose expression lowers mitochondrial superoxide (O2ⁱ⁻) production. UCP2, therefore, has potential to contribute to antioxidant response. It is inducible in macrophages. OBJECTIVES AND METHODS: We hypothesized that induction of UCP2 occurred in response to pulmonary hyperoxia in vivo and that expression localized to pulmonary macrophages. We then investigated mechanisms of UCP2 regulation in hyperoxia-exposed macrophages in vitro and correlated changing UCP2 expression with mitochondrial membrane potential (Δψm) and O2ⁱ⁻ production. RESULTS: UCP2 is induced in lungs of mice within 1 h of hyperoxia exposure. Induction occurs in pulmonary alveolar macrophages in vivo, and can be replicated in vitro in isolated macrophages. UCP2 mRNA does not change. UCP2 increases quickly after the first hyperoxia-induced burst of mitochondrial O2ⁱ⁻ generation. Suppression of Δψm and mitochondrial O2ⁱ⁻ production follow and persist while UCP2 is elevated. DISCUSSION AND CONCLUSIONS: Induction of UCP2 is an early response to hyperoxia in pulmonary macrophages. The mechanism is post-transcriptional. UCP2 induction follows a transient rise in mitochondrial ROS generation. The subsequent falls in Δψm and mitochondrial O2ⁱ⁻ support the notion that regulable UCP2 expression in macrophages acts to contain mitochondrial ROS generation. That, in turn, may limit inappropriate pro-inflammatory activation in HALI.

Paclitaxel inhibits osteoclast formation and bone resorption via influencing mitotic cell cycle arrest and RANKL-induced activation of NF-κB and ERK.

Pathological bone destruction (osteolysis) is a hallmark of many bone diseases including tumor metastasis to bone, locally osteolytic giant cell tumor (GCT) of bone, and Paget's disease. Paclitaxel is frequently prescribed in the treatment of several malignant tumors where it has been shown to exert beneficial effects on bone lesions. However, the mechanism(s) through which paclitaxel regulates osteoclast formation and function remain ill defined. In the present study, we demonstrate that paclitaxel dose-dependently inhibits receptor activator of nuclear factor-kappa B ligand (RANKL)-induced osteoclastogenesis in both RAW264.7 cells and mouse bone marrow macrophage (BMM) systems. In addition, paclitaxel treatment reduces the bone resorptive activity of human osteoclasts derived from GCT of bone, and attenuates lipopolysaccharide (LPS)-induced osteolysis in a mouse calvarial model. Complementary cellular and biochemical analyses revealed that paclitaxel induces mitotic arrest of osteoclastic precursor cells. Furthermore, luciferase reporter gene assays and western blot analysis indicate that paclitaxel modulates key RANKL-induced activation pathways that are essential to osteoclast formation including NF-κB and ERK. Collectively, our findings demonstrate a role for paclitaxel in the regulation of osteoclast formation and function and uncover potential mechanism(s) through which paclitaxel alleviates pathological osteolysis.

Therapeutic inhibition of the SRC-kinase HCK facilitates T cell tumor infiltration and improves response to immunotherapy.

Although immunotherapy has revolutionized cancer treatment, many immunogenic tumors remain refractory to treatment. This can be largely attributed to an immunologically "cold" tumor microenvironment characterized by an accumulation of immunosuppressive myeloid cells and exclusion of activated T cells. Here, we demonstrate that genetic ablation or therapeutic inhibition of the myeloid-specific hematopoietic cell kinase (HCK) enables activity of antagonistic anti-programmed cell death protein 1 (anti-PD1), anti-CTLA4, or agonistic anti-CD40 immunotherapies in otherwise refractory tumors and augments response in treatment-susceptible tumors. Mechanistically, HCK ablation reprograms tumor-associated macrophages and dendritic cells toward an inflammatory endotype and enhances CD8+ T cell recruitment and activation when combined with immunotherapy in mice. Meanwhile, therapeutic inhibition of HCK in humanized mice engrafted with patient-derived xenografts counteracts tumor immunosuppression, improves T cell recruitment, and impairs tumor growth. Collectively, our results suggest that therapeutic targeting of HCK activity enhances response to immunotherapy by simultaneously stimulating immune cell activation and inhibiting the immunosuppressive tumor microenvironment.

c-Cbl promotes T cell receptor-induced thymocyte apoptosis by activating the phosphatidylinositol 3-kinase/Akt pathway.

The ability of thymocytes to assess T cell receptor (TCR) signaling strength and initiate the appropriate downstream response is crucial for determining their fate. We have previously shown that a c-Cbl RING finger mutant knock-in mouse, in which the E3 ubiquitin ligase activity of c-Cbl is inactivated, is highly sensitive to TCR-induced death signals that cause thymic deletion. This high intensity signal involves the enhanced tyrosine phosphorylation of the mutant c-Cbl protein promoting a marked increase in the activation of Akt. Here we show that this high intensity signal in c-Cbl RING finger mutant thymocytes also promotes the enhanced induction of two mediators of TCR-directed thymocyte apoptosis, Nur77 and the pro-apoptotic Bcl-2 family member, Bim. In contrast, a knock-in mouse harboring a mutation at Tyr-737, the site in c-Cbl that activates phosphatidylinositol 3-kinase, shows reduced TCR-mediated responses including suppression of Akt activation, a reduced induction of Nur77 and Bim, and greater resistance to thymocyte death. These findings identify tyrosine-phosphorylated c-Cbl as a critical sensor of TCR signal strength that regulates the engagement of death-promoting signals.

Uncoupling protein-2 accumulates rapidly in the inner mitochondrial membrane during mitochondrial reactive oxygen stress in macrophages.

Uncoupling protein-2 (UCP2) is a member of the inner mitochondrial membrane anion-carrier superfamily. Although mRNA for UCP2 is widely expressed, protein expression is detected in only a few cell types, including macrophages. UCP2 functions by an incompletely defined mechanism, to reduce reactive oxygen species production during mitochondrial electron transport. We observed that the abundance of UCP2 in macrophages increased rapidly in response to treatments (rotenone, antimycin A and diethyldithiocarbamate) that increased mitochondrial superoxide production, but not in response to superoxide produced outside the mitochondria or in response to H2O2. Increased UCP2 protein was not accompanied by increases in ucp2 gene expression or mRNA abundance, but was due to enhanced translational efficiency and possibly stabilization of UCP2 protein in the inner mitochondrial membrane. This was not dependent on mitochondrial membrane potential. These findings extend our understanding of the homeostatic function of UCP2 in regulating mitochondrial reactive oxygen production by identifying a feedback loop that senses mitochondrial reactive oxygen production and increases inner mitochondrial membrane UCP2 abundance and activity. Reactive oxygen species-induction of UCP2 may facilitate survival of macrophages and retention of function in widely variable tissue environments.

Caffeic acid phenethyl ester, an active component of honeybee propolis attenuates osteoclastogenesis and bone resorption via the suppression of RANKL-induced NF-kappaB and NFAT activity.

Receptor activator NF-kappaB ligand (RANKL)-activated signaling is essential for osteoclast differentiation, activation and survival. Caffeic acid phenethyl ester (CAPE), a natural NF-kappaB inhibitor from honeybee propolis has been shown to have anti-tumor and anti-inflammatory properties. In this study, we investigated the effect of CAPE on the regulation of RANKL-induced osteoclastogenesis, bone resorption and signaling pathways. Low concentrations of CAPE (<1 microM) dose dependently inhibited RANKL-induced osteoclastogenesis in RAW264.7 cell and bone marrow macrophage (BMM) cultures, as well as decreasing the capacity of human osteoclasts to resorb bone. CAPE inhibited both constitutive and RANKL-induced NF-kappaB and NFAT activation, concomitant with delayed IkappaBalpha degradation and inhibition of p65 nuclear translocation. At higher concentrations, CAPE induced apoptosis and caspase 3 activities of RAW264.7 and disrupts the microtubule network in osteoclast like (OCL) cells. Taken together, our findings demonstrate that inhibition of NF-kappaB and NFAT activation by CAPE results in the attenuation of osteoclastogenesis and bone resorption, implying that CAPE is a potential treatment for osteolytic bone diseases.

Oxidative stress causes a decline in lysosomal integrity during hypothermic incubation of rat hepatocytes.

Oxidative stress during cold preservation has been identified as a significant cause of cell injury but the process by which injury occurs is poorly understood. We examined loss of lysosomal integrity as a possible cause of cell injury during extended cold storage of isolated rat hepatocytes. After 21 h of hypothermia, there was a marked decline in lysosomal integrity, which was correlated with an increase in lipid peroxidation. When lipid peroxidation was prevented with the antioxidant Trolox (a vitamin E analog) or the iron chelator desferrioxamine, lysosomal integrity was preserved. In contrast, increasing lysosomal iron with ferric chloride caused an increase in lipid peroxidation and decreased lysosomal integrity. Loss of lysosomal integrity during cold preservation in this experimental model was consistent with iron-initiated oxidative stress. The progressive loss of lysosomal integrity during hypothermic incubation has the potential to affect liver function after transplantation.

Monocyte-derived macrophages from men and women with Type 2 diabetes mellitus differ in fatty acid composition compared with non-diabetic controls.

We examined whether macrophages from men and women with Type 2 diabetes mellitus (T2DM) exhibited differences in expression of key genes involved in fatty acid metabolism and in fatty acid composition compared with macrophages from non-diabetic controls. Peripheral blood monocytes from subjects with T2DM (n=9) and non-diabetic controls (n=10) were differentiated into macrophages in 10% autologous serum and normal (5mM) or high (22mM) glucose. Levels of PPARalpha, PPARgamma, LXRalpha, SCD and ABCA1 mRNAs were similar in macrophages from subjects with T2DM and controls. At 5mM glucose, macrophage stearic acid (C18:0) was 12.6+/-1.0% of total fatty acids for T2DM compared with 18.1+/-2.0% for controls (p=0.03). Macrophage linoleic acid (C18:2) was 15.5+/-0.8% for T2DM and 9.3+/-2.0% for controls (p=0.005). The ratio of macrophage stearic acid (C18:0)/oleic acid (C18:1) was 0.29 [0.25,0.48] for T2DM versus 0.54 [0.36,0.82] for controls (p=0.04). Compared with non-diabetic controls, macrophages from men and women with T2DM had significantly different fatty acid profiles consistent with increased stearoyl-CoA desaturase (SCD) activity and increased C18:2 accumulation. This pattern of altered macrophage fatty acid composition may be relevant to diabetic atherogenesis.

A novel mutation (K378X) in the sequestosome 1 gene associated with increased NF-kappaB signaling and Paget's disease of bone with a severe phenotype.

UNLABELLED: Sequestosome 1/p62 (p62) mutations are associated with PDB; however, there are limited data regarding functional consequences. We report a novel mutation in exon 7 (K378X) in a patient with polyostotic Paget's disease of bone. p62 mutants increased NF-kappaB activation and significantly potentiated osteoclast formation and bone resorption in human primary cell cultures. INTRODUCTION: Sequestosome 1/p62 (p62) mutations are associated with Paget's disease of bone (PDB); however, there are limited data regarding functional consequences. One report has linked the common P392L mutation in the p62 ubiquitin binding associated (UBA) domain with increases in NF-kappaB activity, a transcription factor essential for osteoclastogenesis. To further clarify the functional impact of p62 mutations associated with PDB, we assessed the effect of p62 mutation (a novel mutation: K378X, and previously reported mutations: P392L and E396X) on RANK-induced NF-kappaB activation and compared this with the effect of wildtype p62. In addition, we studied the effect of p62 mutation on osteoclast formation and bone resorption. MATERIALS AND METHODS: We performed co-transfection experiments with expression plasmids for p62 (wildtype or mutated) and RANK and an NF-kappaB luciferase reporter gene. Luciferase activities were recorded after addition of luciferin to cellular lysates. RAW(264.7) cells stably expressing enhanced green fluorescent protein (EGFP)-tagged p62 (wildtype, K378X, or P392L) or EGFP alone were assessed for changes in cell proliferation. Additionally, these cells were stimulated with RANKL to produce osteoclast-like cells (OLCs). Primary human monocytes collected from the K378X-affected patient and a control subject were stimulated to form OLCs and bone resorption data were obtained. RESULTS: The novel mutation introduces a premature stop codon in place of Lys-378 and thereby eliminates the entire p62 UBA domain; this and two additional natural mutations (P392L, E396X) increased NF-kappaB activation compared with wildtype p62. Wildtype p62 consistently inhibited NF-kappaB activation compared with empty vector. UBA mutations (K378X and P392L) significantly increased the number of OLCs formed in response to RANKL and also the number of nuclei of the OLCs. K378X-affected human monocytes formed more OLCs with more nuclei and increased bone resorption compared with control monocytes. CONCLUSIONS: Our data show that mutation of the p62 UBA domain results in increased activation of NF-kappaB and osteoclast formation and function compared with wildtype p62. These results may partially explain the mechanism by which p62 mutation contributes to the pathogenesis of PDB.

Moraxella catarrhalis stimulates the release of proinflammatory cytokines and prostaglandin E from human respiratory epithelial cells and monocyte-derived macrophages.

The outer membrane proteins of Moraxella catarrhalis, a bacterial pathogen which causes disease in both children and adults, play an important role in its phenotypic properties. However, their proinflammatory potential with regard to respiratory epithelium and macrophages is unclear. To this end, we examined the cytokine- and mediator-inducing capacity of a heat-killed wild-type M. catarrhalis strain and a nonautoagglutinating mutant as well as their outer membrane proteins and secretory/excretory products using the A549 respiratory epithelial cell line. The outer membrane proteins and secretory/excretory products from both isolates as well as the heat-killed bacteria all induced interleukin (IL)-6, IL-8 and prostaglandin E2, but not IL-1beta, from the A549 cell line in a dose- and time-dependent manner. Heat-killed bacteria and secretory/excretory products stimulated the release of IL-1beta, IL-6, IL-8 and prostaglandin E2 from human monocyte-derived macrophages. Both heat-killed isolates also stimulated nuclear translocation and transactivation of nuclear factor-kappaB. The heat-killed wild-type autoagglutinating isolate induced significantly greater amounts of IL-6 and IL-8 from A549 cells than the nonautoagglutinating mutant compared with the monocyte-derived macrophages but no significant differences in the amounts induced by the two strains were observed. These differences were also evident when the respiratory cell line was stimulated with outer membrane proteins as well as in the degree of nuclear factor-kappaB transactivation. There was little difference in the stimulatory activity of the secretory/excretory products. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis analyses revealed some differences in the outer membrane proteins and secretory excretory products between the two isolates. Combined, these data show that M. catarrhalis secretory excretory products and outer membrane proteins are associated with the induction of inflammatory responses in both respiratory epithelium and macrophages.

Alexidine Dihydrochloride Attenuates Osteoclast Formation and Bone Resorption and Protects Against LPS-Induced Osteolysis.

Aseptic loosening and periprosthetic infection leading to inflammatory osteolysis is a major complication associated with total joint arthroplasty (TJA). The liberation of bacterial products and/or implant-derived wear particles activates immune cells that produce pro-osteoclastogenic cytokines that enhance osteoclast recruitment and activity, leading to bone destruction and osteolysis. Therefore, agents that prevent the inflammatory response and/or attenuate excessive osteoclast (OC) formation and bone resorption offer therapeutic potential by prolonging the life of TJA implants. Alexidine dihydrochloride (AD) is a bisbiguanide compound commonly used as an oral disinfectant and in contact lens solutions. It possesses antimicrobial, anti-inflammatory and anticancer properties; however, its effects on OC biology are poorly described. Here, we demonstrate that AD inhibits OC formation and bone resorption in vitro and exert prophylatic protection against LPS-induced osteolysis in vivo. Biochemical analysis demonstrated that AD suppressed receptor activator of NF-κB ligand (RANKL)-induced activation of mitogen-activated protein kinases (ERK, p38, and JNK), leading to the downregulation of NFATc1. Furthermore, AD disrupted F-actin ring formation and attenuated the ability of mature OC to resorb bone. Collectively, our findings suggest that AD may be a promising prophylactic anti-osteoclastic/resorptive agent for the treatment of osteolytic diseases caused by excessive OC formation and function.

Thonzonium bromide inhibits RANKL-induced osteoclast formation and bone resorption in vitro and prevents LPS-induced bone loss in vivo.

Osteoclasts (OCs) play a pivotal role in a variety of lytic bone diseases including osteoporosis, arthritis, bone tumors, Paget's disease and the aseptic loosening of orthopedic implants. The primary focus for the development of bone-protective therapies in these diseases has centered on the suppression of OC formation and function. In this study we report that thonzonium bromide (TB), a monocationic surface-active agent, inhibited RANKL-induced OC formation, the appearance of OC-specific marker genes and bone-resorbing activity in vitro. Mechanistically, TB blocked the RANKL-induced activation of NF-κB, ERK and c-Fos as well as the induction of NFATc1 which is essential for OC formation. TB disrupted F-actin ring formation resulting in disturbances in cytoskeletal structure in mature OCs during bone resorption. Furthermore, TB exhibited protective effects in an in vivo murine model of LPS-induced calvarial osteolysis. Collectively, these data suggest that TB might be a useful alternative therapy in preventing or treating osteolytic diseases.

Src family kinase expression and subcellular localization in macrophages: implications for their role in CSF-1-induced macrophage migration.

A major role of colony-stimulating factor-1 is to stimulate the differentiation of mononuclear phagocytic lineage cells into adherent, motile, mature macrophages. The colony-stimulating factor-1 receptor transduces colony-stimulating factor-1 signaling, and we have shown previously that phosphatidylinositol 3-kinase p110δ is a critical mediator of colony-stimulating factor-1-stimulated motility through the colony-stimulating factor-1 receptor pY721 motif. Src family kinases are also implicated in the regulation of macrophage motility and in colony-stimulating factor-1 receptor signaling, although functional redundancy of the multiple SFKs expressed in macrophages makes it challenging to delineate their specific functions. We report a comprehensive analysis of individual Src family kinase expression in macrophage cell lines and primary macrophages and demonstrate colony-stimulating factor-1-induced changes in Src family kinase subcellular localization, which provides clues to their distinct and redundant functions in macrophages. Moreover, expression of individual Src family kinases is both species specific and dependent on colony-stimulating factor-1-induced macrophage differentiation. Hck associated with the activated colony-stimulating factor-1 receptor, whereas Lyn associated with the receptor in a constitutive manner. Consistent with this, inhibitor studies revealed that Src family kinases were important for both colony-stimulating factor-1 receptor activation and colony-stimulating factor-1-induced macrophage spreading, motility, and invasion. Distinct colony-stimulating factor-1-induced changes in the subcellular localization of individual SFKs suggest specific roles for these Src family kinases in the macrophage response to colony-stimulating factor-1.

Thapsigargin modulates osteoclastogenesis through the regulation of RANKL-induced signaling pathways and reactive oxygen species production.

UNLABELLED: The mechanism by which TG modulates osteoclast formation and apoptosis is not clear. In this study, we showed a biphasic effect of TG on osteoclast formation and apoptosis through the regulation of ROS production, caspase-3 activity, cytosolic Ca2+, and RANKL-induced activation of NF-kappaB and AP-1 activities. INTRODUCTION: Apoptosis and differentiation are among the consequences of changes in intracellular Ca2+ levels. In this study, we investigated the effects of the endoplasmic reticular Ca2+-ATPase inhibitor, thapsigargin (TG), on osteoclast apoptosis and differentiation. MATERIALS AND METHODS: Both RAW264.7 cells and primary spleen cells were used to examine the effect of TG on RANKL-induced osteoclastogenesis. To determine the action of TG on signaling pathways, we used reporter gene assays for NF-kappaB and activator protein-1 (AP-1) activity, Western blotting for phospho-extracellular signal-related kinase (ERK), and fluorescent probes to measure changes in levels of intracellular calcium and reactive oxygen species (ROS). To assess rates of apoptosis, we measured changes in annexin staining, caspase-3 activity, and chromatin and F-actin microfilament structure. RESULTS: At concentrations that caused a rapid rise in intracellular Ca2+, TG increased caspase-3 activity and promoted apoptosis in osteoclast-like cells (OLCs). Low concentrations of TG, which were insufficient to measurably alter intracellular Ca2+, unexpectedly suppressed caspase-3 activity and enhanced RANKL-induced osteoclastogenesis. At these lower concentrations, TG potentiated ROS production and RANKL-induced NF-kappaB activity, but suppressed RANKL-induced AP-1 activity and had little effect on ERK phosphorylation. CONCLUSION: Our novel findings of a biphasic effect of TG are incompletely explained by our current understanding of TG action, but raise the possibility that low intensity or local changes in subcellular Ca2+ levels may regulate intracellular differentiation signaling. The extent of cross-talk between Ca2+ and RANKL-mediated intracellular signaling pathways might be important in determining whether cells undergo apoptosis or differentiate into OLCs.

Sesquiterpene lactone parthenolide blocks lipopolysaccharide-induced osteolysis through the suppression of NF-kappaB activity.

UNLABELLED: Effective treatment for bacteria-induced bone lytic diseases is not yet available. In this study, we showed that PAR, an NF-kappaB inhibitor found in medicinal herbs, can block LPS-induced osteolysis. PAR does this by inhibiting osteoclastogenesis and bone resorption and promoting apoptosis of osteoclasts through the suppression of NF-kappaB activity. INTRODUCTION: Osteolysis induced by chronic gram-negative bacterial infection underlies many bone diseases such as osteomyelitis, septic arthritis, and periodontitis. Drugs that inhibit lipopolysaccharide (LPS)-induced osteolysis are critically needed for the prevention of bone destruction in infective bone diseases. In this study, we investigated the effect of parthenolide (PAR) on LPS-induced osteolysis in vivo and studied its role in osteoclastogenesis, bone resorption, apoptosis, and NF-kappaB activity. MATERIALS AND METHODS: The LPS-induced osteolysis in the mouse calvarium model was used to examine the effect of PAR in vivo. RANKL-induced osteoclast differentiation from RAW264.7 cells and bone resorption assays were used to assess the effect of PAR in vitro. Assays for NF-kappaB activation, p65 translocation, and IkappaB-alpha degradation were used to determine the mechanism of action of PAR in osteoclasts and their precursors. Flow cytometry and confocal microscopic analysis were used to examine cell apoptosis. Semiquantitative RT-PCR was performed to examine the effect of PAR on gene expression of RANK and TRAF6. RESULTS: We found that PAR (0.5 and 1 mg/kg), injected simultaneously with LPS (25 mg/kg) or 3 days later, blocked the LPS-induced osteolysis in the mouse calvarium model. In vitro studies showed that low concentrations of PAR (<1 microM) inhibited in vitro osteoclastogenesis and osteoclastic bone resorption, whereas higher concentrations (>5 microM) triggered apoptotic cell death of osteoclasts and their precursor cells in a dose-dependent manner. Furthermore, PAR inhibited LPS-induced NF-kappaB activation, p65 translocation, and IkappaB-alpha degradation both in mature osteoclasts and their precursors in a time- and dose-dependent manner. In addition, PAR inhibited NF-kappaB activation induced by osteoclastogenic factors RANKL, interleukin (IL)-1beta, or TNF-alpha to varying degrees and reduced the gene expression of RANK and TRAF6. CONCLUSION: The NF-kappaB pathway is known to mediate both osteoclast differentiation and survival. These findings indicate that PAR blocks LPS-induced osteolysis through the suppression of NF-kappaB activity and suggest that it might have therapeutic value in bacteria-induced bone destruction.

12-O-tetradecanoylphorbol-13-acetate (TPA) inhibits osteoclastogenesis by suppressing RANKL-induced NF-kappaB activation.

UNLABELLED: The mechanism by which TPA-induced PKC activity modulates osteoclastogenesis is not clear. Using a RAW(264.7) cell culture system and assays for NF-kappaB nuclear translocation, NF-kappaB reporter gene activity, and MAPK assays, we demonstrated that TPA inhibits osteoclastogenesis through the suppression of RANKL-induced NF-kappaB activation. INTRODUCTION: The protein kinase C (PKC) pathway has been suggested to be an important regulator of osteoclastic bone resorption. The role of PKC in RANKL-induced osteoclastogenesis, however, is not clear. In this study, we examined the effects of 12-O-tetradecanoylphorbol-13-acetate (TPA), a PKC activator, on osteoclastogenesis and studied its role in RANKL-induced signaling. MATERIALS AND METHODS: RANKL-induced RAW(264.7) cell differentiation into osteoclast-like cells was used to assess the effect of TPA on osteoclastogenesis. Assays for NF-kappaB nuclear translocation, NF-kappaB reporter gene activity, protein kinase activity, and Western blotting were used to examine the effects of TPA on RANKL-induced NF-kappaB, c-Jun N-terminal kinase (JNK), and MEK/ERK and p38 signal transduction pathways. RESULTS: We found that TPA inhibited RANKL-induced RAW(264.7) cell differentiation into osteoclasts in a dose-dependent manner. Time course analysis showed that the inhibitory effect of TPA on RANKL-induced osteoclastogenesis occurs predominantly at an early stage of osteoclast differentiation. TPA alone had little effect on NF-kappaB activation in RAW(264.7) cells, but it suppresses the RANKL-induced NF-kappaB activation in a dose-dependent fashion. Interestingly, the suppressive effect of TPA on RANKL-induced NF-kappaB activation was prevented by a conventional PKC inhibitor, Go6976. Supershift studies revealed that the RANKL-induced DNA binding of NF-kappaB complexes consisted of C-Rel, NF-kappaB1 (p50), and RelA (p65). In addition, TPA induced the activation of JNK in RAW(264.7) cells but had little effect on RANKL-induced activation of JNK. TPA also inhibited RANKL-induced activation of ERK but had little effect on p38 activation. CONCLUSION: Given that NF-kappaB activation is obligatory for osteoclast differentiation, our studies imply that inhibition of osteoclastogenesis by TPA is, at least in part, caused by the suppression of RANKL-induced activation of NF-kappaB during an early stage of osteoclastogenesis. Selective modulation of RANKL signaling pathways by PKC activators may have important therapeutic implications for the treatment of bone diseases associated with enhanced bone resorption.

Pro-inflammatory effects of Burkholderia cepacia on cystic fibrosis respiratory epithelium.

Burkholderia cepacia causes pulmonary infection with high mortality in cystic fibrosis (CF) patients which is likely to involve interaction with respiratory epithelium. In this study the pro-inflammatory properties of B. cepacia were examined using a range of respiratory epithelial cell lines. B. cepacia and cell-free culture supernatants were used to stimulate cell lines with (SigmaCFTE29o- and IB3) and without (A549) the CF transmembrane conductance regulator mutation (CFTR), together with corrected cell lines (C38 and S9). Interleukin (IL)-6 and IL-8, but not GM-CSF or IL-1beta, were released from all the cell lines whereas PGE(2) (prostaglandin E(2)) was released from the A549, IB3 and S9 cell lines only. Nuclear factor (NF)-kappaB activation preceded cytokine release and suppression of NF-kappaB activity diminished cytokine release. These studies indicated that B. cepacia secretory products are potent pro-inflammatory agents for respiratory epithelium and suggest functional CFTR is not required for cytokine or prostanoid responses.

Tumor necrosis factor-alpha promotes survival in methotrexate-exposed macrophages by an NF-kappaB-dependent pathway.

INTRODUCTION: Methotrexate (MTX) induces macrophage apoptosis in vitro, but there is not much evidence for increased synovial macrophage apoptosis in MTX-treated patients. Macrophage apoptosis is reported, however, during clinical response to anti-tumor necrosis factor-alpha (TNF-α) treatments. This implies that TNF-α promotes macrophage survival and suggests that TNF-α may protect against MTX-induced apoptosis. We, therefore, investigated this proposal and the macrophage signaling pathways underlying it. METHODS: Caspase-3 activity, annexin-V binding/7-aminoactinomycin D (7-AAD) exclusion and cell-cycle analysis were used to measure steps in apoptosis of primary murine macrophages and cells of the RAW264.7 macrophage cell line that had been exposed to clinically-relevant concentrations of MTX and TNF-α. RESULTS: MTX induces apoptosis in primary murine macrophages at concentrations as low as 100 nM in vitro. TNF-α, which has a context-dependent ability to increase or to suppress apoptosis, efficiently suppresses MTX-induced macrophage apoptosis. This depends on NF-κB signaling, initiated through TNF Receptor Type 1 ligation. Macrophage colony stimulating factor, the primary macrophage survival and differentiation factor, does not activate NF-κB or protect macrophages from MTX-induced apoptosis. A weak NF-κB activator, Receptor Activator of NF-κB Ligand (RANKL) is likewise ineffective. Blocking NF-κB in TNF-α-exposed macrophages allowed pro-apoptotic actions of TNF-α to dominate, even in the absence of MTX. MTX itself does not promote apoptosis through interference with NF-κB signaling. CONCLUSIONS: These findings provide another mechanism by which TNF-α sustains macrophage numbers in inflamed tissue and identify a further point of clinical complementarity between MTX and anti-TNF-α treatments for rheumatoid arthritis.