Bacterial lipopolysaccharide induces osteoclast formation in RAW 264.7 macrophage cells

Lipopolysaccharide (LPS) is a potent bone resorbing factor. The effect of LPS on osteoclast formation was examined by using murine RAW 264.7 macrophage cells. LPS-induced the formation of multinucleated giant cells (MGC) in RAW 264.7 cells 3 days after the exposure. MGCs were positive for tartrate-resistant acid phosphatase (TRAP) activity. Further, MGC formed resorption pits on calcium-phosphate thin film that is a substrate for osteoclasts. Therefore, LPS was suggested to induce osteoclast formation in RAW 264.7 cells. LPS-induced osteoclast formation was abolished by anti-tumor necrosis factor (TNF)-alpha antibody, but not antibodies to macrophage-colony stimulating factor (M-CSF) and receptor activator of nuclear factor (NF)-kappaB ligand (RANKL). TNF-alpha might play a critical role in LPS-induced osteoclast formation in RAW 264.7 cells. Inhibitors of NF-kappaB and stress activated protein kinase (SAPK/JNK) prevented the LPS-induced osteoclast formation. The detailed mechanism of LPS-induced osteoclast formation is discussed.     

Prostaglandin E2 enhances osteoclastic differentiation of precursor cells through protein kinase A-dependent phosphorylation of TAK1

Prostaglandin E2 (PGE2) synergistically enhances the receptor activator for NF-kappa B ligand (RANKL)-induced osteoclastic differentiation of the precursor cells. Here we investigated the mechanisms of the stimulatory effect of PGE2 on osteoclast differentiation. PGE2 enhanced osteoclastic differentiation of RAW264.7 cells in the presence of RANKL through EP2 and EP4 prostanoid receptors. RANKL-induced degradation of I kappa B alpha and phosphorylation of p38 MAPK and c-Jun N-terminal kinase in RAW264.7 cells were up-regulated by PGE2 in a cAMP-dependent protein kinase A (PKA)-dependent manner, suggesting that EP2 and EP4 signals cross-talk with RANK signals. Transforming growth factor beta-activated kinase 1 (TAK1), an important MAPK kinase kinase in several cytokine signals, possesses a PKA recognition site at amino acids 409-412. PKA directly phosphorylated TAK1 in RAW264.7 cells transfected with wild-type TAK1 but not with the Ser412 --> Ala mutant TAK1. Ser412 --> Ala TAK1 served as a dominant-negative mutant in PKA-enhanced degradation of I kappa B alpha, phosphorylation of p38 MAPK, and PGE2-enhanced osteoclastic differentiation in RAW264.7 cells. Furthermore, forskolin enhanced tumor necrosis factor alpha-induced I kappa B alpha degradation, p38 MAPK phosphorylation, and osteoclastic differentiation in RAW264.7 cells. Ser412 --> Ala TAK1 abolished the stimulatory effects of forskolin on those cellular events induced by tumor necrosis factor alpha. Ser412 --> Ala TAK1 also inhibited the forskolin-induced up-regulation of interleukin 6 production in RAW264.7 cells treated with lipopolysaccharide. These results suggest that the phosphorylation of the Ser412 residue in TAK1 by PKA is essential for cAMP/PKA-induced up-regulation of osteoclastic differentiation and cytokine production in the precursor cells.     

Signal transduction mechanisms of CD137 ligand in human monocytes

Bidirectional signalling, i.e. simultaneous signalling through a receptor as well as its cell surface-bound ligand has been identified for several members of the TNF and TNF receptor family members. Reverse signalling through the ligands offers the advantage of an immediate feed-back and a more precise fine tuning of biological responses. Little is known about the molecular nature of reverse signalling through the ligands. CD137 ligand, member of the TNF family is expressed on monocytes and induces activation, migration, prolongation of survival and proliferation of monocytes. Here we show that reverse signalling by CD137 ligand is mediated by protein tyrosine kinases, p38 mitogen activated protein kinase (MAPK), extracellular signal-regulated kinase (ERK)1,2, MAP/ERK kinase (MEK), Phosphoinositide-3-kinase (PI3-K) and protein kinase A (PKA) but not by protein kinase C (PKC). This study also shows that reverse signalling relies on the same signal transduction molecules as signalling through classical receptors and is in its nature not different from it.     

Casein kinase I epsilon associates with and phosphorylates the tight junction protein occludin

Occludin is an integral-membrane protein that contributes to tight junction function. We have identified casein kinase I epsilon (CKI epsilon) as a binding partner for the C-terminal cytoplasmic domain of occludin by yeast two-hybrid screening. CKI epsilon phosphorylated occludin and co-localised and co-immunoprecipitated with occludin from human endothelial cells. Amino acids 265-318 of occludin were sufficient for CKI epsilon binding and phosphorylation. Deletion of the C-terminal 48 amino acids of occludin increased CKI epsilon binding and phosphorylation, suggesting that this region inhibits CKI epsilon binding. These data identify CKI epsilon as a novel occludin kinase that may be important for the regulation of occludin.     

The ups and downs of MEK kinase interactions

MEK kinases (MEKKs) comprise a family of related serine–threonine protein kinases that regulate mitogen-activated protein kinase (MAPK) signalling pathways leading to c-Jun NH₂-terminal kinase (JNK) and p38 activation, induced by cellular stress (e.g., UV and γ irradiation, osmotic stress, heat shock, protein synthesis inhibitors), inflammatory cytokines (e.g., tumour necrosis factor , TNF, and interleukin-1, IL1) and G protein-coupled receptor agonists (e.g., thrombin). These stress-activated kinases have been implicated in apoptosis, oncogenic transformation, and inflammatory responses in various cell types. At present, the signalling events involving MEKKs are not well understood. This review summarises our current knowledge concerning the regulation and function of MEKK family members, with particular emphasis on those factors capable of directly interacting with distinct MEKK isoforms.     

NF-kappaB overrides the apoptotic program of TNF receptor 1 but not CD40 in carcinoma cells

The activation of NF-kappaB and phosphatidylinositol-3 (PI3) kinase by TNF-alpha and TRAIL overrides the pro-apoptotic effects of these ligands in carcinoma cells and hinders their therapeutic application. In this report we show that CD40 ligand, another member of the TNF superfamily, also triggers the activation of these signalling pathways but, importantly, utilises only the PI3 kinase cascade for anti-apoptotic responses, inasmuch as suppression of PI3 kinase but not NF-kappaB sensitises carcinoma cells to CD40L-induced apoptosis. Therefore, NF-kappaB activation does not always confer anti-apoptotic effects. Moreover, no cross-talk between the two pathways was observed, as the specific suppression of PI3 kinase with chemical inhibitors did not influence CD40-mediated IkappaBalpha phosphorylation and degradation or NF-kappaB binding and transactivation. Similarly, whilst suppression of Akt expression by RNA interference sensitised tumour cells to CD40L-induced apoptosis, it had no effect on CD40-mediated IkappaBalpha degradation. These data provide new evidence for the role of NF-kappaB and PI3 kinase/Akt in phenotypic effects mediated by CD40 ligation and highlight differences in the mechanisms by which TNF family members regulate apoptosis in carcinoma cells.     

The tumor necrosis factor receptor stalk regions define responsiveness to soluble versus membrane-bound ligand

The family of tumor necrosis factor receptors (TNFRs) and their ligands form a regulatory signaling network that controls immune responses. Various members of this receptor family respond differently to the soluble and membrane-bound forms of their respective ligands. However, the determining factors and underlying molecular mechanisms of this diversity are not yet understood. Using an established system of chimeric TNFRs and novel ligand variants mimicking the bioactivity of membrane-bound TNF (mTNF), we demonstrate that the membrane-proximal extracellular stalk regions of TNFR1 and TNFR2 are crucial in controlling responsiveness to soluble TNF (sTNF). We show that the stalk region of TNFR2, in contrast to the corresponding part of TNFR1, efficiently inhibits both the receptor's enrichment/clustering in particular cell membrane regions and ligand-independent homotypic receptor preassembly, thereby preventing sTNF-induced, but not mTNF-induced, signaling. Thus, the stalk regions of the two TNFRs not only have implications for additional TNFR family members, but also provide potential targets for therapeutic intervention.     

Engagement of tumor necrosis factor mRNA by an endotoxin-inducible cytoplasmic protein.

BACKGROUND: Tumor necrosis factor (TNF) production by macrophages plays an important role in the host response to infection. TNF-alpha gene expression in RAW 264.7 macrophages is predominantly regulated at the translational level. A key element in this regulation is an AU-rich (AUR) sequence located in the 3' untranslated region (UTR) of TNF mRNA. In unstimulated macrophages, the translation of TNF mRNA is inhibited via this AUR sequence. Upon stimulation with LPS, this repression is overcome and translation occurs. In this study, we attempted to identify cellular proteins that interact with the AUR sequence and thereby regulate TNF mRNA translation. MATERIALS AND METHODS: RNA probes corresponding to portions of TNF mRNA 3' UTR were synthesized. These labeled RNAs were incubated with cytoplasmic extracts of either unstimulated or lipopolysaccharides (LPS)-stimulated RAW 264.7 macrophages. The RNA/protein complexes formed were analyzed by gel retardation. Ultraviolet (UV) cross-linking experiments were performed to determine the molecular weight of the proteins involved in the complexes. RESULTS: TNF mRNA AUR sequence formed two complexes (1 and 2) of distinct electrophoretic mobilities. While the formation of complex 1 was independent of the activation state of the macrophages from which the extracts were obtained, complex 2 was detected only using cytoplasmic extracts from LPS-stimulated macrophages. Upon UV cross-linking, two proteins, of 50 and 80 kD, respectively, were capable of binding the UAR sequence. The 50-kD protein is likely to be part of the LPS-inducible complex 2, since its binding ability was enhanced upon LPS stimulation. Interestingly, complex 2 formation was also triggered by Sendaï virus infection, another potent activator of TNF mRNA translation in RAW 264.7 macrophages. In contrast, complex 2 was not detected with cytoplasmic extracts obtained from B and T cell lines which are unable to produce TNF in response to LPS. Protein tyrosine phosphorylation is required for LPS-induced TNF mRNA translation. Remarkably, the protein tyrosine phosphorylation inhibitor herbimycin A abolished LPS-induced complex 2 formation. Complex 2 was already detectable after 0.5 hr of LPS treatment and was triggered by a minimal LPS dose of 10 pg/ml. CONCLUSIONS: The tight correlation between TNF production and the formation of an LPS-inducible cytoplasmic complex suggests that this complex plays a role in the translational regulation of TNF mRNA.     

Hydrogen peroxide induces the production of tumor necrosis factor-alpha in RAW 264.7 macrophage cells via activation of p38 and stress-activated protein kinase

The effect of hydrogen peroxide (H(2)O(2)) on production of tumor necrosis factor (TNF)-alpha was examined in RAW 264.7 murine macrophage cells. H(2)O( 2) led to production of TNF-alpha up to 24 h after the treatment, but not nitric oxide in RAW 264.7 cells. H(2)O(2) induced TNF-alpha production in mouse peritoneal macrophages as well as RAW 264.7 cells. The H(2)O(2)induced TNF-alpha production was prevented by inhibitors of p38 and stress-activated protein kinase (SAPK/JNK), and H(2)O( 2) induced the phosphorylation of p38 and SAPK. Further, H(2)O( 2) significantly augmented the AP-1 activity, but not nuclear factor (NF)-kappaB activity in RAW 264.7 cells. A high level of intracellular reactive oxygen radicals (ROS) was detected in H(2)O(2)-exposed RAW 264.7 cells. Ebselen, a cell permeable antioxidant, prevented the H( 2)O(2)-induced TNFalpha production. H(2)O(2) significantly enhanced lipopolysaccharide (LPS)-induced TNF-alpha production. Therefore, H( 2) O(2) was suggested to induce TNF-alpha production in macrophages via activating p38 and SAPK/JNK as oxidative stress-related signal pathways.     

TRAF-6 dependent signaling pathway is essential for TNF-related apoptosis-inducing ligand (TRAIL) induces osteoclast differentiation

Human osteoclast formation from mononuclear phagocyte precursors involves interactions between tumor necrosis factor (TNF) ligand superfamily members and their receptors. Recent evidence indicates that in addition to triggering apoptosis, the TNF-related apoptosis-inducing ligand (TRAIL) induces osteoclast differentiation. To understand TRAIL-mediated signal transduction mechanism in osteoclastogenesis, we demonstrated that TRAIL induces osteoclast differentiation via a Tumor necrosis factor receptor-associated factor 6 (TRAF-6)-dependent signaling pathway. TRAIL-induced osteoclast differentiation was significantly inhibited by treatment with TRAF-6 siRNA and TRAF6 decoy peptides in both human monocytes and murine RAW264.7 macrophage cell lines, as evaluated in terms of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells and bone resorption activity. Moreover, TRAIL-induced osteoclast differentiation was also abolished in TRAF6 knockout bone marrow macrophages. In addition to induction of NFATc1, treatment of TRAIL also induced ubiquitination of TRAF6 in osteoclast differentiation. Thus, our data demonstrate that TRAIL induces osteoclastic differentiation via a TRAF-6 dependent signaling pathway. This study suggests TRAF6-dependent signaling may be a central pathway in osteoclast differentiation, and that TNF superfamily molecules other than RANKL may modify RANK signaling by interaction with TRAF6-associated signaling.     

Ganglioside GM3 suppresses lipopolysaccharide‐induced inflammatory responses in rAW 264.7 macrophage cells through NF‐κB,AP‐1, and MAPKs signaling

Gangliosides are known to specifically inhibit vascular leukocyte recruitment and consequent interaction with the injured endothelium, the basic inflammatory process. In this study, we have found that the production of nitric oxide (NO), a main regulator of inflammation, is suppressed by GM3 on murine macrophage RAW 264.7 cells, when induced by LPS. In addition, GM3 attenuated the increase in cyclooxyenase‐2 (COX‐2) protein and mRNA levels in lipopolysaccharide (LPS)‐activated RAW 264.7 cells in a dose‐dependent manner. Moreover, GM3 inhibited the expression and release of pro‐inflammatory cytokines of tumor necrosis factor‐alpha (TNF‐α), interleukin‐6 (IL‐6), and interleukin‐1β (IL‐1β) in RAW 264.7 macrophages. At the intracellular level, GM3 inhibited LPS‐induced nuclear translocation of nuclear factor kappa‐light‐chain‐enhancer of activated B cells (NF‐κB) and activator protein (AP)‐1 in RAW 264.7 macrophages. We, therefore, investigated whether GM3 affects mitogen‐activated protein kinase (MAPK) phosphorylation, a process known as the upstream signaling regulator. GM3 dramatically reduced the expression levels of the phosphorylated forms of ERK, JNK, and p38 in LPS‐activated RAW 264.7 cells. These results indicate that GM3 is a promising suppressor of the vascular inflammatory responses and ganglioside GM3 suppresses the LPS‐induced inflammatory response in RAW 264.7 macrophages by suppression of NF‐κB, AP‐1, and MAPKs signaling. Accordingly, GM3 is suggested as a beneficial agent for the treatment of diseases that are associated with inflammation.     

Localization of Sites in the Tail Domain of the Middle Molecular Mass Neurofilament Subunit Phosphorylated by a Neurofilament-Associated Kinase and by Casein Kinase I

Abstract: We have shown previously that a neurofilament (NF)-associated kinase (NFAK) extracted from chicken NF preparations phosphorylates selectively the middle molecular mass NF subunit (NF-M). Here we show that the major kinase activity in NFAK is indistinguishable from enzymes of the casein kinase I (CKI) family based on the following criteria: (1) inhibition of NFAK phosphorylation by the selective CKI inhibitor CKI-7, (2) the similarity in substrate specificity of NFAK and authentic CKI, (3) the correspondence of two-dimensional phosphopeptide maps of NF-M phosphorylated in vitro by NFAK with those generated by CKI under similar conditions, and (4) immunological cross-reactivity of NFAK with an antibody raised against CKI. We have also identified Ser⁵⁰², Ser⁵²⁸, and Ser⁵³⁶ as phosphorylation sites by NFAK/CKI in vitro, each of which is also phosphorylated in vivo. All three serines are found in peptides with CKI phosphorylation consensus sequences, and Ser⁵²⁸ and Ser⁵³⁶ and flanking amino acids are highly conserved in higher vertebrate NF-M sequences. Neither Ser⁵⁰² nor Ser⁵³⁶ has been identified previously as NF-M phosphorylation sites.     

Heterotrimers formed by tumor necrosis factors of different species or muteins

Incubation of murine tumor necrosis factor (mTNF) at subnanomolar concentrations results in partial dissociation of the trimers, coinciding with a decrease in bioactivity. Using size-exclusion chromatography, we observed that the conversion of labeled mTNF to monomers is not only prevented by coincubation with an excess of unlabeled mTNF but also with unlabeled human TNF (hTNF). Moreover, after coincubation of mTNF and hTNF four different TNF complexes were revealed by native polyacrylamide gel electrophoresis, viz. homotrimeric mTNF and hTNF, as well as two complexes with an intermediate migration pattern. Analytical gel filtration in combination with native polyacrylamide gel electrophoresis and Western blot immunodetection indicated that these new complexes consisted of heterotrimeric TNF molecules. We conclude that an exchange of monomers takes place during coincubation of two different species of TNF, which results in homotrimeric and heterotrimeric TNF. To assess receptor interaction in vitro, TNF heterotrimeric molecules were used as obtained after incubation of mTNF with labeled hTNF (which only binds to mTNF receptor I) or with labeled mutein mTNF75 (specific for mTNF receptor II). These heterotrimers were retained by both mTNF receptors, which means that the mTNF subunits incorporated in heterotrimeric complexes still can bind to both types of TNF receptor. In addition, the gradual decrease in mTNF bioactivity during preincubation at subnanomolar concentrations was prevented by the presence of mutein mTNF75, which is inactive in an L929 cytotoxicity assay, indicating that heterotrimerization can influence the overall bioactivity.     

hCEACAM1-4L downregulates hDAF-associated signalling after being recognized by the Dr adhesin of diffusely adhering Escherichia coli

Human decay accelerating factor (hDAF, CD55) and members of the c arcino e mbryonic- a ntigen-related c ell- a dhesion m olecules (hCEACAMs) family are recognized as receptors by Gram-negative, diffusely adhering Escherichia coli (DAEC) strains expressing Afa/Dr adhesins. We report here that hCEACAM1-4L has a key function in downregulating the protein tyrosine Src kinase associated with hDAF signalling. After infecting HeLa epithelial cells stably transfected with hCEACAM1-4L cDNA with Dr adhesin-positive E. coli , the amount of the pTyr⁴¹⁶-active form of the Src protein decreased, whereas that of the pTyr⁵²⁷-inactive form of Src protein did not increase. This downregulation of the Src protein implies that part of the hCEACAM1-4L protein had been translocated into lipid rafts, the protein was phosphorylated at Tyr residues in the cytoplasmic domain, and it was physically associated with the protein tyrosine phosphatase, SHP-2. Finally, we found that the hCEACAM1-4L-associated SHP-2 was not phosphorylated and lacked phosphatase activity, suggesting that the downregulation of Src protein associated with hDAF signalling results from the absence of dephosphorylation of the pTyr⁵²⁷-inactive form necessary for Src kinase activation.     

Regulatory roles and molecular signaling of TNF family members in osteoclasts

The tumor necrosis factor (TNF) family has been one of the most intensively studied families of proteins in the past two decades. The TNF family constitutes 19 members that mediate diverse biological functions in a variety of cellular systems. The TNF family members regulate cellular functions through binding to membrane-bound receptors belonging to the TNF receptor (TNFR) family. Members of the TNFR family lack intrinsic kinase activity and thus they initiate signaling by interacting intracellular signaling molecules such as TNFR associated factor (TRAF), TNFR associated death domain (TRADD) and Fas-associated death domain (FADD). In bone metabolism, it has been shown that numerous TNF family members including receptor activator of nuclear factor kappaB ligand (RANKL), TNF-alpha, Fas ligand (FasL) and TNF-related apoptosis-inducing ligand (TRAIL) play pivotal roles in the differentiation, function, survival and/or apoptosis of osteoclasts, the principal bone-resorbing cells. These TNF family members not only regulate physiological bone remodeling but they are also implicated in the pathogenesis of various bone diseases such as osteoporosis and bone loss in inflammatory conditions. This review will focus on our current understanding of the regulatory roles and molecular signaling of these TNF family members in osteoclasts.     

Association of CPI-17 with protein kinase C and casein kinase I

The protein kinase C-potentiated inhibitor protein of 17kDa, called CPI-17, specifically inhibits myosin light chain phosphatase (MLCP). Phosphorylation of Thr-38 in vivo highly potentiates the ability of CPI-17 to inhibit MLCP. Thr-38 has been shown to be phosphorylated in vitro by a number of protein kinases including protein kinase C (PKC), Rho-associated coiled-coil kinase (ROCK), and protein kinase N (PKN). In this study we have focused on the association of protein kinases with CPI-17. Using affinity chromatography and Western blot analysis, we found interaction with all PKC isotypes and casein kinase I isoforms, CKIalpha and CKI. By contrast, ROCK and PKN did not associate with CPI-17, suggesting that PKC may be the relevant kinase that phosphorylates Thr-38 in vivo. CPI-17 interacted with the cysteine-rich domain of PKC and was phosphorylated by all PKC isotypes. We previously found that CPI-17 co-purified with casein kinase I in brain suggesting they are part of a complex and we now show that CPI-17 associates with the kinase domain of CKI isoforms.     

Analysis of amphotericin B-induced cell signaling with chemical inhibitors of signaling molecules

Although amphotericin B (AmB) is a major polyene antibiotic against invasive fungal infection, administration to patients sometimes causes inflammatory side effects, which limits the usage of the antibiotic. We studied the intracellular signaling that was induced by AmB. p65 (RelA) of nuclear factor‐κB (NF‐κB), a well‐known signaling molecule as an inducer of proinflammatory cytokines, was phosphorylated by AmB in RAW264.7 cells, a monocyte‐like cell line. Among chemical inhibitors of signaling molecules, U‐73122 (phospholipase C (PLC) inhibitor), Gö6976 (protein kinase C (PKC) inhibitor), BAPTA‐AM (calcium chelator), LFM‐A13 (Bruton's tyrosine kinase (Btk)‐specific inhibitor), and PP2 (c‐Src kinase inhibitor) suppressed AmB‐induced phosphorylation of p65 and translocation of p65 into the nucleus. U‐73122 and Gö6976 reduced AmB‐mediated induction of proinflammatory cytokines (tumor necrosis factor (TNF)‐α and interleukin (IL)‐6) in RAW264.7 cells. Furthermore, AmB‐induced activation of NF‐ κ B was observed in toll‐like receptor (TLR) 2‐expressed cells, and the activation of NF‐κB was inhibited by U‐73122, whereas peptidoglycan‐induced NF‐κB activation, which was also dependent on TLR2, was not inhibited by U‐73122. Finally, U‐73122 partially suppressed in vivo production of TNF‐α and IL‐6 induced by AmB administration in BALB/c mice. These results suggested that the signaling from AmB stimulation to proinflammatory cytokine production is mediated by TLR2, Btk, PLC, PKC, c‐Src and NF‐κB. These signaling molecules may become a target for chemotherapy suppressing AmB‐induced proinflammatory cytokine production.     

Site-specific casein kinase 1epsilon-dependent phosphorylation of Dishevelled modulates beta-catenin signaling

Careful regulation of the Wnt-Beta-catenin signaling pathway is critical to many aspects of development and cancer. Casein kinase Iepsilon is a Wnt-activated positive regulator of this pathway. Members of the Dishevelled family have been identified as key substrates of casein kinase I (CKI). However, the specific sites phosphorylated in vivo by CKI and their relative importance in the physiologic regulation of these proteins in the canonical Wnt-beta-catenin signaling pathway remain unclear. To address this question, recombinant mouse Dishevelled (mDvl-1) was phosphorylated by CKIin vitro and phosphorylation sites were identified by MS. CKI phosphorylation of mDvl-1 at two highly conserved residues, serines 139 and 142, was observed by MS and confirmed by phosphopeptide mapping of in vivo phosphorylated protein. Phosphorylation of these sites is dependent on casein kinase I epsilon activity in vivo. Phenotypic analysis of mutant mDvl-1 indicates that phosphorylation of these sites stimulates the Dvl-activated beta-catenin-dependent Wnt signaling pathway in both cell culture and in Xenopus development. Casein kinase I epsilon is a Wnt-regulated kinase, and regulated phosphorylation of Dvl allows fine tuning of the Wnt-beta-catenin signaling pathway.