Necrostatin-7 suppresses RANK-NFATc1 signaling and attenuates macrophage to osteoclast differentiation.

Osteoclasts play a crucial role in osteolytic bone diseases, such as osteoporosis, rheumatoid arthritis, periodontitis, Paget's disease of bone and bone metastatic tumors. Therefore, controlling osteoclast differentiation and function has been considered a promising therapeutic strategy. Here, we show that necrostatin (Nec)-7, an inhibitor of programmed necrosis, strongly suppressed receptor activator of nuclear factor (NF)-κB ligand (RANKL)-induced osteoclastogenesis and bone resorption, without compromising macrophage colony-stimulating factor (M-CSF)-supported survival and growth of osteoclast precursor cells. Accordingly, Nec-7 significantly decreased the levels of RANKL-induced osteoclastogenic marker genes, such as cathepsin K. Mechanistically, Nec-7 neither affected MAPK nor NF-κB activation; however, it strongly inhibited the RANKL receptor (RANK) to nuclear factor of activated T cells c1 (NFATc1) signaling. Lentiviral expression of RANK in bone marrow-derived macrophages significantly restored osteoclastogenesis and NFATc1 amplification in Nec-7-treated cells. In this study, we revealed that Nec-7-sensitive pathways are crucially involved in osteoclast formation and function. Investigation of the molecular mechanism(s) through which Nec-7 inhibits RANK-NFATc1 signaling axis may lead to the development of new therapeutic strategies for bone disease.

Actin-binding protein coronin 1A controls osteoclastic bone resorption by regulating lysosomal secretion of cathepsin K.

Osteoclasts degrade bone matrix proteins via the secretion of lysosomal enzymes. However, the precise mechanisms by which lysosomal components are transported and fused to the bone-apposed plasma membrane, termed ruffled border membrane, remain elusive. Here, we identified coronin 1A as a negative regulator of exocytotic release of cathepsin K, one of the most important bone-degrading enzymes in osteoclasts. The modulation of coronin 1A expression did not alter osteoclast differentiation and extracellular acidification, but strongly affected the secretion of cathepsin K and osteoclast bone-resorption activity, suggesting the coronin 1A-mediated regulation of lysosomal trafficking and protease exocytosis. Further analyses suggested that coronin 1A prevented the lipidation-mediated sorting of the autophagy-related protein LC3 to the ruffled border and attenuated lysosome-plasma membrane fusion. In this process, the interactions between coronin 1A and actin were crucial. Collectively, our findings indicate that coronin 1A is a pivotal component that regulates lysosomal fusion and the secretion pathway in osteoclast-lineage cells and may provide a novel therapeutic target for bone diseases.

Inhibition of MMP-2-Mediated Mast Cell Invasion by NF-κB Inhibitor DHMEQ in Mast Cells.

BACKGROUND: Stimulation with antigen and IgE is known to activate NF-κB in mast cells. In the present research, we studied the role of NF-κB on cellular migration in mast cell-like RBL-2H3 cells and bone marrow-derived mast cells (BMMCs) using the NF-κB inhibitor (-)-DHMEQ. METHODS: A Matrigel invasion chamber was used to evaluate cell migration. A PCR array was used to screen the expression of 84 key genes involved in cell migration. RESULTS: (-)-DHMEQ inhibited antigen/IgE-induced NF-κB activation and expressions of its target genes such as IL-6 and TNF-α. (-)-DHMEQ was found to inhibit in vitro invasion toward the antigen without any toxicity. We then looked for NF-κB-dependent genes that would be important for mast cell invasion using the PCR array. (-)-DHMEQ was found to lower the expression of matrix metalloproteinase (MMP)-2. The MMP inhibitor GM6001 also inhibited cellular invasion toward the antigen. These effects of (-)-DHMEQ were obtained in both RBL-2H3 cells and BMMCs. CONCLUSIONS: These findings indicate that (-)-DHMEQ suppressed mast cell migration via the inhibition of NF-κB-regulated MMP-2 expression.

Necrostatin-1 protects against reactive oxygen species (ROS)-induced hepatotoxicity in acetaminophen-induced acute liver failure.

Excessive acetaminophen (APAP) use is one of the most common causes of acute liver failure. Various types of cell death in the damaged liver are linked to APAP-induced hepatotoxicity, and, of these, necrotic cell death of hepatocytes has been shown to be involved in disease pathogenesis. Until recently, necrosis was commonly considered to be a random and unregulated form of cell death; however, recent studies have identified a previously unknown form of programmed necrosis called receptor-interacting protein kinase (RIPK)-dependent necrosis (or necroptosis), which is controlled by the kinases RIPK1 and RIPK3. Although RIPK-dependent necrosis has been implicated in a variety of disease states, including atherosclerosis, myocardial organ damage, stroke, ischemia-reperfusion injury, pancreatitis, and inflammatory bowel disease. However its involvement in APAP-induced hepatocyte necrosis remains elusive. Here, we showed that RIPK1 phosphorylation, which is a hallmark of RIPK-dependent necrosis, was induced by APAP, and the expression pattern of RIPK1 and RIPK3 in the liver overlapped with that of CYP2E1, whose activity around the central vein area has been demonstrated to be critical for the development of APAP-induced hepatic injury. Moreover, a RIPK1 inhibitor ameliorated APAP-induced hepatotoxicity in an animal model, which was underscored by significant suppression of the release of hepatic enzymes and cytokine expression levels. RIPK1 inhibition decreased reactive oxygen species levels produced in APAP-injured hepatocytes, whereas CYP2E1 expression and the depletion rate of total glutathione were unaffected. Of note, RIPK1 inhibition also conferred resistance to oxidative stress in hepatocytes. These data collectively demonstrated a RIPK-dependent necrotic mechanism operates in the APAP-injured liver and inhibition of this pathway may be beneficial for APAP-induced fulminant hepatic failure.

Cilostazol attenuates hepatic stellate cell activation and protects mice against carbon tetrachloride-induced liver fibrosis.

AIM: Liver fibrosis is a common pathway leading to cirrhosis. Cilostazol, a clinically available oral phosphodiesterase-3 inhibitor, has been shown to have antifibrotic potential in experimental non-alcoholic fatty liver disease. However, the detailed mechanisms of the antifibrotic effect and its efficacy in a different experimental model remain elusive. METHODS: Male C57BL/6J mice were assigned to five groups: mice fed a normal diet (groups 1 and 2); 0.1% or 0.3% cilostazol-containing diet (groups 3 and 4, respectively); and 0.125% clopidogrel-containing diet (group 5). Two weeks after feeding, groups 2-5 were intraperitoneally administered carbon tetrachloride (CCl4 ) twice a week for 6 weeks, while group 1 was treated with the vehicle alone. To investigate the effects of cilostazol on hepatic cells, in vitro studies were conducted using primary hepatic stellate cells (HSC), Kupffer cells and hepatocytes with cilostazol supplementation. RESULTS: Sirius red staining revealed that groups 3 and 4 exhibited a lesser fibrotic area (2.49 ± 0.43% and 2.31 ± 0.30%, respectively) than group 2 (3.17 ± 0.67%, P < 0.05 and P < 0.001, respectively). In vitro studies showed cilostazol dose-dependently suppressed HSC activation (assessed by morphological change, cell proliferation, and the expression of HSC activation markers), suggesting the therapeutic effect of cilostazol is mediated by its direct action on HSC. CONCLUSION: Cilostazol could alleviate CCl4 -induced hepatic fibrogenesis in vivo, presumably due, at least partly, to its direct effect to suppress HSC activation. Given its clinical availability and safety, it may be a novel therapeutic intervention for chronic liver diseases.

Structure-activity relationship of 9-methylstreptimidone, a compound that induces apoptosis selectively in adult T-cell leukemia cells.

We previously reported that 9-methylstreptimidone, a piperidine compound isolated from a culture filtrate of Streptomyces, induces apoptosis selectively in adult T-cell leukemia cells. It was screened for a compound that inhibits LPS-induced NF-kappaB and NO production in mouse macrophages. However, 9-methystreptimidone is poorly obtained from the producing microorganism and difficult to synthesize. Therefore, in the present research, we studied the structure-activity relationship to look for new selective inhibitors. We found that the structure of the unsaturated hydrophobic portion of 9-methylstreptimidone was essential for the inhibition of LPS-induced NO production. Among the 9-methylstreptimidone-related compounds tested, (+/-)-4,alpha-diepi-streptovitacin A inhibited NO production in macrophage-like cells as potently as 9-methylstreptimidone and without cellular toxicity. Moreover, this compound selectively induced apoptosis in adult T-cell leukemia MT-1 cells.

Involvement of DNA binding domain in the cellular stability and importin affinity of NF-κB component RelB.

NF-κB is a transcription factor for the immune activation and tissue stability, but excess activation of NF-κB often causes inflammation and cancer. An NF-κB component RelB is involved in B-cell maturation and autoimmunity. In the present research we studied the role of the RelB DNA binding domain on cellular stability and importin affinity. We prepared a RelB protein mutated at Arg141 to Ala and Tyr142 to Ala (AA mutant) having no DNA binding activity. The stability of this mutant protein was greatly reduced compared with that of the wild-type protein. We also constructed a nuclear localization signal-inactivated mutant of RelB, and found that this mutant was also unstable in the cells. Thus, RelB destabilization was caused by the loss of DNA binding possibly because of the change in cellular localization. The mutation also decreased the affinity to importin-α5 decreasing the nuclear localization. Our newly discovered NF-κB inhibitor (-)-DHMEQ binds to a specific Cys residue in RelB to inhibit DNA binding and also decreased the stability and importin affinity. These findings would indicate that the DNA binding activity of this transcription factor is a crucial for its stability and intracellular localization.

Synthesis and biological evaluation of molecular probes based on the 9-methylstreptimidone derivative DTCM-glutarimide.

Molecular probes based on 3-[(dodecylthiocarbonyl)methyl]glutarimide (DTCM-glutarimide) were synthesized and assessed for inhibitory activity against LPS-induced NO production. Among the probes examined, several derivatives exhibited potential for use in determining the target proteins of DTCM-glutarimide.

Design and synthesis of biotinylated DHMEQ for direct identification of its target NF-κB components.

The design and synthesis of dehydroxymethylepoxyquinomicin (DHMEQ) derivatives were carried out to investigate the intracellular targets. The synthetic biotin probe exhibited membrane permeability and combined selectively with the target protein p65.

Inhibition of macrophage activation and suppression of graft rejection by DTCM-glutarimide, a novel piperidine derived from the antibiotic 9-methylstreptimidone.

OBJECTIVE: We have previously synthesized a novel piperidine compound, 3-[(dodecylthiocarbonyl)methyl]glutarimide (DTCM-glutarimide), that inhibits LPS-induced NO production, and in the present research we studied further the anti-inflammatory activity of DTCM-glutarimide in a macrophage cell line and in mice bearing transplanted hearts. MATERIALS AND METHODS: Mouse macrophage-like RAW264.7 cells were employed for the evaluation of cellular inflammatory activity. DTCM-glutarimide was synthesized in our laboratory. The AP-1 activity was measured by nuclear translocation and phosphorylation. For the heart transplantation experiment, male C57BL/6 (H-2b) and BALB/c (H-2d) mice were used as donor and recipient, respectively. DTCM-glutarimide was administered intraperitoneally. RESULTS: DTCM-glutarimide inhibited the LPS-induced expression of iNOS and COX-2 in macrophages; but, unexpectedly, it did not inhibit LPS-induced NF-κB activation. Instead, it inhibited the nuclear translocation of both c-Jun and c-Fos. It also inhibited LPS-induced c-Jun phosphorylation. Moreover, it inhibited the mixed lymphocyte reaction in primary cultures of mouse spleen cells; and furthermore, in mice it prolonged the graft survival in heart transplantation experiments. CONCLUSION: The novel piperidine compound, DTCM-glutarimide, was found to be a new inhibitor of macrophage activation, inhibiting AP-1 activity. It also inhibited graft rejection in mice, and thus may be a candidate for an anti-inflammatory agent.

A new NF-κB inhibitor based on the amino-epoxyquinol core of DHMEQ.

The amino-epoxyquinols 6a and 6b were synthesized as soluble derivatives of an NF-κB inhibitor DHMEQ (1). In spite of the opposite configuration from 1, 6b rather than 6a affected the deactivation of NF-κB, based on NO secretion and MALDI-TOF MS analysis. It was indicated that 6b inhibited the activation by different manner from that of 1.

Inactivation of NF-kappaB components by covalent binding of (-)-dehydroxymethylepoxyquinomicin to specific cysteine residues.

Previously, we designed and synthesized a potent NF-kappaB inhibitor, DHMEQ. Although DHMEQ showed potent anti-inflammatory and anticancer activities in animals, its molecular target has not been elucidated. In the present study, its target protein was found to be p65 and other Rel homology proteins. We found that (-)-DHMEQ bound to p65 covalently with a 1:1 stoichiometry by conducting SPR and MALDI-TOF MS analyses. MS analysis of the chymotrypsin-digested peptide suggested the binding of (-)-DHMEQ to a Cys residue. Formation of Cys/(-)-DHMEQ adduct in the protein was supported by chemical synthesis of the adduct. Substitution of specific Cys in p65 and other Rel homology proteins resulted in the loss of (-)-DHMEQ binding. (-)-DHMEQ is the first NF-kappaB inhibitor that was proven to bind to the specific Cys by chemical methodology. These findings may explain the highly selective inhibition of NF-kappaB and the low toxic effect of (-)-DHMEQ in cells and animals.