Prenylated quinolinecarboxylic acid derivative prevents neuronal cell death through inhibition of MKK4.

The development of neuroprotective agents is necessary for the treatment of neurodegenerative diseases. Here, we report PQA-11, a prenylated quinolinecarboxylic acid (PQA) derivative, as a potent neuroprotectant. PQA-11 inhibits glutamate-induced cell death and caspase-3 activation in hippocampal cultures, as well as inhibits N-Methyl-4-phenylpyridinium iodide- and amyloid ß1-42-induced cell death in SH-SY5Y cells. PQA-11 also suppresses mitogen-activated protein kinase kinase 4 (MKK4) and c-jun N-terminal kinase (JNK) signaling activated by these neurotoxins. Quartz crystal microbalance analysis and in vitro kinase assay reveal that PQA-11 interacts with MKK4, and inhibits its sphingosine-induced activation. The administration of PQA-11 by intraperitoneal injection alleviates 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced degeneration of nigrostriatal dopaminergic neurons in mice. These results suggest that PQA-11 is a unique MKK4 inhibitor with potent neuroprotective effects in vitro and in vivo. PQA-11 may be a valuable lead for the development of novel neuroprotectants.

Mitochondrial reactive oxygen species suppress humoral immune response through reduction of CD19 expression in B cells in mice.

Reactive oxygen species (ROS) are implicated in the modulation of diverse processes including immune responses. To evaluate the effects of metabolic ROS produced by mitochondria on B-cell function and development, we created transgenic (Tg) mice expressing a phosphorylation-defective mutant of succinate dehydrogenase A in B cells (bSDHAY215F ). Splenic B cells in male, but not female, bSDHAY215F mice produced three times more ROS than those in the control mice, and had decreased production of IgM, IgG1 , and IgG3 , and affinity maturation of IgG1 against T-cell-dependent antigens. Following immunization, the male bSDHAY215F mice further displayed suppressed germinal center (GC) formation, and proliferation of GC B cells. Signaling analysis revealed defects in the intrinsic BCR responses, such as activation of Lyn, Btk, and PLCγ2, thus resulting in reduced intracellular Ca2+ mobilization. Notably, the expression levels of B-cell co-receptor CD19 and its interaction with Lyn after BCR ligation were significantly reduced in B cells from male bSDHAY215F mice. These results suggest that mitochondrial ROS suppress humoral immune responses through reduction of CD19 expression and resultant BCR signaling in B cells. Therefore, B-cell immunity may be more labile to oxidative stress in male mice than in female mice.

Enhanced cAMP-stimulated protein kinase A activity in human fibrolamellar hepatocellular carcinoma.

BACKGROUND: Fibrolamellar hepatocellular carcinoma (FL-HCC) affects children without underlying liver disease. A consistent mutation in FL-HCCs leads to fusion of the genes encoding a heat shock protein (DNAJB1) and the catalytic subunit of protein kinase A (PRKACA). We sought to characterize the resultant chimeric protein and its effects in FL-HCC. METHODS: The expression pattern and subcellular localization of protein kinase A (PKA) subunits in FL-HCCs were compared to paired normal livers by quantitative polymerase chain reaction (qPCR), immunoblotting, and immunofluorescence. PKA activity was measured by radioactive kinase assay, and we determined whether the FL-HCC mutation is present in other primary liver tumors. RESULTS: The fusion transcript and chimeric protein were detected exclusively in FL-HCCs. DNAJB1-PRKACA was expressed 10-fold higher than the wild-type PRKACA transcript, resulting in overexpression of the mutant protein in tumors. Consequently, FL-HCCs possess elevated cAMP-stimulated PKA activity compared to normal livers, despite similar Kms between the mutant and wild-type kinases. CONCLUSION: FL-HCCs in children and young adults uniquely overexpress DNAJB1-PRKACA, which results in elevated cAMP-dependent PKA activity. These data suggest that aberrant PKA signaling contributes to liver tumorigenesis.

Prenylated quinolinecarboxylic acid derivative suppresses immune response through inhibition of PAK2.

Development of new immunosuppressing agents is necessary in organ transplantation or immune diseases. Because Ppc-1 exhibits a suppressing effect on interleukin-2 (IL2) production in Jurkat cells, we synthesized and screened Ppc-1 derivatives that preserve prenylated quinolinecarboxylic acid (PQA) structure, and identified compound 18 (PQA-18) as a novel molecule with immunosuppressing effect. PQA-18 suppressed not only IL2 but also IL4, IL6, and tumor necrosis factor-α production in human peripheral lymphocytes without affecting cell viability. Two-dimensional gel electrophoresis analysis and in vitro kinase assay revealed that PQA-18 inhibits kinase activity of p21-activated kinase 2 (PAK2). Administration of PQA-18 by intraperitoneal injection suppressed the population of a subset of regulatory T cells and the immunoglobulin (Ig) production against T cell-dependent antigens in mice. Treatment with the PQA-18 ointment on Nc/Nga mice, a model of human atopic dermatitis, improved skin lesions and serum IgE levels. These results suggest that PQA-18 is a unique PAK2 inhibitor with potent immunosuppressing effects in vitro and in vivo. PQA-18 may be a valuable lead for the development of novel immunosuppressants.

Weight loss by Ppc-1, a novel small molecule mitochondrial uncoupler derived from slime mold.

Mitochondria play a key role in diverse processes including ATP synthesis and apoptosis. Mitochondrial function can be studied using inhibitors of respiration, and new agents are valuable for discovering novel mechanisms involved in mitochondrial regulation. Here, we screened small molecules derived from slime molds and other microorganisms for their effects on mitochondrial oxygen consumption. We identified Ppc-1 as a novel molecule which stimulates oxygen consumption without adverse effects on ATP production. The kinetic behavior of Ppc-1 suggests its function as a mitochondrial uncoupler. Serial administration of Ppc-1 into mice suppressed weight gain with no abnormal effects on liver or kidney tissues, and no evidence of tumor formation. Serum fatty acid levels were significantly elevated in mice treated with Ppc-1, while body fat content remained low. After a single administration, Ppc-1 distributes into various tissues of individual animals at low levels. Ppc-1 stimulates adipocytes in culture to release fatty acids, which might explain the elevated serum fatty acids in Ppc-1-treated mice. The results suggest that Ppc-1 is a unique mitochondrial regulator which will be a valuable tool for mitochondrial research as well as the development of new drugs to treat obesity.

Synthesis of prenylated quinolinecarboxylic acid derivatives and their anti-obesity activities.

Mitochondrial uncoupling is one of the therapeutic strategies used to control energy metabolism in various metabolic diseases and in obesity. Ppc-1 (1), a prenylated quinolinecarboxylic acid isolated from cellular slime molds, shows uncoupling activity in vitro and anti-obesity activity in vivo. In this study, we synthesized Ppc-1 (1) and its derivatives, and revealed the structure-activity relationship of uncoupling activities. The triprenylated compound 18 showed mitochondrial uncoupling activity that was more potent than that of Ppc-1 (1). Compound 18 also suppressed weight gain in mice without undesired effects such as lesions on tissues. These results indicate that compound 18 could be used as a seed compound for new anti-obesity drugs.

Phosphorylation of flotillin-1 by mitochondrial c-Src is required to prevent the production of reactive oxygen species.

We have shown that mitochondrial c-Src regulates reactive oxygen species (ROS) production by phosphorylating the succinate dehydrogenase A of respiratory complex II (CxII). To elucidate the molecular mechanisms underlying ROS production regulated by c-Src in the CxII, we investigated the CxII protein complex derived from cells treated with Src family kinase inhibitor PP2. We identified flotillin-1 as a c-Src target that prevents ROS production from CxII. Phosphorylation-site analysis suggests Tyr56 and Tyr149 on flotillin-1 as sites for phosphorylation by c-Src. A comparison of cells expressing flotillin-1 and its phosphorylation defective mutants confirms the requirement for flotillin-1 phosphorylation for its interaction with CxII and subsequent reduction in ROS production. Our findings suggest a critical role of flotillin-1 in ROS production mediated by c-Src.

Mitochondrial c-Src regulates cell survival through phosphorylation of respiratory chain components.

Mitochondrial protein tyrosine phosphorylation is an important mechanism for the modulation of mitochondrial functions. In the present study, we have identified novel substrates of c-Src in mitochondria and investigated their function in the regulation of oxidative phosphorylation. The Src family kinase inhibitor PP2 {amino-5-(4-chlorophenyl)-7-(t-butyl) pyrazolo [3,4d] pyrimidine} exhibits significant reduction of respiration. Similar results were obtained from cells expressing kinase-dead c-Src, which harbours a mitochondrial-targeting sequence. Phosphorylation-site analysis selects c-Src targets, including NDUFV2 (NADH dehydrogenase [ubiquinone] flavoprotein 2) at Tyr(193) of respiratory complex I and SDHA (succinate dehydrogenase A) at Tyr(215) of complex II. The phosphorylation of these sites by c-Src is supported by an in vivo assay using cells expressing their phosphorylation-defective mutants. Comparison of cells expressing wild-type proteins and their mutants reveals that NDUFV2 phosphorylation is required for NADH dehydrogenase activity, affecting respiration activity and cellular ATP content. SDHA phosphorylation shows no effect on enzyme activity, but perturbed electron transfer, which induces reactive oxygen species. Loss of viability is observed in T98G cells and the primary neurons expressing these mutants. These results suggest that mitochondrial c-Src regulates the oxidative phosphorylation system by phosphorylating respiratory components and that c-Src activity is essential for cell viability.

Dysregulation of very long chain acyl-CoA dehydrogenase coupled with lipid peroxidation.

Idiopathic pulmonary fibrosis (IPF) is a chronic progressive lung disease of unknown etiology. We previously revealed increased oxidative stress and high expression of antioxidant proteins in culture cell lines established from lesional lung tissues with IPF (Kabuyama Y, Oshima K, Kitamura T, Homma M, Yamaki J, Munakata M, Homma Y. Genes Cells 12: 1235-1244, 2007). In this study, we show that IPF cells contain high levels of free cholesterol and its peroxidized form as compared with normal TIG7 lung fibroblasts, suggesting that radical oxygen species (ROS) are generated within specific organelles. To understand the molecular basis underlying the generation of ROS in IPF cells, we performed proteomic analysis of mitochondrial proteins from TIG and IPF cells. This analysis shows that the phosphorylation of Ser586 of very long chain acyl-CoA dehydrogenase (VLCAD) is significantly reduced in IPF cells. Similar results are obtained from immunoblotting with anti-pS586 antibody. Kinase activity toward a peptide containing Ser586 from IPF cells is significantly lower than that from TIG cells. Furthermore, a phosphorylation-negative mutant (S586A) VLCAD shows reduced electron transfer activity and a strong dominant-negative effect on fatty acid beta-oxidation. The ectopic expression of the S586A mutant induced human embryonic kidney (HEK) 293 cells to produce significantly high amounts of oxidized lipids and hydrogen peroxide. HEK293 cells expressing the S586A mutant exhibit a reduction in cell growth and an enhancement in apoptosis. These results suggest a novel regulatory mechanism for homeostatic VLCAD activity, whose dysregulation might be involved in the production of oxidative stress and in the pathogenesis of IPF.

Involvement of thioredoxin reductase 1 in the regulation of redox balance and viability of rheumatoid synovial cells.

Rheumatoid arthritis (RA), a chronic and systemic disease of unknown etiology, is characterized by hyperplasia of synovial cells, which ultimately lead to the destruction of cartilage and bone. To elucidate the molecular mechanisms that lead to RA, we analyzed synovial cells established from patients with RA by oligonucleotide microarrays. Gene expression profiles clearly suggested that oxidative stress is enhanced in RA synovial cells, which was confirmed by measuring cellular levels of reactive oxygen species. One of the highly up-regulated proteins in RA synovial cells was thioredoxin reductase 1 (TRXR1), a protein that plays an important role in antioxidant defense system. Subsequent analysis demonstrated that TRXR1 suppresses hydrogen peroxide and inhibits apoptosis of RA synovial cells. Thus, our results reveal a novel pathophysiologic function of RA synovial cells as a generator of oxidative stress, and a self-defense mechanism against self-generated oxidative stress.

Enhancement of lymphocyte migration and cytokine production by ephrinB1 system in rheumatoid arthritis.

Although the etiology of early events in rheumatoid arthritis (RA) remains undefined, an anomaly in T cell homeostasis and hyperproliferation of synovial-lining cells are involved in the disease process. Since it has been reported that the ephrin/Eph receptor system plays important signaling roles in inflammation processes, we attempted to examine ephrinB molecules in T cells and synovial cells derived from RA in this study. The expression level of ephrinB1 was significantly high in synovial fibroblasts and CD3-positive exudate lymphocytes in synovial tissues derived from patients with RA compared with those in osteoarthritis (OA). Protein and mRNA levels of ephrinB1 were also higher in peripheral blood lymphocytes (PBLs) prepared from patients with RA than those from normal controls. Similar results were obtained from an animal model of human RA, collagen antibody-induced arthritis mice. Moreover, a recombinant ephrinB1/Fc fusion protein stimulated normal PBLs to exhibit enhanced migration and production of TNF-alpha. EphrinB1/Fc also activated synovial cells established from patients with RA to produce IL-6. Tyrosine phosphorylation of EphB1 was induced in these cells by ephrinB1/Fc. The CpG islands in the 5' upstream regulatory region of the ephrinB1 gene were hypomethylated in RA patients compared with those of normal donors. These results suggest that ephrinB1 and EphB1 receptors play an important role in the inflammatory states of RA, especially by affecting the population and function of T cells. Inhibition of the ephrinB/EphB system might be a novel target for the treatment of RA.

Involvement of EphB1 receptor/EphrinB2 ligand in neuropathic pain.

STUDY DESIGN: We investigated involvement of EphB/ephrinB system in neuropathic pain. OBJECTIVE: Using immunoblotting, immunohistochemistry, and RNA interference techniques, we examined the expression levels of EphB receptors and ephrinB ligands in neuropathic pain. We also explored the effect of ephrinB siRNA for neuropathic pain. SUMMARY OF BACKGROUND DATA: It has been reported that EphB2 regulates the development of synaptic plasticity in the hippocampus by interacting with N-methyl-D-aspartate (NMDA) receptors. In acute pain models, it has been clear that EphB1/ephrinB2 interactions via the NMDA receptor modulates synaptic efficacy in spinal cord. METHODS: Adult female Sprague-Dawley rats were used in this study. A crush injury model was prepared by crushing the left L5 spinal nerve distal to dorsal root ganglions (DRG) under deep anesthesia. The sham operation was subjected as control. Expression of ephrinB2 and EphB1 were examined by immunoblotting and immunohistochemical analyses with anti-EphB and anti-ephrinB antibodies. To assess involvement of ephrinB in neuropathic pain, we examined the effect of small interference RNA (siRNA) on mechanical allodynia. RESULTS: Among EphB and ephrinB isoforms tested, ephrinB2 and EphB1 were predominant in DRG and spinal cord. Results showed that the expression of ephrinB2 was enhanced in neurons in DRG and spinal cord by the injury in a time-dependent manner. EphB1 was expressed in neurons of spinal cord. Administration of ephrinB2 siRNA reduced the expression of ephrinB2 and mechanical allodynia. CONCLUSION: Expression of ephrinB2 is enhanced by nerve injury in neurons in DRG and spinal cord, while its receptor EphB1 is expressed in spinal cord. These results suggest that induction of ephrinB2 might activate EphB1/ephrinB2 signaling pathway to regulate synaptic plasticity and reorganization, and that ephrinB2 siRNA could be a potential therapeutic agent for neuropathic pain.

Early signaling events induced by 280-nm UV irradiation.

The depletion of stratospheric ozone results in increased UV (ultraviolet) light below 300 nm, and has significant effects on biological systems. To better understand the effects of UV in this range, early signaling events induced by monochromatic UV light were investigated using the chicken B cell line DT40 and mutants lacking protein tyrosine kinases (PTKs). Among MAP kinase family proteins, P38 MAP kinase (P38) was selectively and immediately activated by 280 nm UV light in cultured DT40 cells. Activation of P38 was completely inhibited in cells deficient in Lyn and Btk. Introduction of wild-type Btk, but not kinase-inactive Btk, restored the P38 activation. In contrast, P38 activation was not affected in Syk-deficient cells. Tyrosine phosphorylation of Lyn was induced by 280 nm UV light, and pretreatment of cells with orthovanadate, an inhibitor of protein tyrosine phosphatase (PTP), enhanced both Lyn phosphorylation and P38 activation. These results show that Lyn and Btk are upstream regulators of the P38 signaling pathway activated by 280 nm UV light and that the triggering event likely involves inactivation of PTP. Furthermore, cell death induced by 280 nm UV irradiation were augmented by Btk depletion or a specific inhibitor for P38, and partially blocked in Lyn-deficient cells, suggesting that the Lyn-Btk-P38 pathway promotes cell survival while other Lyn pathways stimulate cell death.