Makio Saeki

Histone H1.2 is a substrate for denitrase, an activity that reduces nitrotyrosine immunoreactivity in proteins

Several reports have described an activity that modifies nitrotyrosine-containing proteins and their immunoreactivity to nitrotyrosine Abs. Without knowing the product of the reaction, this new activity has been called a “denitrase.” In those studies, some nonspecific proteins, which have multiple tyrosine residues, e.g., albumin, were used as a substrate. Therefore, the studies were based on an unknown mechanism of reaction and potentially a high background. To solve these problems, one of the most important things is to find a more suitable substrate for assay of the enzyme. We developed an assay strategy for determining the substrate for denitrase combining 2D-gel electrophoresis and an on-blot enzyme assay. The resulting substrate from RAW 264.7 cells was Histone H1.2, an isoform protein of linker histone. Histone H1.2 has only one tyrosine residue in the entire molecule, which ensures the exact position of the substrate to be involved. It has been reported that Histones are the most prominent nitrated proteins in cancer tissues. It was also demonstrated that tyrosine nitration of Histone H1 occurs in vivo. These findings lead us to the idea that Histone H1.2 might be an intrinsic substrate for denitrase. We nitrated recombinant and purified Histone H1.2 chemically and subjected it to an on-blot enzyme assay to characterize the activity. Denitrase activity behaved as an enzymatic activity because the reaction was time dependent and was destroyed by heat or trypsin treatment. The activity was shown to be specific for Histone H1.2, to differ from proteasome activity, and to require no additional cofactors.

Gingival Fibroblasts as a Promising Source of Induced Pluripotent Stem Cells

Background Induced pluripotent stem (iPS) cells efficiently generated from accessible tissues have the potential for clinical applications. Oral gingiva, which is often resected during general dental treatments and treated as biomedical waste, is an easily obtainable tissue, and cells can be isolated from patients with minimal discomfort. Methodology/Principal Findings We herein demonstrate iPS cell generation from adult wild-type mouse gingival fibroblasts (GFs) via introduction of four factors (Oct3/4, Sox2, Klf4 and c-Myc; GF-iPS-4F cells) or three factors (the same as GF-iPS-4F cells, but without the c-Myc oncogene; GF-iPS-3F cells) without drug selection. iPS cells were also generated from primary human gingival fibroblasts via four-factor transduction. These cells exhibited the morphology and growth properties of embryonic stem (ES) cells and expressed ES cell marker genes, with a decreased CpG methylation ratio in promoter regions of Nanog and Oct3/4. Additionally, teratoma formation assays showed ES cell-like derivation of cells and tissues representative of all three germ layers. In comparison to mouse GF-iPS-4F cells, GF-iPS-3F cells showed consistently more ES cell-like characteristics in terms of DNA methylation status and gene expression, although the reprogramming process was substantially delayed and the overall efficiency was also reduced. When transplanted into blastocysts, GF-iPS-3F cells gave rise to chimeras and contributed to the development of the germline. Notably, the four-factor reprogramming efficiency of mouse GFs was more than 7-fold higher than that of fibroblasts from tail-tips, possibly because of their high proliferative capacity. Conclusions/Significance These results suggest that GFs from the easily obtainable gingival tissues can be readily reprogrammed into iPS cells, thus making them a promising cell source for investigating the basis of cellular reprogramming and pluripotency for future clinical applications. In addition, high-quality iPS cells were generated from mouse GFs without Myc transduction or a specific system for reprogrammed cell selection.

Nitration of PPARγ inhibits ligand‐dependent translocation into the nucleus in a macrophage‐like cell line, RAW 264

Nitration of tyrosine residues in proteins has been observed in many inflammatory tissues of arthritis, ulcerative colitis, septic shock and ischemia‐reperfusion injury. Although several studies have been carried out, it is still unclear what type of protein is nitrated and whether tyrosine nitration interferes with protein function. Peroxisome proliferator‐activated receptor gamma (PPARγ) is a nuclear receptor whose activation is linked to several physiological pathways including regulation of insulin sensitivity and control of inflammation. PPARγ possesses several tyrosine residues, which might be potential targets for nitration by peroxynitrite during inflammatory responses. Here we have investigated whether PPARγ is nitrated in macrophage‐like RAW 264 cells and the effect of nitration on the translocation of PPARγ into the nucleus. Western blot analysis showed that tumor necrosis factor‐α, lipopolysaccharide or peroxynitrite treatment significantly increases the nitration of PPARγ. Cell fractionation analysis and immunofluorescence coupled with confocal laser microscopy revealed that nitration of PPARγ inhibits its ligand‐dependent translocation from the cytosol into the nucleus. Together, these results indicate that nitration of PPARγ during inflammation may be involved in a reduction in the control of inflammatory responses and also in the development of resistance to PPARγ ligand‐based therapies against inflammation.

Enhanced bone regeneration via multimodal actions of synthetic peptide SVVYGLR on osteoprogenitors and osteoclasts

Recently, the binding sequence Ser-Val-Val-Tyr-Gly-Leu-Arg (SVVYGLR) was found adjacent to the RGD sequence in osteopontin, suggesting involvement in osteo-immune cross-talk. The aim of this study was to investigate bioactive functions of a synthetic SVVYGLR peptide in osteoprogenitor cells and osteoclasts, and to examine potential applications in bone regeneration. The SVVYGLR peptide significantly enhanced the adhesion and proliferation of several human mesenchymal cells including bone marrow-derived mesenchymal stem cells, and alphavbeta3 integrin was involved in cell attachment to the peptide. Additionally, the peptide reduced the number of TRAP-positive multinucleated cells during osteoclastogenesis of RAW264.7 cells and normal murine pre-osteoclasts, and also suppressed NFAT activity and expression of osteoclastogenesis-related mRNAs. When standardized bone defects in rat calvariae were filled with a collagen sponge containing the peptide or PBS (control), the number of TRAP-positive osteoclasts in the grafted sites after 3 weeks was significantly lower in the peptide group. By the 5th week, significantly enhanced resorption of the grafted collagen sponge and new bone formation was observed within and surrounding the sponge in the peptide group. These data suggest that SVVYGLR is an effective bioactive peptide for bone tissue regeneration that promotes attachment and proliferation of osteogenic cells while also suppressing osteoclastogenesis.

Insulin-like growth factor-1 protects peroxynitrite-induced cell death by preventing cytochrome c-induced caspase-3 activation.

We investigated the effect of IGF‐1 on cell death induced by peroxynitrite in human neuroblastoma SH‐SY5Y cells. Exposure of the cells to 3‐morpholinosydnonimine (SIN‐1), a peroxynitrite donor, caused cytochrome c release from the mitochondria, caspase‐3‐like activation, and cell death. Pre‐incubation of the cells with the caspase‐3 inhibitor partially prevented SIN‐1‐induced cell death. Simultaneous addition of IGF‐1 reduced SIN‐1‐induced caspase‐3‐like activation and cell death, whereas IGF‐1 failed to reduce the release of cytochrome c. IGF‐1 increased Akt phosphorylation, and Akt phosphorylation was inhibited by wortmannin, an inhibitor of phosphatidylinositol 3‐kinase. In addition, wortmannin prevented IGF‐1‐evoked inhibition of cell death and caspase‐3‐like activation. In a cell‐free system, addition of cytochrome c to cytosolic fraction resulted in caspase‐3‐like activation. The activation was reduced when the cytosolic fraction prepared from IGF‐1‐treated cells was used. These results suggest that IGF‐1 protects peroxynitrite‐induced cell death downstream of cytochrome c release through the inhibition of caspase‐3‐like activation. J. Cell. Biochem. 84: 708–716, 2002.

Morphine prevents peroxynitrite-induced death of human neuroblastoma SH-SY5Y cells through a direct scavenging action

N-ethyl-2-(1-ethyl-2-hydroxy-2-nitrosohydrazino)-ethanamine (NOC12), a nitric oxide donor, 3-morpholinosydnonimine (SIN-1), a generator of peroxynitrite (ONOO-), and peroxynitrite induced cell death accompanied by DNA fragmentation in human neuroblastoma SH-SY5Y cell cultures. Morphine prevented the cell death induced by SIN-1 or peroxynitrite, but not that induced by NOC12. The protective effect of morphine was concentration-dependent (10-100 microM), but was not antagonized by naloxone. The selective ligands for opioid receptor subtypes, [D-Ala2, N-Me-Phe4, Gly-ol5]enkephalin (DAMGO, micro-opioid receptor agonist), [D-Pen2,5]enkephalin (DPDPE, delta-opioid receptor agonist) and trans-(+/-)-3,4-dichloro-N-methyl-N-(2-[1-pyrrolidinyl]-cyclohexyl)benze neacetamide (U-50488, kappa-opioid receptor agonist) even at the concentration of 100 microM did not prevent the cell death induced by SIN-1. From measurement of the absorbance spectrum of peroxynitrite, the decomposition of peroxynitrite in 0.25 M potassium phosphate buffer (pH 7.4) was very rapid and complete within seconds. However, the absorbance was very stable in the presence of morphine. In addition, morphine inhibited peroxynitrite-induced nitration of tyrosine in a concentration-dependent manner. These results indicate that morphine rapidly reacts with peroxynitrite. The present study showed that morphine prevented peroxynitrite-induced cell death through its direct scavenging action, suggesting that morphine can protect cells against damage caused by peroxynitrite.

Calcineurin potentiates the activation of procaspase-3 by accelerating its proteolytic maturation.

We have previously shown that procaspase-3 exists in a high molecular weight complex in neonatal rat brain. Here, we purify and identify the protein that interacts with procaspase-3 from rat neonatal cortex. We searched binding proteins to procaspase-3 from a cytosolic extract of neonatal rat brain using chromatogram, two-dimensional gel electrophoresis, and far Western immunoblot. Analysis by tandem mass spectrometry identified the protein as a regulatory subunit of calcineurin (calcineurin B). Overexpression of calcineurin B in HEK293 cells potentiated processing of caspase-3 and apoptosis triggered by tumor necrosis factor-α and cycloheximide treatment. In a cell-free system, overexpression of calcineurin B in HEK293 cells markedly increased processing of caspase-3 by cytochrome c. Immunodepletion of calcineurin B from cytosolic extracts from Jurkat cells decreased processing of caspase-3 by cytochrome c. Knockdown of calcineurin B by RNA interference resulted in reduced apoptosis in HEK293 cells but not in caspase-3-deficient MCF-7 cells. These results suggest that calcineurin B potentiates the activation of procaspase-3 by accelerating its proteolytic maturation.

Inhibition by nifedipine of adherence- and activated macrophage-induced death of human gingival fibroblasts

The effects of nifedipine on the death and proliferation of gingival fibroblasts were investigated to elucidate the mechanism of gingival overgrowth that is associated with chronic administration of Ca2+ channel blockers. The number of adhered viable and dead fibroblasts obtained from healthy human gingiva increased after confluence, whereas cell death was inhibited by nifedipine in a concentration-dependent manner. A similar inhibition was also observed in the presence of other calcium channel blockers, such as nicardipine, diltiazem, and verapamil. When gingival fibroblasts were co-cultured with RAW264 (macrophage-like) cells, lipopolysaccharide (LPS) caused the concentration-dependent death of fibroblasts. Nifedipine significantly inhibited the LPS-induced cell death. Although neither LPS nor N-ethyl-2-(1-ethyl-2-hydroxy-2-nitroso-hydrazino)-ethanamine, a nitric oxide donor, directly caused fibroblast death, 3-morpholino-sydnonimine (SIN-1), a peroxynitrite donor, induced fibroblast death, regardless of the presence of RAW cells. The cell death induced by SIN-1 was not affected by nifedipine treatment. LPS stimulation caused an increase in the immunoreactivity of inducible nitric oxide synthase (iNOS) and in the nitrite concentration in the incubation medium of RAW cells. The induction of iNOS was completely prevented by the incubation with nifedipine. The inhibition by nifedipine of nitrite production in RAW cells was also observed after treatment with nicardipine, but not with either diltiazem or verapamil. Therefore, the inhibition by nifedipine of both adherence- and LPS-stimulated macrophage-induced death of fibroblasts may be the mechanism of gingival overgrowth seen during chronic treatment with Ca(2+) channel blockers.

p130cas is a cellular target protein for tyrosine nitration induced by peroxynitrite.

We found that the exposure of human neuroblastoma SH-SY5Y cells to the peroxynitrite donor 3-morpholinosydnonimine (SIN-1) induced tyrosine nitration of a 130-kDa protein, and prevented tyrosine phosphorylation of the 130-kDa protein. The focal adhesion protein p130cas was identified as a component of the 130-kDa protein using specific antibody. These results suggest that p130cas is a new target protein for nitration induced by SIN-1.

RPAP3 interacts with Reptin to regulate UV‐induced phosphorylation of H2AX and DNA damage

We have previously reported that Monad, a novel WD40 repeat protein, potentiates apoptosis induced by tumor necrosis factor‐α and cycloheximide. By affinity purification and mass spectrometry, RNA polymerase II‐associated protein 3 (RPAP3) was identified as a Monad binding protein and may function with Monad as a novel modulator of apoptosis pathways. Here we report that Reptin, a highly conserved AAA + ATPase that is part of various chromatin‐remodeling complexes, is also involved in the association of RPAP3 by immunoprecipitation and confocal microscopic analysis. Overexpression of RPAP3 induced HEK293 cells to death after UV‐irradiation. Loss of RPAP3 by RNAi improved HeLa cell survival after UV‐induced DNA damage and attenuated the phosphorylation of H2AX. Depletion of Reptin reduced cell survival and facilitated the phosphorylation on H2AX. These results suggest that RPAP3 modulates UV‐induced DNA damage by regulating H2AX phosphorylation. J. Cell. Biochem. 106: 920–928, 2009.