Jae-Ryong Kim

Exploration of replicative senescence-associated genes in human dermal fibroblasts by cDNA microarray technology

The aging process is known to be regulated by specific genes in various organisms, including yeast, the nematode C. elegans, fruitflies and mice. To explore the novel genes involved in aging process, we applied cDNA microarray technology to a replicative senescence model of human dermal fibroblasts (HDF). Eighty-four genes, including inflammatory genes, cell cycle regulatory genes, cytoskeletal genes, and metabolic genes were found to show more than two fold expressional differences in young and old fibroblasts. Furthermore, 31 genes were confirmed to be up- or down-regulated during replicative senescence by semi-quantitative RT-PCR. The overexpressions of several genes including CD36, putative lymphocyte G0/G1 switch gene (G0S2), tumor protein D52-like 1 (TPD52L1), chemokine (C-X-C motif) ligand 6, myxovirus resistant gene 1 (MX1), and the down-regulation of the immunoglobulin superfamily containing leucine-rich repeat (ISLR), neurotrimin, insulin-like growth factor 2 associated protein (IGF2A), and apoptosis-related RNA binding protein (NAPOR3) were newly identified. These results suggest that fibroblasts show the deregulation of various cellular processes, such as inflammatory response, mitosis, cell adhesion, transport, signal transduction, and metabolism during replicative senescence.

Interferon-γ induces cellular senescence through p53-dependent DNA damage signaling in human endothelial cells

Cellular senescence is a stress-response phenomenon in which cells lose the ability to proliferate; it is induced by telomere shortening, activation of oncogenes or tumor suppressor genes, or exposure to a sub-lethal dose of DNA damaging agents or oxidative stresses. cDNA microarray analysis reveals that the levels of interferons (IFNs) and IFN-inducible genes were altered during replicative senescence in human umbilical vascular endothelial cells (HUVECs). However, the role of IFNs in cellular senescence of HUVECs remains unidentified. This study demonstrated that prolonged treatment with IFN-gamma induced cellular senescence in HUVECs, as confirmed by G0/G1 cell cycle arrest, up-regulation of p53 and p21 protein levels, increased SA-beta-gal staining, and the accumulation of phospho-H(2)AX foci. IFN-gamma-induced cellular senescence was observed only in p16-knockdown cells or p16-null mouse embryonic fibroblasts (MEFs), but not in p53-knockdown cells or p53-null MEFs. IFN-gamma treatment increased ROS production, and an antioxidant, N-acetylcysteine, inhibited IFN-gamma-induced cellular senescence. Knockdown of ATM kinase or IFI16 rescued IFN-gamma-induced cellular senescence. Therefore, these results suggest that IFN-gamma might play an important role in cellular senescence through a p53-dependent DNA damage pathway and contribute to the pathogenesis of atherosclerosis via its pro-senescent activity.

Resveratrol inhibits foam cell formation via NADPH oxidase 1- mediated reactive oxygen species and monocyte chemotactic protein-1.

Resveratrol is a polyphenolic compound in red wine that has anti-oxidant and cardioprotective effects in animal models. Reactive oxygen species (ROS) and monocyte chemotactic protein-1 (MCP-1) play key roles in foam cell formation and atherosclerosis. We studied LPS-mediated foam cell formation and the effect of resveratrol. Resveratrol pretreatment strongly suppressed LPS-induced foam cell formation. To determine if resveratrol affected the expression of genes that control ROS generation in macrophages, NADPH oxidase 1 (Nox1) was measured. Resveratrol treatment of macrophages inhibited LPS-induced Nox1 expression as well as ROS generation, and also suppressed LPS-induced MCP-1 mRNA and protein expression. We investigated the upstream targets of Nox1 and MCP-1 expression and found that Akt-forkhead transcription factors of the O class (FoxO3a) is an important signaling pathway that regulates both genes. These inhibitory effects of resveratrol on Nox1 expression and MCP-1 production may target to the Akt and FoxO3a signaling pathways.

A combination of Lox-1 and Nox1 regulates TLR9-mediated foam cell formation.

The formation of foam cells is the hallmark of early atherosclerotic lesions, and the uptake of modified low-density lipoprotein (LDL) by macrophage scavenger receptors is thought to be a key process in their formation. In this study, we examined the role of lectin-like oxLDL receptor-1 (Lox-1) and NADPH oxidase 1 (Nox1) in toll-like receptor 9 (TLR9)-mediated foam cell formation. TLR9 activation of Raw264.7 cells or mouse primary peritoneal macrophages by CpG ODN treatment enhanced Lox-1 gene and protein expression. In addition, CpG ODN-induced Nox1 mRNA expression, which in turn increased foam cell formation. The inhibition of CpG ODN-induced reactive oxygen species (ROS) generation by treatment with antioxidants, as well as with knockdown of Nox1 using siRNA, suppressed the formation of foam cells. The induction of Lox-1 and Nox1 by CpG ODN was regulated via the TLR9-p38 MAPK signaling pathway. CpG ODN also increased NFkappaB activity, and a potent inhibitor of NFkappaB that significantly blocked CpG-induced Nox1 expression, suggesting that Nox1 regulation is mediated through an NFkappaB-dependent mechanism. Taken together, these results suggest that a combination of Lox-1 and Nox1 plays a key role in the TLR9-mediated formation of foam cells via the p38 MAPK pathway.

Identification of amyloid

Amyloid β-peptide (Aβ), a causative molecule in the pathogenesis of Alzheimers disease and the main component of senile plaques, is known to be neurotoxic in vitro and in vivo. The mechanisms involved in this Aβ-mediated neurotoxicity are not fully understood, although there is evidence to suggest the involvement of oxidative stress, alterations in calcium homeostasis, and/or of CDK activators. Many studies have suggested that Aβ may exert its toxic effect via the activation of transcription factors. Therefore, we investigated Aβ- responsive genes in human neuroblastoma CHP134 cells using 3.1K human DNA microarrays. Among the several genes overexpressed or repressed by Aβ, RTP801, Hi95/sestrin 2, and stanniocalcin 2 were confirmed to be Aβ-mediated overexpression in the cells by semiquantitative RT-PCR. Transient expression of the sense RTP801 gene in CHP134 cells increased sensitivity to Aβ cytotoxicity and the expression of the antisense RTP801 gene protected the cells from the Aβ toxicity. These results suggest that RTP801 might play important roles in Aβ toxicity and the pathogenesis of Alzheimers disease.

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Previous reports described thioredoxin (Trx) as a very poor reductant for mammalian MsrB2 and MsrB3, which lack a resolving Cys residue. In contrast, we here report that Trx could reduce both MsrB2 and MsrB3 enzymes, similarly to the reduction of mammalian MsrA. We demonstrated that functional Trx is required for the reduction of these enzymes. We further identified MsrB2- or MsrB3-Trx complexes formed through intermolecular disulfide bonds involving catalytic residue of Trx. The present study provides evidence that the sulfenic acid intermediate of oxidized MsrBs lacking resolving Cys could interact with Trx and be directly reduced by this protein.

responsive genes by cDNA microarray technology: Involvement of RTP801 in amyloid

Non-enzymatic glycation of serum apolipoproteins is a main feature of diabetes mellitus under hyperglycemia. Advanced glycation end products are implicated in the development of aging and metabolic syndrome, including premature atherosclerosis in diabetic subjects. ApoA-I is the principal protein constituent of HDL. In this study, glycated human apoA-I (gA-I) by fructation was characterized on functional and structural correlations in lipid-free and lipid-bound states. The gA-I showed more spontaneous multimeric band formation up to pentamer and exhibited slower elution profile with more degraded fragments from fast protein liquid chromatography. The gA-I showed modified secondary structure from fluorescence and circular dichroism analysis. Reconstituted high-density lipoprotein (rHDL) containing the gA-I had less content of phospholipid with a much smaller particle size than those of rHDL-containing nA-I (nA-I-rHDL). The rHDL containing gA-I (gA-I-rHDL) consisted of less molecular number of apoA-I than nA-I-rHDL with decreased alpha-helical content. Treatment of the gA-I-rHDL induced more atherogenic process in macrophage cell and premature senescence in human dermal fibroblast cell. Conclusively, fructose-mediated apoA-I glycation resulted in severe loss of several beneficial functions of apoA-I and HDL regarding anti-senescence and anti-atherosclerosis activities due to a lack of anti-oxidant activity with increased susceptibility of protein degradation and structural modification.

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Unmethylated CpG oligodeoxynucleotides (CpG ODNs) activate immune cells to produce immune mediators. This study demonstrates that in murine macrophage RAW 264.7 cells, CpG ODN-mediated matrix metalloproteinase-9 (MMP-9) expression is regulated at transcriptional level and requires de novo protein synthesis. Inhibition of ERK and p38 MAPK, but not JNK, results in significant decrease of CpG ODN-induced MMP-9 expression. We found that endosomal maturation inhibitors, chloroquine and bafilomycin A, block CpG ODN-induced ERK and p38 MAPK activation and the subsequent MMP-9 expression. We also observed that CpG ODN induces NF-κ B activation and NF-κ B is a downstream target of p38 MAPK. Taken together, our data demonstrate that CpG ODN triggers MMP-9 expression via TLR-9 dependent ERK and p38 MAPK activation followed by NF-κ B activation.

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The induction of inducible NO synthase (iNOS) by group IIA phospholipase A2 (PLA2) involves the stimulation of a novel signaling cascade. In this study, we demonstrate that group IIA PLA2 up-regulates the expression of iNOS through a novel pathway that includes M-type secretory PLA2 receptor (sPLA2R), phosphatidylinositol 3-kinase (PI3K), and Akt. Group IIA PLA2 stimulated iNOS expression and promoted nitrite production in a dose- and time-dependent manner in Raw264.7 cells. Upon treating with group IIA PLA2, Akt is phosphorylated in a PI3K-dependent manner. Pretreatment with LY294002, a PI3K inhibitor, strongly suppressed group IIA PLA2-induced iNOS expression and PI3K/Akt activation. The promoter activity of iNOS was stimulated by group IIA PLA2, and this was suppressed by LY294002. Transfection with Akt cDNA resulted in Akt protein overexpression in Raw264.7 cells and effectively enhanced the group IIA PLA2-induced reporter activity of the iNOS promoter. M-type sPLA2R was highly expressed in Raw264.7 cells. Overexpression of M-type sPLA2R enhanced group IIA PLA2-induced promoter activity and iNOS protein expression, and these effects were abolished by LY294002. However, site-directed mutation in residue responsible for PLA2 catalytic activity markedly reduced their ability to production of nitrites and expression of iNOS. These results suggest that group IIA PLA2 induces nitrite production by involving of M-type sPLA2R, which then mediates signal transduction events that lead to PI3K/Akt activation.

Thioredoxin as a reducing agent for mammalian methionine sulfoxide reductases B lacking resolving cysteine

This study examined the hypothesis that the control of NADPH oxidase‐2 (Nox2)‐mediated reactive oxygen species (ROS) regulates the expression of matrix metalloproteinases (MMPs) and the migration of macrophages. Lipopolysaccharide (LPS) stimulation of Raw264.7 cells and mice peritoneal macrophages increased the expression of MMP‐9, 10, 12 and 13 mRNA, and also increased Raw264.7 cell migration. Treatment with an antioxidant (N‐acetyl cysteine) or Nox inhibitors strongly inhibited the expression of MMPs by LPS and inhibited cell migration. LPS caused ROS production in macrophages and increased the mRNA expression of Nox isoforms Nox1 and Nox2 by 20‐fold and two‐fold, respectively. While Nox1 small interfering RNA (siRNA) did not inhibit LPS‐mediated expression of MMPs, Nox2 siRNA inhibited the expressions of MMP‐9, 10 and 12. Neither Nox1 nor Nox2 siRNA influenced the LPS‐mediated expression of MMP‐13. In addition, NAC or apocynin attenuated LPS‐induced ROS production and MMP‐9 expression. MMP‐9 expression and cell migration were controlled by ERK1/2–ROS signaling. Collectively, these results suggest that LPS stimulates ROS production via ERK and induce various types of MMPs expression and cell migration.