Phenyl 2‐pyridyl ketoxime induces cellular senescence‐like alterations via nitric oxide production in human diploid fibroblasts

Yang, Kyeong Eun,Jang, Hyun&#x2010,Jin,Hwang, In&#x2010,Hu,Chung, Young&#x2010,Ho,Choi, Jong&#x2010,Soon,Lee, Tae&#x2010,Hoon,Chung, Yun&#x2010,Jo,Lee, Min&#x2010,Seung,Lee, Mi Young,Yeo, Eui&#x2010,J BLACKWELL PUBLISHING 2016 AGING CELL Vol.15 No.2

<P><B>Summary</B></P><P>Phenyl‐2‐pyridyl ketoxime (PPKO) was found to be one of the small molecules enriched in the extracellular matrix of near‐senescent human diploid fibroblasts (HDFs). Treatment of young HDFs with PPKO reduced the viability of young HDFs in a dose‐ and time‐dependent manner and resulted in senescence‐associated β‐galactosidase (SA‐β‐gal) staining and G2/M cell cycle arrest. In addition, the levels of some senescence‐associated proteins, such as phosphorylated ERK1/2, caveolin‐1, p53, p16<SUP>ink4a</SUP>, and p21<SUP>waf1</SUP>, were elevated in PPKO‐treated cells. To monitor the effect of PPKO on cell stress responses, reactive oxygen species (ROS) production was examined by flow cytometry. After PPKO treatment, ROS levels transiently increased at 30 min but then returned to baseline at 60 min. The levels of some antioxidant enzymes, such as catalase, peroxiredoxin II and glutathione peroxidase I, were transiently induced by PPKO treatment. SOD II levels increased gradually, whereas the SOD I and III levels were biphasic during the experimental periods after PPKO treatment. Cellular senescence induced by PPKO was suppressed by chemical antioxidants, such as N‐acetylcysteine, 2,2,6,6‐tetramethylpiperidinyloxy, and L‐buthionine‐(<I>S</I>,<I>R</I>)‐sulfoximine. Furthermore, PPKO increased nitric oxide (NO) production via inducible NO synthase (iNOS) in HDFs. In the presence of NOS inhibitors, such as L‐NG‐nitroarginine methyl ester and L‐NG‐monomethylarginine, PPKO‐induced transient NO production and SA‐β‐gal staining were abrogated. Taken together, these results suggest that PPKO induces cellular senescence in association with transient ROS and NO production and the subsequent induction of senescence‐associated proteins<B>.</B></P>

3′‐Sialyllactose as an inhibitor of p65 phosphorylation ameliorates the progression of experimental rheumatoid arthritis

Kang, Li&#x2010,Jung,Kwon, Eun&#x2010,Soo,Lee, Kwang Min,Cho, Chanmi,Lee, Jae&#x2010,In,Ryu, Young Bae,Youm, Tae Hyun,Jeon, Jimin,Cho, Mi Ra,Jeong, Seon&#x2010,Yong,Lee, Sang&#x2010,Rae,Kim, Wook,Yang John Wiley and Sons Inc. 2018 British journal of pharmacology Vol.175 No.23

<P><B>Background and Purpose</B></P><P>3′‐Sialyllactose (3′‐SL) is a safe compound that is present in high levels in human milk. Although it has anti‐inflammatory properties and supports immune homeostasis, its effect on collagen‐induced arthritis (CIA) is unknown. In this study, we investigated the prophylactic and therapeutic effect of 3′‐SL on the progression of rheumatoid arthritis (RA) in <I>in vitro</I> and <I>in vivo</I> models.</P><P><B>Experimental Approach</B></P><P>The anti‐arthritic effect of 3′‐SL was analysed with fibroblast‐like synoviocytes <I>in vitro</I> and an <I>in vivo</I> mouse model of CIA. RT‐PCR, Western blotting and ELISA were performed to evaluate its effects <I>in vitro</I>. Histological analysis of ankle and knee joints of mice with CIA was performed using immunohistochemistry, as well as safranin‐O and haematoxylin staining.</P><P><B>Key Results</B></P><P>3′‐SL markedly alleviated the severity of CIA in the mice by reducing paw swelling, clinical scores, incidence rate, serum levels of inflammatory cytokines and autoantibody production. Moreover, 3′‐SL reduced synovitis and pannus formation and suppressed cartilage destruction by blocking secretion of chemokines, pro‐inflammatory cytokines, https://en.wikipedia.org/wiki/Matrix_metalloproteinases and osteoclastogenesis <I>via</I> NF‐κB signalling. Notably, phosphorylation of p65, which is a key protein in the NF‐κB signalling pathway, was totally blocked by 3′‐SL in the RA models.</P><P><B>Conclusions and Implications</B></P><P>3′‐SL ameliorated pathogenesis of CIA by suppressing catabolic factor expression, proliferation of inflammatory immune cells and osteoclastogenesis. These effects were mediated <I>via</I> blockade of the NF‐κB signalling pathway. Therefore, 3′‐SL exerted prophylactic and therapeutic effects and could be a novel therapeutic agent for the treatment of RA.</P>

Interleukin‐21 promotes osteoclastogenesis in humans with rheumatoid arthritis and in mice with collagen‐induced arthritis

Kwok, Seung&#x2010,Ki,Cho, Mi‐,La,Park, Mi‐,Kyung,Oh, Hye&#x2010,Joa,Park, Jin&#x2010,Sil,Her, Yang&#x2010,Mi,Lee, Seon&#x2010,Yeong,Youn, Jeehee,Ju, Ji Hyeon,Park, Kyung Su,Kim, Sung&#x20 Wiley Subscription Services, Inc., A Wiley Company 2012 Vol.64 No.3

<P><B>Abstract</B></P><P><B>Objective</B></P><P>Bone destruction is a critical pathology involved in the functional disability caused by rheumatoid arthritis (RA). Osteoclasts, which are specialized bone‐resorbing cells regulated by cytokines such as RANKL, are implicated in bone destruction in RA. The aim of this study was to determine whether interleukin‐21 (IL‐21), a potent immunomodulatory 4–α‐helical bundle type 1 cytokine, has osteoclastogenic activity in patients with RA and in mice with collagen‐induced arthritis (CIA).</P><P><B>Methods</B></P><P>The expression of IL‐21 in synovial tissue was examined using immunohistochemistry. The concentrations of IL‐21 in serum and synovial fluid were determined by enzyme‐linked immunosorbent assay. The levels of RANKL and osteoclastogenic markers were measured using real‐time polymerase chain reaction. CD14+ monocytes from patients with RA or mouse bone marrow cells were cocultured with fibroblast‐like synoviocytes (FLS) from patients with RA or CD4+ T cells from mice with CIA in the presence of IL‐21 and subsequently stained for tartrate‐resistant acid phosphatase activity to determine osteoclast formation.</P><P><B>Results</B></P><P>IL‐21 was up‐regulated in the synovium, synovial fluid, and serum of patients with RA and in the synovium and serum of mice with CIA. IL‐21 induced RANKL expression in mixed joint cells and CD4+ T cells from mice with CIA and in CD4+ T cells and FLS from patients with RA. Moreover, IL‐21 enhanced in vitro osteoclastogenesis without the presence of RANKL‐providing cells and by inducing RANKL expression in CD4+ T cells and FLS.</P><P><B>Conclusion</B></P><P>Our data suggest that IL‐21 promotes osteoclastogenesis in RA. We believe that therapeutic strategies targeting IL‐21 might be effective for the treatment of patients with RA, especially in preventing bone destruction.</P>

Interleukin‐22 promotes osteoclastogenesis in rheumatoid arthritis through induction of RANKL in human synovial fibroblasts

Kim, Kyoung&#x2010,Woon,Kim, Hae&#x2010,Rim,Park, Jin&#x2010,Young,Park, Jin&#x2010,Sil,Oh, Hye&#x2010,Jwa,Woo, Yun&#x2010,Ju,Park, Mi‐,Kyung,Cho, Mi‐,La,Lee, Sang&#x2010,Heon Wiley Subscription Services, Inc., A Wiley Company 2012 Vol.64 No.4

<P><B>Abstract</B></P><P><B>Objective</B></P><P>To examine the regulatory role of interleukin‐22 (IL‐22) in the expression of RANKL and induction of osteoclastogenesis in rheumatoid arthritis (RA).</P><P><B>Methods</B></P><P>Concentrations of IL‐22 and RANKL in the serum and synovial fluid of RA patients were measured using enzyme‐linked immunosorbent assay. RA synovial fibroblasts were treated with recombinant human IL‐22 (rhIL‐22), and the expression of RANKL messenger RNA (mRNA) and protein was measured using real‐time polymerase chain reaction, Western blotting, and intracellular immunostaining. Human monocytes were cocultured with IL‐22–prestimulated RA synovial fibroblasts and macrophage colony‐stimulating factor, and osteoclastogenesis was assessed by counting the multinucleated cells (those staining positive for tartrate‐resistant acid phosphatase).</P><P><B>Results</B></P><P>The IL‐22 concentration in the synovial fluid was higher in RA patients than in patients with osteoarthritis (OA). The serum IL‐22 concentration was also higher in RA patients than in OA patients and healthy volunteers, and this correlated with serum titers of rheumatoid factor and anti–cyclic citrullinated peptide antibodies. In RA synovial fibroblasts treated with rhIL‐22, the expression of RANKL mRNA and protein was increased in a dose‐dependent manner. IL‐22–induced RANKL expression was down‐regulated significantly by the inhibition of p38 MAPK/NF‐κB or JAK‐2/STAT‐3 signaling. In human monocytes cocultured with IL‐22–prestimulated RA synovial fibroblasts in the absence of exogenous RANKL, the monocytes differentiated into osteoclasts, but this osteoclastogenesis decreased after p38 MAPK/NF‐κB or JAK‐2/STAT‐3 signaling was inhibited.</P><P><B>Conclusion</B></P><P>These results show that IL‐22 up‐regulates RANKL expression in RA synovial fibroblasts and induces osteoclastogenesis. These effects are mediated by the p38 MAPK/NF‐κB and JAK‐2/STAT‐3 signaling pathways.</P>

Compressive mechanical force augments osteoclastogenesis by bone marrow macrophages through activation of c‐Fms‐mediated signaling

Cho, Eui&#x2010,Sic,Lee, Keun&#x2010,Soo,Son, Young&#x2010,Ok,Jang, Yong&#x2010,Suk,Lee, Seung&#x2010,Youp,Kwak, So&#x2010,Yeong,Yang, Yeon&#x2010,Mi,Park, Seung&#x2010,Moon,Lee, Jeong&#x2010,Chae Wiley Subscription Services, Inc., A Wiley Company 2010 Journal of cellular biochemistry Vol.111 No.5

<P><B>Abstract</B></P><P>Little is known about the effects of mechanical forces on osteoclastogenesis by bone marrow macrophages (BMMs) in the absence of mechanosensitive cells, including osteoblasts and fibroblasts. In this study, we examined the effects of mechanical force on osteoclastogenesis by applying centrifugal force to BMMs using a horizontal microplate rotor. Our findings, as measured by an in vitro model system, show that tumor necrosis factor (TNF)‐α is capable of inducing osteoclast differentiation from BMMs and bone resorption in the presence of macrophage‐colony stimulating factor (M‐CSF) and is further facilitated by receptor activator of nuclear factor‐kappaB (NF‐κB) ligand (RANKL). Application of force to BMMs accelerated TNF‐α‐induced osteoclastogenesis; this was inhibited either by anti‐TNF‐α or anti‐TNF‐α receptor but not by OPG. TNF‐α also increased c‐Fms expression at both mRNA and protein levels in BMMs. An anti‐c‐Fms antibody completely inhibited osteoclast differentiation and bone resorption induced by TNF‐α but partially blocked osteoclastogenesis stimulated in combination with RANKL. These results suggest that TNF‐α (in the presence of M‐CSF) is capable of inducing osteoclastogenesis from BMMs, and that osteoclastogenesis is significantly stimulated by force application through the activation of c‐Fms‐mediated signaling. Overall, the present study reveals the facilitating effect of mechanical force on osteoclastic differentiation from BMMs without the addition of mechanosensitive cells. J. Cell. Biochem. 111: 1260–1269, 2010. © 2010 Wiley‐Liss, Inc.</P>

22‐ S ‐Hydroxycholesterol protects against ethanol‐induced liver injury by blocking the auto/paracrine activation of MCP ‐1 mediated by LXRα

Na, Tae&#x2010,Young,Han, Young&#x2010,Hyun,Ka, Na&#x2010,Lee,Park, Han&#x2010,Su,Kang, Yun Pyo,Kwon, Sung Won,Lee, Byung&#x2010,Hoon,Lee, Mi‐,Ock John WileySons, Ltd 2015 The Journal of pathology Vol.235 No.5

<P><B>Abstract</B></P><P>Chronic ethanol consumption causes hepatic steatosis and inflammation, which are associated with liver hypoxia. Monocyte chemoattractant protein‐1 (MCP‐1) is a hypoxia response factor that determines recruitment and activation of monocytes to the site of tissue injury. The level of MCP‐1 is elevated in the serum and liver of patients with alcoholic liver disease (ALD); however, the molecular details regarding the regulation of MCP‐1 expression are not yet understood completely. Here, we show the role of liver X receptor α (LXRα) in the regulation of MCP‐1 expression during the development of ethanol‐induced fatty liver injury, using an antagonist, 22‐S‐hydroxycholesterol (22‐S‐HC). First, administration of 22‐S‐HC attenuated the signs of liver injury with decreased levels of MCP‐1 and its receptor CCR2 in ethanol‐fed mice. Second, hypoxic conditions or treatment with the LXRα agonist GW3965 significantly induced the expression of MCP‐1, which was completely blocked by treatment with 22‐S‐HC or infection by shLXRα lentivirus in the primary hepatocytes. Third, over‐expression of LXRα or GW3965 treatment increased MCP‐1 promoter activity by increasing the binding of hypoxia‐inducible factor‐1α to the hypoxia response elements, together with LXRα. Finally, treatment with recombinant MCP‐1 increased the level of expression of LXRα and LXRα‐dependent lipid droplet accumulation in both hepatocytes and Kupffer cells. These data show that LXRα and its ligand‐induced up‐regulation of MCP‐1 and MCP‐1‐induced LXRα‐dependent lipogenesis play a key role in the autocrine and paracrine activation of MCP‐1 in the pathogenesis of alcoholic fatty liver disease, and that this activation may provide a promising new target for ALD therapy.Copyright © 2014 The Authors. <I>The Journal of Pathology</I> published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.</P>

Plant‐expressed Fc‐fusion protein tetravalent dengue vaccine with inherent adjuvant properties

Kim, Mi Young,Copland, Alastair,Nayak, Kaustuv,Chandele, Anmol,Ahmed, Muhammad S.,Zhang, Qibo,Diogo, Gil R.,Paul, Matthew J.,Hofmann, Sven,Yang, Moon&#x2010,Sik,Jang, Yong&#x2010,Suk,Ma, Julian K&#x20 BLACKWELL 2018 PLANT BIOTECHNOLOGY JOURNAL Vol.16 No.7

<P><B>Summary</B></P><P>Dengue is a major global disease requiring improved treatment and prevention strategies. The recently licensed Sanofi Pasteur Dengvaxia vaccine does not protect children under the age of nine, and additional vaccine strategies are thus needed to halt this expanding global epidemic. Here, we employed a molecular engineering approach and plant expression to produce a humanized and highly immunogenic poly‐immunoglobulin G scaffold (PIGS) fused to the consensus dengue envelope protein III domain (cEDIII). The immunogenicity of this IgG Fc receptor‐targeted vaccine candidate was demonstrated in transgenic mice expressing human FcγRI/CD64, by induction of neutralizing antibodies and evidence of cell‐mediated immunity. Furthermore, these molecules were able to prime immune cells from human adenoid/tonsillar tissue <I>ex vivo</I> as evidenced by antigen‐specific CD4<SUP>+</SUP> and CD8<SUP>+</SUP> T‐cell proliferation, IFN‐γ and antibody production. The purified polymeric fraction of dengue PIGS (D‐PIGS) induced stronger immune activation than the monomeric form, suggesting a more efficient interaction with the low‐affinity Fcγ receptors on antigen‐presenting cells. These results show that the plant‐expressed D‐PIGS have the potential for translation towards a safe and easily scalable single antigen‐based tetravalent dengue vaccine.</P>

Anti‐inflammatory mechanism of ginsenoside Rh1 in lipopolysaccharide‐stimulated microglia: critical role of the protein kinase A pathway and hemeoxygenase‐1 expression

Jung, Ji&#x2010,Sun,Shin, Jin A.,Park, Eun&#x2010,Mi,Lee, Jung&#x2010,Eun,Kang, Young&#x2010,Sook,Min, Sung&#x2010,Won,Kim, Dong&#x2010,Hyun,Hyun, Jin&#x2010,Won,Shin, Chan&#x2010,Young,Kim, Hee&#x201 Blackwell Publishing Ltd 2010 Journal of Neurochemistry Vol.115 No.6

<P> <I>J. Neurochem.</I> (2010) <B>115,</B> 1668–1680.</P><P><B>Abstract</B></P><P>Microglia activation plays a pivotal role in neurodegenerative diseases, and thus controlling microglial activation has been suggested as a promising therapeutic strategy for neurodegenerative diseases. In the present study, we showed that ginsenoside Rh1 inhibited inducible nitric oxide synthase, cyclooxygenase‐2, and pro‐inflammatory cytokine expression in lipopolysaccharide (LPS)‐stimulated microglia, while Rh1 increased anti‐inflammatory IL‐10 and hemeoxygenase‐1 (HO‐1) expression. Suppression of microglial activation by Rh1 was also observed in the mouse brain following treatment with LPS. Subsequent mechanistic studies revealed that Rh1 inhibited LPS‐induced MAPK phosphorylation and nuclear factor‐κB (NF‐κB)‐mediated transcription without affecting NF‐κB DNA binding. As the increase of pCREB (cAMP responsive element‐binding protein) is known to result in suppression of NF‐κB‐mediated transcription, we examined whether Rh1 increased pCREB levels. As expected, Rh1 increased pCREB, which was shown to be related to the anti‐inflammatory effect of Rh1 because pre‐treatment with protein kinase A inhibitors attenuated the Rh1‐mediated inhibition of nitric oxide production and the up‐regulation of IL‐10 and HO‐1. Furthermore, treatment of HO‐1 shRNA attenuated Rh1‐mediated inhibition of nitric oxide and reactive oxygen species production. Through this study, we have demonstrated that protein kinase A and its downstream effector, HO‐1, play a critical role in the anti‐inflammatory mechanism of Rh1 by modulating pro‐ and anti‐inflammatory molecules in activated microglia.</P>

Vitamin C deficiency attenuates liver fibrosis by way of up‐regulated peroxisome proliferator‐activated receptor‐gamma expression in senescence marker protein 30 knockout mice

Park, Jin&#x2010,Kyu,Ki, Mi‐,Ran,Lee, Hye&#x2010,Rim,Hong, Il&#x2010,Hwa,Ji, Ae&#x2010,Ri,Ishigami, Akihito,Park, Se&#x2010,Il,Kim, Ji&#x2010,Min,Chung, Hae&#x2010,Young,Yoo, Sung&#x2010,Eun,Jeo Wiley Subscription Services, Inc., A Wiley Company 2010 Hepatology Vol.51 No.5

<P><B>Abstract</B></P><P>Senescence marker protein 30 (SMP30), an important aging marker molecule that is highly expressed in the liver, has been known to protect hepatocytes from apoptosis by the synthesis of vitamin C. To explore the function of SMP30 in liver fibrosis, the effect of SMP30 deficiency on liver fibrosis was investigated in SMP30 knockout (KO) mice. Moreover, the <I>in vivo</I> results were further confirmed by way of hepatic stellate cell (HSC) isolation. We demonstrated that carbon tetrachloride (CCl<SUB>4</SUB>)‐induced liver fibrosis and the nuclear translocation of p‐Smad2/3, the immediate downstream of transforming growth factor beta (TGF‐β), were significantly inhibited in the liver of SMP30 KO mice compared with wildtype (WT) mice. We also confirmed that both WT and SMP30 KO HSCs did not express SMP30. Finally, we further confirmed that up‐regulation of peroxisome proliferator‐activated receptor‐gamma (PPAR‐γ) caused by a lack of vitamin C was the pivotal factor in the mechanisms for attenuated liver fibrosis of SMP30 KO mice, and feeding with vitamin C restored CCl<SUB>4</SUB>‐induced liver fibrosis in SMP30 KO mice. <I>Conclusion:</I> Vitamin C deficiency by SMP30 depletion attenuated liver fibrosis by way of up‐regulated PPAR‐γ expression in SMP30 KO mice. Our results provide, for the first time, the possible mechanisms underlying inhibition of HSC activation associated with vitamin C and PPAR‐γ up‐regulation in liver fibrosis of SMP30 KO mice. (H<SMALL>EPATOLOGY</SMALL> 2010.)</P>

The restoration of myeloid‐derived suppressor cells as functional antigen‐presenting cells by NKT cell help and all‐<i>trans</i>‐retinoic acid treatment

Lee, Jung&#x2010,Mi,Seo, Jeong&#x2010,Hwan,Kim, Yeon&#x2010,Jeong,Kim, Yun&#x2010,Sun,Ko, Hyun&#x2010,Jeong,Kang, Chang&#x2010,Yuil Wiley Subscription Services, Inc., A Wiley Company 2012 International journal of cancer: Journal internati Vol.131 No.3

<P><B>Abstract</B></P><P>Myeloid‐derived suppressor cells (MDSCs), which accumulate during tumor progression, have been shown to function as important suppressor cells. In a previous study, we showed that immunosuppressive MDSCs could function as immunogenic antigen‐presenting cells (APCs) with the help of activated natural killer T (NKT) cells. In the current study, however, we found that MDSCs harvested at a late time point after tumor injection (late MDSCs) were poorly immunogenic even when stimulated with activated NKT cells. As tumor growth progressed, the expression of MHC and costimulatory molecules on MDSCs was gradually down‐regulated. Late MDSCs also had innate defects in activation and differentiation mediated by cytokine stimuli. Although late MDSCs treated only with all‐<I>trans‐</I>retinoic acid (ATRA), a stimulating agent for MDSC differentiation, could not become immunogenic, NKT ligand‐loaded, ATRA‐treated late MDSCs could be converted into immunogenic APCs to induce incremental immune responses. Furthermore, these effects were mediated by NKT cells secreting IFNγ, and ATRA‐mediated increases in glutathione (GSH) levels. Thus, combined treatment with differentiating and activating agents is a prerequisite for the conversion of late MDSCs into immunogenic APCs. Collectively, these results suggest that combined treatments are required for the differentiation and activation of late MDSCs in late stage cancer.</P>