Eun-Kyung Song

Overexpression of SIRT1 Protects Pancreatic β-Cells Against Cytokine Toxicity by Suppressing the Nuclear Factor-κB Signaling Pathway

OBJECTIVE—SIRT1, a class III histone/protein deacetylase, is known to interfere with the nuclear factor-κB (NF-κB) signaling pathway and thereby has an anti-inflammatory function. Because of the central role of NF-κB in cytokine-mediated pancreatic β-cell damage, we postulated that SIRT1 might work in pancreatic β-cell damage models. RESEARCH DESIGN AND METHODS—RINm5F (RIN) cells or isolated rat islets were treated with interleukin-1β and interferon-γ. SIRT1 was activated by resveratrol, a pharmacological activator, or ectopic overexpression. The underlying mechanisms of SIRT1 against cytokine toxicity were further explored. RESULTS—Treatment of RIN cells with cytokines induced cell damage, and this damage was well correlated with the expression of the inducible form of nitric oxide (NO) synthase (iNOS) and NO production. However, SIRT1 overexpression completely prevented cytokine-mediated cytotoxicity, NO production, and iNOS expression. The molecular mechanism by which SIRT1 inhibits iNOS expression appeared to involve the inhibition of the NF-κB signaling pathway through deacetylation of p65. In addition, SIRT1 activation by either resveratrol or adenoviral-directed overexpression of SIRT1 could prevent cytokine toxicity and maintain normal insulin-secreting responses to glucose in isolated rat islets. CONCLUSIONS—This study will provide valuable information not only into the mechanisms underlying β-cell destruction but also into the regulation of SIRT1 as a possible target to attenuate cytokine-induced β-cell damage.

NF‐κB‐dependent lymphocyte hyperadhesiveness to synovial fibroblasts by hypoxia and reoxygenation: potential role in rheumatoid arthritis

Hypoxia/reoxygenation has been incriminated as a major factor in the pathogenesis of ischemia/reperfusion injury in various ischemic diseases such as rheumatoid arthritis (RA). In this study, we have investigated the effect of hypoxia/reoxygenation on the expression of intercellular adhesion molecule‐1 (ICAM‐1) in synovial fibroblasts and adherence of lymphocytes to synovial fibroblasts. Hypoxia/reoxygenation strongly activated nuclear factor‐κB (NF‐κB) in synovial fibroblasts to the levels produced by phorbol 12‐myristate 13‐acetate and caused lymphocyte hyperadhesiveness to synovial fibroblasts as well as up‐regulation of ICAM‐1, both of which were completely blocked by a NF‐κB antagonist (pyrrolidine dithiocarbamate). These results indicate that hypoxia/reoxygenation has a major role in sequestration of inflammatory cells to synovium mediated by the activation of NF‐κB. Our data suggest that hypoxia/reoxygenation could be an important target for the development of new, therapeutic strategies in RA.

Connexin-43 Hemichannels Mediate Cyclic ADP-ribose Generation and Its Ca2+-mobilizing Activity by NAD+/Cyclic ADP-ribose Transport

Background: The ADP-ribosyl cyclase CD38 produces cyclic ADP-ribose from NAD+ in the extracellular space. Cyclic ADP-ribose induces intracellular Ca2+ mobilization. Results: We demonstrate that connexin 43 hemichannels import cyclic ADP-ribose to the intracellular target ryanodine receptor. Conclusion: Connexin 43 hemichannels mediate the extracellular production and Ca2+-mobilizing action of cyclic ADP-ribose. Significance: We show that connexin 43 hemichannels resolve the topological hindrance between CD38 and ryanodine receptor. The ADP-ribosyl cyclase CD38 whose catalytic domain resides in outside of the cell surface produces the second messenger cyclic ADP-ribose (cADPR) from NAD+. cADPR increases intracellular Ca2+ through the intracellular ryanodine receptor/Ca2+ release channel (RyR). It has been known that intracellular NAD+ approaches ecto-CD38 via its export by connexin (Cx43) hemichannels, a component of gap junctions. However, it is unclear how cADPR extracellularly generated by ecto-CD38 approaches intracellular RyR although CD38 itself or nucleoside transporter has been proposed to import cADPR. Moreover, it has been unknown what physiological stimulation can trigger Cx43-mediated export of NAD+. Here we demonstrate that Cx43 hemichannels, but not CD38, import cADPR to increase intracellular calcium through RyR. We also demonstrate that physiological stimulation such as Fcγ receptor (FcγR) ligation induces calcium mobilization through three sequential steps, Cx43-mediated NAD+ export, CD38-mediated generation of cADPR and Cx43-mediated cADPR import in J774 cells. Protein kinase A (PKA) activation also induced calcium mobilization in the same way as FcγR stimulation. FcγR stimulation-induced calcium mobilization was blocked by PKA inhibition, indicating that PKA is a linker between FcγR stimulation and NAD+/cADPR transport. Cx43 knockdown blocked extracellular cADPR import and extracellular cADPR-induced calcium mobilization in J774 cells. Cx43 overexpression in Cx43-negative cells conferred extracellular cADPR-induced calcium mobilization by the mediation of cADPR import. Our data suggest that Cx43 has a dual function exporting NAD+ and importing cADPR into the cell to activate intracellular calcium mobilization.

NAADP Mediates Insulin-Stimulated Glucose Uptake and Insulin Sensitization by PPARγ in Adipocytes

Insulin stimulates glucose uptake through the membrane translocation of GLUT4 and GLUT1. Peroxisome proliferator-activated receptor γ (PPARγ) enhances insulin sensitivity. Here, we demonstrate that insulin stimulates GLUT4 and GLUT1 translocation, and glucose uptake, by activating the signaling pathway involving nicotinic acid adenine dinucleotide phosphate (NAADP), a calcium mobilizer, in adipocytes. We also demonstrate that PPARγ mediates insulin sensitization by enhancing NAADP production through upregulation of CD38, the only enzyme identified for NAADP synthesis. Insulin produced NAADP by both CD38-dependent and -independent pathways, whereas PPARγ produced NAADP by CD38-dependent pathway. Blocking the NAADP signaling pathway abrogated both insulin-stimulated and PPARγ-induced GLUT4 and GLUT1 translocation, thereby inhibiting glucose uptake. CD38 knockout partially inhibited insulin-stimulated glucose uptake. However, CD38 knockout completely blocked PPARγ-induced glucose uptake in adipocytes and PPARγ-mediated amelioration of glucose tolerance in diabetic mice. These results demonstrated that the NAADP signaling pathway is a critical molecular target for PPARγ-mediated insulin sensitization.

Peroxisome Proliferator-Activated Receptor γ and Retinoic Acid Receptor Synergistically Up-Regulate the Tumor Suppressor PTEN in Human Promyeloid Leukemia Cells

Peroxisome proliferator-activated receptor γ (PPARγ) and retinoic acid receptors (RARs) have been a focus in chemotherapy for human cancers. The tumor suppressor PTEN plays a pivotal role in the growth of human cancer cells. We investigated whether costimulation of PPARγ and RAR could synergistically up-regulate PTEN in human leukemia cells and consequently potentiate the inhibition of growth and cell cycle progression of these cells. We found that overexpression of PTEN with the adenoviral vector Ad/PTEN caused growth arrest at the G1 phase of the cell cycle of HL-60 cells. HL-60 cells treated with either a PPARγ ligand (ciglitazone) or a RAR ligand(all-trans retinoic acid [ATRA]) up-regulated PTEN in HL-60 cells. The 2 compounds in combination showed synergistic effects on PTEN expression at the protein and messenger RNA levels. Moreover, the combination of ciglitazone and ATRA synergistically reduced cell growth rates and cell cycle arrest at the G1 phase. Our results suggest that, PPARγ and RAR play an important role in controlling the growth of leukemia cells via the up-regulation of PTEN.

Induction of G1 phase arrest and apoptosis in MDA-MB-231 breast cancer cells by troglitazone, a synthetic peroxisome proliferator-activated receptor γ (PPARγ) ligand

Peroxisome proliferator‐activated receptor γ (PPARγ) ligands inhibit cell proliferation and induce apoptosis in cancer cells. Here we wished to determine whether the PPARγ ligand induces apoptosis and cell cycle arrest of the MDA‐MB‐231 cell, an estrogen receptor α negative breast cancer cell line. The treatment of MDA‐MB‐231 cell with PPARγ ligands was shown to induce inhibition of cell growth in a dose‐dependent manner as determined by MTT assay. Cell cycle analysis showed a G1 arrest in MDA‐MB‐231 cells exposed to troglitazone. An apoptotic effect by troglitazone demonstrated that apoptotic cells elevated by 2.5‐fold from the control level at 10 μM, to 3.1‐fold at 50 μM and to 3.5‐fold at 75 μM. Moreover, troglitazone treatment, applied in a dose‐dependent manner, caused a marked decrease in pRb, cyclin D1, cyclin D2, cyclin D3, Cdk2, Cdk4 and Cdk6 expression as well as a significant increase in p21 and p27 expression. These results indicate that troglitazone causes growth inhibition, G1 arrest and apoptotic death of MDA‐MB‐231 cells.

TNF-α upregulates PTEN via NF-κB signaling pathways in human leukemic cells

TNF-α plays a variety of biological functions such as apoptosis, inflammation and immunity. PTEN also has various cellular function including cell growth, proliferation, migration and differentiation. Thus, possible relationships between the two molecules are suggested. TNF-α has been known to downregulate PTEN via NF-κB pathway in the human colon cell line, HT-29. However, here we show the opposite finding that TNF-α upregulates PTEN via activation of NF-κB in human leukemic cells. TNF-α increased PTEN expression at HL-60 cells in a time- and dose-dependent manner, but the response was abolished by disruption of NF-κB with p65 anisense phosphorothioate oligonucleotide or pyrrolidine dithiocarbamate. We found that TNF-α activated the NF-κB pathways, evidenced by the translocation of p65 to the nucleus in TNF-α-treated cells. We conclude that TNF-α induces upregulation of PTEN expression through NF-κB activation in human leukemic cells.

Tumor necrosis factor-α enhances DMSO-induced differentiation of HL-60 cells through the activation of ERK/MAPK pathway

The differentiation of promyelocytic leukemic cells into mature cells is the major strategy for drug-based treatment of leukemia. Higher efficient methods to differentiate promyelocytic leukemic cells have been developed using various differentiation inducers including interferon-α, interleukin-4, tumor necrosis factor-α (TNF-α), and dimethyl sulfoxide (DMSO) as a single agent or in combination with each other. Here, we show that a combination of TNF-α with DMSO shows a synergic effect on HL-60 cell differentiation through the activation of ERK pathway. TNF-α enhanced CD11b expression and percent of cell population in the G1 phase induced by DMSO, which are hallmarks for HL-60 cell differentiation. Inhibition of ERK pathway abolished the synergic effect of TNF-α in combination with DMSO on HL-60 differentiation, but the inhibition NF-κB pathway did not. These results suggest that TNF-α synergistically increases DMSO-induced differentiation of HL-60 cells through the activation of ERK/MAPK-signaling pathway.

Interferon β protects against lethal endotoxic and septic shock through SIRT1 upregulation

Lipopolysaccharide (LPS), an endotoxin derived from gram-negative bacteria, promotes the secretion of proinflammatory cytokines and mediates endotoxemia through activation of mitogen activated protein kinases, NF-κB, and interferon regulatory factor-3. Silent information regulator transcript-1 (SIRT1), an NAD-dependent deacetylase, mediates NF-κB deacetylation, and inhibits its function. SIRT1 may affect LPS-mediated signaling pathways and endotoxemia. Here we demonstrate that SIRT1 blocks LPS-induced secretion of interleukin 6 and tumor necrosis factor α in murine macrophages, and protects against lethal endotoxic and septic shock in mice. We also demonstrate that interferon β increases SIRT1 expression by activating the Janus kinase – signal transducer and activator of transcription (JAK-STAT) pathway in mouse bone marrow derived macrophages. In vivo treatment of interferon β protects against lethal endotoxic and septic shock, which is abrogated by infection with dominant negative SIRT1-expressing adenovirus. Our work suggests that both SIRT1 and SIRT1-inducing cytokines are useful targets for treating patients with sepsis.

Troglitazone enhances tamoxifen-induced growth inhibitory activity of MCF-7 cells.

Peroxisome proliferator-activated receptor gamma (PPARgamma) ligands have been identified as a potential source of therapy for human cancers. However, PPARgamma ligands have a limitation for breast cancer therapy, since estrogen receptor alpha (ER(alpha)) negatively interferes with PPARgamma signaling in breast cancer cells. Here we show that ER(alpha) inhihits PPARgamma transactivity and ER(alpha)-mediated inhibition of PPARgamma transactivity is blocked by tamoxifen, an estrogen receptor blocker. The activation of ER(alpha) with 17-beta-estradiol blocked PPRE transactivity induced by troglitazone, a PPARgamma ligand, indicating the resistance of ER(alpha)-positive breast cancer cells to troglitazone. Indeed, troglitazone inhibited the growth of ER(alpha)-negative MDA-MB-231 cells more than that of ER(alpha)-positive MCF-7 cells. Combination of troglitazone with tamoxifen led to a marked increase in growth inhibition of ER(alpha)-positive MCF-7 cells compared to either agent alone. Our data indicates that troglitazone enhances the growth inhibitory activity of tamoxifen in ER(alpha)-positive MCF-7 cells.