Glycogen Synthase Kinase 3 Inactivation Induces Cell Senescence through Sterol Regulatory Element Binding Protein 1-Mediated Lipogenesis in Chang Cells

BACKGROUND: Enhanced lipogenesis plays a critical role in cell senescence via induction of expression of the mature form of sterol regulatory element binding protein 1 (SREBP1), which contributes to an increase in organellar mass, one of the indicators of senescence. We investigated the molecular mechanisms by which signaling molecules control SREBP1-mediated lipogenesis and senescence. METHODS: We developed cellular models for stress-induced senescence, by exposing Chang cells, which are immortalized human liver cells, to subcytotoxic concentrations (200 microM) of deferoxamine (DFO) and H2O2. RESULTS: In this model of stress-induced cell senescence using DFO and H2O2, the phosphorylation profile of glycogen synthase kinase 3alpha (GSK3alpha) and beta corresponded closely to the expression profile of the mature form of SREBP-1 protein. Inhibition of GSK3 with a subcytotoxic concentration of the selective GSK3 inhibitor SB415286 significantly increased mature SREBP1 expression, as well as lipogenesis and organellar mass. In addition, GSK3 inhibition was sufficient to induce senescence in Chang cells. Suppression of GSK3 expression with siRNAs specific to GSK3alpha and beta also increased mature SREBP1 expression and induced senescence. Finally, blocking lipogenesis with fatty acid synthase inhibitors (cerulenin and C75) and siRNA-mediated silencing of SREBP1 and ATP citrate lyase (ACL) significantly attenuated GSK3 inhibition-induced senescence. CONCLUSION: GSK3 inactivation is an important upstream event that induces SREBP1-mediated lipogenesis and consequent cell senescence.

Glycogen Synthase Kinase 3 Inactivation Induces Cell Senescence through Sterol Regulatory Element Binding Protein 1-Mediated Lipogenesis in Chang Cells

BACKGROUND: Enhanced lipogenesis plays a critical role in cell senescence via induction of expression of the mature form of sterol regulatory element binding protein 1 (SREBP1), which contributes to an increase in organellar mass, one of the indicators of senescence. We investigated the molecular mechanisms by which signaling molecules control SREBP1-mediated lipogenesis and senescence. METHODS: We developed cellular models for stress-induced senescence, by exposing Chang cells, which are immortalized human liver cells, to subcytotoxic concentrations (200 microM) of deferoxamine (DFO) and H2O2. RESULTS: In this model of stress-induced cell senescence using DFO and H2O2, the phosphorylation profile of glycogen synthase kinase 3alpha (GSK3alpha) and beta corresponded closely to the expression profile of the mature form of SREBP-1 protein. Inhibition of GSK3 with a subcytotoxic concentration of the selective GSK3 inhibitor SB415286 significantly increased mature SREBP1 expression, as well as lipogenesis and organellar mass. In addition, GSK3 inhibition was sufficient to induce senescence in Chang cells. Suppression of GSK3 expression with siRNAs specific to GSK3alpha and beta also increased mature SREBP1 expression and induced senescence. Finally, blocking lipogenesis with fatty acid synthase inhibitors (cerulenin and C75) and siRNA-mediated silencing of SREBP1 and ATP citrate lyase (ACL) significantly attenuated GSK3 inhibition-induced senescence. CONCLUSION: GSK3 inactivation is an important upstream event that induces SREBP1-mediated lipogenesis and consequent cell senescence.

Identification of Plasma Coagulation Factor XIII, Transglutaminase 3 and N epsilon-(gamma-glutamyl) lysine cross-Link in the Silicotic Nodule by Immunohistochemistry / 대한산업의학회지

OBJECTIVES: This study was performed to examine the immunohistochemical distribution of TGase 1, 2, 3, coagulation factor XIII and N epsilon-(gamma-glutamyl) lysine cross-link in the silicotic nodules formed after an intratracheal instillation of the silica. METHODS: The immunohistochemical examinations used antibodies against TGase 1, 2, 3, coagulation factor XIII and N epsilon-(gamma-glutamyl) lysine isopeptide in the silicotic nodules induced after an intratracheal instillation of 50 mg of size fractionated, crystalline silica. RESULTS: A high level of TGase 3 was related to the severity of fibrosis in silicotic nodules and extracellular coagulation factor XIII was detected around the nodules. Expressions of both membrane-bound TGase 1 and TGase 2 were barely detected in the nodules although high expressions were detected in the intact lung. Formation of N epsilon-(gamma-glutamyl) lysine cross-links was increased in severe fibrotic nodules. CONCLUSIONS: TGase 3 might contribute to the eventual stone-like fibrosis via formation of N epsilon-(gamma-glutamyl) lysine cross-links. Futhermore, coagulation factor XIII plays a role in the formation of a provisional matrix which results in fibrogenesis during silicotic nodule formation.

Identification of Cross-linked 46 KDa Protein in Experimentally Induced Silicotic Nodule in Rat Lung / 대한산업의학회지

OBJECTIVES: This study was conducted in order to understand the cellular events associated with silica-induced pathogenesis of the rat lung. METHODS: Silicosis was induced by an intratracheal instillation of 50 mg of silica (SiO2, 0.15 - 10 micrometer) suspended in 500 microliter of a sterile saline solution in Sprague-Dawley rats weighing 200g. Silicotic nodules were excised from the rat lungs 4 weeks after silica instillation, then boiled for 4 days at 110 degrees in solution containing 2% SDS, 10 M urea and 40 mM DTT. The insoluble cellular encapsulates were electrophoresed on 4-12 % gradient SDS-PAGE, and the amino acid composition was analyzed. Affinity chromatographies of the homogenate supernatants of the control lung, silicotic nodule, and normal rat plasma were performed using rabbit IgG, anti-rat, cross-linked protein from the silicotic nodule. The amounts of N epsilon-(gamma-glutamyl) lysine cross-linked in the control lungs and silicotic nodules were determined using HPLC analysis. RESULTS: The remaining cross-linked protein was insoluble in the 10 M urea and 40 mM sulfhydryl reagents even under prolonged boiling conditions. The encapsulate revealed the retention of silica particles within the protein whose amino acid composition showed a high percentage of alanine, leucine and glycine. A 46 KDa protein was identified as a cross-linked protein in the silicotic nodule by affinity chromatography. The level of N epsilon-(gamma-glutamyl) lysine dipeptide in the nodule digest was prominently increased compared with that in the control lung. CONCLUSIONS: Transglutaminase (TGase)-catalyzed cross-linking appears to be involved in the silicotic nodule formation, and the 46 KDa protein may be cross-linked to itself and other extracellular matrix proteins during fibrosis and the formation of eventually insoluble nodule.