Methylation of eukaryotic elongation factor 2 induced by basic fibroblast growth factor via mitogen-activated protein kinase

Protein arginine methylation is important for a variety of cellular processes including transcriptional regulation, mRNA splicing, DNA repair, nuclear/cytoplasmic shuttling and various signal transduction pathways. However, the role of arginine methylation in protein biosynthesis and the extracellular signals that control arginine methylation are not fully understood. Basic fibroblast growth factor (bFGF) has been identified as a potent stimulator of myofibroblast dedifferentiation into fibroblasts. We demonstrated that symmetric arginine dimethylation of eukaryotic elongation factor 2 (eEF2) is induced by bFGF without the change in the expression level of eEF2 in mouse embryo fibroblast NIH3T3 cells. The eEF2 methylation is preceded by ras-raf-mitogen-activated protein kinase kinase (MEK)-extracellular signal-regulated kinase (ERK1/2)-p21(Cip/WAF1) activation, and suppressed by the mitogen-activated protein kinase (MAPK) inhibitor PD98059 and p21(Cip/WAF1) short interfering RNA (siRNA). We determined that protein arginine methyltransferase 7 (PRMT7) is responsible for the methylation, and that PRMT5 acts as a coordinator. Collectively, we demonstrated that eEF2, a key factor involved in protein translational elongation is symmetrically arginine-methylated in a reversible manner, being regulated by bFGF through MAPK signaling pathway.

Methylation of eukaryotic elongation factor 2 induced by basic fibroblast growth factor via mitogen-activated protein kinase

Protein arginine methylation is important for a variety of cellular processes including transcriptional regulation, mRNA splicing, DNA repair, nuclear/cytoplasmic shuttling and various signal transduction pathways. However, the role of arginine methylation in protein biosynthesis and the extracellular signals that control arginine methylation are not fully understood. Basic fibroblast growth factor (bFGF) has been identified as a potent stimulator of myofibroblast dedifferentiation into fibroblasts. We demonstrated that symmetric arginine dimethylation of eukaryotic elongation factor 2 (eEF2) is induced by bFGF without the change in the expression level of eEF2 in mouse embryo fibroblast NIH3T3 cells. The eEF2 methylation is preceded by ras-raf-mitogen-activated protein kinase kinase (MEK)-extracellular signal-regulated kinase (ERK1/2)-p21(Cip/WAF1) activation, and suppressed by the mitogen-activated protein kinase (MAPK) inhibitor PD98059 and p21(Cip/WAF1) short interfering RNA (siRNA). We determined that protein arginine methyltransferase 7 (PRMT7) is responsible for the methylation, and that PRMT5 acts as a coordinator. Collectively, we demonstrated that eEF2, a key factor involved in protein translational elongation is symmetrically arginine-methylated in a reversible manner, being regulated by bFGF through MAPK signaling pathway.

Clusterin protects H9c2 cardiomyocytes from oxidative stress-induced apoptosis via Akt/GSK-3beta signaling pathway

Clusterin is a secretory glycoprotein, which is highly up-regulated in a variety of normal and injury tissues undergoing apoptosis including infarct region of the myocardium. Here, we report that clusterin protects H9c2 cardiomyocytes from H2O2-induced apoptosis by triggering the activation of Akt and GSK-3beta. Treatment with H2O2 induces apoptosis of H9c2 cells by promoting caspase cleavage and cytochrome c release from mitochondria. However, co-treatment with clusterin reverses the induction of apoptotic signaling by H2O2, thereby recovers cell viability. The protective effect of clusterin on H2O2-induced apoptosis is impaired by PI3K inhibitor LY294002, which effectively suppresses clusterin-induced activation of Akt and GSK-3beta. In addition, the protective effect of clusterin is independednt on its receptor megalin, because inhibition of megalin has no effect on clusturin-mediated Akt/GSK-3beta phosphoylation and H9c2 cell viability. Collectively, these results suggest that clusterin has a role protecting cardiomyocytes from oxidative stress and the Akt/GSK-3beta signaling mediates anti-apoptotic effect of clusterin.

SIRT1 promotes DNA repair activity and deacetylation of Ku70

Human SIRT1 controls various physiological responses including cell fate, stress, and aging, through deacetylation of its specific substrate protein. In processing DNA damage signaling, SIRT1 attenuates a cellular apoptotic response by deacetylation of p53 tumor suppressor. The present study shows that, upon exposure to radiation, SIRT1 could enhance DNA repair capacity and deacetylation of repair protein Ku70. Ectopically over-expressed SIRT1 resulted in the increase of repair of DNA strand breakages produced by radiation. On the other hand, repression of endogenous SIRT1 expression by SIRT1 siRNA led to the decrease of this repair activity, indicating that SIRT1 can regulate DNA repair capacity of cells with DNA strand breaks. In addition, we found that SIRT1 physically complexed with repair protein Ku70, leading to subsequent deacetylation. The dominant-negative SIRT1, a catalytically inactive form, did not induce deacetylation of Ku70 protein as well as increase of DNA repair capacity. These observations suggest that SIRT1 modulates DNA repair activity, which could be regulated by the acetylation status of repair protein Ku70 following DNA damage.

Stem Cell In Adult Pancreas: Its Activation and Induction of Beta Cell Differentiation / 대한해부학회지

Stem cells in adult pancreas and their specific marker are poorly characterized. We hypothesized that pancreatic stem cells could evolve from the duct system in response to neogenic stimulation. Following partial pancreatectomy (Px), we found extensive formation of ductules consisting of nestin-positive epithelial cells with higher replicating ability in the neogenic foci after Px. The neogenic ductules were isolated for the culture of nestin-positive duct cells. These nestinpositive duct cells were numerous and displayed extensive self-replication in the duct cell explants, thus depicted as nestin-positive duct stem (NPDS) cells. Endocrine cells, mostly insulin cells were present in the explants at day 2 as single cells or as small clusters adjacent to the NPDS cells, and formed islet-like masses at day 3 of culture, implying islet cell differentiation from NPDS cells. We found transient up-regulation of PDX-1 expression by RT-PCR at day 3 after Px in pancreatic tissue. We investigated the effect of clusterin overexpression on differentiation of insulin beta cells from duct cells We found that the number of insulin producing cells increased 11.5 fold when clusterin was overexpressed. Insulin expression, both insulin mRNA and peptide levels, was increased in clusterin cDNA transfected cells. In conclusion, we suggest that NPDS cells could be generated from adult pancreas by neogenic motivations and they may differentiate into insulin-secreting-cells, and clusterin could stimulate not only differentiation of precursor cells in the pancreatic duct, but also proliferation of predifferentiated beta cells. Those differentiated beta cells are functional cells secreting insulin in response to glucose stimulation.

Cyto-protective Effects of Aqueous Extract from the Gall of Rhus chinensis on Pancreatic Beta-cell / 대한해부학회지

We have previously reported that aqueous extract of gall from Rhus chinensis, known as "Obaeja", inhibited rat intestinal alpha-glucosidase and suppressed postprandial hyperglycemia by delaying digestion and absorption of intestinal carbohydrate (Shim et al., 2003). This led us to speculate that obaeja could be involved in ameliorating beta-cell injury by lowering glucotoxicity. In the present study, we thus examined the protective effect of obaeja on pancreatic beta-cell damage along with its anti-diabetic effect in streptozotocin-induced animal models. Streptozotocin was administered to rat pups (neonate/STZ model), or to adult rats with a lower dose using osmotic pump (osmotic pump/STZ model) for inducing beta cell death and diabetes. Obaeja was given to those rat pups after weaning in neonate/STZ model, or 2 weeks before subcutaneous implantation of osmotic pump to rats of the other latter model. In the diabetic control rats of the neonate/STZ model, which were not fed with obaeja, some pancreatic islets demonstrated a destruction of beta cell mass with insulitis 2 weeks after weaning, while some larger and irregular islets were formed by proliferation of alpha cells. In particular, we found some pancreatic lobules showing a severe inflammation and degeneration of islet and acinar tissues in this model. Islets in these inflammatory lobules were smaller in size with only few cells. In contrast, any inflammatory responses and insulitis were not observed in pancreas of the rats fed obaeja in this model. The islets in those rats maintained their normal profiles and islet cell population. Such anti-cytotoxic effect was also monitored in the diabetic rats of osmotic pump/STZ model. Especially, occurrence of hyperglycemia in the obaeja fed rats was delayed by 25~30 days than that of diabetic control rats in this model. Taken together, these results imply that regulation of postprandial blood glucose level by obaeja feeding may ameliorate a secondary injury caused by glucotoxicity.

Stimualtion of Beta Cell Neogenesis by SBTI (Soybean Trypsin Inhibitor) or Soybean Diet in the Subtotal Pancreatectomy Models / 대한해부학회지

The present study was performed to corroborate our hypothesis that soybean diet or SBTI treatment could stimulate neogenic regeneration of pancreatic beta cells, but also increase insulin synthesis and secretion from the beta cells for correction of hyperglycemia and diabetic symptoms. We, thus, monitored the beta cell regeneration in the neogenic pancreas as well as the changes of the blood glucose and insulin levels after subtotal pancreatectomy. The diabetic animals with hyperglycemia induced by the subtotal pancreatectomy showed recovery of blood glucose level toward the normal range (<150 mg/dl) by giving raw soybean for 3~4 weeks. Most animals treated with SBTI remained in euglycemic condition in spite of diabetic induction by subtotal pancreatectomy. Their serum insulin level was also recovered to the level of normal control, indicating the increased insulin synthesis and secretion from the neogenic beta cells. Neogenic area was enlarged at least 2 times in the pancreatectomized rats with dietary soybean or SBTI treatment, when compared with their pancreatectomized controls without any dietary treatment. In neogenic tissue, few endocrine cells were detected as a single cell or cell cluster at 3 days, and they formed primitive islet at 7 days after pancreatectomy in non-treated controls. The numbers of beta cells as well as alpha cells were considerably increased in the SBTI treated rats, and early formation of primitive islets were found in the neogenic tissue of those animals at 3 days after pancreatectomy. Those beta cells demonstrated a strong immunoreactivity for insulin, indicating their bioactive insulin secretion. Clusterin, a marker protein for pancreatic neogenesis, was expressed in the wider pancreatic area and at earlier stage after pancreatectomy when compared with non-treated control rats, indicating acceleration and stimulation of neogenesis of pancreas by stimulating proliferation and differentiation of the functional pancreatic cells. Taken together, we concluded that dietary soybean and SBTI could stimulate beta cell neogenesis and induce activation of insulin synthesis and secretion from the neogenic beta cells for correction of glucose homeostasis in diabetic subjects.

Regulatory Effects of Nitric Oxide (NO) on Pancreatic Exocrine Secretion / 대한해부학회지

Recently nitric oxide (NO) is known as a bioactive molecule modulating secretory activity in various glandular tissues. Previously we have localized bNOS, a neuronal isoform of nitric oxide synthase, in the pancreatic tissue, particularly in the pancreatic islet of Langerhans and in the neurons of intrapancreatic ganglia. It implies that NO may play the important roles in regulation of pancreatic secretion by transmitting the neuronal signals from autonomic nervous system to endocrine and/or exocrine system of pancreas. We also revealed that NO is involved in regulation of insulin secretion and its synthesis. The present study was designed to elucidate the regulatory effect of NO on the pancreatic exocrine secretion by way of insulo-acinar axis. For the experiment, we observed modification of amylase secretion in the rats treated with N(G)-nitro-L-arginine-methyl ester (NAME), a potent NOS inhibitor. In addition, we observed the expression of clusterin which is known to be a protein associated with cell viability in order to assess the cytotoxic effect of NO. The present study showed that the intra-pancreatic NO is involved in regulation of amylase secretion of pancreatic acinar cells. Amylase immunoreactivity was significantly decreased at 60 and 90 min after NAME injection, although little change was seen during 30 min after treatment. However, the amylase immunoreaction was recovered toward the normal range at 120 min after NAME treatment. In electron-immunolabeling experiment, we observed the secretory granules with higher electron density, but less immunolabeling for amylase at 60~90 min after NAME treatment, while they restored normal feature and labeling density at 120 min. Clusterin expression increased along with the time course of experiment and demonstrated a highest level at 120 min after NAME injection. Taken together, the above results indicate that lowered level of NO induced by NAME treatment reduces amylase secretion of acinar tissue. It implies that increased level of NO in physiological range may stimulate pancreatic exocrine secretion.

Transplantation of Langerhans islet into digestive organs of the diabetic rat / 대한해부학회지

Although replacement therapy with insulin can prevent acute metabolic disorder in patients with IDDM (insulin dependent diabetes mellitus), it does not permanently restore glucose homeostasis. Recently it has been reported that islet transplantation could completely correct the glucose metabolic abnormalities and prevent further progression of the secondary complications of IDDM. For successful transplantation, the isolated islets should be prepared without loss of viability, while their immunogenicity being suppressed to reduce graft rejection. The present study was aimed to determine the optimal condition of islet culture, and to transplant them into the digestive organs including gastroin-testinal wall and salivary gland. For islet culture, pancreatic islets were isolated by a modified collagenase digestion technique from rats and cultured for 24, 48 and 72 hours in RPMI-1640 containing 0, 5.6 and 16.7 mM glucose. The viability of islets was evaluated by detection of insulin mRNA expressed in islet beta-cells using the in-situ hybridization and northern blot analysis, while their insulin content was examined by immunocytochemistry. Insulin mRNA was significantly reduced after 48 hours of culture in the islets incubated with absence of glucose, while distinct immunoreaction for insulin remained in the same islet. On the other hand, the islets cultured with normoglycemic (5.6 mM glucose) and hyperglycemic (16.7 mM glucose) conditions showed a normal or excessive transcription of insulin gene after 72 hours, respectively. These results indicate that biosynthetic activity of islets could be maintained longer than 72 hours without alteration of viability when they were cultured in normoglycemic condition. Therefore, we used islets cultured for 72 hours with 5.6 mM glucose for transplantation. The islets were implanted into the submucosal wall of the stomach and duodenum as well as into the parenchyme of the submandibular gland of the streptozotocin-induced diabetic rats. The transplanted islets in the gastrointestinal wall were abolished in 72 hours, while the islets injected into the submandibular gland retained normal cellular structure with viability for longer period. The beta-cell in the submandibular gland showed similar immunoreactivity for insulin compared to that of normal islets. However, they showed gradual infiltration of lymphocytes and beta-cell destruction at 10~14 days after transplantation. We suggested that the submandibular gland could be recommended as an alternative site for islet transplantation, because it is very easy to access for transplantation and provides the structural and functional similarities to pancreas in which the islets spontaneously grow.

Effects of dexamethasone and DHEA on the changes of glutamate and polyamine uptake in rat astrocytes by lipopolysaccharide and antimycin A

Interactions among dexamethasone, dehydroepiandrosterone (DHEA), lipopolysaccharide (LPS), and antimycin A on the glutamate uptake and the polyamine uptake were investigated in primary cultures of rat cerebral cortical astrocytes to examine the effects of dexamethasone and DHEA on the regulatory role of astrocytes in conditions of increased extracellular concentrations of glutamate or polyamines. 1. (3H)Glutamate uptake: LPS and antimycin A decreased Vmax, but both drugs had little effect on Km. Dexamethasone also decreased basal Vmax without any significant effect on Km. And dexamethasone further decreased the antimycin A-induced decrease of Vmax. DHEA did not affect the kinetics of basal glutamate uptake and the change by LPS or antimycin A. 2. (14C)Putrescine uptake: LPS increased Vmax, and antimycin A decreased Vmax. They showed little effect on Km. Dexamethasone decreased Vmax of basal uptake and further decreased the antimycin A-induced decrease of Vmax, and also decreased Vmax to less than control in LPS-treated astrocytes. DHEA did not affect Km and the change of Vmax by LPS or antimycin A. 3. (14C)Spermine uptake: Antimycin A decreased Vmax, and LPS might increase Vmax. Km was little affected by the drugs. Dexamethasone decreased basal Vmax and might further decrease the antimycin A-induced decrease of Vmax. And dexamethasone also decreased Vmax to less than control in LPS-treated astrocytes. DHEA might increase basal Vmax and Vmax of LPS-treated astrocytes. 4. Vmax of glutamate uptake by astrocytes was increased by putrescine (1000 muM & 2000 muM) and spermidine (200 muM, 500 muM & 2000 muM). Spermine, 200 muM (and 100 muM), also increased Vmax, but a higher dose of 2000 muM decreased Vmax. Km of glutamate uptake was not significantly changed by these polyamines, except that higher doses of spermine showed tendency to decrease Km of glutamate uptake. In astrocytes, dexamethasone inhibited the glutamate uptake and the polyamine uptake in normal or hypoxic conditions, and the polyamine uptake might be stimulated by LPS and DHEA. Polyamines could aid astrocytes to uptake glutamate.

Molecular Cloning of Mouse Interleukin-2 Receptor bata-Chain Gene Promoter / 대한면역학회지

No abstract available.

Association of dexamethasone-induced apoptosis of G|1-arrest of human leukemic CEM cells with polyamine deficit

The effects of DFMO or/and putrescine on the dexamethasone-induced apoptosis of CEM cells were studied to investigate the role of polyamines in anti-leukemic glucocorticoid action. Dexamethasone-induced apoptosis was preceded by significant decreases of cellular polyamine contents and putrescine uptake activity. But DFMO produced decreases of putrescine and spermidine contents and marked increase of putrescine uptake activity, but did not induce apoptosis. However, dexamethasone and DFMO, respectively, induced G|1-arrest in cell cycle and hypophosphorylation of pRb, resulting in the increase of G|1 to S ratio and decrease of CEM cell count. DFMO enhanced the dexamethasone-induced apoptosis and G|1-arrest. On the other hand, putrescine little affected the apoptotic and G|1-arresting activities of dexamethasone, but almost suppress the effects of DFMO and also the DFMO-dependent enhancement of dexamethasone effects. These results suggested that the dexamethasone-induced apoptosis to be associated with pRb hypophosphorylation and G|1-arrest in CEM cells might be ascribed to the concomitant decreases of cellular polyamine contents and putrescine uptake activity.

Sequence analysis of polyhedrin gene promoter and construction of an expression vector of hyphantria cunea nuclear polyhedrosis virus

No abstract available.