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ABSTRACT: Transforming growth factor (TGF)-β1 regulates diverse cellular functions. Particularly, TGF-β1 induces monocyte migration to sites of injury or inflammation in early period, whereas TGF-β1 inhibits cell migration in late phase. In this study, we attempted to understand how TGF-β1 suppresses cell migration in late phase. We found that TGF-β1 of short exposure induces the production of chemokines, such as macrophage inflammatory protein (MIP)-1α, by Raw 264.7 cells. However, knock-down of small GTPase RhoA by sh-RhoA inhibited the production of MIP-1α and macrophage migration, suggesting that RhoA is essential for expression of this chemokine. An activator of Epac (exchange proteins directly activated by cAMP; a guanine nucleotide exchange factor of Rap1), 8CPT-2Me-cAMP which leads to Rap1 activation abrogated MIP-1α expression and macrophage migration. Indeed, GTP-RhoA and GTP-Rap1 levels were reciprocally regulated in a time-dependent manner following TGF-β1 stimulation. 8CPT-2Me-cAMP suppressed GTP-RhoA levels, whereas si-Rap1 augmented GTP-RhoA levels and cell migration. TGF-β1 produced cAMP in late period and si-RNAs of Epac1 (exchange protein directly activated by cAMP 1) and Epac2 reduced GTP-Rap1 levels leading to promotion of GTP-RhoA levels. Furthermore, si-RNA of ARAP3 (Rap-dependent RhoGAP) increased GTP-RhoA level and cell migration. Therefore, we propose the mechanism that prolonged TGF-β1 treatment produce cAMP, which activates sequentially Epac, Rap1 and ARAP3, resulting in suppression of RhoA, chemokine expression, and macrophage migration. Contrary to the general concept that Rap1 stimulates cell migration, we demonstrated in this study that Rap1 inhibits cell migration by suppression of RhoA activity in response to TGF-β1. © 2013 Wiley Periodicals, Inc.
Journal of Cellular Physiology 04/2013; · 3.87 Impact Factor
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Jee-In Heo,
Soo-Jin Oh,
Yoon-Jung Kho,
Jeong-Hyeon Kim,
Hong-Joon Kang,
Seong-Hoon Park,
Hyun-Seok Kim,
Jong-Yeon Shin,
Min-Ju Kim,
Minju Kim, Sung Chan Kim,
Jae-Bong Park,
Jaebong Kim,
Jae-Yong Lee
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ABSTRACT: DNA damage in eukaryotic cells induces signaling pathways mediated by the ATM, p53 and ERK proteins, but the interactions between these pathways are not completely known. To address this issue, we performed a time course analysis in human embryonic fibroblast cells treated with DNA-damaging agents. DNA damage induced the phosphorylation of p53 at Ser 15 (p-p53) and the phosphorylation of ERK (p-ERK). Inhibition of p53 by a dominant negative mutant or in p53(-/-) fibroblast cells abolished ERK phosphorylation. ERK inhibitor prevented p53 phosphorylation, indicating that phosphorylations of p53 and p-ERK are interdependent each other. A time course analysis showed that ATM interacted with p-p53 and p-ERK in early time (0.5 h) and interaction between ATM-bound p-p53 and p-ERK or ATM-bound p-ERK and p-p53 occurred in late time (3 h) of DNA damage. These results indicate that ATM mediates interdependent activation of p53 and ERK through formation of a ternary complex between p-p53 and p-ERK in response to DNA damage to cause growth arrest.
Molecular Biology Reports 05/2012; 39(8):8007-14. · 2.93 Impact Factor
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ABSTRACT: Phagocytic NADPH oxidase plays a critical role in superoxide generation in macrophage cells. Small GTPases, including Rac1 and Rac2, have been implicated in the regulation of NADPH oxidase activity. Rap1, which has no effect in a cell-free system of oxidase activation, recently has been proven to colocalize with cytochrome b(558). In addition, neutrophils from rap1A(-/-) mice reduce fMLP-stimulated superoxide production. Here, we tried to determine whether Rap1 also plays a role in the production of superoxide. IgG-opsonized zymosan (IOZ) particles treatment induced Rap1 activation and superoxide generation. Knock-down of Rap1 by si-Rap1 suppressed IOZ-induced superoxide formation. Sh-RhoA also reduced superoxide levels, but 8CPT-2Me-cAMP, an activator of Epac1 (a guanine nucleotide exchange factor (GEF) of Rap1), could recover the levels to the control value. When cells were stimulated by IOZ, Rap1 and Rac1 were translocated to the membrane, and then interacted with p22(phox). 8CPT-2Me-cAMP rescued sh-RhoA-induced reduction of the interaction between Rac1 and p22(phox), and enhanced lysophosphatidic acid (LPA)-induced increase of their interaction. Moreover, Rac1 activity was increased by both LPA and 8CPT-2Me-cAMP when treated with IOZ particles. Si-Vav2 impaired GTP-Rac1 levels in response to 8CPT-2Me-cAMP/IOZ. Phosphorylation of RhoA activates Rac1 in response to IOZ by the enhanced binding of phospho-RhoA to RhoGDI, leading to the release of Rac1 from the Rac1-RhoGDI complex. In conclusion, IOZ treatment induces Rap1 activation and phosphorylation of RhoA, which in turn cause Rac1 activation and promote Rac1 translocation to the membrane leading to binding with p22(phox) that activates NADPH oxidase and produces superoxide.
Free radical biology & medicine 02/2012; 52(9):1796-805. · 5.42 Impact Factor
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ABSTRACT: Phagocytosis occurs primarily through two main processes in macrophages: the Fcγ receptor- and the integrin αMβ2-mediated processes. Complement C3bi-opsonized particles are known to be engulfed through integrin αMβ2-mediated process, which is regulated by RhoA GTPase. C3 toxin fused with Tat-peptide (Tat-C3 toxin), an inhibitor of the Rho GTPases, was shown to markedly inhibit the phagocytosis of serum (C3bi)-opsonized zymosans (SOZs). However, 8CPT-2Me-cAMP, an activator of exchange protein directly activated by cAMP (Epac, Rap1 guanine nucleotide exchange factor), restored the phagocytosis of the SOZs that was previously inhibited by the Tat-C3 toxin. In addition, a constitutively active form of Rap1 GTPase (CA-Rap1) also restored the phagocytosis that was previously reduced by a dominant negative form of RhoA GTPase (DN-RhoA). This suggests that Rap1 can replace the function of RhoA in the phagocytosis. Inversely, CA-RhoA rescued the phagocytosis that was suppressed by DN-Rap1. These findings suggest that both RhoA and Rap1 GTPases collectively regulate the phagocytosis of SOZs. In addition, filamentous actin was reduced by the Tat-C3 toxin, which was again restored by 8CPT-2Me-cAMP. Small interfering profilin suppressed the phagocytosis, suggesting that profilin is essential for the phagocytosis of SOZs. Furthermore, 8CPT-2Me-cAMP increased the co-immunoprecipitation of profilin with Rap1, whereas Tat-C3 toxin decreased that of profilin with RhoA. Co-immunoprecipitations of profilin with actin, Rap1, and RhoA GTPases were augmented in the presence of GTPγS rather than GDP. Therefore, we propose that both Rap1 and RhoA GTPases regulate the formation of filamentous actin through the interaction between actin and profilin, thereby collectively inducing the phagocytosis of SOZs in macrophages.
Journal of Biological Chemistry 12/2011; 287(7):5145-55. · 4.77 Impact Factor
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ABSTRACT: Neural tissue is arisen from presumptive ectoderm via inhibition of bone morphogenetic protein (BMP) signaling during Xenopus early development. Previous studies demonstrate that ectopic expression of dominant negative BMP4 receptor (DNBR) produces neural tissue in animal cap explants (AC) and also increases the expression level of various genes involved in neurogenesis. To investigate detail mechanism of neurogenesis in transcriptional level, we analyzed RNAs increased by DNBR using total RNA sequencing analysis and identified several candidate genes. Among them, xCITED2 (Xenopus CBP/p300-interacting transcription activator) was induced 4.6 fold by DNBR and preferentially expressed in neural tissues at tadpole stage. Ectopic expression of xCITED2 induced anterior neural genes without mesoderm induction and reduced BMP downstream genes, an eye specific marker and posterior neural marker. Taken together, these results suggest that xCITED2 may have a role in the differentiation of anterior neural tissue during Xenopus early development.
Experimental neurobiology. 09/2011; 20(3):123-9.
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Seong-Hoon Park,
Hong-Jun Kang,
Hyun-Seok Kim,
Min-Ju Kim,
Jee-In Heo,
Jeong-Hyeon Kim,
Yoon-Jung Kho, Sung Chan Kim,
Jaebong Kim,
Jae-Bong Park,
Jae-Yong Lee
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ABSTRACT: Since the detailed comparison of DNA repair activities among mammalian embryonic fibroblast cells with different replicative life spans has not been investigated, we tested DNA repair activities in embryonic fibroblast cells derived from mammals including human, dog, rat, and mouse. The cell viability after treatment of four DNA damage agents appeared to be decreased in the order of human embryonic fibroblasts (HEFs) > dog embryonic fibroblasts (DEFs) > rat embryonic fibroblasts (REFs) > mouse embryonic fibroblasts (MEFs) although statistical significance was lacking. The amounts of strand breaks and AP (apurinic/apyrimidinic) sites also appear to be decreased in the order of HEFs > DEFs > REFs ≥ MEFs after treatment of DNA damage agents. The DNA repair activities and rates including base excision repair (BER), nucleotide excision repair (NER) and double-strand break repair (DSBR) including non-homologous end-joining (NHEJ) decreased again in the order of HEFs > DEFs > REFs ≥ MEFs. BER and NHEJ activities in 3% O(2) also decreased in the order of HEFs > DEFs > REFs > MEFs. This order in DNA repair activity appears to be coincident with that of replicative life span of fibroblasts and that of life span of mammals. These results indicate that higher DNA repair activity is related with longer replicative life span in embryonic fibroblast cells.
Biogerontology 08/2011; 12(6):565-79. · 3.34 Impact Factor
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Samuel Beck,
Xun Jin,
Jinlong Yin,
Sung-Hak Kim,
Nam-Kyung Lee,
Se-Yeong Oh,
Xiong Jin,
Min-Kook Kim,
Eun-Bae Kim,
Jee-Soo Son, Sung-Chan Kim,
Do-Hyun Nam,
Se-Hyuk Kim,
Sang-Kee Kang,
Hyunggee Kim,
Yun-Jaie Choi
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ABSTRACT: Glioma stem cells (GSCs) are presumably major culprits for brain tumor initiation, progression, and recurrence after conventional therapies. Thus, selective targeting and eradication of GSCs may provide a promising and effective therapeutic approach. Here, we isolated a GSC-targeting (GSCT) peptide that demonstrated selective binding affinity for many undifferentiated GSCs using in vitro phage display technology. This GSCT peptide binds to isotypes of Nestin proteins specifically expressed in GSCs, enabling it to target Nestin-positive cells in human glioblastoma tissues. In human glioblastoma tissue specimens, the fluorescence-conjugated GSCT peptide could visualize putative GSC populations, showing its possible use as a diagnostic agent. GSCT peptide is also internalized into undifferentiated GSCs specifically in vitro, and moreover, intravenously injected GSCT peptide effectively penetrated into tissues, specifically accumulated in gliomas that arise from subcutaneous and orthotopic implantation, and predominantly targeted Nestin-positive cells in these tumors. Thus, our GSCT peptide may be useful for the development of more promising therapeutic and diagnostic modalities that target GSCs in brain tumors.
Biomaterials 08/2011; 32(33):8518-28. · 7.40 Impact Factor
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ABSTRACT: In early vertebrate development, mesoderm induction is a crucial event regulated by several factors including the activin, BMP and FGF signaling pathways. While the requirement of FGF in Nodal/activin-induced mesoderm formation has been reported, the fate of the tissue modulated by these signals is not fully understood. Here, we examined the fate of tissues when exogenous activin was added and FGF signaling was inhibited in animal cap explants of Xenopus embryos. Activin-induced dorsal mesoderm was converted to ventral mesoderm by inhibition of FGF signaling. We also found that inhibiting FGF signaling in the dorsal marginal zone, in vegetal-animal cap conjugates or in the presence of the activin signaling component Smad2, converted dorsal mesoderm to ventral mesoderm. The expression and promoter activities of a BMP responsive molecule, PV.1 and a Spemann organizer, noggin, were investigated while FGF signaling was inhibited. PV.1 expression increased, while noggin decreased. In addition, inhibiting BMP-4 signaling abolished ventral mesoderm formation induced by exogenous activin and FGF inhibition. Taken together, these results suggest that the formation of dorso-ventral mesoderm in early Xenopus embryos is regulated by a combination of FGF, activin and BMP signaling.
Differentiation 06/2011; 82(2):99-107. · 2.81 Impact Factor
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Samuel Beck,
Xun Jin,
Young-Woo Sohn,
Jun-Kyum Kim,
Sung-Hak Kim,
Jinlong Yin,
Xumin Pian, Sung-Chan Kim,
Do-Hyun Nam,
Yun-Jaie Choi,
Hyunggee Kim
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ABSTRACT: Telomerase reverse transcriptase (TERT), the catalytic subunit of the enzyme telomerase, is robustly expressed in cancer cells. TERT enables cells to avoid chromosome shortening during repeated replication by maintaining telomere length. However, several lines of evidence indicate that many cancer cells exhibit shorter telomere length than normal tissues, implying an additional function of TERT in tumor formation and progression. Here, we report a telomerase activity-independent function of TERT that induces cancer stemness in glioma cells. Overexpression of TERT712, a telomerase activity-deficient form of TERT, in U87MG cells promoted cell self-renewal in vitro, and induced EGFR expression and formation of gliomas exhibiting cellular heterogeneity in vivo. In patients with glioblastoma multiforme, TERT expression showed a high correlation with EGFR expression, which is closely linked to the stemness gene signature. Induction of differentiation and TERT-knockdown in glioma stem cells led to a marked reduction in EGFR expression, cancer stemness, and anticancer drug resistance. Together, our findings indicate that TERT plays a crucial role in tumor progression by promoting cancer stemness through expression of EGFR.
Molecules and Cells 12/2010; 31(1):9-15. · 2.18 Impact Factor
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Jee-In Heo,
Soo-Jin Oh,
Yoon-Jung Kho,
Jeong-Hyeon Kim,
Hong-Joon Kang,
Seong-Hoon Park,
Hyun-Seok Kim,
Jong-Yeon Shin,
Min-Ju Kim, Sung Chan Kim,
Jae-Bong Park,
Jaebong Kim,
Jae-Yong Lee
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ABSTRACT: Since anti-apoptotic effect of ERK has not been elucidated clearly in DNA-damage-induced cell death, the role of ERK was examined in normal HEF cells treated with mild DNA damage using etoposide or camptothecin. ERK was activated by DNA damage in HEF cells. PD98059 increased apoptosis and reduced DNA-damage-induced p21Waf1/Cip1/Sdi level. Depletion of p21Waf1/Cip1/Sdi induced cell death and PD98059 induced additional cell death. DNA-damage-induced increase in cytoplasmic localization and phosphorylation of threonine residues of p21Waf1/Cip1/Sdi was reversed by PD98059. Thus, the results suggest that ERK pathway mediates anti-apoptotic effects through phosphorylation and cytoplasmic localization of p21Waf1/Cip1/Sdi in response to mild DNA damage.
Molecular Biology Reports 11/2010; 38(4):2785-91. · 2.93 Impact Factor
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Soo-Jin Oh,
Jee-In Heo,
Yoon-Jung Kho,
Jeong-Hyeon Kim,
Hong-Joon Kang,
Seong-Hoon Park,
Hyun-Seok Kim,
Jong-Yeon Shin,
Min-Ju Kim, Sung Chan Kim,
Jae-Bong Park,
Jaebong Kim,
Jae-Yong Lee
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ABSTRACT: Nitric oxide (NO) regulates proliferation, differentiation and survival of neurons. Although NO is reported to involve in NGF-induced differentiation of PC12 cells, the role of NO has not been characterized in primary neuron cells. Therefore, we investigated the role of NO in neuronal differentiation of primary cortical neuron cells. Primary cortical neuron cells were prepared from rat embryos of embryonic day 18 and treated with NMMA (NOS inhibitor) or PTIO (NO scavenger). Neurite outgrowth of neuron cells was counted and the mRNA levels of p21, p27, c-jun and c-myc were measured by RT-PCR. Neurite outgrowth of primary cortical neuron cells was inhibited a little by NOS inhibitor and completely by NO scavenger. The mRNA levels of p21 and p27, differentiation-induced growth arrest genes were increased during differentiation, but they were decreased by NOS inhibitor or NO scavenger. On the other hand, the level of c-jun mRNA was not changed and the level of c-myc mRNA was increased during differentiation differently from previously reported. The levels of these mRNA were reversed in NOS inhibitor- or NO scavenger-treated cells. The level of nNOS protein was not changed but NOS activity was inhibited largely by NOS inhibitor or NO scavenger. These results suggest that NO is an essential mediator for neuronal differentiation of primary cortical neuron cells.
Experimental neurobiology. 09/2010; 19(2):83-9.
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ABSTRACT: The rat pheochromocytoma cell line PC12 has been widely used as a model to study neuronal differentiation. PC12 cells give rise to neurites in response to basic fibroblast growth factor (bFGF). However, it is unclear whether bFGF promotes neurite outgrowth by inducing RhoA inactivation, and a mechanism for RhoA inactivation in PC12 cells in response to bFGF has not been reported. Lysophosphatidic acid (LPA) treatment and the expression of constitutively active (CA)-RhoA (RhoA V14) impaired neurite formation in response to bFGF, while Tat-C3 exoenzyme and the expression of dominant negative (DN)-RhoA (RhoA N19) stimulated neurite outgrowth. GTP-bound RhoA levels were reduced in response to bFGF, which suggests that the inactivation of RhoA is essential to neurite outgrowth in response to bFGF. To investigate the mechanism of RhoA inactivation, this study examined the roles of p190RhoGAP and Rap-dependent RhoGAP (ARAP3). DN-p190RhoGAP prevented neurite outgrowth, while WT-p190RhoGAP and Src synergistically stimulated neurite outgrowth; these findings suggest that bFGF promotes the inactivation of RhoA and subsequent neurite outgrowth through p190RhoGAP and Src. Furthermore, DN-Rap1 and DN-ARAP3 reduced neurite formation in PC12 cells. These results suggest that RhoA is likely to be inactivated by p190RhoGAP and ARAP3 during neurite outgrowth in response to bFGF.
Journal of Cellular Physiology 09/2010; 224(3):786-94. · 3.87 Impact Factor
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ABSTRACT: Although human telomerase catalytic subunit (TERT) has several cellular functions including telomere homeostasis, genomic stability, cell proliferation, and tumorigenesis, the molecular mechanism underlying anti-apoptosis regulated by TERT remains to be elucidated. Here, we show that ectopic expression of TERT in spontaneously immortalized human fetal fibroblast (HFFS) cells, which are a telomerase- and p53-positive, leads to increases of cell proliferation and transformation, as well as a resistance to DNA damage response and inactivation of p53 function. We found that TERT and a mutant TERT (no telomerase activity) induce expression of basic fibroblast growth factor (bFGF), and ectopic expression of bFGF also allows cells to be resistant to DNA-damaging response and to suppress activation of p53 function under DNA-damaging induction. Furthermore, loss of TERT or bFGF markedly increases a p53 activity and DNA-damage sensitivity in HFFS, HeLa and U87MG cells. Therefore, our findings indicate that a novel TERT-bFGF axis accelerates the inactivation of p53 and consequent increase of resistance to DNA-damage response.
Experimental and Molecular Medicine 08/2010; 42(8):574-82. · 2.48 Impact Factor
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ABSTRACT: Rat pheochromocytoma (PC12) cells have been used to investigate neurite outgrowth. Nerve growth factor (NGF) has been well known to induce neurite outgrowth from PC12 cells. RhoA belongs to Ras-related small GTP-binding proteins, which regulate a variety of cellular processes, including cell morphology alteration, actin dynamics, and cell migration. NGF suppressed GTP-RhoA levels after 12 h in PC12 cells and was consistently required for a long time to induce neurite outgrowth. Constitutively active (CA)-RhoA suppressed neurite outgrowth from PC12 cells in response to NGF, whereas dominant-negative (DN)-RhoA stimulated it, suggesting that RhoA inactivation is essential for neurite outgrowth. Here, we investigated the mechanism of RhoA inactivation. DN-p190RhoGAP abrogated neurite outgrowth, whereas wild-type (WT)-p190RhoGAP and WT-Src synergistically stimulated it along with accelerating RhoA inactivation, suggesting that p190RhoGAP, which can be activated by Src, is a major component in inhibiting RhoA in response to NGF in PC12 cells. Contrary to RhoA, Rap1 was activated by NGF, and DN-Rap1 suppressed neurite outgrowth, suggesting that Rap1 is also essential for neurite outgrowth. RhoA was co-immunoprecipitated with Rap1, suggesting that Rap1 interacts with RhoA. Furthermore, a DN-Rap-dependent RhoGAP (ARAP3) prevented RhoA inactivation, abolishing neurite formation from PC12 cells in response to NGF. These results suggest that NGF activates Rap1, which, in turn, up-regulates ARAP3 leading to RhoA inactivation and neurite outgrowth from PC12 cells. Taken together, p190RhoGAP and ARAP3 seem to be two main factors inhibiting RhoA activity during neurite outgrowth in PC12 cells in response to NGF.
Experimental and Molecular Medicine 03/2010; 42(5):335-44. · 2.48 Impact Factor
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Jinlong Yin,
Xun Jin,
Samuel Beck,
Dong Ho Kang,
Zhongshan Hong,
Zhehu Li,
Yongcheng Jin,
Qiankun Zhang,
Yun-Jaie Choi, Sung-Chan Kim,
Hyunggee Kim
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ABSTRACT: Our current understanding of muscle and adipose tissue development has been largely restricted to the study of murine myogenic and adipogenic cell lines, since attempts to establish these cell lines from other species have met with only limited success. Here we report that a spontaneously immortalized bovine embryonic fibroblast cell line (BEFS) undergoes differentiation into adipogenic or myogenic lineages when ectopically transduced with PPARgamma2 (an adipogenic lineage determinant) or MyoD (a myogenic lineage determinant) and grown in adipogenic and myogenic differentiation culture media (ADCM and MDCM, respectively). We also found that PPARgamma2-overexpressing BEFS cells (BEFS-PPARgamma2) grown in ADCM with or without the PPARgamma2 ligand, troglitazone, preferentially differentiate into adipogenic cells in the presence of ectopic MyoD expression. Ectopic expression of PPARgamma2 in the inducible MyoD-overepxressing BEFS cells (BEFS-TetOn-MyoD) completely suppresses myogenic differentiation and leads to a significant increase in adipogenic differentiation, suggesting that the adipogenic differentiation program might be dominant. Therefore, BEFS, BEFS-PPARgamma2, and BEFS-TetOn-MyoD would be a valuable biological model for understanding a fundamental principle underlying myogenic and adipogenic development, and for isolating various genetic and chemical factors that enable muscle and adipocyte differentiation.
Biotechnology Letters 10/2009; 32(2):195-202. · 1.68 Impact Factor
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Min-Ju Kim,
Kyungsook Ahn,
Seong-Hoon Park,
Hong-Jun Kang,
Bong Geom Jang,
Soo-Jin Oh,
Sun-Mee Oh,
Yu-Jin Jeong,
Jee-In Heo,
Jun-Gyo Suh,
Soon Sung Lim,
Yoon-Jung Ko,
Sung-Oh Huh, Sung Chan Kim,
Jae-Bong Park,
Jaebong Kim,
Jong-Il Kim,
Sangmee Ahn Jo,
Jae-Yong Lee
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ABSTRACT: To examine the function of SIRT1 in neuronal differentiation, we employed all-trans retinoic acid (ATRA)-induced differentiation of neuroblastoma cells. Nicotinamide inhibited neurite outgrowth and tyrosine hydroxylase (TH) expression. Inhibition of PARP or histone deacetylase did not inhibit TH expression, showing the effect to be SIRT1 specific. Expression of FOXO3a and its target proteins were increased during the differentiation and reduced by nicotinamide. FOXO3a deacetylation was increased by ATRA and blocked by nicotinamide. SIRT1 and FOXO3a siRNA inhibited ATRA-induced up-regulation of TH and differentiation. Taken together, these results indicate that SIRT1 is involved in ATRA-induced differentiation of neuroblastoma cells via FOXO3a.
FEBS letters 05/2009; 583(7):1183-8. · 3.54 Impact Factor
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ABSTRACT: Amyloid-beta (Abeta) is one of the main factors to cause Alzheimer's disease. Although fibrillar Abeta (fAbeta) activates microglial cells that release toxic compounds to induce partial neuronal death, the mechanism of interaction between Abeta and microglia remains unclear. Therefore, we examined the interaction of microglial cells (BV2) and fAbeta on a gelatin-precoated plate. The binding was markedly enhanced by RhoA inactivation using Tat-C3, dominant negative RhoA, and si-RhoA. To identify the receptor for fAbeta, we tested various antibodies to mask receptors. Among them, anti-beta2-integrin antibody mostly suppressed cell binding to fAbeta. The incremental binding of cells induced by RhoA inhibition was also blocked by addition of anti-beta2-integrin antibody. These results suggest that RhoA inhibition stimulates beta2-integrin-mediated cell interaction to fAbeta.
Neuroreport 12/2008; 19(17):1661-5. · 1.66 Impact Factor
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ABSTRACT: Febrile seizure (FS) is the most common type of seizure that occurs during early childhood. It has been proposed that atypical FS (prolonged, multiple, or lateralized) results in the development of recurrent complex partial seizures accompanied by Ammon's horn sclerosis or mesial temporal sclerosis, which is the most common of the intractable epilepsy. To elucidate the characteristics of epileptogenesis or acquired epilepsy following FS, we performed prospective long-term studies using hyperthermia-induced seizure model. Rat pups (postnatal 11 day old) were induced to hyperthermia (41-43 degrees C in core temperature) by exposure to a 175 W mercury vapor lamp. Six-nine weeks after hyperthermic seizure, the dentate gyrus showed impairments of paired-pulse inhibitions and excitability ratio. In addition, newly generated granule cells and synaptogenesis were observed in this region. Ten-twelve weeks after hyperthermic seizure, animals (approximately 68%) showed electroencephalographic seizure activity with increased VGLUT-1 immunoreactivity in the dentate gyrus. Parvalbumin immunoreactivity was markedly reduced in the hilus. These findings indicate that in this model the epileptogenic changes in the dentate gyrus may be based on the persistent alterations in excitability via neurogenesis, synaptogenesis, and impaired GABA(B) receptor-mediated inhibition.
Brain Research 07/2008; 1216:1-15. · 2.73 Impact Factor
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ABSTRACT: Ascorbic acid has been reported to extend replicative life span of human embryonic fibroblast (HEF). Since the detailed molecular mechanism of this phenomenon has not been investigated, we attempted to elucidate. Continuous treatment of HEF cells with ascorbic acid (at 200 microM) from 40 population doubling (PD) increased maximum PD numbers by 18% and lowered SA-beta-gal positive staining, an aging marker, by 2.3 folds, indicating that ascorbic acid extends replicative life span of HEF cells. Ascorbic acid treatment lowered DCFH by about 7 folds and Rho123 by about 70%, suggesting that ascorbic acid dramatically decreased ROS formation. Ascorbic acid also increased aconitase activity, a marker of mitochondrial aging, by 41%, indicating that ascorbic acid treatment restores age-related decline of mitochondrial function. Cell cycle analysis by flow cytometry revealed that ascorbic acid treatment decreased G1 population up to 12%. Further western blot analysis showed that ascorbic acid treatment decreased levels of p53, phospho-p53 at ser 15, and p21, indicating that ascorbic acid relieved senescence-related G1 arrest. Analysis of AP (apurinic/apyrimidinic) sites showed that ascorbic acid treatment decreased AP site formation by 35%. We also tested the effect of hydrogen peroxide treatment, as an additional oxidative stress. Continuous treatment of 20 microM of hydrogen peroxide from PD 40 of HEF cells resulted in premature senescence due to increased ROS level, and increased AP sites. Taken together, the results suggest that ascorbic acid extends replicative life span of HEF cells by reducing mitochondrial and DNA damages through lowering cellular ROS.
Nutrition research and practice 01/2007; 1(2):105-12. · 1.08 Impact Factor
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Seung Ha Kang,
Kwang Keun Cho,
Jin Duck Bok, Sung Chan Kim,
Jaie Soon Cho,
Peter Chang-Whan Lee,
Sang Kee Kang,
Hong Gu Lee,
Jung Hee Woo,
Hyun Jeong Lee,
Sang Cheol Lee,
Yun Jaie Choi
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ABSTRACT: A gene, phoI, coding for a phosphatase from Enterobacter sp. 4 was cloned in Escherichia coli and sequenced. Analysis of the sequence revealed one open reading frame (ORF) that encodes a 269-amino acid protein with a calculated molecular mass of 29 kDa. PhoI belongs to family B acid phosphatase and exhibits 49.4% identity and 62.4% homology to the hel gene from Heamophilus influenzae, which encoded an outer membrane protein (P4). The optimum pH and temperature for phosphatase activity were pH 5.5 and 40 degrees C, respectively. Its specific activity on rho-nitrophenyl phosphatate was 70 U/mg at pH 5.5 and 40 degrees C. Enzyme activity was inhibited by Al3+, EDTA, and DTT, but fivefold activated by Cu2+ ion (350 U/mg). PhoI showed a strong synergistic effect when used with a purified E. coli phytase, AppA, to estimate combination effects.
Current Microbiology 05/2006; 52(4):243-8. · 1.82 Impact Factor
