Over-activation of microglia cells in the brain contributes to neurodegenerative processes promoted by the production of various neurotoxic factors including pro-inflammatory cytokines and nitric oxide. Recently, accumulating evidence has suggested that mitochondrial dynamics are an important constituent of cellular quality control and function. However, the role of mitochondrial dynamics in microglial activation is still largely unknown. In this study, we determined whether mitochondrial dynamics are associated with the production of pro-inflammatory mediators in lipopolysaccharide (LPS)-stimulated immortalization of murine microglial cells (BV-2) by a v-raf/v-myc carrying retrovirus (J2). Excessive mitochondrial fission was observed in lentivirus-transfected BV-2 cells stably expressing DsRed2-mito following LPS stimulation. Furthermore, mitochondrial localization of dynamin-related protein 1 (Drp1) (a key regulator of mitochondrial fission) was increased and accompanied by de-phosphorylation of Ser637 in Drp1.Interestingly, inhibition of LPS-induced mitochondrial fission and reactive oxygen species (ROS) generation by Mdivi-1 and Drp1 knock-down attenuated the production of pro-inflammatory mediators via reduced nuclear factor kappa-light-chainenhancer of activated B cells (NF-jB) and mitogen-activated protein kinase (MAPK) signaling. Our results demonstrated for the first time that mitochondrial fission regulates mitochondrial ROS production in activated microglial cells and influences the expression of pro-inflammatory mediators through the activation of NF-jB and MAPK. We therefore suggest that mitochondrial dynamics may be essential for understanding pro-inflammatory mediator expression in activated microglial cells. This could represent a new therapeutic approach for preventing neurodegenerative diseases. Keywords: Drp1, lipopolysaccharide, Mdivi-1, microglia, mitochondrial dynamics, neuroinflammation. Address correspondence and reprint requests to Dong-Seok Lee, College of Natural Sciences, Kyungpook National University, Daegu, Republic of Korea. E-mail: lee1@knu.ac.kr 1 These authors contributed equally to this work. Abbreviations used: CMVie, cytomegalovirus immediate early; Cox-2, cyclooxygenase-2; COXIV, cytochrome c oxidase; cPPT, central polypurine tract; DMEM, Dulbecco's modified Eagle's medium; Drp1, dynamin-related protein 1; DsRed2, discosoma sp. red fluorescent; ERK, extracellular signal-regulated kinase; FBS, fetal bovine serum; Fis1, mitochondrial fission 1; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; iNOS, inducible nitric oxide synthase; JNK, c-jun N-terminal kinase; LPS, lipopolysaccharide; LTR, long terminal repeat; MAPK, mitogen-activated protein kinase; Mdivi-1, 3-(2,4-dichloro-5-methoxyphenyl)-2,3-dihydro-2-thioxo-4(1H)-quinazolinone3-(2,4-dichloro-5-methoxyphenyl)-2-sulfanyl-4(3H)-quinazolinone; Mfn1, mitofusin 1; Mfn2, mitofusin 2; MOI, multiplicity of infection; MTS, mitochondrial targeting sequence; NF-jB, nuclear factor kappa-light-chainenhancer of activated B cells...
Various source-derived mesenchymal stem cells (MSCs) with multipotent capabilities were considered for cell therapeutics of incurable diseases. The applicability of MSCs depends on the cellular source and on their different in vivo functions, despite having similar phenotypic and cytological characteristics. We characterized MSCs from different sources, including human bone marrow (BM), placenta (PL), and adipose tissue (AT), in terms of the phenotype, surface antigen expression, differentiation ability, proteome reference map, and blood flow recovery in a hindlimb ischemic disease model. The MSCs exhibit different differentiation potentials depending on the cellular source despite having similar phenotypic and surface antigen expression. We identified approximately 90 differentially regulated proteins. Most up- or down-regulated proteins show cytoskeletal or oxidative stress, peroxiredoxin, and apoptosis roles according to their functional involvement. In addition, the PL-MSCs retained a higher therapeutic efficacy than the BM- and AT-MSCs in the hindlimb ischemic disease model. In summary, we examined differentially expressed key regulatory factors for MSCs that were obtained from several cellular sources and demonstrated their differentially expressed proteome profiles. Our results indicate that primitive PL-MSCs have biological advantages relative to those from other sources, making PL-MSCs a useful model for clinical applications of cell therapy.
Green tea is a highly popular beverage globally. Green tea contains a number of polyphenol compounds referred to as catechins, and (−)-epigallocatechin gallate (EGCG) is believed to be the major biologically active compound found in green tea. EGCG has been reported to suppress lung cancer, but the molecular mechanisms of the inhibitory effects of EGCG are not clear. We found that EGCG interacted with the Ras-GTPase-activating protein SH3 domain-binding protein 1 (G3BP1) with high binding affinity (K d = 0.4 μmol/L). We also showed that EGCG suppressed anchorage-independent growth of H1299 and CL13 lung cancer cells, which contain an abundance of the G3BP1 protein. EGCG was much less effective in suppressing anchorage-independent growth of H460 lung cancer cells, which express much lower levels of G3BP1. Knockdown shG3BP1-transfected H1299 cells exhibited substantially decreased proliferation and anchorage-independent growth. shG3BP1 H1299 cells were resistant to the inhibitory effects of EGCG on growth and colony formation compared with shMock-transfected H1299 cells. EGCG interfered with the interaction of G3BP1 and the Ras-GTPase-activating protein and further suppressed the activation of Ras. Additional results revealed that EGCG effectively attenuated G3BP1 downstream signaling, including extracellular signal-regulated kinase and mitogen-activated protein kinase/extracellular signal-regulated kinase kinase, in wild-type H1299 and shMock H1299 cells but had little effect on H460 or shG3BP1 H1299 cells. Overall, these results strongly indicate that EGCG suppresses lung tumorigenesis through its binding with G3BP1. Cancer Prev Res; 3(5); 670-9. ©2010 AACR.
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