title: Netrin-1 protects hypoxia-induced mitochondrial apoptosis through HSP27 expression via DCC- and integrin α6β4-dependent Akt, GSK-3β, and HSF-1 in mesenchymal stem cells.
abstract: Netrin (Ntn) has the potential to be successfully applied as an anti-apoptotic agent with a high affinity for tissue, for therapeutic strategies of umbilical cord blood-derived mesenchymal stem cells (UCB-MSC), although the mechanism by which Ntn-1 protects hypoxic injury has yet to be identified. Therefore, the present study examined the effect of Ntn-1 on hypoxia-induced UCB-MSC apoptosis, as well as the potential underlying mechanisms of its protective effect. Hypoxia (72 h) reduced cell viability (MTT reduction, and [(3)H]-thymidine incorporation) and cell number, and induced apoptosis (annexin and/or PI positive), which were reversed by Ntn-1 (10 ng/ml). Moreover, Ntn-1 decreased the increase of hypoxia-induced Bax, cleaved caspase-9, and -3, but blocked the decrease of hypoxia-reduced Bcl-2. Next, in order to examine the Ntn-1-related signaling cascade in the protection of hypoxic injury, we analyzed six Ntn receptors in UCB-MSC. We identified deleted in colorectal cancer (DCC) and integrin (IN) α6β4, except uncoordinated family member (UNC) 5A-C, and neogenin. Among them, IN α6β4 only was detected in lipid raft fractions. In addition, Ntn-1 induced the dissociation of DCC and APPL-1 complex, thereby stimulating the formation of APPL-1 and Akt2 complex. Ntn-1 also reversed the hypoxia-induced decrease of Akt and glycogen synthase kinase 3β (GSK-3β) phosphorylation, which is involved in heat shock factor-1 (HSF-1) expression. Ntn-1-induced phospho-Akt and -GSK-3β were inhibited by DCC function-blocking antibody, IN a6b4 function-blocking antibody, and the Akt inhibitor. Hypoxia and/or Ntn-1 stimulated heat shock protein (HSP)27 expression, which was blocked by HSF-1-specific small interfering RNA (siRNA). Furthermore, HSP27-specific siRNA reversed the Ntn-1-induced increase of phospho-Akt. Additionally, HSP27-specific siRNA attenuated the Ntn-1-reduced loss of mitochondrial membrane injury via the inhibition of cytochrome c (cyt c) release and formation of cyt c and HSP27 complex. Moreover, the inhibition of each signaling protein attenuated Ntn-1-induced blockage of apoptosis. In conclusion, Ntn-1-induced HSP27 protected hypoxic injury-related UCB-MSC apoptosis through DCC- and IN α6β4-dependent Akt, GSK-3β, and HSF-1 signaling pathways.
authors.affiliation: Department of Veterinary Internal Medicine, College of Veterinary Medicine and Research Institute for Veterinary Science, Seoul National University, Seoul, Korea.
authors.forename: T W
authors.initials: TW
authors.lastname: Son
authors.forename: S P
authors.initials: SP
authors.lastname: Yun
authors.forename: M S
authors.initials: MS
authors.lastname: Yong
authors.forename: B N
authors.initials: BN
authors.lastname: Seo
authors.forename: J M
authors.initials: JM
authors.lastname: Ryu
authors.forename: H Y
authors.initials: HY
authors.lastname: Youn
authors.forename: Y M
authors.initials: YM
authors.lastname: Oh
authors.forename: H J
authors.initials: HJ
authors.lastname: Han
mesh_terms: D017209:Apoptosis / Q000187:drug effects; D015687:Cell Hypoxia / Q000235:genetics; D002478:Cells, Cultured; D000075389:DCC Receptor; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects; D038362:Glycogen Synthase Kinase 3 / Q000235:genetics* / Q000378:metabolism; D000071679:Glycogen Synthase Kinase 3 beta; D055551:HSP27 Heat-Shock Proteins / Q000235:genetics* / Q000378:metabolism; D000076249:Heat Shock Transcription Factors; D006360:Heat-Shock Proteins; D006801:Humans; D039161:Integrin alpha6beta4 / Q000235:genetics* / Q000378:metabolism; D059630:Mesenchymal Stem Cells / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D018832:Molecular Chaperones; D009414:Nerve Growth Factors / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D000075388:Netrin-1; D010100:Oxygen / Q000494:pharmacology; D051057:Proto-Oncogene Proteins c-akt / Q000235:genetics* / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000235:genetics* / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism; D025521:Tumor Suppressor Proteins / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology*; D014470:Umbilical Cord / Q000166:cytology / Q000187:drug effects / Q000378:metabolism
chemical_list: D000075389:DCC Receptor; C501909:DCC protein, human; D004268:DNA-Binding Proteins; D055551:HSP27 Heat-Shock Proteins; C057725:HSPB1 protein, human; D000076249:Heat Shock Transcription Factors; D006360:Heat-Shock Proteins; D039161:Integrin alpha6beta4; D018832:Molecular Chaperones; C000617351:NTN1 protein, human; D009414:Nerve Growth Factors; D011956:Receptors, Cell Surface; D014157:Transcription Factors; D025521:Tumor Suppressor Proteins; D000075388:Netrin-1; C000605506:GSK3B protein, human; D000071679:Glycogen Synthase Kinase 3 beta; D051057:Proto-Oncogene Proteins c-akt; D038362:Glycogen Synthase Kinase 3; D010100:Oxygen
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