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

Cell Death Dis. 2013 Mar 28;4(3):e563. doi: 10.1038/cddis.2013.94.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Apoptosis / drug effects
  • Cell Hypoxia / genetics
  • Cells, Cultured
  • DCC Receptor
  • DNA-Binding Proteins / genetics*
  • DNA-Binding Proteins / metabolism
  • Gene Expression Regulation / drug effects
  • Glycogen Synthase Kinase 3 / genetics*
  • Glycogen Synthase Kinase 3 / metabolism
  • Glycogen Synthase Kinase 3 beta
  • HSP27 Heat-Shock Proteins / genetics*
  • HSP27 Heat-Shock Proteins / metabolism
  • Heat Shock Transcription Factors
  • Heat-Shock Proteins
  • Humans
  • Integrin alpha6beta4 / genetics*
  • Integrin alpha6beta4 / metabolism
  • Mesenchymal Stem Cells / cytology
  • Mesenchymal Stem Cells / drug effects*
  • Mesenchymal Stem Cells / metabolism
  • Mitochondria / drug effects
  • Mitochondria / metabolism
  • Mitochondria / pathology
  • Molecular Chaperones
  • Nerve Growth Factors / genetics
  • Nerve Growth Factors / metabolism
  • Nerve Growth Factors / pharmacology*
  • Netrin-1
  • Oxygen / pharmacology
  • Proto-Oncogene Proteins c-akt / genetics*
  • Proto-Oncogene Proteins c-akt / metabolism
  • Receptors, Cell Surface / genetics*
  • Receptors, Cell Surface / metabolism
  • Signal Transduction / drug effects
  • Transcription Factors / genetics*
  • Transcription Factors / metabolism
  • Tumor Suppressor Proteins / genetics*
  • Tumor Suppressor Proteins / metabolism
  • Tumor Suppressor Proteins / pharmacology*
  • Umbilical Cord / cytology
  • Umbilical Cord / drug effects
  • Umbilical Cord / metabolism


  • DCC Receptor
  • DCC protein, human
  • DNA-Binding Proteins
  • HSP27 Heat-Shock Proteins
  • HSPB1 protein, human
  • Heat Shock Transcription Factors
  • Heat-Shock Proteins
  • Integrin alpha6beta4
  • Molecular Chaperones
  • NTN1 protein, human
  • Nerve Growth Factors
  • Receptors, Cell Surface
  • Transcription Factors
  • Tumor Suppressor Proteins
  • Netrin-1
  • GSK3B protein, human
  • Glycogen Synthase Kinase 3 beta
  • Proto-Oncogene Proteins c-akt
  • Glycogen Synthase Kinase 3
  • Oxygen