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: T W Son, S P Yun, M S Yong, B N Seo, J M Ryu, H Y Youn, Y M Oh, H J Han
• Journal: Cell death & disease (Cell Death Dis) Vol. 4 Pg. e563 (Mar 28 2013) ISSN: 2041-4889 [Electronic] England
• PMID: 23538444 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• 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

Topics

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