Protective effect of netrin-1 on hypoxia-induced mitochondrial apoptosis through HSP27expression in human umbilical cord blood derived mesenchymal stem cells

Abstract

Netrin (Ntn) has the potential to provide an anti-apoptotic agent with a high affinity for tissue, for therapeutic strategies of human umbilical cord blood derived mesenchymal stem cell (hUCB-MSC), although the mechanism by which Ntn-1protects hypoxic injury has yet to be identified. Therefore, the present study examined the effect of Ntn-1 on hypoxia-induced hUCB-MSC apoptosis, as well as the potential underlying mechanisms.Hypoxia (72 h) induced a decrease in the rate of MTT reduction and [3H]-thymidine incorporation, which were inhibited by Ntn-1 (10 ng/ml). Also, Ntn-1 decreased the increase of hypoxia-induced Bax, cleaved caspase-9, and cleaved caspase-3, but blocked the decrease of hypoxia-reduced Bcl-2.Next, in order to examine the Ntn-1-related signaling cascade in protection of hypoxic injury,six Ntn receptors in hUCB-MSC were analyzed. DCC and integrin(IN) α6β4 except UNC5A-C, and neogenin were existed in hUCB-MSC. Among them, IN α6β4 only was detected in lipid raft fractions. In addition, Ntn-1 induced the dissociation of DCC and APPL-1 complexthereby stimulating the formation of APPL-1 and Akt2 complex. Ntn-1 also reversed the hypoxia-induced decrease ofAkt and GSK-3β phosphorylation, which is involved in HSF-1 expression. Ntn-1-induced phospho-Akt and -GSK-3β was inhibited by DCC and IN α6β4 function-blocking antibody, and Akt inhibitor. Hypoxia and/or Ntn-1 stimulated HSP27 expression, which was blocked by HSF-1-specific siRNA. Furthermore,HSP27-specific siRNA reversedthe Ntn-1-induced increase of phospho-Akt.Additionally,HSP27-specific siRNA attenuated the Ntn-1-reduced loss of mitochondrial membrane injury viainhibition of cytochrome c (cyt c) release and formation of cyt c and HSP27 complex. Moreover, the inhibition of each signaling proteins attenuated Ntn-1-induced blockage of apoptosis. In conclusion, Ntn-1-induced HSP27 protected hypoxic injury-relatedhUCB-MSC apoptosis through DCC- and IN α6β4-dependent Akt, GSK-3β, and HSF-1signaling pathways.