Vitamin K2 suppresses rotenone-induced microglial activation in vitro.

AIM: Increasing evidence has shown that environmental factors such as rotenone and paraquat induce neuroinflammation, which contributes to the pathogenesis of Parkinson's disease (PD). In this study, we investigated the molecular mechanisms underlying the repression by menaquinone-4 (MK-4), a subtype of vitamin K2, of rotenone-induced microglial activation in vitro. METHODS: A microglial cell line (BV2) was exposed to rotenone (1 µmol/L) with or without MK-4 treatment. The levels of TNF-α or IL-1ß in 100 µL of cultured media of BV2 cells were measured using ELISA kits. BV2 cells treated with rotenone with or without MK4 were subjected to mitochondrial membrane potential, ROS production, immunofluorescence or immunoblot assays. The neuroblastoma SH-SY5Y cells were treated with conditioned media (CM) of BV2 cells that were exposed to rotenone with or without MK-4 treatment, and the cell viability was assessed using MTT assay. RESULTS: In rotenone-treated BV2 cells, MK-4 (0.5-20 µmol/L) dose-dependently suppressed the upregulation in the expression of iNOS and COX-2 in the cells, as well as the production of TNF-α and IL-1ß in the cultured media. MK-4 (5-20 µmol/L) significantly inhibited rotenone-induced nuclear translocation of NF-κB in BV2 cells. MK-4 (5-20 µmol/L) significantly inhibited rotenone-induced p38 activation, ROS production, and caspase-1 activation in BV2 cells. MK-4 (5-20 µmol/L) also restored the mitochondrial membrane potential that had been damaged by rotenone. Exposure to CM from rotenone-treated BV2 cells markedly decreased the viability of SH-SY5Y cells. However, this rotenone-activated microglia-mediated death of SH-SY5Y cells was significantly attenuated when the BV2 cells were co-treated with MK-4 (5-20 µmol/L). CONCLUSION: Vitamin K2 can directly suppress rotenone-induced activation of microglial BV2 cells in vitro by repressing ROS production and p38 activation.

Overexpression of the ß2AR gene improves function and re-endothelialization capacity of EPCs after arterial injury in nude mice.

BACKGROUND: Proliferation and migration of endothelial progenitor cells (EPCs) play important roles in restoring vascular injuries. ß2 adrenergic receptors (ß2ARs) are widely expressed in many tissues and have a beneficial impact on EPCs regulating neoangiogenesis. The aim of the present study was to determine the effect of overexpressing ß2ARs in infused peripheral blood (PB)-derived EPCs on the re-endothelialization in injured vessels. METHODS: Induction of endothelial injury was performed in male nude mice that were subjected to wire-mediated injury to the carotid artery. Human PB-derived EPCs were transfected with an adenovirus serotype 5 vector expressing ß2AR (Ad5/ß2AR-EPCs) and were examined 48 h later. ß2AR gene expression in EPCs was detected by real-time polymerase chain reaction and Western blot analysis. In vitro, the proliferation, migration, adhesion, and nitric oxide production of Ad5/ß2AR-EPCs were measured. Meanwhile, phosphorylated Akt and endothelial nitric oxide synthase (eNOS), which are downstream of ß2AR signaling, were also elevated. In an in vivo study, CM-DiI-labeled EPCs were injected intravenously into mice subjected to carotid injury. After 3 days, cells recruited to the injury sites were detected by fluorescent microscopy, and the re-endothelialization was assessed by Evans blue dye. RESULTS: In vitro, ß2AR overexpression augmented EPC proliferation, migration, and nitric oxide production and enhanced EPC adhesion to endothelial cell monolayers. In vivo, when cell tracking was used, the number of recruited CM-DiI-labeled EPCs was significantly higher in the injured zone in mice transfused with Ad5/ß2AR-EPCs compared with non-transfected EPCs. The degree of re-endothelialization was also higher in the mice transfused with Ad5/ß2AR-EPCs compared with non-transfected EPCs. We also found that the phosphorylation of Akt and eNOS was increased in Ad5/ß2AR-EPCs. Preincubation with ß2AR inhibitor (ICI118,551), Akt inhibitor (ly294002), or eNOS inhibitor (L-NAME) significantly attenuated the enhanced in vitro function and in vivo re-endothelialization capacity of EPCs induced by ß2AR overexpression. CONCLUSIONS: The present study demonstrates that ß2AR overexpression enhances EPC functions in vitro and enhances the vascular repair abilities of EPCs in vivo via the ß2AR/Akt/eNOS pathway. Upregulation of ß2AR gene expression through gene transfer may be a novel therapeutic target for endothelial repair.

Parkin represses 6-hydroxydopamine-induced apoptosis via stabilizing scaffold protein p62 in PC12 cells.

AIM: Parkin has been shown to exert protective effects against 6-hydroxydopamine (6-OHDA)-induced neurotoxicity in different models of Parkinson disease. In the present study we investigated the molecular mechanisms underlying the neuroprotective action of parkin in vitro. METHODS: HEK293, HeLa and PC12 cells were transfected with parkin, parkin mutants, p62 or si-p62. Protein expression and ubiquitination were assessed using immunoblot analysis. Immunoprecipitation assay was performed to identify the interaction between parkin and scaffold protein p62. PC12 and SH-SY5Y cells were treated with 6-OHDA (200 µmol/L), and cell apoptosis was detected using PI and Hoechst staining. RESULTS: In HEK293 cells co-transfected with parkin and p62, parkin was co-immunoprecipitated with p62, and parkin overexpression increased p62 protein levels. In parkin-deficient HeLa cells, transfection with wild-type pakin, but not with ligase activity-deficient pakin mutants, significantly increased p62 levels, suggesting that parkin stabilized p62 through its E3 ligase activity. Transfection with parkin or p62 significantly repressed ERK1/2 phosphorylation in HeLa cells, but transfection with parkin did not repress ERK1/2 phosphorylation in p62-knockdown HeLa cells, suggesting that p62 was involved in parkin-induced inhibition on ERK1/2 phosphorylation. Overexpression of parkin or p62 significantly repressed 6-OHDA-induced ERK1/2 phosphorylation in PC12 cells, and parkin overexpression inhibited 6-OHDA-induced apoptosis in PC12 and SH-SY5Y cells. CONCLUSION: Parkin protects PC12 cells against 6-OHDA-induced apoptosis via ubiquitinating and stabilizing scaffold protein p62, and repressing ERK1/2 activation.

Protease Omi cleaving Hax-1 protein contributes to OGD/R-induced mitochondrial damage in neuroblastoma N2a cells and cerebral injury in MCAO mice.

AIM: In the penumbra after focal cerebral ischemia, an increase of protease Omi is linked to a decrease of Hs1-associated protein X-1 (Hax-1), a protein belonging to the Bcl-2 family. In this study we investigated the mechanisms underlying the regulation of Hax-1 by protease Omi in cerebral ischemia/reperfusion (I/R) injury. METHODS: Mouse neuroblastoma N2a cells were subjected to oxygen-glucose deprivation and reoxygenation (OGD/R); cell viability was assessed with MTT assay. Mice underwent 2-h middle cerebral artery occlusion (MCAO) and reperfusion, and the infarct volume was determined with TTC staining. The expression of Omi and Hax-1 was detected using immunoblot and immunofluorescence assays. The mitochondrial membrane potential was measured using TMRM staining. RESULTS: In the brains of MCAO mice, the protein level of Omi was significantly increased, while the protein level of Hax-1 was decreased. Similar changes were observed in OGD/R-treated N2a cells, but the mRNA level of Hax-1 was not changed. Furthermore, in OGD/R-treated N2a cells, knockdown of Omi significantly increased Hax-1 protein level. Immunofluorescence assay showed that Omi and Hax-1 were co-localized in mitochondria of N2a cells. OGD/R caused marked mitochondrial damage and apoptosis in N2a cells, while inhibition of Omi protease activity with UCF-101 (10 µmol/L) or overexpression of Hax-1 could restore the mitochondrial membrane potential and attenuate cell apoptosis. Moreover, pretreatment of MCAO mice with UCF-101 (7.15 mg/kg, ip) could restore Hax-1 expression, inhibit caspase activation, and significantly reduce the infarct volume. CONCLUSION: Protease Omi impairs mitochondrial function by cleaving Hax-1, which induces apoptosis in OGD/R-treated N2a cells and causes I/R injury in MCAO mice.

Protease Omi facilitates neurite outgrowth in mouse neuroblastoma N2a cells by cleaving transcription factor E2F1.

AIM: Omi is an ATP-independent serine protease that is necessary for neuronal function and survival. The aim of this study was to investigate the role of protease Omi in regulating differentiation of mouse neuroblastoma cells and to identify the substrate of Omi involved in this process. METHODS: Mouse neuroblastoma N2a cells and Omi protease-deficient mnd2 mice were used in this study. To modulate Omi and E2F1 expression, N2a cells were transfected with expression plasmids, shRNA plasmids or siRNA. Protein levels were detected using immunoblot assays. The interaction between Omi and E2F1 was studied using immunoprecipitation, GST pulldown and in vitro cleavage assays. N2a cells were treated with 20 µmol/L retinoic acid (RA) and 1% fetal bovine serum to induce neurite outgrowth, which was measured using Image J software. RESULTS: E2F1 was significantly increased in Omi knockdown cells and in brain lysates of mnd2 mice, and was decreased in cells overexpressing wild-type Omi, but not inactive Omi S276C. In brain lysates of mnd2 mice, endogenous E2F1 was co-immunoprecipitated with endogenous Omi. In vitro cleavage assay demonstrated that Omi directly cleaved E2F1. Treatment of N2a cells with RA induced marked differentiation and neurite outgrowth accompanied by significantly increased Omi and decreased E2F1 levels, which were suppressed by pretreatment with the specific Omi inhibitor UCF-101. Knockdown of Omi in N2a cells suppressed RA-induced neurite outgrowth, which was partially restored by knockdown of E2F1. CONCLUSION: Protease Omi facilitates neurite outgrowth by cleaving the transcription factor E2F1 in differentiated neuroblastoma cells; E2F1 is a substrate of Omi.

Bcl-2-dependent upregulation of autophagy by sequestosome 1/p62 in vitro.

AIM: To investigate whether sequestosome 1/p62 (p62), a key cargo adaptor protein involved in both the ubiquitin-proteasome system and the autophagy-lysosome system, could directly regulate autophagy in vitro. METHODS: HEK 293 cells or HeLa cells were transfected with p62-expressing plasmids or siRNA targeting p62. The cells or the cell lysates were subsequently subjected to immunofluorescence assay, immunoprecipitation assay, or immunoblot analysis. In vitro pulldown assay was used to study the interaction of p62 with Bcl-2. RESULTS: Overexpression of p62 significantly increased the basal level of autophagy in both HEK 293 cells and HeLa cells, whereas knockdown of p62 significantly decreased the basal level of autophagy. In vitro pulldown assay showed that p62 directly interacted with Bcl-2. It was observed in HeLa cells that p62 co-localized with Bcl-2. Furthermore, knockdown of p62 in HEK 293 cells significantly increased the amount of Beclin 1 that co-immunoprecipitated with Bcl-2. CONCLUSION: p62 induces autophagy by disrupting the association between Bcl-2 and Beclin 1.