| The present study, using cellular and molecular biology techniques and methods including cell culture, RNA interference, fluorescent staining and imaging, immunocytochemistry, Western blotting, etc., and employing PC12 cells, SH-SY5Y cells and primary neurons as experimental objects, systematically investigated the role of rapamycin in prevention against cadmium (Cd)-induced neuronal cell apoptosis, the mechanisms of rapamycin against apoptosis by targeting mTORC1 and mTORC2 pathways, inhibiting activation of JNK and Erkl/2 pathways, blocking mitochondrial ROS-mediated activation of JNK and Erkl/2 pathways, and targeting PTEN/PP2A signaling network involved in suppressing activation of Erkl/2 pathway. In addition, we also preliminary explored the mechanisms of rapamycin intervention autophagy via inhibiting JNK pathway in neuronal cells in response to Cd. The detailed results were sunnarized as follows:1. Rapamycin attenuates Cd-induced apoptotic cell death in neuronal cellsPC 12 cells, SH-SY5Y cells and primary neurons were chosen and treated with Cd (10 and 20 μM) for 4 h or 24 h following pretreatment with/without rapamycin (0.2μg/ml) for 48 h. Cell morphology was detected, live cells were counted by trypan blue exclusion, cell apoptosis was evaluated by DAPI and TUNEL staining, caspase-3/7 activity was assayed, and cell caspase-3 was determined by Western blotting. We showed that Cd induced cell viability reduction and morphological alteration, increased the percentage of cells with nuclear fragmentation and condensation, the number of TUNEL-positive cells, cleaved-caspase-3 expression and caspase-3/7 activity in a dose-dependent manner. Pretreatment with rapamycin significantly attenuated Cd-induced cell viability reduction and apoptosis, and blocked Cd-elicited caspase-3 activation. Our findings indicate that rapamycin obviously prevents Cd-induced apoptotic cell death in neuronal cells.2. Rapamycin prevents cadmium-induced neuronal cell apoptosis via targeting both mTORCl and mTORC2 pathwaysPC12 cells, SH-SY5Y cells and primary neurons were chosen and treated with Cd(10 and 20 μM) for 4 h or 24 h following pretreatment with/without rapamycin (0.2μg/ml) for 48 h, or co-pretreatment with Akt inhibitor X (20 μM) for 2 h. In some cases, PC 12 and/or SH-SY5Y cells, infected with Ad-mTOR-T, Ad-mTOR-TE, Ad-dn-Akt, Ad-4EBP1-5A shRNA Raptor、shRNA Ricto、shRNA Raptor/Rictor or shRNA S6K1, were treated with Cd (20 μM) for 4 h or 24 h following pretreatment with/without rapamycin (0.2 μg/ml) for 48 h. Cell morphology was detected, live cells were counted by trypan blue exclusion, cell apoptosis was evaluated by DAPI staining, the related signal alteration was determined by Western blotting. The results showed that rapamycin blocked Cd-induced phosphorylation of Akt, S6K1 and 4E-BP1 in the cells. Overexpression of mTOR-T conferred resistance to rapamycin inhibition of Cd-induced cell apoptosis, implying that rapamycin prevention against Cd-induced neurotoxicity is mTOR kinase activity-dependent. Both mTORCI and mTORC2 were involved in the inhibitory activity of rapamycin. That is supported by the findings that silencing raptor, rictor or raptor/rictor enhanced rapamycin’s inhibition of Cd-induced cell apoptosis. In addition, downregulation of S6K1, ectopic expression of 4E-BP1-5A, dominant negative Akt, or co-treatment with Akt inhibitor X also potentiated rapamycin’s prevention against apoptosis. The findings indicate that rapamycin prevents neuronal apoptosis via inhibiting Cd-induced activation of mTORCI-mediated S6K1 and 4E-BP1 pathways, as well as mTORC2-mediated Akt pathway.3. Rapamycin prevents cell apoptosis via inhibiting Cd activation of JNK and Erkl/2 pathways in neuronal cellsPC 12 cells, SH-SY5Y cells and primary neurons were chosen and treated with Cd (10 and 20μM) for 4 h or 24 h following pretreatment with/without rapamycin (0.2 μg/ml) for 48 h, or co-pretreatment with/without JNK inhibitor SP600125 (10 μM), Erkl/2 inhibitor U0126 (5 μM) or PD98059 (10 μM) for 1 h, respectively. In some cases, PC12 cells, infected with Ad-dn-c-Jun or shRNA Erkl/2, were pretreated with/without rapamycin for 48 h, followed by exposure to Cd (10 or 20 μM) for 4 h or 24 h. Live cells were counted by trypan blue exclusion, cell apoptosis was evaluated by DAPI staining, the related signal alteration was determined by Western blotting. We showed that pretreatment with rapamcyin for 48 h significantly suppressed Cd-activated MAPKs cascades including JNK, Erkl/2 and p38. JNK inhibitor SP600125 and ectopic expression of dominant negative c-Jun markedly inhibited Cd-induced neuronal apoptosis, and enhanced the inhibitory activity of rapamycin. pharmacological inhibition of Erkl/2 by U0126 or PD98059, or silencing Erkl/2 by shRNA interference also powerfully reversed Cd-induced neuronal apoptosis, and strengthened rapamycin prevention against Cd-triggered apoptosis The findings reveal that rapamycin inhibits Cd-activated JNK and Erkl/2 pathways, thereby preventing neuronal apoptosis.4. Rapamycin inhibits Cd-induced mitochondrial ROS-mediated activation of JNK and Erkl/2 pathways in neuronal cellsPC12 cells and primary neurons were chosen and treated with Cd (10 and 20 μM) for 4 h or 24 h following pretreatment with/without rapamycin (0.2 μg/ml) for 48 h, or co-pretreatment with/without TTFA (10 μM), antimycin A (50μM), Mito-TEMPO (10 pM), SP600125 (20 μM) or U0126 (50 μM) for 1 h, respectively. In some cases, PC12 cells and primary neruons, infected with Ad-dn-c-Jun or Ad-MKK1-K97M, were pretreated with/without rapamycin for 48 h or Mito-TEMPO for 1 h, followed by exposure to Cd (20 μM) for 24 h. Fluorescence imaging for reactive oxygen species (ROS) intensity was captured by using ROS probe CM-H2DCFDA, the related signal alteration was determined by Western blotting. The results showed that rapamycin suppressed Cd-induced ROS generation in neuronal cells. TTFA and Mito-TEMPO reduced Cd-induced ROS production. This effects was further reinforced, whereas antimycin A elevated ROS and resisted rapamycin’s inhibition of ROS, implying that rapamycin inhibits Cd-induced mitochondrial ROS. Mito-TEMPO might suppressed and strengthened rapamycin’s blockage of Cd-activated JNK, Erkl/2 and p38 pathways in neuronal cells. SP600125 and U0126 inhibited and enhanced rapamycin’s or Mito-TEMPO’s inhibition of Cd-induced cell ROS generation, indicating that there exists a close relationship between mitochondrial ROS and activated JNK/Erkl/2 pathways in the cells induced by Cd, and regulating JNK and Erkl/2 also affects ROS production. of pretreatment with by, or expression of dominant negative c-Jun or MKK1 synergized the inhibitory effects of rapamycin or Mito-TEMPO on Cd-induced ROS. The findings suggest that rapamycin inhibitsCd-induced mitochondrial ROS-mediated activation of JNK and Erkl/2 pathways in neuronal cells.5. Rapamycin prevents Cd activation of Erkl/2 from neuronal apoptosis through targeting PTEN/PP2A signaling networkPC 12 cells, SH-SY5Y cells and primary neurons were chosen and treated with Cd (10 and 20 μM) for 4 h or 24 h following pretreatment with/without rapamycin (0.2 μg/ml) for 48 h, or co-pretreatment with/without Erkl/2 inhibitor PD98059 (10 μM) or PP2A inhibitor okadaic acid (OA,100 nM) for 1 h, or Akt inhibitor X (20 pM) for 2 h, respectively. In some cases, we also employed PC 12 cells with dominant negative PP2A and Akt activity, or with overexpression of PP2A and PTEN, with rapamycin-resistant and kinase-active mTOR (mTOR-T) by adenovirus interference, respectively, or the cells with silencing mTOR by lentiviral shRNA interference for further experiments. Live cells were counted by trypan blue exclusion, cell apoptosis was evaluated by DAPI staining, the related signal alteration was determined by Western blotting. The results showed that rapamycin attenuated Cd-increased phospho-PP2A and demethylated-PP2A, and Cd-decreased expression of PTEN. Inhibition of PP2A by OA or ectopic expression of dominant negative PP2A resisted rapamycin’s inhibition of Cd-induced phospho-Erkl/2 and apoptosis in the neuronal cells, whereas overexpression of PP2A enhanced the inhibitory activity of rapamycin. Overexpression of PTEN or dominant negative Akt, or inhibition of Akt and Erkl/2 by Akt inhibitor X or PD98059, respectively, potently strengthened rapamycin’s inhibition of Cd-induced phospho-Erkl/2 and cell apoptosis. Moreover, expression of mTOR-T conferred resistance to rapamycin’s attenuation of Cd-induced reduction of PP2A activity and PTEN expression, activation of Akt and Erkl/2 activation, as well as apoptosis in neuronal cells, whereas silencing mTOR mimicked rapamycin’s effects. The results uncover that rapamycin prevents Cd activation of Erkl/2 from apoptosis through targeting PTEN/PP2A signaling network in neuronal cells.6. Rapamycin intervenes to autophagy via inhibiting Cd-activated JNK pathway in neuronal cellsPC 12 cells were chosen and treated with Cd (10 and 20 μM) for 4 h following pretreatment with/without rapamycin (0.2 μg/ml) for 6-48 h or 48 h, or co-pretreatment with/without JNK inhibitor SP600125 (20 pM) for 1 h. In some cases, PC 12 cells, infected with Ad-dn-c-Jun or Ad-PP5, were pretreated with/without rapamycin for 48 h, followed by exposure to Cd (10μM) for 4 h. The related signal alteration was determined by Western blotting. The results exhibited that pretreatment with rapamycin for 12-48 h significantly inhibited Cd-induced cell LC3-II elevation. JNK inhibitor SP600125 or ectopic expression of dominant negative c-Jun obviously attenuated Cd-induced cell LC3-Ⅱ increase and enhanced rapamycin’s inhibitory effects. Overexpression of PP5 markedly suppressed Cd-induced phosphorylation of JNK/c-Jun and increase of LC3-Ⅱ, but failed to further potentiate rapamycin inhibition of Cd-induced LC3-Ⅱ. Our data suggest that rapamycin intervenes to autophagy via targeting inhibition of Cd-activated JNK pathway in neuronal cells. |
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