Objective The present study aims to investigate the role of protein kinase C 5 subtype （PKCS） phosphorylation in the process of 6-hydroxydopamine （6-OHDA）-induced dopaminergic cell death, and demonstrate the molec...Objective The present study aims to investigate the role of protein kinase C 5 subtype （PKCS） phosphorylation in the process of 6-hydroxydopamine （6-OHDA）-induced dopaminergic cell death, and demonstrate the molecular basis of neurological disorders, such as Parkinson＇ s disease. Methods The pheochromocytoma （PC 12） cell line was employed in the present study. Cells were treated with 2 μmol/L PKC5 inhibitor Rottlerin, 10 nmol/L protein kinase C α subtype （PKCα） inhibitor bisindolylmaleimide 1, or 5 nmol/L G66976 that could specifically inhibit the calcium-dependent PKC isoforms, respectively. PKC8 activator phorbol-12-myristate-13-acetate （PMA, 100 nmol/L） was also used in this study. All these agents were added to the medium before cells were incubated with 6-OHDA. Cells with no treatment served as control. The cytotox- icity of 6-OHDA was determined by methyl thiazolyl tetrazolium （MTT） reduction assay and PKCδ phosphorylation levels in various groups were measured by western blotting. Results Bisindolylmaleimide I and Go6976 exerted no significant attenuation on the cytotoxicity of 6-OHDA, nor any effects on PKCδ phosphorylation in PC 12 cells. However, Rottlerin could inhibit the phosphorylation of PKC5 and attenuate 6-OHDA-induced cell death, and the cell viability was raised to 69.6 ±2.63% of that in control group （P 〈 0.05）. In contrast, PMA induced a significant increase in PKC5 phosphorylation and also strengthened the cytotoxic effects of 6-OHDA. The cell viability of PMA-treated PC12 cells decreased to 49.8±5.06% of that in control group （P 〈 0.001）. Conclusion Rottlerin can protect PC 12 cells from cytotoxicity of 6-OHDA probably by inhibiting PKC δ phosphorylation. The results suggest that PKCδ may be a key regulator of neuron loss in Parkinson＇s disease.展开更多
基金supported by the International Cooperation Project of Science and Technology Department of Heilongjiang Province, China (No.WB04301, No.WB08B05)the Science and Technology Foundation of Education Department of Heilongjiang Province, China (No.11521076)
文摘Objective The present study aims to investigate the role of protein kinase C 5 subtype （PKCS） phosphorylation in the process of 6-hydroxydopamine （6-OHDA）-induced dopaminergic cell death, and demonstrate the molecular basis of neurological disorders, such as Parkinson＇ s disease. Methods The pheochromocytoma （PC 12） cell line was employed in the present study. Cells were treated with 2 μmol/L PKC5 inhibitor Rottlerin, 10 nmol/L protein kinase C α subtype （PKCα） inhibitor bisindolylmaleimide 1, or 5 nmol/L G66976 that could specifically inhibit the calcium-dependent PKC isoforms, respectively. PKC8 activator phorbol-12-myristate-13-acetate （PMA, 100 nmol/L） was also used in this study. All these agents were added to the medium before cells were incubated with 6-OHDA. Cells with no treatment served as control. The cytotox- icity of 6-OHDA was determined by methyl thiazolyl tetrazolium （MTT） reduction assay and PKCδ phosphorylation levels in various groups were measured by western blotting. Results Bisindolylmaleimide I and Go6976 exerted no significant attenuation on the cytotoxicity of 6-OHDA, nor any effects on PKCδ phosphorylation in PC 12 cells. However, Rottlerin could inhibit the phosphorylation of PKC5 and attenuate 6-OHDA-induced cell death, and the cell viability was raised to 69.6 ±2.63% of that in control group （P 〈 0.05）. In contrast, PMA induced a significant increase in PKC5 phosphorylation and also strengthened the cytotoxic effects of 6-OHDA. The cell viability of PMA-treated PC12 cells decreased to 49.8±5.06% of that in control group （P 〈 0.001）. Conclusion Rottlerin can protect PC 12 cells from cytotoxicity of 6-OHDA probably by inhibiting PKC δ phosphorylation. The results suggest that PKCδ may be a key regulator of neuron loss in Parkinson＇s disease.
