| Objective:Function defect in hot shock proteins (HSPs), e.g. Hsp70, has been reported to play a key role in Parkinson's disease (PD)?And overexpressed HSP70 refolds the aggregated ?-synuclein to non-toxic and non-aggregated form. Thus, Hsp 70 is a well defined therapeutic target, and Hsp 70 promotion is an efficient stragegy, to prevent and even reverse the ?-synuclein-induced toxicity in PD. In present study, we investigated the promotion of Hsp70 expression in SH-SY5Y neuroblastoma cells by glutamine (Gln), which has recently been recognized to induce Hsp70 expression, and then we investigated the role of HSF-1 in the Hsp70 promotion by Gln. Moreover, we determined the regulatory role of Gln in the ?-synuclein degradation in ?-synuclein-overexpressed SH-SY5Y cells.Methods:1 Study the effect of glutamine on expression of heat shock protein 70 in SH-SY5Y neuroblastoma cellsRecently, Gln has been shown to safely enhance HSP expression in vitro and in vivo settings. This time we study the infuluenc of glutamine on heat shock protein expression.Given the high key regulatory role of Hsp70 in the ?-synuclein degradation, we investigated the possible regulation of Gln on the Hsp70 expression in SH-SY5Y neuroblastoma cells. Firstly, the Hsp70 expression in mRNA level was determined by quantitative PCR method in SH-SY5Y cells post Gln treatment,and the Hsp70 protein expression levels of glutamine treatment analyzed by Western blotting (Western blot analysis).We study the effect of different dose of glutamine on expression of Hsp70. The effect of time on the expression of Hsp70 in SH-SY5Y cells was studied by different treatment time of glutamine.2 In SH-SY5Y cells,study the relationship between the upregulation of Hsp70 and HSF-1 induced by glutamine Heat shock factor (HSF) has been shown to bind to the target sequence in a heat shock element (HSE), which is located in the hot promoter inducible genes and to promote the expression of heat shock proteins (HSPs), including Hsp70. In order to clarify the SH-SY5Y cell signaling pathway glutamine promote Hsp70 expression, then we study the HSF-1 knock on effect except in glutamine on expression of Hsp70. In SH-SY5Y cells, first, the expression of HSF-1 was knocked out by RNAi technique, Then the effect of siRNA-HSF-1 transfection on HSF-1mRNA level was detected by quantitative PCR, and the effect of siRNA-HSF-1 transfection on the level of HSF-1 protein was detected by blot Western technique.3 In SH-SY5Y cells, study the effect ofa-synuclein overexpression on the Hsp70 and HSF-13.1 Constructed an SH-SY5Y cell clone which overexpressed wild-type a-synucleinFunction defect of HSPs is believed to play a key role in PD. And the Hsp70 is the most investigated molecular chaperone, which negative regulates the a-synuclein aggregation in PD and in a-synuclein-induced toxicity in cells. To explore the influence on the a-synuclein degradation by the Gln-promoted Hsp70 upregulation, we then constructed an SH-SY5Y cell clone which overexpressed wild-type a-synuclein, SH-SY5Y (Syn+). The coding sequence of wild-type a-synuclein was cloned into the eukaryotic expression vector, pcDNA3.1(+).. 3.2 Effect ofa-synuclein overexpression on the expression of HSP70 and HSF-1 In order to further explore the effect of a-synuclein overexpression on the expression of HSP70 and HSF-1, we quantified the expression of Hsp70 and HSF-1 in mRNA level, by quantitative PCR, and in protein level by western blot assay.Then we compared them.4 In SH-SY5Y (Syn+) cells, study the effect of glutamine on the expression of Hsp70First, in SH-SY5Y (SYN+) cells, the regulation of glutamine on the expression level of alpha-mRNA was studied by quantitative PCR method. Then in SH-SY5Y (SYN+) cells, blotting Western was used to detect the protein levels ofa-synuclein. And in order to study the effect of glutamine dose on the expression of the a-synuclein, we compared the protein levels of thea-synuclein between 4 mM,8 mM and 16 mM. In addition, in SH-SY5Y (SYN+) cells, we studied the effect of HSF-1 knockdown ona-synuclein in the treatment of glutamine (8mM).Result:1 Glutamine upregulates the Hsp70 expression in SH-SY5Y neuroblastoma cellsFirstly, the Hsp70 expression in mRNA level was determined by quantitative PCR method in SH-SY5Y cells post Gln treatment. Figure 1A demonstrated that Gln (4-16 mM) treatment for 24 hours significantly upregulated the Hsp70 mRNA level. And there was a dose-dependence of the Gln upregulation on Hsp70 expression, there was significant difference in Hsp70 mRNA level between 4 mM and 8 mM groups, or between 8 mM and 16 mM groups. The time-dependence was also confirmed of the Hsp70 upregulation by Gln, Figure 1B indicated that Hsp70 mRNA was upregulated since 12 hours post Gln treatment with 4 mM, and peaked 24 hours later, and there was significant difference between cells treated for 12 hours and cells treated for 24 hours. To reconfirm the upregulation, we examined the Hsp70 protein level in SH-SY5Y cells post Gln treatment. Gln treatment with 4 to 16 mM for 24 hours also promoted the Hsp70 protein level. Taken together, Glutamine upregulates the Hsp70 expression in SH-SY5Y cells.2 Upregulation of Hsp70 by glutamine in SH-SY5Y cells is HSF-1-dependentFirstly RNAi technology was adopted to knockdown the HSF-1 expression, and Figure 2A demonstrated that HSF-1-specific siRNA, siRNA-HIF-1, significantly downregulated the HIF-1 mRNA level in in SH-SY5Y cells during the Gln treatment with 8 mM. And the HIF-1 protein level was also downregulated by the siRNA-HIF-1 transfection.And what's more, the Hsp70 was a lso significantly downregulated in both protein and mRNA levels, compared to the siRNA control-transfected cells. Thus, we confirmed that the Gln-promoted Hsp70 expression was HSF-1-dependent.3 a-synuclein overexpression in SH-SY5Y cells has no influence on Hsp70 and HSF-1 expression3.1 we constructed an SH-SY5Y cell clone which overexpressed wild-type a-synuclein, SH-SY5Y (Syn+). The coding sequence of wild-type a-synuclein was cloned into the eukaryotic expression vector, pcDNA3.1(+). a-syn-overexpressed SH-SY5Y cell clone was selected under 800?g/ml G418 and maintained in complete medium containing 500?g/ml G418. A significantly higher and stable level of a-Syn mRNA was observed in the SH-SY5Y (Syn+) cells post various passages. And the a-Syn expression in protein level was also significantly upregulated in the SH-SY5Y (Syn+) cells by western blot analysis. Moreover, the a-Syn overexpression did not vary among various passages.3.2 To further investigate the influence of overexpressed a-Syn on Hsp70 and HSF-1, we also quantified the expression of Hsp70 and HSF-1 in mRNA level, by quantitative PCR, and in protein level by western blot assay. Figure 3A-D demonstrated that there was no difference in bothe mRNA and protein levels of Hsp70 and HSF-1 between SH-SY5Y cells transfected CAT-pcDNA3.1(+) and SH-SY5Y (Syn+) cells, and among various passages of SH-SY5Y (Syn+) cells. Thus, the a-Syn-overexpressed SH-SY5Y cells are stable and are qualified to investigate the influence of Hsp70 and HSF-1 on the a-Syn degradation.4 Upregulation of Hsp70 by glutamine increases the a-synuclein degradation in SH-SY5Y (Syn+) cellsFirstly, the possible regulation of Gln on the ?-Syn expression in mRNA level was investigated by RT-qPCR, it was shown in Figure 4A that the 8 mM Gln had no influence on ?-Syn mRNA level in the SH-SY5Y (Syn+) cells, from 6 to 12 hours post treatment. Then we examined the protein level of ?-Syn in the SH-SY5Y (Syn+) cells post Gln treatment. Figure 4B demonstrated that the Gln treatment for 24 hours reduced the protein level of ?-Syn, compared to the internal control ?-actin. And there was a dose-dependence of the ?-Syn reduction, there was a significant difference between the 4 mMand 8 mM groups, or between the 8 mM and 16 mM groups. Moreover, we investigated the HSF-1 knockdown on the ?-Syn reduction by Gln treatment, and it was shown that 25 or 50 nM siRNA-HIF-1 inhibited the ?-Syn reduction by Gln treatment, compared to the control siRNA. Thus, we confirmed that Gln treatment promoted the ?-synuclein degradation in the SH-SY5Y (Syn+) cells, as was abrogated by HSF-1 knockdown.Conclusion:1. After glutamine treatment, in the SH-SY5Y cells the expression of heat shock protein 70 increases. It increases both in mRNA level and in protein level,and in the mRNA expression levels,the increasing of heat shock protein 70 in a dose-dependent and time-dependent manner.2 in SH-SY5Y cells, Gln-induce Hsp70 upregulation is HSF-1-dependent.3 ?-Syn overexpressed in SH-SY5Y (Syn+) cells has on influence on Hsp70 and HSF-1 expression, therefore, ?-Syn overexpressed SH-SY5Y (Syn+) cells are stable and can be used to study ?-synuclein degradation by glutamine, Hsp70 and HSF-1.4 Upregulation of Hsp70 by glutamine increases the ?-synuclein degradation in SH-SY5Y (Syn+) cells, and therefore can be considered as a target for the treatment of Parkinson's disease. |
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