| Parkinson's disease (PD) is a progressive neurodegenerative disorder characterized by the loss of dopaminergic neurons in the substantia nigra (SN), giving rise to dopamine (DA) depletion in the striatum. A number of factors have been implicated in the pathogenesis of cell death in PD, which include genetic mutations, environmental factors and oxidative stress. However the precise pathogenic mechanism leading to neurodegeneration in PD is not known. Extensive studies have demonstrated that iron plays a key role in the pathogenesis of PD. Because iron levels increase in the SN of patients with PD and animal models. Iron is a potential toxin because excessive iron can react with hydrogen peroxide to produce hydroxyl radicals and thus lead to cell death; Neuroprotection is achieved by pharmocological chelation of iron in animal models of PD.Our previous studies demonstrated that 6-OHDA administration increased nigral iron levels, thus leading to dopaminergic neuron loss; decreased expression of Ferroportinl(Fpnl) can account for the increased iron levels; and the decreased Fpnl expression is caused by increased expression of iron responsive proteinl (IRP1). IRP1 is regulated by PKC, but the regulation is tissue specific and the changes in PKC activities did not accompany changes in IRP1 activity. Therefore, in the present study, we aim to investigate IRP1 can be activated by PKC-dependent phosphorylation in dopaminergic cell line MES23.5 cells. Further, PKC activation induced by 6-OHDA can regulate IRP1 expression, thus achieving the regulation of cellusar iron levels. The results were as follows:1. The viability of cells treated with PMA (0.01- 2.0μM) for 24h was unchanged compared with control(F=3.050,p>0.05)2. IRP1 expression increased significantly after MES23.5 cells were treated with 0.2μM PMA for 24h compared with control(p<0.01). IRP1 expression decreased when MES23.5 cells were treated with 2μmol/L PKC inhibitor Bisindolylmaleimide I Hydrochloride (BisI) for 24h, compared with control (p<0.05) and PMA treatment group(p<0.01). IRP1 expression did not change compared with control (p>0.05), while IRP1 expression decreased compared with PMA treatment group (p<0.01), when MES23.5 cells were co-treated with 0.2μM PMA and 2μmol/L BisI for 24h.3. Fpnl expression decreased significantly after MES23.5 cells were treated with 0.2μM PMA for 24h compared with control(p<0.05). Fpnl expression increased when MES23.5 cells were treated with 2μmol/L BisI for 24h, compared with control (p<0.01) and PMA treatment group(p<0.01). Fpnl expression did not change compared with control (p>0.05), while Fpnl expression increased compared with PMA treatment group (p<0.05), Fpnl expression decreased compared with BisI treatment group (p<0.01), when MES23.5 cells were co-treated with 0.2μM PMA and 2μmol/L BisI for 24h.4. Iron efflux was decreased significantly after MES23.5 cells were treated with 0.2μM PMA for 24h compared with control(P<0.05). Iron efflux was enhanced when MES23.5 cells were treated with 2μmol/L BisI for 24h, compared with control (p<0.01) and PMA treatment group(p<0.01). Iron efflux did not change compared with control (p>0.05), Iron efflux was decreased compared with BisI treatment group P<0.01), when MES23.5 cells were co-treated with 0.2μM PMA and 2μmol/L BisI for 24h.5. PKCδphosphorylation increased as soon as 1h after 10umol/L6-OHDA administration compared with control (p<0.05). PKCδphosphorylation did not change after 6-OHDA were treated for 3h,6h,12h and 24h compared with control(P>0.05).6. PKCζphosphorylation increased at 6h after 10μmol/L6-OHDA administration compared with control (p<0.05), still evident after 12h(p<0.05). PKCδphosphorylation did not change after 6-OHDA were treated for 3h and 24h compared with control(p>0.05).7. PKCs phosphorylation did not change after 6-OHDA were treated for lh,3h,6h,12h,and 24h compared with control(p>0.05).8. PKCδphosphorylation increased 1h after 10μmol/L6-OHDA administration compared with control (p<0.05). PKCδphosphorylation decreased compared with 6-OHDA treatment group (p<0.05), PKCδphosphorylation did not change compard to the control (p>0.05), when MES23.5 cells were pretreatment with Rottlerin (PKCδinhibitor) for 1h, then treated with 6-OHDA for 1h.9. PKCζphosphorylation increased 6h after 10μmol/L6-OHDA administration compared with control (p<0.05). PKCζphosphorylation decreased compared with control and 6-OHDA treatment group (p<0.01) when MES23.5 cells were treated with 1μg/mL PKCζPseudo-substrate inhibitor for 1h. PKCζphosphorylation decreased compared with 6-OHDA treatment group (p<0.01), PKCζphosphorylation did not change compard to the control (p>0.05), PKCζphosphorylation increased compared with PKCζPseudo-substrate inhibitor treatment group(P<0.05), when MES23.5 cells were pretreatment with PKCζPseudo-substrate inhibitor for 1h, then treated with 6-OHDA for 6h.10. IRP1 expression increased significantly after MES23.5 cells were treated with 10μmol/L6-OHDA for 24h compared with control(p<0.01). IRP1 expression decreased (p<0.05) when MES23.5 cells were treated with 1μmol/LRottlerin for 24h. IRP1 expression did not change compared with control (p>0.05), while IRP1 expression decreased compared with 6-OHDA treatment group (p<0.05), when MES23.5 cells were pretreatment with Rottlerin for 1h, then treated with 6-OHDA for 24h.11.IRP1 expression increased significantly after MES23.5 cells were treated with 10μmol/L6-OHDA for 24h compared with control (p<0.01). IRP1 expression did not change compared with control (p>0.05), while IRP1 expression decreased compared with 6-OHDA treatment group (p<0.01), when MES23.5 cells were pretreatment with PKCζPseudo-substrate inhibitor for 1h, then treated with 6-OHDA for 24h.The above results suggest that IRP1 is activated by PKC-dependent phosphorylation in dopaminergic cell line MES23.5 cells. Fpnl expression and iron efflux are decreased when IRP1 is activated; PKC inhibitor BisI can abolish the activation of IRP1. The above results indicate that IRP1 can be activated by PKC-dependent phosphorylation in dopaminergic cell line MES23.5 cells. PKCδand PKCζare activated by 6-OHDA, while PKCεcan not be activated by 6-OHDA; PKCδinhibitor Rottlerin inhibites PKCδphosphorylation, abolishes IRP1 up-regulation induced by 6-OHDA; PKCζPseudo-substrate inhibitor also inhibites PKCζphosphorylation and abolishes IRP1 up-regulation induced by 6-OHDA. The above results indicates that PKC activation induced by 6-OHDA can regulate IRP1 expression, thus achieving the regulation of cellusar iron levels. Investigations of the role of PKC in influencing IRP1 functions suggested a relationship between PKC and iron metabolism. Since activation of PKC is a common signal transduction mechanism in a variety of physiological or pathological processes, the results of this study provide novel evidence supporting the use of PKC inhibitor in the treatment of PD, with the goal of inhibiting iron accumulation by regulation of IRP1 expression. |
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