Next, we set out to determine which signaling pathway underlies the D2R-mediated increase of Zif268 expression. Activation of D2R, a Gαi-protein-coupled receptor, has been shown to stimulate the Erk1/2 signaling pathway through a mechanism involving the Gβγ subunit complex (Faure et al. 1994; Choi et al. 1999; Ghahremani et al. 2000; Beaulieu and Gainetdinov 2011). In addition, increases in the level of Zif268 in vitro in response to cholinergic agonists (Greenwood and Dragunow 2002), calcium influx (Rusanescu et al. 1995), or growth factor treatments (Kumahara et al. 1999; Shin et al. 2009) are dependent on the Erk1/2 signaling pathway (Knapska and Kaczmarek 2004). We therefore tested whether the D2R-mediated up-regulation of Zif268 is a consequence of a Gβγ-mediated activation of Erk1/2. First, we established that Erk1/2 is activated in response to quinpirole treatment in our model system. To do so, we measured levels of phosphorylated, and thus activated, Erk1/2 (pErk1/2) in the presence and absence of quinpirole. As shown in Fig. 5a, quinpirole treatment caused a significant increase in the levels of pErk1/2 within 15 min. To test whether the D2R-mediated activation of Erk1/2 requires Gβγ, we pre-treated the cells with gallein, a small molecule that binds to a domain on Gβγ that is essential for its association with downstream targets (Bonacci et al. 2006). As shown in Fig. 5b, we found that pre-treatment of cells with gallein (20 μM) blocked the increase in Erk1/2 phosphorylation in response to quinpirole. Together, these results show that D2R activation leads to a Gβγ-dependent activation of the Erk1/2 signaling pathway. To determine whether the D2R-mediated up-regulation of Zif268 expression and the subsequent increase in GDNF are the consequence of the Gβγ-mediated activation of the Erk1/2 signaling pathway, cells were pre-treated for 10 min with either gallein (20 μM), or the mitogen-activated protein kinase kinase (MEK, the kinase immediately upstream of Erk1/2) inhibitors U0126 (10 μM), or PD98059 (10 μM) prior to a 30- or 240-min treatment with 50 μM quinpirole, and the levels of Zif268 and GDNF mRNA, respectively, were assessed. As shown in Fig. 6a, the Gβγ inhibitor attenuated the D2R-mediated increase of Zif268, as compared with the vehicle-pre-treated cells. In addition, both MEK inhibitors lowered the basal level of Zif268 expression and blocked the D2R-mediated up-regulation of Zif268 mRNA, as compared with the vehicle-pre-treated controls (Fig. 6a). Moreover, the expression levels of GDNF after D2R activation were also attenuated in the presence of either the MEK or the Gβγ inhibitors (Fig. 6b). Taken together, our findings suggest that quinpirole-mediated activation of the D2R leads to a Gβγ-dependent activation of the Erk1/2 signaling pathway, which is necessary for both Zif268 and GDNF expression.
Figure 5. Activation of the Dopamine D2 receptor in SH-SY5Y cells results in a Gβγ-dependent increase in extracellular signal–regulated kinase 1/2 (Erk1/2) phosphorylation, (a) Cells were treated with quinpirole (QP, 50 μM) for 5, 15, and 30 min (black bars) or saline (white bar). Western blot was used to measure levels of phosphorylated Erk1/2 (pErk1/2). Image is a representative of four independent experiments. Levels of pErk2 (lower band in the upper panel) were normalized to the total Erk2 protein levels. Glyceraldehyde-3-phosphate dehydrogenase was used as a loading control. Bar graph represents the mean immunoreactivity signal of pErk2/Erk2 ± SEM. *p < 0.05, as compared with saline (one-way anova with post hoc Student–Newman–Keul's). (b) Cells pre-treated for 10 min with the Gβγ inhibitor gallein (20 μM, in dimethylsulfoxide (DMSO) vehicle) or an equal volume of DMSO at a final concentration of 0.1%. Cells were then treated with 50 μM quinpirole (black bars) or saline (white bars) for 15 min, the time point of maximal Erk1/2 pathway activation determined in (a). Western blot analysis was used to assess the level of pErk1/2, as in (a). Image is a representative of four independent experiments. Bar graph represents the mean immunoreactivity signal of pErk2/Erk2 ± SEM. *p < 0.05, as compared with the saline-treated control (two-way anova with post hoc Bonferroni comparisons within each pre-treatment).
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Figure 6. Dopamine D2 receptor-mediated up-regulation of zinc-finger protein 268 (Zif268) and glial cell line–derived neurotrophic factor (GDNF) is Gβγ and extracellular signal–regulated kinase 1/2 dependent. Cells were pre-treated for 10 min with the Gβγ inhibitor gallein (20 μM, in dimethylsulfoxide (DMSO) vehicle), or the MEK inhibitors U0126 or PD98059 (both at a concentration of 10 μM, in DMSO vehicle), or an equal volume of DMSO at a final concentration of 0.1%. Cells were then incubated for 30 min (a) or 240 min (b) with quinpirole (QP; 50 μM; black bars) or saline (white bars). The level of Zif268 (a) and GDNF (b) expression was determined by RT-PCR. Images are representative of four independent experiments. Zif268 and GDNF levels (upper panels) were normalized to those of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (lower panels). Bar graphs depict the mean Zif268 or GDNF/GAPDH ± SEM (white bars: saline-treated groups; black bars: QP-treated groups). ***p < 0.001, as compared with the saline-treated control (two-way anova with post hoc Bonferroni comparisons within each pre-treatment).
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