Activating transcription factor 3 up-regulated by c-Jun NH2-terminal kinase/c-Jun contributes to apoptosis induced by potassium deprivation in cerebellar granule neurons
Section snippets
Neuronal culture and potassium deprivation
Rat CGNs were prepared from 7 to 8-day-old Sprague–Dawley rat pups (15–19 g) as described previously (Li et al., 2000, Ma et al., 2007). Briefly, neurons were dissociated from freshly dissected cerebella by mechanical disruption in the presence of trypsin and DNase and then seeded at a density of 1.5×106 cells/ml in basal modified Eagle medium (BME) containing 10% fetal bovine serum and 25 mM KCl (25 K+S). Cytosine arabinoside (10 μM) was added 24 h after seeding to limit the growth of
JNK/c-Jun is activated and ATF3 is up-regulated during apoptosis induced by potassium deprivation in CGNs
To determine whether potassium deprivation evokes the activation of JNK/c-Jun in CGNs, DIV7 neurons were switched to 25 K or 5 K for various duration of time (0.5, 1, 2, 4 h), and then processed for Western blotting with antibodies against phospho-JNK and phospho-c-Jun. Consistent with previous studies (Coffey et al., 2002, Ma et al., 2007), potassium deprivation (5 K vs. 25 K) led to an increase in the level of phospho-JNK starting at 0.5 h without detectable changes in total JNK level (Fig. 1
Discussion
Although the induction of ATF3 during neuronal apoptosis has been widely reported, the role of ATF3 in neuronal apoptosis has not been well studied. To the best of our knowledge, this is the first report that utilizes siRNA targeting ATF3 for analysis of the role of endogenous ATF3 during neuronal apoptosis. By knockdown of ATF3, we demonstrate that the up-regulation of ATF3 mediated by JNK/c-Jun pathway contributes to apoptosis induced by potassium deprivation in CGNs. JNK/c-Jun pathway plays
Conclusion
In summary, our results suggest that JNK/c-Jun mediates the up-regulation of ATF3 and ATF3 contributes to apoptosis induced by potassium deprivation in CGNs.
Acknowledgment
This work was supported by the National Natural Science Foundation of China (grants 30570562, 30629002 and U0632006); the Natural Science Foundation of Guangdong Province (grant 5100982); Y.-P.C. was financially supported by Hong Kong University/NFSC Young Research Award.
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