| ObjectiveThe present experiment aims to study the effect of the interaction between p38 MAPK-dependent phosphorylation and degradation of SRC-3 and RARa-mediated transcription in all-trans-retinoic acid (ATRA) treated embryonic cortical neurons.MethodsHealthy adult BALB/c mice on embryonic day 16 were selected. Embryos were extracted and their embryonic corticals were collected following the method of NEOCORTICAL CELLDISSECTION. When cells reach 70-90% confluence, cultures were treated with ATRA (1μM) for different time. Western-immunoblotting was adopted to examine the proteins including p38 MAPK, phosphorylated p38MAPK, SRC-3, phosphorylated SRC-3 and RARα. EMSA technology was performed to examine RARαtranscriptional activity. Gene expression of HOXd3 was analyzed by RT-PCR . In this experiment, cultures were treated with ATRA (1μM) as well as MG 132(5μM), the inhibitor of ubiquitin-proteasome, at 16 hr, Western-immuno- blotting was used to detect the total proteins of SRC-3. Cultures were also treated with ATRA (1μM) as well as the specific p38MAPK inhibitor SB203580 (5μM), at 16hr, Western- immunoblotting was adopted to examine the proteins including SRC-3 and phosphorylated SRC-3. EMSA technology was performed to examine RARαtranscriptional activity. Gene expression of HOXd3 was analyzed by RT-PCR . Cultures were also treated with ATRA (1μM) as well as the specific ERK (p42/44) MAPK inhibitor PD98059 (5μM), at 16 hr, Western- immunoblotting was adopted to examine the proteins including SRC-3 and phosphorylated SRC-3.Results1. Activation of p38MAPK involves ATRA-induced phosphorylation of SRC-3Following treatment of the cells with 1μM ATRA, the phosphorylation of p38MAPK was detected as early as 2 hr treatment, and the phosphorylation remained increase throughout the period of 48 hr treatment. We treated the neurons with 1μM ATRA, and the phosphorylation of SRC-3 was analyzed. Results showed that ATRA induced a time-dependent increase in phosphorylation of SRC-3. When the cells were treated with both ATRA (1μM) and SB203580 (5μM), a specific inhibitor for p38MAPK, the phosphorylation of SRC-3 was abolished by SB203580. However, when the cells were treated with both ATRA (1μM) and PD98059 (5μM), a specific inhibitor for ERK (p42/44) MAPK, the phosphorylation of SRC-3 was not abolished.2. Inhibition of p38MAPK blocks ATRA-induced SRC-3 degradationWe treated the neuron cells with 1μM of ATRA for different period of time, and the total cellular SRC-3 was analyzed by western blotting. Results showed that the total SRC-3 was not changed up to 8 hr treatment, but reduced when the cultures were treated with ATRA for 16 hr, and the total cellular SRC-3 almost disappeared following 48 hr ATRA treatment. Similar to the phosphorylation of SRC-3, ATRA-induced decrease in the total SRC-3 was reversed by specific p38MAPK inhibitors, SB203580 (5μM) (or PD169316), but was not affected by specific ERK (p42/44)MAPK inhibitor, PD98059 (5μM). Furthermore, the ATRA-induced total SRC-3 reduction was blocked when MG132 (5μM), a specific proteasome inhibitor was added to the cultures with ATRA.3. Inhibition of P38MAPK enhances RARαinteraction with RAREThe relevance of SRC-3 phosphorylation by p38 MAPK for RARα-mediated transcription activity was investigated using Electrophoretic Mobility Shift Assay (EMSA). Nuclear extracts from normal cells were subject to EASA for the specific binding of RARαto DR5 RARE. Results showed that both cool probe of DR5 RARE and anti-RARαprevented DR5 RARE complex formation, indicating that RARαspecifically interacts with DR5 RARE to form a complex. The specific binding of RARαto DR5 RARE was significantly increased in ATRA-treated fetal cortical neurons. The increased binding activity of RARαto DR5 RARE in response to ATRA (1μM) treatment started at 2 hr with a peak at 16 hr during a period of 48 hr treatment. When the neuron cells were treated with both ATRA (1μM) and SB203580 (5μM) at the same time for 16 hr, a significant increase in the ATRA-induced binding of RARαto DR5 RARE was observed.The above experiments showed a time-dependent increase in the interaction of RARαwith DR5 RARE following ATRA exposure, we investigated whether ATRA treatment increased expression of RARα. Unexpectedly, results showed that ATRA treatment decreased cellular RARαprotein. The decrease of RARαexpression occurred as early as 2 hr, and remained at the same level up to 8 hr following ATRA (1μM) addition to the cultures, the expression of RARαwas slightly further reduced when the neurons were treated with ATRA (1μM) for 16 hr and 48 hr. The decrease of RARαby ATRA treatment was not reversed when SB203580 (5μM), the specific p38MAPK inhibitor was added to the cultures.4. Inhibition of p38 MAPK increases the expression of HOXd3 geneWe used HOXd3 as a target gene in response to ATRA to investigate whether p38MAPK was involved in ATRA-induced gene expression. The expression of HOXd3 gene was analyzed using RT-PCR. The results showed that the pattern of changes in HOXd3 expression in response to ATRA was similar to that of RARαbinding to DR5 RARE. In fetal cortical neurons, the expression of HOXd3 gene was increased as early as 2 hr with a peak at 16 hr following ATRA (1μM) treatment. Treatment of the cultures with SB203580 (5μM) alone also increased expression of HOXd3, and the increase of HOXd3 expression in response to ATRA (1μM) in these cells was further enhanced by SB203580 (5μM).Conclusion1. In ATRA treated neuron cells, ATRA induces the phosphorylation of both SRC-3 and p38MAPK. Further more, ATRA induces the phosphorylation of SRC-3 via the activation of p38MAPK.2. In ATRA treated neuron cells, ATRA-induced decrease in SRC-3 results from a degradation process involving the ubiquitin-proteasome, and the phosphorylation of SRC-3 by p38 MAPK signals its degradation.3. In ATRA treated neuron cells, initially, p38MAPK increases RARα-mediated transcription through SRC-3 phosphorylation, via the control of the dynamics of the interactions of SRC-3 with RARα. Then, the inhibition of the p38MAPK-mediated phosphorylation/ degradation of SRC-3 contributes to increase RARα-mediated transcription via reversing the degradation of SRC-3 or involving other mechanisms.4. ATRA could induce the expression variation of HOXd3 gene along with RARαtranscriptional activity variation in embryonic cortical neurons. Which further demonstrate that it is p38MAPK modulates RARα-mediated transcription.
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