Subcellular localization of DAXX influence ox-LDL induced apoptosis in macrophages.

Here we aimed to evaluate the effects of DAXX subcellular localization on ox-LDL induced macrophages apoptosis. Cytoplasmic localization vector DAXX-W621A and nuclear localization vector DAXX-S667A were constructed by point mutation in DAXX. Blank vector, full length DAXX, DAXX-W621A, DAXX-S667A was transfect into RAW264.7 cells, respectively. Then the cells were incubated with 100 mg/ml ox-LDL for 48 h. Immunofluorescent assay was used to assay the localization of DAXX. MTT and Flow cytometry was used to determine cellular viability and apoptosis. RT-PCR and Western blot were used to analyze the expression levels. A significantly increased expression of DAXX was found in transfected cells of DAXX. The content of DAXX in nucleus was significantly increased in DAXX(S667A), and DAXX was significantly increased in cytoplasm of DAXX(W621A). Besides, we found DAXX was mainly expressed in nucleus with a low-level expression in cytoplasm through immunofluorescence. However in DAXX(W621A) group, the DAXX began to transferred to cytoplasm, which exhibited significant florescence. After treated with ox-LDL, the cytoactive of DAXX-W621A exhibited significantly decreased level when compared DAXX group. However, after added inhibitor LMB, the inhibition was relieved. The cell viability was also significantly increased in DAXX-S667A group. The results of apoptosis rates were similar in each group. Furthermore, we found the expression of ASK1 and JNK was also consistent with the apoptosis rates. Cytoplasmic localization of DAXX can increase injury sensitivity of ox-LDL on cells, and nuclear localization can antagonise the effect of ox-LDL. Besides, it is certified ox-LDL induced apoptosis is mainly through ASK1-JNK pathway.