Palladin Plays an Important Role in Pulmonary Cellular Infiltration in Differentiation Syndrome

Background

Acute promyelocytic leukemia (APL) is characterized by a specific t(15;17) chromosomal translocation, which fuses the promyelocytic leukemia (PML) gene on chromosome 15 to the retinoic acid receptor-(RARα ) gene on chromosome 17. The introduction of all-trans retinoic acid (ATRA) into the treatment strategy of APL fundamentally changed the management and outcome of this disease. Treatment with ATRA relieves this blockage, resulting in terminal differentiation of the APL blasts.

Aims

To investigate the molecular mechanisms of ATRA induced differentiation, we have described a series of novel genes up-regulated by ATRA. One of them, Rig-K (Retinoic-acid-induced gene-K), was the human homolog of palladin. This study is mainly focus on the function of palladin gene during the process of differentiation.

Methods

1. Lentivirus shRNA expression system was used to generate stable palladin konckdown NB4 cell lines. Cells morphological and the expression of differentiation markers were observed；

2. Use flow cytometry and immunofluorescence to compare the difference of cell cycle, cell proliferation, apoptosis, cell migration and granulocyte related functions such as adhesion, phagocytosis between control and palladin knockdown group;

3. Real-time PCR and chemokines antibody array were used to detect the effect of palladin reduction on the levels of chemokines secreted by NB4 cells；

4. Use microgravity rotary cell culture system to study the infiltration capacity of differentiated NB4 cells to lung tissues. Observe whether palladin knockdown play a role in this processes.

Results

1. Palladin knockdown has minor effect on NB4 differentiation based on cell morphology and granulocytic differentiation-related antigens. Palladin knockdown can partly relieved the proliferation inhibition effect during differentiation. Cycle arrest can partly relieved by palladin knock down manifest as reduced G0/G1 arrest. Palladin knock down can also remarkable reduce apoptosis rate compared with control group at 96 hours time point;

2. The phagocytosis level was about 50% dropped when the palladin was knocked down, suggest palladin play important roles in phagocytosis progress. Besides, we also observed the localization of palladin in the phagocytic cup, and palladin is colocalizated with F-actin in phagocytic cup. The migration level was also about 50% dropped when the palladin was knocked down in differentiated NB4 cells. Palladin colocalization with F-actin and vinculin, knockdown of palladin alter the distribution of F-actin and vinculin, suggest palladin take part in the formation of actin related structures;

3. Differentiation induction of APL cells is associated with increased expression of specific adhesion molecules and inflammatory cytokines, palladin knockdown decrease the upregulation of many chemokines;

4. Palladin knockdown also reduced infiltration ability of differentiated NB4 cells into lung tissues.

Conclusion

Palladin knockdown significantly decreased the granulocyte related function of differentiated APL cells, especially cell motile ability and secretion of chemokines. In vitro model shows that palladin knockdown can inhibit the invasion ability of differentiated APL cells into lung tissues, suggest palladin may play important roles in retinoic acid syndrome.